Developments in Bee Products & Apitherapy Research 2019 P 213

Developments in Bee Products Research & Apitherapy 2019
1. Rediscovery of Apitherapy Mechanisms: A Novel Challenge in Cardiovascular Autonomic

Neuropharmacology
Miran KR* Pharmacology and Biochemistry Department, Faculty of Pharmacy, The British University in Egypt, Cairo, Egypt, June 2019
2. Apitherapy Have a Role in Treatment of Multiple Sclerosis

Suzette I. Helal1*, Ahmad Hegazi2, Khaled Al-Menabbawy3 1National Research Center - Research on Children with Special
Needs Department, Giza, Egypt; 2National Research Center – Child Health, Cairo, Egypt; 3National Research Center – Microbiology and
Immunology, Cairo, Egypt, June, 2014
3. Novel Therapeutic Modality Employing Apitherapy for Controlling of Multiple Sclerosis
Ahmad G Hegazi*, Khaled Al-Menabbawy, Eman H Abd El Rahman and Suzette I Helal Department of
Zoonotic Diseases, National Research Center, Dokki, Giza, Egypt, Feb 2015
4. Bee Venom: Overview of Main Compounds and Bioactivities for Therapeutic Interests
Rim Wehbe 1,y , JacintheFrangieh 1,2,y, MohamadRima 3,4, DanyElObeid 5, Jean-Marc Sabatier 6
and ZiadFajloun 1,7,* Aug 2019
5. Comparison of Physicochemical, Microbiological Properties and Bioactive Compounds

Content of Grassland Honey and other Floral Origin Honeys
Laura AgripinaScripca, Liliana Norocel and Sonia Amariei* Faculty of Food Engineering, Stefancel Mare University of Suceava,
720229 Suceava, Romania * Correspondence:sonia@usm.ro;Tel.: +40-230-216-147. Aug 2019
6. Correlation Study of Honey Regarding their Physicochemical Properties and Sugars and
Cyclitols Content
Ileana AndreeaRatiu 1,2,3 , HossamAl-Suod 1,2, MałgorzataBukowska 1,2, MagdalenaLigor 2 and BogusławBuszewski Dec 2019
7. Volatile Profile and Physico-Chemical Analysis of Acacia Honey for Geographical Origin
and Nutritional Value Determination
Niculina M.Mada¸s 1,2, LiviuA.Marghita¸s 1, DanielS.Dezmirean 1, VictoritaBonta 3, Otilia Bobi¸s 3,*, Marie- LaureFauconnier 4 ,
Frédéric Francis 2, EricHaubruge 2 and Kim B.Nguyen 2 Sept 2019
8. Dissecting the Antimicrobial Composition of Honey
Victoria C. Nolan, James Harrison and Jonathan A. G. Cox *, School of Life and Health Sciences, Aston University, Aston
Triangle, Birmingham B4 7ET, UK; Dec 2019
9. Organic Bee Pollen: Botanical Origin, Nutritional Value, Bioactive Compounds,
Antioxidant Activity and Microbiological Quality
Xesús Feás 1,*, M. Pilar Vázquez-Tato 1, Leticia Estevinho 2, Julio A. Seijas 1 and Antonio Iglesias. Department of Organic Chemistry,
Faculty of Science, University of Santiago de Compostela. CIMO-Mountain Research Center, Agricultural College of Bragança,
Polytechnic Institute of Bragança, Campus Santa Apolónia. Department of Anatomy and Animal Production, Faculty of Veterinary
Medicine, University of Santiago de Compostela. July 2012
10. Antioxidant properties of bee pollen
1Damir Aličić1*, Drago Šubarić2, Midhat Jašić3, Hatidža Pašalić3, Đurđica Ačkar2. 1High Secondary
School ''Čelić'', 75246 Čelić, Bosnia and Herzegovina. 2Josip Juraj Strossmayer University of Osijek, Faculty of
Food Technology Osijek, Franje Kuhača 20, HR-31000 Osijek, Croatia. 3Faculty of Pharmacy, University of
Tuzla, Univerzitetska 8, 75000 Tuzla, Bosnia and Herzegovina. 2014
11. Phenolic Composition Influences the Health-Promoting Potential of Bee-Pollen
1 University ofBelgrade—FacultyofChemistryP.O.Box51,11158Belgrade,Serbia; mosicm@chem.bg.ac.rs
(M.M.);jvelicko@chem.bg.ac.rs(J.T.);ztesic@chem.bg.ac.rs(Ž.T.) 2 Department of Food Chemistry, National Institute of
Chemistry, Hajdrihova19,SI-1000 Ljubljana, Slovenia. Nov 2019
12. Bee Collected Pollen and Bee Bread: Bioactive Constituents and Health Benefits
Rodica Margaoan 1 , MirelaStrant, 2, AlinaVaradi 2, ErkanTopal 3 , BanuYücel 4, Mihaiela Cornea-Cipcigan 5,*, MariaG.Campos 6,7,* and
Dan C.Vodnar 8. November 2019
13. Royal Jelly: An ancient remedy with remarkable antibacterial properties
Filippo Fratini a,b,∗, Giovanni Cilia a, Simone Mancinia, Antonio Felicioli a,b June 2016
14. Apitherapy Hospital in Russia
Compiled by Harihar Adhikari, Chief, Centre for Industrial Entomology Development, DOA, Nepal
Neurology & Neurotherapy Open Access Journal
ISSN: 2639-2178
Rediscovery of Apitherapy Mechanisms: A Novel Challenge in

Cardiovascular Autonomic Neuropharmacology
Miran KR*
Review Article
Pharmacology and Biochemistry Department, Faculty of Pharmacy, The British
Volume 4 Issue 1
University in Egypt, Cairo, Egypt Received Date: May 30, 2019
Published Date: June 18, 2019
*Corresponding author: Dr. Miran KR, Pharmacology and Biochemistry Department, DOI: 10.23880/nnoaj-16000134
Faculty of Pharmacy, The British University in Egypt, El Sherouk City, Suez Desert Road,
Cairo 11837, Egypt, Email: miran.rakha@bue.edu.eg; mirankhalil@hotmail.com
Abstract
Apitherapy is the scientific term which refers to the medical use of honey, bee venom, propolis, royal jelly, bee pollen,
beeswax and apilarnil. Bee venom therapy for arthritis, autoimmune and neurodegenerative diseases is the most well-
known. Numerous studies supported honey for healing of wounds and burns, and even as an anticancer agent. Moreover,
several studies evaluating the cardiovascular effects of honey bee products revealed very promising results,
paradoxically, apitherapy is still not in focus with a cardiovascular neuropharmacological viewpoint. In an effort to
provide extensive information about the cardiovascular bioactivity of natural honey, as the major honey bee product,
both in terms of familiarity and profit, this review overviews, for the first time; (1) cardioactive and vascoactive effects of
natural honey, (2) mechanisms underlying cardioactive and vasoactive potentials of natural honey, and (3) how
improving our knowledge of the mechanisms mediating protective and ameliorative outcomes of natural honey may lead
to novel therapeutic strategies for the treatment of cardiovascular diseases? Future challenge remaining will be to permit
full exploitation of apitherapy potency to expand novel horizons in the cardiovascular autonomic neuropharmacology.
Keywords: Cardiovascular Autonomic Neuropharmacology; Antihypertensive Effect; Antiarrhythmic Effect;

Ameliorative Effect to Cardiovascular Risk Factors; Biotherapy; Apitherapy
Introduction of honey in healing of wounds and burns are supported by

numerous studies [9-12]. Recently, evidence is growing
Apitherapy or bee therapy is the medicinal use of the that honey have the potential to be anticancer agent [13-
natural products made by bees such as honey, bee venom, 15].
propolis, royal jelly, bee pollen, beeswax and apilarnil to
prevent or treat illness and promote healing [1]. A wide Several studies evaluating the cardiovascular effects of
variety of conditions and diseases have been suggested as honey bee products revealed very promising results,
candidates for apitherapy, the most well-known being bee paradoxically, apitherapy is still not in focus with a
venom therapy for arthritis, autoimmune and cardiovascular neuropharmacological viewpoint. Despite
neurodegenerative diseases [2-8]. The potential benefits researchers who are interested in this field of study
Rediscovery of Apitherapy Mechanisms: A Novel Challenge in Neurol Neurother

Cardiovascular Autonomic Neuropharmacology
2
produced studies which are still considered scarce and may ameliorate oxidative stress via upregulation of Nrf2
there is paucity in their reports, the majorities of their activity or expression [17,18].
emerged results are of special interest and deserve to be
taken into consideration. Therefore, we should give them A combination of diabetes mellitus and hypertension
their due recognition. In the recent years, more attention is associated with substantially increased cardiovascular
was paid to the cardioactive and vasoactive effects of risk factors [42]. Besides other factors, evidence suggests
natural honey revealing its antihypertensive effect, its that diabetes mellitus may exacerbate hypertension via
antiarrhythmic effect, and its ameliorative effect to increased oxidative stress [43]. Induction of experimental
cardiovascular risk factors [16-38]. In an effort to provide diabetes by streptozotocin (STZ) in animal models has
extensive information about the cardiovascular been used extensively to study the relationship between
bioactivity of natural honey, as the major honey bee diabetes and autonomic cardiovascular dysfunction [44].
product, both in terms of familiarity and profit, and to the Recently, the effects of honey on blood pressure,
best of our knowledge, this review overviews, for the first antioxidant enzymes, and markers of oxidative stress in
time, the influence of natural honey on the cardiovascular the kidneys of STZ-induced diabetic Wistar-Kyoto (WKY)
neurophysiology, toward opening of a novel gateway to and spontaneously hypertensive rats (SHR) were
the cardiovascular autonomic neuropharmacology. investigated [19]. It was indicated that STZ-induced
diabetes mellitus caused reduced systolic blood pressure
The Antihypertensive Effect of Natural (SBP), total antioxidant status (TAS), activities of
Honey glutathione peroxidase (GPx) and glutathione reductase
(GR) in SHR. In contrast, SBP, activities of GPx and GR
The role of oxidative stress in hypertension is a were elevated while TAS was reduced in diabetic WKY.
subject of much research interest [16]. Oxidative stress is Honey treatment further reduced SBP in diabetic SHR but
implicated in the pathology of hypertension, however not in diabetic WKY. It also increased TAS, reduced
some evidence also indicates that hypertension generates glutathione (GSH)/oxidized glutathione (GSSG) ratio, GSH,
oxidative stress [39,40]. These lines of evidence support a activities of GPx and GR in diabetic SHR. It was revealed
role of oxidative stress as an important determinant in the that oxidative stress is implicated in the pathogenesis
imbalance between vasoconstrictor and vasodilator and/or complications of hypertension and/or diabetes
mechanisms [17,39,40]. The beneficial effects of mellitus. Moreover, if hyperglycemia is not a prerequisite
antioxidants in ameliorating oxidative stress and for the development of elevated blood pressure in
suppressing or reducing elevated blood pressure in diabetic normotensive rats, then oxidative stress in
experimental and clinical studies further corroborate the diabetic normotensive rats may be a consequence of
role of oxidative stress in hypertension [41]. A recent hypertension and/or diabetes. Furthermore, it was
study investigated the effect of honey on elevated systolic elucidated that oxidative stress, unlike in SHR, may not
blood pressure (SBP) in spontaneously hypertensive rats contribute considerably to elevated SBP in STZ-treated
(SHR). It also evaluated the effect of honey on the diabetic rats. The inability of honey to decrease elevated
amelioration of oxidative stress in the kidney of SHR as a SBP of diabetic WKY further substantiates this view. The
possible mechanism of its antihypertensive effect [17]. It effectiveness of honey not only in ameliorating oxidative
was reported that honey supplementation reduced SBP stress but also in reducing elevated SBP in diabetic SHR
and renal malondialdehyde (MDA) levels in SHR. corroborate the prominent role of oxidative stress in
Suppressed elevation in SBP was also accompanied by elevated SBP of SHR. Therefore, it was assumed that
reduced activities of glutathione S-transferase (GST) and differences in types, severity, and
catalase (CAT) in the kidney of SHR. Findings from this combinations/complications of diseases (hypertension
study also indicated that mRNA expression of nuclear and/or diabetes), as well as strains, may influence
factor erythroid 2-related factor 2 (Nrf2), a potential responses to blood pressure and antioxidant enzymes in
renoprotective transcription factor, was markedly response to changes in redox status [19,45].
reduced or impaired in the kidney of SHR. Besides, the
results of study revealed that the induction of antioxidant The Antiarrhythmic Effect of Natural
enzymes, perhaps as a compensatory mechanism, may Honey
occur in spite of impaired or reduced mRNA expression of
Nrf2. Consequently, it was indicated that honey Hazards of antiarrhythmic drugs (such as lethal
supplementation in SHR elicits antihypertensive effect via arrhythmias in some patients) have led to a limitation on
amelioration of renal oxidative stress. Moreover, honey the administration of antiarrhythmic drugs. Hence, there
Miran KR. Rediscovery of Apitherapy Mechanisms: A Novel Copyright© Miran KR.

Challenge in Cardiovascular Autonomic Neuropharmacology.
Neurol Neurother 2019, 4(1): 000134.
3
is a tendency to use drugs which have less adverse effects Na+/Ca2+ exchange, prevents the accumulation of Ca2+ ions
and more efficacies [46]. On one hand, the administration within the cardiac cells [61], reducing the occurrence of
of exogenous catecholamines, for example, is mandatory ventricular arrhythmias such as ventricular extrasystole
to support the failing circulation in acutely ill patients. In resulting from increased sympathetic activity under the
contrast to the short-term benefits, prolonged adrenergic influence of catecholamines [20-22,59]. Chlorine is
stress is detrimental to the cardiovascular system by essential for the process of Cl-/HCO3- exchange which is
initiating a series of adverse effects triggering significant concerned with pH regulation in cardiac cells [60] and is
cardiotoxicity [47]. On the other hand, it was evident that activated by natural honey in response to its alkaline
sympathetic overactivity holds a key neurophysiological potentiality. Therefore, it can suppress or reduce the
role in hypertension, heart failure, acute coronary activity of Na+/HCO3- symporter which is stimulated by β-
syndromes, Takotsubo (stress) cardiomyopathy, agonists leading to the cardiac arrhythmias produced by
metabolic syndrome, phaeochromocytoma (an adrenaline catecholamines as a result of calcium overload [20-22,59].
secreting tumor), and arrhythmias [48-58].
In this respect, the role that fructose (the major
For the first time, using a novel rat model for constituent in natural honey) plays in the antiarrhythmic
hyperadrenergic activity, the first evidence was provided effect of natural honey cannot be overlooked. For the first
that natural wild honey not only can be used to protect time, a recent study demonstrated an acute influence of
against the deleterious effects of epinephrine fructose on isolated cardiomyocyte excitation-contraction
(adrenaline), as a chemical mediator and an endogenous coupling [62]. The findings indicated cardiomyocyte
hormone, which can be a hidden menace in cases of capacity to transport and functionally utilize exogenously
hyperadrenergic activity, but also can be used to treat the supplied fructose. In a ‘proof-of-principle’ manner, the
side adverse effects of epinephrine, as an exogenous study provided functional demonstration that fructose
sympathomimetic and β-agonist, thereby facilitating its may serve as a substrate to support cardiomyocyte
safe use as an inotrope [21]. It was proven that natural excitation-contraction coupling in an acute setting.
wild honey exerts its cardioprotective and therapeutic Furthermore, it was established that the fructose-specific
effects against epinephrine-induced cardiac disorders and transporter, GLUT5, is expressed in cardiomyocytes and
vasomotor dysfunction directly, via its very pronounced may provide a route of cardiomyocyte fructose entry. The
total AOC and its great wealth of both enzymatic and investigation suggested that fructose may provide ATP for
nonenzymatic antioxidants involved in cardiovascular this transporter thus increasing the driving force for
defense mechanisms, besides its substantial quantities of Na+/Ca2+ exchange and promoting Ca2+ extrusion, which
mineral elements such as magnesium, sodium, and may prevent the accumulation of Ca2+ ions within the
chlorine, and/or indirectly, via the enhancement of cardiac cells [61], reducing the occurrence of ventricular
endothelium-derived relaxing factor nitric oxide arrhythmias [20-22,59].
(EDRF/NO) release through the influence of ascorbic acid
(vitamin C) [20,21]. Ischemic heart disease (IHD) causes more deaths and
disability and incurs greater economic costs than any
In isolated toad hearts, the cardiac arrhythmias other illness in the developed world. Cardiac arrhythmias
induced by epinephrine, included; extrasystoles, and myocardial infarction are serious manifestations of
tachyarrhythmias and bradyarrhythmias as well as IHD. In the course of cardiac surgery and myocardial
abnormalities of both P-wave and ST segment [22,57]. infarction, ventricular arrhythmias such as ventricular
The in vitro study elucidated that natural wild honey tachycardia and ventricular fibrillation are the most
managed to suppress or attenuate the cardiotoxicity important causes of mortality [63]. A group of evidence
produced by epinephrine. At the same time, it potentiated supported potential cardioprotective effects of natural
the powerful inotropic effect of epinephrine [22,59]. The honey against ischemia/reperfusion (I/R)-induced
antiarrhythmic effect of natural wild honey against injuries, and consequently against cardiac arrhythmias
catecholamine cardiotoxicity was mainly attributed to its and myocardial infarction [23-31]. It was articulated that
minerals such as magnesium, sodium and chlorine [20- short-term perfusion of isolated rat heart with natural
22,59]. Magnesium, by inhibiting the voltage-dependent honey as a pharmacologic preconditioning agent has
calcium channels, suppresses the delayed-after- prophylactic effects on I/R-induced cardiac arrhythmias
depolarization (DAD) which is produced by and myocardial infarction [31]. Besides, it could reduce
catecholamines and it is considered the cause of their left ventricle perfusion pressure [27]. Also, prolonged
cardiac arrhythmias [60]. Sodium, through the process of (long-term) preconditioning of isolated rat heart with

4
natural honey has antiarrhythmic and anti-infarct inflammatory and antioxidant properties [36]. On one
activities against I/R-induced injuries [24]. In addition, hand, consumption of natural honey could lower
chronic administration of natural honey produced concentrations of inflammatory mediators such as plasma
antiarrhythmic effects against I/R-induced cardiac and urinary prostaglandin E2 (PGE2), prostaglandin F2
arrhythmias in isolated rat heart [29]. Moreover, acute alpha (PGF2 alpha) and thromboxane B2 (TXB2) of
administration of natural honey in normothermic healthy individuals [68,69]. On the other hand, many
ischemic conditions could protect the isolated rat heart as reports have demonstrated lowering effects of
the reduction of cardiac arrhythmias and myocardial antioxidants on CRP levels [70,71]. Supporting this is that
infarction size [25]. On the other hand, preischemic a wide range of phenolic constituents, which act as
administration of natural honey before zero flow global natural antioxidants, is present in honey like quercetin,
ischemia showed protective effects against I/R-induced caffeic acid, acacetin, kaempferol, galangin and have a
cardiac arrhythmias and myocardial infarction in isolated promising pharmacological role in preventing
rat heart [28]. Furthermore, postischemic administration cardiovascular diseases [72]. Many epidemiological
of natural honey in global ischemia protected isolated rat studies have shown that regular flavonoid intake is
heart against I/R-induced injuries [26]. Antioxidant and associated with a reduced risk of cardiovascular diseases.
free radical scavenging activity, reduction of necrotized In coronary heart disease, the protective effects of
tissue, existence of rich energy sources such as glucose phenolic compounds include mainly antithrombotic, anti-
and fructose, and improvement of some hemodynamic ischemic, antioxidant, and vasorelaxant. It was suggested
functions were supposed to be responsible for these that flavonoids decrease the risk of coronary heart
cardioprotective effects of natural honey [24- disease by three major actions: improving coronary
26,28,29,31]. vasodilatation, decreasing the ability of platelets in the
blood to clot, and preventing low-density lipoproteins
The Ameliorative Effect of Natural Honey (LDLs) from oxidizing. Additionally, it was elucidated that
to Cardiovascular Risk Factors CRP, at concentrations known to predict adverse vascular
events, directly quenches the production of nitric oxide
Currently, there is much evidence linking (NO), in part, through the posttranscriptional effect on the
cardiovascular autonomic functions and the most well- endothelial NO synthase (eNOS) mRNA stability.
established major cardiovascular risk factors including Diminished NO bioactivity, in turn, inhibits angiogenesis,
dyslipidemia, hypertension, diabetes mellitus, and obesity an important compensatory mechanism in chronic
[64-67]. Various clinical and preclinical studies focused ischemia. Through decreasing NO synthesis, CRP may
on potential influence of natural honey on cardiovascular facilitate the development of diverse cardiovascular
risk factors [33]. Emerging results of these studies should diseases. Thereby, risk reduction strategies designed to
be taken into consideration as indicators of the indirect lower plasma CRP may be effective by improving NO
effect of natural honey on the cardiovascular autonomic bioavailability [73]. In this regard, it was reported that
functions. Especially, it is now substantiated that natural natural honey increased the contents of plasma and
honey ameliorates cardiovascular risk factors in healthy urinary nitrite/nitrate, the stable NO metabolites or end
individuals and in patients with elevated cardiovascular products, in healthy individuals as well as experimental
risk factors [36-38], as well as in experimental animals animals [68,74]. Therefore, natural honey may partly
[32,34,35]. It was indicated that consumption of honey, affect the CRP levels through NO production [36].
particularly in hyperlipidemic, hypertensive, diabetic and
overweight or obese individuals, reduces the levels of Of great importance is that honey contains a number
total cholesterol (TC), low-density lipoprotein cholesterol of mineral elements such as potassium, calcium, sodium,
(LDL-C), triglycerides (TG), C-reactive protein (CRP), magnesium, phosphorus, zinc, copper, iron, manganese,
fasting blood glucose (FBG), homocysteine (HC) and blood chromium, and selenium [75]. Some of these minerals
pressure (BP) in hypertensive patients. Moreover, it such as chromium are recognized for their role in
increases the levels of high-density lipoprotein lowering plasma total cholesterol, reduction of elevated
cholesterol (HDL-C) and plasma insulin (PI). Furthermore, blood glucose, maintenance of normal glucose tolerance
it does not increase body weight in overweight or obese and insulin secretion from the pancreatic beta-cells
subjects. [76,77]. Other studies have also demonstrated that zinc
and copper can improve insulin sensitivity thereby
The ameliorative effect of natural honey to CRP level, decreasing blood glucose levels [78,79]. Even though the
as a marker of inflammation, was explained by its anti- amounts of these minerals in honey may be low, it is

5
suggested that the daily ingestion of honey may achieve several studies, reveals that natural honey has very
adequate concentrations of these minerals and thus exert promising protective and ameliorative outcomes which
pharmacological responses [80]. Coupled with the cannot be ignored. Consequently, more in-depth in vitro
evidence which proved that the daily consumption of and in vivo studies jointly with clinical trials should be
honey increased serum concentrations of zinc and copper initiated to objectively substantiate efficacy of natural
in healthy subjects, these ions may contribute to the honey as an important complementary and/or alternative
antidiabetic effect of honey [78,79,81]. Surprisingly, it was apitherapy, its rationale, evidence supporting its use, its
reported that fructose feeding in mice was associated possible interaction with conventional medicines, and
with lower blood glucose levels [82]. Increased C-peptide where possible, what is known about its safety and
and insulin secretion and modulation of appetite- efficacy to further validate natural honey in medical
regulating hormones such as leptin, ghrelin and peptide applications as a promising biotherapy.
YY may also contribute to improved glycemic control as a
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Open Access Macedonian Journal of Medical Sciences. 2014 Jun 15; 2(2):265-270.
http://dx.doi.org/10.3889/oamjms.2014.044
Clinical Science
Apitherapy Have a Role in Treatment of Multiple Sclerosis

1* 2 3
Suzette I. Helal , Ahmad Hegazi , Khaled Al-Menabbawy
1 2
National Research Center - Research on Children with Special Needs Department, Giza, Egypt; National Research Center
3
– Child Health, Cairo, Egypt; National Research Center – Microbiology and Immunology, Cairo, Egypt
Abstract
Citation: Helal SI, Hegazi A, Al-Menabbawy K. AIM: Multiple sclerosis (MS) is an inflammatory disease in which the fatty myelin sheaths around
Apitherapy Have a Role in Treatment of Multiple the axons of the brain and spinal cord are damaged. We Study the effect of Apitherapy in treatment
Sclerosis. OA Maced J Med Sci. 2014 Jun 15;
2(2):265-270. of MS.
http://dx.doi.org/10.3889/oamjms.2014.044
Key words: Apitherapy; Multiple Sclerosis; Bee
MATERIAL AND METHODS: Fifty patients with MS, their ages ranged between 26-71 years, were
venom; Food Supplements. subjected to complete clinical and neurological history and examination to confirm the diagnosis. All
*
Correspondence: Suzette Helal, Children with cases were under their regular treatment they were divided into two main groups, Group I received
Special Needs Department, National Research honey, pollen, royal jelly and propolis and were treated with apiacupuncture 3 times weekly, for 12
Centre, El-Bohouth Street, Dokki, 2311, Cairo, months, in addition to their medical treatment, while group II remains on their ordinary medical
Egypt. Fax: +202 3370931, Tel:
+201001004562. E-mail: treatment only. Apiacupuncture was done by bee stings for regulating the immune system.
suzettehelal@yahoo.com
Received: 07-Apr-2014; Revised: 18-Apr-
RESULTS: Results revealed that 4 patients showed some improvement regarding their defects in
2014; Accepted: 05-May-2014; Online first: gait, bowel control, constipation and urination, while 12 cases, showed some mild improvement in
22-May-2014 their movement in bed, and better improvement in bed sores, sensation, and better motor power,
Copyright: © 2014 Helal et al. This is an open only two cases of them were able to stand for few minutes with support.
access article distributed under the terms of
the Creative Commons Attribution License, CONCLUSION: Although Apitherapy is not a curable therapy in MS, but it can be used to minimize
which permits unrestricted use, distribution,
and reproduction in any medium, provided the the clinical symptoms of MS, and can be included among programs of MS therapy.
original author and source are credited.
Competing Interests: The authors have
declared that no competing interests exist.
Introduction 5]. The primary aims of therapy are returning function

after an attack, preventing new attacks, and
Multiple sclerosis (MS) is an inflammatory preventing disability [6].
disease in which the fatty myelin sheaths around the
axons of the brain and spinal cord are damaged, Apitherapy is the medical use of honey
leading to demyelination and scarring as well as a bee products. This can include the use of honey,
broad spectrum of signs and symptoms [1]. MS is an pollen, bee bread, propolis, royal jelly, and apilarnil
important problem both for people with the disease and bee venom. Most claims of apitherapy have not
and for society. There is no cure, and alleviation of been proved to the scientific standards of evidence-
symptoms forms the cornerstone of care. Excessive based medicine and are anecdotal in nature. Bee
fatigue that severely limits activity is experienced by at venom therapy is an alternative form of healing. Bee
least two-thirds of the estimated 60,000 people with venom therapy is the part of apitherapy which utilizes
MS in the UK [2]. There is no known cure for multiple bee venom in the treatment of health conditions.
sclerosis. Treatments attempt to return function after However, bee venom is a complex mix of a variety of
an attack, prevent new attacks, and prevent disability peptides and proteins, some of which have strong
[3]. MS medications can have adverse effects or be neurotoxic and immunogenic effects [7]. It has been
poorly tolerated, and many people pursue alternative used since ancient times to treat arthritis, rheumatism,
treatments, despite the lack of supporting scientific back pain, skin diseases and in this modern age as an
study. The prognosis is difficult to predict; it depends alternative therapy to treat autoimmune diseases,
on the subtype of the disease, the individual's disease Lyme disease and chronic fatigue syndrome [8, 9].
characteristics, the initial symptoms and the degree of Some reports have shown beneficial effects of bee
disability the person experiences as time advances [4, venom in postherpetic neuralgia [10], swine flu [11],
_______________________________________________________________________________________________________________________________
OA Maced J Med Sci. 2014 Jun 15; 2(2):265-270. 265

Clinical Science
_______________________________________________________________________________________________________________________________
fibromyalgia and multiple sclerosis [12]. There is no (roughly 5 tbsp) is composed of Energy 1,272 kJ
standardized practice for the administration of bee (304 kcal), Carbohydrates 82.4 g, Sugars 82.12 g,
venom. Dietary fiber 0.2 g, Fat 0 g, Protein 0.3 g, Water 17.10
g, Riboflavin (vit. B2) 0.038 mg (3%), Niacin (vit. B3)
Thus this study aimed to evaluate the role of
0.121 mg (1%), Pantothenic acid (B5) 0.068 mg (1%),
apitherapy (bee venom, honey, pollen, royal jelly and
Vitamin B6 0.024 mg (2%), Folate (vit. B9) 2 μg (1%),
propolis) among cases with MS.
Vitamin C 0.5 mg (1%), Calcium 6 mg (1%), Iron 0.42
mg (3%), Magnesium 2 mg (1%), Phosphorus 4 mg
(1%), Potassium 52 mg (1%), Sodium 4 mg (0%), Zinc
Material and Methods 0.22 mg (2%) [16].
This study was carried on Fifty patients known Laboratory studies: Three milliliters of fasting
to be with MS. Diagnosis was confirmed by clinical venous blood were drawn with a sterile plastic syringe
examination and radiological studies [13, 14]. Twelve from each case and control. One milliliter of the blood
males and thirty eight females, their ages ranged was gently placed in a dry clean plastic tube
between 26-71 years, with a mean of 38.7 ± 4.8, two, containing EDTA as a blood anticoagulant for the
from those attending the outpatient clinic of Adult estimation of blood Hb concentration, using
Neurology in National Research Centre, Dokki, cyanomethe-hemoglobin method according to
Egypt,” over a period of three years from September the procedure described by Betke and Savelsberg,
2008 till April 2011. All cases were subjected to 1950 using Drabkin’s solution as a diluent [17]. The
complete clinical and neurological history and rest 2 ml. blood were gently placed in a dry clean
examination to confirm the diagnosis. There were 32 plastic tube and left for 15 minutes to clot then
cases with quadriparesis, (8 males and 24 female) centrifuged at 3500 rpm for separation of serum,
and 18 cases with paraparesis (4 males and 14 which then kept in deep freeze at -70ºC until analysis
females). All cases were under their regular treatment for estimation of serum levels of calcium, zinc,
either by corticosteroids, or interferon. These cases copper, vitamin E and Folat. All these investigations
were divided into two main groups, each group were done at the beginning of the study and by the
consists of 25 cases (6 males and 19 females), Group end of one year of supplementation and bee sting
I received honey, pollen, royal jelly and propolis and sessions.
were treated with bee stings 3 times weekly, for 12
months, started gradually by one sting then gradually Statistical analysis: All data obtained were
increase up to 25 stings per session - “after sensitivity statistically analyzed using Microsoft Excel and SPSS
test was done” - in addition to their medical treatment, 11.5 for windows software package including, “t test,
while group II remains on their ordinary medical non-parametric Qui square, Mann witney and anova
treatment only. Informed consent was signed by the test” as (Steel and Torrie, 1980) [18].
patients and their caregivers to participate in the study
including the following:
Results
Full general and neurological assessment,
including full history and examination according to In this study were included fifty patients
sheet prepared for the study, neurological diagnosed as MS, twelve males and thirty eight
examination on the beginning of the study to confirm females, their ages ranged between 26-71 years, with
the diagnosis then examination was repeated every a mean of 38.7 ± 4.8. Thirty two cases with
two months to examine and detect the changes which quadriparesis, (8 males and 24 female) and 18 cases
may be happened for each case, and recorded in the with paraparesis (4 males and 14 females). During the
form of scores starting from 0 for normal with no period of the study there was gradual assessment of
abnormal signs up to 5 for complete disability. signs and symptoms every 2 months for both groups
of patients. This periodic assessment for both groups
Assessment parameters: General condition, showed the improvement in the scores of signs
depression, no energy fatigue, sleeping, heat recorded every two months as shown in Table (1 & 2).
tolerance, attention span, memory, rigidity, spasms Results revealed that 4 patients out of 9 (44.4% of
and tremors were evaluated by scoring from 1 up 7, paraparesis cases), showed significant improvement
the condition scores starting from1 for normal with no regarding their defects in gait, bowel control,
abnormal signs up to 7 for bad by asking the patient constipation and urination, while 12 cases out of 16
and devalued during diagnosis. cases (75% of quadriparesis cases), showed mild
Apiacupuncture done by bee stings in the improvement in their movement in bed, and better
following points Du 13, 14, Li 11, S6, S9, points for improvement in bed sores, sensation, and better
cervical area and lumber area and vision points GB2 motor power, only two cases of them (12.5%) were
and Li3 [15]. able to stand for few minutes with support among
patients of group I. Also Fig. 1 showed mild
Supplementation: All patients of both groups improvement.
were ordered to receive 50 gm twice daily. For 100 g,
_______________________________________________________________________________________________________________________________
266 http://www.mjms.mk/
http://www.id-press.eu/mjms/
Helal et al. Apitherapy Have a Role in Treatment of Multiple Sclerosis
_______________________________________________________________________________________________________________________________
Table 1: Scores of signs recorded every two months (1-12), for patients of group (I).
Months 2 Months 4 Months 6 Months 8 Months 10 Months 12 Months
General Condition 4.87 ± 0.440 4.57 ± 0.278 3.87 ± 0.398 3.71 ± 0.267 2.85 ± 0.243 2.37 ± 0.182
Depression 3.50 ± 0.707 3.62 ± 0.625 3.37 ± 0.652 3.00 ± 0.534 2.62 ± 0.323 1.75 ± 0.25
No Energy Fatigue 3.75 ± 0.647 3.37 ± 0.595 3.62 ± 0.893 4.25 ± 0.883 2.87 ± 0.665 2.75 ± 0.311
Sleeping 3.75 ± 0.490 3.37 ± 0.679 2.50 ± 0.327 2.37 ± 0.323 1.75 ± 0.25 1.37 ± 0.182
Heat tolerance 3.75 ± 0.725 3.37 ± 0.818 2.50 ± 0.590 2.37 ± 0.534 1.75 ± 0.462 1.37 ± 0.365
Attention span 2.12 ± 0.479 2.00 ± 0.534 1.87 ± 0.398 1.50 ± 0.267 1.25 ± 0.163 1.25 ± 0.163
Memory 2.12 ± 0.350 2.00 ± 0.462 1.75 ± 0.365 1.50 ± 0.188 1.62 ± 0.182 1.50 ± 0.188
Rigidity 2.50 ± 0.50 2.50 ± 0.654 3.00 ± 0.731 2.25 ± 0.411 2.12 ± 0.479 1.62 ± 0.323
Spasms 2.37 ± 0.595 1.87 ± 0.610 2.50 ± 0.462 1.75 ± 0.313 2.50 ± 0.707 1.87 ± 0.398
Tremors 2.37 ± 0.32 2.50 ± 0.654 2.75 ± 0.674 2.25 ± 0.453 2.37 ± 0.679 2.01 ± 0.15
Headaches 1.87 ± 0.639 2.62 ± 0.625 2.12 ± 0.398 1.62 ± 0.263 1.75 ± 0.313 1.37 ± 0.263
Eye sight 2.50 ± 0.566 2.50 ± 0.566 2.25 ± 0.490 1.87 ± 0.440 1.87 ± 0.398 1.75 ± 0.313
Speech 1.62 ± 0.263 1.62 ± 0.263 1.87 ± 0.295 1.50 ± 0.267 1.50 ± 0.267 1.37 ± 0.182
Swallowing 1.37 ± 0.263 1.37 ± 0.182 1.37 ± 0.182 1.12 ± 0.125 1.25 ± 0.163 1.12 ± 0.125
Numbness 2.62 ± 0.375 2.62 ± 0.375 2.25 ± 0.453 1.87 ± 0.295 1.62 ± 0.375 1.25 ± 0.163
Balance 4.62 ± 0.800 3.87 ± 0.685 3.00 ± 0.843 2.50 ± 0.707 1.87 ± 0.692 1.50 ± 0.834
Walking 4.37 ± 0.595 4.37 ± 0.595 4.50 ± 0.707 4.25 ± 0.75 3.87 ± 0.742 2.87 ± 0.639
Hand coordination 2.62 ± 0.532 2.62 ± 0.460 1.87 ± 0.226 1.75 ± 0.25 1.50 ± 0.188 1.37 ± 0.182
Writing 2.62 ± 0.679 2.87 ± 0.789 2.12 ± 0.548 1.87 ± 0.479 1.75 ± 0.365 1.62 ± 0.263
Bladder & Bowel 4.12 ± 0.580 4.12 ± 0.580 3.50 ± 0.597 3.25 ± 0.526 2.37 ± 0.323 2.12 ± 0.226
As shown in Table 3, the scores of symptoms [1]. Several subtypes, or patterns of progression, have
recorded at start and the end of the investigation been described. Subtypes use the past course of the
among patients for both groups. There was no disease in an attempt to predict the future course.
statistical significant differences between group I and They are important not only for prognosis but also for
group II at the start of the study, regarding their therapeutic decisions [5]. Although there is no known
general condition, depression, fatigue, sleeping heat cure for multiple sclerosis, several therapies have
tolerance, attention span, memory, rigidity, spasms proven helpful.
and tremors, while by the end of the study there were
Bee sting therapy is increasingly used to treat
significant improvement regarding general condition,
patients with multiple sclerosis (MS) in the belief that it
depression sleeping, heat tolerance attention span,
can stabilize or ameliorate the disease. However,
memory as well as rigidity and muscle spasms.
there are no clinical studies to justify its use.
Table 4 shows the comparison between the Wesselius et al, 2005 found that there was no
mean plasma and serum concentrations of improvement of disability, fatigue, and quality of life
micronutrients for both groups of MS at the start and [20]. Bee sting therapy was well tolerated, and there
end of the study. There were no statistical significant were no serious adverse events. They concluded that
differences between group I and group II at the start of the treatment with bee venom in patients with
the study, regarding their mean plasma and serum relapsing multiple sclerosis did not reduce disease
concentrations of micronutrients for both groups of MS activity, disability, or fatigue and did not improve
at the start and end of the study. quality of life.
The therapeutic benefits of honeybee venom
have been known for a long time to relieve pain and to
Discussion treat inflammatory diseases particularly for treatment
MS affects the ability of nerve cells in the of arthritic and rheumatic conditions in humans [21,
brain and spinal cord to communicate with each other 22] and in animals [23-25]. Specific immunotherapy
effectively [3]. Theories include genetics or infections. with bee venom can result in an almost complete
Different environmental risk factors have also been protection against adverse (or allergic) reactions from
found [19]. Life expectancy of people with MS is 5 to stings in the great majority of cases [26].
10 years lower than that of the unaffected population
Table 2: Scores of signs recorded every two months (1-12), for patients of group (II).
Months 2 Months 4 Months 6 Months 8 Months 10 Months 12 Months
General Condition 3.73 ± 0.5 3.49 ± 0.26 3.17 ± 0.23 3.01 ± 0.22 2.83 ± 0.13 2.8 ± 0.12
Depression 3.62 ± 0.24 3.6 ± 0.35 3.29 ± 0.12 3.05 ± 0.72 2.76 ± 0.45 2.45 ± 0.33
No Energy Fatigue 3.24 ± 0.6 3.25 ± 0.35 3.24 ± 0.38 3.28 ± 0.28 2.95 ± 0.85 2.85 ± 0.35
Sleeping 3.4 ± 0.58 3.39 ± 0.46 3.07 ± 0.12 2.8 ± 0.62 2.35 ± 0.25 2.07 ± 0.12
Heat tolerance 3.5 ± 0.75 3.39 ± 0.16 3.06 ± 0.590 2.73 ± 0.63 2.5 ± 0.42 2.37 ± 0.365
Attention span 2.1 ± 0.45 2.06 ± 0.32 1.93 ± 0.66 1.67 ± 0.21 1.53 ± 0.62 1.38 ± 0.22
Memory 2.3 ± 0.35 2.13 ± 0.27 2.05 ± 0.05 1.92 ± 0.27 1.82 ± 0.15 1.72 ± 0.13
Rigidity 2.48 ± 0.52 2.39 ± 0.52 2.17 ± 0.33 2.06 ± 0.18 1.92 ± 0.72 1.84 ± 0.23
Spasms 2.4 ± 0.58 2.26 ± 0.19 2.19 ± 0.25 2.03 ± 0.18 1.89 ± 0.09 1.74 ± 0.398
Tremors 2.36 ± 0.43 2.3 ± 0.52 2.28 ± 0.45 2.2 ± 0.32 2.17 ± 0.66 2.12 ± 0.718
Headaches 1.86 ± 0.19 1.72 ± 0.23 1.66 ± 0.32 1.59 ± 0.28 1.49 ± 0.19 1.42 ± 0.23
Eye sight 2.48 ± 0.36 2.39 ± 0.34 2.31 ± 0.09 2.27 ± 0.62 2.13 ± 0.18 2.01 ± 0.13
Speech 1.59 ± 0.63 1.55 ± 0.17 1.49 ± 0.24 1.44 ± 0.15 1.4 ± 0.28 1.37 ± 0.08
Swallowing 1.45 ± 0.21 1.41 ± 0.22 1.38 ± 0.42 1.32 ± 0.19 1.29 ± 0.33 1.26 +0.25
Numbness 2.5 ± 0.5 2.42 ± 0.34 2.23 ± 0.66 2.07 ± 0.35 1.91 ± 0.07 1.72 ± 0.12
Balance 3.49 ± 0.26 3.37 ± 0.63 3.02 ± 0.42 2.71 ± 0.17 2.47 ± 0.32 2.05 ± 0.47
Walking 3.3 ± 0.62 3.27 ± 0.25 3.14 ± 0.45 3.07 ± 0.37 2.96 ± 0.52 2.87 ± 0.639
Hand coordination 2.7 ± 0.28 2.6 ± 0.34 2.47 ± 0.56 2.35 ± 0.77 2.19 ± 0.28 2.04 ± 0.12
Writing 2.65 ± 0.25 2.57 ± 0.09 2.42 ± 0.52 2.31 ± 0.08 2.18 ± 0.33 2.02 ± 0.26
Bladder & Bowel 3.09 ± 0.35 2.96 ± 0.65 2.83 ± 0.42 2.79 ± 0.67 2.69 ± 0.44 2.63 ± 0.16
_______________________________________________________________________________________________________________________________

Clinical Science
_______________________________________________________________________________________________________________________________
In our study we found that there is a various cytokines like interleukin (IL)-1β, IL-6, tumor
statistically significant improvement regarding their necrosis factor-α, etc. In a mouse model using the
immunity and the improvement in their health and subcutaneous route, rapid increases in serum alanine
general conditions and this is explained by [27, 28], aminotransferase and aspartate aminotransferase
that Bee stings cause hemoconcentration which might transaminases, creatinine, urea nitrogen, uric acid,
be related to the marked edema induced by the sodium and chloride electrolytes, and creatine kinase
venom. Following bee stings there is an increase in were recorded [27, 28].
Table 3: Comparison between patients of group I & group II regarding the signs at the start and end of the study.
Mean ± SE of signs at the end of the

Mean ± SE of signs at the start of the study
study
Clinical signs P - value P - value
Group I Group II Group I Group II
(n= 25) (n= 25) (n= 25) (n= 25)
General Condition 4.87 ± 0.440 3.73 ± 0.5 0.06 2.37 ± 0.182 2.8 ± 0.12 0.05
Depression 3.5 ± 0.707 3.62 ± 0.24 0.1 1.75 ± 0.25 2.45 ± 0.33 0.004
No Energy Fatigue 3.75 ± 0.647 3.24 ± 0.6 0.05 2.75 ± 0.311 2.85 ± 0.35 0.04
Sleeping 3.75 ± 0.49 3.4 ± 0.58 0.07 1.37 ± 0.182 2.07 ± 0.12 0.003
Heat tolerance 3.75 ± 0.725 3.5 ± 0.75 0.05 1.37 ± 0.365 2.37 ± 0.365 0.005
Attention span 2.12 ± 0.479 2.1 ± 0.45 0.2 1.25 ± 0.163 1.38 ± 0.22 0.04
Memory 2.12 ± 0.350 2.3 ± 0.35 0.1 1.50 ± 0.188 1.72 ± 0.13 0.02
Rigidity 2.5 ± 0.5 2.48 ± 0.52 0.2 1.62 ± 0.323 1.84 ± 0.23 0.05
Spasms 2.37 ± 0.595 2.4 ± 0.58 0.3 1.87 ± 0.398 1.74 ± 0.398 0.05
Tremors 2.37 ± 0.32 2.36 ± 0.43 0.5 2.01 ± 0.15 2.12 ± 0.718 0.1
Headaches 1.87 ± 0.639 1.86 ± 0.19 0.02 1.37 ± 0.263 1.42 ± 0.23 0.02
Eye sight 2.50 ± 0.566 2.48 ± 0.36 0.005 1.75 ± 0.313 2.01 ± 0.13 0.005
Speech 1.62 ± 0.263 1.59 ± 0.63 0.04 1.37 ± 0.182 1.37 ± 0.08 0.04
Swallowing 1.37 ± 0.263 1.45 ± 0.21 0.05 1.12 +0.125 1.26 +0.25 0.05
Numbness 2.62 ± 0.375 2.5 ± 0.5 0.05 1.25 ± 0.163 1.72 ± 0.12 0.05
Balance 4.62 ± 0.800 3.49 ± 0.26 0.005 1.50 ± 0.834 2.05 ± 0.47 0.005
Walking 4.37 ± 0.595 3.3 ± 0.62 0.04 2.87 ± 0.639 2.87 ± 0.639 0.04
Hand coordination 2.62 ± 0.532 2.7 ± 0.28 0.005 1.37 ± 0.182 2.04 ± 0.12 0.02
Writing 2.62 ± 0.679 2.65 ± 0.25 0.04 1.62 ± 0.263 2.02 ± 0.26 0.005
Bladder & Bowel 4.12 ± 0.580 3.09 ± 0.35 0.05 2.12 ± 0.226 2.63 ± 0.16 0.04
According to Janik et al, 2007 [10], course of arthritis, lupus and scleroderma. It is also used for a
treatment starts with testing the patient for allergy, number of other diseases and conditions, including
which is known to occur in 1% of the general depression, skin conditions, menstrual cramps and
population. Bee venom is administered in the form of varicose veins [27, 28]. It is claimed that bee venom
a direct bee sting or else by injection of a venom therapy works with the patient's own body to reduce
extract, the treatment is usually given twice a week, inflammation. The theory is that because the stings
this is agreed with our study we tested the patients for produce inflammation, the body mounts an anti-
allergy, and we used the direct bee sting twice weekly inflammatory response. Presumably, this would then
for 6 months for each patients. work to reduce inflammation where the myelin is being
attacked by the immune system in a person with MS
Bee venom acupuncture (BVA), as a kind of
[27, 28]
herbal acupuncture, exerts not only pharmacological
actions from the bioactive compounds isolated from The location of the sting is important, with the
bee venom but also a mechanical function from sting acting as a sort of acupuncture in combination
acupuncture stimulation. BVA is growing in popularity, with the effects of the venom, while others report the
especially in Korea, and is used primarily for pain location is not important. The number of stings also
relief in many kinds of diseases [29]. varies widely from a few to hundreds and they may be
administered either by live bees or by injection. This
We used Bee venom therapy for patients with
treatment can cause pain, and even result in death if
MS and this is agreed with Park et al., 2010 and
the subject has an allergy to bee venom, which can
Prado et al., 2010, they stated that Bee venom
produce anaphylactic shock [20]. For honeybee
therapy is used by people with many different
venom subcutaneous immunotherapy 100 or 200 μg
autoimmune disorders, including MS, rheumatoid
doses are considered effective.
Table 4: Shows the comparison between the mean plasma and serum concentrations of micronutrients for both groups of MS at
the start and end of the study.
Mean ± SE of plasma concentration among Mean ± SE of plasma concentration among
cases at the start of the study cases at the end of the study
Nutrient Level P – value P - value
Group I Group II Group I Group II
(n= 25) (n= 25) (n= 25) (n= 25)
Iron (µmol/l) 9.26 ± 3.2 9.14 ± 1.7 0.1 12.1 ± 0.9 10.6 ± 1.4 0.02
Calcium ( mg/dl ) 1.83 ± 0.04 1.85 ± 0.02 0.12 2.09 ± 0.02 1.9 ± 0.04 0.005
Zinc (µg/dl) 14.2 ± 1.3 13.9 ± 1.5 0.06 16.7 ± 2.03 14.1 ± 2.02 0.04
Copper (µg/dl) 17.9 ± 3.4 18.1 ± 2.6 0.07 20.05 ± 0.6 18.9 ± 1.9 0.05
Vit. E (mg/dl) 20.8 ± 3.5 21.1 ± 2.8 0.1 23.9 ± 2.6 22.3 ± 2.4 0.05
Folate (nmcl/l) 32.2 ± 2.7 31.9 ± 7.8 0.3 49.6 ± 6.4 34.6 ± 5.9 0.005
Magnesium ( mmol/l ) 0.64 ± 0.05 0.65 ± 0.02 0.05 1.04 ± 0.03 0.98 ± 0.04 0.04
_______________________________________________________________________________________________________________________________
Helal et al. Apitherapy Have a Role in Treatment of Multiple Sclerosis
_______________________________________________________________________________________________________________________________
The median lethal dose (LD50) for honeybee progressive MS. While the treatment was well-
venom has been reported in a number of reports [30,
tolerated, no beneficial effects were seen on the MRIs
31] as 2.8 mg venom/kg body weight for intravenous
or clinically among these patients [3, 5]. In our study
and 3.8 mg venom/kg body weight for intraperitonealy
there was a statistical significant improvement
delivery in mice.
regarding the clinical findings while results of MRIs
A 2004 randomized crossover study was done for follow up for our patients showed that there is
conducted in the Netherlands among 24 people with no more changes in the demyelinating lesions which
either relapsing-remitting MS or secondary- means that demyelination shows no progression.
Fig. 1 showed CT at the start and by the end of the study.
Despite a lack of scientific evidence, bee for fatigue in multiple sclerosis: a rapid and systematic review.
Health Technol Assess. 2000;4(27):1-61.
venom therapy has been reported by people with MS
to increase stability, as well as reduce fatigue and 3. Compston A, Coles A. Multiple sclerosis. Lancet. 2002 Apr
spasticity. More than 1,300 people with MS have sent 6;359(9313):1221-31. Review. Erratum in: Lancet
2002;360(9333):648.
testimonials to the American Apitherapy Society in
support of the therapy [3], this is agreed by our study 4. Weinshenker BG. Natural history of multiple sclerosis. Ann
as patients under bee venom therapy showed marked Neurol. 1994;36 Suppl:S6-11.
improvement in stature, stability, and spasticity, While 5. Lublin FD, Reingold SC. Defining the clinical course of multiple
our study is disagreed with Castro et al, 2005 [7] they sclerosis: results of an international survey. National Multiple
Sclerosis Society (USA) Advisory Committee on Clinical Trials
found that there were no definite conclusions of New Agents in Multiple Sclerosis. Neurology.
regarding efficacy and therefore there was little 1996;46(4):907-11.
evidence to support the use of honeybee venom in the
6. Feinstein A. The clinical neuropsychiatry of multiple
treatment of MS. Larger and more carefully conducted sclerosis (2nd ed. ed.). Cambridge: Cambridge University
multicenter studies will be required to establish Press, 2007: p. 20.
efficacy.
7. Castro HJ, Mendez-Lnocencio JI, Omidvar B, Omidvar J,
In conclusion, although Apitherapy is not a Santilli J, Nielsen HS Jr, Pavot AP, Richert JR, Bellanti JA. A
phase I study of the safety of honeybee venom extract as a
curable therapy in MS, but it can be used to minimize possible treatment for patients with progressive forms of
the clinical symptoms of MS, and can be included multiple sclerosis. Allergy Asthma Proc. 2005;26(6):470-6.
among programs of MS therapy.
8. Hegazi AG. Medical importance of bee products. ARI BİLİMİ /
BEE SCIENCE. 2013; 12(4): 136-146.
9. Hegazi AG. Propolis an over view. J. Bee Informed. 1998;
Acknowledgment 5:5,22 –23; 6:723-28.
Many thanks for Prof. Dr. Iman Refaat and 10. Janik JE, Wania-Galicia L, Kalauokalani D. Bee stings--a
David Fakhry from the department of Medical remedy for postherpetic neuralgia? A case report. Reg Anesth
Pain Med. 2007;32(6):533-5.
Biochemistry in NRC.
11. Singla RK, Bhat VG. Honey bee sting and venom offering
active as well as passive immunization could reduce swine flu
pandemic A (H1N1). Med Hypotheses. 2010;74(3):617-8.
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_______________________________________________________________________________________________________________________________
Journal of Clinical & Cellular
Immunology Hegazi et al., J Clin Cell Immunol 2015, 6:1
http://dx.doi.org/10.4172/2155-9899.1000299
Research Article Open Access
Novel Therapeutic Modality Employing Apitherapy for Controlling of Multiple

Sclerosis
Ahmad G Hegazi*, Khaled Al-Menabbawy, Eman H Abd El Rahman and Suzette I Helal
Department of Zoonotic Diseases, National Research Center, Dokki, Giza, Egypt
*Corresponding authors: Ahmed Hegazi, Department of Zoonotic Diseases, National Research Center, Egypt, Tel: +201001440063; Fax: +20237749222; E-mail:
ahmedhegazi128@gmail.com
Received date: November 23, 2014, Accepted date: February 18, 2015, Published date: February 25, 2015
Copyright: © 2015 Hegazi AG, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Objective: Study the effect of bee sting therapy (Apitherapy) in the treatment of Multiple Sclerosis.
Methods: Fifty patients with MS, their ages ranged between 26-71 years, were subjected to complete clinical and
neurological history and examination to confirm the diagnosis. All cases were under their regular treatment they
were divided into two main groups, Group I received honey, pollen, royal jelly and Propolis and were treated with
acupuncture 3 times weekly, for 12 months, in addition to their medical treatment, while group II remains on their
ordinary medical treatment only. Acupuncture was done by bee stings for regulating the immune system.
Results: Results revealed that 4 patients showed some improvement regarding their defects in gait, bowel
control, constipation and urination, while 12 cases, showed some mild improvement in their movement in bed, and
better improvement in bed sores, sensation, and better motor power, only two cases of them were able to stand for a
few minutes with support. Interleukin (IL) 1β, tumor necrosis factor alpha (TNFα, and IL-6 were detected. The level
of TNF-α was significantly elevated in patients in Group II, while IL-1β was reduced in Group II than Group I and no
significant differences were found for IL-6 between the 2 groups. The mean values of IgE level in both groups of
M.S. Patients were low, but with no statistical significance, while by the end of the study there were an elevation in
the levels of IgE for both groups, which was statistically significant.
Conclusion: Although Apitherapy is not a curable therapy in MS, but it can be used to minimize the clinical
symptoms of MS, and can be included among programs of MS therapy.
Keywords: Multiple sclerosis; Apitherapy; Bee venom; Cytokine; diseases, Lyme disease and chronic fatigue syndrome [5-7]. Some
Food supplements reports have shown beneficial effects of bee venom in post herpetic
neuralgia [8], fibromyalgia and multiple sclerosis [9]. Interleukin (IL)
Introduction 1β, tumor necrosis factor alpha (TNFα), and IL-6 are cytokines which
mediate cellular responses during immune activation and
Multiple sclerosis (MS) is a chronic and putatively immune inflammation. In Multiple Sclerosis (MS) they might be responsible
mediated inflammatory disease of the central nervous system [1]. MS for T-cell activation (IL-1β), for demyelination (TNFα), and for
altered immune function in patients. It exhibits many of the hallmarks immunoglobulin (Ig) synthesis (IL-6) within the central nervous
of an inflammatory autoimmune disorder including breakdown of the system [10]. There is no standardized practice for the administration
blood-brain barrier, the recruitment of lymphocytes, microglia, and of bee venom. The aim of this investigation was to evaluate the effect
macrophages to lesion sites, as well as the presence of multiple lesions of bee venom therapy (Bee Sting) and other bee products on the
[2]. It is characterized by the damage of fatty myelin sheaths around immunological status of cases with MS.
the axons of the brain and spinal cord, leading to demyelination and
scarring as well as a broad spectrum of signs and symptoms [3]. Materials and Methods
Apitherapy is the medical use of honey bee products. This includes
The aim of this study was to determine the immunological status of
the use of honey, pollen, bee bread, propolis, royal jelly, and apilarnil
MS patients. Patients had to be free of immunomodulatory treatment
and bee venom. Most claims of apitherapy have not been proved to the
during the study period and for at least the preceding 12 months. This
scientific standards of evidence-based medicine and are anecdotal in
study was carried out on fifty MS patients diagnosed and confirmed by
nature. Bee venom therapy is an alternative form of healing. Bee
clinical examination and radiological studies [11-13,7]. Twelve males
venom therapy is the part of apitherapy which utilizes bee venom in
and thirty eight females, their ages ranged between 26-71 years, with a
the treatment of health conditions. However, bee venom is a complex
mean of 38.7 ± 4.8. 2, were selected from patients attending the
mix of a variety of peptides and proteins, some of which have strong
outpatient clinic of Adult Neurology in National Research Centre,
neurotoxic and immunogenic effects [4]. It has been used since
Dokki, Egypt,” over a period of three years from September 2008 till
ancient times to treat arthritis, rheumatism, back pain, skin diseases
April 2011. All cases were subjected to complete clinical and
and in this modern age as an alternative therapy to treat autoimmune
neurological history and examination to confirm the diagnosis. They
J Clin Cell Immunol Volume 6 • Issue 1 • 1000299

ISSN:2155-9899 JCCI, an open access journal
Citation: Hegazi AG, Al-Menabbawy K, Abd El Rahman E, Helal SI (2015) Novel Therapeutic Modality Employing Apitherapy for Controlling of
Multiple Sclerosis. J Clin Cell Immunol 6: 299. doi:10.4172/2155-9899.1000299
Page 2 of 7
were signed consent according to medical ethic committee (14146). Statistical analysis
There were 32 cases with quadriparesis, (8 males and 24 females) and
18 cases with paraparesis (4 males and 14 females). All cases were All data obtained were statistically analyzed using Microsoft Excel
under their regular treatment either by corticosteroids, or interferon. and SPSS 11.5 for windows software package including, “to test, non-
These cases were divided into two main groups, each group consists of parametric Qui square, Mann Witney and anova test”.[18].
25 cases (6 males and 19 females), Group I received honey, pollen,
royal jelly and Propolis and were treated with bee stings (from Results
honeybee (Apis mellifera L.) workers of pure Carniolan race) 3 times
In the current study, fifty patients diagnosed as MS, twelve males
weekly, for 12 months, started gradually by one sting, then gradually
and thirty eight females, their ages ranged between 26-71 years, with a
increase up to 25 stings per session-“after a sensitivity test was done”-
mean of 38.7 ± 4.8. Thirty two cases with quadriparesis, (8 males and
in addition to their medical treatment, while group II remains on their
24 female) and 18 cases with paraparesis (4 males and 14 females).
ordinary medical treatment only. Informed consent was signed by the
During the period of the study there was a gradual assessment of signs
patients and their caregivers to participate in the study including the
and symptoms every 2 months for both groups of patients. These
following:
periodic assessments for both groups showed the improvement in the
Full general and neurological assessment, including full history and scores of signs recorded every two months as shown in Table 1 as well
examination according to sheet prepared for the study, neurological as in the scores of symptoms recorded during the same period among
examination on the beginning of the study to confirm the diagnosis, patients for both groups.
then the examination was repeated every two months to examine and
Results revealed that 4 patients out of 9 (44.4% of paraparesis cases),
detect the changes which may be happened for each case, and recorded
showed significant improvement regarding their defects in gait, bowel
in the form of scores starting from 0 for normal with no abnormal
control, constipation and urination, while 12 cases out of 16 cases
signs up to 5 for complete disability.
(75% of quadriparesis cases), showed mild improvement in their
Apiacupuncture done by bee stings in the following points Du 13, movement in bed, and better improvement in bed sores, sensation,
14, Li 11, S6, S9, points for cervical area and lumbar area and vision and better motor power, only two cases of them (12.5%) were able to
points GB2 and Li3 [14]. stand for a few minutes with support among patients of group I. As
shown in Table 1 there was no statistically significant differences
Supplementation between group I and group II at the start of the study, regarding their
general condition, depression, fatigue, sleeping heat tolerance,
All patients of both groups were ordered to receive one tablet attention span, memory, rigidity, spasms and tremors, while by the
(Kirkland signature, USA) according to Bowling and Stewart [15]. end of the study there were significant improvement regarding general
Each Tablet Contains-% Daily Value: Vitamin A 2,500 I.U. 50% as condition, depression sleeping, heat tolerance attention span, memory
Beta Carotene-40%, Vitamin C 90 mg-150%, Vitamin D3 500 as well as rigidity and muscle spasms.
I.U.-125%, Vitamin E 50 I.U.-167%, Vitamin K 30 mcg-38%, Thiamin
(Vitamin B1) 1.5 mg-100%, Riboflavin (Vitamin B2) 1.7 mg-100%, In this study, the mean values of IgE in both groups of M.S. Patients
Niacin 20 mg-100%, Vitamin B6 3 mg-150%, Folate (Folic Acid) 500 (Table 2) were low, but with no statistical significance, while by the
mcg-125%, Vitamin B12 25 mcg-417%, Biotin 30 mcg-10%, end of the study there were an elevation in the levels of IgE for both
Pantothenic Acid 10 mg-100%, Calcium 220 mg-22%, Phosphorus 110 groups, which was statistically significant among cases of group (I).
mg-11%, Iodine 150 mcg-100%, Magnesium 50 mg-13%, Zinc 11 The aims of the present study were to evaluate the pro-inflammatory
mg-73%, Selenium 55 mcg-79%, Copper 0.9 mg-45%, Manganese 2.3 tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and IL-6.
mg-115%, Chromium (as Chromium Picolinate) 45 mcg-38%, Results are shown as the mean ± standard error in pictograms per
Molybdenum 45 mcg-60%, Chloride 72 mg-2%, Potassium 80 mg-2%, milliliter (Table2). All patients with MS had significantly higher
Boron 150 mcg, Nickel 5 mcg, Silicon 2 mg, Vanadium 10 mcg, Lutein cytokine concentrations. The level of TNF-α was significantly elevated
(flower) 250 mcg, Lycopene 300 mcg. in Group II patients (6.1 ± 1.3 pg/mL) vs control Group I subjects (4.3
± 0.1 pg/mL) at the end of experiment (12 months), as was the level of
IL-1β (123.0 ± 2.2 vs 224.1 ± 1.4 pg/mL; P ≤ .0001) at the end of
Laboratory studies
experiment respectively. In contrast, no significant differences were
Three milliliters of venous blood were drawn from fasting patients found for IL-6 between the group I and group II.
and controls with a sterile plastic syringe. A total of 2 ml. blood was
gently placed in a dry clean plastic tube and left for 15 minutes to clot, Discussion
then centrifuged at 3500 r.p.m for separation of serum, which then
kept in deep freeze at -70°C until analysis. Serum samples were MS affects the ability of nerve cells in the brain and spinal cord to
obtained from patients with clinically definite MS for estimation of communicate with each other effectively [19]. Theories include
serum levels of immunoglobulin E [16] using commercially available genetics or infections as causes for MS. Different environmental risk
ELISA kits according to the manufacturers' directions. Serum cytokine factors have also been found [20]. Life expectancy of people with MS is
levels (Interleukin 1β, tumor necrosis factor alpha and IL-6) were 5 to 10 years lower than that of the unaffected population [3]. Several
assessed using enzyme linked-immunosorbent assays [17] using subtypes, or patterns of progression, have been described. Subtypes
commercially available ELISA kits according to the manufacturer’s use the past course of the disease in an attempt to predict the future
directions (kits produced by the Bender MED System, Vienna, course. They are important not only for prognosis, but also for
Austria). All these investigations were done at the beginning of the therapeutic decisions [21]. Although there is no known cure for
study and by the end of one year of supplementation and bee sting multiple sclerosis, several therapies have proven to be helpful.
sessions.

Page 3 of 7
2 Months 4 Months 6 Months 8 Months 10 Months 12 Months
G1 G 1I G1 G 1I G1 G 1I G1 G 1I G1 G 1I G1 G 1I
General 4.87 ± 3.73 ± 0.5 4.57 ± 3.49 ± 3.87 ± 3.17 ± 3.71 ± 3.01 ± 2.85 ± 2.83 ± 2.37 ± 2.8 ± 0.12
Condition 0.440 0.278 0.26 0.398 0.23 0.267 0.22 0.243 0.13 0.182
Depression 3.50 ± 3.62 ± 3.62 ± 3.6 ± 3.37 ± 3.29 ± 3.00 ± 3.05 ± 2.62 ± 2.76 ± 1.75 ± 2.45 ±
0.707 0.24 0.625 0.35 0.652 0.12 0.534 0.72 0.323 0.45 0.25 0.33
No Energy 3.75 ± 3.24 ± 0.6 3.37 ± 3.25 ± 3.62 ± 3.24 ± 4.25 ± 3.28 ± 2.87 ± 2.95 ± 2.75 ± 2.85 ±
Fatigue 0.647 0.595 0.35 0.893 0.38 0.883 0.28 0.665 0.85 0.311 0.35
Sleeping 3.75 ± 3.4 ± 0.58 3.37 ± 3.39 ± 2.50 ± 3.07 ± 2.37 ± 2.8 ± 1.75 ± 0.25 2.35 ± 1.37 ± 2.07 ±
0.490 0.679 0.46 0.327 0.12 0.323 0.62 0.25 0.182 0.12
Heat tolerance 3.75 ± 3.5 ± 0.75 3.37 ± 3.39 ± 2.50 ± 3.06 ± 2.37 ± 2.73 ± 1.75 ± 2.5 ± 0.42 1.37 ± 2.37 ±
0.725 0.818 0.16 0.590 0.590 0.534 0.63 0.462 0.365 0.365
Attention span 2.12 ± 2.1 ± 0.45 2.00 ± 2.06 ± 1.87 ± 1.93 ± 1.50 ± 1.67 ± 1.25 ± 1.53 ± 1.25 ± 1.38 ±
0.479 0.534 0.32 0.398 0.66 0.267 0.21 0.163 0.62 0.163 0.22
Memory 2.12 ± 2.3 ± 0.35 2.00 ± 2.13 ± 1.75 ± 2.05 ± 1.50 ± 1.92 ± 1.62 ± 1.82 ± 1.50 ± 1.72 ±
0.350 0.462 0.27 0.365 0.05 0.188 0.27 0.182 0.15 0.188 0.13
Rigidity 2.50 ± 2.48 ± 2.50 ± 2.39 ± 3.00 ± 2.17 ± 2.25 ± 2.06 ± 2.12 ± 1.92 ± 1.62 ± 1.84 ±
0.50 0.52 0.654 0.52 0.731 0.33 0.411 0.18 0.479 0.72 0.323 0.23
Spasms 2.37 ± 2.4 ± 0.58 1.87 ± 2.26 ± 2.50 ± 2.19 ± 1.75 ± 2.03 ± 2.50 ± 1.89 ± 1.87 ± 1.74 ±
0.595 0.610 0.19 0.462 0.25 0.313 0.18 0.707 0.09 0.398 0.398
Tremors 2.37 ± 2.36 ± 2.50 ± 2.3 ± 2.75 ± 2.28 ± 2.25 ± 2.2 ± 2.37 ± 2.17 ± 2.01 ± 2.12 ±
0.32 0.43 0.654 0.52 0.674 0.45 0.453 0.32 0.679 0.66 0.15 0.718
Headaches 1.87 ± 1.86 ± 2.62 ± 1.72 ± 2.12 ± 1.66 ± 1.62 ± 1.59 ± 1.75 ± 1.49 ± 1.37 ± 1.42 ±
0.639 0.19 0.625 0.23 0.398 0.32 0.263 0.28 0.313 0.19 0.263 0.23
Eye sight 2.50 ± 2.48 ± 2.50 ± 2.39 ± 2.25 ± 2.31 ± 1.87 ± 2.27 ± 1.87 ± 2.13 ± 1.75 ± 2.01 ±
0.566 0.36 0.566 0.34 0.490 0.09 0.440 0.62 0.398 0.18 0.313 0.13
Speech 1.62 ± 1.59 ± 1.62 ± 1.55 ± 1.87 ± 1.49 ± 1.50 ± 1.44 ± 1.50 ± 1.4 ± 0.28 1.37 ± 1.37 ±
0.263 0.63 0.263 0.17 0.295 0.24 0.267 0.15 0.267 0.182 0.08
Swallowing 1.37 ± 1.45 ± 1.37 ± 1.41 ± 1.37 ± 1.38 ± 1.12 ± 1.32 ± 1.25 ± 1.29 ± 1.12 ± 1.26
0.263 0.21 0.182 0.22 0.182 0.42 0.125 0.19 0.163 0.33 0.125 +0.25
Numbness 2.62 ± 2.5 ± 0.5 2.62 ± 2.42 ± 2.25 ± 2.23 ± 1.87 ± 2.07 ± 1.62 ± 1.91 ± 1.25 ± 1.72 ±
0.375 0.375 0.34 0.453 0.66 0.295 0.35 0.375 0.07 0.163 0.12
Balance 4.62 ± 3.49 ± 3.87 ± 3.37 ± 3.00 ± 3.02 ± 2.50 ± 2.71 ± 1.87 ± 2.47 ± 1.50 ± 2.05 ±
0.800 0.26 0.685 0.63 0.843 0.42 0.707 0.17 0.692 0.32 0.834 0.47
Walking 4.37 ± 3.3 ± 0.62 4.37 ± 3.27 ± 4.50 ± 3.14 ± 4.25 ± 3.07 ± 3.87 ± 2.96 ± 2.87 ± 2.87 ±
0.595 0.595 0.25 0.707 0.45 0.75 0.37 0.742 0.52 0.639 0.639
Hand 2.62 ± 2.7 ± 0.28 2.62 ± 2.6 ± 1.87 ± 2.47 ± 1.75 ± 2.35 ± 1.50 ± 2.19 ± 1.37 ± 2.04 ±
coordination 0.532 0.460 0.34 0.226 0.56 0.25 0.77 0.188 0.28 0.182 0.12
Writing 2.62 ± 2.65 ± 2.87 ± 2.57 ± 2.12 ± 2.42 ± 1.87 ± 2.31 ± 1.75 ± 2.18 ± 1.62 ± 2.02 ±
0.679 0.25 0.789 0.09 0.548 0.52 0.479 0.08 0.365 0.33 0.263 0.26
Bladder & 4.12 ± 3.09 ± 4.12 ± 2.96 ± 3.50 ± 2.83 ± 3.25 ± 2.79 ± 2.37 ± 2.83 ± 2.12 ± 2.63 ±
Bowel 0.580 0.35 0.580 0.65 0.597 0.42 0.526 0.67 0.323 0.13 0.226 0.16
Table 1: Scores of signs recorded every two months (1-12), for patients of both groups.
Bee sting therapy is increasingly used to treat patients with MS in was well tolerated, and there were no serious adverse events. They
the belief that it can stabilize or ameliorate the disease. However, there concluded that the treatment with bee venom in patients with
are no clinical studies to justify its use [22]. Found that there were no relapsing multiple sclerosis did not reduce disease activity, disability,
improvement of disability, fatigue, and quality of life. Bee sting therapy or fatigue and did not improve quality of life.

Page 4 of 7
Groups Mean at the start of the study Mean at the end of the study
IgE (IL) 1β (TNF) α, IL-6 IgE (IL) 1β (TNF) α, IL-6
Group (I) 664 ± 32 153 ± 3.73 4.5 ± 0.001 4.8 ± 0.01 1031 ± 14 123.0 ± 2.2 6.1 ± 1.3 14.8 ± 0.05
Group (II) 491± 42 222 ± 4.13 2.6 ± 0.002 4.6 ± 0.04 1045 ± 32 224.1 ± 1.4 4.3 ± 0.1 13.6 ± 0.04
Table 2: Mean levels of IgE, Interleukin (IL) 1β, tumor necrosis factor alpha (TNF) α, and IL-6 for both groups at the start of the study and by the
end of the study
The therapeutic benefits of honeybee venom have been known for a there was a statistically significant improvement regarding the clinical
long time to relieve pain and to treat inflammatory diseases, findings while the results of MRIs done to follow up for our patients
particularly for treatment of arthritic and rheumatic conditions in showed that there is no more changes in the demyelinating lesions
humans [23,24] and in animals [25-27]. Specific immunotherapy with which means that demyelination shows no progression as shown in
bee venom can result in an almost complete protection against adverse other previous investigation [7].
(or allergic) reactions from stings in the great majority of cases [28].
BV administration was reported to stimulate the function of
In this study, there was a statistically significant improvement in immune system [34] and to affect the release of cortisol production
patients regarding their immunity, their health and general conditions which is known as natural anti-inflammatory agent [35]. Melittin
and this is in agreement to that explained by Park et al. and Prado et al. which is the major component of BV was found to suppress
[29,30] that bee stings cause hemoconcentration which might be inflammation by inhibiting Phospholipase (PLA) enzymatic activity
related to the marked edema induced by the venom. According to [36]. This enzyme was abundantly released in severe inflammatory
Janik et al. [8], course of treatment starts with testing the patient for disorders and actively found to cause tissue and organ degradation
allergy, which is known to occur in 1% of the general population. Bee which will lead to the loss of their functions [37]. Melittin was also
venom is administered in the form of a direct bee sting or else by found to block the production of neutrophil superoxide [38]. Bee
injection of a venom extract, and the treatment is usually given twice a venom (inflammation, allergy, cytotoxic, haemolysis), antibacterial,
week. antiinflammatory, immunoactivating, immunosupressive, analgesic,
radioprotective, anticarcinogenic, accelerates heartbeat, increases
Bee Venom Acupuncture (BVA), as a kind of herbal acupuncture,
blood circulation, lowers blood pressure, improves haemoglobin
exerts not only pharmacological actions from the bioactive
synthesis, anticoagulant, lowers cholesterol levels, membrane effects
compounds isolated from bee venom but also a mechanical function
on blood cells, influences immuno-active blood cells and hormone
from acupuncture stimulation. BVA is growing in popularity,
levels, antiarhythmic, heart stimulating therapeutic effects,
especially in Korea, and is used primarily for pain relief in many kinds
improvement in hypertonia and artherosclerosis [38-40].
of diseases [29-31]. Park et al. and Prado et al. stated that bee venom
therapy is used by people with many different autoimmune disorders, The mechanism of action of bee venom was clarified as follows: bee
including MS, rheumatoid arthritis, lupus and scleroderma. It is also venom blocks the building of proinflammatory substances and inhibits
used for a number of other diseases and conditions, including the proliferation of rheumatoid synovial cells. Today, bee venom is
depression, skin conditions, menstrual cramps and varicose veins. It is applied directly via sting or injection. This practice was initiated in
claimed that bee venom therapy works with the patient's own body to 1964 in Russia [41] and has been further developed since then, mostly
reduce inflammation. The theory is that because the stings produce in the Far East [42].
inflammation, the body mounts an anti-inflammatory response.
Apamin accounts for less than 2% of venom dry weight, presents a
Presumably, this would then work to reduce inflammation where the
neurotoxic action [42] and possesses unusual functional as well as
myelin is being attacked by the immune system in a person with MS
structural properties [44]. It is remarkable among peptides in its ability
[29,30].
to cross the blood-brain barrier and act on the central nervous system.
The location of the sting is important, with the sting acting as a sort Apamin is known to block calcium dependent potassium fluxes by
of acupuncture in combination with the effects of the venom, while binding to a Ca2þ-dependent potassium channel [45]. Apamin is the
others reported that the location is not important. The number of smallest neurotoxic polypeptide known. It has been isolated from bee
stings also varies widely from a few to hundreds and they may be venom [46].
administered either by live bees or by injection. This treatment can
Nam et al. [47] who stated that bee venom is a mixture of many
cause pain, and even result in death if the subject has an allergy to bee
substances. In this study we found that there is a statistically
venom, which can produce anaphylactic shock [22]. For honeybee
significant improvement regarding their immunity and the
venom subcutaneous immunotherapy 100 or 200 μg doses are
improvement in their health and general conditions and this is
considered effective. The median lethal dose (LD50) for honeybee
explained by Park et al. and Prado et al. [29,30] bee stings cause
venom has been reported in a number of reports [32,33] as 2.8 mg
hemoconcentration which might be related to the marked edema
venom/kg body weight for intravenous and 3.8 mg venom/kg body
induced by the venom. Following bee stings there is an increase in
weight for intraperitoneal delivery in mice. A 2004 randomized
various cytokines like interleukin (IL)-1β, IL-6, tumor necrosis factor-
crossover study was conducted in the Netherlands among 24 people
α, etc. In a mouse model using the subcutaneous route, rapid increases
with either relapsing-remitting MS or secondary-progressive MS.
in serum alanine aminotransferase and aspartate aminotransferase
While the treatment was well-tolerated, no beneficial effects were seen
transaminases, creatinine, urea nitrogen, uric acid, sodium and
on the MRIs or clinically among these patients [21,19]. In our study

Page 5 of 7
chloride electrolytes, and creatine kinase were recorded [48,28,29]. with control subjects, with 9 of the 13 cytokines or markers showing
The pain and swelling of the sting are caused by histamine, dopamine, highly significant differences in these 2 groups. Serum TNFα was
serotonin, and norepinephrine. Several toxins are also present, significantly higher in depressed and MS patients than in normal
including apamin, melittin, monamine, and mast-cell degranulating controls interferon (IFN)-γ; interleukins (ILs)-1β, 6 and tumor
peptide. Lastly, the substances responsible for the allergic response necrosis factor (TNF)-α. Significant increases between patients and
include hyaluronidase and phospholipase-A2, enzymes that work to control subjects were found for IL-1β (mean, 26.0 vs 14.3 pg/mL; P ≤ .
activate immune cells and produce Immunoglobulin E [47]. 0001), IL-6 (mean, 16.8 vs 7.5 pg/mL; P=0.03) and TNF-α (mean, 4.5
vs 1.6 pg/mL; P=0.01) [56].
Despite a lack of scientific evidence, bee venom therapy has been
reported by people with MS to increase stability, as well as reduce Concerning to the levels of IgE which showed significant low levels
fatigue and spasticity. More than 1,300 people with MS have sent among cases of both groups (I and II), while by the end of the study
testimonials to the American Apitherapy Society in support of the marked elevation of IgE levels. The previous study was assessed and
therapy [19,7] this is agreed by our study as patients under bee venom they can be explained by Nam et al. [47] who stated that bee venom is
therapy showed marked improvement in stature, stability, and a mixture of many substances. In this study, we found that there is a
spasticity, while our study is disagreed with Castro et al. [4] as they statistically significant improvement regarding their immunity and the
found that there were no definite conclusions regarding efficacy and improvement in their health and general conditions and this is
therefore there was little evidence to support the use of honeybee explained by Park et al. and Prado et al. [29,30] bee stings cause
venom in the treatment of MS. hemoconcentration which might be related to the marked edema
induced by the venom. Following bee stings there is an increase in
The present study was conducted to evaluate the pro-inflammatory
various cytokines like interleukin (IL)-1β, IL-6, tumor necrosis factor-
Tumor Necrosis Factor (TNF)-α and interleukin (IL)-1β, and IL-6.
α, etc. The pain and swelling of the sting is caused by histamine,
Results are shown as the mean ± standard error in pictograms per
dopamine, serotonin, and norepinephrine. Several toxins are also
milliliter (Table2). All patients with MS had significantly higher
present, including albumin, melting, Menominee, and mast-cell
cytokine concentrations. The level of TNF-α was significantly elevated
degranulation peptide. Lastly, the substances responsible for the
in Group II patients (6.1 ± 1.3 pg/mL) vs control Group I subjects (4.3
allergic response include hyaluronidase and phospholipase-A2,
± 0.1 pg/mL), as was the level of IL-1β (123.0 ± 2.2 vs 224.1 ± 1.4
enzymes that work to activate immune cells and produce
pg/mL; P ≤ .0001), respectively. In contrast, No significant differences
immunoglobulin E [47].
were found for IL-6 between the 2 groups. These findings supported
by the previous findings of different authors as Martins et al. [10] who
found that 10 of the 13 cytokines or markers were significantly Conclusions
different between patients and age-matched control subjects. The From the results we conclude that bee venom injected intradermally
increase in proinflammatory and down-regulatory cytokines in could be a potential new therapeutic agent in the treatment of MS
patients with MS is consistent with the progression of the disease patients, with minimal tolerable side effects. Interleukin-1β, 6 and
because inflammatory and restorative processes seem to occur TNF could be considered as an indicator in the treatment of MS with
simultaneously. Following bee stings there is an increase in various intradermal injection of bee venom. Larger randomized controlled
cytokines like interleukin (IL)-1β, IL-6, tumor necrosis factor-α, etc. In complementary studies are needed to explore their efficacy. This work
a mouse model using the subcutaneous route, rapid increases in serum is a potential starting point for larger studies with wider scales of
alanine aminotransferase and aspartate aminotransferase applications to confirm our findings.
transaminases, creatinine, urea nitrogen, uric acid, sodium and
chloride electrolytes, and creatine kinase were recorded Park et al. and
Prado et al. Cytokines are significantly elevated in patients with MS are Acknowledgement
consistent with the pathologic features of this disease. Interleukin (IL) The authors are grateful for the financial support and to the Clinic
1β, Tumor Necrosis Factor alpha (TNF-α), and IL-6 are cytokines, of Adult Neurology in National Research Centre, Dokki, Egypt.
which are increased during immune activation and inflammation
[48,49]. IL-1β is released from macrophages and endothelial cells and
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51. Takahashi JL, Giuliani F, Power C, Imai Y, Yong VW (2003)
Interleukin-1beta promotes oligodendrocyte death through glutamate
27. Park HJ, Lee SH, Son DJ, Oh KW, Kim KH, et al. (2004) Antiarthritic excitotoxicity. Ann Neurol 53: 588-595.
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suppression of NF-kappaB through interaction with the p50 subunit.
52. Cash E, Minty A, Ferrara P, Caput D, Fradelizi D, et al. (1994)
Macrophage-inactivating IL-13 suppresses experimental autoimmune
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(2008) Sublingual immunotherapy for large local reactions caused by
53. Imitola J, Chitnis T, Khoury SJ (2005) Cytokines in multiple sclerosis:
from bench to bedside. Pharmacol Ther 106: 163-177.
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29. Park S, Chun HJ, Keum B, Seo YS, Kim YS, et al. (2010) Anaphylactic
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molecules
Review
Bee Venom: Overview of Main Compounds and
Bioactivities for Therapeutic Interests
Rim Wehbe 1,† , Jacinthe Frangieh 1,2,† , Mohamad Rima 3,4 , Dany El Obeid 5 ,
Jean-Marc Sabatier 6 and Ziad Fajloun 1,7, *
1 Laboratory of Applied Biotechnology (LBA3B), Azm Center for Research in Biotechnology and its
Applications, EDST, Lebanese University, Tripoli 1300, Lebanon
2 Mitochondrial and Cardiovascular Pathophysiology—MITOVASC, Team 2, Cardiovascular
Mechanotransduction, UMR CNRS 6015, INSERM U1083, Angers University, 49045 Angers, France
3 Department of Neuroscience, Institut de Biologie Paris Seine (IBPS), INSERM, CNRS, Sorbonne Université,
F-75005 Paris, France
4 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS U7104,
Université de Strasbourg, 67400 Illkirch, France
5 Faculty of Agriculture & Veterinary Sciences, Lebanese University, Dekwaneh, Beirut 2832, Lebanon
6 Institute of NeuroPhysiopathology, UMR 7051, Faculté de Médecine Secteur Nord, 51, Boulevard Pierre
Dramard-CS80011, 13344-Marseille Cedex 15, France
7 Faculty of Sciences 3, Michel Slayman Tripoli Campus, Lebanese University, Ras Maska 1352, Lebanon
* Correspondence: ziad.fajloun@ul.edu.lb; Tel.: +961-6-21-3255
† These authors contributed equally to this work.

Received: 24 July 2019; Accepted: 16 August 2019; Published: 19 August 2019
Abstract: Apitherapy is an alternate therapy that relies on the usage of honeybee products, most
importantly bee venom for the treatment of many human diseases. The venom can be introduced
into the human body by manual injection or by direct bee stings. Bee venom contains several
active molecules such as peptides and enzymes that have advantageous potential in treating
inflammation and central nervous system diseases, such as Parkinson’s disease, Alzheimer’s disease,
and amyotrophic lateral sclerosis. Moreover, bee venom has shown promising benefits against different
types of cancer as well as anti-viral activity, even against the challenging human immunodeficiency
virus (HIV). Many studies described biological activities of bee venom components and launched
preclinical trials to improve the potential use of apitoxin and its constituents as the next generation
of drugs. The aim of this review is to summarize the main compounds of bee venom, their
primary biological properties, mechanisms of action, and their therapeutic values in alternative
therapy strategies.
Keywords: bee venom; apitoxin; apitherapy; Parkinson’s disease; Alzheimer’s disease; amyotrophic
lateral sclerosis; cancer; HIV
1. Generalities about Honeybees

Among honeybees, Apis mellifera (Figure 1) is the main species used for crop pollination in the
world [1]. The usage of all bee products, including bee venom and honey, dates back thousands of
years as their medicinal properties were cited in religious books like the Bible and the Quran [2–4].
Apitherapy is a branch of alternative medicine that relies on the usage of honeybee products that
consists of honey, pollen, propolis, royal jelly, and mainly bee venom (BV), which is also known as
apitoxin [5,6].
Molecules 2019, 24, 2997; doi:10.3390/molecules24162997 www.mdpi.com/journal/molecules

Molecules 2019, 24, 2997 2 of 13
Molecules2019,
Molecules 2019,24,
24,xxFOR
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Figure1.
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Figure 1.1.Apis
Apismellifera
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mellifera (copyrightDany
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Obeid).
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Bee venomtherapy
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BV is produced by female worker bees and is known to contain many active components including:
BVBV isis produced
produced by by female
female worker
worker beesbees andand isis known
known toto contain
contain manymany active
active components
components
(i) peptides like melittin, apamin, mast cell degranulating (MCD) peptide, and adolapin, (ii) enzymes,
including:(i)
including: (i)peptides
peptideslike likemelittin,
melittin,apamin,
apamin,mastmastcellcelldegranulating
degranulating(MCD) (MCD)peptide,
peptide,andandadolapin,
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such as phospholipase A2 (PLA2) and hyaluronidase, and (iii) amino acids and volatile compounds.
(ii)enzymes,
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phospholipaseA2 A2(PLA2)
(PLA2)and andhyaluronidase,
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acidsand
andvolatile
volatile
Several studies assessed the therapeutic potential of these components in treating human inflammatory
compounds. Several
compounds. Several studies
studies assessed
assessed the the therapeutic
therapeutic potential
potential ofof these
these components
components inin treating
treating
diseases as well as central nervous system diseases, such as Parkinson’s disease (PD), Alzheimer’s
humaninflammatory
human inflammatorydiseases
diseasesas aswell
wellasascentral
centralnervous
nervoussystem
systemdiseases,
diseases,suchsuchas asParkinson’s
Parkinson’sdisease
disease
disease (AD), and amyotrophic lateral sclerosis (ALS), as well as many other conditions [9,10].
(PD),Alzheimer’s
(PD), Alzheimer’sdisease
disease(AD),(AD),andandamyotrophic
amyotrophiclaterallateralsclerosis
sclerosis(ALS),
(ALS),as aswell
wellasas many
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Interestingly, bee venom, in similarity to other animal venoms, has also shown beneficial anti-cancer
conditions [9,10]. Interestingly, bee venom, in similarity to other animal
conditions [9,10]. Interestingly, bee venom, in similarity to other animal venoms, has also shown venoms, has also shown
and anti-viral potential against ovarian and prostate cancer, as well as HIV [11–14].
beneficialanti-cancer
beneficial anti-cancerand andanti-viral
anti-viralpotential
potentialagainst
againstovarian
ovarianandandprostate
prostatecancer,
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wellas
asHIV
HIV[11–[11–
Bee venom is characterized by inducing allergic reactions following the sting. These reactions
14].
14].
can take place in the skin, the respiratory track, the cardiovascular system, and the gastrointestinal
Beevenom
Bee venomisischaracterized
characterizedby byinducing
inducingallergic
allergicreactions
reactionsfollowing
followingthe thesting.
sting.These
Thesereactions
reactions
system. Subsequently, severe anaphylactic shock could lead to cerebral or myocardial ischemia [15,16].
cantake
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theskin,
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thecardiovascular
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These allergic responses are due to the presence, within the venom, of multiple protein allergens, most
system. Subsequently,
system. Subsequently, severe severe anaphylactic
anaphylactic shockshock could
could lead
lead toto cerebral
cerebral or or myocardial
myocardial ischemia
ischemia
of which possess an enzymatic activity [9]. The major BV allergens and specific Immunoglobulin E
[15,16].These
[15,16]. Theseallergic
allergicresponses
responsesare aredueduetotothethepresence,
presence,within
withinthethevenom,
venom,ofofmultiple
multipleprotein
protein
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PLA2,
which melittin,
possessand anhyaluronidase.
enzymaticactivity Apart[9].
activity fromTheIgE-mediated
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BVallergens
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andspecific
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suggest that allergens
Immunoglobulin can also involveareIgE-independent reactions, such as a bradykinin from(BK) mediator,
Immunoglobulin EE(IgE)
(IgE) inducers
inducers are PLA2,melittin,
PLA2, melittin,andand hyaluronidase.
hyaluronidase. Apartfrom
Apart IgE-mediated
IgE-mediated
leading to various
mechanisms,studies anaphylactic
studiessuggest
suggestthat symptoms
thatallergens [17,18].
allergenscan canalsoThe production
alsoinvolve of this
involveIgE-independent non-immune
IgE-independentreactions, mediator
reactions,such
suchas can
asaa
mechanisms,
be induced by
bradykinin melittin,
(BK) mediator,known as a PLA2
leading activator that can mimic BK’s effects
[17,18].on tracheal tone [17,19].
bradykinin (BK) mediator, leading totovarious
various anaphylactic
anaphylactic symptoms
symptoms [17,18]. The
The production
production ofofthis
this
non-immune mediator can be induced by melittin, known as a PLA2
non-immune mediator can be induced by melittin, known as a PLA2 activator that can mimic BK’s activator that can mimic BK’s
Molecules 2019, 24, 2997 3 of 13
In addition, MCD-peptide or peptide 401 is able to induce an anaphylactoid reaction by degranulating

mast cells [9,20].
Beside molecular studies investigating the possible mechanisms behind inflammatory bee sting
responses, many clinical studies are deeply looking into the potential use of BV for treating chronic
diseases. Hence, the following parts of the review aim to highlight the primary biological properties of
BV and its bioactive molecules that have potential in developing therapeutic strategies.
2. Main Compounds of Bee Venom

BV is an odorless and transparent liquid containing a hydrolytic mixture of proteins with acid
pH (4.5 to 5.5) that bees often use as a defense tool against predators. One drop of BV consists of 88%
of water and only 0.1 µg of dry venom [10]. The latter is an extremely complex blend of peptides
including melittin, adolapin, apamin, and MCD-peptide. It also contains enzymes, most importantly
PLA2, and compounds of low molecular weight like bioactive amines (e.g., histamine and epinephrine)
and minerals [9].
2.1. Melittin
Melittin, a 26-residue peptide, is the main component of BV and accounts for 40–60% of its
composition [21]. The carboxyl-terminal region of the peptide is hydrophilic and responsible for the
lytic action, while the amino-terminal region of its sequence is predominantly hydrophobic with no
lytic activity [22]. The amphipathic property of melittin makes it soluble in water in both its monomeric
and tetrameric forms. It also allows melittin to be easily inserted into membranes by disrupting both
natural and synthetic phospholipid bilayers. Previous studies have shown that the mechanism of
action of melittin in disrupting membranes is mediated by pore formation lysing both prokaryotic
and eukaryotic cells in a non-selective matter. In fact, melittin binds to membranes as monomers but
acts on the membrane inclusively. Depending on its concentration, this biopeptide can induce either
transient or stable pores [23]. When a transient pore is formed, only ions are able to diffuse through
the membrane. However, in the case of stable pore formation, the membrane becomes permeable to
relatively large molecules, such as glucose [24]. The pore formation induced by melittin is responsible
of its hemolytic, anti-microbial, anti-fungal, and antitumor activities [12,25]. Lately, melittin has been
shown to cause neural plastic changes along pain-signaling pathways by activation and sensitization
of nociceptor cells. The mechanism involves the phosphorylation of mitogen-activated protein kinases
(MAPK) as well as the activation of thermal nociceptive channels like TRPV1 (transient receptor
potential vanilloid receptor 1), ATP-gated P2X and P2Y purinergic receptors. Likewise, melittin can
act as an activator of PLA2 [26]. It is also a major biologically active substance of BV that produces
anti-nociceptive, anti-inflammatory, and anti-arthritic effects once administrated to the acupoint of the
patient [27].
2.2. Apamin
Apamin is an 18-amino acid peptide containing two disulfide bridges. It is the smallest neurotoxin
in BV [28]. This polypeptide is able to cross the blood-brain barrier and therefore it affects the central
nervous system functioning via different modes of action. For example, it causes neurotoxic effects in
the mammalian spinal cord, resulting in hyperactivity and seizures, as it has been shown in rats. By
blocking calcium-activated K+ channels, apamin is also able to affect the permeability of cell membrane
toward potassium ions (K+ ). In the vascular smooth muscle, the toxin is able to inhibit vascular
smooth muscle cell proliferation and migration via the Akt and Erk signaling pathways [29]. This
finding highlights the potential of apamin in atherosclerosis therapy strategies. Another study assessed
the consequences of K+ channels sensitivity to apamin and showed that the neurotoxin can inhibit
NO-induced relaxation of the spontaneous contractile activity of the myometrium in non-pregnant
women [30].
Molecules 2019, 24, 2997 4 of 13
2.3. Mast Cell Degranulating (MCD) Peptide

The MCD peptide, also known as peptide 401, is a BV polypeptide containing 22 amino acids with
similar structure to apamin, as they both contain two disulfide bonds. It accounts for 2–3% of BV’s
dry weight. The name MCD echoes the biological action in histamine release from mast cells. It is an
epileptogenic neurotoxin, an important inhibitor of K+ channels, and can cause a significant reduction
in the blood pressure of rats [31]. Some of MCD biological activities seem to have distinct mechanisms
and may represent a good illustration of the structure-function relationship. Studies describe MCD as
a powerful anti-inflammatory agent and may serve as a potential candidate for the study of secretory
mechanisms of inflammatory cells, such as mast cells, basophils, and leukocytes, leading to the design
of compounds with therapeutic applications [32].
2.4. Adolapin
Adolapin is a basic polypeptide with 103 amino acids residues. It corresponds to 1% of the dry
weight of BV. Researchers have shown that adolapin possesses anti-inflammatory, anti-nociceptive,
and antipyretic effects by blocking prostaglandin synthesis and inhibiting cyclooxygenase activity [33].
The polypeptide can inhibit lipoxygenase from human platelets and may exert an analgesic effect
according to Jung et al. [34].
2.5. Phospholipase A2
PLA2, the most lethal enzyme in BV, is a single polypeptide chain of 128 amino acids containing
four disulfide bridges. Bee venom phospholipase A2 (bvPLA2) belongs to the group III sPLA2
enzymes and can act as a ligand for specific receptors. BvPLA2 represents 12–15% of BV’s dry weight
and is extremely alkaline. BvPLA2 is a hydrolytic enzyme, able to specifically cleave the sn-2 acyl
bond of phospholipids at the water/lipid interface [35]. Interestingly, its activity can be improved by
melittin. This has been shown to occur during the process of erythrocyte lysis, proving the presence
of synergistic action between both bvPLA2 and melittin [36,37]. In fact, it has been demonstrated
that melittin helps in exposing membrane phospholipids to the catalytic site of enzymes via opening
melittin-induced channels [38]. Additionally, new experimental data have demonstrated protective
immune responses of bvPLA2 against a broad range of diseases, such as asthma, Alzheimer’s disease,
and Parkinson’s disease [39–41]. BvPLA2 plays a neuroprotective role by inducing the microglial
deactivation and reducing CD4+ T cell infiltration in the MPTP-induced mouse model of PD (MPTP:
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin) [42].
2.6. Hyaluronidase
Hyaluronidase represents 1.5–2% of BV dry weight and is known to break down hyaluronic
acid in tissues, such as in synovial bursa in rheumatoid arthritis. BV hyaluronidase allows the active
components of BV to diffuse effectively into a victim’s tissue by affecting its structural integrity and
increasing blood flow in the area. These two actions combine to intensify the wide spreading of the
venom [43,44].
3. Bioactivities and Therapeutic Applications of Bee Venom and Its Major Compounds
3.1. Anti-Inflammatory Potential

Inflammation is a protective process for the body in response to harmful stimuli. Chronic
inflammation can lead to the development of several diseases like rheumatoid arthritis (RA), diabetes,
cardiovascular disease, obesity, asthma, skin diseases, and CNS-related diseases, such as PD, AD, and
ALS [45].
Melittin, when administrated at high doses, causes local pain, itching, and inflammation. However,
low doses of this BV compound can induce wide anti-inflammatory effects. Many reports investigated
Molecules 2019, 24, 2997 5 of 13
the anti-inflammatory mechanisms of melittin in different diseases such as RA and ALS [46,47]. In
fact, it acts by inhibiting inflammatory cytokines like interleukin-6 (IL-6), IL-8, tumor necrosis factor-α
(TNF-α), and interferon-γ (IFN-γ). Moreover, melittin decreases signaling pathways that activate
inflammatory cytokines, including nuclear factor-kappa B (NF-κB), protein kinase Akt, and extracellular
signal-regulated kinases (ERK1/2) in porphyromonas gingivalis lipopolysaccharide (PgLPS)-treated
human keratinocytes. These findings indicate that, by blocking their primary signaling pathways,
melittin inhibits inflammatory cytokines leading to a reduced inflammation in skin, liver, joint, and
neuronal tissue [48].
Regarding skin diseases, a recent study by Kim et al. showed that BV reduces Atopic Dermatitis,
the most common allergic chronic inflammatory skin disease [49]. In fact, the venom stimulates
CD55 production by triggering ERK1/2 pathways, which leads to the alleviation of the disease’s
symptoms [50]. Interestingly, a previous study by Shin et al. described the anti-inflammatory potential
of bvPLA2 in skin diseases by showing that the enzyme attenuates Atopic skin inflammation through
interaction with CD206 [51].
3.2. BV Application for the Treatment of Neurodegenerative Diseases
3.2.1. Parkinson’s Disease

PD is a degenerative movement disorder that leads to progressive disability in patients. The
pathological hallmarks of the disease are the progressive loss of dopaminergic neurons in the substantia
nigra (a basal ganglia structure found in the human brain), and the presence of Lewy bodies that contains
aggregates of alpha-synuclein, a widely distributed protein in the brain [52,53]. Abnormal microglial
activation is also a pathological sign in different neurodegenerative diseases, including PD [54]. Many
preclinical trials investigated the effect of BV on the migration of leukocytes or microglial activation in
animal and cellular models. Other tests evaluated the neuroprotective potential of BV acupuncture
therapy (BVA) against rotenone-induced oxidative stress, neuroinflammation, and apoptosis in PD
mice models [55]. Rotenone is a pesticide that may affect pathophysiological mechanisms that are
implicated in PD [55]. Interestingly, BV proved its ability to prevent dopamine depletion after the
administration of rotenone. In addition, the locomotor activity was re-established after treating PD
mice models with BV. The treatment effectively repressed DNA damage and inhibited the expression of
the apoptotic Bax, Bcl-2, and caspase-3 genes in the brain of PD mice. These findings demonstrate that
BV normalized all the apoptotic and neuroinflammatory markers and restored brain neurochemistry
after rotenone injury [56]. It has also been shown that BV can protect doparminergic neurons from
degeneration in experimental PD models. Along with this finding, acupoint stimulation of lower hind
limbs with BV was found to be protective in the MPTP (1-Methyl-4-Phenyl-1,2,3,6-TetrahydroPyridine)
mouse model of PD [57].
3.2.2. Alzheimer’s Disease

AD is the most common neurodegenerative disease and many pathological processes are involved
in its emergence [58]. However, the hypothesis of amyloid cascade and the toxicity of amyloid beta
(Aβ) peptides have dominated research so far due to advanced studies showing that aggregates of this
peptide are characteristic signs of the disease [59–61]. Although the etiology of AD remains unknown,
the evidences suggest that inflammatory responses may play a crucial role in its pathogenesis [62,63].
The current treatments for cognitive loss related to AD rely on the usage of muscarinic or nicotinic
receptor ligands and the acetylcholinesterase (AChE) inhibitor [64]. As an alternative strategy, Ye et al.
showed that bvPLA2 can be used as a treatment to block the progression of AD in transgenic mice [40].
This is due to the ability of bvPLA2 to reduce the accumulation of Aβ and improve cognitive functioning
in mice brains. The same study similarly shows that bvPLA2 can increase glucose brain metabolism and
reduce neuroinflammatory responses in the hippocampus, which can limit AD pathogenesis [40]. A
recent study also showed that regulatory T-cells populations could be modulated by bvPLA2 treatment
Molecules 2019, 24, 2997 6 of 13
in a 3xTg-AD mouse model. Therefore, authors suggested a new therapeutic approach to reduce the
progression of AD by combining bvPLA2 treatment along with Aβ vaccination therapy to prevent its
adverse inflammatory response [60].
3.2.3. Amyotrophic Lateral Sclerosis

ALS is a CNS disease that causes the death of motor neurons [65]. A significant trait of ALS
is the abnormal accumulation of mutant SOD1 (mtSOD1) protein aggregates [66]. A mice model
of ALS carrying the mutated mtSOD1 gene with a Glycine to Alanine substitution (SOD1G93A ) was
characterized by Jaarsma et al., facilitating the understanding of ALS etiology [67]. Both in vitro and
in vivo studies using the mutant SOD1 transgenic mice demonstrated various cellular pathogenic
events in motor neurons like protein misfolding, dysfunction of mitochondria, and accumulation of
neurofilament [67]. Interestingly, BV showed some potential for counteracting this disease. In fact, the
administration of BV, at a precise and symptomatic stage of the progression of ALS, leads to an increase
in motor activity in SOD1G93A mutant mice and a prolongation in life expectancy when compared
with age-matched control mice. This could be caused by the blockage of activated microglia usually
found in mice models of ALS [68]. Another study demonstrated that bee venom acupuncture (BVA) at
ST36 inhibits neuroinflammation in the spinal cord of symptomatic ALS mice by significantly reducing
the levels of inflammatory proteins like TLR4, CD14, and TNF-α [69].
3.3. BV and/or Melittin Applications in Cancer

The use of apitoxin, especially its main compound melittin, as a novel cancer-treatment strategy has
gained wide importance recently [70,71]. In fact, melittin is known to be a nonspecific cytolytic peptide
that can attack the lipid bilayer, thus leading to a significant toxicity when injected intravenously [72].
Nevertheless, many optimization approaches, including the use of nanoparticle-based delivery of
melittin, have been exploited. Remarkably, the crude BV as well as melittin have shown antitumor
activities against different cancer cell types including breast, liver, leukemia, lung, melanoma, and
prostate cancer cells [70,72–75]. Wang et al. [76] investigated the mechanism behind melittin antitumor
activity and showed that melittin can induce apoptosis of hepatocellular carcinoma cells (HCC) through
the activation of the CAMKII-TAK1-JNK/p38 signaling pathway (CAMKII: Ca2+ /calmodulin-dependent
protein kinase; TAK1: Transforming growth factor-beta-activated kinase 1; JNK/p38: Mitogen-activated
protein kinases). Moreover, melittin can sensitize TRAIL-resistant HCC cells (TRAIL: Tumor necrosis
factor-related apoptosis-inducing ligand) to TRAIL-induced apoptosis, probably via activating the
CAMKII-TAK1-JNK/p38 pathway and inhibiting the IKK-NFκB pathway (Figure 3). These findings
are in agreement with the activation of calcium channels by melittin that leads on to the increase
of intracellular Ca2+ concentration and the activation of calcium sensitive CaMKII, as seen also in
Figure 3 [76].
Park et al. [13] also reported that BV and its major component, melittin, induce an inhibition of
cancer cells growth both in vitro and in vivo via the activation of caspases (3 and 9) pathways and
the inhibition of NF-κB signaling and its downstream proliferative and anti-apoptotic gene products
like Bcl-2, cIAP-2, iNOS, COX-2, and cPLA2 (Figure 3) [13]. Similarly, Zheng et al. [77] demonstrated
that BV exerts an anti-proliferative effect and induces apoptosis via the activation of death receptors
(DR4 and DR5). Another interesting finding emerged about melittin by highlighting its anti-metastatic
and antigrowth properties [73]. In cancer, metastasis and the invasion of malignant cells are the main
reasons behind the progression of the disease. Therefore, researchers in the cancer field focus on
understanding the molecular mechanisms that regulate malignant cell migration and the possible way
to prevent it, as a crucial step in their fight against cancer [78,79]. In this context, it has been found that
melittin inhibits in vitro and in vivo HCC cells motility by suppressing Rac1-dependent pathways [73].
On the other hand, a recent study proved that the combination of melittin with a chemotherapeutic
agent like temozolomide remarkably decreases growth along with the invasion of melanoma cells,
compared to conditions where TMZ or melittin were used alone [71].
Molecules 2019, 24, 2997 7 of 13
These findings show the great potential of melittin in cancer treatment by acting on different key
points of
Molecules the24,disease
2019, and REVIEW
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asan
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Despite theconvincing
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applicability to humans remains very very
remains challenging because
challenging of its non-specific
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inmelittin
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ordersuch problems.
to avoid suchDue to nanotechnology,
problems. it has been possible
Due to nanotechnology, to develop
it has been andtoeffectively
possible develop
test conjugates of melittin against a broad range of human cancer types in preclinical
and effectively test conjugates of melittin against a broad range of human cancer types in preclinical models [81].
Cheng et al. aimed to develop an efficient yet safe delivery system for melittin,
models [81]. Cheng et al. aimed to develop an efficient yet safe delivery system for melittin, which which can reduce its
hemolytic
can activity
reduce its whileactivity
hemolytic conserving
whileits cytotoxic its
conserving advantages. Therefore, aTherefore,
cytotoxic advantages. dual secured
a dualnano-sting
secured
(DSNS) was designed via the combination of a zwitterionic glycol chitosan
nano-sting (DSNS) was designed via the combination of a zwitterionic glycol chitosan and disulfide and disulfide bonds.
Melittin loaded DSNS showed almost complete cytotoxic effect on many
bonds. Melittin loaded DSNS showed almost complete cytotoxic effect on many cancer cells types cancer cells types at very
at
low low
very concentrations
concentrations while leaving
while redred
leaving blood
bloodcells
cellsunharmed
unharmed[82]. [82].Furthermore,
Furthermore, itit has been shown
has been shown
that intravenous
that intravenousadministration
administrationof of melittin
melittin prodrug-loaded
prodrug-loadednanoparticles,
nanoparticles,using
usingper
per fluorocarbon
fluorocarbon
nanoparticles,in
nanoparticles, inaamelanoma
melanomamouse mousemodel
modelefficiently
efficientlyreduced
reducedthe thetumor
tumorgrowth
growthrate
ratecompared
comparedto to
saline and blank nanoparticle treatment
saline and blank nanoparticle treatment [83]. [83].
3.4.Antiviral
3.4. Antiviraland andAntibacterial
AntibacterialProperties
Properties
ItIt is
is well
wellknown
known thatthat
BV and
BV its
andtwoitsmajor
twocomponents (melittin and
major components PLA2) and
(melittin present
PLA2)antimicrobial
present
activities and thus can be used as complementary anti-bacterial agents
antimicrobial activities and thus can be used as complementary anti-bacterial agents [84–87]. [84–87]. These compounds
These
exert their effects against bacteria by inducing pores through their membranes
compounds exert their effects against bacteria by inducing pores through their membranes leading leading to their cleavage
and
to then
their lysis [36].
cleavage and then lysis [36].
Nevertheless,
Nevertheless, the the antiviral
antiviral effect
effectofofBVBVhas
hasnotnot
beenbeenmentioned
mentioned muchmuchin literature. A recent
in literature. study
A recent
investigated
study BV antiviral
investigated potential
BV antiviral and came
potential out with
and came interesting
out with findings
interesting both both
findings in vivo and in
in vivo andvitro.
in
This study showed that BV and melittin have significant antiviral effects against
vitro. This study showed that BV and melittin have significant antiviral effects against numerous numerous enveloped
viruses (vesicular
enveloped viruses stomatitis virus, influenza
(vesicular stomatitis virus, A virus, herpes
influenza simplex
A virus, herpes virus,
simplexetc.)virus,
and non-enveloped
etc.) and non-
viruses (enterovirus-71 and coxsackie virus) in vitro [88]. The study also
enveloped viruses (enterovirus-71 and coxsackie virus) in vitro [88]. The study also showed showed that melittin protected
that
mice that were exposed to lethal doses of influenza A H1N1 virus. Although
melittin protected mice that were exposed to lethal doses of influenza A H1N1 virus. Although the the precise mechanism of
action by which BV and melittin act as antiviral agents remains unclear, it
precise mechanism of action by which BV and melittin act as antiviral agents remains unclear, it has has been confirmed that
BV interacts
been confirmed directly
that BV with the viral
interacts surface.
directly Moreover,
with the viral BV and its
surface. components
Moreover, can its
BV and stimulate type I
components
interferon (IFN), and therefore suppress viral replication in the host cell [89].
can stimulate type I interferon (IFN), and therefore suppress viral replication in the host cell [89].
Additionally, researchers
Additionally, researchers at at Washington
Washington University
University School
School of of Medicine
Medicine in in St. Louis have
St. Louis have
reported the possible application of nanoparticles loaded with melittin
reported the possible application of nanoparticles loaded with melittin in destroying the human in destroying the human
immunodeficiencyvirus
immunodeficiency viruswhile
whileleaving
leavingnon-infected
non-infectedcellscellsunharmed.
unharmed.In Inthis
thisapproach,
approach,the theauthors
authors
suggest a preventive strategy in which these nanoparticles are used in developing a vaginal gel that
inhibits the spread of HIV. Its theoretical principle is as follows: Melittin molecules present on
nanoparticles fuse with the viral envelope forming pore-like attack complexes, thus breaking the viral
envelope [14]. Another study showed that bvPLA2 can also block the replication of the virus. The
Molecules 2019, 24, 2997 8 of 13
suggest a preventive strategy in which these nanoparticles are used in developing a vaginal gel
that inhibits the spread of HIV. Its theoretical principle is as follows: Melittin molecules present on
nanoparticles fuse with the viral envelope forming pore-like attack complexes, thus breaking the viral
envelope [14]. Another study showed that bvPLA2 can also block the replication of the virus. The
same team further identified the peptide sequence of bvPLA2 responsible of the inhibition of HIV
replication [89–92].
4. Conclusions
The use of BV for medical applications can be traced back thousands of years. Here, the therapeutic
interests of crude bee venom and/or its main compounds, particularly melittin, are discussed. The latter
grants broad anti-inflammatory properties by affecting primary inflammation signaling pathways and
inducing the inhibition of pro-inflammatory genes expression. BV also possesses a neuroprotective
potential in neurodegenerative diseases such as PD, AD, and ALS by significantly blocking their
progression and improving cognitive functioning in mice models. In terms of antitumor activity,
both melittin and BV have a cytotoxic effect on cancer cells and a significant anti-metastatic activity.
Optimization approaches are currently focusing on the possible use of nanoparticle-based delivery of
melittin, or even BV, in order to avoid their nonspecific cytotoxic effect. The antiviral activity of BV is
also promising since BV and melittin have notable toxic effects against a broad spectrum of enveloped
viruses, including the challenging HIV, and few non-enveloped viruses. Finally, the clinical application
of BV therapy is still a long way ahead, but researchers believe that the ongoing work on this topic will
eventually allow BV and its compounds to be considered as definitive candidates in various therapies
in upcoming years.
Author Contributions: Conceptualization, Z.F. and J.-M.S.; funding acquisition, Z.F. and D.E.O.; writing—original
draft preparation, R.W.; writing—review and editing, J.F., R.W., M.R., and Z.F.
Funding: This research was funded by the Lebanese University.
Acknowledgments: The authors would like to thank Elie N. Mahfoud for his diligent proofreading of the paper.
They also thank Cesar Mattei and Christian Legros for their helpful discussion.
Conflicts of Interest: The authors declare no conflict of interest.
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
molecules
Article
Comparison of Physicochemical, Microbiological
Properties and Bioactive Compounds Content of
Grassland Honey and other Floral Origin Honeys
Laura Agripina Scripcă, Liliana Norocel and Sonia Amariei *
Faculty of Food Engineering, Stefan cel Mare University of Suceava, 720229 Suceava, Romania
* Correspondence: sonia@usm.ro; Tel.: +40-230-216-147
Academic Editor: Juraj Majtan

Received: 30 June 2019; Accepted: 9 August 2019; Published: 13 August 2019
Abstract: The aim of this study was to compare the physicochemical, the microbiological, and
the antioxidant characteristics of unifloral honey, polyfloral honey, honeydew, and hay meadows
honey. Hay meadow is type of semi-natural grassland with a great floral diversity, an important
resource for pollinators. Grasslands are the source of the spring nectar honey obtained in May
and June. Water content, sugars (fructose, glucose, sucrose, trehalose, melezitose, maltose, erlose,
turanose, and raffinose), electrical conductivity, phenolic content (gallic acid, protocatechuic acid,
4-hydrxybenzoic acid, vanilic acid, chlorogenic acid, caffeic acid, p-coumaric acid, rosmarinic acid,
myricetin, quercitin, luteolin, kaempferol), color, viscosity, and microbiological characteristics were
performed for all samples of honey. The total polyphenols content was significant for grassland honey
(21.50 mg/100 g) and honeydew (30.49 mg/100 g) and less significant for acacia (0.08 mg/100 g) and
rape honey (0.14 mg/100 g). All samples were microbiologically safe, and standard plate count (SPC)
values were <10 cfu/g for all the samples, but the grassland honey had the highest microbiological
quality: 33.3% of samples without microorganisms, 50.0% with the presence of yeast under limit,
and 16.7% with yeast and mold under limit, a situation that does not meet other types of honey.
The results of statistical analysis obtained with principal component analysis (PCA) showed a major
difference between the grassland honey and the other types of honey.
Keywords: grassland honey; bioactive compounds; sugar composition; phenolic compounds
1. Introduction
According to Directive 2001/110 EC, honey is defined as the naturally occurring natural substance
produced by Apis melifera bees [1]. Honey is a sweet substance naturally produced by the transformation
and the processing of the nectar of bees or dew, which is stored in the cells of the honeycombs [2].
Honey has extremely varied composition due to geographical and botanical origin. Honey contains
about 80% sugars [3,4] (fructose, glucose, maltose, sucrose, isomaltose, melezitose, raffinose, erlose,
turanose, trehalose), and the other 20% is water, organic acids (acetic, butanoic, formic, citric, succinic,
lactic, malic, pyroglutamic, gluconic, and a large number of aromatic acids) [5], amino acids (proline,
asparagine acid and asparagine, calcium alginate, serine, methionine, tyrosine, leucine, lysine,
arginine, histidine, ornithine, isoleucine, valine), minerals (calcium, iron, zinc, potassium, phosphorus,
magnesium, selenium, chromium and manganese), group B vitamins (riboflavin, niacin, folic acid,
pantothenic acid, pyridoxine and vitamin C), and enzymes (diastase, invertase, glucose oxidase,
catalase) [6]. The sources of nectar are the spontaneous flora, the culture, and the sweet excretions of
aphids or sweet and viscous substances that are secreted in certain periods by leaves and stems of trees.
This diversity of sources results in a wide variety of types of honey with special chemical compositions,
microbiological properties, and sensory qualities. Moreover, honey has anti-inflammatory properties,

Molecules 2019, 24, 2932 2 of 17
and its use as an antiviral, antibacterial, antiparasitic, antimutagenic, and anticancer agent has often
been suggested [7–11]. Researchers proved that the properties of honey make it an important natural
antioxidant due to its phenolic content [12,13]. In addition, correlations were reported between
the color and the biological properties of honey, including phenolic content, antioxidant capacity,
antimicrobial activity, and other parameters of honey [14,15]. Grassland honey has a special chemical
composition due to the floral richness of the areas that act as sources of nectar and the soil composition.
The grasslands are generally protected areas with dozens of flower species, where they come from the
high content of polyphenols and their variety, transmitting to honey their special flavors and colors.
The aim of this study was to draw attention to this type of honey and to promote efforts to keep this
protected area from expansion of agricultural crops, which are treated with fertilizers and pesticides.
2. Results
Physicochemical parameters of six types of honey (rape, honeydew, linden, polyfloral, acacia,
and grassland) were investigated. Tables 1–3 present the quality parameters (water content, color,
electrical conductivity, viscosity, sugars, and polyphenols) of 14 rape, nine honeydews, 16 polyfloral,
21 lindens, 21 acacia, and 12 grassland honey samples.
Molecules 2019, 24, 2932 3 of 17
Table 1. Physicochemical parameters of different types of honey from Romania.
Water Hue angle, Yellow Conductivity Viscosity

Honey Variety L* a* b* Chroma
Content, % (degrees) Index (mS/cm) (Pa·s)
Rape (n = 14) Mean ± SD 17.32 ± 0.45 26.06 ± 5.55 −0.72 ± 0.64 7.83 ± 4.12 3.51 ± 0.93 84.99 ± 3.36 42.48 ± 6.05 0.14 ± 0.02 12.69 ± 1.12
Min 16.70 17.61 −2.19 4.56 2.66 76.77 25.03 0.11 10.96
Max 18.00 38.07 −0.14 18.61 5.79 88.81 78.93 0.19 14.73
Honeydew (n = 9) Mean ± SD 16.62 ± 0.71 21.71 ± 1.38 7.06 ± 1.20 8.36 ± 1.45 5.53 ± 0.38 49.72 ± 5.55 55.24 ± 10.10 0.64 ± 0.05 8.46 ± 0.74
Min 15.70 19.67 5.35 6.17 4.80 41.82 36.97 0.57 7.14
Max 17.90 23.84 9.29 10.24 5.94 57.29 69.40 0.73 9.47
Polyfloral (n = 16) Mean ± SD 16.78 ± 0.65 36.13 ± 2.16 4.69 ± 0.74 13.77 ± 0.57 5.87 ± 0.22 71.26 ± 2.51 54.67 ± 4.43 0.38 ± 0.05 6.63 ± 1.30
Min 15.90 31.90 3.24 12.74 5.46 66.28 48.87 0.28 4.18
Max 17.90 39.65 5.98 14.82 6.21 76.39 62.14 0.45 8.62
Linden (n = 21) Mean ± SD 17.01 ± 0.51 33.06 ± 1.76 −0.49 ± 0.31 13.85 ± 0.69 4.39 ± 0.21 87.96 ± 1.32 60.02 ± 4.50 0.61 ± 0.04 6.37 ± 0.96
Min 16.00 27.92 −1.21 12.43 4.02 85.05 51.29 0.55 4.76
Max 17.90 35.27 −0.12 14.94 4.84 89.52 70.20 0.72 8.06
Acacia (n = 21) Mean ± SD 17.13 ± 0.48 49.26 ± 2.23 −1.11 ± 0.32 15.05 ± 1.07 4.92 ± 0.17 85.73 ± 1.30 43.77 ± 4.01 0.28 ± 0.04 3.35 ± 0.65
Min 16.30 45.28 −1.69 13.25 4.56 83.20 35.98 0.22 2.12
Max 18.00 52.61 −0.45 16.38 5.29 88.43 51.27 0.35 4.27
Grassland (n = 12) Mean ± SD 17.03 ± 0.38 32.77 ± 1.94 1.81 ± 1.74 13.31 ± 0.78 4.87 ± 0.52 82.22 ± 7.46 58.15 ± 3.68 0.31 ± 0.04 7.71 ± 0.72
Min 16.30 29.34 0.31 12.15 4.35 62.63 52.04 0.24 6.39
Max 17.60 35.23 6.29 14.59 5.99 88.78 63.39 0.35 8.64
SD: standard deviation; Min: minimum value; Max: maximum value. L*: lightness of honey, a*—from red(+) to green (-), b*—from yelow(+) to blue(-), chroma-saturation.
Molecules 2019, 24, 2932 4 of 17
Table 2. Sugar content of different types of honey from North Romania.
Sugars (g/100 g)
Honey Variety
Glucose Fructose Sucrose Trehalose Melezitose Maltose Erlose Turanose Raffinose G/F G/W
Rape (n = 14) Mean ± SD 39.95 ± 1.54 30.26 ± 1.06 0.93 ± 0.66 0.01 ± 0.00 0.01 ± 0.00 0.29 ± 0.43 0.00 ± 0.02 0.17 ± 0.13 0.05 ± 0.12 1.31 ± 0.09 2.30 ± 0.12
Min. 38.02 28.58 0.00 0.00 0.00 0.00 0.00 0.01 0.00 1.20 2.16
Max. 42.55 31.54 1.76 0.02 0.02 1.24 0.10 0.44 0.43 1.48 2.49
Honeydew (n = 9) Mean ± SD 36.41 ± 0.54 32.41 ± 0.19 1.36 ± 0.28 0.04 ± 0.00 4.89 ± 1.56 0.27 ± 0.33 0.42 ± 0.53 0.10 ± 0.06 0.01 ± 0.01 1.12 ± 0.02 2.19 ± 0.09
Min. 35.68 32.14 0.74 0.03 1.24 0.00 0.00 0.03 0.00 1.09 2.03
Max. 37.21 32.68 1.80 0.04 5.84 0.76 1.54 0.20 0.02 1.15 2.34
Polyfloral (n = 16) Mean ± SD 31.96 ± 0.26 38.55 ± 0.61 1.12 ± 0.57 0.25 ± 0.02 0.71 ± 0.14 0.01 ± 0.02 1.23 ± 2.66 0.23 ± 0.20 0.03 ± 0.01 0.82 ± 0.02 1.90 ± 0.07
Min. 31.62 37.54 0.19 0.23 0.58 0.00 0.00 0.00 0.01 0.80 1.79
Max. 32.31 39.35 1.95 0.28 1.12 0.08 8.85 0.71 0.05 0.86 2.02
Linden (n = 21) Mean ± SD 30.88 ± 0.41 40.48 ± 0.53 0.51 ± 0.44 0.32 ± 0.03 1.98 ± 0.42 0.20 ± 0.53 0.08 ± 0.16 0.11 ± 0.12 0.05 ± 0.09 0.76 ± 0.02 1.81 ± 0.06
Min. 30.30 39.43 0.08 0.28 1.13 0.00 0.00 0.00 0.00 0.73 1.70
Max. 31.56 41.15 1.67 0.38 2.56 2.34 0.68 0.40 0.38 0.80 1.92
Acacia (n = 21) Mean ± SD 26.21 ± 1.62 44.11 ± 1.03 0.63 ± 0.51 1.85 ± 0.95 0.04 ± 0.01 0.05 ± 0.14 0.53 ± 1.15 0.12 ± 0.14 0.02 ± 0.03 0.59 ± 0.05 1.53 ± 0.10
Min. 23.63 42.65 0.04 0.83 0.03 0.00 0.00 0.00 0.00 0.51 1.32
Max. 28.26 45.98 1.58 3.74 0.07 0.62 4.28 0.45 0.13 0.66 1.66
Grassland (n = 12) Mean ± SD 36.82 ± 1.91 36.64 ± 3.66 0.60 ± 0.28 0.29 ± 0.20 1.88 ± 1.36 0.18 ± 0.31 0.07 ± 0.15 0.19 ± 0.16 0.02 ± 0.01 1.02 ± 0.14 2.16 ± 0.12
Min. 34.26 31.85 0.27 0.03 0.03 0.00 0.00 0.01 0.00 0.83 1.99
Max. 39.98 41.23 0.99 0.47 3.11 0.87 0.52 0.44 0.04 1.19 2.34
SD: standard deviation; Min: minimum value; Max: maximum value. G/F: glucose to fructose ratio; G/W: glucose/water content ratio.
Molecules 2019, 24, 2932 5 of 17
Table 3. The results of the concentration of the twelve phenolic compounds.

Phenolic Compound (mg/100 g)
Honey Variety
Total
4-Hydroxy
Gallic Protocatechuic Vanilic Chlorogenic Caffeic P-Coumaric Rosmarinic Total Poliphenols
Benzoic Myricetin Quercitin Luteolin Kaempferol
Acid Acid Acid Acid Acid Acid Acid Poliphenols Content, mg
Acid
GAE/100 g
Rape
Mean ± SD 0.00 ± 0.01 0.01 ± 0.05 0.01 ± 0.03 0.01 ± 0.03 0.02 ± 0.05 0.04 ± 0.05 0.03 ± 0.05 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.14 ± 0.04 0.17 ± 0.02
(n = 14)
Min 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.08 0.12
Max 0.07 0.21 0.14 0.14 0.17 0.15 0.16 0.00 0.00 0.00 0.00 0.00 0.21 0.22
Honeydew 14.01 ±
Mean ± SD 0.14 ± 0.22 2.44 ± 4.62 0.18 ± 0.37 5.74 ± 9.19 0.37 ± 0.65 0.74 ± 1.80 0.12 ± 0.09 3.47 ± 6.47 2.91 ± 6.95 0.08 ± 0.24 0.27 ± 0.40 30.49 ± 22.13 32.5 ± 0.8
(n = 9) 23.95
Min 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 14.87 14.95
Max 0.61 13.32 0.99 24.81 1.78 5.53 0.23 60.60 19.75 21.21 0.71 1.18 80.38 80.47
Polyfloral
Mean ± SD 1.27 ± 2.10 0.15 ± 0.24 0.08 ± 0.18 1.20 ± 1.03 0.13 ± 0.31 0.14 ± 0.45 0.29 ± 0.55 3.80 ± 2.37 0.50 ± 0.85 1.23 ± 1.89 0.07 ± 0.28 0.24 ± 0.35 9.13 ± 1.84 9.36 ± 1.82
(n = 16)
Min 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6.54 6.78
Max 5.23 0.65 0.52 3.10 1.16 1.84 2.32 6.57 2.26 6.13 1.12 1.21 12.81 12.94
Linden
Mean± SD 1.10 ± 1.28 0.22 ± 0.43 0.10 ± 0.19 0.00 ± 0.00 0.15 ± 0.47 0.10 ± 0.28 0.12 ± 0.29 0.57 ± 1.36 0.31 ± 0.59 0.03 ± 0.10 0.00 ± 0.00 0.01 ± 0.04 2.71 ± 1.64 2.83 ± 1.63
(n = 21)
Min 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.94 1.06
Max 3.97 1.72 0.54 0.00 2.15 1.20 1.25 4.67 1.92 0.48 0.00 0.20 5.44 5.49
Acacia (n
Mean± SD 0.02 ± 0.05 0.00 ± 0.00 0.00 ± 0.01 0.00 ± 0.02 0.01 ± 0.03 0.03 ± 0.03 0.02 ± 0.06 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.08 ± 0.06 0.09 ± 0.05
= 21)
Min 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.02 0.04
Max 0.20 0.00 0.02 0.09 0.14 0.09 0.29 0.00 0.00 0.00 0.00 0.00 0.28 0.32
Grassland
Mean ± SD 0.53 ± 1.50 2.31 ± 3.52 0.52 ± 1.40 7.35 ± 12.48 0.41 ± 1.34 0.44 ± 1.08 3.78 ± 8.60 3.35 ± 2.81 0.92 ± 1.10 1.16 ± 1.52 0.14 ± 0.32 0.60 ± 1.27 21.50 ± 10.73 22.16 ± 11.01
(n = 12)
Min 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 12.97 13.01
Max 5.23 8.85 4.78 44.81 4.67 3.56 24.23 6.23 2.67 4.26 0.92 4.53 45.85 45.88
SD: standard deviation; Min: minimum value; Max: maximum value.

Molecules 2019, 24, 2932 6 of 17
2.1. Water Content Determination

Water content determination is important because it influences the shelf-life of honey. High water
content could accelerate crystallization in certain types of honey and increase its water activity to
ferment and deteriorate its quality [16,17].
The Codex Alimentarius [18] standard specified that water content should not exceed 20% in honey
to ensure safety against fermentation caused by the action of osmotolerant yeasts during storage [19].
High water content indicates extraction of a product in high humidity conditions or premature
extraction. If the content is smaller, the honey crystallizes faster. Glucose/water content ratio (G/W)
is another indicator for honey crystallization. If the G/W ratio is greater than 2.1, honey crystallizes
faster [20]. All water content values in the tested honey samples were normal and did not exceed 20%.
All results of water content were in accordance with the legislation established by the Codex [18].
The lowest water content was found in the honeydew (H) sample—H8 with 15.7%—and the highest
content was found in acacia (A) honey and rape (R) honey, A11 and R9, respectively, with 18.0% each.
For rape honey and acacia honey, water content varied from 16.7% in R3 to 18.0% in R9, and from
16.3% in A20 to 18.0% in A11.
The values of water content for linden (L) honey samples ranged from 16.0% in L15 to 17.9% in L2
and L18. The water content of polyfloral (P) honey ranged from 15.9 % in P6 and P14 to 17.9% in P2.
In honeydew and grassland (G) honeys, the water content varied from 15.7 % in H8 to 17.9% in H4 and
from 16.3% in G3 to 17.6% in G1 and G6.
2.2. Electrical Conductivity (EC)

The electrical conductivity (EC) indicates the ability of honey to conduct an electric current,
and tests were carried by ions and chemical modification in the honey. The Codex Alimentarius
requires honey to have an electrical conductivity no greater than 0.8 mS/cm. The EC is an important
physicochemical parameter in unifloral honey authentication.
This parameter is influenced by storage condition, temperature, water content, minerals, and ions
content. The lowest level of electrical conductivity was found in rape honey (R1 with 0.11 mS/cm),
and the highest level was 0.73 mS/cm in honeydew H4. The highest value of EC in rape samples
analyzed was 0.19 mS/cm in R9, and the lowest was 0.11 mS/cm in R1.
For linden honey, EC varied between 0.55 mS/cm in L15 and 0.72 mS/cm in L2. EC content in
acacia and polyfloral honey varied from 0.22 in A4 and A10 to 0.35mS/cm in A11 and from 0.28 mS/cm
in P11 to 0.45 mS/cm in P12. In honeydew and grassland honey, EC ranged between 0.57 mS/cm in H8
to 0.73 mS/cm in H4 and between 0.24 mS/cm in G6 to 0.35 mS/cm in G12. Similar results were found
by Alves et al. [21]. The conductivity value of linden honey (0.64 mS/cm) fell within the established
limits of Directive 2001/110 EU (<0.8 mS/cm) [22].
2.3. Color
The color of honey is one of the most important indicators for consumers. Lightly colored honey
is preferred oven dark honey. The color of honey is dependent on factors such as floral origin and
nectar source. In the current study, differences were determined for grassland characteristics and the
other assortments of honey.
The color parameters analyzed were L*, a*, b*, (L*—luminosity, a*—from red(+) to green(-),
b*—from yelow(+) to blue(-)), the chrome (saturation)and the hue angle (H) were also calculated.
The L* parameter represents the lightness of honey, and the parameter ranged between 19.67 in
honeydew and 52.61 in acacia honey.
The a* parameter represents the green compound (negative a* values), which was present in
all samples of rape honey, acacia honey, and linden honey. Higher values of the green compound
were found for rape honey (−2.19). The red parameter (positive a* values) was found in grassland,
honeydew, and polyfloral honey. Higher values of the red parameter were found for honeydew (9.29).
Molecules 2019, 24, x 5 of 17
found for rape honey (−2.19). The red parameter (positive a* values) was found in grassland,
honeydew,
Molecules 2019, and polyfloral honey. Higher values of the red parameter were found for honeydew
24, 2932 7 of 17
(9.29).
The yellow parameter (positive b* values) was found in all samples and in all types of honey
The yellow
analyzed. parameter
These values (positive
ranged betweenb* 4.56
values)
andwas found
18.61 in honey.
in rape all samples and in all types of honey
analyzed. These values ranged between 4.56 and 18.61 in rape honey.
2.4. Viscosity
2.4. Viscosity
Honey viscosity is another important parameter for the evaluation of state, fluidity, and
Honey viscosity is another important parameter for the evaluation of state, fluidity,
crystallization.
and crystallization.
The highest value of viscosity (14.73 Pa·s) was found in rape honey, and the lowest level (4.18
The highest value of viscosity (14.73 Pa·s) was found in rape honey, and the lowest level (4.18 Pa·s)
Pa·s) of this parameter was found in some samples of polyfloral honey.
of this parameter was found in some samples of polyfloral honey.
In order to emphasize the difference between the six types of honey, we performed principal
In order to emphasize the difference between the six types of honey, we performed principal
component analysis (PCA), and the results are presented in the biplot from Figure 1. The PCA
component analysis (PCA), and the results are presented in the biplot from Figure 1. The PCA
method limited all data into two main components covering 83.82% of the variability (PC1-50.05%
method limited all data into two main components covering 83.82% of the variability (PC1-50.05% and
and PC2-33.76%). Analyzed parameters such as electrical conductivity, chroma, a*, and yellow index
PC2-33.76%). Analyzed parameters such as electrical conductivity, chroma, a*, and yellow index were
were grouped around the polyfloral honey and the honeydew honey. Water content and viscosity
grouped around the polyfloral honey and the honeydew honey. Water content and viscosity had higher
had higher values for rape honey, and hue angle, luminosity, and b* were more representative for
values for rape honey, and hue angle, luminosity, and b* were more representative for acacia honey.
acacia honey. Grassland and linden honey had almost medium values and are located in the center
Grassland and linden honey had almost medium values and are located in the center of the biplot.
of the biplot.
Figure 1. Principal component analysis (PCA) of the dataset consisting of analyzed parameters of each
Figure 1. Principal component analysis (PCA) of the dataset consisting of analyzed parameters of
honey sample (R-rape, H-honeydew, P-polyfloral, L-linden, A-acacia, G-grassland).
each honey sample (R-rape, H-honeydew, P-polyfloral, L-linden, A-acacia, G-grassland).
2.5. Sugars
2.5. Sugars
Nine sugars were investigated from 98 samples of six types of honey. The results obtained are
Nine sugars
shown in Table 2. were investigated from 98 samples of six types of honey. The results obtained are
shown Glucose and2.fructose were the predominant compounds in the honey samples analyzed but were
in Table
found in different ratios from one assortment to the next. Although the concentration of both sugars
varies depending on the origin of the honey, it is generally expected that fructose will be found in
a higher proportion than glucose [23,24].
Glucose to fructose ratio (G/F) should be smaller than 1.2 (this ratio is used to evaluate honey
granulation, because glucose is less soluble in water than fructose) [25].
Honeys with a high G/F ratio would remain liquid for longer periods because of the modification
of the saturated level of glucose by the presence of the larger amount of fructose.
In rape, honeydew, and some samples of grassland honey, the predominant sugar found was
glucose, and in acacia, polyfloral, linden, and some samples of grassland honey, the higher concentration
of sugar was represented by fructose.
The lowest level of glucose concentration was found in acacia sample A20 with 23.63 g/100 g
honey, and the highest concentration was 42.55 g/100 g honey in rape sample R1. Fructose content
ranged from 28.58 g/100 g honey in rape sample R1 to 45.98 g/100 g honey in acacia sample A21.
Molecules 2019, 24, 2932 8 of 17
Others sugar identified were sucrose, trehalose, maltose, melezitose, raffinose, erlose, and turanose,
which were found in lower concentrations than glucose and fructose. The sucrose content is an important
parameter of the authentication of honey. The presence of a high level of sucrose in honey indicates
adulteration with different syrups by harvesting the product before maturation, and this content can
be reduced by the action of the invertase enzyme.
The highest levels of sucrose and trehalose were in P9 with 1.94 g/100 g honey and in A21 with
3.74 g/100 g honey, respectively, and in R2, sucrose was not detectable. The lowest concentration of
trehalose was found in A1 (0.83 g/100 g honey).
Maltose, melezitose, erlose, turanose, and raffinose contents were not detectable in some samples
of rape honey, linden honey, polyfloral honey, acacia honey, and grassland honey. The highest content
of maltose was in linden sample L6 with 2.34 g/100 g honey. Erlose, turanose, and raffinose were
found in higher quantities in A17 with 4.28 g/100 g honey, in P2 with 8.84 g/100 g honey, and in
R9 with 0.43 g/100 g honey, respectively. The high content of melezitose (1.98 ± 0.42) in the linden
honey was explained by the presence of honeydew honey. The color of linden honey is light yellow to
amber, and in the case of the presence of honeydew honey (it is possible in linden forests), it becomes
a greenish-gray color, demonstrated by the values of the parameters a* −0.49 ± 0.31 and b* 13.85 ± 0.69,
which indicated the color sample in the field of yellow-yellow-green (a* negative, b* positive, quadrant
II). Usually, linden honey is rich in maltose and contains about 1.2–1.9% of it [26,27]. A content of
0.20 ± 0.53 maltose, as was obtained in the analyzed samples, shows that the linden honey was not
matured—a process in which the maltose is formed and the product darkens. At a ratio of 0.875,
as indicated by the literature, honey crystallizes after three months; at a ratio of 0.76 ± 0.02 for the
analyzed honey, it would crystallize over a longer period.
The G/F ratio was smaller than 1.0 for the analyzed acacia, polyfloral, and linden honeys. These honeys
have a smaller tendency to crystallize. The higher ratio was for acacia and was equal to 0.51.
Some samples of the grassland honey had a G/F < 1.0, and some samples had a G/F ratio > 1.
Rape samples and honeydew samples were characterized by a greater G/F ratio (about 1.48 for rape
honey), indicating rapid crystallization.
A higher G/W ratio was found for rape honey (2.30), and a lower ratio was found for acacia honey (1.53).
The method for the reduction of variables, PCA, was performed also for the sugars content.
The PCA method narrowed the data into two main components covering 74.90% of the variability,
thus PC1 represented 54.04% of data, and PC2 represented 20.86%. The results are illustrated in
Figure 2. It can be observed that acacia honey was predominately fructose and trehalose, and a slightly
lower concentration was found in linden honey. In the opposite quadrant, the grassland and the
honeydew honeys were characterized by melesitose, glucose content, G/W ratio, G/F ratio, and sucrose.
Rape honey and linden honey were characterized by maltose and raffinose. Polyfloral honey was
distinguished from other types of honey by its erlose and turanose contents. Regarding the grassland
honey, there was no specific sugar for this; all the sugars were present in medium concentrations
compared to the other honeys.
Molecules
Molecules 2019,
2019, 24,
24, xx 88 of
of 17
17
grassland
Molecules
grassland honey,
2019, 24, 2932there
honey, there was
was nono specific
specific sugar
sugar for
for this;
this; all
all the
the sugars
sugars were
were present
present in
in medium
9 of 17
medium
concentrations compared to the other
concentrations compared to the other honeys.honeys.
Figure 2.
Figure 2. Principal
2. Principal component
Principalcomponent analysis
componentanalysis of of
analysis of the
the dataset
dataset
the consisting
consisting
dataset of
of sugar
of sugar
consisting concentrations
concentrations
sugar of eachof
concentrations each
ofhoney
each
honey
honey sample.
sample.sample. (R-rape,
(R-rape,(R-rape, H-honeydew,
H-honeydew, P-polyfloral,
P-polyfloral,
H-honeydew, L-linden,
L-linden,
P-polyfloral, A-acacia,
A-acacia,
L-linden, G-grassland).
G-grassland).
A-acacia, G-grassland).
2.6. Phenolic Compounds

2.6. Phenolic
Phenolic Compounds
Compounds
Polyphenols
Polyphenolsgenerally
generallyact
generally as as
act
act primary
as primary
primary antioxidants,
antioxidants,providing
antioxidants, providing
providingprotection by removing
protection
protection by free radicals,
by removing
removing free
free
thus ending
radicals, thusthe reactions
ending the in the oxidative
reactions in thechain [28,29].
oxidative Honey
chain phenolic
[28,29]. profiles
Honey
radicals, thus ending the reactions in the oxidative chain [28,29]. Honey phenolic profiles vary vary
phenolic depending
profiles on
vary
geographical
depending onorigin and
geographical floral source
origin and [30,31].
floral
depending on geographical origin and floral source [30,31].source [30,31].
The
The phenolic
phenoliccompounds
phenolic compoundsfound
compounds foundinin
found thethe
in honey
the analyzed
honey
honey analyzed
analyzed included
included
includedgallic acid,acid,
gallic
gallic protocatechuic
acid, acid,
protocatechuic
protocatechuic
4-hydrxybenzoic
acid,
acid, 4-hydrxybenzoic
4-hydrxybenzoic acid, acid,
vanillic acid, acid,
acid, vanillic
vanillic chlorogenic
acid, acid, acid,
chlorogenic
chlorogenic caffeic
acid, acid, acid,
caffeic
caffeic p-coumaric acid, acid,
acid, p-coumaric
p-coumaric rosmarinic
acid, acid,
rosmarinic
rosmarinic
myricetin,
acid, quercitin,
acid, myricetin, luteolin,
myricetin, quercitin, and
quercitin, luteolin,kaempferol.
luteolin, and The
and kaempferol. total
kaempferol. The polyphenols
The total quantities
total polyphenols were significant
polyphenols quantities
quantities were for
were
grassland
significanthoney
significant for and honeydew
for grassland
grassland honey
honey and andhoneydew
and less significant
honeydew and
and lessfor significant
less acacia andfor
significant rape
for honey.
acacia
acacia andThe
and rape
raperesults
honey.ofThe
honey. the
The
twelve
results phenolic compounds analyzed from honeys are presented in Table 3.
results of the twelve phenolic compounds analyzed from honeys are presented in Table 3. Figure 33
of the twelve phenolic compounds analyzed from honeys are Figure
presented in3 presents
Table 3. the type
Figure
and the quantities
presents
presents the type of
the type andpolyphenols
and the
the quantitiesfromof
quantities the
of honey samples
polyphenols
polyphenols from
fromand theallows
the honeyforsamples
honey an evaluation
samples and of thefor
and allows
allows major
for an
an
compounds
evaluation ofthat
the contribute
major to
compoundscolor and
that flavor.
contribute to
evaluation of the major compounds that contribute to color and flavor. color and flavor.
Figure
Figure 3.
3. Quantities
Quantities of polyphenols in
in honey
honey samples
samples (mg/100
(mg/100 g).
Figure 3. Quantities of
of polyphenols
polyphenols in honey samples (mg/100 g).
g).
The gallic acid was representative for polyfloral honey. Honeydew was rich in caffeic acid,
protocatechuic acid, quercitin, and rosmarinic acid. Grassland honey was characterized by vanillic
acid, 4-hydroxybenzoic acid, p-coumaric acid, myricetin, luteolin, and kaempferol chlorogenic acid.
In accordance with specialty literature that previously proved this, after honeydew, grassland honey
The gallic acid was representative for polyfloral honey. Honeydew was rich in caffeic acid,
protocatechuic acid, quercitin, and rosmarinic acid. Grassland honey was characterized by vanillic
acid, 4-hydroxybenzoic acid, p-coumaric acid, myricetin, luteolin, and kaempferol chlorogenic acid.
In accordance
Molecules 2019, 24,with
2932 specialty literature that previously proved this, after honeydew, grassland honey 10 of 17
had the highest antioxidant activity. The high level of gallic acid was 5.23 mg/100 g honey for
polyfloral honey and grassland honey. The maximum concentration of caffeic acid was found in
had the highest
honeydew antioxidant
(5.53 mg/100 activity.
g honey), Theashigh
as well level of gallic
protocatechuic acidacid was
(13.32 5.23 mg/100
mg/100 g honey),g honey
quercitinfor
polyfloral honey and grassland honey. The maximum concentration of
(21.21 mg/100 g honey), and rosmarinic acid (60.60 mg/100 g honey). The maximum concentration of caffeic acid was found in
honeydew
vanillic acid(5.53
was mg/100
found ing grassland
honey), ashoneywell as protocatechuic
(44.80 acid (13.32
mg/100 g honey), mg/100tog4-hydroxybenzoic
in addition honey), quercitin
(21.21(4.77
acid, mg/100 g honey),
mg/100 and rosmarinic
g honey), p-coumaric acid (60.60
acid, mg/100
(24.23 mg/100g honey).
g honey),The maximum
myricetin, concentration
(2.67 mg/100 of g
vanillic acid was found in grassland honey (44.80 mg/100 g honey), in addition
honey), luteolin (0.91 mg/100 g honey), kaempferol (4.52 mg/100 g honey), and chlorogenic acid (4.67 to 4-hydroxybenzoic
acid, (4.77
mg/100 mg/100 g
g honey). Inhoney), p-coumaric
rape samples were acid,
gallic(24.23
acid, mg/100 g honey),
protocatechuic myricetin,
acid, (2.67 mg/100
4-hydroxybenzoic acid,g
caffeic acid, vanillic acid, p-coumaric acid, chlorogenic acid, and 4-hydroxybenzoic acid wasacid
honey), luteolin (0.91 mg/100 g honey), kaempferol (4.52 mg/100 g honey), and chlorogenic the
(4.67 mg/100
dominant g honey).
phenolic In rape samples
compound for thesewere gallic(0.21
samples acid,mg/100
protocatechuic
g honey).acid,For 4-hydroxybenzoic
acacia honey samples, acid,
caffeic acid,acid
p-coumaric vanillic
was acid, p-coumaric
dominant acid, chlorogenic
(0.29 mg/100 g honey). The acid, andphenolics
other 4-hydroxybenzoic acid was
found in acacia honeysthe
dominant phenolic compound for these samples (0.21 mg/100 g honey). For
were gallic acid, caffeic acid, vanillic acid, p-coumaric acid, and chlorogenic acid. The majority of acacia honey samples,
p-coumaric
linden honeysacid wasrepresented
were dominant (0.29 mg/100 g honey).
by rosemarinic The other
acid (4.67 mg/100 phenolics
g honey),found
andinluteolin
acacia honeys were
and vanillic
gallicwere
acid acid,not
caffeic acid, vanillic
detectable. acid, p-coumaric
Rosemarinic acid, and
acid (6.57 mg/100 chlorogenic
g honey) acid. The majority
was representative of linden
for polyfloral
honeys were represented by rosemarinic acid (4.67 mg/100 g honey), and luteolin and vanillic acid
honeys.
werePCAnot detectable. Rosemarinic
analysis regarding theacid (6.57 mg/100
polyphenols as gactive
honey)variables
was representative
and the honeyfor polyfloral
type ashoneys.
active
observations was performed, and the two main components covering 90.30% of the variabilityactive
PCA analysis regarding the polyphenols as active variables and the honey type as were
observations(PC1-71.27%
determined was performed, andand the two main
PC2-19.03%). components
From Figure 4,covering
it can be90.30% of thethat
observed variability
there waswere a
determined difference
significant (PC1-71.27% and PC2-19.03%).
between From Figure
the six samples. 4, it canhoney
Grassland be observed that there
was rich was a significant
in p-coumaric acid,
difference betweenacid,
4-hydroxybenzoic the six samples. Grassland
kaempferol, honeyacid,
luteolin, vanilic was and
rich chlorogenic
in p-coumaric acid,
acids 4-hydroxybenzoic
content. Honeydew
acid,characterized
was kaempferol, luteolin, vanilic acid,
by protocatechuic andcaffeic
acid, chlorogenic acids content.
acid, quercitin, Honeydew
myricetic, was characterized
and rosmarinic acid. The
by protocatechuic
total acid, caffeic
polyphenols content was acid,
foundquercitin, myricetic,
to be higher and rosmarinic
in honeydew samples.acid.
In theThe total polyphenols
opposite quadrant,
content rape,
acacia, was found to be higher
and linden honeyin had
honeydew
lower samples. In the opposite
concentrations quadrant, acacia,
of all polyphenols, and rape,
gallicand linden
acid was
honey had lower
concentrated concentrations
in polyfloral honey. of all polyphenols, and gallic acid was concentrated in polyfloral honey.
Figure
Figure 4.
4. Principal
Principal component
component analysis
analysis of
of the
the dataset
dataset consisting
consisting of
of polyphenols
polyphenols concentration
concentration of
of each
each
honey
honey sample (R-rape, H-honeydew, P-polyfloral, L-linden, A-acacia, G-grassland).
A-acacia, G-grassland).
2.7. Microbiological
2.7. Microbiological Analysis
Analysis
The values obtained for parameters SPC, total coliforms (TC), Bacillus cereus, yeasts, and molds
for each sample of honey investigated as shown in Supplementary Materials Table S1 were within
acceptable limits according to standards in force. Pathogenic microflora was not detectable for any type
of honey analyzed. The quality of samples was superior in correlation with the values obtained for
parameters SPC, TC, Bacillus cereus, yeasts, and molds. SPC values varied from <10 to 40 cfu/g. TC and
Molecules 2019, 24, 2932 11 of 17
Bacillus cereus were not detectable for any sample analyzed. The higher values of yeasts and molds
were 20 cfu/g and 10 cfu/g, respectively. Clostridium botulinum was absent, which is very important
because its presence in honey is dangerous for babies under one year old [32].
In conclusion, all samples had superior microbiological characteristics, but the grassland honey
had the highest microbiological quality. The microbiological characteristics were correlated with the
phenolic compound content. A higher amount of polyphenols meant a better microbiological stability.
Pearson correlation was performed in order to correlate the analyzed parameters (polyphenols,
sugars, color parameters, conductivity, and water content) for all the samples. In the case of rape
honey, a strong negative correlation was found between glucose content and total polyphenols
(−0.958), and there was a strong positive correlation between glucose (0.956), trehalose (0.900),
melezitose (0.950), and fructose content. Also, a perfect correlation was obtained between the erlose
concentration and the vanilic acid concentration (1.000). Person correlation for honeydew showed
a stronger positive relationship between total polyphenols and melezitose (0.912), fructose (0.832),
trehalose (0.793), and maltose (0.720). Other positive correlations were obtained for chlorogenic
acid and 4-hydroxybenzoic acid (0.992), erlose and 4-hydroxybenzoic acid (0.839), and conductivity
and water content (0.921). Negative correlations resulted between total polyphenols and G/F ratio
(−0.808) and glucose content (−0.828). Polyfloral honey indicated a positive correlation between total
polyphenols and fructose (0.955), trehalose (0.952), and melezitose (0.919), as well as one between erlose
and caffeic acid (0.746). There was a negative correlation in the case of gallic acid with chlorogenic acid
(−0.723) and rosmarinic acid (−0.760) as well as total polyphenols and glucose (−0.955). Linden honey
only evidenced strong correlations between sugar and polyphenols content (negative for glucose with
fructose, trehalose, and melezitose, and positive for total polyphenols with these sugars). In contrast to
the other types of honey, acacia had no correlation between total polyphenols content and sugar content.
The results of the Pearson correlation for grassland honey indicated that, besides the correlations
obtained for the other types of honey, there were correlations given by phenolic acids such as chlorogenic
and 4-hydroxybenzoic acid (0.952), p-coumaric and 4-hydroxybenzoic acid (0.695), and protocatechuic
and gallic acids (0.577).
3. Discussion
The study draws attention to the special qualities of grassland honey with its high content
of polyphenols and microbiological characteristics. The important role of polyphenols in honey
composition has been emphasized by all researchers in the field. For the first time, a well-defined
link was demonstrated between the global honey structure and its biological activities [33].
Complex structures of proteins and polyphenols are involved in honey antioxidant capacity, antibacterial
activity, and hydrogen peroxide production. According to M. Bucekova and co-authors, the polyphenols
promote antimicrobial activity by producing H2 O2 in their autoxidation and by influencing the Fenton
reaction to create the reactive hydroxyl radicals [34,35]. The highest content of polyphenols and
flavonoids with an average of 30.49 mg/100 g was determined to be in honeydew honey followed by
grassland honey with an average of 21.50 mg/100 g. Similar to grassland honey, values of 20–40 mg/100 g
were presented by Kuś and co-authors for heather honey, and values of 27.4 mg GAE/100 g were
found by Bucekova and co-authors for wildflowers [14,35]. The values of total phenolic compounds
(TPC) obtained for Romanian monofloral honey were smaller than those obtained by other authors.
The difference is greater in the case of honeydew honey, as the analyzed samples had values within
the range of 14.95–80.47 mg GAE/100 g, and in the case of honeydew honey analyzed by Marchitas
and co-authors [36], the samples had values within the range of 35–130 mg GAE/100 g. The values of
the total phenolic content were slightly lower than those obtained by Małgorzata Dzugan et al. [37] of
34.59–75.49 mg GAE/100 g for the samples of honeydew honey from southeastern Poland (Podkarpacie,
Poland) with the same climate. The variation in TPC was most likely due to differences in geographical
location, harvesting time, and storage conditions. Honeydew honeys showed the highest value of
total phenolic compounds (phenolic acids and flavonoids) of all the honey studied, followed by
Molecules 2019, 24, 2932 12 of 17
grassland honey, polyfloral honey, and linden honey, respectively, but these values were also lower
than those reported by Ciucure et al. [38]. The TPCs of Irish multi-floral urban and rural honeys
were within the range of 10–50 mg GAE/100 g of honey [39]. The results obtained for TPC in rural
honey were similar to those obtained for grassland honey in northern Romania, within the range
of 13.01–45.88 mg GAE/100 g of honey, with values higher than unifloral honey, most likely due to
differences in geographical location. The grassland honey had the highest microbiological quality
and was free of microorganisms in more than 33% of the samples, and the other samples were well
below the allowed limit. Pathogenic microflora was not detectable for grassland honey as they were
for other types of honey analyzed. All samples were analyzed in terms of the presence of B. cereus,
which is widespread in nature and frequently isolated from soil and growing plants. The Bacillus cereus
group can cause two different types of foodborne illness: the diarrhoeal type caused by enterotoxin(s)
produced during vegetative growth of B. cereus in the small intestine, and the emetic type [40]. At the
species level, the vast majority (>60%) of the isolates belonging to the genus Bacillus fall into the
B. cereus group or in class Bacillus subtilis. According to ISO 7932:2004, the confirmatory stage does not
enable the distinction of B. cereus from other closely related Bacillus species, such as Bacillus anthracis,
Bacillus thuringiensis, Bacillus weihenstephanensis, and Bacillus mycoides. It is necessary to perform
a motility test to differentiate B. cereus from the other Bacillus species when their presence is suspected.
In our study, microorganisms were isolated from various types of honeys. Only the application of the
spectrometric technique of Paweł Pomastowski and co-authors allowed for an unambiguous distinction
between species/species groups, e.g., B. subtilis or B. cereus groups [41]. The method is very important
because it allows for the isolation of B. subtilis spores, which enjoy GRAS (Generally Regarded As
Safe) status from the U.S. Food and Drug Administration (FDA) and are included in the European
Food Safety Authority (EFSA) list of Qualified Presumption of Safety (QPS) with applications in
biomedicine and biotechnology (as oral vaccines, disinfectants, probiotics, or display systems) [42,43].
The grassland honey is a special honey sourced from the nectar in wild flowers that exist as hundreds
of unique species in the meadows of hilly areas in the north of the Romania. These meadows are the
borders of rural areas, where traditional farming is the main activity. Intensive agricultural practices,
monocultures, and the use of pesticides and fertilizers change the destination of land and determine the
decrease of these areas and the degradation of bees’ habitats. Promoting this type of honey due to its
qualities also means issuing a warning about the essential role of bees as pollinators and in maintaining
biodiversity and ecosystems, protecting crops, as well as their essential role in food production and
food safety. The authors of the paper intend to continue studying this type of honey as well as other
hive products such as wax, pollen, royal jelly, and propolis.
4. Materials and Methods
4.1. Materials
The honey assortments analyzed were acacia honey (A, n = 21), linden honey (L, n = 21), rape honey
(R, n = 14), polyfloral honey (P, n = 16), honeydew honey (H, n = 9), and grassland honey (G, n = 12)
and were purchased from authorized local producers from North Romania (Apicola Suceava trading
company, Suceava, Romania). The reagents used were analytically pure from Fluka (Charlotte, NC,
United States), Lachner (Neratovice, Czech Republic), Merck KGaA (Darmstadtcity, Germany),
Scharlau (Barcelona, Spania), Sigma-Aldrich (Steinheim, Germany), Santa Cruz Biotechnology
Inc. (Dallas, TX, USA), LGC Labor GmbH, todylaborotories.com. Hydrochloric acid 35% was
purchased from Lachner (Neratovice, Czech Republic). Glacial acetic acid was purchased from Merck
KGaA, Darmstadt, Germany. Agar plates, potato-dextrose agar (PDA), and Sabouroud agar were
purchased by Sigma-Aldrich Inc., and agar Deoxycholate-Citrate Lactose (ADCL) was provided by
todylaborotories.com. Sugar standards composed of d (+) glucose, d (-) fructose, d (+) sucrose, and d (+)
trahalose dihydrate were provided by Santa Cruz Biotechnology Inc (Dallas, TX, USA), and mannose,
Molecules 2019, 24, 2932 13 of 17
maltose monohydrate, melezitose, raffinose pentahydrate, erlose, and d (+) turanose were purchased
from LGC Labor GmbH (Augsburg, Germany).
Phenolic standards comprising gallic acid, protocatechuic acid, 4-hydrxybenzoic acid, vanilic
acid, chlorogenic acid, caffeic acid, p-coumaric acid, rosmarinic acid, myricetin, quercitin, luteolin,
and kaempferol were purchased by Sigma-Aldrich Inc., Germany. Methanol was provided by Fluka
(Charlotte, NC, United States), and acetonitrile was provided by Scharlau (Barcelona, Spania).
4.2. Methods
4.2.1. Water Content

The refractometer analysis (Refractometer RE40, Mettler Toledo, OH, United State of America)
was based on the direct correlation between the refractive index and the solids content of honey,
which allowed for the calculation of water content [1] according to the Harmonized Methods of the
International Honey Commission [44].
4.2.2. Sugars
Glucose, fructose, sucrose, trehalose, mannose, maltose, melezitose, raffinose, erlose, and turanose
were analyzed by HPLC 10 AD VP Shimadzu (Shimadzu Corp., Kyoto, Japan) with an RI detector
(Refractive Index Detector) in accordance with Bogdanov [44]. A column of 150 × 4.6 mm i.d. and
particle size 3 µm was used to separate the compounds. Each analyzed sample consisted of 5 g of
honey dissolved in 40 mL water. The samples were transferred in a volumetric flask of 100 mL and
filled with water to the mark. The samples were filtered through a membrane of 0.45 µm and were
collected in vials. The conditions for the analysis were: acetonitrile/water (75:25, v/v) as mobile phase,
flow rate 1.0 mL/min, temperature 40 ◦ C, volume of sample 10 µL. Standard solutions of 1 mg/mL
were used for the calibration curve of each analyzed sugar. The calibration curves obtained for all
sugars were characterized by a linear regression factor higher than 0.9980. The peak area obtained for
each analyzed sugar was compared with standard sugar, allowing their quantitative determination.
Sugar contents were expressed as g/100 g honey. Each result was the mean of triplicate determinations.
4.2.3. Phenolic Compounds

Phenolic compounds were analyzed by HPLC 10 AD VP Shimadzu-Japan with a diode array
detector. A column Alltech 250 × 4.6 mm i.d. and particle size of 5 µm was used for phenolic
compounds separation. Acidified water with hydrochloric acid at pH equal to 2.00 was used. With this,
a solution of 40% methanol was prepared. This was the solute for the honey. Each sample consisted
of 1 g of honey dissolved in 5 mL of solute, which was shacked on the magnetic stirrer and filtered
through a membrane of 0.45 µm [45]. The conditions for the analysis were: a flow rate of 1.0 mL/min,
acetic acid 0.1% and acetonitrile as the two mobile phases, temperature of 30 ◦ C, volume of sample
10 µL. Using standard solutions of 1 mg/mL for each phenolic compound, a calibration curve was
obtained. The linear regression R2 was higher than 0.9976. The peak area obtained for each analyzed
phenolic compound was compared with the standard, allowing for their quantitative determination of
polyphenols at 280 and 320 nm wavelengths. Polyphenols contents were expressed as mg/100 g honey.
Each result was the mean of triplicate determinations.
Phenolic compounds were analyzed by the Folin–Ciocalteu method according to Pontis.
The compounds were dissolved with 5 g of honey in 20 mL of distilled water and transferred
to 50 mL flasks and made up to the stock to form the stock solution. Then, 0.5 mL of the stock solution
was mixed with 0.3 mL of Folin–Ciocalteu reagent and 2 mL of 15% sodium carbonate. Then, 5 mL
was added to volumetric flasks and filled up to the mark with distilled water. The mixture was kept in
the dark for 2 h, after which the absorbance was measured with the Perkin Elmer LAMBDA EZ201
UV-VIS Spectrophotometer (Waltham, MA, United States) at a wavelength of 798 nm [46]. Results were
expressed as mg of gallic acid equivalents (GAE)/100 g of honey.
Molecules 2019, 24, 2932 14 of 17
4.2.4. Color was Determined by the Chromometer CR 410—Konica Minolta

CIE L*a*b* is a color space defined by the International Commission on Illumination (CIE) and it
expresses color as tree values: L*—the lightness parameter; a*—the red/green parameter, with +a*
indicating red and −a* indicating green; and b*—the yellow/blue parameter, with +b* indicating
yellow and −b* indicating blue. The L*, a*, and b* coordinate axis defined the three dimensional CIE
color space [47]. Chromameter was calibrated with a reference white porcelain tile (L* = 91.59; a* =
0.31; b* = 0.33) before the determinations. The illuminant used was D 65 [47]. The chroma parameter
represented color saturation and varied from brilliant to bright. Hue angle was the color attribute by
which the color was perceived [48].
4.2.5. Electrical Conductivity

Electrical conductivity was determined with conductometer Mettler Toledo MPC 227 with
thermostatic control. The conductivity was calculated using the formula [44]:
S=K×G (1)
where K is the cell constant in cm−1 and G is the electrical conductance in mS.
4.2.6. Viscosity
Viscosity was determined with the Brookfield viscometer. The results were expressed in Pa·s.
4.2.7. Microbiologic Analysis

Microbiological analysis was performed according to ISO 4831:2006. Microbiology of food and
animal feeding stuffs: horizontal method for the detection and enumeration of coliforms, ISO 18593:2018.
Microbiology of food and animal feeding stuffs: horizontal method for sampling techniques from
surfaces using contact plates and swabs, ISO 7932:2004. Microbiology of food and animal feeding stuffs:
horizontal method for the enumeration of presumptive Bacillus cereus, ISO 21527:2008. Microbiology of
food and animal feeding stuffs: horizontal method for the enumeration of yeasts and molds.
The samples analyzed were prepared by mixing 10 g of each honey sample with 90 mL of peptone
saline solution (8.5 g/L) to achieve the initial dilution. The mixture was then used for the other dilutions.
All colonies appearing after incubation were counted. Microbial counts were expressed in colony
forming units/gram of honey (cfu/g).
• Number of standard counts. Serial dilutions from 10−1 to 10−3 of the initial dilution inoculated on
agar plates were recommended. The plates were incubated for 48 h at 30 ◦ C.
• Bacillus. The initial dilution was brought to 80 ◦ C and held at this temperature for 10 min,
after which it was cooled rapidly in ice. Bacteria for aerobic spore formation were inoculated on
agar medium. The plates were incubated for 48 h at 30 ◦ C.
• Number of coliforms. Inoculation was carried out on Agar Deoxycholate-Citrate Lactose medium
(ADCL- todylabrotaories.com). The plates were incubated for 24 h at 37 ◦ C.
• Yeast. From the initial dilution, they were inoculated on agar potato-dextrose medium (PDA) and
incubated for 72 h at 25 ◦ C.
• Molds. The mold was inoculated on Sabouroud agar medium and incubated for 7 days at 25 ◦ C [20].
4.3. Statistical Analysis

The resultant data were analyzed by PCA, Pearson correlation, and descriptive analyses with
XLSTAT software (2018, trial version, Addinsoft, NY, United States). PCA evaluated the correlations
between the honey type, and the physico-chemical parameters analyzed the variation and extracted
the main components.
Molecules 2019, 24, 2932 15 of 17
5. Conclusions
Physicochemical and microbiological parameters were found to be in the required standards for
all honey samples. High content of polyphenols was found in grassland honey and in honeydew.
Grassland honey is characterized not only by the high content of polyphenols but also by the presence
of all phenolic compounds analyzed.
Supplementary Materials: The following are available online, Table S1: Results of microbiological determination
of honey samples, Table S2: Physicochemical parameters of different type of honey from Romania, Table S3:
Sugars content of different type of honey from the North of Romania, Table S4 The results of the concentration of
the twelve phenolic compounds.
Author Contributions: Conceptualization, S.A., L.S., and L.N.; methodology, L.S. and S.A.; software, L.N.;
validation, S.A., L.S., and L.N.; formal analysis, L.S.; investigation, L.S., L.N.; resources, S.A.; data curation, L.N.,
L.S.; writing—original draft preparation, L.S.; writing—review and editing, S.A. and L.N.; visualization, S.A.;
supervision, S.A.; project administration, L.S.; funding acquisition, S.A.
Funding: This work was supported from contract no. 18PFE/16.10.2018 funded by Ministry of
Research and Innovation within Program 1—Development of national research and development system,
Subprogram 1.2—Institutional Performance—RDI excellence funding projects.
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Sample Availability: The honey samples are available from the authors for limited time.
molecules
Article
Correlation Study of Honey Regarding their
Physicochemical Properties and Sugars and
Cyclitols Content
Ileana Andreea Ratiu 1,2,3 , Hossam Al-Suod 1,2 , Małgorzata Bukowska 1,2 , Magdalena Ligor 2
and Bogusław Buszewski 1,2, *
1 Interdisciplinary Centre of Modern Technologies, Nicolaus Copernicus University, Wileńska 4, 87-100 Toruń,
Poland; andreea_ratiu84@yahoo.com (I.A.R.); hossamalsoud@hotmail.com (H.A.-S.);
malgorzatabukowska72@gmail.com (M.B.)
2 Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus
University, Gagarina 7, 87-100 Toruń, Poland; magdalena.ligor@umk.pl
3 Babeş-Bolyai University, Faculty of Chemistry and Chemical Engineering, 11 Arany Janos,
RO-400028 Cluj-Napoca, Romania
* Correspondence: bbusz@chem.umk.pl

Received: 22 November 2019; Accepted: 18 December 2019; Published: 20 December 2019
Abstract: Honey is a natural sweetener, with an osmotic effect on microorganisms due to the increased
sugar content and low amount of water. Cyclitols are minor constituents of honey. They play a
defensive role in plants against unfavorable environmental conditions. Honey’s physicochemical
properties can vary, resulting in a wide range of colors, flavors, scents, antioxidant activity, dissimilar
values of pH, acidity, electrical conductivity, etc. Some literature regarding correlation between
honey types is already available, but a comprehensive study displaying an ample evaluation of
multifarious aspects is still needed. This study focuses on the correlation between 18 honey types,
originating from 10 countries, collected during four years, summarizing a total of 38 samples. A total
of 6 physicochemical properties and 18 target components (sugars and cyclitols) were considered
as variables. A correlation analysis is presented between the investigated parameters and between
honey types, together with the statistical analysis which allowed for observation of the clusters’
distribution according with the investigated variables.
Keywords: honey; sugars and cyclitols; physicochemical properties; correlation analysis
1. Introduction
Honeybees (Apis mellifera L.) are herbivorous pollinator insects, consuming nectar and pollen
throughout their life cycles. The nectar is primarily an energy source which, in addition to sugars
contains various other components with important nutritional properties. However, honey is a natural
product, known for its antibacterial properties [1] and medical uses [2], with osmotic effect due to the
increased sugar content and low amount of water [3]. Honey is quite acidic (with values between 3.2 and
4.5), a fact that inhibits the pathogens, while the hydrogen peroxide produced due to glucose oxidase
also helps the preservation of this foodstuff [4]. Besides being a high-energy carbohydrate product
with easy digestible sugars, such as those in fruits, honey contains around 180 substances, including:
amino acids, enzymes, proteins, vitamins, minerals, phenolic compounds, etc [5]. The properties and
composition of honey are dependent on its geographical origin, type of flowers, harvesting season,
environmental factors, and treatments administered by beekeepers [6].
Cyclitols (sugar alcohols) are additional minor constituents of honey, and are widely unexplored
compared with those mentioned previously. Cyclitols are important antioxidant, anti-cancer and

Molecules 2020, 25, 34 2 of 15
anti-inflammatory agents [7]. However, they are secondary metabolites, naturally occurring in
plant material, playing an important role in plant self-defense against unfavorable environmental
conditions [8]. Moreover, cyclitols are responsible for the cell’s good functioning, cell wall formation,
phosphate storage, and osmoregulation. In the pharmaceutical industry, they are used in the treatment
of psychiatric dysfunctions, panic attacks, depression, or obsessive-compulsive disorders [8]. It is
seldom acknowledged that, despite its bactericidal effect [3], honey can sometimes contain pathogens
(bacteria, fungi, or yeasts) that can withstand concentrated sugar and acidity [9]. The pathogens can
arrive in honey either via pollen, or due to improper honey manipulation and storage. Moreover,
pesticides or their residues can often be present in honey [10].
Some of the chromatographic techniques generally used for the composition analysis of honey
samples, can also be employed for the analysis of sugars and cyclitols, the most widely used being high
performance liquid chromatography (HPLC) [11,12]. Gas chromatography with mass spectrometry
(GC-MS) is quite often used as well, but this technique involves a derivatization step [13–15]. In
terms of the sample preparation for the detection of cyclitols and sugars, honey samples can be easily
handled, since just a dilution in water is required. Conversely, for the analysis of cyclitols and sugars
from plant material, an elaborated procedure is required in terms of both, the solvent used and the
extraction techniques [15–20].
The current study presents a comprehensive comparison of 18 types of honey, coming from 10
countries and collected over four years in terms of the correlation between their assortment as well as
the content of sugars and cyclitols. A map presenting the samples origins is presented in Figure 1.
As variables, six physico-chemical properties (Pfund value, color, pH, acidity, electric conductivity,
and antioxidant activity), along with seven sugars and 11 cyclitols detected and quantified in honey
samples were used. Consequently, a complex correlation analysis was developed between both the
investigated parameters and honey types. Our obtained results concluded that there is a relevant
correlation between different physical properties and types of honey as well. The samples cultivated in
2015 and 2016 a presented distinctive correlation compared with those harvested in 2017 and 2018,
due to the natural changes which occur in honey during the storage period. Clusters analyses based
on detected sugars and cyclitols segregated the samples based on the strength of the total amount of
detected targets. Hierarchical clustering analyses with respect to the 23 variables selected, led to the
formation of 11 groups, including five main clusters with high significance and another six more, with
lower levels of significance, or simply fused together in an aleatory way.
Figure 1. Map presenting the origin of samples.

Molecules 2020, 25, 34 3 of 15
2. Results and Discussion
2.1. Role and Assessment of Physico-Chemical Properties of Honey

The investigated samples were collected from 10 different countries, over four years (2015 to 2018),
summarizing 18 varieties. Generally, the honey color was amber, ranging from a very light to dark
amber shade. However, less common color like: white, extra white, yellow-white or green were the
subject of analyses. Pfund value (mm) was between 10.2 in the case of extra white Acacia honey and
514.55 for the green honey of Ivy vine (corresponding to dark amber, according with Pfund scale). In
Figure 2 a distribution of the 38 samples in terms of honey type, colors and cultivability year was
drawn. The number for a specific category is presented between brackets, each set summarizing the
number 38.
Figure 2. The distribution of the 38 samples, in terms of honey type, color and cultivability year.
The electrical conductivity of honey is connected with the content of mineral and organic acids.
It is part of the routine analysis for honey control and often a method involved for honey origin
identification [21]. However, the maximum approved value of electrical conductivity for edible honey
is 800 S/cm, (the equivalent of 0.8 mS/cm), as stipulated by the EU Directive [22]. Four samples
presented in Table 1, namely samples 2, 22, 32, and 38, exceeded the maximum allowed value of
0.8 mS/cm. However, electrical conductivity ranged from 0.041± 0.02 (in spring flowers honey, collected
from Poland, 2018) up to 1.221 ± 0.05 (Linden/Multiflora honey, from Poland, 2016).
Honey acidity is linked with the presence of organic acids naturally occurring in this foodstuff,
which is closely connected to, and balanced with the content of lactones, esters, phosphates ions,
sulfates ions and chlorides ions [21]. According with the EU Directive [22], the maximum allowed
value of acidity is 50 meq/kg. An increased value of acidity denotes the beginning of the fermentation
process, through which the produced alcohols are transformed into organic acids [21]. In eight of the
38 investigated honeys (samples 5, 14, 25, 26, 28, 29, 34, and 35) the maximum acidity value allowed
by the European standards was exceeded (as presented in Table 1). These samples belong to the
categories: multiflora, buckwheat, ivy vine, and honeydew/buckwheat. Moreover, all of them had a
dark amber color. Due to the observation that four out of seven buckwheat honeys, two out of five
multiflora honeys, and only one sample from both honeydew/buckwheat and ivy vine honey presented
Molecules 2020, 25, 34 4 of 15
increased values of acidity, we concluded that these honeys are more easily subject to fermentation,
and/or that it is generally possible that in dark honeys the fermentation process is facilitated faster.
This hypothesis was confirmed by other researchers as well [21]. On the opposite side were the white,
extra-white, yellow-white and light amber honeys (rape, linden, acacia, clover, and sunflower) which
presented considerably low values of acidity, generally ranging from 12 to 24 meq/kg. These findings
indicate that in the honeys with light colors considerably lower contents of organic acids are present in
comparison with dark honeys. Nevertheless, it was highlighted that the content of organic and amino
acids, on which the acidity is dependent, are contingent on the botanical origin of honey [23,24]. In
our investigated samples the acidity ranged from 12 to 114 meq/kg. Notably, the two extreme values
were recorded for the same type of honey, a multiflora variety coming from Greece and Kameron
respectively, denoting the clear degradation of the sample from Kameron.
The honey pH value is connected with the existence and growth of microorganisms. The EU
Directive [22] does not impose a maximum allowed value for honey pH, however, a low pH will
prevent microbiological spoilage. In our samples, the pH was ranging from 3.20 ± 0.01 to 4.49 ± 0.01, a
fact that apparently denotes the absence of bacteria.
Regarding antioxidant activity, there are a couple of methods used for testing this parameter in
honey, such as: free radical scavenging activity (DPPH), ferric reducing/antioxidant power (FRAP),
oxygen radical absorbance capacity (ORAC), ascorbic acid content (AEAC), and Trolox equivalent
antioxidant activity (TEAC). Notwithstanding, each one of them allows the measurement of a different
group of antioxidants, and consequently it cannot be affirmed that there is an ideal one which can fully
evaluate the antioxidant activity [25]. It is supposed constituents in honey like: flavonoids, phenolic
acids, vitamins, enzymes, as well as a small amount of mineral content, particularly copper and iron can
be responsible for antioxidant activities [26]. TEAC antioxidant activity (expressed in trolox equivalent
antioxidant activity) has been tested in this study. Values between 2.53 (Acacia honey coming from
Romania) and 7.03 (a mixture of honeydew and buckwheat, coming from a private beekeeper from
Janowiec, Lubelskie, Poland) were obtained. It was observed that the dark amber honey generally
showed higher antioxidant activity compared with the honey with lighter color, as presented in Table 1.
2.2. The Content of Sugars and Cyclitols in the Investigated Honey Samples
The targets detected (sugars and cyclitols) were simultaneously separated in one chromatographic
run by a single GC column. Some components (fructose and glucose) appeared as different isomers in
the form of two or three peaks. The peaks of fructose can be identified with α-furanose, β-furanose, and
β-pyranose, while the D-glucose peaks are represented by α-pyranose and β-pyranose, as confirmed
by other researchers previously [27]. In Figure 3, a heat map combined with a dendrogram is presented
for cyclitols (part A) and sugars (part B). The heat map was built to express a snapshot of the quantified
concentration of sugars and cyclytols in honey samples. The real amounts are expressed in mg/g
and highlighted in Supplementary Table S1. Just the concentrations of cyclitols and main sugars
are shown (glucose, fructose, and maltose) in Supplementary Table S1. The hierarchical clustering
model based on detected cyclitols (Figure 3A, horizontal part) highlighted the formation of six main
clusters. Generally, epi-inositol, cis-inositol, bornesitol, D-pinitol and chiro-inositol were detected in
lower amounts compared with other cyclitols, or not detected at all in some samples. Moreover, they
fused together in one cluster with similar distance levels, as shown in the left part of Figure 3A. On the
other extreme were grouped: ononitol, neo-inositol, quebrachitol and muco-inositol, which presented a
lower level of similarities compared with those previously mentioned. Nevertheless, they were present
in all investigated samples. The lowest total amount of cyclitols (8.38 mg/g) was detected in sample no
12, Sunflower honey, collected in 2016 in Poland and the highest total amount (59.51 mg/g) in sample
no 30, Spring flower, from Poland, 2018. However, we could not conclude that the detected amount of
cyclitols is connected with honey type while, for example, in the case of Buckwheat honey, for which
seven samples were analyzed, the detected quantities ranged between 14.49 (sample no 14) up to 50.58
Molecules 2020, 25, 34 5 of 15
(sample no 37). Other examples, such as the case of multiflora, raspberry or acacia honey, can be added
to this one.
Figure 3. Heat maps presenting the quantity of cyclitols (part A) and sugars (part B) detected in honey
samples. The sample numbers from the vertical dendrograms were allotted similar to those presented
in Table 1.
In the case of sugar concentrations (part B), it is worth mentioning that fructose and glucose were
the most important sugars quantified, present in all samples in far higher concentration compared with
others. As can be observed in the horizontal dendrogram of sugars, glucose and fructose clustered
separately from other sugars. Fructose was detected in greatest proportion than glucose, except in
some honeys such as rape, dandelion, ivy vine, goldenrod, three form seven buckwheat honeys and
two from five multiflora honeys. Notwithstanding, some other researchers reported as well that the
glucose is higher than the fraction in rape and dandelion honey, fact that causes the rapid crystallization
of honey [28]. Regarding the quantities detected, fructose amount was between 243.3 ± 1.99 mg/g (in
sample 8) and 422.65 ± 1.64 mg/g (in sample 3). Glucose amount was between 218.3 ± 0.77 mg/g (in
sample 34) up to 449.9 ± 1.35 mg/g (in sample 29).
The dendrograms presented in the vertical parts showed the formation of many clusters, but
no clustering according with the honey types was observed. However, the samples alignment
corresponded broadly to the total quantity detected. Consequently, alignment the samples, from up to
down presented an increasing trend according with the total amount of detected sugars or cyclitols
respectively. Each number presented in the vertical dendrograms corresponds to one honey sample
and was allotted similar with those presented in Table 1.
2.3. Correlation Analyses

In order to run the correlation analysis for the investigated samples, two different approaches
were used: non-parametric tests (Spearman correlation) and parametric tests (Pearson correlation).
Molecules 2020, 25, 34 6 of 15
The decision regarding the method chosen was made based on the available data set. However, the
obtained correlation for both approaches is presented below.
2.3.1. Correlation Between the Investigated Variables

In the case of the correlation between investigated variables, the determined physico-chemical
values, total amount of sugars, total amount of cyclitols and the individual targets quantified (seven
sugars and 11 cyclitols) of each sample were used. For this data set we chose a non-parametric
Spearman correlation, which presents a monotonic relationship between dissimilar variables and is
suitable to compare samples with various measurement units. Maltose was the only one variable
which did not present correlation with others, and consequently it was removed from the matrix.
The correlation between the investigated variables is presented in Figure 4 in the form of a heat map
combined with a dendrogram of clusters’ analysis, which shows the formation of five main clusters
(which were labeled from #a to #e).
Figure 4. Heat map presenting correlation between the investigated variables together with hierarchical
clusters analyses, where ** = correlation significant at the 0.01 level, * = correlation significant at the
0.05 level, a, b, c, d, e = label of the main clusters.
By examination of the relationships between the mentioned groups, a strongly positively correlation
related to physical properties: acidity, electrical conductivity, pH and Pfund value, r(21) = 0.76 up to
0.90, p = 0.01 was highlighted. Moreover, hierarchical cluster analyses presented all four physical
properties fused together in one cluster with similar distance level (cluster #a, Figure 4). Positioned at
the opposite site in a rather arbitrarily manner was the cluster corresponding to antioxidant activity.
Low correlation was found between acidity and antioxidant activity r(21) = 0.38, p = 0.05. Furthermore
the antioxidant activity was moderately negative correlated with the sucrose level. This finding
indicates that a high level of sucrose will decrease the antioxidant activity potential, while a high
acidity will increase the antioxidant activity. Another main cluster (#b) with almost the same level of
similarity was segregated from muco-inositol, epi-inositol, cis-inositol and bornesitol. Muco-inositol was
Molecules 2020, 25, 34 7 of 15
moderately positive correlated with epi-inositol, which in its turn was moderately positive correlated
with cis-inositol. Cis-inositol was howsoever positively related to bornesitol. Moreover, the four
mentioned targets have been observed to have low negative correlation with some other compounds
(Figure 4).
Two more equal groups of clusters were formed by: sucrose, lactose, turanose (#c) and allo-inositol,
total cyclitols, and quebrachitol (#d). They presented various low to strong correlations both positively
and negatively, between them and with other targets, as presented in Figure 4. Finally, the last main
cluster (#e) was formed by segregation of glucose with total sugars (which presented the same level of
similarity) and fructose with chiro-inositol. A generally low (r(21) = 0.32, p = 0.05) up to very strong
correlation (r(21) = 0.92, p = 0.01) was found between the targets which formed the clusters #c, #d and
#e. Figure 4 shows too the formation of other secondary clusters with higher distance levels, which
presented lower similarities with the five discussed previously, or they simply fused together in an
arbitrary way. As a general conclusion, these findings indicate that there is relevant correlation between
honey’s properties, even if these characteristics are far different and in appearance not linked-up with
each other.
2.3.2. Correlation Between Honey Types

Pearson moment product correlation was used to highlight the similarities between honey types,
while as variables we considered the 38 investigated samples. The Pearson test was chosen because it
is a parametric statistical tool expecting a linear correlation between the investigated variables, suitable
when those variables are coming from the same source, and/or they have the same measure unit. The
correlation analysis is presented in Figure 5.
In Part A, the correlation matrix is shown presenting the level of significance, while a mirror of
this matrix in a heat map form is designed in Part B, in order to highlight the difference between the
correlation values. However, we can confirm that the correlation values significant at 0.01 level (light
dots) were between r(36) = 0.7 to 0.99, which pointed to a strong to very strong correlation. Moreover,
the correlation values significant at a 0.05 level (dark dots), ranged from r(36) = 0.41 to 0.65, denotative
of a moderate correlation.
By analyzing the matrix correlation, it was observed that samples 1 to 3, originating from Australia,
were generally not correlated with samples 4 to 11, originating from different European countries,
Brazil, Kameron or Russia. However, a correlation between Australian samples and some samples
coming from Poland did exist, as presented in Figure 5. It was observed that nine out of twelve samples
(corresponding to the samples no 12 to 23), which presented strong correlation with the samples from
Australia were cultivated in 2015 and 2016.
Notably that all the samples coming from Lenah Valley, Tasmania, Australia were cultivated in
2016, while the other non-correlated samples from European countries, Brazil, Kameron, or Russia
were all cultivated in 2017 and 2018. Going forward, we observed that the non-correlated samples
(24–30 and 34–36) were all cultivated in 2017 and 2018. Generally, we observed that the samples
cultivated in 2015 and 2016 were not correlated with those harvested in 2017 and 2018. However, some
notable exceptions were remarked. Samples 14, 31, and 37, belonging to buckwheat type, cultivated in
2016 presented moderate up to very strong correlation with almost all investigated samples. Moreover,
clover and spring flower types presented some correlation with all categories as well. All these findings
may indicate that the samples cultivated in the same year present a strong correlation, or that during
storage honey can change its composition and properties.
No studies were found to confirm or disprove the correlation related to year of cultivability.
Instead, other researchers confirmed that during prolonged storage time the honey changes its
composition and characteristics. Thus, the literature study outlined the changes in sugar composition,
accounting for increasing, and decreasing amounts, as well as sugar degradation and conversion in
furans derivatives [29]. Some of the changes occuring in honey during storage that may influence
nutritional and sensory properties can be associated with the Maillard reaction, which occurs either
Molecules 2020, 25, 34 8 of 15
slowly during storage, or rapidly by heating. The Maillard reaction is a chemical response between a
reducing sugar and a primary amino group, resulting in browning and reduction of nutritional value
inhoney [5]. Strecker degradation, another reaction occuring in honey, contributes to the loss of amino
acids [5]. Alcohol concentration can increase during storage as well. Their occurrence is generally due
to lipid oxidative degradation or reduction processes catalyzed by aldehyde reductase from honey
contaminated with yeasts, molds or bacteria [30,31]. Another pathway of alcohols occurence in honey
is the transformation of hydrocarbons into smaller molecules due to oxidative processes [30]. The
oxidation of fatty acids in honey, especially linoleic and linolenic acids, results in the formation of
aldehydes and ketones creating a rancid flavor [31]. However, other constituents reported to undergo
changes during storage are: proteins, organic acids, vitamins, minerals, phenolic compounds and
volatile compounds [5]. One study regarding Lithuanian honey stability, in terms of the chemical
classes described above, concluded that qualitative and quantitative changes started to be evident only
after three months of storage. Physical properties such as consistency, crystallization, and rheology
were also revealed to be subject to changes during storage in Lithuanian honey [31].
Figure 5. Correlation matrix presenting the significance between honey types (part A) and heat map
highlighting the difference in correlation values (part B).The numbers from 1 to 38 are similar with
those presented in Table 1.
3.1. Honey Samples Collection and Chemicals Involved

Most of the honey samples were collected from different regions of Poland (27 samples) from
private beekeepers and manufacturers. Other samples were collected from Australia (3 samples),
Romania (2 samples), Greece, Kameron, France, Czech Republic, Russia and Brazil (1 sample from
each mentioned country). A full list of samples, including place of cultivation, year, manufacturer, and
their physicochemical properties is presented in Table 1.
Molecules 2020, 25, 34 9 of 15
Table 1. Physical-chemical properties and origin of honey samples.
Pfund Value Color According Acidity Electrical Conductivity Antioxidant Manufacturer/

No Honey Type pH
(mm) to Pfund Scale (meq/kg) (mS/cm) Activity Place & Origin Country/Year
1 Bush 129.66 dark amber 4.20 ± 0.03 26.0 0.677 ± 0.03 4.43 ± 0.4
“Peter & Trisha Norris”, Lenah Valley, Tasmania, Australia, 2016
2 Leatherwood 88.81 amber 4.32 ± 0.03 21.0 0.704 ± 0.02 4.25 ± 0.05
3 Clover 20.10 yellow- white 3.60 ± 0.01 22.0 0.255 ± 0.01 3.95 ± 0.27
4 Multiflower 77.05 light amber 4.30 ± 0.02 12.0 0.067 ± 0.00 2.95 ± 0.16 ”Fragrant Greece”, Greece, 2017
5 Multiflower 183.73 dark amber 4.15 ± 0.05 114.0 0.322 ± 0.04 4.9 ± 0.06 ”Adam Gardynik”, Nyaoundere Kameron, 2018
6 Acacia 33.10 white 3.80 ± 0.02 20.0 0.103 ± 0.04 2.53 ± 0.21 Private beekeeper, Cluj County, Romania, 2017
7 Rapeseed 94.13 amber 3.66 ± 0.03 36.6 0.266 ± 0.05 4.75 ± 0.41 Private beekeeper, Cluj County, Romania, 2017
8 Multiflower 100.45 amber 3.49 ± 0.01 24.0 0.073 ± 0.01 2.74 ± 0.11 “Lume de miel”, certified by Famille Michand, France, 2018
9 Raspberry 101.56 amber 3.53 ± 0.02 30.0 0.076 ± 0.03 3.15 ± 0.19 “Medokomerc”, Čestín, Czech Republic, 2017
yellow-
10 Clover 18.99 3.74 ± 0.04 20.0 0.046 ± 0.06 4.09 ± 0.24 “Pykoht”, Dubna, Russia, 2017,
white
11 Multiflower 170.76 dark amber 4.04 ± 0.02 44.0 0.140 ± 0.01 4.63 ± 0.33 “Isis Mel”, Embu-Guaçu, Brazil, 2018
12 Sunflower 62.07 light amber 3.88 ± 0.01 21.0 0.404 ± 0.04 3.8 ± 0.33 Private beekeeper, Olekszyn, Wielkopolskie, Poland, 2016
13 Raspberry 91.29 amber 3.65 ± 0.02 21.0 0.292 ± 0.03 3.48 ± 0.09 Private beekeeper, Olekszyn, Wielkopolskie, Poland, 2016
14 Buckwheat 190.7 dark amber 3.59 ± 0.03 72.5 0.556 ± 0.02 6.01 ± 0.03 ”Pasieka Andrzej Kuś”, Kujawsko-Pomorskie, Poland, 2016
Private beekeeper, Solec Kujawski, Kujawsko- Pomorskie,
15 Rapeseed 34.34 white 4.02 ± 0.06 12.8 0.187 ± 0.01 3.85 ± 0.09
Poland, 2016
16 Buckwheat 107.5 amber 3.89 ± 0.02 35.0 0.310 ± 0.03 5.38 ± 0.29 “Sadecki
˛ Bartnik”, Stróże, Małopolskie, Poland, 2016
17 Goldenrod 104.53 amber 3.67 ± 0.01 49.0 0.669 ± 0.05 5.36 ± 0.08 “Jakubiec gospodarstwo”, Bielsko-Biała,Ślaskie,
˛ Poalnd, 2015
18 Sunflower 114.44 dark amber 3.68 ± 0.03 25.0 0.374 ± 0.04 3.91 ± 0.22 “Sadecki
Private beekeeper, Karczowiska Górne, Warmińsko-Mazurskie,
19 Acacia 10.2 extra white 3.58 ± 0.05 22.0 0.235 ± 0.01 3.55 ± 0.2
Poland, 2017
20 Acacia 21.47 white 3.61 ± 0.02 16.5 0.213 ± 0.05 4.08 ± 0.16 Private beekeeper, Janowiec, Lubelskie, Poland 2017
21 Linden 68.01 light amber 4.02 ± 0.01 23.0 0.678 ± 0.03 4.46 ± 0.1 “Sadecki
22 Multifloral/linden 59.72 light amber 4.27 ± 0.03 30.0 0.854 ± 0.02 4.75 ± 0.21 Private beekeeper, Wilga Mazowieckie, Poland, 2016
23 Goldenrod 58.85 light amber 3.31 ± 0.02 42.7 0.331 ± 0.01 4.43 ± 0.06 ”Słoneczna Pasieka”, Stryków, Łódzkie, Poland, 2016
24 Dandelion 101.31 amber 3.99 ± 0.00 22.0 0.050 ± 0.04 3.57 ± 0.14 Private beekeeper, Białowieża, Podlaskie, Poland, 2017
25 Ivy vine 514.55 green 3.80 ± 0.02 53.0 0.258 ± 0.01 5.29 ± 0.23 ”Piotr Nowakowski”, Wrocław, Dolnoślaskie,
˛ Poland, 2018
26 Buckwheat 117.04 dark amber 3.2 ± 0.01 100.0 0.100 ± 0.02 5.33 ± 0.14 Private beekeeper, Białystok, Podlaskie, Poland, 2018
27 Raspberry 87.45 amber 3.82 ± 0.03 33.0 0.061 ± 0.02 6.01 ± 0.22 Private beekeeper, Białowieża, Podlaskie, Poland 2017
28 Buckwheat 182.15 dark amber 3.78 ± 0.04 65.0 0.088 ± 0.01 6.24 ± 0.06 Private beekeeper, Białowieża, Podlaskie, Poland 2017
Molecules 2020, 25, 34 10 of 15
Table 1. Cont.
Pfund Value Color According Acidity Electrical Conductivity Antioxidant Manufacturer/

No Honey Type pH
(mm) to Pfund Scale (meq/kg) (mS/cm) Activity Place & Origin Country/Year
29 Multiflower 219.42 dark amber 3.66 ± 0.01 80,0 0.087 ± 0.05 5.62 ± 0.02 Private beekeeper, Lubelskie, Poland, 2017
30 Spring flowers 39.42 very light amber 3.60 ± 0.03 23.0 0.041 ± 0.02 2.76 ± 0.11 Private beekeeper, Janowiec, Lubelskie, Poland, 2018
31 Buckwheat 184.63 dark amber 3.59 ± 0.02 44.0 0.393 ± 0.02 5.73 ± 0.21 “Barć Świ˛etokrzyska” Daleszyce, Świ˛etokrzyskie, Poland, 2016
32 Honeydew 151.08 dark amber 4.49 ± 0.01 31.0 1.221 ± 0.05 5.21 ± 0.27 “Sadecki
33 Rape 114.07 amber 3.64 ± 0.04 15.0 0.181 ± 0.02 2.72 ± 0.19 “Sadecki
Private beekeeper, Karczowiska Górne, Warmińsko-Mazurskie,
34 Honeydew/buckwheat 408.08 dark amber 3.58 ± 0.02 84.0 0.114 ± 0.03 7.03 ± 0.17
Poland, 2017
35 Buckwheat 160.12 dark amber 3.28 ± 0.03 95.0 0.106 ± 0.01 5.17 ± 0.07 Private beekeeper, Sosnówka, Dolnoślaskie,
˛ Poland, 2018
36 Rape 85.47 light amber 3.59 ± 0.02 24.0 0.035 ± 0.02 3.48 ± 0.27 Private beekeeper, Miłków, Dolnoślaskie,
˛ Poland, 2018
Private beekeeper, Krzeczyn Mały k. Lubina, Dolnoślaskie,
˛
37 Buckwheat 231.05 dark amber 4.04 ± 0.01 29.0 0.462 ± 0.03 4.97 ± 0.28
Poland, 2016
Private beekeeper, Bobrowniki, Kujawsko- Pomorskie,
38 Linden/Multiflora 76.8 light amber 4.27 ± 0.02 33.3 0.812 ± 0.03 4.21 ± 0.16
Poland, 2016
Molecules 2020, 25, 34 11 of 15
Standard purity ≥95% D-pinitol, myo-inositol, D-chiro-inositol, ononitol, bornesitol, allo-inositol,

cis-inositol, epi-inositol, D-glucose, D-fructose, D-maltose, lactose, D-sorbitol, and D-(+)-turanose
and trimethylsilylimidazole (TMSI) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Standards of sucrose, xylose, quebrachitol, neo-inositol, muco-inositol with purity ≥98%, and
pyridine were bought from Avantor (Gliwice, Poland). DPPH (2,2-Diphenyl-1-picrylhydrazyl),
trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), and 70% EtOH, were purchased from
Sigma Aldrich (St. Louis, MO, USA). Ultra-pure water was obtained from a Milli-Q water system
(Millipore Bedford, MS, Boston, Massachusetts, USA).
3.2. Samples Analysis

For the analysis of sugars and cyclitols analysis, 0.5 g from each honey sample was measured in
50 mL plastic vials and dissolved in 25 mL of water. From each obtained solution, 5 mL were transferred
to 10 mL glass vials and evaporated to dryness under a nitrogen gas flow. The obtained residuum
was resolved in 2 mL of pyridine. From the obtained pyridine solution 100 µL was derivatized using
TMSI (ratio 1:1) at 80 ◦ C for 120 min. At the end of the derivatization 1 µL was taken from each sample
and injected into the GC injection port. The GC-MS analysis was carried out using an AutoSystem XL
gas chromatograph coupled with mass spectrometer TurboMass (both from Perkin Elmer, Norwalk,
CT, USA. He at 1 mL/min was used as carrier gas. An RTX-5MS capillary column (30 m × 0.25mm ×
0.250 µm, Restek, Bellefonte, PA, USA) was used. The oven temperature was programmed as follows:
initial temperature of 90 ◦ C was kept for 1 min, increased at a rate of 10.0 ◦ C/min to 300 ◦ C and
maintained for 5 min. The injector temperature was 260 ◦ C and injections were made in the split mode,
with a split flow of 1:25. The mass spectrometer was operating as follows: ion source temperature
280 ◦ C, ionization energy 70 eV (electron impact ionization), and m/z scanning range 35–650 Da. The
acquisition of chromatographic data was performed by means of TurboMass (Perkin Elmer) and
mass spectrum library NIST 2005 (National Institute of Standards and Technology Gaithersburg,
Montgomery County, Maryland, USA).
3.3. Validation Parameters

Standard solutions with known concentrations were prepared and analyzed to determine the areas
corresponding to each concentration. To generate calibration curves, a minimum of five concentrations
of each individual standard were measured. In the cases where the determined area of peaks detected
in samples did not fit the initial considered scale (it was the case of glucose and fructose), more points
were added to the calibration curves. Calibration data, including retention time (Rt), retention index,
calibration equations, linearity presented as a correlation coefficient (R2) of the calibration curves,
limits of detection (LOD), limits of quantification (LOQ) and precision (RSD) are presented in Table 2.
Retention indexes were calculated using Kovats retention index equation and established based on
mixed alkane standards from C9 to C27. LOD ranged from 1.5 to 19.92 ng*mL−1 and LOQ from 4.55
to 60.35 ng*mL−1 . The accuracy was evaluated as a recovery at each concentration over 80–120% of
the analyte range concentrations. The results showed that average recovery at different concentration
levels ranged from 93.4% to 97.2%, while the RSD was 3%. The calibration curve parameters had a
good linearity, with a correlation coefficient R2 ranging between 0.9977 and 0.9988. The amount of
each identified target calculated using calibration curves is highlighted in Suplementary Table S1. For
the construction of calibration curves, three repetitions were realised for each concentration, and the
same protocol was followed for each honey sample.
Molecules 2020, 25, 34 12 of 15
Table 2. Calibration data of detected components including: retention time (Rt), calibration equations,
linearity coefficient (R2), LOD, LOQ, and precision (RSD).
Retention Retention LOD LOQ

Standard Regression Equation R2 RSD%
Time (Rt ) Index (Ri ) (ng*mL−1 ) (ng*mL−1 )
9.51 1728 y = 0.1136x − 0.0836 0.9990 2.42 10.81 32.75
Xylose
9.68 1735 y = 0.2014x + 0.0347 0.9998 2.34 19.92 60.35
10.08 1832 y = 0.274x − 1.3427 0.9992 1.03 8.66 26.25
D-fructose 10.14 1840 y = 0.1245x − 0.5283 0.9995 2.15 3.68 11.14
10.24 1847 y = 0.0788x − 0.5207 0.9990 1.90 3.87 11.73
D-Pinitol 10.44 1861 y = 0.4234x + 0.0917 0.9998 0.15 2.72 8.25
Quabrachitol 10.86 1878 y = 0.1074x − 0.0521 0.9993 1.08 4.12 12.47
Allo-inositol 10.98 1902 y = 0.4181x − 0.3954 0.9983 1.07 19.06 57.76
11.15 1922 y = 0.2911x − 4.8439 0.9977 1.11 2.69 8.17
D-Glucose
12.25 2006 y = 0.0807x − 0.4037 0.9983 3.43 4.12 12.47
Neo-inositol 11.25 1934 y = 0.2969x − 0.1251 0.9995 0.93 10.89 33.00
Muco-inositol 11.49 1941 y = 0.2338x − 0.1819 0.9994 1.12 7.29 22.09
D-Chiro-inositol 11.93 1965 y = 1.279x + 0.8057 0.9994 0.10 6.02 18.25
Sequoyitol 12.01 1973 y = 0.167x − 0.0591 0.9993 1.46 7.13 21.60
Ononitol 12.12 1988 y = 0.0929x − 0.0288 0.9996 1.15 4.12 12.47
Bornesitol 12.51 2035 y = 0.2315x − 0.0338 0.9996 1.16 11.22 33.99
Epi-inositol 12.70 2070 y = 0.2038x + 0.1189 0.9993 0.53 5.61 17.00
Cis-inositol 13.00 2095 y = 0.1617x + 0.0601 0.9979 0.84 9.72 29.44
Myo-inositol 13.53 2120 y = 1.0889x − 0.1878 0.9997 0.56 19.42 58.88
Sucrose 18.67 2686 y = 0.0259x + 0.0075 0.9988 2.52 2.69 8.17
Maltose 18.77 2702 y = 0.1744x + 0.048 0.9981 1.86 6.16 18.65
Lactose 18.91 2730 y = 0.0151x + 0.0138 0.9987 1.90 1.50 4.55
D-(+)-turanose 19.14 2747 y = 0.1222x − 0.0637 0.9996 3.08 1.75 5.31
3.4. Determination of Physico-Chemical Properties
3.4.1. Pfund Value and Honey Color

For the determination of Pfund value 4 g of honey was dissolved 8 mL of distilled water, heated
up to 50 ◦ C, and kept under stirring until the total solvation of sugar crystals was achieved. The
absorbance of the obtained solution was measured with a UV-Vis spectrophotometer (NanoDrop
2000c; Thermo Fisher Scientific, Waltham, MA USA) at wave length λ = 635 nm. The honey color was
determined based on Pfund scale. The Pfund value was calculated with the formula (1).
Pfund [mm] = −38.7 + 371.39 × Abs (1)
where Pfund = honey color value in the Pfund scale and Abs = absorbance at the wave length of
635 nm.
3.4.2. Acidity and pH

From each honey sample 5 g was dissolved in 37.5 mL of water. The pH of obtained solutions
was measured in triplicate using a pH-meter CPC-501 (Elmetron, Chorzow, Poland) with a glass
electrode. The acidity (expressed in miliequivalent of acid per kilogram of honey) was determined by
Molecules 2020, 25, 34 13 of 15
the potentiometric titration of honey solution, previously prepared for pH measurement, with NaOH
(0.1M) until the pH value of 8.3 was obtained. The acidity was calculated using the formula (2).
A [meq/kg] = VNaOH × 10 (2)
where A = total acidity and VNaOH = the volume of NaOH (0.1M) solution used for the titration.
3.4.3. Electrical Conductivity

The electrical conductivity measurement of the honey was performed as follow: 2 g of sample was
dissolved in 10 mL of deionized water. From the obtained solution 1 mL was taken, the temperature
was adjusted at 20 ◦ C, and the solution was subsequently transferred in a conductivity cell. The
electrical conductivity of each sample was measured with the equipment CPC-501 equipment (Elmetron,
Chorzow, Poland) in triplicate and expressed in milisiemens/centimeter.
3.4.4. Antioxidant Activity

The antioxidant activity of honey was determined using the DPPH (2,2-Diphenyl-1-picrylhydrazyl)
free radical scavenging assay. In brief, 5 mL of honey solution (obtained by dilution 1:50) was evaporated
to dryness and redissolved in 2 mL of 70% ethanol. From ethanoic solution, 50 µL were mixed with
200 µL of 0.1 mM DPPH solution. The obtained mixtures were incubated under stirring for 30 min
at room temperature in darkness. The absorbance was measured at 517 nm using a Varioskan Lux
spectrophotometer (Thermo Scientific, Vantaa, Finland). The results were expressed in trolox units
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). The equivalent antioxidant capacity was
calculated using calibration curve of trolox solutions prepared at desired concentrations (between
0.025 to 0.3 µol/mL) in 70% EtOH.
3.5. Statistical Approach

IBM SPSS Statistical package, version 21 was used for hierarchical clustering analysis and
correlation analysis. Microsoft Excel 2016 and Microsoft Power Point 2010 were used to prepare and
integrate the figures composed from multiple parts.
4. Conclusions
The obtained results concluded that there is multiple-scale correlation between the physicochemical
properties and carbohydrate contents of 18 types of honey. Consequently, it was highlighted by
non-parametric tests that between different preset variables, i.e physical properties, the contents of
major constituents (glucose, fructose, etc.) and minor constituents (cyclitols), demonstrated a relevant
correlation. Moreover, parametric tests revealed a generally strong positive correlation between honey
types, when the contents of sugars and cyclitols were assigned as variables. Nevertheless, we found
that samples cultivated in 2015 and 2016 presented a distinctive correlation compared with those
harvested in 2017 and 2018, due to the natural changes which occur in honey during a prolonged
storage period. Hierarchical cluster analyses based on the amount of detected targets congregated the
samples broadly based on the strength of the total amount. However, the dendrograms built with
respect to the 23 variables selected, led to the formation of five main clusters with high significance,
and six more with lower levels of significance.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/1/34/s1,

Table S1: The amount of sugars and cyclitols (in mg/mL) quantified in honey samples, where nd–not detected.
Author Contributions: Conceptualization: I.A.R. and B.B.; Methodology: I.A.R. and H.A.-S.; Software: I.A.R.;
Validation: I.A.R. and H.A.-S.; Formal Analysis: I.A.R., H.A.-S., M.B., M.L. and B.B.; Investigation I.A.R., H.A.-S.
and M.B.; Resources: B.B.; Data Curation I.A.R. and H.A.-S.; Writing: I.A.R.; Writing—Review & Editing: I.A.R.,
B.B. and H.A.-S.; Visualization: I.A.R.; Supervision: I.A.R. and B.B.; Project Administration, B.B. and M.L.; Funding
Acquisition: B.B. All authors have read and agreed to the published version of the manuscript.
Molecules 2020, 25, 34 14 of 15
Funding: This work was financed in the framework of the grant entitled: “Cultivated plants and natural products
as a source of biologically active substances destined for the production of cosmetic and pharmaceutical products
as well as diet supplements” (No. BIOSTRATEG2/298205/9/NCBR/2016) attributed by the National Center for
Research and Development (Warsaw, Poland).
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foods
Article
Volatile Profile and Physico-Chemical Analysis of
Acacia Honey for Geographical Origin and
Nutritional Value Determination
Niculina M. Mădaş 1,2 , Liviu A. Mărghitaş 1 , Daniel S. Dezmirean 1 , Victorita Bonta 3 ,
Otilia Bobiş 3, * , Marie-Laure Fauconnier 4 , Frédéric Francis 2 , Eric Haubruge 2 and
Kim B. Nguyen 2
1 Department of Apiculture and Sericulture, University of Agricultural Sciences and Veterinary Medicine,
Mănăştur st, 3-5, 400372 Cluj-Napoca, Romania; mmadas@gmail.com (N.M.M.);
lmarghitas@usamvcluj.ro (L.A.M.); ddezmirean@usamvcluj.ro (D.S.D.)
2 Department of Functional and Evolutionary Entomology, University of Liège, Gembloux Agro-Bio Tech,
Passage des Déportés, 2, 5030 Gembloux, Belgium; frederic.francis@ulg.ac.be (F.F.);
e.haubruge@ulg.ac.be (E.H.); kim.nguyen@beeodiversity.com (K.B.N.)
3 Life Science Institute, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Manastur st.
3-5, 400372 Cluj-Napoca, Romania; victorita.bonta@usamvcluj.ro
4 Laboratory of Chemistry of Natural Molecules, University of Liège, Gembloux Agro-Bio Tech, Passage des
Déportés, 2, 5030 Gembloux, Belgium; marie-laure.fauconnier@uliege.be
* Correspondence: obobis@usamvcluj.ro; Tel.: +40-746027940

Received: 25 August 2019; Accepted: 24 September 2019; Published: 27 September 2019
Abstract: Honey composition and color depend greatly on the botanical and geographical origin.
Water content, water activity and color of 50 declared acacia samples, collected from three different
geographical zones of Romania, together with chromatographic determination of sugar spectrum
were analyzed. A number of 79 volatile compounds from the classes of: Alcohols, aldehydes,
esters, ketones, sulphur compounds, aliphatic hydrocarbons, nitrogen compounds, carboxylic acids,
aromatic acids and ethers were identified by solid-phase micro-extraction and gas-chromatography
mass spectrometry. The overall volatile profile and sugar spectrum of the investigated honey samples
allow the differentiation of geographical origin for the acacia honey samples subjected to analysis. The
statistical models of the chromatic determination, physicochemical parameters and volatile profile
was optimal to characterize the honey samples and group them into three geographical origins, even
they belong to the same botanical origin.
Keywords: acacia honey; gas-chromatography; HPLC; sugar spectrum; volatile organic compounds
1. Introduction
Nutritional value of honey [1] is due to the chemical composition of simple sugars and
all the substances (vitamins, enzymes, minerals, free amino-acids, phenolic and volatile organic
compounds—VOCs), that have been described in many studies made over the time [2–5].
The bioactive properties of honey are related to the botanical and geographical origin, being
influenced mainly by the plants from where the bees collect the nectar, and thus by the place where
these plants grow [2,5–9]. When talking about botanical origin, honey may be classified as floral
(derived from nectar of melliferous plants) or non-floral (honeydew) (derived from sweet substances
released by aphids on different plant parts which bees collect and transform similarly to floral nectar).
Botanical and geographical authentication of honey is a market feature, and for this reason different
Foods 2019, 8, 445; doi:10.3390/foods8100445 www.mdpi.com/journal/foods

2 of 25
ups and specialized Foods 2019, 8, 445 have established a list of methods and
laboratories 2 of 24
, for the correct classification of honey samples.

haracterized by a research
requiredgroups
percentage of specificlaboratories
and specialized pollen, different in
have established a list of methods and parameters to be
nging between 10determined,
and 20% (orange honey) to 70–90% for eucalyptus
for the correct classification of honey samples.
the monoflorality of honeyMonofloralhave honey
also economic importance,
is characterized becausepercentage of specific pollen, different in every
by a required
and pollen belonging predominantly to a certain species,
national standard, ranging between 10 and 20% (orange may have honey) to 70–90% for eucalyptus honey [10–13].
eepers have higher incomes obtained from its marketing.
Establishing the monoflorality of honey have also economic importance, because specific honey, with
nalysis may causenectar
classification
and pollen problems,
belonging duepredominantly
to the fact thattocertain
a certain species, may have higher selling price and
d in pollen (lavender, citrus) and additional
beekeepers have higher incomes obtained analysis are from
needed to
its marketing.
ty decision. These However,analysis only maypollenbe analysis
physico-chemical [14,15],
may cause classification problems, due to the fact that certain plants
omatographic techniques for possible markers determination (sugars,
are underrepresented in pollen (lavender, citrus) and additional analysis are needed to complement
]. the uniflorality decision. These analysis may be physico-chemical [14,15], spectrophotometric and
by volatile organic compounds coming
chromatographic fromfor
techniques plant nectar,
possible developed
markers determination (sugars, phenols or volatiles) [9,16–22].
etic pathways, thus Honey is closely related to botanical origin
aroma is given by volatile organic compounds [20]. coming from plant nectar, developed
avor constituents may come from the bees in the enzymatic
through different biosynthetic pathways, thus is closely process of related to botanical origin [20]. Additionally,
honey [20]. some of the flavor constituents may come from the bees in the enzymatic process of transforming the
tile compounds are reported
nectar in honey,
into honey [20]. the main classes being: furans,
terpenes, nor-isoprenoids, acidsclasses
Different and pyrene derivatives
of volatile [9,19,20,22].
compounds are reported in honey, the main classes being: furans,
orms and a very rich flora, Romania have the possibility
alcohols, aldehydes, ketones, terpenes, nor-isoprenoids, to produce acids and pyrene derivatives [9,19,20,22].
monofloral (acacia, linden,
Having rape, sunflower,
different landforms raspberry, heather,
and a very mint, Romania have the possibility to produce many
rich flora,
n, meadow) and last but of
types nothoney,
least honeydew
from monofloralhoney [5,14,16,21,23–26].
(acacia, linden, rape, sunflower, raspberry, heather, mint, etc.), to
les were studied until now [21,27,28], but
multifloral (mountain, meadow) the comparative
and last but analysis of honeydew honey [5,14,16,21,23–26].
not least
declared honey, fromRomanian
different geographical origins of Romania, was
honey volatiles were studied until now [21,27,28], but the comparative analysis of
in purpose of the volatiles
present study was totype
of the same assess the volatile
of declared composition
honey, from different geographical origins of Romania, was not
sent in acacia samples
made until now. The main purpose of the of
from different geographical regions Romania,
present study was to assess the volatile composition on the
xtraction; gas chromatography, mass spectrometry (SPME/GC/MS)
creation of aroma present in acacia samples from different geographical regions of Romania, using
solid phase micro-extraction; gas chromatography, mass spectrometry (SPME/GC/MS) technique.

emical methods used Most
in honey
of theanalysis are mainly
physico-chemical used for
methods quality
used in honey analysis are mainly used for quality control,
ike electrical conductivity
but some of them, like electrical conductivity and be
and sugar spectrum determination, may sugar spectrum determination, may be used for
phical origin determination.
botanical or geographical origin determination.
2.1. Honey Samples

ective of this study, the strategy
Based on thewas
maintoobjective
collect honey samples
of this study, thefrom
strategy was to collect honey samples from different
in order to study the typicity of Robinia pseudoacacia honey from
meliferous areas, in order to study the typicity of Robinia pseudoacacia honey from Romania. Fifty honey
es were collected samples
directly were
from collected
beekeepers; the selection
directly was made
from beekeepers; theinselection was made in three distinct geographic
egions with different
regionspedological characteristics
with different pedological(diverse landforms,
characteristics (diverse landforms, proportionally distributed and
nd concentrically concentrically
arranged around Transylvania
arranged aroundPlateau), and climate
Transylvania Plateau), and climate (temperate-continental, with
regional climaticregional
variation due variation
climatic to the variated topography),
due to the with
variated topography), with predominant Robinia pseudoacacia
cacia trees as nectar
treessources. Zone
as nectar 1 wasZone
sources. represented by Transilvania
1 was represented by Transilvania region (Alba, Cluj, Covasna, Harghita,
Harghita, MuresMuresand Satu
and Mare counties),
Satu Mare Zone Zone
counties), 2 was2 the
wassouthern
the southern part of Romania (Călăraşi, Dolj, Giurgiu,
Dolj, Giurgiu, Teleorman
Teleorman and V Vȃlcea
and lcea counties)
counties) andand
ZoneZone 3 were
3 were mainly eastern part of Romania (Bacău, Botoşani,
Buzău, Galaţi, Suceava, Tulcea and Vaslui counties) (Figure 1). Samples were kept in glass jars, at 4 ◦ C,
mania (Bacău, Botoşani, Buzău, Galaţi, Suceava, Tulcea and Vaslui
were kept in glassinjars,
darkatplaces
4 °C, in dark
until places until analysis. \â
analysis.
Foods 2019, 8, 445 3 of 24
Foods 2019, 8, x FOR PEER REVIEW 3 of 25
Figure1.1.Geographic
Figure Geographicorigin
originofofinvestigated
investigatedacacia
acaciahoney
honeysamples
samples(Green
(Greentriangle
triangle– –Zone
Zone1;1;Blue
Blue
triangle – Zone 2; Red triangle – Zone 3 of sample harvesting).
triangle – Zone 2; Red triangle – Zone 3 of sample harvesting).
2.2. Chemicals and Reagents

2.2. Chemicals and Reagents
All chemicals and reagents were analytically grade purity. Ultrapure water was made with
All chemicals and reagents were analytically grade purity. Ultrapure water was made with
MilliQ Integral SG Wasseraufbreitung (Darmstadt, Germany). Sigma Aldrich (Steinheim, Germany)
MilliQ Integral SG Wasseraufbreitung (Darmstadt, Germany). Sigma Aldrich (Steinheim, Germany)
provided fructose, glucose, sucrose, maltose, isomaltose, trehalose and erlose. A mixture of homologues
provided fructose, glucose, sucrose, maltose, isomaltose, trehalose and erlose. A mixture of
n-alkanes (C7 –C30 ) (Sigma, Darmstadt, Germany) was used for gas chromatographic determinations
homologues n-alkanes (C7–C30) (Sigma, Darmstadt, Germany) was used for gas chromatographic
of VOCs.
determinations of VOCs.
2.3. Physico-Chemical Analysis
2.3. Physico-Chemical Analysis
Selective physicochemical parameters were determined according to Romanian standard [29]
Selective physicochemical parameters were determined according to Romanian standard [29]
and Harmonized Methods of International Honey Commission [30]. Water content was determined
and Harmonized Methods of International Honey Commission [30]. Water content was determined
refractometrically (Abbe digital refractometer, WYA-S, Selecta, km. 585, ABRERA Barcelona, Spain)
refractometrically (Abbe digital refractometer, WYA-S, Selecta, km. 585, ABRERA Barcelona, Spain)
Spain). The respective content was expressed as mg/100 g; the water activity was measured using
Spain). The respective content was expressed as mg/100 g; the water activity was measured using a
a water activity meter Decagon, AquaLab CX-3 (Pullman, WA, USA). Measuring cells were placed
water activity meter Decagon, AquaLab CX-3 (Pullman, WA, USA). Measuring cells were placed
minimul 2 h into a thermostatated chambre “Zanotti” type at 20 ◦ C and measured afterwards.
minimul 2 h into a thermostatated chambre “Zanotti” type at 20 °C and measured afterwards.
Equipment etalonation was made by measuring distilled water activity (aw = 1.000 ± 0.003 La 20 ◦ C)
Equipment etalonation was made by measuring distilled water activity (aw = 1.000 ± 0.003 La 20 °C)
and a standard of lithium chloride (aw 0.120 ± 0.003 La 20 ◦ C). Color has been determined using
and a standard of lithium chloride (aw 0.120 ± 0.003 La 20 °C). Color has been determined using
refractrometric method in uniform color space CIELAB (Supplementary Figure S1), where any color
refractrometric method in uniform color space CIELAB (Supplementary Figure S1), where any color
may be specified by using rectangular coordinates L*, a*, b*. Axes a* and b* represent color tonality,
may be specified by using rectangular coordinates L*, a*, b*. Axes a* and b* represent color tonality,
and L* axis represent the luminosity.
and L* axis represent the luminosity.
Measurement principle is placing the sample in a three-dimensional space, in a position in
Measurement principle is placing the sample in a three-dimensional space, in a position in
three-axis rapping as follows:
three-axis rapping as follows:
L* which place the sample on the axis that has black and white;
L* which place the sample on the axis that has black and white;
a* which place the sample on the axis that has green and red;
a* which place the sample on the axis that has green and red;
b* which place the sample on the axis that has blue and green.
b* which place the sample on the axis that has blue and green.
Measuring equipment, HUNTERLAB miniscan XE (Reston, VA, USA), measures by
Measuring equipment, HUNTERLAB miniscan XE (Reston, VA, USA), measures by
spectrophotometry the reflectance of the sample color.
spectrophotometry the reflectance of the sample color.
L* value indicate the degree of brightness, positive values of a* indicates red zone, negative values
L* value indicate the degree of brightness, positive values of a* indicates red zone, negative
of a*, green component; positive values of b* indicates yellow Zone and negative values of b* indicates
values of a*, green component; positive values of b* indicates yellow zone and negative values of b*
blue component.
indicates blue component.
Foods 2019, 8, 445 4 of 24
The HPLC analysis of the carbohydrates is carried out on a modified Alltima Amino 100Å stainless
steel column (Hicrom, Berkshire, UK) (4.6 mm diameter, 250 mm length, particle size 5 µm) following
IHC method modified in APHIS-DIA Laboratory [16]. The SHIMADZU instrument (LC–10AD VP
model, Shimadzu, Kyoto, Japan) was equipped with degasser, two pumps, autosampler, thermostat
oven, controller and refractive index detector. The injection volume was 10 µL and the flow rate
1.3 mL/min. The mobile phase is a solution of HPLC purity acetonitrile and ultrapure water (80/20
v/v). Briefly, 5 g of honey were dissolved in water (40 mL) and transferred quantitatively into a 50 mL
volumetric flask, containing 25 mL methanol HPLC grade purity and filled up to the mark with water.
The solution was filtered through a 0.45 µm membrane filter, collected in sample vials and placed in
autosampler for analysis. For the quantification of main sugars, different calibration curves in the range
50–0.25 g/100 g (fructose 50–20 g/100 g; glucose 10-40 g/100 g; sucrose 0.3–15 g/100 g; turanose, maltose,
isomaltose, erlose 0.25–5 g/100 g), with regression coefficients (R2 ) higher of 0.998 were obtained. The
results are expressed in g/100 g honey.
2.4. Solid-Phase Microextraction and GC-MS Analysis

For the evaluation of the VOC profile in honey, a SPME automatic support was used, equipped
with a fiber type 50/30 divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) (Supelco,
Bellefonte, PA, USA). Thus, 4 g of each honey sample in a 30 mL headspace vial, sealed afterwards with
a screwed cap with PTFE/silicon septa. Samples were homogenized and heated to 40 ◦ C for 30 min.
After equilibration, the fiber was introduced into the vial headspace through the septum and exposed
to the sample for 30 min.
Volatile analyses were performed using a GC Agilent 7890A system (Wilmington, DE, USA) with
injectors both with and without mobile phase flux dividers and an AGILENT 5675C inert XLEO/CI
MSD mass spectrometry with a triple-axis detector. The SPME fiber was desorbed at 250 ◦ C for 5 min
without dividing of the mobile phase. The separation of VOC was performed on a Hewlett-Packard
SP-5-DBWAX (30 m × 0.25 mm i.d. and 0.25 µm film thickness) (Bellefonte, PA, USA).
The column temperature was maintained in the column oven following the gradient: the initial
temperature of 35 ◦ C was kept constant for 5 min, raised with 3 ◦ C/min until it reached 50 ◦ C. After
this, it raised with 5 ◦ C/min until 180 ◦ C, with 6 ◦ C/min up to 250 ◦ C and held at this value for 10 min.
The temperatures of the injector and detector were set at 250 ◦ C and 230 ◦ C, respectively. Helium was
used as carrier gas (>90% purity).
2.5. Data Analysis

The identification of the compounds was made by retention time and by comparing the mass
spectra obtained with standard mass spectra from the “Pal 600” spectra library. Also, the retention
indices (RI) were calculated by injecting a mixture of homologues n-alkanes (C7–C30) under the same
chromatographic conditions (Sigma, Darmstadt, Germany) [31]. Furthermore, the indices calculated
for the selected compounds were compared with the literature data (www.pherobase.com). For a
better quantification of the results, the method of normalization area was used, each compound’s
concentration being calculated according to the following expression:
X% = (AX /ΣAi ) (1)
where: Ax : The compounds surface, ΣAi : The sum of all identified compound’s surface.

Physico-chemical analysis were performed in triplicate, data being expressed as mean ± SD.
Differences between means were determined using one-way ANOVA test (IBM SPSS Statistics for
Windows, Version 25.0, IBM Corp., Armonk, NY, USA). Variance analysis, ANOVA, calculates the ratio
Foods 2019, 8, 445 5 of 24
between the variation caused by intergroup and intragroup differences and establishes whether this
ratio is large enough in order to distinguish the groups.
3.1. Physicochemical Parameter Values for Acacia Honeys

Acacia honey in general has a low water content [14,32–35], which can be observed also in this
study. The values obtained are within the limits pertaining to the honeys found in the temperate
climate, especially in Europe.
In order to carry out a primary interpretation of the data obtained for this parameter and to have
a clear image of its variability in accordance with the geographical origin, a box plot graph of these
values was constructed for each zone. The box plot diagram summarizes the charts for the five values
specific to their spread in each Zone (the minimum, the first quartile, the median, the third quartile
and the maximum) and the extreme values. In this chart, one can observe a central line for each box
plot, representing the median of the values measured for each zone. If the median is closer to the lower
margin, the value spread leans towards the left, and if it is closer to the higher margin, the spread
leans towards the right. For Zone 1, the maximum of the median was found to be 16.704, for Zone 2,
17.416, and for Zone 3, the median value was 17.088. The box plot shows 50% of the values, and its size
shows result variability. The horizontal lines above and below the box are traced from between the
minimum and the maximum for each zone, so that the minimum values are 15.4, 15.8 and 17.1 for
zones 1, 2, 3, respectively. The extreme values are placed outside the box plot and are marked with “*”,
accompanied by the sample number. In the case of water content determination, two extreme values
have been found, samples S15 (belonging to Zone 1) and S25 (Zone 2) (Supplementary Figure S2).
The values obtained for water activity ranged between 0.540 (S51, Zone 1) and 0.690 (S25, Zone 2)
(Table 1).
A water activity value of 0.562 was recorded for Slovakian acacia honey [36], 0.490 for Czech
honey [33] and 0.81 for Romanian honey [37].
The L* value shows the brightness level, positive values of the parameter a* show red zone,
negative values for a* show the green component, positive values for b* show the yellow Zone and
negative values for b* indicate the blue component. The values for L* (Table 2) were between 50.19
and 62.70, and the average obtained after measuring all the samples was 58.96. These values are in
accordance with those measured by Bertoncelj et al. [38] for Slovenian honey (64.4), some Spanish
honeys (51.62–56.11) [38] but are much higher than those found for Slovakian honey (11.29) [36] and
other Romanian acacia honeys (48.96) [37].
The value for parameter a* (Table 2) lies between –1.28 and 5.80. The average obtained by the
measuring of all the samples was 0.08 ± 1.12. A wide distribution can be observed when determining
this parameter, as samples were situated in the green Zone (negative a*), as well as in the red
Zone (positive a*). Only positive values were found for this parameter by Kasperová et al. (4.07) [35]
in the case of Slovakian honey, Romanian honey [37], whereas Bertoncelj et al. [38] found only negative
values for Slovenian acacia honey (−2.82), as well as Karabagias et al. [8] for Portuguese honey. As it
can be observed in Table 2, the positive and negative values obtained for this parameter cannot be
linked to geographical origin or with the sample year of harvest. Each Zone has shown both negative
and positive values. The same characteristic presented Spanish honey [39].
Foods 2019, 8, 445 6 of 24
Table 1. Selective physico-chemical parameter values for investigated honey samples (water content (%) and water activity (aw )).
Zone 1 Zone 2 Zone 3

Obtained Values Obtained Values Obtained Values
Sample Code Sample Code Sample Code
Water Content (%) Water Activity (aw ) Water Content (%) Water Activity (aw ) Water Content (%) Water Activity (aw )
S1 16.8 ± 0.1 0.599 ± 0.000 S4 16.1 ± 0.2 0.568 ± 0.001 S5 17.3 ± 0.1 0.565 ± 0.001
S2 15.5 ± 0.1 0.573 ± 0.006 S6 17.4 ± 0.0 0.578 ± 0.001 S7 15.8 ± 0.2 0.593 ± 0.001
S3 16.9 ± 0.0 0.607 ± 0.001 S11 16.4 ± 0.0 0.578 ± 0.001
S8 16.0 ± 0.2 0.578 ± 0.001 S14 17.3 ± 0.1 0.594 ± 0.001 S21 16.8 ± 0.2 0.686 ± 0.004
S9 16.3 ± 0.1 0.602 ± 0.003 S29 17.2 ± 0.1 0.610 ± 0.001
S10 15.4 ± 0.0 0.575 ± 0.001 S19 17.8 ± 0.3 0.621 ± 0.001 S30 15.5 ± 0.2 0.574 ± 0.001
S12 15.7 ± 0.3 0.558 ± 0.003 S31 16.1 ± 0.1 0.615 ± 0.001
S13 17.5 ± 0.0 0.603 ± 0.001 S25 20.4 ± 0.1 0.690 ± 0.004 S32 16.5 ± 0.1 0.599 ± 0.000
S15 21.3 ± 0.2 0.600 ± 0.000 S33 16.9 ± 0.1 0.601 ± 0.000
S16 15.7 ± 0.1 0.562 ± 0.001 S27 17.0 ± 0.0 0.602 ± 0.000 S34 20.4 ± 0.1 0.656 ± 0.001
S17 18.0 ± 0.2 0.610 ± 0.003 S39 15.4 ± 0.1 0.555 ± 0.003
S18 17.1 ± 0.1 0.621 ± 0.000 S35 16.7 ± 0.2 0.598 ± 0.001 S40 18.9 ± 0.2 0.580 ± 0.001
S20 18.0 ± 0.1 0.616 ± 0.001
S22 15.7 ± 0.1 0.559 ± 0.001 S36 16.8 ± 0.2 0.595 ± 0.001 S41 16.9 ± 0.3 0.574 ± 0.001
S23 16.6 ± 0.1 0.591 ± 0.000
S24 16.7 ± 0.1 0.597 ± 0.001 S37 18.7 ± 0.1 0.620 ± 0.001 S42 16.9 ± 0.3 0.573 ± 0.000
S28 16.7 ± 0.1 0.592 ± 0.001 S38 16.7 ± 0.1 0.580 ± 0.002 S52 14.8 ± 0.2 0.537 ± 0.003
S48 17.1 ± 0.1 0.578 ± 0.001
S51 15.0 ± 0.2 0.540 ± 0.001 S50 18.0 ± 0.2 0.596 ± 0.000 S55 17.2 ± 0.2 0.587 ± 0.000
S53 16.8 ± 0.1 0.570 ± 0.000
S58 16.1 ± 0.0 0.552 ± 0.00 S57 16.1 ± 0.1 0.558 ± 0.001 S56 18.8 ± 0.1 0.611 ± 0.001
Average 16.705 ± 0.1 0.585 ± 0.001 Average 17.416 ± 0.13 0.600 ± 0.001 Average 17.088 ± 0.15 0.591 ± 0.01
Every value is a mean of three determinations (n = 3) ± standard deviations.
Foods 2019, 8, 445 7 of 24
Table 2. The color parameter values of acacia honey samples (L*, a*, b*) (n = 50).
Zone 1 Zone 2 Zone 3

Sample Obtained Values Sample Obtained Values Sample Obtained Values
Code L* a* b* Code L* a* b* Code L* a* b*
S1 60.62 ± 0.02 −0.76 ± 0.01 13.02 ± 0.01 S4 54.11 ± 0.08 2.03 ± 0.02 25.99 ± 0.06 S5 60.48 ± 0.13 −0.39 ± 0.01 10.98 ± 0.03
S2 59.42 ± 0.03 −0.69 ± 0.02 13.06 ± 0.02 S6 50.19 ± 021 0.39 ± 0.02 19.60 ± 0.03 S7 56.20 ± 0.12 0.22 ± 0.02 24.91 ± 0.00
S3 51.28 ± 0.09 1.98 ± 0.02 26.07 ± 0.03 S11 59.60 ± 0.04 −0.62 ± 0.01 12.81 ± 0.04
S8 59.62 ± 0.09 −0.54 ± 0.04 15.32 ± 0.05 S14 56.94 ± 0.26 0.98 ± 0.03 21.53 ± 0.06 S21 56.96 ± 0.10 0.58 ± 0.03 21.17 ± 0.15
S9 60.24 ± 0.08 −0.42 ± 0.01 13.34 ± 0.03 S29 60.10 ± 0.07 −0.76 ± 0.01 13.68 ± 0.09
S10 58.16 ± 0.06 −0.37 ± 0.02 19.27 ± 0.09 S19 57.76 ± 0.03 0.50 ± 0.03 17.23 ± 0.05 S30 59.17 ± 0.14 0.29 ± 0.02 17.45 ± 0.08
S12 59.52 ± 0.05 −0.11 ± 0.03 15.85 ± 0.01 S31 57.03 ± 0.19 0.44 ± 0.04 20.23 ± 0.39
S13 58.96 ± 0.22 0.08 ± 0.02 16.79 ± 0.04 S25 57.56 ± 0.11 0.52 ± 0.01 17.73 ± 0.09 S32 55.73 ± 0.37 1.18 ± 0.01 22.19 ± 0.28
S15 54.48 ± 0.18 2.31 ± 0.01 28.22 ± 0.05 S33 55.88 ± 0.18 0.98 ± 0.02 21.84 ± 0.14
S16 61.87 ± 0.19 −0.36 ± 0.03 9.47 ± 0.11 S27 57.82 ± 0.28 0.57 ± 0.03 20.82 ± 013 S34 55.42 ± 0.16 1.20 ± 0.02 23.23 ± 0.07
S17 60.51 ± 0.17 −0.23 ± 0.03 13.07 ± 0.10 S39 59.84 ± 0.11 −0.40 ± 0.00 18.19 ± 0.09
S18 50.92 ± 0.19 5.80 ± 0.06 30.41 ± 0.59 S35 59.69 ± 0.11 0.08 ± 0.03 13.08 ± 0.03 S40 60.46 ± 0.08 −0.60 ± 0.01 12.05 ± 0.09
S20 60.98 ± 0.07 0.68 ± 0.03 11.20 ± 0.11
S22 59.68 ± 0.20 0.23 ± 0.02 16.56 ± 0.08 S36 60.98 ± 0.40 −0.29 ± 0.00 11.39 ± 0.18 S41 62.60 ± 0.13 −0.81 ± 0.02 7.03 ± 0.03
S23 60.54 ± 0.04 −0.37 ± 0.01 11.75 ± 0.02
S24 60.12 ± 0.02 −0.11 ± 0.02 14.76 ± 0.03 S37 57.48 ± 0.07 0.62 ± 0.03 19.82 ± 0.18 S42 61.34 ± 0.10 −0.70 ± 0.02 10.38 ± 0.06
S28 59.28 ± 0.16 0.02 ± 0.02 16.73 ± 0.14 S38 60.97 ± 0.38 −0.62 ± 0.01 12.07 ± 0.22 S52 62.70 ± 0.18 −0.99 ± 0.03 9.87 ± 0.07
S48 64.00 ± 0.12 −0.66 ± 0.01 6.23 ± 0.04
S51 59.88 ± 0.38 −0.71 ± 0.02 20.67 ± 0.32 S50 62.59 ± 0.03 −1.05 ± 0.03 7.21 ± 0.03 S55 61.05 ± 0.12 −0.73 ± 0.01 11.25 ± 0.08
S53 61.11 ± 0.06 −0.68 ± 0.01 13.60 ± 0.10
S58 61.96 ± 0.43 −1.28 ± 0.04 11.37 ± 0.05 S57 62.03 ± 0.35 −0.82 ± 0.02 8.66 ± 0.04 S56 59.90 ± 0.00 −0.27 ± 0.03 14.07 ± 0.10
Average 59.31 ± 0.14 0.12 ± 0.02 15.16 ± 0.10 Average 58.18 ± 0.18 0.24 ± 0.02 16.26 ± 0.09 Average 59.06 ± 0.13 −0.08 ± 0.02 15.96 ± 0.11
L* - degree of brightness (luminosity); a*, b* - color tonality.
Foods 2019, 8, 445 8 of 24
As for b* parameter values (Table 2), the recorded minimum was 6.23, and the maximum 30.41.
The measurement average for all zones was 15.70. Despite the fact that the Zone between the minimum
and maximum values of the b* parameter is relatively vast, the average obtained for acacia honey in
correlated to the value found for Slovenian acacia honey [38] (17.95) and to that of Slovakian honey
(14.66) [36], but much higher than other Romanian acacia honeys [37].
The highest median value for the L* parameter was recorded in the case of the samples from
Zone 1 (59.315), a value close to the one from Zone 3 (59.062), while the value for Zone 2 has shown
the lowest value (58.177). Variance values are higher than one and show a greater degree of variation.
The results of the ANOVA p = 0.5662 and F = 0.575 test show that there are no significant differences
between samples when measuring this parameter. Thus, the samples are homogenous (Table 3).
The results obtained for each sugar are presented in Figure 2A, B. Each identified and quantified
The results obtained for each sugar are presented in Figure 2A, B. Each identified and quantified
sugar
sugar waswas individually
individually discussed,
discussed, as
as the
the values
values obtained
obtained are
are extremely
extremely important
important to
to determine
determine and
and
authenticate the botanical origin of the honey samples.
authenticate the botanical origin of the honey samples.
Figure
Figure2.2.Glucose
Glucoseand
andfructose
fructosecontent
content(A)
(A)and
andminor
minorsugars
sugars(B)
(B)ofofthe
theanalyzed
analyzedsamples
samples(mean
(meanvalue
valueof
three repetitions
of three ± SD).
repetitions ± SD).
As far as the glucose content is concerned, the values obtained for the 50 analyzed samples ranged
between 22.32% and 43.71%. The mean value obtained for every zone was: 30.57 (Zone 1), 30.40 (Zone 2)
and 30.37% (Zone 3).
Generally, acacia honey has an average glucose content of 26.3% [40]. Romanian acacia honey has
values ranging from 30.87% [41] and 32.58% [14]. The results obtained by analyzing these samples fell
within these limits and proved to be very similar to other acacia honeys in Europe [42,43].
The fructose content is a very important parameter in analyzing acacia honey. The fluid state of this
type of honey is due to a high fructose content and implicitly to a higher than 1.2 fructose/glucose ratio.
The results obtained following the HPLC analysis of fructose in the honey samples (Figure 2A) ranged
between 33.80% and 48.16%. The mean value of the fructose content for each zone was: 43.55 (Zone 1),
Foods 2019, 8, 445 9 of 24
Table 3. Analysis of variance for water content, water activity and color (L, a*, b*).
Parameter/Zone Analysis of Variance Parameters

Water Content (%)
Groups Count Average Variance
Zone 1 21 16.70476 1.789726
Zone 2 12 17.41667 1.463333
Zone 3 17 17.08824 2.327353
ANOVA F p-value F crit
1.06635 0.352444 3.195056
Water activity (aw )
Zone 1 21 0.585333 0.000510
Zone 2 12 0.6000 0.001154
Zone 3 17 0.591412 0.001423
0.848591 0.43447 3.195056
Zone 1 21 59.31476 9.517026
Zone 2 12 58.17667 12.29979
Zone 3 17 59.06235 5.631544
0.575627 0.566267 3.195056
a*
Zone 1 21 0.118095 2.189636
Zone 2 12 0.2425 0.72793
Zone 3 17 −0.08118 0.544336
0.306413 0.737542 3.195056
b*
Zone 1 21 15.16219 40.39225
Zone 2 12 16.26417 32.77234
Zone 3 17 15.96059 30.49048
0.156758 0.855356 3.195056
As far as the glucose content is concerned, the values obtained for the 50 analyzed samples
ranged between 22.32% and 43.71%. The mean value obtained for every Zone was: 30.57 (Zone 1),
30.40 (Zone 2) and 30.37% (Zone 3).
Generally, acacia honey has an average glucose content of 26.3% [40]. Romanian acacia honey has
values ranging from 30.87% [41] and 32.58% [14]. The results obtained by analyzing these samples fell
within these limits and proved to be very similar to other acacia honeys in Europe [42,43].
The fructose content is a very important parameter in analyzing acacia honey. The fluid state of
this type of honey is due to a high fructose content and implicitly to a higher than 1.2 fructose/glucose
ratio. The results obtained following the HPLC analysis of fructose in the honey samples (Figure 2A)
ranged between 33.80% and 48.16%. The mean value of the fructose content for each Zone was: 43.55
(Zone 1), 43.58 (Zone 2) and 43.15% (Zone 3). While determining this parameter, we could observe the
fact that only 5 samples out of 50 had a lower than 40% fructose content. Similar values were obtained
Foods 2019, 8, 445 10 of 24
in the case of other acacia honey samples from Romania [14,41] or other acacia honey samples in
Europe [42,43]. As it has already been mentioned, the fructose/glucose ratio is very important in acacia
honey analysis. Sabatini et al. [40] found about a 1.66 ± 0.14 ratio in the case of acacia honey. The results
obtained following the HPLC analysis of individual carbohydrates in the analyzed samples highlight
(with three exceptions) the supraunitary values of this ratio, while 50% of the samples showed a >
1.5 ratio.
Sucrose is a very important indicator of honey authenticity, as standards require a maximum of
5% in authentic honey. The samples analyzed have significantly lower values, under no suspicion of
falsification. The values registered ranged between 0.0 and 5.51%, with an average value of 1.0% (mean
of all zones) (Figure 2B). Only one sample had a concentration higher than 5% (sample S58, Zone 1).
Some of the analyzed samples had a sucrose content that proved to be under the detection limit of the
method and device (0.02%). The results obtained in this study confirmed earlier studies [41,44–47].
The ANOVA analysis (the ratio between the variance produced by differences inside the groups
and the variance produced by differences outside the group) shows that there are no statistic differences
(p = 0.3010 > 0.05 and F = 1.23174 < Fcrit ) between the samples harvested from the three production zones.
Maltose is an important disaccharide found in honey with determined values ranging between
1.3% [40], 1.9% [48] and 2.5% [49] or a medium value of 3.53% obtained by Mărghitaş et al. [14] for
acacia honey in Romania (Transylvania). It is generally found in nectar honey and particularly in
acacia honey.
The values obtained after analyzing the samples in this study varied between 0.68 and 4.16%. The
ANOVA variance analysis shows that the value of p is lower than 0.05 (p = 0.012177), but F is bigger
than Fcrit . (F = 4.848788 and Fcrit = 3.195056). Consequently, the hypothesis fails because there are
significant differences between the samples.
Isomaltose is a disaccharide found in a relatively small quantity in honey, generally less than
1%. Depending on the carbohydrate determination method chosen (HPLC, GC or FTIR), but also on
the botanical origin of honey, the quantity may differ, but remains low. The values obtained in the
case of acacia honey varied between 0.14 and 0.79% and the mean value of the 50 samples was 0.39%.
Similar values were obtained for the acacia honey in Italy [40], other acacia honeys in Romania [14]
or the honey in Poland [49]. However, it was easy to observe that this disaccharide presented values
two times higher for the samples collected in geographical Zone 1 (Figure 2B). The two other zones
presented similar values, although geographically they are very different (Zone 2 being located on the
south-west of the country, while Zone 3 represents the eastern part of the country).
Turanose is a sugar found in small quantities in honey. The use of high performance liquid
chromatography, gas chromatography or nuclear magnetic resonance, made it possible for this
disaccharide to be found by different researchers in small quantities in Portuguese heather honey [50],
in Polish nectar honey [49], in 5 types of Italian honey [51], in Hungarian acacia honey [52] and in
Turkish nectar and honeydew honey [53].
Turanose was quantified between 0.38 and 2.77%, with an average of 2.08% in the acacia honey
samples used in this study. These values are generally higher than the ones found by the aforementioned
authors who have analyzed acacia honey, but similar to other honey types analyzed.
Erlose is a trisaccharide found in honey in different quantities, in accordance with its botanical
origin. The studies conducted at the APHIS Laboratory (USAMV Cluj-Napoca) have shown a rather
high concentration of erlose in acacia honey, compared to other honey types [41].
Although Goldschmidt and Burckert [54] highlighted erlose as a honey component since 1955,
many researchers did not report this trisaccharide when using high performance liquid chromatography
as a determination method [49,52,55]. The values obtained for the analyzed honey samples ranged
between 0.09 and 3.19%.
The glucidic spectrum analyzed by refractive index high performance liquid chromatography
has shown the presence of seven saccharides specific to acacia honey. The samples fall between the
Foods 2019, 8, 445 11 of 24
limits established for this honey type, due to the high fructose content, >1.2 fructose/glucose (F/G)
ratio, small sucrose content and higher than one maltose quantities [40,42,43].
In conclusion, the glucidic spectrum for acacia honey was established, and carbohydrate
concentrations, especially fructose and F/G ratio, which show high values for this honey type.
In orderthe
illustrate tocorrelations
illustrate the correlations
between betweenand
carbohydrates carbohydrates
to determineand to determine
which samples are which samples
outliers, are
a graphical
outliers, a graphical
representation was maderepresentation was madecalled
for all carbohydrates, for alla carbohydrates, called
matrix plot (Figure 3).a matrix plot (Figure 3).
S1
S10
S11
20 30 40 0 2 4 1 2 3 1,5 3,0 4,5
0,0 0,5 1,0
0,0 1,5 3,0 S12
S13
S14
45
S15
Fructose 40 S16
S17
35 S18
40 S19
S2
30 S20
Glucose S21
20 S22
S23
4 S24
S25
Sucrose 2 S27
0 S28
3 S3
S30
2
S31
Turanose 1 S32
S33
4,5 S34
S35
3,0 S36
Maltose S37
1,5 S38
1,0 S39
S4
S40
0,5
Isomaltose S41
S42
0,0 S48
S5
S50
Erlose S51
S52
S53
S57
S58
S6
Figure3.3. Graphic
Figure Graphic representation
representation matrix
matrix plot
plot of
of sugars.
sugars.
Due
Due totothe
thevast
vastdatabase,
database,high
highsample
sample and
and analysis
analysis number, itit isisfitfittotouse
use a Pearson
a Pearson correlation
correlation
matrix in order to identify the correlation
matrix in order to identify the correlation indices. These indices have a value ranging between
have a value ranging between (–1 (–1toto
1).1).
Table 4 4–6
Tables Tableshow5 Table 6 show
positive values,positive values,that
which means which
there means that there
is a positive associationis a positive association
of parameters, while of
parameters, while negative
negative values show an values
inverseshow an inverse
dependence of dependence of the two
the two parameters. The parameters. The values
values colored in blue colored
are
insignificant
blue are significant for the 0.01 level, while the ones in yellow are significant
for the 0.01 level, while the ones in yellow are significant of the 0.05 level.of the 0.05 level.
For Zone 1, the best correlation was established between ash content and color. Also,
positive correlations were found between different sugars, 0.72 for turanose–maltose and 0.79 for
saccharose–erlose. The most significant negative correlation found for Zone 1 was between water
activity–color (−0.841).
For Zone 2, significant positive correlations were found between sugars, 0.93 for turanose–maltose
and 0.90 for sucrose–erlose. The most significant negative correlation was identified between the color
parameters L*–a* (−0.94).
For Zone 3, the best correlation was established between water content and water activity, 0.92.
The negative correlation found for Zone 3 was between color parameters (−0.80).
Foods 2019, 8, 445 12 of 24
Table 4. Pearson correlation matrix of physico-chemical parameters for Zone 1.
Parameters Water Content Fructose Glucose Sucrose Turanose Maltose Isomaltose Erlose Water Activity Color L* Color a* Color b*
Water content 1.000
Fructose −0.133 1.000
Glucose 0.071 −0.476 1.000
Sucrose 0.277 0.136 −0.539 1.000
Turanose −0.034 0.425 −0.371 −0.078 1.000
Maltose −0.209 0.419 −0.190 −0.045 0.721 1.000
Isomaltose 0.098 −0.325 0.294 −0.527 0.333 0.110 1.000
Erlose 0.167 0.374 −0.592 0.790 0.209 0.281 −0.441 1.000
Water activity 0.581 −0.321 0.624 −0.359 −0.120 −0.139 0.471 −0.393 1.000
Color L* −0.009 0.378 −0.518 0.161 0.270 0.208 −0.185 0.369 −0.447 1.000
Color a* 0.167 −0.612 0.708 −0.262 −0.385 −0.511 0.292 −0.527 0.549 −0.841 1.000
Color b* 0.027 −0.411 0.435 −0.021 −0.379 −0.350 0.020 −0.266 0.288 −0.818 0.723 1.000
L* - degree of brightness (luminosity); a* , b* - color tonality; Blue: significant for the 0.01 level; Yellow: significant of the 0.05 level.
Water content 1.000
Fructose −0.271 1.000
Glucose 0.343 −0.590 1.000
Sucrose −0.457 −0.194 0.332 1.000
Turanose −0.555 0.602 −0.584 −0.139 1.000
Maltose −0.601 0.211 0.178 0.372 0.589 1.000
Isomaltose −0.117 −0.107 0.078 −0.264 0.577 0.519 1.000
Erlose −0.768 0.133 −0.270 0.519 0.640 0.695 0.376 1.000
Water activity 0.921 −0.321 0.500 −0.367 −0.676 −0.522 −0.182 −0.762 1.000
Color L* −0.095 0.564 −0.160 −0.011 0.286 0.092 −0.163 −0.083 −0.014 1.000
Color a* 0.048 −0.263 0.403 0.157 −0.220 0.292 0.153 0.177 0.137 −0.728 1.000
Color b* 0.092 −0.336 0.382 0.059 −0.175 0.299 0.264 0.175 0.134 −0.803 0.962 1.000
Foods 2019, 8, 445 13 of 24
Water content 1.000
Fructose −0.097 1.000
Glucose −0.013 −0.891 1.000
Sucrose −0.482 0.106 0.049 1.000
Turanose −0.414 0.655 −0.693 0.450 1.000
Maltose −0.367 0.664 −0.611 0.485 0.947 1.000
Isomaltose −0.359 0.273 −0.202 0.200 0.669 0.745 1.000
Erlose −0.264 0.684 −0.666 0.513 0.908 0.935 0.566 1.000
Water activity 0.744 −0.428 0.337 −0.750 −0.745 −0.723 −0.411 −0.719 1.000
Color L* −0.334 0.157 −0.165 0.667 0.565 0.572 0.243 0.733 −0.716 1.000
Color a* 0.363 −0.077 0.022 −0.796 −0.473 −0.464 −0.125 −0.619 0.708 −0.944 1.000
Color b* 0.177 −0.299 0.255 −0.576 −0.548 −0.580 −0.224 −0.749 0.624 −0.949 0.876 1.000
Foods 2019, 8, 445 14 of 24
3.2. Volatile Compounds

The GC-MS analysis method is a combination of two analytical techniques: capillary column
GC, which separates the components of a mixture and mass spectrometry (MS), which supplies the
information necessary in the structural determination of each constituent. The results obtained following
the SPME/GC-MS analysis is shown as chromatograms (Supplementary Figure S3). Their interpretation
is achieved by identifying mass spectrum and the retention time. These two parameters significantly
contribute to the identification of each volatile compound found in honey. Also, retention time has a
determining role in many cases where the constituents show identical mass spectra. Following the
SPME/GC-MS analysis, 79 constituents were identified the honey samples: 72 compounds samples from
Transylvania (Zone 1), 57 compounds in samples from Southern Romania (Zone 2) and 66 compounds
in samples from Eastern Romania (Zone 3). From all identified compounds, only 54 are common to all
50 samples. The chemotype changes from one region to another, and the difference is constituted by
the 6 compounds found only in one region and 9 compounds in two of the regions.
The interpretation of the chromatograms is achieved using the “Data Analysis” program. The
noise level is set to 50 for the x-coordinate and the program goes over the chromatogram signal by
signal according to this ratio. Identification of the compounds is achieved by comparing standard
mass spectra from the “Pal 600” spectra library. The surface is calculated following the “apexing mass”
model, which means measuring the surfaces in its entire mass.
The next stage in the qualitative analysis is the calculation of the Kovats indexes. We have used
this retention index in order to eliminate the relativity of the retention parameters by inserting some
reference points in order to compare the retention times of chromatographic separation for each volatile
compound found in honey. In this respect, a mixture of homologues n-alkanes (C7 –C30 ) was used
under the same chromatographic conditions (Sigma, Darmstadt, Germany), whose retention indices
showed values between 600 and 2200. The retention indices specific to each compound is calculated
as follows:
RI = 100 ((log tri − log trn )/(log trn+1 − log trn )), (2)
where: RI: compound retention index, tri : compound retention time, trn : retention time of the adjacent
and anterior alkane, trn+1 : retention time of the adjacent and subsequent alkane.
The expression of the retention index uses two reference points: the alkane with the same number
of carbon atoms in its molecule (n) as the volatile compound identified as i, which will elute before the
compound, and the second reference will be the alkane with n+1 carbon atoms in its molecule, which
will elute after the i compound. Table 7 comparatively shows the retention indices calculated RIb and
the retention indexes found in literature. RIa corresponds to each volatile compound found and the
type of column used (polar column). The main bibliographical source used was the PHEROBASE
database, which is one of the most comprehensive and up-to-date.
The main volatile compounds of acacia honey identified undoubtedly are: 3-methyl-3-buten-1-ol
for Zone 1, ethanol, acetic acid, 5-ethenyldihydro-5-furanone for Zone 2, acetone, 3-methyl-3-buten-1-ol,
trans-linalool oxide, benzemethanol for Zone 3. Among the secondary compounds identified in a
percentage of 80–90% of the samples one can find:
Dimethyl sulfide, acetone, 3-hydroxy-2-butanone, linalool oxide, acetic acid, 2-furancarboxaldehyde,
benzaldehyde, linalool L, ho-trienol, 4-ketoisophorone, epoxylinalool, benzenemethanol for Zone 1;
Dimethyl sulfide, acetone, 3-hydroxy-2-butanone, 2-methyl-2-buten-1-ol, nonanal, linalool,
2-furancarboxaldehyde, 5-methyl-2-furancarboxaldehyde, butanoic acid, epoxy-linalool, 3-hydroxy-2-
butanone, methylpentanoic acid, benzenethanol for Zone 2;
Dimethyl sulfide, ethanol, isoamyl alcohol, 2-methyl-2-buten-1-ol, cis-3-hexen-1-ol, linalool
oxide, acetic acid, 2-furancarboxaldehyde, benzaldehyde, propanoic acid, hotrienol, butyrolactone,
benzeneacetaldehyde, phenol for Zone 3.
Thus, once the qualitative analysis was completed, the volatile profile of the Romanian acacia
honey was outlined.
Foods 2019, 8, 445 15 of 24
Table 7. Retention index from literature (Ria ) and calculated retention index (RIb ) of volatile compounds
in honey samples of the three investigated zones (Z1–Z3).
Z1 Z2 Z3
Rank Volatile Compound (Usual and/or IUPAC Name) RIa
RIb RIb RIb
1 dimethyl sulfide/methylsulfanylmethane 734
2 acetone/2-propanone 810 807 806 805
3 ethyl acetate 872 862
4 3-methylbutanal/Isovaleraldehyde 912 906 906 904
5 isopropyl alcohol/2-propanol 917 915 919 913
6 ethanol 928 923 923 921
7 2,2,4,6,6-pentamethylheptane 945
8 2,3-butanedione/diacetyl 977 969 967 968
9 2-methylpropanenitrile/isobutyronitrile 985 976
10 alfa-pinene/2,6,6-trimethylbicyclo[3.1.1]hept-2-ene 999 999 1000
11 2-butanol/sec-butanol 1022 1010
12 3-methylpentanal/3-methylvaleraldehyde 1016 1010 1011
13 2-methyl-3-buten-2-ol/dimethylvinylcarbinol 1036 1041 1024 1013
14 3,6-dimethyldecane 1051 1054
15 hexanal 1067 1061
16 2-methyl-2-butenal 1069
17 2-methylpropan-1-ol/isobutyl alcohol/isobutanol 1085 1077
18 3-pentanol 1110 1090 1084 1086
19 delta-3-carene/3,7,7-trimethylbicyclo[4.1.0]hept-3-ene 1148 1122 1127 1104
20 5-methyl-2-hexanone 1129
21 heptanal 1184 1162 1155 1164
22 2-heptanone 1160 1169
23 limonene 1198 1171 1169
24 3-methyl-2-butenal 1174 1173 1174
25 dodecane 1200 1181 1181 1180
26 isoamyl alcohol/3-methylbutan-1-ol 1230 1183 1184 1183
27 3-methyl-3-buten-1-ol 1240 1223 1223 1221
28 3-hydroxy-2-butanone/acetoin/3-hydroxy-2-butanone 1272 1269 1254 1255
29 octanal 1278 1269
30 2-methyl-2-buten-1-ol 1315 1294 1294 1293
31 6-methyl-5-hepten-2-one 1329 1312 1350 1304
32 3-hydroxy-3-methylbutanoic acid 1347 1350 1345
33 cis-3-hexene-1-ol 1351 1360 1362 1358
34 nonanal 1382 1371 1377 1369
35 linalool oxide/6-ethenyl-2,2,6-trimethyloxan-3-ol 1423 1415 1412 1412
36 acetic acid 1432 1434 1432 1430
37 2-furancarboxaldehyde/furfural 1434 1441 1440 1435
38 trans-linalool oxide 1451 1445 1442 1443
39 2-ethyl-1-hexanol 1472 1477 1473
40 decanal 1485 1481 1480 1478
41 2-acetylfuran 1491 1482 1482 1480
42 benzaldehyde 1502 1495 1494 1493
43 lilac aldehyde B 1519 1524 1516
44 propanoic acid/propionic acid 1523 1526 1528 1520
45 linalool L 1544 1532 1527 1528
46 lilac aldehyde A 1545 1544 1543
47 5-methyl-2-furancarboxaldehyde 1563 1553 1551 1551
48 2-methylpropanoic acid 1584 1555 1558 1553
49 2,2-dimethylpropanoic acid/pivalic acid 1585
50 ho-trienol 1586 1594 1592
51 butyrolactone 1600 1603 1596
52 butanoic Acid 1628 1615 1617 1612
53 benzenacetaldehyde/acetaldehyde 1646 1622 1622 1618
54 5-ethenyldihydro-5-methyl-2(3H)-Furanone 1647 1644 1641
55 2-methylbutanoic acid/DL-2-methylbutyric acid 1667 1661 1662 1662
56 4-ketoisophorone/4-oxoisophorone 1672 1669
57 alfa-terpineol/(S)-2-(4-Methyl-3-cyclohexenyl)-2-propanol 1688 1675 1677 1680
58 3-pyridinecarboxaldehyde/nicotinic aldehyde 1676 1682
59 borneol 1698 1684
60 naphtalene 1718 1718
61 epoxylinalool 1728 1732 1722
62 pentanoic acid/valeric acid 1729
63 3-methylpentanoic acid 1780 1783 1784 1786
Foods 2019, 8, 445 16 of 24
Table 7. Cont.
Z1 Z2 Z3
Rank Volatile Compound (Usual and/or IUPAC Name) RIa
RIb RIb RIb
64 beta-damascenone 1790 1804 1801
65 hexanoic acid 1814 1836 1837 1835
66 para-cymen-8-ol/2-(4-methylphenyl)propan-2-ol 1847 1838 1838
67 trans-geranylacetone/(5E)-6,10-dimethylundeca-5,9-dien-2-one 1841 1846 1837
68 benzenemethanol/phenylmethanol 1844 1862 1860 1847
69 benzeneethanol/phenethyl alcohol 1878 1895 1893 1891
70 benzyl nitrile/2-Phenylacetonitrile
71 sabinene/4-methylidene-1-propan-2-ylbicyclo[3.1.0]hexane
72 2,5-furandicarboxaldehyde/furan-2,5-dicarbaldehyde
73 phenol 1932
74 octanoic acid/caprylic acid 2083
75 nonanoic acid/pelargonic acid 2110
76 3-phenyl-2-propenal/cinnamaldehyde
77 2-methoxy-4-vinylphenol
78 benzoic acid
79 HMF/5-hydroxymethil-2-Furfural
The results revealed that there is a remarkable influence of the production Zone on the volatile
profile of the honey samples. Of all the identified compounds, only 56 are common to all the 50
samples. The chemotype changes from one region to another, and the difference resides in the 23
compounds found only in certain regions. The compounds found only in Zone 1 are: 2-butanol,
5-methyl-2-hexanone, 2-heptanone, octanal, 2,2-dymethyl propanoic acid, naphthalene, nonanoic and
octanoic acids. The origin of these compounds can be either vegetal (nectar and poliniferous plants)
or animal, products secreted by the bee. For example, octanol is found in Houttuynia cordata and
in Lavandula angustifolia [56]. The 2-heptanone compound was identified in the secretion of bees by
Shearer [57], but it is also found in Artemisia dracunculus and Larrea tridentate [58,59]. In regard to Zone 2,
borneol and HMF compounds have been identified. Borneol is a monoterpenic compound found in
medicinal plants [60]. Borneol is used as an additive in cosmetic products due to its analgesic and
antibacterial effects [61]. The presence of the HMF compound is a hint towards honey freshness [62].
In Zone 3, pentanoic acid was found. Also, it was found that Zone 1 shows a volatile profile similar
to Zone 3, a relevant example being the existing common compounds, beta-damascenone, hotrienol,
benzyl nitrile, 2,5-furancarboxaldehyde. Out of these compounds, beta-damascenone was found in
Ipomoea pes caprae with an antispastic effect [63]. Additionally, hotrienol was found to be a component
of many honey varieties, and of essential oils of many plants. This compound was found to be specific
to citrus, lavender, and mint honey [64–66].
By using the peak area, it is possible to determine the percentage of each compound, compared to
the total surface of the areas.
The calibration and concentration determination methods for volatile compounds are:
Gauging of the calibration curve method (area normalization);
Internal standard method [67].
For a better interpretation of the results, the first method was used, the concentration of each
compound being calculated according to the following expression:
X
X% = (Ax / Ai ), (3)
P
where: Ax : compound surface, Ai : the sum of all identified compound surfaces.
Table 8 shows the semi-quantification results for each product, as well as product chemical
classification. The volatile compounds identified in the acacia honey samples stemming from the
three geographical areas are classified into 10 chemical subclasses: Sulphur compounds, ketones,
esters, aldehydes, alcohols, nitrate compounds, aliphatic hydrocarbons, carboxylic acids, aromatic
acids and ethers. From the collected data, we can see that the honey samples harvested from Zone 1
Foods 2019, 8, 445 17 of 24
(Transylvania) are the richest in volatile compounds, as all of these compound classes are present
in the samples. Keeping in mind that the number of compounds was so high, only the majorities
were discussed.
Regarding the presence of acetic acid, this compound was found in samples from all three zones,
but in different quantities. Acetic and butyric acids could be produced by bee metabolism [68]. This is
the main compound of the honey samples, and its percentages vary between 12.26% for Zone 3, 16.64%
for Zone 1 and 24.73% for Zone 2. The previous studies show that the percentage of this compound
can vary between 17.32% and 36.15% for different Chilean honey types [19], while having a percentage
of 0.01% in Spanish honey [64]. We must mention that the two studies have used different extraction
methods, ultrasound extraction and thermal desorption. Kadar et al. [21] have found that Romanian
acacia honey has linalool oxide as the main compound, without having identified any acetic acid.
Ethanol is another important constituent of honey, all honey samples have shown a high ethanol
content, especially those from Zone 2 and Zone 3, as their content reached 17.91% and 13.07%,
respectively. In the case of Transylvanian honey (Zone 1), there was a relatively low ethanol content
found, of 4.11%. Bastos and Alves [68] issue the hypothesis that the high ethanol content is due to the
presence of ferments. Ethanol was found to be a marker for lavender honey [65]. Furthermore, ethanol
was found in honeys of different geographical and botanical origins [8,9,22,64].
The volatile profile found depends on the extraction method. Therefore, the fiber used shows a
great affinity for compounds with low molecular mass, like ethanol or acetic acid [69], which justifies
the significant presence of all these compounds in the honey samples, from a quantitative point of view.
Another major compound was 2-propanone, present in a high concentration in Zone 3 (20.60%),
as opposed to its presence in honey stemming from Zone 1 and Zone 2, where its values were 2.83%
and 5.11%, respectively. Transylvanian honeys (Zone 1) have shown the lowest 2-propanone or acetone
content, these differences being the result of the discrepancy between geographical, climatic and
especially floral conditions. In a recent study, acetone concentration in Chilean honey was determined,
according to geographical area. In the eastern part, the Zone percentage was between 1.57–17.15%,
in the central area, 7.8–9.63%, and in the western zone, 5.00–13.13% [19]. Acetone is considered a
marker for pine honey (Albies spp.) [68]. Additionally, acetone was found to be a major compound in
acacia and rosemary monofloral honey [65]. Linalool oxide and 2–furancarboxaldehyde are usually
found in the honey samples collected from Zone 2 (8.61% and 9.73). Linalool oxide is a compound of
floral origin (Pherobase) and is considered to be a secondary product of linalool. Jerkovic et al. [70]
determined a 2.23% concentration of this product in acacia honey, whereas in chestnut honey, the
concentration was of 0.40%.
Furfural is a compound derived from furan, which is considered to be an indicator of the thermal
and storage processes [64]. On the other hand, furfural was identified as being a relevant compound
in unifloral lime tree, lavender and acacia honey [65]. Furfural is present in all regions with an area
percentage of 9.73% for Zone 2, 7.26% for Zone 1 and 5.24% for Zone 3. Radovic et al. [71] established
furfural as being a marker for rape honey. These volatile compounds, furfural and 5-methyl-furfural
were identified in fresh citrus honey. Moreover, the evolution of the concentration of these compounds
was observed and it intensified during storage and temperature increase from 10 to 40 ◦ C [64]. The mild
heating of the samples during the SPME analysis recommended for the improvement of the extraction
result and the reduction of the balance time, may be responsible for some of these compounds [72].
Foods 2019, 8, 445 18 of 24
Table 8. Relative area of volatile organic compounds (VOC) of honey samples from the three investigated
zones (Z1 – Z3).
Zone 1 Zone 2 Zone 3 %COV %COV %COV

Rank Compound Class
(%) (%) (%) Z1 Z2 Z3
Sulphur
1 dimethyl sulfide/methylsulfanylmethane 1.578 2.867 2.171 1.578 2.867 2.171
compounds
2 acetone/2-propanone 2.830 5.119 20.609
8 2,3-butanedione/diacetyl 1.387 1.420 0.765
20 5-methyl-2-hexanone 0.066 0.000 0.000
22 2-heptanone 0.542 0.000 0.000 Ketone 7.463 7.433 23.058
28 3-hydroxy-2-butanone/acetoin 0.708 0.646 0.853

31 6-methyl-5-hepten-2-one 0.188 0.171 0.125
56 4-ketoisophorone/4-oxoisophorone 0.963 0.000 0.510
64 beta-demascenone 0.209 0.000 0.072
67 trans-geranylacetone 0.570 0.077 0.124
3 ethyl acetate 0.000 0.000 2.720 Esters 0.000 0.000 2.720
4 3-methyl butanal/isovaleraldehyde 1.753 0.538 0.002
15 hexanal 0.178 0.000 0.000
17 2-methyl-2-butenal 0.447 0.370 0.000
21 heptanal 0.074 0.020 0.004
24 3-methyl-2-butenal 1.514 0.522 0.165
29 octanal 0.020 0.000 0.000
34 nonanal 1.382 1.183 0.575 Aldehyde 27.639 22.344 13.439
37 2-Furancarboxaldehyde / furfural 7.262 9.737 5.241

41 decanal 0.648 4.464 0.070
42 benzaldehyde 7.237 3.890 4.686
43 lilac aldehyde B 1.898 0.177 0.141
46 lilac aldehyde A 2.430 0.017 0.046
47 5-methyl-2-furancarboxaldehyde 0.321 0.516 0.156
53 benzenacetaldehyde/acetaldehyde 1.088 0.809 1.945
3-pyridinecarboxaldehyde/nicotinic
59 0.706 0.000 0.095
aldehyde
2,5-furandicarboxaldehyde/
72 0.670 0.000 0.292
furan-2,5-dicarbaldehyde
76 3-phenyl-2-propenal/cinnamaldehyde 0.011 0.097 0.021
79 HMF/5-hydroxymethyl-2-Furfural 0.000 0.004 0.000
5 isopropyl alcohol/2-propanol 0.864 0.003 0.265
6 ethanol 4.144 13.077 17.910
11 2-butanol/sec-butanol 0.007 0.000 0.000
2-methyl-3-buten-2-ol/
13 0.442 0.493 0.122
dimethylvinylcarbinol
16 2-methyl-1-propanol/isobutanol 0.000 0.000 0.101
18 3-pentanol 0.007 0.096 0.011
25 isoamyl alcohol 0.405 0.510 1.171 Alcohols 19.130 23.876 28.359
27 3-methyl-3-buten-1-ol 2.090 1.335 0.565

30 2-methyl-2-buten-1-ol 0.716 0.713 0.208
33 cis-3-hexene-1-ol 0.078 0.193 0.145
39 2-ethyl-1-hexanol 0.174 0.028 0.098
45 linalool L 3.101 1.418 1.930
50 ho-trienol 2.636 0.000 1.340
57 alfa-terpineol 0.158 0.344 0.039
58 borneol 0.000 0.005 0.000
61 epoxylinalol 0.532 0.584 0.292
para-cymen-8-ol/2-
66 0.007 0.006 0.003
(4-methylphenyl)propan-2-ol
68 benzenemethanol 0.850 1.279 1.623
69 benzeneethanol/phenethyl alcohol 2.780 3.683 2.378
73 Phenol 0.139 0.109 0.158
Foods 2019, 8, 445 19 of 24
Table 8. Cont.
Zone 1 Zone 2 Zone 3 %COV %COV %COV

Rank Compound Class
(%) (%) (%) Z1 Z2 Z3
7 2,2,4,6,6-pentamethylheptane 0.000 0.000 0.021
alfa-pinene/4,7,7-
10 0.026 0.026 0.012
trimethylbicyclo[3.1.1]hept-3-ene
Aliphatic
12 3-methylpentanal/3-methylvaleraldehyde 0.608 0.155 0.498 0.937 0.263 0.608
hydrocarbons
14 3,6 dimethyldecane 0.156 0.000 0.024
delta-3-carene/3,7,7-
19 0.035 0.006 0.018
trimethylbicyclo[4.1.0]hept-3-ene
23 limonene 0.056 0.073 0.016
26 dodecane 0.003 0.003 0.019
sabinene/4-methylidene-1-propan-
71 0.053 0.000 0.000
2-bicyclo[3.1.0]hexane
Nitrogen
9 2-methylpropanenitrile 0.326 0.000 0.045 0.916 0.000 1.610
compounds
70 benzylnitrile 0.590 0.000 1.565
32 3-hydroxy-3-methylbutanoic acid 0.025 0.074 0.070
36 acetic acid 16.641 24.733 12.269
44 propanoic acid/propionic acid 0.862 0.697 0.687
48 2-methylpropanoic acid 1.335 0.203 1.385
49 2,2-dimethyl propanoic acid/Pivalic acid 0.052 0.000 0.000 Carboxylic
32.225 30.369 21.723
acids
52 butanoic acid 1.646 0.750 0.644
2-methylbutanoic
55 5.094 1.771 1.219
acid/DL-2-methylbutyric Acid
62 pentanoic acid/valeric acid 0.000 0.000 0.108
63 3-methylpentanoic acid 5.336 1.629 4.497
65 hexanoic acid 0.661 0.507 0.265
74 octanoic acid 0.172 0.000 0.000
75 nonanoic acid 0.092 0.000 0.000
78 benzoic acid 0.309 0.005 0.579
linalool
35 6.883 8.618 4.188
oxide/6-ethenyl-2,2,6-trimethyloxan-3-ol
38 trans-linalool oxide 1.564 2.162 1.115 Aromatic
9.698 12.790 6.211
40 2-acetylfuran 0.121 0.435 0.267 acids
51 butyrolactone 0.224 0.174 0.274

54 5-ethenyldihydro-5-methyl-2(3H)-furanone 0.888 1.401 0.367
60 naphtalene 0.018 0.000 0.000
77 2-methoxy-4-vinylphenol 0.085 0.057 0.103 Ethers 0.085 0.057 0.103
We can also observe a higher benzaldehyde content, especially in Zone 1 honey (7.23%), as
compared to the one in Zone 2 (3.89%) and Zone 3 (4.68%). Benzaldehyde was identified as being
a relevant compound in lavender, acacia and rosemary honey [65,72]. Yang et al. [73] identified
benzaldehyde as being a volatile marker in Corsican chestnut honey with a 10.8% concentration.
Furthermore, we can also note the presence of benzeneethanol in significant amounts in the case of the
honey samples from Zone 2 (3.58%), Zone 1 (2.78%) and Zone 3 (2.37%). Benzenethanol and benzoic
acid were identified as rape honey markers [74].
Alcohols constitute an important part of Romanian honey samples reaching values of 28.35%
in Zone 3, 23.87% in Zone 2, 19.13% in Zone 1. The alcohols present in all the zones were ethanol,
2-propanol, 2-methyl-3-butene-2-ol, isoamyl alcohol, 3-methyl-3-butene-1-ol, 2-methyl-2-butene-1-ol,
cis-3-hexen-1-ol, 2-ethyl-1-hexanol, linalool, hotrienol, alfa-terpienol, benzeneethanol, benzenemethanol,
phenol. Among the identified alcohols, ethanol, benzeneethanol, 3-methyl-3-butene-1-ol proved to
have the highest area percentage of all the analyzed samples (Table 8).
Alcohols represent important markers of honey; alcohols, such as 3-methyl-3-butene-1-ol and
2-methyl-2-butene-1-ol were described as adding a fresh aroma, their presence in honey being associated
with different floral origins [64]. Ethanol, 2-methyl-propanol and 3-methyl-3-butene-1-ol were
Foods 2019, 8, 445 20 of 24
described as being very important compounds in monofloral lavender honey [65]. The identification
of 3-methyl-3-butene-1-ol was also associated with monofloral rosemary honey [75].
As far as aldehydes are concerned, it can be observed that the honey samples from
Zone 1 present the highest concentration, 27.63%, followed by the ones from Zone 2 with
a 22.34% concentration. The samples in Zone 3 have a relative aldehyde concentration of
13.43%. The aldehydes identified in the samples from all the three production areas are:
3-methyl-butanal, heptanal, 3-methyl-2-butanal, nonanal, furfural, decanal, benzaldehyde, lilac
aldehyde, 5-methyl-2-furancarboxaldehyde, acetaldehyde and 3-phenylfenil-propenal. Plutowska et
al. [76] have established hexanal as a specific marker for the acacia honey produced in Poland, while
methyl butanol and lilac aldehyde proved to be a specific marker for the Polish honeydew honey. The
following substances from the aldehyde group were also identified in the composition of these honeys:
hexanal, nonanal, decanal, acetaldehyde and lilac aldehydes.
Ketones, sulfur compounds, esters and aliphatic hydrocarbons were also identified in small
amounts, but none of them could be considered markers for acacia honey.
As other studies presented the idea that minor compounds also influence honey flavor, the table
was organized in order to summarize the specific aromas of all the identified volatile compounds.
Thus, it can be observed that the honey aroma is very complex and depends on the specific flora of
each production zone, but also on the compounds secreted by the bees or on the transformations that
occur during storage.
4. Conclusions
In accordance with the motivation and the objectives proposed, the quality indices and the
biomarkers specific to acacia honey were established. However, pure acacia honey represents only a
low percentage of the total honey production, as the majority of honeys have a varied composition of
nectar or honeydew, being thus considered polyfloral honeys. Therefore, distinguishing monofloral
honeys from polyfloral honeys is a significant challenge that involves assessing the botanical origin of
honey and its correct labelling. Volatile compounds can be useful in classifying acacia honeys, but they
are insufficient in order to distinguish between the samples produced in different areas. As such, the
use of multiple volatile compounds is recommended, which will constitute the volatile fingerprint of a
certain area, so that the results are less susceptible to the variation of individual components.
Many consumers seek for products with special aromas, specific to production zones. In conclusion,
consumer affinity for the special aroma of the acacia honey, which have an important nutritional value,
being recommended in small amounts even for diabetes patients [77], is legitimate and this study aims
at scientifically explaining the fact that honey flavor is influenced by its origin.
Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/8/10/445/s1,

Figure S1: CIELAB color space (www.printroot.com); Figure S2: Descriptive statistics for water content of
Romanian acacia honey and box plot graph for the three production zones, Figure S3: GC-MS chromatogram of
VOC in sample S1.
Author Contributions: Conceptualization, N.M.M.; data curation, K.B.N.; formal analysis, N.M.M.; funding
acquisition, D.S.D.; investigation, M.-L.F.; methodology, M.-L.F, V.B.; review, M.-L.F; supervision, L.A.M., E.H.;
validation, K.B.N.; visualization, F.F.; writing—original draft, N.M.M., O.B.; writing—review and editing, O.B.
Funding: The research did not receive external funding.
Acknowledgments: The manuscript is part of NMM PhD thesis. Financial support for the manuscript was
granted by project SAFE-HONEY (PN-III=P2-890/12.09.2017 (to VB and OB) and Program 1 - Development of the
National Research and Development System, Subprogram 1.2 - Institutional Performance - Projects for Financing
the Excellence in CDI, Contract no. 37PFE/06.11.2018. Title of the project: “Increasing the institutional performance
through consolidation and development of research directions within the USAMVCN” (to DSD and BO).
Foods 2019, 8, 445 21 of 24
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antibiotics
Review
Dissecting the Antimicrobial Composition of Honey
Victoria C. Nolan, James Harrison and Jonathan A. G. Cox *
School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK;
180208508@aston.ac.uk (V.C.N.); j.harrison11@aston.ac.uk (J.H.)
* Correspondence: J.a.g.cox@aston.ac.uk; Tel.: +44-121-204-5011

Received: 28 October 2019; Accepted: 3 December 2019; Published: 5 December 2019
Abstract: Honey is a complex sweet food stuff with well-established antimicrobial and antioxidant
properties. It has been used for millennia in a variety of applications, but the most noteworthy
include the treatment of surface wounds, burns and inflammation. A variety of substances in honey
have been suggested as the key component to its antimicrobial potential; polyphenolic compounds,
hydrogen peroxide, methylglyoxal and bee-defensin 1. These components vary greatly across honey
samples due to botanical origin, geographical location and secretions from the bee. The use of medical
grade honey in the treatment of surface wounds and burns has been seen to improve the healing
process, reduce healing time, reduce scarring and prevent microbial contamination. Therefore, if
medical grade honeys were to be included in clinical treatment, it would reduce the demand for
antibiotic usage. In this review, we outline the constituents of honey and how they affect antibiotic
potential in a clinical setting. By identifying the key components, we facilitate the development of an
optimally antimicrobial honey by either synthetic or semisynthetic production methods.
Keywords: honey; antimicrobials; methylglyoxal; hydrogen peroxide; bee-defensin 1;

wound treatment
1. Introduction
Honey has been established as an effective antimicrobial and antioxidant for millennia [1]. Used
mainly for the treatment of surface wounds, burns and inflammation, it has since been developed into
medical treatments in the form of medical grade honey [2,3]. Despite this, the initial interest into honey
as an antimicrobial therapy was drastically diminished upon the discovery and implementation of
antibiotics. However, with the alarming rise in the prevalence of antimicrobial-resistant organisms,
in particular the increase in multi-drug resistance (MDR), the number of effective antibiotic compounds
is shrinking at a greater rate than new drugs are being developed [4,5]. This grave predicament has
many researchers looking back to the pre-antibiotic era for solutions, sparking more recent interest into
the mechanisms of action of honey as an antimicrobial [6]. Throughout history, honey has been used in a
variety of cultures, with differing applications. The ancient Egyptians used honey as a topical ointment,
a wound dressing and for embalming their dead, whereas the ancient Greeks used it as a remedy for
gout, pain, fever and also wound healing [7]. The first observations of the antimicrobial activity of
honey were made in 1892, and since then honey has been observed to have a broad spectrum of activity,
inhibiting both Gram positive and Gram negative organisms, including: Escherichia coli, Pseudomonas
aeruginosa, Klebsiella pneuomniae, Staphylococcus aureus, Bacillus subtilis and Listeria monocytogens and
their multidrug-resistant counterparts (Table 1) [8,9]. The efficacy of honey against these organisms is
dependent on the honey used, due to variations in botanical origin, bee health, geographical location
and the processing of honey [1,10,11]. Manuka honey, from the Australian Leptospermum sp., has
been identified to inhibit the Gram positive organism Enterococcus faecalis, whereas the Gram negative
E. coli was more resistant to honey treatment [12]. Observations of Manuka and Chinese Buckwheat
Antibiotics 2019, 8, 251; doi:10.3390/antibiotics8040251 www.mdpi.com/journal/antibiotics

Antibiotics 2019, 8, 251 2 of 16
(Fagopyrum esculentum) honey identified a minimum inhibitory concentration (MIC) of 5% (w/v) against
S. aureus and 60% (w/v) against P. aeruginosa [13]. Similar results of linen vine honey showed S. aureus
was more susceptible than P. aeruginosa [14]. Another study observing the effectiveness of honey
from a variety of botanical origins identified greater susceptibility overall towards the Gram positive
organisms, S. aureus and Staphylococcus epidermidis, and either no effect or reduced susceptibility to
the Gram negative organisms, E. coli and P. aeruginosa [15]. Further to this, one study observing the
antimicrobial activity of Polish honey against S. aureus found an MIC of only 1.56% (v/v) of honey was
required [16]. However, other studies have identified that Gram positive bacteria are more resistant to
honey [17–19]. One study identified that Gram negative organisms were more susceptible to honey
than Gram positives, suggesting this could be due to the higher hydrogen peroxide content and
osmolality of the samples [20]. In regards to Rubus honey, from Southwest Spain, Proteus mirabilis
was the most susceptible organism tested, exhibiting an MIC range of 7.8 to 31.3 mg/mL, yet S. aureus
had an MIC range of up to 125 mg/mL [17]. Further to this, honeys of monofloral origin (algarrobo
and citrus) and mulitfloral origin exhibited greater efficacy against the Gram negative organisms than
the Gram positive organisms, with P. aeruginosa having an MIC of 100 mg/mL, whereas S. aureus MIC
ranged from to 250 mg/mL and E. feacalis ranging from 200 to 250 mg/mL with some honey samples
having no effect on either Gram positive organism tested [18]. Moreover, a study observing the effect of
Egyptian honey identified the only effective honey against S. aureus was Sidr honey at an MIC of 100%
and only four out of six honey samples were effective against Streptococcus mutans. All honey samples
tested were effective against P. mirabilis and K. pneumoniae with MIC values of 50% or less. Only one
honey was not effective against E. coli and three out of six were not effective against P. aeruginosa, but
the MIC values for those that were inhibitory were 50% or less [21]. Furthermore, it has been identified
that Acinetobactor calcoaceticus was the most affected organism, compared to E. coli, P. aeruginosa and
S, aureus, when treated with a range of Scottish honey samples [19]. This variety of results suggests
that not all honeys are equal and their effectiveness is largely variable, outlining the significance of
botanical origin and geographical location on the antimicrobial activity exhibited by a specific honey.
Table 1. Antimicrobial effect of honey from different geographical locations.
Geographical Variation in Honeys Antimicrobial Activity

Country of Origin Honey Sample Organisms
Australia
New Zealand [13] Manuka Staphylococcus aureus, Pseudomonas aeruginosa
S. aureus, MRSA, MSSA, coagulase-negative
New Zealand [22] Manuka Staphylococcus epidermidis, Klebsiella pneumonia, ESBL
E. coli
Australia [23] Leptospermum based honey S. aureus
North America
Canada [24] Canadian Honey E. coli, Bacillus subtilis
Christmas vine, Morning glory,
Cuba [14] Black mangrove, Linen vine, S. aureus, P. aeruginosa, E. coli and B. subtilis
Singing bean
South America
Chile [11] Ulmo Honey MRSA, E. coli and P. aeruginosa
Algarrobo, citrus and multifloral S. aureus, Enterococcus faecalis, E. coli, Morganella
Argentina [18]
honey morganii and P. aeruginosa
Europe
Blossom, heather, Highland, Acinetobactor calcoaceticus, S. aureus, P. aeruginosa and
Scotland [19]
Portobello Orchard E. coli
S. aureus, S. epidermidis, Micrococcus luteus, E. faecalis,
Northwest Spain [17] Rubus Honey B. cereus, Proteus mirabilis, E. coli, P. aeruginosa and
Salmonella. typhimurium
Heather, Rasberry, Rapeseed,
Denmark [15] S. aureus, P. aeruginosa and E. coli
Hawthorn and White Clover
Slovakia [25] Honeydew Honey P. aeruginosa and S. aureus
Asia
China [13] Buckwheat Honey S. aureus and P. aeruginosa
S. aureus, Streptococcus pyogenes, Corynebacteria
Saudi Arabia [26] Sider Honey pseudotuberculosis, K. pneumonia, P. aeruginosa and
E. coli
Africa
Astragalus, Wall-rocket,
Algeria [9] Eucalyptus, Legume, Peach, Clostridium perfringens, S. aureus, E. coli and B. subtilis.
Juniper, Buckthorn and multifloral
S. typhimurium, Shigella dysenteriae, E. coli, B. cereus
Nigeria [27] Wildflower and Bitter leaf Honey
and S. aureus
Cotton, Blackseed, Orange,
E. coli, S. aureus, Streptococcus mutans, P. mirabilis,
Egypt [21] Eucalyptus, Sidr and Clover
P. aeruginosa and K. pnuemoniae
Honey
S. aureus, S. pyogenes, Corynebacteria
Acacia, Citrus, Clover, Coriander,
Egypt [26] pseudotuberculosis, K. pneumonia, P. aeruginosa and
Cotton and Palm Honey
E. coli
Interestingly, it has been observed that no organism has gained resistance to honey [28].
Furthermore, sub inhibitory doses of honey have been shown to restore oxacillin susceptibility
in methicillin-resistant Staphylococcus aureus (MRSA) [29]. Initial studies into honey have outlined
some key factors contributing to its antimicrobial effects, these were high sugar content, low pH,
hydrogen peroxide, polyphenolic compounds and the identification of an inhibine (Figure 1) [2,8,30].
Further studies exploring why honey is a powerful antimicrobial identified that inhibine was a
1,2-dicarbonyl compound in the form of methylglyoxal, a potent antimicrobial, found mainly in
Manuka honey [31]. More recent studies have also identified a bee-derived protein, bee defensin-1, as
a potential antimicrobial component within honey (Figure 1) [25]. This furthers the argument that
honey samples contain various antimicrobial compounds and their activity cannot be attributed to a
single antimicrobial agent. Moreover, honey contains multiple components that act synergistically,
studies exploring why honey is a powerful antimicrobial identified that inhibine was a 1,2-dicarbonyl
compound in the form of methylglyoxal, a potent antimicrobial, found mainly in Manuka honey [31].
More recent studies have also identified a bee-derived protein, bee defensin-1, as a potential
antimicrobial component within honey (Figure 1) [25]. This furthers the argument that honey samples
contain
Antibiotics various
2019, 8, 251antimicrobial compounds and their activity cannot be attributed to a single 4 of 16
antimicrobial agent. Moreover, honey contains multiple components that act synergistically,
enhancing its potency as an antimicrobial. This review aims to explore the different components that
enhancing
are attributed its to
potency asantimicrobial
honey’s an antimicrobial. Thisand
activity review aims to explore
its potential the different components that
applications.
are attributed to honey’s antimicrobial activity and its potential applications.
Figure 1. The main constituents attributed to honey’s antimicrobial activity and their mechanism of
action. Direct inhibitory factors affect cellular mechanisms (blue), indirect inhibitory factors have a
wider ranging effect on the bacterial cell (green).
2. Composition and Classification

Honey is a complex food substance, comprised of 180 to 200 different substances, including sugar,
water, proteins, vitamins, minerals, polyphenolic compounds and plant derivatives [10,25]. Depending
on origin, honey can be classified as honeydew or blossom. Honeydew honey is produced by the
collection of living plant, aphid and insect secretions [32], whereas blossom honey is produced by the
collection of flower nectar and characterised by pollen content. Blossom honey can be further divided
into unifloral, where the botanical origin is predominantly from one flower species, or multifloral,
where multiple sources of flower species can be identified [33]. The botanical origin of honey can have
the biggest influence on its antioxidant activity [34]. One honey that has been of great significance, due
to its broad spectrum of antimicrobial activity, is Manuka honey, derived from Leptospermum sp. [1].
This unifloral honey is used within the pharmaceutical industry and has been developed into medical
grade honey. The antimicrobial activity of Manuka honey has been attributed to phytochemicals
produced by the Leptospermum sp. plant and subsequently transferred to the honey. Recently however,
honeydew honey has been investigated as a more potent antimicrobial than unifloral honey, furthering
the importance of honey origin [35]. Furthermore, the composition of active compounds present within
plant nectar can vary, depending on geographical location and climate conditions [34]. All of these
different components can influence the quality of the honey and, subsequently, the antimicrobial activity.
3. Carbohydrates
Carbohydrates, predominantly monosaccharides such as glucose and fructose, constitute up to
82.4% of the chemical make-up of all varieties of honey [36]. The next largest component of honey is
water, ranging from 13–23% [37]. These two factors impose a stressful environment for microorganisms,
as a result of low pH and high osmotic pressure, preventing food spoilage due to unsuitable growth
conditions (Figure 1) [2,37]. It is considered that this unfavourable environment largely contributes to
the antimicrobial activity of honey. Wahdan (1998) demonstrated that an undiluted sugar solution,
mimicking the same sugar and water percentage of honey, exhibited bacteriostatic and bactericidal
activity, indicating that these parameters play an important role in the antimicrobial activity of
honey [38]. Conversely, Brady, Molan and Harfoot (1996) created an artificial honey, representative of
sugar content and acidity, and tested it against a range of dermatophytes, a pathogenic fungus that
is the cause of cutaneous mycoses [39]. They observed no inhibitory activity against any organism
tested. However, they did observe activity for Manuka honey, suggesting that high sugar levels and
low acidity are not the sole source of antimicrobial activity. Further to this, Wahdan (1998) found
significant differences between the activity of the sugar solution and honey, indicating there are other
components
Antibiotics 2019, 8, x within honey that attribute to its antimicrobial activity [38]. In 1937, the antimicrobial 6 of 15
activity of honey was linked to the presence of an ‘inhibine’, a previously unidentified component
to bindof to
honey, the discovery
glucose of which
more readily, supported
resulting in the theory that sugar
a continuous content of
production alone was not peroxide
hydrogen responsible[24]. It
forbeen
has also the antimicrobial
suggested thatactivity exhibitedcrowding
molecular by honey [38].
couldStudies
play aexploring the mechanisms
role in hydrogen behind
peroxide the
production,
antimicrobial activity of honey identified a variety of other possibilities, including
provided the concentration of glucose was high enough [60]. The levels of hydrogen peroxide the presence of in
polyphenols, hydrogen peroxide 1,2-dicarbonyls and bee defensin-1 (Figure 1).
honey vary between samples and are dependent on two factors: the amount of glucose oxidase added
and the presence of Compounds:
4. Polyphenolic pollen-derived catalase [61]. Since glucose oxidase catalyses the reaction, it is
assumed that higher levels of glucose oxidase result in more hydrogen peroxide production. This can
Polyphenolic compounds are a diverse group of chemicals that include flavonoids and phenolic
be influenced by honey beedefined
acids (non-flavonoids), healthbyand
thediversity
presence ofofphenolic
foraged diet [62].
structures Conversely,
[40]. Produced asmoreplantrecent research
secondary
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metabolites,that the
these levels compounds
bioactive of glucoseareoxidase present
transferred areplant
from the not to
directly
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(Figureto2),the
andvolume
have of
hydrogen peroxideasproduced,
been identified althoughof these
a major component non-enzymatic
the health-promoting methods
properties of of production
honey are yet to be
[41]. Furthermore,
elucidated [25]. Additionally,
the phenolic acids identifiedcatalase
in honey is known
have for the
been used breakdown
to identify of hydrogen
the botanical peroxideorigin
and geographical into water
of a given honey sample [17]. Therefore, the botanical origin of honey is significant
and oxygen, therefore it is of no surprise that catalase concentration is proportional to hydrogen because it can
influence the phytochemicals
peroxide content [61]. present, and consequently impact the antimicrobial capacity [11,32].
FigureFigure
2. Acquisition
2. Acquisitionofof antimicrobial compounds
antimicrobial compounds withinwithin
honey. honey. (A) Polyphenolic
(A) Polyphenolic compounds
compounds derived
from
derived the plant
from are transferred
the plant by the bee.
are transferred by (B)
theSucrose from
bee. (B) the flower
Sucrose from is ingested by the
the flower bee and broken
is ingested by the bee
down into glucose and fructose upon addition of diastase and invertase by the
and broken down into glucose and fructose upon addition of diastase and invertase by the bee. bee. The glucose is The
oxidised by glucose oxidase upon the addition of oxygen, producing D-glucono-δ-lactone and hydrogen
glucose is oxidised by glucose oxidase upon the addition of oxygen, producing D-glucono-δ-lactone
peroxide. The hydrogen peroxide has antimicrobial activity. (C) Bee defensin-1 is added to honey
and hydrogen peroxide. The hydrogen peroxide has antimicrobial activity. (C) Bee defensin-1 is
by the bee (Swissmodel 6mry.5.A). (D) Dihydroxyacetone is harvested from Leptospermum sp. and
addedconverted
to honey by the bee (Swissmodel
non-enzymatically 6mry.5.A).
to methylglyoxal (D) Dihydroxyacetone
through dehydration reaction. is harvested from
Leptospermum sp. and converted non-enzymatically to methylglyoxal through dehydration reaction.
Polyphenolic compounds have been identified in honey, a variety of which have been identified
Hydrogenantimicrobial
as having peroxide is activity and the mechanisms
a well-established of action
antimicrobial have largely
agent. Classed been elucidated
as an (Table
oxidative 2,
biocide, it
Figure 1). The concentrations at which these polyphenolic compounds are active
removes electrons from chemical structures, resulting in oxidation [63]. The oxidation action causes are much lower
within honey, however a similar occurrence has been observed with regards to hydrogen peroxide.
inhibition of microbial growth and irreversible DNA damage through the generation of hydroxyl
radicals [3,6465]. The generation of hydroxyl radicals in honey is produced in a Fenton-like reaction
through hydrogen peroxide. It is noteworthy that, upon the addition of Fe2+ or Cu2+ ions, an improved
bacteriostatic effect against MRSA and VRE (Vancomycin-resistance Enterococci) was observed due
to the increased decomposition of hydrogen peroxide to hydroxyl radicals, whereas the removal of
Furthermore, polyphenols are typically responsible for destroying free radicals and inhibiting oxidation,
and have been suggested to be involved in the generation of hydrogen peroxide [25,42–44]. The testing
of honey phenolic extracts against S. aureus, E. coli and K. pneumoniae identified an antimicrobial
affect [43]. Further investigations into the role of polyphenolic compounds and their direct antimicrobial
impact on honey are required.
Table 2. Common polyphenolic compounds found within honey and their antimicrobial mechanism
of action.
Phenolic Acids Mechanism Flavonoids Mechanism

2-cis,A-trans Abscisic
Unknown Apigenin Inhibits DNA gyrase [44]
acid
2-Hydroxycinnamic
Unknown Catechin Hydrogen peroxide generation [45]
acid
Caffeic acid Oxidative Stress [46] Chrysin Inhibits DNA gyrase [47]
Increase in membrane
permeability resulting in Inhibition of peptidoglycan and
Chlorogenic acid Galangin
cytoplasmic and ribosome synthesis [49]
nucleotide leakage [48]
Disruption to topoisomerase-II DNA
Cinnamic acid Unknown Genistein
cleavage complex [50]
Ellagic acid Unknown Isorhamnetin Unknown
Cell membrane
Ferulic acid dysfunction and changes Kaempferol Inhibits DNA gyrase [47]
in cell morphology [51]
Cell membrane
Inhibits FAS-I in Mycobacteria and
disruption resulting in
Gallic acid Luteolin inhibits DNA helicase DnaB and
pore formation and
RecBCD [47]
intracellular leakage [52]
Cell membrane
p-Coumaric acid disruption and binding Myricetin Inhibits DNA B helicase [54]
to bacterial DNA [53]
p-Hydroxybenzoic acid Unknown Naringenin Unknown
Protocatechuic acid Unknown Pinobanksin Unknown
Sinapic acid Unknown Pinocembrin Induces cell lysis [47]
Cell membrane Disrupts membranes, transport and
Syringic acid Quercetin
dysfunction [55] motility [56]
Induces topoisomerase IV mediated
Vannilic acid Unknown Rutin
DNA cleavage [57]
5. Hydrogen Peroxide
The presence of hydrogen peroxide within honey has been well established and is considered one
of the main antimicrobial constituents in honey. It is produced as a by-product during nectar harvest
by the honey bee (Apis mellifera). Upon harvest, bee-derived enzymes are added, including diastase,
invertase and glucose oxidase. The diastase and invertase break down the larger disaccharides, mainly
sucrose, into monosaccharides, glucose and fructose [58]. Upon the addition of oxygen, glucose oxidase
catalyses the oxidation of glucose to D-glucono-δ-lactone and hydrogen peroxide, the latter of which
has antimicrobial activity (Figure 2) [59]. Interestingly, the antimicrobial effect of hydrogen peroxide in
honey increases upon dilution, enabling the glucose oxidase enzyme to bind to glucose more readily,
resulting in a continuous production of hydrogen peroxide [24]. It has also been suggested that
molecular crowding could play a role in hydrogen peroxide production, provided the concentration of
glucose was high enough [60]. The levels of hydrogen peroxide in honey vary between samples and
are dependent on two factors: the amount of glucose oxidase added and the presence of pollen-derived
catalase [61]. Since glucose oxidase catalyses the reaction, it is assumed that higher levels of glucose
oxidase result in more hydrogen peroxide production. This can be influenced by honey bee health and
diversity of foraged diet [62]. Conversely, more recent research has suggested that the levels of glucose
oxidase present are not directly related to the volume of hydrogen peroxide produced, although these
non-enzymatic methods of production are yet to be elucidated [25]. Additionally, catalase is known for
the breakdown of hydrogen peroxide into water and oxygen, therefore it is of no surprise that catalase
concentration is proportional to hydrogen peroxide content [61].
Hydrogen peroxide is a well-established antimicrobial agent. Classed as an oxidative biocide, it
removes electrons from chemical structures, resulting in oxidation [63]. The oxidation action causes
inhibition of microbial growth and irreversible DNA damage through the generation of hydroxyl
radicals [3,64,65]. The generation of hydroxyl radicals in honey is produced in a Fenton-like reaction
through hydrogen peroxide. It is noteworthy that, upon the addition of Fe2+ or Cu2+ ions, an improved
bacteriostatic effect against MRSA and VRE (Vancomycin-resistance Enterococci) was observed due
to the increased decomposition of hydrogen peroxide to hydroxyl radicals, whereas the removal of
hydrogen peroxide with catalase restored bacterial growth, outlining the relationship between hydroxyl
radical generation and hydrogen peroxide production [66].
Hydrogen peroxide levels within honey can range between 0.5 and 2.5 mM, however, a minimum
level of 2.7 mM hydrogen peroxide is required to cause DNA degradation in E. coli [60]. Regardless
of this, honey containing less than 2.5 mM hydrogen peroxide can exhibit the ability to induce DNA
degradation in bacteria, suggesting that hydrogen peroxide is not the only antimicrobial component of
honey. A relationship between hydrogen peroxide, polyphenols and DNA degradation induced by
honey has been outlined, suggesting higher levels of polyphenols in the presence of hydrogen peroxide
improved the oxidative stress imposed on bacterial cells [67]. However, Manuka honey maintains
DNA degradation after removal of hydrogen peroxide and exhibits no change in antimicrobial activity,
indicating that hydrogen peroxide is not the only antimicrobial component within honey [31,59].
6. 1,2-dicarbonyls
Antimicrobial activity observed in honey that contains reduced hydrogen peroxide, or after the
removal of hydrogen peroxide, has been defined as non-peroxide activity. Non-peroxide activity has
been attributed to a variety of different substances, one of which is a group of compounds known as
1,2-dicarbonyls. The 1,2-dicarbonyls are highly reactive compounds, generated in carbohydrate-rich
foods through caramelization or Maillard reactions [68]. These are achieved through heat treatment or
prolonged storage and are associated with aroma, colour and taste [69]. 1,2-dicarbonyls are formed as an
intermediate of a non-enzymatic reaction with glucose and free amino groups, resulting in the formation
of advanced glycation end products (AGEs) [70]. Those formed by hexoses include 3-deoxyglucosone
(3-DG) and glucoson; formation by disaccharides and oligosaccharides results in 3-deoxypentosone
(3-DP) [68]. Breakdown products of 3-DG result in the generation of 5-hydroxymethalfurfural, indicating
honey freshness [9]. Other breakdown products of antimicrobial significance are methylglyoxal
and glyoxal.
Methylglyoxal (MGO) has been identified as the main antimicrobial component of Manuka
honey [71]. The MGO content of Manuka honey has been directly correlated to the ‘Unique Manuka
Factor’ (UMF) rating, indicating this is the main antimicrobial component of Manuka honey [72].
The presence of MGO in Manuka honey is determined by the concentration of dihydroxyacetone.
Adams, Manley-Harris and Molan (2009) identified that all nectar collected from Leptospermum sp.
contains varying levels of dihydroxyacetone and no measurable MGO [73]. To further investigate
this, they added dihydroxyacetone to clover honey and observed production of MGO. Furthermore,
the addition of arginine and lysine resulted in greater production of MGO, consistent with findings
that the non-enzymatic production of MGO requires these amino acids [74]. Within the hive, low
amounts of MGO can be detected, but high levels of dihydroxyacetone are present. Once harvested,
the conversion of dihydroxyacetone into MGO takes place, resulting in increased MGO levels and a
reduction in dihydroxyacetone [75]. Interestingly, heating of the honey to 37 ◦ C results in increased
MGO, however, heating to 50 ◦ C causes a loss of both MGO and dihydroxyacetone [73].
The conversion of dihydroxyacetone into MGO is considered to happen non-enzymatically in

honey (Figure 2). However, in the methylglyoxal pathway, dihydroxyacetone-phosphate is converted
to MGO by methylglyoxal synthase [73]. Further research into the production of MGO within honey
could elucidate the exact mechanisms behind its production in honey.
The mechanism of action of MGO is due to its ability to alter the structure of bacterial fimbriae
and flagella (Figure 1). Observations were made that increased concentrations of MGO result in less
fimbriae and flagella, and a concentration of 2 mM results in the loss of all fimbriae and flagella, as well
as inducing damage to cell membranes and the shrinking and rounding of bacterial cells [76]. However,
bacteria without fimbriae and flagella have also been observed to be inhibited by Manuka honey, such
as S. aureus. In Manuka honey, a variety of polyphenolic compounds have been identified, including
apigenin, quercetin and caffeic acid (Table 2), which inhibit bacteria through different mechanisms [13].
This further supports that honey possesses multiple antibacterial properties and does not act through a
single mechanism. Additionally, these multiple components could be the reason no bacteria have been
observed to gain resistance to honey.
7. Bee defensin-1
Bee defensin-1 is an antimicrobial peptide (AMP) identified in bee hemolymph (the bee blood
system) and hypopharyngeal glands [6]. It is one of four AMPs, others include apidaecin, abaecin,
hymenoptaecin and defensin [77]. Their role within the bee is as an innate immune response, exhibiting
activity against fungi, yeast, protozoa and both Gram positive and Gram negative bacteria [78].
Importantly, bee defensin-1 is mainly effective against Gram positive bacteria, most notably B. subtilis,
S. aureus and Paenibacillus larvae, however, it has limited effectiveness against multidrug-resistant
organisms [79]. Levels of bee defensin-1 vary between honey samples, this is a result of its production
from glands of individual bees, whose production of AMP varies [80]. Although the full mechanism
of action for bee defensin-1 has not been elucidated, defensin proteins from other species have been
shown to create a pore within the bacterial cell membrane, resulting in cell death [81]. Furthermore,
bee defensin-1 has been shown to be important in the role of wound healing, through stimulation of
MMP-9 secretions from keratinocytes [82].
8. Antibiotic Residue
A variety of antibiotic residues have been identified in honey, including sulphonamides, macrolides,
tetracyclines and aminoglycosides [83]. The occurrence of this is attributed to the use of antibiotics
in apiculture, environmental contamination and improper beekeeping [84]. Within the EU, no trace
elements of antibiotics are permitted, however there is no determined maximum residue level and
traces can be found in honey samples worldwide [83]. Further to this, it is illegal to use antibiotics
in beekeeping in some EU countries. Al-Waili et al., (2012) has suggested that antibiotics in honey
could potentially increase the instance of antibiotic resistance, however there is little evidence of
this [84]. Increases in antimicrobial resistance are often attributed to the misuse and improper use
of antibiotics, as well as their wide applications within the veterinary industry, extending to the
meat and dairy industry [85]. One study identified that antibiotic residues in milk were higher than
the minimum residue level, and, overall, commercial farms had higher levels of antibiotic residue
than local farms [86]. Another study, focused on determining levels of ciprofloxacin, streptomycin,
sulphonamide and tetracycline within meat, found levels of ciprofloxacin and streptomycin to be above
the MRL (maximum residue level), with the overall traces of all antibiotics ranging between 20.7 and
952.2 µg/kg [87]. However, traces observed within honey are drastically lower than these values and
observations have shown these trace amounts diminish over time. One hive treated with lincomycin
identified 24 µg of the antibiotic in honey three days after treatment, however traces lowered to 3.5 µg
129 days after treatment [88]. Therefore, the occurrence of antibiotic residue within honey should not
be a cause for concern at present. Furthermore, these amounts are sufficiently low that they could not
be attributed to the antimicrobial activity observed in honey.
9. Antibiofilm Properties
Biofilms are formed by bacteria upon adhesion to a surface, resulting in the production of an
extracellular matrix [89]. This matrix allows for protection of the bacterial community, preventing
penetration of antimicrobials and avoiding host defences [90]. Honey has been observed to effectively
inhibit and kill a range of planktonic bacteria, but, more interestingly, honey has the ability to disrupt
biofilms. The antibiofilm properties of honey have been attributed to its ability to disrupt quorum
sensing and penetrate the biofilm itself [91]. Honey has been shown to effectively kill single-species
biofilms, including those of P. aeruginosa, S. aureus at a one in two dilution, and Streptococcus pyogenes
at 30% Manuka honey (w/v) [92,93]. However, multispecies biofilms are more common, especially in
regard to honeys used within a clinical setting, indicating a more important area of antibiofilm research.
One study explored the effects of Manuka, honeydew and artificial honey at a concentration of 100%
on multispecies biofilms formed of Streptococcus agalactiae, S. aureus, P aeruginosa and E. faecalis. They
identified that Manuka and honeydew honey had antibiofilm efficacy against P. aeruginosa, S. aureus
and S. agalactiae, but no effect was observed from artificial honey against S. aureus and S. agalactiae.
Moreover, all honey varieties were able to successfully inhibit P. aeruginosa, including the artificial
honey, whereas no sample was able to inhibit E. faecalis [94]. This shows promise for the use of
honey against multispecies biofilms, especially within wounds. However, the concentration of honey
administered needs to be considered, as it has been demonstrated that sub-inhibitory concentrations of
honey can improve biofilm formation in S. aureus, rather than inhibit it [16,95]. Therefore, more research
is required to define the appropriate concentrations of honey to be administered for this purpose.
The ability of honey to disrupt biofilms has been attributed to two main components: bee defensin-1
and MGO [94,96]. The production of biofilms is achieved through external signals, followed by the
activation of specific genes [97]. Therefore, the ability of bee defensin-1 to disrupt membranes, resulting
in the inhibition of DNA, RNA and protein synthesis, identifies it as an obvious candidate for biofilm
disruption [81]. Furthermore, the capability of MGO to alter bacterial fimbriae and flagella, ultimately
preventing adhesion to surfaces, would impair biofilm formation [76]. Thus, it is unsurprising that the
presence of either bee defensin-1 or MGO results in antibiofilm action. In addition, this suggests that
Manuka honey not only has potent antimicrobial activity, but antibiofilm activity as well.
10. Honey and Antibiotic Synergy

Observing the broad spectrum of activity exhibited by honey, especially against drug-resistant
organisms, has led to investigations of honey–antibiotic synergy. A variety of antibiotics and honey
combinations have now been explored, with some promising results. The pairing of Manuka honey
with tetracycline exhibited an increased antimicrobial affect against P. aeruginosa and S. aureus. The
broad spectrum activity of tetracycline, and the enhancement of its activity upon the addition of Manuka
honey, makes the combination a strong candidate for wound healing [98]. Another combination,
in which sub-inhibitory concentrations of Medihoney were used alongside rifampicin, detected no
rifampicin resistance of S. aureus, including MRSA and clinical isolates [99]. This is not the first
instance of honey reversing resistance to antibiotics. Jenkins and Cooper (2012) identified that
sub-inhibitory concentrations of honey, with the addition of oxacillin, restored the susceptibility of
MRSA to oxacillin [29]. These findings provide a strong basis for the use of honey in clinical settings,
especially for persistent or chronic infections. Additionally, combinations of honey and antibiotics have
been shown to have synergistic and additive actions against biofilms. This was demonstrated by the
combination of vancomycin with Manuka honey against S. aureus, and gentamicin with Manuka honey
against P. aeruginosa [100]. Furthermore, one study has observed the synergistic effects of Portuguese
honey and phage therapy, identifying that 25% (w/v) honey paired with phage was equally as effective
in E. coli biofilm destruction as 50% (w/v) honey alone [101]. This highlights the exciting potential
and possibilities of the use of honey, and the need for further research into its synergistic effects and
clinical applications.
Antibiotics 2019, 8, 251 10 of 16
11. Honey in Medical Settings

The main applications of honey within a medical setting are for the treatment of surface wounds
and burns. Two distinct types of honey have been developed into medical grade honey, Medihoney
and Revamil. Medihoney is developed from Manuka honey, whereas Revamil honey is produced in
greenhouses under standardised conditions [80]. Interestingly, the active components of these two
honeys differ. The Medihoney activity is based on MGO activity, where hydrogen peroxide activity
is variable, with no noted activity of bee defensin-1 [102]. More recently, it has been suggested the
defensin-1 in Manuka honey is altered by the presence of MGO, which could have prevented detection
of the protein in previous studies [74]. However, Revamil acts primarily through hydrogen peroxide
and bee defensin-1 activity [103].
The honey can be applied directly to the surface of a wound. This provides a physical barrier
between the wound and the environment, preventing contamination [104]. The secondary effects
provided by application are the antimicrobial properties, including both bacteriostatic and bactericidal
activity, further preventing wound contamination [80]. Additionally, an osmotic gradient is generated
due to the high sugar content and low water activity, generating a flow of bacteria, necrotic tissue and
debris out of the wound [91]. Finally, the phenolic content in honey aids in inflammation, helping
to improve wound healing [105]. Overall, this has been observed to improve both the healing of the
wound, and the time taken to heal and reduce scarring [106]. This can reduce the use of antibiotics,
while still aiding wound treatment.
A case study involving two patients deployed the use of honey to aid in wound healing and
clearing of infection. The first patient had a persistent self-inflicted wound that showed no sign of
healing; upon daily treatment with Manuka honey the wound showed signs of re-epithelialisation, and,
after six weeks, it had fully healed, demonstrating the ability of honey to promote angiogenesis. The
second patient had incurred two large haematomas which became infected with P. aeruginosa and S.
aureus, later confirmed to be MRSA. After failure to heal, Manuka honey was used to clear the infection
and promote healing. After eight weeks, the infection had been cleared [107]. Another study which
explored the use of honey to aid the healing of skin grafts identified increased healing and reduced
pain in comparison to the vaseline control [108]. Additionally, honey can be used to heal burns. In a
study observing the effects of honey and 1% silver sulfadiazine, they found that honey reduced the
healing time and cleared the burn of infection and pain quicker than the 1% silver sulfadiazine [109].
These case studies outline the different uses of honey within a clinical setting, outlining that honey
should be implemented in a variety of wound healing applications, not only to prevent infection, but
also to reduce healing times and patient discomfort.
12. Conclusions
Honey is a potent antimicrobial agent, exhibiting a broad spectrum of activity. A variety of
components contribute to the antimicrobial potential of honey, including sugar content, polyphenolic
compounds, hydrogen peroxide, 1,2-dicarbonyl compounds and bee defensin-1. All of these are
present in varying levels, depending on nectar source, honey bee and storage. These components act
synergistically, allowing honey to be effective against a variety of microorganisms. The variation in the
quantity and structural modifications of components is also a major contributing factor as to why some
honeys can be more effective at inhibiting bacterial growth than others, furthering the requirement for
continued research.
Within a medical setting, honey can be used as an effective wound treatment, removing the need
for antibiotics. Honey has the potential to vastly reduce the requirement of drugs of last resort for
highly drug-resistant bacterial infections, since current resistance to antimicrobial mechanisms of honey
is largely unseen. This is likely due to the multiple mechanisms of antibacterial action from the plethora
of antimicrobial compounds, resulting in a unique combination therapy, which has yet to be identified
as a source of antimicrobial resistance. As the authors of this review, we feel that the use of honey will
likely be expanded on in the future. This is largely due to the rise in MDR organisms causing infections
Antibiotics 2019, 8, 251 11 of 16
that are extensively untreatable by multiple classes of antibiotic, particularly since honey has been
shown to be capable of reversing certain mechanisms of antibiotic resistance. Therefore, the revival
of this alternative antimicrobial agent represents a promising therapeutic avenue to help curb the
increasing incidence of antibiotic-resistant bacterial infections. Furthermore, the complete elucidation
of the mechanisms of activity and synthesis of all components of honey could lead to the generation of
an optimally antimicrobial synthetic or semisynthetic honey. Discovery of the precise concentrations
of these synergistic components would enable us to develop the most effective, broad-spectrum honey
with activity against a wide range of antibiotic-resistant bacterial species.
Author Contributions: V.C.N., J.H. and J.A.G.C. reviewed the literature and wrote the manuscript.
Funding: Jonathan A. G. Cox is grateful to the Academy of Medical Sciences and Global Challenges Research Fund
for supporting the Mycobacterial Research Group at Aston University with a Springboard Grant (SBF003\1088:).
VCN is supported with a PhD Studentship jointly funded by Give A Child Health Fund and Aston University.
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Molecules 2012, 17, 8359-8377; doi:10.3390/molecules17078359
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Organic Bee Pollen: Botanical Origin, Nutritional Value,

Bioactive Compounds, Antioxidant Activity and
Microbiological Quality
Xesús Feás 1,*, M. Pilar Vázquez-Tato 1, Leticia Estevinho 2, Julio A. Seijas 1
and Antonio Iglesias 3
1
Department of Organic Chemistry, Faculty of Science, University of Santiago de Compostela,
E-27080, Lugo, Spain; E-Mails: pilar.vazquez.tato@usc.es (M.P.V.-T.); julioa.seijas@usc.es (J.A.S.)
2
CIMO-Mountain Research Center, Agricultural College of Bragança, Polytechnic Institute of Bragança,
Campus Santa Apolónia, E-5301-855, Bragança, Portugal; E-Mail: leticia@ipb.pt
3
Department of Anatomy and Animal Production, Faculty of Veterinary Medicine, University of
Santiago de Compostela, E-27002, Lugo, Galicia, Spain; E-Mail:antonio.iglesias@usc.es
* Author to whom correspondence should be addressed; E-Mail: xesus.feas@usc.es;

Tel.: +34-982-285-900; Fax: +34-982-285-872.
Received: 17 May 2012; in revised form: 29 June 2012 / Accepted: 2 July 2012 /
Published: 11 July 2012
Abstract: Organic bee pollen (BP, n = 22) harvested from the Douro International Natural
Park (DINP, Portugal) was studied. Nine botanical families were found in the mixture of
the samples. The water activity and pH ranged 0.21–0.37 and 4.3–5.2, respectively. The BP
analyses averaged 67.7% carbohydrates, 21.8% crude protein, 5.2% crude fat and 2.9% ash.
The energy ranged from 396.4 to 411.1 kcal/100 g. The principal fatty acid found was
linolenic, followed by linoleic acid, palmitic acid and oleic acid. The phenolic and flavonoid
contents varied from 12.9 to 19.8 mg of gallic acid equivalents/g of extract and from 4.5 to
7.1 mg of catechin equivalents/g of extract, respectively. The scavenger activity and β-carotene
bleaching assays values (EC50) were 3.0 ± 0.7 mg/mL and 4.6 mg/mL ± 0.9 mg/mL,
respectively. E. coli, sulphite-reducing Clostridia, Salmonella and S. aureus were not
found. Since there are studies indicating appreciable differences among BPs from different
regions, the full characterization of BP from diverse origins still appears to be a sound
research priority in order to obtain reliable data about this beehive product.
Molecules 2012, 17 8360
Keywords: bee pollen; antioxidant capacity; bioactive compound; fatty acids;

microbiological safety; organic food
1. Introduction
Bee honey has been used by man since the beginning of Humanity. Although this is the most
common beehive product, there are other products, such as bee pollen (BP), royal jelly, propolis and
beeswax. These natural goods are well appreciated by consumers due to the high number of quality
checks they go through, as well as for their dietetic and therapeutic qualities.
BP is the result of the agglutination of flower pollens; it is made by worker honey bees with nectar
and salivary substances and stored at the hive entrance [1]. The collection of BP is a relatively recent
development, dependent primarily on the basic concept of scraping pollen off of the bees’ legs as they
enter the hive. When analyzing and studying the nutritional and therapeutic properties of BP, modern
science has made it possible to specify its valuable antimicrobial [2], antifungal [3], antioxidant [4],
anti-radiation [5], hepatoprotective [6], chemopreventive [7], anticancer [8] and antiinflammatory
activities [9].
The major components of BP are carbohydrates, crude fibers, proteins and lipids at proportions
ranging between 13 and 55%, 0.3 and 20%, 10 and 40%, 1 and 10%, respectively. Other minor
components are minerals and trace elements, vitamins and carotenoids, phenolic compounds,
flavonoids, sterols and terpenes [10]. In fact, BP is referred to as the “only perfectly complete food”, as
it contains all the essential amino acids needed for the human organism. However, the composition of
BP depends strongly on the plant source and geographic origin, together with other factors such as
climatic conditions, soil type, and beekeeper activities.
Apiculture is valuable in social, environmental and economic terms and the conservation and
preservation of this practice is essential. The quality and diversity of Portuguese’s landscape should be
considered a valuable and supporting resource for apiculture to achieve international prominence and
competitiveness. Nowadays, honey represents the most highly valued product, since Portugal further
regulates the registration of honey (9 of 18), bearing the European Protected Designation of Origin
(PDO) [11,12]. However, the collection of BP, which as a high quality product and used to be
appreciated, currently suffers from marketing problems because there is a large sector of the public
who are misinformed about its properties.
Presently, in the Portuguese continental territory there are 29 Special Protected Areas and 60 Sites
of Community Importance classified according to the Council Directive 92/43/EEC which deals with
the conservation of natural habitats and wild fauna and flora that are considered to be threatened in the
European Union [13]. The management of such areas must be ecological, economical and socially
sustainable; which makes apiculture one of the most promising activities to develop.
Organic apiculture is an ecologically based system, which encourages the use of good agricultural
practices to maintain the balance and diversity of the agricultural ecosystem, and also promotes the
sustainable use of natural resources, environmental quality, animal welfare and human health [14].
Organic beehive products are free from many problems, such as pollution fallout and chemical
Molecules 2012, 17 8361
residues. Moreover, the use of the beehive products for therapeutic purposes demands it be harvested
in areas with no organic contamination sources [15]. Today, concerns about traces of numerous toxic
substances have prompted some demand for beehive products that are certified as organic [16].
However full characterization of BP is scarce and there is a lack of information about the
characteristics of the product certified as organic [17].
The present study aims to characterize, for the first time, organic AP with respect to: (i) floral origin;
(ii) physico-chemical (water activity and pH), nutritional (ash, protein, fat and carbohydrate) and
energy value; (iii) fatty acid profile; (iv) bioactive compounds (phenolics and flavonoids); (v) antioxidant
activity; and (vi) microbial safety (aerobic mesophiles, moulds and yeasts, fecal coliforms, Escherichia
coli, sulphite-reducing Clostridia, Salmonella and Staphylococcus aureus).
2.1. Palynological Identification
The BP profile analysis results allow us to determinate its floral origin. Table 1 shows the frequency
of occurrence, range and mean values of the 11 pollen types identified in the 23 samples. The BP
analyzed have between three (samples 3 and 4) and seven (sample 13) pollen types; the mean number
is 4.8 with a SD of 1.0. Nine families of PL were found in the BP mixture of: Cistaceae,
Boraginaceae, Rosaceae, Fagaceae, Asteraceae, Fabaceae, Ericaceae, Mimosaceae and Myrtaceae.
None of the botanical families is represented in all the samples studied, since PL can vary according to
the region where they are offered, a factor that depends on the available surrounding bee pasture in the
apiary vegetation.
Table 1. Frequency classes (presence, range and mean ± SD) of the pollen types in the
organic apian pollen.
Family Pollen type Found (n a) Frequency (%) Range (%) Mean ± SD b (%)
Cistaceae Cistus 17 77.3 5.2–90.6 44.0 ± 30.0
Boraginaceae Echium 16 72.7 24.5–60.5 24.5 ± 18.7
Rosaceae Prunus 12 54.5 0.8–10.3 5.8 ± 2.7
Fagaceae Castanea 11 50.0 1.2–65.8 23.6 ± 22.6
Asteraceae Leontodon 10 45.5 3.2–49.5 21.6 ± 12.3
Fabaceae Trifollium 10 45.5 4.4–45.6 13.3 ± 12.3
Ericaceae Erica 8 36.4 6.4–68.0 32.7 ± 24.4
Fagaceae Quercus 7 31.8 1.2–16.0 8.3 ± 4.9
Mimosaceae Mimosa 7 31.8 1.2–11.2 5.3 ± 3.6
Myrtaceae Eucalyptus 4 18.2 1.3–5.6 3.2 ± 1.8
Rosaceae Rubus 3 13.6 2.1–5.6 4.0 ± 1.8
a
sample size; b SD = standard deviation.
Molecules 2012, 17 8362
Table 2. Palynological spectrum of the total organic apian pollen.

Samples
Pollen Types
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
5.0 9.8 0.8 5.4 2.8 5.9 7.1 3.8 10.3 6.0 6.7 6.7
Prunus ND ND ND ND ND ND ND ND ND ND
I I I I I I I I I I I I
Rosaceae
3.1 5.6 4.4
Rubus ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
I I I
68.2 56.0 5.4 80.6 54.8 90.6 16.8 69.8 6.5 5.2 11.5 32.8 13.2 65.0 67.9 25.7 74.5
Cistaceae Cistus ND ND ND ND ND
D D P D D D A D I I I A I D P A D
2.5 23.2 26.6 18.2 5.4 19.8 2.6 18.7 48.9 53.6 49.6 17.1 21.2 18.5 6.9 60.5
Boraginaceae Echium ND ND ND ND ND ND
I A A A I A I A D D D A I A I D
19.9 10.8 1.2 18.8 65.8 24.8 4.5 54.6 5.2 48.9 5.4
Castanea ND ND ND ND ND ND ND ND ND ND ND
A I I A D A I D I D I
Fagaceae
1.2 9.4 7.8 16.0 5.3 12.5 5.6
Quercus ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
I I I A I I I
4.4 5.6 7.8 45.6 9.0 14.7 12.5 19.8 6.9 6.8
Fabaceae Trifolium ND ND ND ND ND ND ND ND ND ND ND ND
I I I D I I I A I I
10.0 19.6 17.6 3.2 28.4 25.4 17.8 23.6 21.3 49.5
Asteraceae Leontondon ND ND ND ND ND ND ND ND ND ND ND ND
I A A I A A A A A D
68.0 46.8 54.3 6.5 6.8 45.9 6.4 27.2
Ericaceae Erica ND ND ND ND ND ND ND ND ND ND ND ND ND ND
D D D I I D I A
5.6 3.4 1.3 2.5
Myrtaceae Eucalyptus ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
I I I I
1.2 11.2 5.2 1.3 8.2 4.4 5.4
Mimosaceae Mimosa ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
I I I I I I I
D: Dominant Pollen (>45%); A: Acessory Pollen (15%–45%), I: Isolated Pollen (<15%) and ND: not detected.
Molecules 2012, 17 8363
A full spectrum analysis of the total BP is given in Table 2. On the basis of palynological analysis,
most of the samples were found to be heterofloral, due to their different colours and consequently
different pollen types. However, in two samples the occurrence of over 80% of Cistus pollen type
(samples 4 and 7) characterized them as unifloral. From the economical standpoint, the assessment of a
monofloral origin may increase the commercial value of these BPs. In fact, it has been reported that
bee pollen from Cistus sp. has anabolic and stimulatory effects on bone components in rats in vitro and
in vivo [18–20], a potent anti-inflammatory activity [9], antiallergic action [21] and high antioxidative
and scavenging abilities [22,23].
Bees forage different plants; thus, BP is always a mixture of different sources. However, in food
control, pollen analysis is very efficient for the differentiation of BP produced in distinctly different
geographical and climatic areas, as well as to ascertain the monofloral origin of BP obtained from
intensively cultivated crops.
Moreover, palynology also allows scientists to infer the vegetation present in an area, and to date
and ascertain any biodiversity changes, as for example the presence and distribution of invasive and/or
exotic plants. Results showed that BP from the DINP contained Mimosa and Eucalyptus pollen types,
found in 7 and 4 samples, respectively.
2.2. Water Activity (aw), pH and Nutritional Composition
The aw of BP samples is 0.31 (average) with a range of 0.21–0.37 and a SD of 0.04 (Table 3). The
range obtained was typical of dehydrated foods and similar when compared to BP from Brazil
(0.3–0.5; [24]) or from Spain (0.261–0.280; [25]). All the BP samples analysed were acidic, with a pH
in the range of 4.3 and 5.2, with an average of 4.8 (±SD = 0.2).
Table 3. Physico-chemical, nutritional and energetic values of organic bee pollen samples.
Sample aw pH Ash Proteins Fat Carbohydrates Energy
S1 0.44 4.9 2.4 23.1 5.7 66.2 407.9
S2 0.45 4.6 3.6 21.4 5.0 67.5 400.5
S3 0.44 4.7 3.4 25.6 6.1 62.0 405.2
S4 0.35 5.2 2.6 23.0 4.5 67.2 401.3
S5 0.45 5.0 2.1 21.9 5.5 67.9 408.9
S6 0.54 5.1 2.1 25.6 4.7 64.7 402.8
S7 0.22 4.6 3.3 27.1 5.2 61.2 400.1
S8 0.21 4.3 2.0 23.0 5.0 67.3 406.3
S9 0.22 4.5 4.0 19.1 6.3 68.4 406.6
S10 0.41 5.1 2.0 21.0 4.9 69.6 406.7
S11 0.42 4.8 2.2 19.2 5.8 70.6 411.1
S12 0.41 4.8 3.0 21.1 4.3 69.2 399.9
S13 0.40 5.0 2.4 20.3 5.0 69.9 406.1
S14 0.40 4.5 2.9 19.5 4.8 70.5 403.3
S15 0.40 4.6 2.5 22.2 5.3 67.4 406.0
S16 0.32 4.9 2.8 20.6 4.8 69.5 403.2
S17 0.44 4.8 4.0 21.3 5.0 67.2 399.1
S18 0.51 4.5 4.0 19.7 4.3 69.7 396.4
Molecules 2012, 17 8364
Table 3. Cont.
Sample aw pH Ash Proteins Fat Carbohydrates Energy
S19 0.44 5.1 4.0 19.3 5.7 68.8 403.2
S20 0.32 4.8 2.4 23.7 6.3 64.8 411.0
S21 0.52 4.9 3.0 22.2 5.4 66.9 404.8
S22 0.37 4.5 3.2 19.4 5.0 70.2 403.1
Mean 0.39 4.8 2.9 21.8 5.2 67.6 404.3
a
SD 0.09 0.2 0.7 2.2 0.6 2.6 3.8
b
Vmax 0.54 5.2 4.0 27.1 6.3 70.6 411.1
c
Vmin 0.21 4.3 2.0 19.1 4.3 61.2 396.4
a
SD = standard deviation; b Vmax = maximum value; and c Vmin = minimum value.
Knowledge about the aw in BP is useful to improve its conservation and storage by preventing the
growth of molds and yeasts. If mold grows, it then ferments, resulting in a product with an off-taste,
high levels of dead yeast, and ethanol that reduces the quality of this product. Furthermore, among the
risks of consuming BP with high aw and commonly stored at room temperature, one is contamination
by fungi, which might produce carcinogenic mycotoxins [26].
The low pH and aw inhibits the presence and growth of microorganisms and makes BP compatible
with many food products. Both parameters are of great importance during the storage of BP as it
influences its texture, stability and shelf life.
2.3. Nutrients Composition
The results of the basic nutrient composition and estimated energetic value (expressed on dry
weight basis) obtained for the analyzed BP are shown in Table 3. The analysis of BP from DINP
averaged 67.7% CH, 21.8% CP, 5.2% CF and 2.9% ash. The energy value of the analyzed BP ranged
from 396.4 to 411.1 kcal/100 g (mean value ± SD = 404.3 ± 3.8 kcal/100 g). Those values confirm that
BP is an excellent source of energy.
The chemical composition and nutritional value of BP shows considerable variability between plant
species. For example, pollens of pine, corn and bulrush contain 13.92; 36.59; and 31.93% total
carbohydrates, 13.45; 20.32; and 18.90% proteins; 1.80; 3.7; and 1.16% lipids and 2.35; 4.90; and
3.80% total ash respectively [27]. Many factors are known to affect the nutrient content of BP,
including climate, geography, apicultural practices and the genetic composition of the plant species.
Generic BP composition data were considered sufficient for most purposes, but now the usefulness
of BP-specific composition data is increasingly being acknowledged. We decided to use BP in the
same form as it appears when commercialized by beekeepers, because it would be economically
impossible for them to separate the pollens into families before selling it. It should be clear that when
nutrient contents are significantly different among foods of the same species, those foods should be
reported independently in food composition databases and other printed materials-including food
labels-with their unique nutrient profiles [28].
Molecules 2012, 17 8365
Table 4. Fatty acid profile of the organic apian pollen.

ω6/ ω3/
Sample C10:0 C16:0 C18:1n9c + t C18:2n6c C18:3n3 C20:0 C20:1c Total a Other ∑SFA ∑MUFA ∑PUFA ∑TUFA PUFA/SFA TUFA/SFA SFA/TUFA
ω3 ω6
S1 5.39 10.15 9.85 14.79 50.71 3.15 1.20 95.24 4.76 18.69 11.05 65.50 76.55 3.50 0.29 3.43 4.10 0.24
S2 7.68 7.83 9.42 17.02 48.19 0.51 3.10 93.74 6.26 16.02 12.52 65.21 77.72 4.07 0.35 2.83 4.85 0.21
S3 3.24 9.02 16.68 20.11 42.84 1.90 1.30 95.09 4.91 14.16 17.98 62.94 80.93 4.44 0.47 2.13 5.71 0.17
S4 3.87 10.45 20.61 18.66 40.08 ND ND 93.66 6.34 14.32 20.61 58.74 79.34 4.10 0.47 2.15 5.54 0.18
S5 3.65 7.90 15.65 20.08 41.64 2.12 1.65 92.70 7.31 13.67 17.30 61.72 79.02 4.51 0.48 2.07 5.78 0.17
S6 3.87 10.45 20.61 18.66 40.08 ND ND 93.66 6.34 14.32 20.61 58.74 79.34 4.10 0.47 2.15 5.54 0.18
S7 3.87 10.00 14.80 24.80 35.82 ND 2.15 91.43 8.57 13.87 16.95 60.61 77.57 4.37 0.69 1.44 5.59 0.18
S8 ND 30.05 4.63 5.94 55.73 ND ND 96.35 3.65 30.05 4.63 61.67 66.29 2.05 0.11 9.39 2.21 0.45
S9 2.98 12.54 11.69 7.89 56.90 1.54 1.23 94.77 5.23 17.06 12.92 64.79 77.71 3.80 0.14 7.21 4.56 0.22
S10 ND 17.50 6.97 15.06 55.73 2.10 ND 97.36 2.64 19.60 6.97 70.79 77.76 3.61 0.27 3.70 3.97 0.25
S11 3.55 10.56 13.34 19.08 42.69 1.12 2.01 92.35 7.66 15.23 15.35 61.77 77.12 4.06 0.45 2.24 5.06 0.20
S12 7.68 8.12 11.40 17.80 45.70 0.51 ND 91.21 8.79 16.31 11.40 63.50 74.90 3.89 0.39 2.57 4.59 0.22
S13 8.27 20.31 11.22 23.26 30.25 0.10 ND 93.40 6.60 28.68 11.22 53.51 64.73 1.87 0.77 1.30 2.26 0.44
S14 6.87 17.15 4.80 24.80 30.24 0.23 0.33 84.42 15.58 24.25 5.13 55.04 60.17 2.27 0.82 1.22 2.48 0.40
S15 3.25 10.56 17.88 22.11 37.53 ND 3.35 94.66 5.34 13.81 21.22 59.63 80.86 4.32 0.59 1.70 5.86 0.17
S16 6.87 22.15 4.80 24.80 25.82 ND ND 84.43 15.57 29.02 4.80 50.61 55.42 1.74 0.96 1.04 1.91 0.52
S17 3.33 16.45 9.56 17.76 44.65 0.45 0.22 92.42 7.58 20.23 9.78 62.41 72.19 3.08 0.40 2.51 3.57 0.28
S18 5.24 20.64 7.89 24.46 34.15 ND ND 92.37 7.63 25.88 7.89 58.60 66.49 2.26 0.72 1.40 2.57 0.39
S19 4.24 10.62 11.98 21.11 42.33 2.12 2.31 94.71 5.29 16.98 14.30 63.44 77.73 3.74 0.50 2.01 4.58 0.22
S20 8.47 20.15 5.80 20.80 28.83 1.04 1.33 86.42 13.58 29.66 7.13 49.63 56.76 1.67 0.72 1.39 1.91 0.52
S21 4.57 8.95 18.91 17.79 43.11 ND ND 93.31 6.69 13.51 18.91 60.90 79.80 4.51 0.41 2.42 5.91 0.17
S22 6.00 10.54 12.79 20.36 40.53 ND 0.34 90.56 9.44 16.54 13.13 60.89 74.02 3.68 0.50 1.99 4.48 0.22
Mean 4.68 13.73 11.88 18.96 41.52 0.77 0.93 92.47 7.53 19.18 12.81 60.48 73.29 3.44 0.50 2.65 4.23 0.27
* SD 2.33 5.93 5.04 4.91 8.65 0.96 1.09 3.43 3.43 5.89 5.34 4.92 7.94 0.99 0.21 1.98 1.41 0.12
** Vmax 8.47 30.05 20.61 24.80 56.90 3.15 3.35 97.36 15.58 30.05 21.22 70.79 80.93 4.51 0.96 9.39 5.91 0.52
*** Vmin ND 7.83 4.63 5.94 25.82 ND ND 84.42 2.64 13.51 4.63 49.63 55.42 1.67 0.11 1.04 1.91 0.17
a
(Calculated value). * SD = standard deviation. ** Vmax = maximum value. *** Vmin = minimum value and ND = no detected. Capric acid (C10:0); Palmitic acid (C16:0); Oleic acid (C18:1n9c + t);
Linoleic acid (C18:2n6c); α-Linolenic acid (C18:3n3); Arachidic acid (C20:0); Eicosenoic acid (C20:1c). SFA: Saturated fatty acids (C10:0 + C16:0 + C20:0); MUFA: Monounsaturated fatty acids
(C18:1n9c + t + C20:1c); PUFA: Polyunsaturated fatty acids (C18:2n6 + C18:3n3); TUFA: Total unsaturated fatty acids (∑MUFA + ∑PUFA).
Molecules 2012, 17 8366
2.4. Fatty Acid Profile Determination
The percentage of fatty acid (FA) composition (%) of BP samples from DINP are shown in Table 4.
Seven FAs were identified and quantified in the pollen samples: capric acid (CPA, C10:0); palmitic
acid (PA, C16:0); oleic acid (OA, C18:1n9c + t) linoleic acid (LA, C18:2n6c); linolenic acid (LNA,
C18:3n3); arachidic acid (AC, C20:0) and eicosenoic acid (EA, C20:1c). The principal fatty acid in the
BP samples was LNA, ranging between 25.82% and 56.90%. It was followed by LA (5.94% to
24.80%), PA (7.83 to 30.05) and OA (4.63 to 20.61). CA, EA and AC were not found in all AP
samples and averaged 4.68%, 0.93% and 0.77%, respectively. In the literature, there are a number of
studies indicating appreciable differences among FA compositions of BPs from different regions or
countries. Therefore, the full characterization of BPs of diverse origins still appears to be a sound
research priority to obtain a reliable data on this valuable beehive product.
The total saturated FA (SFA = C10:0 + C16:0 + C20:0), monounsaturated FA (MUFA = C18:1n9c +
t + C20:1c), polyunsaturated FA (PUFA = C18:2n6 + C18:3n3), total unsaturated FA (TUFA = ∑MUFA +
∑PUFA) and the PUFA/SFA, SFA/TUFA ratios, and ω−6/ω−3, ω−3/ω−6, obtained for the AP samples
are shown in Table 4. The analysed samples present a level of TUFA between 55.42% and 88.93% of
the total (calculated value) FA. In all cases, PUFA is significantly higher than MUFA and SFA. The
profiles obtained for the analyzed organic BP from the Portuguese DINP were similar to the data from
a report by Shawer et al. [29], on Spanish pollens but are slightly different from those of Poland, Korea
and China [30]. The LA and LNA are key compounds for cell membranes and are associated
with brain function and neurotransmission. These FA also play an important role in the transference of
the O2 to blood plasma, in the synthesis of hemoglobin and in cellular division [31,32]. Moreover,
FA from ω−6 series are biogenetic precursors of some physiologically important thromboxanes,
leukotrienes and prostaglandins hormones, which are related to the inflammatory response. The
nutritional value of essential ω−3 and ω−6 FA is also widely known for its health benefits [33].
2.5. Bioactive Compounds and Antioxidant Activity
The BPs were screened for their bioactive compounds, namely phenolics and flavonoids, and results
are presented in Figure 1. Concentrations of TPC and TFC varied from 12.9 to 19.8 (mean value ± SD =
16.4 ± 2.0 GAEs) and from 4.5 to 7.1 (mean value ± SD = 5.8 ± 0.8 CAEs) respectively. Results showed
that the concentration of TFC shows a minor variation, if we compare it to the results obtained for TPC.
Previous works verified that antioxidant activity is related to the amount of flavonoids and phenolic
compounds [34]. In this research, the correlations found were: TPC-DPPH (R = 0.60), TPC-BCB
(R = 0.29), TFC-DPPH (R = 0.54) and TFC-BCB (R = 0.58). Figure 2 summarises the results obtained for
antioxidant activity of the MeOH-APE. The use of at least two methods is recommended to assess and
compare the antioxidant capacity of a sample [35]. In the present work antioxidant properties of the
MeOH-APE were evaluated by the DPPH method and BCB assay. The scavenger activity of the free
DPPH• was expressed in terms of EC50, that is the amount of antioxidant necessary to decrease by 50% the
initial DPPH concentration. Lower values indicate better antioxidant capacity of the MeOH-APE. The
EC50 values ranged from 2.0 mg/mL to 4.3 mg/mL, with a mean value of 3.0 ± 0.7 mg/mL. The results
obtained (EC50) in BCB assay averaged 4.6 mg/mL with a range of 3.1 to 5.9 mg/mL and a SD of 0.9.
Molecules 2012, 17 8367
Figure 1. Total phenolics [TPC; in mg of gallic acid equivalents per g of organic apian
pollen extract (GAEs)] and total flavonoids [TFC; in mg of catechin equivalents per g of
organic bee pollen extract (CAEs)] of the analyzed samples.
Figure 2. EC50 values (mg/mL) obtained for the antioxidant activity by scavenging of
2,2-diphenyl-1-picryl-hydrazyl radicals (DPPH) and β-carotene bleaching assay (BCB) in
the extracts from organic apian pollen samples.
As expected the β-carotene bleaching protection of \ the samples was lower than that provided by
the TBHQ standard (79.3% at 2 mg/mL). In the DPHH assay, the results are expressed as the ratio
percentage of the absorbance decrease of DPPH· solution in the presence of MeOH-APE, and the free
radical-scavenging activity of the extracts is attributed to their hydrogen-donating ability [36]. In the
BCB assay, the LA free radical, formed upon the abstraction of a hydrogen atom from one of its
diallylic methylene groups, attacks the highly unsaturated β-carotene and undergoes rapid discolouration
in the absence of an antioxidant. The presence of antioxidants in the MeOH-APE can hinder the extent
of BCB by neutralizing the linoleate-free radical and other free radicals formed in the system [37].
As the molecules lose their double bonds by oxidation, the compound loses its characteristic orange
colour, a fact that can be monitored spectrophotometrically.
The experimental results of antioxidant activity of MeOH-APE from the BP from DINP were
similar to the data by Campos et al. [34] from pollen harvested in Portugal and New Zeland (EC50 of
Molecules 2012, 17 8368
40 to 500 μg/mL) but superior to those found by Leblanc et al. [38] in Sonoran Desert pollen,
(antioxidant rates from 19% to 90%).
Comparing the results obtained from BP in the present work with values previously observed in
honey, it is possible to conclude that the antioxidant activity of MeOH-APE is higher than honey (EC50
of 37.03, 39.25 and 75.51 mg/mL for dark, amber and light honeys, respectively) [39].
2.6. Microbial Quality
BP samples were subjected to study in what concerns their aerobic mesophiles, moulds and yeasts,
fecal coliforms, E. coli, clostridia spores, Salmonella and S. aureus on different selective microbiological
media. Based on the obtained results (Table 5) it could be concluded that organic BP samples from DINP
represent a product of high microbiological quality. This could be attributed to the nature of such
foodstuffs in relation to their origin, processing and handling. Examined samples did not show any
presence of E. coli, sulphite-reducing Clostridia, Salmonella and S. aureus. Fecal coliforms were only
detected in two samples. In the work of Serra and Escola [40], honeybee-collected pollens were
contaminated with high number of moulds, coliforms and Lancefield group D Streptococci.
Table 5. Microbial analyses of the organic apian pollen.

Aerobic Moulds and Fecal E. coli Sulphite-reducing Salmonella S. aureus
Sample
mesophiles yeasts coliforms (in 1 g) clostridia (in 0.01 g) (in 25 g) (in 1 g)
S1 900 320 <1 absent absent absent absent
S7 1700 2800 20 absent absent absent absent
S8 650 3560 30 absent absent absent absent
S10 <10 <10 <1 absent absent absent absent
S11 <10 450 <1 absent absent absent absent
S17 670 <10 <1 absent absent absent absent
S22 <10 <10 <1 absent absent absent absent
Taking into account the nutrient content, a variety of microorganisms could grow in BP. If harvest,
storage and marketing practices are not appropriate, microorganisms might develop in it as happens in
dehydrated foods. The quantification of quality parameters for commercial purposes (aerobic mesophiles
Molecules 2012, 17 8369
and moulds and yeasts) is generally lower than that reported by other authors. Hervatin [41] found mould
and yeast levels greater (104 CFU/g) than those obtained in the present study. From the microbiological
point of view, the low values of moulds and yeasts are most probably related to environmental
conditions, and are indicative of an appropriate management of organic apiaries.
Microbiological criteria provide guidance on the acceptability of foodstuffs and their manufacturing
processes. From the hygienic point of view, microbiological safety is the main quality criterion in BP.
Destruction of bacteria by irradiation, ozone treatments or chemical fumigants is not necessary and can
lead to toxic residues [10]. The microbiological content should correspond to the hygienic standards:
Salmonella (absent/10 g), S. aureus (absent/1 g), Enterobaceteriaceae (Max. 100/g), E. coli (absent/1 g),
total aerobic plate count (<100,000/g) and moulds and yeast (<50,000/g) [1].
Most primary producers are not directly affected, as specific microbiological criteria have not been
set for beehive products. However, beekeepers may be affected indirectly if their customers require
changes to specifications, as a result of improvements in production hygiene and selection of raw
materials for health purposes.
3. Experimental
3.1. Chemicals
2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA, USA).
3,4,5-Trihydroxybenzoic acid (gallic acid; GA), tert-butylhydroquinone (TBHQ), butylated
hydroxyanisole (BHA), polyoxyethylene (20) sorbitan monooleate (Tween 80), α-tocopherol,
β-carotene, petroleum ether and ethanol were obtained from Sigma Chemical Co. (St. Louis, MO,
USA). Sodium sulfate (Na2SO4, anhydrous, powder, extra pure), H2SO4, KOH, aluminium chloride
(AlCl3), NaNO2 and NaOH were purchased from Acros Organic (Geel, Belgium). Capric acid (CPA,
C10:0) [CAS 334-48-5]; palmitic acid (PA, C16:0) [CAS 57-10-3]; oleic acid (OA, C18:1n9c + t)
[CAS 112-80-1 CAS 112-79-8]; linoleic acid (LA, C18:2n6c) [CAS 60-33-3]; linolenic acid (LNA,
C18:3n3) [CAS 463-40-1]; arachidic acid (AC, C20:0) [CAS 506-30-9] and eicosenoic acid
(EA, C20:1c) [CAS 5561-99-9]) were purchased from Sigma-Aldrich (Tres Cantos, Spain). The
Folin-Ciocalteu reagent (FCR), chloroform (CHCl3) and sodium carbonate (Na2CO3), were obtained
from Merck (Darmstadt, Germany). Methanol (MeOH) and hexane were obtained from Pronolab
(Lisboa, Portugal). High purity water (18 MΩ cm), which was used in all experiments, was obtained
from a Milli-Q purification system (Millipore, Bedford, MA, USA).
3.2. Apian Pollen Material
Twenty-two (n = 22) typical bee pollen samples (AP), from Apis mellifera, were collected by
beekeepers from different apiaries, located inside the Portuguese territory declared as Douro
International Natural Park (IDNP). They were obtained using bottom-fitted pollen traps in May of 2010.
A 10-day-cycle of pollen trapping installation was employed in order to avoid making pollen trapping
difficult to bees: pollen was trapped for 5 days and after that period it was stopped for the next 5 days [42].
After the beekeepers dried the harvested material, a single 200 g jar of bee pollen was delivered to
the Microbiology Lab, where it was stored in a dark place at room temperature (±20 °C) until analysis,
Molecules 2012, 17 8370
which occurred no longer than one month after the extraction from the hives by beekeepers. All BP
samples showed no sign of fermentation or spoilage.
3.3. Sample Floral-Type Identification
The determination of the frequency of pollen load (PL) classes, were determined in the BP, in order
to ascertain the floral origin and to obtain a complete pollen spectrum. The botanical origin of the BP
was based on the method proposed by Almeida-Muradian et al. [43]. Briefly, the analyses are based on
the separation according to colour of PL from 2 g of BP. The PL counted were from 287 to 387 (mean
value ± standard deviation = 335 ± 29 %). Each subsample was weighed to calculate its percentage in
the main BP. Three slides of each subsample were prepared by washing the PL in 50% ethanol and
using glycerin and paraffin for permanent preparations. The examination of the slides was carried out
with a Leitz Diaplan microscope (Leitz Messtechnik GmbH, Wetzlar, Germany) at ×400 and ×1,000.
In order to recognise the pollen grains, we used the reference collection of the CIMO-Mountain
Research Center (Agricultural College of Bragança) and different pollen morphology guides.
3.4. Water Activity and pH
The water activity (aw) was measured using a model Rotronic Hygroskop DP. For pH, 5 g of
grounded AP were diluted with 20 mL of distilled water and mixed thoroughly. The pH values for
these samples were measured using a digital pH Meter (pH 526 Multical, WTW, Weilheim, Germany).
3.5. Chemical Composition and Nutritional Value
Chemical composition of the BP were analysed according to the AOAC procedures [44]. The ash
content was determined after incineration at 600 ± 15 °C, in a SNOL 8.2/1100-1 electric laboratory
furnace (AB “Umega”, Utena, Lithuania). Nitrogen content (N) was determined using the Kjeldahl
method (230-Hjeltec Analyzer, Foss Tecator, Höganäs, Sweden). The crude protein (CP) content was
calculated using the conversion factor of 5.6 (N × 5.6). The crude fat (CF) was determined by
gravimetry after extraction with petroleum ether using an automatic Soxtec device (FOSS, SoxtecTM
2050, Höganäs, Sweden). The total carbohydrate (CH) contents were obtained by difference. Ash, CP,
CF and CH contents of AP were expressed as a percentage of the original sample on a dry weight basis
(g/100 g sample). The total energy (kcal/100 g) was estimated using the Atwater coefficients (4 kcal/g
for CP and CH, 9 kcal/g for CF) [45].
3.6. Fatty Acid Profile Determination
Fatty acid methyl esters (FAMEs) were prepared from the extracted CF fraction by transesterification
using MeOH in the presence of H2SO4 as follows: A sample containing 20 ± 50 mg of lipids was
redissolved in 0.75 mL n-hexane; then 0.1 mL of 2 N KOH in MeOH was added and the solution was
mixed for 2 min in a vortex mixer (Model Reax 2000, Schwabach, Germany), dried over anhydrous
Na2SO4 and left for 25 min. After phase separation the upper layer of n-hexane containing the FAMEs
was removed and immediately injected into the gas-chromatograph (GC). Quantitative and qualitative
analysis of FAMEs, was performed on a DANI model GC 1000 coupled flame-ionization detector
Molecules 2012, 17 8371
(FID) equipped with a Macherey-Nagel (OPTIMA 225: 50% cyanopropyl-methyl–50%

phenylmethylpolysiloxane) column (30 m × 0.32 mm ID × 0.25 μm df). The carrier gas was H2 at a
pressure of 0.61 bar, and the split ratio was 1:40. The flow rate of the carrier gas was set
at 4.0 mL/min. A thermal gradient from 170 to 240 °C at 3.5 °C/min was used with the injector and
FID temperatures at 240 °C. The injection volume was 1 µL. FAMEs were identified by comparing the
retention times of the peaks of the sample with those of known reference esters. Fatty acid (FA)
composition was expressed as percent of major FAMEs from the peak areas with an integrator. Results
were recorded and processed using CSW 1.7 software (DataApex 1.7).
3.7. Bioactive Compounds Quantification
3.7.1. Extracts Preparation
Extracts were prepared according to procedures from Morais et al. [4] with modifications. Briefly,
BPs were ground and a portion of the obtained powder was mixed (1:2) (w/v) with MeOH, sonicated
for 30 min. and left to macerate for 48 h at room temperature. After this time, the solution was
centrifuged for 5 min at 13,000 g (4 °C). The centrifugal supernatant was evaporated under reduced
pressure by a rotavapor system consisting of a rotary vacuum evaporator (Heidolph VV. 2000, Leuven,
Belgium) equipped with a water bath and a B169 vacuum pump (Büchi, Flawil, Switzerland). The
residue was dissolved in H2O and lyophilised. Finally, the obtained AP extract (APE) was stored at
−80 °C, for further analysis.
3.7.2. Total Phenolic Content (TPC)
The total phenolic content in the BPE were recorded by FCR as described by Moreira et al. [46].
Briefly, a dilute solution of each BPE in MeOH (MeOH-APE; 500 μL of 1:10 g/mL) was mixed with
500 μL of the FCR and 500 μL of Na2CO3 (10% w/v). After incubation in dark at room temperature
for 1 h, the absorbance of the reaction mixture at 700 nm is determined against the blank (the same
mixture without the MeOH-APE) using a Unicam Helios Alpha UV-visible spectrometer (Thermo
Spectronic, Cambridge, UK). GA standard solutions (0.01–0.08 mM) were used for constructing the
calibration curve (y = 2.3727x + 0.0022; R2 = 0.9998). Total phenols content were expressed as mg of
GA equivalents per g of BPE (GAEs).
3.7.3. Total Flavonoid Content (TFC)
For flavonoid contents the aluminium chloride method was used. Briefly, MeOH-BPE (250 μL)
was mixed with 1.25 mL of distilled H2O and 75 μL of a 5% NaNO2 solution. After 5 min, 150 μL of a
10% AlCl3·H2O solution was added. After 6 min, 500 μL of 1 M NaOH and 275 μL of distilled H2O
were added to the mixture and vortexed. The intensity of the pink colour of the reaction mixture at
510 nm is determined against the blank (the same mixture without the MeOH-BPE). Catechin (CA)
standard solutions (0.022–0.34 mM) were used for constructing the calibration curve (y = 0.9689x − 0.0092;
R2 = 0.9987). The total flavonoids content (TFC) was expressed as mg of CA equivalents per g of
BPE (CAEs).
Molecules 2012, 17 8372
3.8. Antioxidant Activity
3.8.1. Scavenging of DPPH Radicals
The scavenging of DPPH· was assayed following the method described by Ferreira et al. [39].
Various concentrations of MeOH-APE (300 μL) were mixed with 2.7 mL of MeOH solution containing
DPPH• (6 × 10−5 mol/L). The mixture was shaken vigorously and left to stand for 60 min in the dark
(until stable absorption values were obtained). The reduction of the DPPH· was measured by continuously
monitoring the decrease of absorption at 517 nm. The radical-scavenging activity (RSA) was calculated
as a percentage of DPPH discoloration using the equation: %RSA = [(ADPPH − AS)/ADPPH] × 100, where
AS is the absorbance of the solution when the MeOH-BPE has been added at a particular level and
ADPPH is the absorbance of the DPPH solution. The MeOH-BPE concentration providing 50% of radical
scavenging activity (EC50) was calculated by interpolation from the graph of RSA percentage against
extract concentration. The standards used were BHA and α-tocopherol.
3.8.2. β-Carotene Bleaching (BCB) Assay
The antioxidant activity of the MeOH-BPE was evaluated by the BCB assay, as described by
Ferreira et al. [39]. A solution was prepared by dissolving 2 mg of β-carotene in 10 mL of CHCl3.
Afterwards 2 mL of the aforesaid solution was pipetted into a 100 mL round-bottom flask. Then the
organic solvent was removed at 40 °C under vacuum. 40 mg of LA, 400 mg of Tween 80 emulsifier
and 100 mL of distilled H2O were added to the flask. The mixture was shaken and 4.8 mL of this
emulsion were transferred into different test tubes containing 200 μL of different concentrations of the
MeOH-BPE. The tubes were shaken and incubated at 50 °C in a water bath. As soon as the emulsion
was added to each tube, the zero time absorbance was measured at 470 nm. Absorbance readings were
then recorded at 20-min intervals until the control sample had changed color. A blank, devoid of
β-carotene, was prepared for background subtraction. Lipid peroxidation inhibition (LPO) was
calculated using the following equation: LPO = (β-carotene content after 2 h of assay/initial β-carotene
content) × 100. The MeOH-BPE concentration providing 50% antioxidant activity (EC50) was calculated
by interpolation from the graph of antioxidant activity percentage. TBHQ was used as standard.
3.9. Microbiological Determinations
Microbiological determinations were carried out as described previously [47]. In short, 10 g of each
BP were aseptically taken and homogenized using a pre-sterilized Stomacher Lab-Blender (Seward
type 400, London, UK) for 3 min with 90 mL of pre-chilled (4 ± 0.5 °C) sterile peptone-physiological
saline solution [0.1% neutral peptone + 0.85% NaCl (Merck, Darmstadt, Germany) in sterile deionized
H2O, pH = 7.0 ± 0.05]. Decimal serial dilutions were prepared from this homogenate in the same
chilled sterile diluents (1:10, by vol). The aerobic mesophile were determined using plate count agar
(PCA), by counting the colony forming units (cfu/g of BP) after incubating the plates at 30 °C for 48 h.
Moulds and yeasts counts followed the protocol of International Organization for Standardization
(ISO) [48]. For sulphite-reducing clostridia counting, aliquots of 10, 5, 1 and 0.1 mL of the initial
suspension were added to an empty tube, thermally treated at 80 °C for 5 min and covered with SPS
Molecules 2012, 17 8373
(sulphite-polymixin-sulfadiazine) agar media, tubes were incubated at 37 °C for 5 days. Fecal coliforms
were enumerated by the Most Probable Number (MPN) technique defined in the protocol ISO [49].
The positive results for fecal coliforms were studied for E. coli. Enumeration was made on Eosin
Methylene Blue Agar-EMB Agar (Oxoid Inc., London, UK) incubated at 35 °C for 24 h. Salmonella
detection followed the ISO protocol [50]. Staphylococcus aureus detection followed the protocol
of [51]. All microbial tests were performed in triplicate.
4. Conclusions
Under European regulations [52], any claims of health or nutritional benefits of a food product must
be supported by science. Different health claims can be made for BP: (a) long term ingestion of pollen
and special pollen preparations can improve the physical performance and fitness of sportsmen and
elderly people and (b) pollen intake can improve gut, gastroenterological and liver health. BP
composition data were considered sufficient for most purposes, but now the usefulness of BP-specific
composition data is increasingly being acknowledged, since the composition of BP varies greatly as a
result of collection from different geographic regions, the time of collection, and the various species of
vegetation from which the pollen is harvested by honeybees. The results obtained in this study
demonstrated that BP constitutes a good source of healthy compounds, namely, phenolics, and
suggests that it might be useful in prevention of diseases in which free radicals are implicated.
Portuguese organic BP from DINP is nutritionally well-balanced and revealed high levels of healthy
fatty acids and good PUFA/SFA ratios. Microbiologically, the commercial quality was considered
good and all samples showed negative results for toxigenic species. The consumption of BP can be
beneficial for the health and, as such, the investigation of its chemical, nutritional and microbiological
composition will contribute to the assessment of this natural product. BP is a food supplement with
potentially beneficial effects for humans.
Conflict of Interest
The authors declare that they have no conflict of interest.
Acknowledgments
We would like to thank the Portuguese beekeepers who kindly supplied us with the samples for this
study. X. F. would also like to thank the Xunta de Galicia (Isidro Parga Pondal Program for Young
Researchers, Grant No.: IPP-020), and JoDee Anderson for the linguistic support she provided.
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(http://creativecommons.org/licenses/by/3.0/).
Hrana u zdravlju i bolesti, znanstveno-stručni časopis za nutricionizam i dijetetiku (2014) 3 (1) 6-12
Antioxidant properties of pollen
Damir Aličić1*, Drago Šubarić2, Midhat Jašić3, Hatidža Pašalić3, Đurđica Ačkar2
1
High Secondary School ''Čelić'', 75246 Čelić, Bosnia and Herzegovina
2
Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology Osijek, Franje Kuhača 20, HR-31000 Osijek,
Croatia
3
Faculty of Pharmacy, University of Tuzla, Univerzitetska 8, 75000 Tuzla, Bosnia and Herzegovina
review
Summary
Today, bee pollen is commonly used in folk medicine and its pharmacy effects have not yet been scientifically proven. The
composition and chemistry of bee pollen are not yet standardized nor defined in pharmacopoeia, and may vary due to its
botanical and geographical origin, the plant species, environmental conditions, age and status of plants. Because of this, the
type of bee pollen depends on the available bee pasture and types of plant species visited by bees. Bee pollen contains
nutritional and essential substances and also significant amounts of polyphenolic substances, mainly flavonoids, that are
considered as the main ingredients of pollen and its antioxidant properties. Researches show that pollen has significant
antioxidant activity and mostly depends on phenol compounds. Large deviations of the this antioxidant activity are
considerable, as well as content of phenolic compounds between pollen grains taken from different plant species and different
geographical regions. The pollen antioxidant activity is usually expressed as the antioxidant capacity, and primarily depends on
its botanical and geographical origin that is the subject of many scientific and research papers. This article gives an overview
of bee pollen, its chemical composition and botanical origin, antioxidant properties and its capacity.
Keywords: honeybee pollen, antioxidant capacity, polyphenols, flavonoids, botanical origin
Introduction the present work was an overview of various pollen

properties such as the chemical composition, the
Honey bee-derived apicultural products such as botanical origin, its collecting, phenolic compounds
pollen have been applied for centuries in traditional and an antioxidant activity.
medicine as well as in food diets and supplementary
nutrition due to their nutritional and physiological Pollen
properties, above all in regard to their health effects
on the human organism (Kroyer & Hegedus, 2001). Pollen grains are microscopic structures found in the
Pharmacological effects of pollen are not yet anthers of stamens in angiosperms (de Arruda et al.,
scientifically based and its chemical composition 2013), they constitute the male reproductive cells in
depends on the available bees pasture and species of plants (Basim et al., 2006), and their purpose is to
plants visited by bees. Pollen is a considerable source transmit their gametes to the female sex organ of the
of compounds with health protective potential, flower (Arruda et al., 2013). Bees, other insects, wind
certain concentration of phytosterols, and is also rich and water pollinate plants by transferring pollen from
in phytochemicals such as phenolic compounds, the stamen to the stigma of another plant (LeBlanc et
which are considered beneficial to human health. al., 2009). According to the regulations of honey and
Researches show that pollen has significant other bee products (Službeni glasnik BiH, br. 37/09),
antioxidant activity that mostly depends on phenolic pollen is a product that worker bees collected in
compounds. However, large deviations of the nature adding to its own specific matter which forms
antioxidant activity are considerable and content of the pellets, and place them in the honeycomb cells.
phenolic compounds between pollen grains taken Special group of worker bees that collect pollen is
from different plant species and different called pollen-bees (Dolovac, 1997).
geographical regions are remarkable. The pollen Pollen is very important in apiculture as a source of
antioxidant activity is usually expressed as the proteins, fats and minerals to bees and as an excess
antioxidant capacity, and primarily depends on its produced from the apiary (de Arruda et al., 2013).
botanical and geographical origin that is the subject The quantity and quality of pollen collected by
of many scientific and research papers. The aim of honeybees affects reproduction, brood rearing and
*
Coressponding author: damir_alicic@yahoo.com
Damir Aličić / Antioxidant properties of ... / (2014) 3 (1) 6-12
longevity, thus ultimately the productivity of the gallic acid, p-coumaric acid, hesperidin, rutin,
colony (Human & Nicolson, 2006). kaempferol, apigenin, luteolin, quercetin, and
The significance of pollen for bee's community is isorhamnetin (Bonvehi et al., 2001).
priceless and is associated with its survival. Bees use
it as food for all the developmental stages in the hive Pollen collection
(Almeida-Muradian et al., 2005; Morais et al., 2011).
Apart from small quantities in nectar honeybees The collection of this natural product is a relatively
obtain all the proteins, lipids, minerals and vitamins recent development, dependent primarily on the basic
they need for brood rearing and adult growth and concept of scraping pollen off of the bees’ legs as
development from pollen (Human & Nicolson, they enter the hive (Feás et al., 2012). Honey bees
2006). The bees place the pollen in honeycombs with collect pollen by adding sugars from nectar and their
their legs and cover this pollen with honey. This own secretions to bind the grains together (Cheng et
pollen reserve is referred to by beekeepers as ‘‘bee al., 2013) and then transfer them back to the colony
bread”. It was determined that an average value of by packing them into hairs on the corbiculae (hind
145 mg of pollen is required to rear just one worker legs) of bees (LeBlanc et al., 2009).
bee (LeBlanc et al., 2009). For the commercial bee pollen collection, indoor or
outdoor pollen collectors can be used. There are
The chemical composition of pollen different versions of these collectors depending on
the type of the hive and the principle of the pollen
Flower pollens' composition can vary due to their subtraction is the same. Bee with pollen must scrape
botanical and geographic origin (Almaraz-Abarca et through small openings in the pollen collector where
al., 2004). The major components of bee pollen are it passes and the balls of pollen fall into the prepared
carbohydrates, crude fibers, proteins and lipids at drawer. The advantage of outdoor pollen collectors is
proportions ranging between 13 % and 55 %, 0.3 % cleaner pollen but its deficiency is smaller amount in
and 20 %, 10 % and 40 %, 1 % and 10 %, respectively comparison with indoor pollen collectors.
(Villanueva et al., 2002). Other minorcomponents are The collected raw pollen with about 20 % moisture
minerals and trace elements, vitamins and carotenoids, content is subject to microbial spoilage and kept in a
phenolic compounds, flavonoids, sterols and terpenes frozen state at - 18 °C up to a certain analysis or dried
(Bogdanov, 2011). Proline, aspartic acid, to 7-8 % moisture content and kept in a cool, dark
phenylalanine and glutamic acid are the primary place. For pollen analyzing, its extracts in different
amino acids in pollen (Roldán et al., 2011). However, solvents and their mixtures are prepared, and most
the composition of bee pollen depends strongly on commonly used solvents are methanol, ethanol and
plant source, together with other factors such as water.
climatic conditions, soil type and beekeeper activities Kroyer & Hegedus (2001) used ethanol,
(Morais et al., 2011). methanol/water and water as pollen solvents, and
Few studies on the active enzymes in bee pollen have reported that the content of polyphenolic compounds
been published (Xue et al., 2012). According de in pollen extract was significantly increased in
Arruda et al. (2013), bee pollen is rich in B complex absolute ethanol.
vitamins (thiamine, niacin, riboflavin, pyridoxine,
pantothenic acid, folic acid and biotin) and Botanical origin of pollen
carotenoids, which can be provitamin A. However,
according to the same author, there is no significant Botanical origin of pollen is determined by
amount of vitamin C or lipid soluble vitamins. palynological (microscopic) analysis respectively by
There are numerous reports of bioactive substances in the microscopic identification and counting of pollen
the pollen such as phenols, flavonoids, anthocyans, grains. Each plant species has its own characteristic
phospholipids and proteins. The main bioactive pollen grain that can be used to determine its
flavonoids are naringenin, isorhamnetin3-O- botanical origin i.e. determining the plants that bees
rutinoside, rhamnetin3-O-neohesperidoside, visited by gathering the pollen.
isorhamnetin, quercetin3-O-rutinoside, quercetin3-O- Pollen grains vary in terms of their morphological
neohesperidoside, kaempferol and quercetin and their characteristics such as form, size, openings/apertures
total amounts are in the range 0.3-1.1 % w/w (Han et and ornamentation, as well as in terms of color and
al., 2012). appearance. Color and other characteristics of pollen
Phenolic compounds are one of the most critical grains can be used to identify the genus of plants and,
ingredients related to antioxidant activity in pollen. sometimes, the plant species (Bačić, 1995; de Arruda
Usually, it contains vanillic acid, protocatechuic acid, et al., 2013).
7
Pollen analysis allows the identification of the major oxidation." Antioxidant opposes oxidation or inhibits
pollen sources used by the bees, as well as the reactions induced by oxygen or peroxides. Thus, the
periods of pollen production in the field and possible presence of antioxidants in the pollen reduces the
times of shortage (de Arruda et al., 2013). harmful effects of the free radicals in the cell and can
Microscopic examination showed that each pellet of slow oxidation reactions in food.
honeybee-collected pollen was largely homogeneous, Antioxidant ability has usually been attributed to the
confirming the observations of Almaraz-Abarca et al. activity of antioxidant enzymes (mainly superoxide
(2004) who observed that pollen pellets dismutase, peroxidase and catalase) as well as to the
predominantly consist of pollen grains from one content of low-molecular antioxidants such as
species. carotenoids, tocopherols, ascorbic acid, phenolic
Research by de Arruda et al. (2013) indicates that substances (Leja et al., 2007). Antioxidants are
bees use a variety of flora for the production of bee considered as possible protection agents reducing
pollen and other bee products. When collecting oxidative damage to important biomolecules,
pollen, bees generally visit the same type of plants to including lipoprotein and DNA (deoxyribonucleic
make pollen grains, and that pollen is mainly acid) from ROS (reactive oxygen species). Oxidative
monofloral origin, with minor additions of pollen stress, the consequence of an imbalance between
grains of other species of plants. According to de ROS generation and antioxidants in the organism,
Arruda et al. (2013), pollen samples that have initiates a series of harmful biochemical events which
amounts exceeding 45 % of a botanical taxon in their are associated with diverse pathological processes
composition can be considered as unifloral pollen. which can lead to various cellular damages and
Morais et al. (2011) proved that pollens with same diseases (Mărghitaş et al., 2009).
color belong to the same family. According to Luz et It is believed that the bee products are large sources
al. (2010) the pollen types observed in the pollen of antioxidants. According to Nagai et al. (2001)
pellets can vary according to the region where they there is significant antioxidant activity in pollen and
are offered, a factor which depends on the available other bee products.
surrounding bee pasture in the apiary vegetation, as Bee pollen, like other bee products (honey, propolis),
well as on the climate conditions for flowering. is due to the abundant and qualitatively and
Therefore, the composition of the pollen may vary quantitatively different contents of phenolic and
due to its botanical and geographical origin flavonoid antioxidants related to botanical species
(Almaraz-Abaraca et al., 2004) and according to and origin, valuable sources of these healthy
Szczesna et al. (2002), the chemical composition of beneficial constituents characterized by high
bee pollen varies according to the plant species, antioxidant activity (de Arruda et al., 2013). This
environmental conditions (different locations, various mechanisms of antioxidant activity permit a
seasons and years), age and status of the plant (when wide range of free radicals scavenging and lipo-
the pollen is developing). peroxidation assays in order to evaluate the complete
For microscopic analysis, homogeneous pollen antioxidant potential (Mărghitaş et al., 2009).
sample is taken in the amount of 2 g, which Many of the present studies are concerned with
corresponds to the number of 300 pollen grains determining the antioxidant activity of pollen
(Almeida-Muradian et al., 2005), which are classified samples of different geographical origin and
into groups with grains of the same color (Mărghitaş establishing a correlation with the content of phenolic
et al., 2009), determining their percentage and other compounds.
participation in the main sample. The colour of the
pollen can be estimated according to the tables Antioxidant capacity
elaborated by Hodges (1984) and Kirk (1994) and
identified by colour and microscope observations of The measure of antioxidant activity can be expressed
pollen grains (Warakomska, 1962). For the by antioxidant capacity. Many factors may affect
determination by the palynological analysis also accurate determination of antioxidant activity (Kukrić
some others standardized taxons for the specific area at al., 2013). A number of methods based on different
or country may be used. mechanisms of antioxidant defense system, are
developed to determine antioxidant capacity, such as
Antioxidant properties of pollen the removal or inhibition of free radicals or chelating
metal ions, which otherwise may lead to the
In the literature, the term "antioxidant" is defined in formation of free radicals (Greblo, 2009). The
many ways. The word antioxidant, as the same name antioxidant properties of the pollen extracts cannot be
indicates, means "something that is opposite to evaluated by just one method due to the complex
8
nature of their constituents. Recent investigations results for DPPH, FRAP and TEAC assay, in regard
show differences between the test systems in with the antioxidant content of bee pollen samples
determining antioxidant capacity. Use of at least two analysed. In conclusion, future analysis is required,
methods is recommended to assess and compare the not only in testing other different systems of
antioxidant capacity of a sample (Sakanaka & evaluating the antioxidant activity, but also in
Ishihara, 2008). There are various methods available separation and identification the specific bioactive
in the assessment of the antioxidant capacity of compounds in bee pollens with different botanical
samples. They provide useful data, however, they are origin, in order to elucidate the differences between
not sufficient to estimate a general antioxidant ability various samples (Mărghitaş et.al., 2009).
of the sample (Filipiak, 2001). These methods differ
in terms of their assay principles and experimental Phenolic compounds in pollen
conditions. Consequently, in different methods,
particular antioxidants have varying contributions to Bee-collected pollen contains significant amounts of
total antioxidant potential (Mărghitaş et al., 2009). polyphenol substances mainly flavonoids which
The enzymatic and non-enzymatic methods are used furthermore are regarded as principal indicating
to determine the antioxidant capacity. Of the non- ingredient substances of pollen and can be used for
enzymatic method, indirect methods (DPPH, ABTS+, setting up quality standards in relation to their
FRAP) and direct methods (ORAC method) (Kukrić nutritional-physiological properties and for quality
at al., 2013) are used mostly. control of commercially distributed pollen
The DPPH method (Brand-Williams, Cuvelier, & preparations (Kroyer & Hegedus, 2001).
Berset, 1995) principle is the reaction of DPPH (2,2- In addition to testing the total phenolic compounds in
diphenyl-1-picrylhydrazyl), a stable free radical, pollen its constituents are tested, such as flavonoids,
which accepts an electron or hydrogen radical to anthocyanins, fenilpropanoids and others. There are
become a stable molecule, and, accordingly, is many studies that explore the contents of phenolics
reduced in presence of an antioxidant. DPPH radical and flavonoids and their common relationship to
is widely used for the preliminary screening of antioxidant activity. Significant and mutual
compounds capable to scavenge activated oxygen dependencies between these components and
species since they are much more stable and easier to antioxidant capacity, botanical and geographical
handle than oxygen free radical (Tominaga et al., origin are established.
2005). The absorbance changing is monitored at 517 The antioxidant activity of polyphenols is mainly due
nm (Parkash, 2001). to their redox properties, which can play an important
The TEAC assay is based on the inhibition of the role in neutralizing free radicals, quenching oxygen,
absorbance of the radical cation of ABTS (2,2’- or decomposing peroxides (Nijveldt et al., 2001).
azinobis(3-ethylbenzothiazoline-6-sulphonate) by According to Carpes et al. (2007), the pollen
antioxidants. Due to its operational simplicity, the collected by bees generally shows characteristic
TEAC assay has been used in many research amounts of total polyphenols due to its botanical and
laboratories for studying antioxidant capacity, and geographical origin.
TEAC values of many compounds and food samples Antioxidant activity is not necessarily correlated with
are reported. high amounts of phenolic compounds and total
The FRAP assay measures the ferric-to-ferrous iron phenolic content, measured by the Folin–Ciocalteu
reduction in the presence of antioxidants and is very procedure, and does not give a full idea of the nature
simple and convenient in terms of its operation of the phenolic constituents in the extracts (Mărghitaş
(Mărghitaş et al., 2009). et al., 2009).
The antioxidant capacity determination results of an Studies by Almaraz-Abarca et al. (2004) and
extract depend greatly on the methodology used, that Mărghitaş et.al. (2009) show that the polyphenol
is the oxidant and the oxidisable substrate used in the composition of pollen, can be a factor in its
measurement. Therefore, it is important to compare determination. Mărghitaş et al. (2009) require the
different analytical methods varying in their detailed examination of phenolic composition in bee
oxidation initiators and targets in order to understand pollen extracts for the comprehensive assessment of
the biological activity of an antioxidant and to obtain individual compounds exhibiting antioxidant activity.
accurate data for a better comparison with other The results in most studies show large variations and
literature. On the other hand, different antioxidants significant differences in the amount and content of
respond differently in various measurement methods phenolic compounds in pollen from different
which involve specific reaction conditions and geographical destinations and different botanical
mechanisms of action. This may explain the various origin.
9
The most important and largest group polyphenols species by reacting with the reactive compound of the
are flavonoids that appear in almost all parts of the radical (Nijveldt et al., 2001).
plant and today approximately 4000-5000 various Total phenols is usually determined
types of flavonoids are known (Kukrić et al., 2013). spectrophotometrically (Moreira et al., 2008; Kroyer
Flavonoids are pigments responsible for the & Hegedus, 2001; Mărghitaş et al, 2009), by
coloration of flowers and leaves and are important for modified Folin-Ciocalteu method which is based on
normal growth, development and defense of plants. phenol coloured reaction with the Folin-Ciocalteu
Each type of pollen has its own specific system of reagent, measuring the resulting intensity of
flavonoids (Crane, 1990). coloration (Kukrić et al., 2013), and total flavonoids
Recent studies have shown that flavonoids derived by colorimetric tests with reference standards (Kim et
from the pollen of different geographical and al., 2003).
botanical origin containing compounds of different
nutritional significance. The reactions of free radicals Health effects of pollen
and scavenging capacity to reactive species of
oxygen in the pollen may be due to differences in Recently, increasing evidence suggests its potential
atmospheric and environmental conditions, soil or therapeutic benefits, including antioxidant (Leja et
plant physiology. Flavonoids have different structural al., 2007), bioactive (Kroyer & Hegedus, 2001;
features and show several biological activities. It Roldán et al., 2001), and antimicrobial properties
appears that they may strongly influence antioxidant (Basim et al., 2006; Carpes et al., 2007; Morais et al.,
activity, gene expression, drug-metabolizing 2011), suggesting that it could be useful in prevention
enzymes, such as cell signaling or cytochrome P450 of diseases in which free radicals are implicated
(CYP) enzymes, express phytoestrogenic potential, (Pascoal et al., 2013).
protect against toxicity of the environmental It is considered to be a natural health food which
contaminant dioxin (Šarić et al., 2009). constitutes a potential source of energy and
It is known that only flavonoids of a certain structure functional components for human consumption (Silva
and particularly hydroxyl position in the molecule, et al., 2006), with a wide range of therapeutic
determine antioxidant properties. In general, these properties, such as antimicrobial, antifungal,
properties depend on the ability to donate hydrogen antioxidant, anti-radiation, hepatoprotective,
or electron to a free radical (Mărghitaş et al., 2009). chemoprotective and/or chemopreventive and anti-
The high ability of phenolic constituents to neutralize inflammatory activities (Pascoal et al., 2013), free
the active oxygen species is strongly associated with radical scavenging activities (Leja et al., 2007; Silva
their structure, such as the conjugated double bonds et al., 2006), inhibition of lipid peroxidation and
and the number of hydroxyl groups in the aromatic suppressing the cellular and humoral response (Xu et
ring, mostly attributed to flavonoids and cinnamic al., 2009). These therapeutic and protective effects
acid derivatives (Leja et al., 2007). have been related to the content of polyphenols by
In addition, the redox properties of polyphenol Almeida-Muradian et al., (2005) and flavonoids by
compounds, especially flavonoids, play an important Šarić et al., (2009).
role in absorbing and neutralising free radicals, The daily ingestion of bee pollen can regulate the
quenching oxygen and decomposing peroxides intestinal functions, effectively reduce capillary
(Damintoti et al., 2005). fragility and has beneficial effects on the
The antioxidant activity of flavonoids is reflected in cardiovascular system, vision and skin (Pietta, 2000).
the inhibitory effect on lipid peroxidation and In addition, it has been reported to trigger beneficial
increasing the activity of antioxidant enzymes effects in the prevention of prostate problems,
(Kukrić et al., 2013). Flavonoids have different arteriosclerosis, gastroenteritis, respiratory diseases,
structural features and show several biological allergy desensitization, improving the cardiovascular
activities (Šarić et al., 2009). and digestive systems, body immunity and delaying
The best-described property of almost every group of aging (Estevinho et al., 2012).
flavonoids, which are the predominant Phytochemicals, such as phenolic compounds are
phenolic class present in honeybee-collected pollen, considered beneficial for human health since they
is their capacity to act as antioxidants (Kroyer & decrease the risk of degenerative diseases by
Hegedus, 2001). One way is the direct scavenging of reducing oxidative stress and inhibiting
free radicals. Flavonoids are oxidised by radicals, macromolecular oxidation. They have been shown to
resulting in a more stable, less-reactive radical. In possess free radical-scavenging and metalchelating
other words, flavonoids stabilize the reactive oxygen activity in addition to their reported anticarcinogenic
properties (Morais et al., 2011).
10
Conclusions Bogdanov, S. (2011): Pollen: Nutrition, Functional

Properties, Health: A Review. Bee Product Science,
Effects of pharmacological bee pollen still have not Available online: http://www.bee-
hexagon.net/files/file/fileE/Health/PollenBook2Revie
been scientifically based and is commonly used in
w.pdf, (accessed on 11 May 2012).
folk medicine. The composition and chemistry of Bonvehí, J.S., Torrentó, M.S., Lorente, E.C. (2001):
pollen are not yet standardized nor defined with Evaluation of polyphenolic and flavonoid compounds
pharmacopoeia, and may vary due to its botanical and in honeybee-collected pollen produced in Spain, J.
geographical origin, the plant species, environmental Agr. Food Chem. 49, 1848-1853.
conditions, age and status of plants. Because of this, Carpes, T., Begnini, R., Matias de Alencar, S., Masson,
the type of bee pollen depends on the available bee M.L. (2007): Study of preparations of bee pollen
pasture, types of plant species visited by bees and the extracts, antioxidant and antibacterial activity, Ciência
period of flowering for characteristic plant species. e Agrotecnologia 31, 1818-1825.
Antioxidant capacity of bee pollen, as well its other Cheng, N., Ren, N., Gao, H., Lei, X., Zheng, J., Cao, W.
(2013): Antioxidant and hepatoprotective effect of
physico-chemical properties primarily depend on its
Schisandra chinensis pollen extract on CCl4 – induced
botanical and geographical origin of and that is the acute liver damage in mice, Food and Chemical
subject of many scientific and research papers. Toxicology 55, 234-240.
Studies have generally shown significant association Crane, E. (1990): Bees and beekeeping: Science, Practice
between antioxidant capacity and total content of and World Resources. Cornstock Publ., Ithaca, NY.,
polyphenols or flavonoids. USA. 593.
Further studies of bee pollen for certain geographic Damintoti, K., Mamoudou, H.D., Simpore, J., Traore, A.S.
region can be directed to the determination of its (2005): Antioxidant and antibacterial activities of
botanical origin, phenolic constituents and to polyphenols from ethnomedicinal plants of Burkina
determine the quality of the characteristic pollen Faso, African Journal of Biotechnology 4 (8), 823-828.
Dolovac, A. (2005): Savremeno pcelarstvo nauka i praksa.
types from the point of antioxidant capacity, and as a
Bemust. Sarajevo.
result, to provide recommendations of applying Estevinho, L.M., Rodrigues, S., Pereira, A.P., Feás, X.
pollen into functional, nutritional and pharmaceutical (2012): Portuguese bee pollen: palynological study
purposes. nutritional and microbiological evaluation,
Pollen should be used preventively in enrichment of International Journal of Food Science and
everyday food or as a natural dietary supplement, due Technology 47, 429-435.
to it contains all essential amino and fatty acids and Feás, X., Vázquez-Tato, M.P., Estevinho, L., Seijas, J.A.,
all the ingredients for a healthy and normal Iglesias, A. (2012): Organic bee pollen: botanical
development of the organism. origin nutritional value bioactive compounds
antioxidant activity and microbiological quality,
Molecules 17, 835-837.
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12
biomolecules
Article
Phenolic Composition Influences the
Health-Promoting Potential of Bee-Pollen
Mirjana Mosić 1 , Jelena Trifković 1 , Irena Vovk 2 , Uroš Gašić 3, * , Živoslav Tešić 1 ,
Branko Šikoparija 4 and Dušanka Milojković-Opsenica 1, *
1 University of Belgrade—Faculty of Chemistry P.O. Box 51, 11158 Belgrade, Serbia;
mosicm@chem.bg.ac.rs (M.M.); jvelicko@chem.bg.ac.rs (J.T.); ztesic@chem.bg.ac.rs (Ž.T.)
2 Department of Food Chemistry, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia;
irena.vovk@ki.si
3 Institute for Biological Research “Siniša Stanković”—National Institute of Republic of Serbia,
University of Belgrade, Bulevar despota Stefana 142, 11060 Belgrade, Serbia
4 BioSense Institute—Research Institute for Information Technologies in Biosystems, University of Novi Sad,
21000 Novi Sad, Serbia; sikoparijabranko@biosense.rs
* Correspondence: uros.gasic@ibiss.bg.ac.rs (U.G.); dusankam@chem.bg.ac.rs (D.M.-O.);
Tel.: +381-112078385 (U.G.); +381-113336766 (D.M.-O.)

Received: 29 October 2019; Accepted: 17 November 2019; Published: 26 November 2019
Abstract: Information on compositional, nutritional and functional properties of bee-pollen, as a

health-promoting food, is essential for defining its quality. Concerning the nutritional importance
of phenolic compounds, the aim of this study was to determine the phenolic profile and
antioxidant activity of twenty-four bee-pollen samples collected from different regions of Serbia.
High-performance thin-layer chromatographic (HPTLC) fingerprinting was used for profiling of
bee-pollen samples according to the botanical type. HPTLC hyphenated with image analysis and a
pattern recognition technique confirmed the grouping of samples caused by the specific phenolic
composition of pollens of different botanical origin. Flavonoid glycosides in bee-pollen samples
were identified by applying ultra-high-performance liquid chromatography (UHPLC) coupled with
linear ion trap-Orbitrap mass spectrometry (LTQ Orbitrap MS). Eight out of twenty-seven flavonol
glycosides were identified in bee-pollen samples for the first time. All analyzed bee-pollen samples
showed a high number of phenolic compounds which may have therapeutic potential.
Keywords: bee-pollen; HPTLC; UHPLC-MS/MS; phenolic profile; antioxidant activity
1. Introduction
Bee-pollen is the main source of important nutrients and phytochemicals that honeybees collect
for the purpose of feeding their larvae. It is a mixture of floral pollens, nectar and mouth secretions of
bees, accumulated as pellets of different color, size and morphology. Due to its beneficial effect in the
human diet, bee-pollen is considered as a health-promoting food. Inter alia, these effects are related
to the antioxidant properties of the phenolic compounds present in pollen [1]. They have a role in
the protection of vital cell components from oxidative damage by neutralizing free radicals and the
prevention of various diseases such as cancer and cardiovascular and neurodegenerative diseases [2].
Phenolic compounds are also recognized as potentially useful taxonomic markers [3]. For this reason,
the characterization of phenolic compounds in bee-pollen has been of increasing interest in recent years.
Recent papers have reported that bee-pollen usually contains flavonoid glycosides, particularly
derivatives of kaempferol, quercetin, and isorhamnetin, as well as hydroxycinnamic acid derivatives [4].
However, due to the high diversity of phenolic substances, quantitative determination of individual
Biomolecules 2019, 9, 783; doi:10.3390/biom9120783 www.mdpi.com/journal/biomolecules

Biomolecules 2019, 9, 783 2 of 14
flavonoid glycosides is difficult. For example, liquid chromatography (LC) coupled with mass
spectrometry (MS) has been applied in recent research for the identification and quantification of
flavonol glycosides in bee-pollen [4–8], red spice paprika from Serbia [9], as well as in Lathyrus cicera L.
seeds [10]. Using advanced LC-MS techniques, the structures of compounds, for which standards are
not commercially available, could be determined by the study of their characteristic fragmentation
patterns thanks to the great number of rules for structural characterization that were summarized in
the past [11–13]. Moreover, tandem MS experiments provide more fragmentations, giving additional
structural information for the identification of different classes of phenolic compounds [14].
The antioxidant capacity of a number of bee-pollen samples from different regions of the world
has been determined. There are lots of methods appropriate for assessing antioxidant activity, e.g.,
2,2-diphenyl-1-picryhydrazyl· (DPPH·) assay, enzymatic or non-enzymatic measurements of lipid
peroxidation inhibition, the ferric reducing antioxidant power (FRAP) assay, or the Trolox equivalent
antioxidant capacity (TEAC) assay [4,15–17].
Due to the high nutritional value and pronounced health-promoting properties, and the potential
use as a supplement to the human diet, bee-pollen represents a valuable natural product. However,
in the absence of clinical studies that would substantiate the medicinal properties, any new information
about the biological activity of bee-pollen is important. Also, a large variability of the chemical
composition of bee-pollen complicates the determination of parameters of authenticity, as well as
the determination of their biological activity. In our previous publications on the characterization of
bee-pollen samples from Serbia, their physicochemical characteristics, techno-functional properties,
mineral content and aflatoxin contamination were described [18–20]. As a continuation of these
studies, the aims of this work were to analyze the phenolic composition of Serbian bee-pollen samples
using high-performance thin-layer chromatographic (HPTLC) and ultra-high-performance liquid
chromatography (UHPLC) coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC–LTQ
Orbitrap MS) techniques. These techniques have already been proven to be reliable for the unambiguous
detection of various phenolic compounds in different samples and matrices. In addition, authentication
of bee-pollen samples was performed by application of appropriate data evaluation procedures.
2.1. Chemicals and Materials

The compound 2-aminoethyl diphenylborinate (NTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH)
and flavonoid standards (myricetin, naringenin, chrysin, and galangin) were purchased from Fluka
(Steinheim, Germany); acetonitrile and formic acid (both of them MS grade), methanol (HPLC grade),
sodium carbonate, hydrochloric acid, toluene, ethyl acetate, and Folin–Ciocalteu reagent from Merck
(KGaA, Darmstadt, Germany); polyethyleneglycol 4000 (PEG), standards of phenolic compounds
(rutin, hyperoside, astragalin, chlorogenic acid, caffeic acid, gallic acid, and ferulic acid) and Trolox
standard from Sigma-Aldrich (Steinheim, Germany). All chemicals which purity is not previously
emphasized were of analytical purity grade.
Methanolic standard mixture of phenolic compounds was prepared with following final standard
concentrations: galangin (50 ng/µL), chlorogenic acid, myricetin, chrysin and caffeic acid (100 ng/µL),
gallic acid, ferulic acid and naringenin (200 ng/µL).
The cartridges for solid-phase extraction (SPE) were Strata C18–E (500 mg/3 mL) obtained from
Phenomenex (Torrance, CA, USA). Ultrapure water was obtained from a TKA MicroPure (Thermo
Fisher Scientific, Bremen, Germany) water purification system, 0.055 µS/cm. Syringe filters (13 mm,
polyfluorotetraethylene (PTFE) membrane 0.45 µm) were purchased from Supelco (Bellefonte, PA, USA).
2.2. Bee-Pollen Samples

A total of 24 samples of bee-pollen collected in different locations in Serbia were provided by
“The Federation of the Beekeeping Organizations of Serbia” (SPOS) (www.spos.info). The frequency of
pollen types in bee-pollen sample was determined and reported in previous study [18]. Two additional
bee-pollen samples (marked as P17 and P24) were analyzed in this study following the methodology
of pollen analysis described in Kostić et al. [18]. For ease of understanding the results obtained, a list
of investigated samples with results of pollen analysis is presented in the Supplementary Materials
(Table S1).
2.3. Preparation of Bee-Pollen Extracts

Bee-pollen samples (1 g) were suspended in 10 mL of methanol/water (7:3, v/v) acidified with
0.1 mL of concentrated formic acid (5 g/100 g)) (7:3, v/v), ultrasonicated for 1 h and centrifuged at
4500 rpm. The supernatant was separated, and re-extraction was performed with the solid residue.
The extracts were pooled, and the methanol was evaporated under vacuum at 40 ◦ C until the entire
solvent was removed. Then, HCl solution (0.1 g/100 g) was added to the aqueous extract to final
volume of 10 mL. In order to remove sugars and polar substances other than phenolic, 2 mL of
prepared solution was cleaned up and concentrated by sorption on a SPE cartridge. The cartridge
was previously activated according to the procedure described in Waisi et al. [21]. Elution of the
phenolic fraction was performed with 1.5 mL acidified methanol (0.1% HCl). Afterwards, this fraction
was dissolved (1:10, v/v) in acetonitrile/water (1:1, v/v). The obtained solutions were stored at –20 ◦ C.
Prior to HPTLC and UHPLC–LTQ Orbitrap MS analysis, bee-pollen extracts were filtered by a 0.45 µm
PTFE membrane filter.
2.4. Determination of the Total Phenolic Content

The total phenolic content (TPC) was estimated spectrophotometrically according to the
Folin–Ciocalteu method [22]. A total of 2.5 mL of Folin–Ciocalteu reagent was added to 0.5 mL of each
pollen extract and left at room temperature for 5 min, after which 2 mL of sodium carbonate solution
(7.5 g/100 mL) was added. After incubation in the dark for 2 h at 25 ◦ C, absorbance was measured at
765 nm against a blank (the same mixture without the sample) using a Cintra 6 spectrophotometer (GBC
Scientific Equipment Ltd., Dandenong, Australia). Gallic acid (20–100 µg/mL) was used as the standard
and TPC was expressed as the mg of gallic acid equivalent (GAE) per gram of bee-pollen sample.
2.5. High-Performance Thin-Layer Chromatography

Chromatographic separation was performed on silica gel HPTLC plates (20 × 10 cm, Art. 5641,
Merck, Darmstadt, Germany). Bee-pollen extracts (2 µL) were applied (as 8 mm bands by an
Automatic Thin-layer chromatography (TLC) sampler 4 (ATS4, CAMAG, Muttenz, Switzerland).
The composition of mobile phases was as follows: toluene/ethyl acetate/formic acid (4/7/1, v/v/v) and
ethyl acetate/water/formic acid (17/2/2, v/v/v) [21]. Twin trough chamber (CAMAG) was saturated
for 20 min with the mobile phase before analysis. The chromatogram was developed upward until a
distance of 80 mm and dried for 2 min in a stream of warm air. After 3 min of heating of the plates
at 100 ◦ C (TLC Plate Heater III, CAMAG), the spot pattern was developed by 0.5% solution of NTS
in ethyl acetate (1 s, Chromatogram Immersion Device III, CAMAG). Distinction of the spots was
performed after 5 min (plates were dried at room temperature in the fume hood), with 5% solution
of PEG 4000 in dichloromethane for 1 s. Images were captured at 366 nm (CAMAG DigiStore 2
documentation system in conjunction with Reprostar 3 (CAMAG) controlled by the winCATS software
(IZASA SCIENTIFIC, Madrid, Spain, Version 1.4.3.6336), four apertures with exposure time of 30 ms
and frame of –2 mm [23], as tagged image file format (TIFF) files.
Image acquisition was performed by Image J processing program [24] using the procedure
described previously [23]. Processing of images included the following steps: denoising (2 pixels median
filter), baseline removal (none, background intensity between images were similar), normalization
(scaling to sum of intensity), warping (correlation optimized warping (COW) algorithm with P16
as target sample (PLS ToolBox, v.6.2.1, for MATLAB 7.12.0 (R2011a) [25], Eigenvector Research, Inc.,
Wenatchee, WA 98801), mean centering.
2.6. UHPLC–LTQ Orbitrap MS

An Accela liquid chromatography (LC) system connected to a linear ion trap–Orbitrap hybrid
mass spectrometer (LTQ OrbitrapXL, Thermo Fisher Scientific) with heated electrospray ionization
(HESI) was used for the identification of flavonoid glycosides in bee-pollen samples. Chromatographic
separation and mass spectrometry parameters were same as previously reported by Guffa et al. [26].

Principal component analysis (PCA) with a singular value decomposition algorithm (SVD) was
performed using PLS ToolBox, v.6.2.1, provided by MATLAB 7.12.0 (R2011a). Kaiser Criterion was
applied for optimal number of principal components (eigenvalues greater than 1 were cut-off).
3.1. Floral Origin of Bee-Pollen Loads

Palynological analysis (Table S1) for 22 samples [18] and for two additional samples identified
24 plant taxa and rust spores in bee-pollen. Eleven samples were monofloral, eight samples were
bifloral and five samples were polyfloral [27].
3.2. HPTLC Fingerprint of Phenolics in Bee-Pollen Samples

Separation and assessment of all the ingredients of complex phenolic mixtures are very difficult to
perform. Instead of targeted analysis of a metabolite, a characteristic chromatographic profile could be
used for comparison which leads to sample recognition. In that sense, screening of phenolic mixtures of
analyzed bee-pollen samples was performed by TLC analysis and obtained chromatograms observed
as a unique multivariate fingerprint. A literature search revealed that no study on the identification of
phenolics in bee-pollen by TLC has been published.
Due to the complexity of bee-pollen extracts, HPTLC systems were optimized in terms of the best
resolution of different polarity phenolic compounds. Different chromatographic systems (CS1 and
CS2) were used for the less polar fraction and for the medium and highly polar fraction, respectively.
The standard mixture was simultaneously analyzed in both systems with substances obtained in the
following ascending order of retention factor (RF ) values: chlorogenic acid, gallic acid, myricetin,
chrysin, caffeic acid, ferulic acid, naringenin, galangin. HPTLC chromatograms of bee-pollen extracts
and the standard mixture are presented in Figure 1.
Figure 1. High-performance thin-layer chromatography (HPTLC) chromatograms of bee-pollen extracts,

(a) chromatographic system (CS)1, (b) CS2. P—Pollen samples (Table S1); SM—standard mixture.
Chromatographic system 1 with low elution strength could not be used for the separation of a
vast number of more polar compounds strongly adsorbed on silica gel (Figure 1a). However, non-polar
flavonoids, such as flavonols, flavanons and isoflavonoids, showed specific patterns with orange and
green colored zones which appear at higher RF values. A different profile of these bands could be
observed. CS2, used for the scanning of profiles of more polar compounds present in bee-pollen
samples, gave chromatograms with good separation efficiency and low background noise. The applied
mobile phase successfully separated medium and highly polar constituents (mainly the more polar
phenolic acids such as chlorogenic, gallic and ellagic acids), and the different flavonoid aglycones
(apigenin, quercetin, kaempferol, and their glycosides) [28,29]. Chromatographic profiles were rich in
well defined, sharp zones which formed two dominant patterns, first with blue colored zones at RF
values between 0.6 and 0.8 which are present in all samples and could be considered as a characteristic
feature of Serbian bee-pollen, and second with orange bands that could be attributed to very polar
glycosides at lower RF values, with profiles that depend on the samples and could be specific to certain
botanical origins.
The chromatographic profiles gave basic information on the phenolic profile of bee-pollen samples,
but could not provide enough detail to define the authenticity of bee-pollen and the parameters which
could be used as markers of certain botanical origins. Further analysis was focused not on the particular
sample but on the matrix of data consisting of all samples and each dot on the chromatograms.
In order to transfer results from TLC chromatograms to numerical values, multivariate image
analysis was performed [30]. Due to the existence of different colored bands of phenolic compounds
at 366 nm, in order to increase selectivity, images were split through red (R), green (G) and blue (B)
channels. Red/green/blue (RGB) images were analyzed with Image J software and raw data were
further processed by a pattern recognition method. The line profile plots of chromatograms for
pollen samples of particular botanical origin, according to its RGB channels, are presented in Figure 2.
Differences in profile plots of the same sample are the result of different color values of each point on the
chromatographic profile depending on the channel applied. The line profile plots of the chromatogram
corresponding to sample P6 (predominantly Apiaceae pollen type) according to RGB channel are
presented in Figure 2a. Visual review revealed that the green channel was the most informative for the
analyzed pollen samples (Figure 2a). Also, phenolic line profile plots obtained for samples in which
one pollen type predominates (samples P1 and P10 with a predomination of Brassicaceae and Fabaceae
pollen type; P6, P18 and P22 with a predomination of Apiaceae, Ranunculaceae and Sophora pollen
type, respectively) were shown to be notably different (Figure 2b,c).
Six data matrices for three channels and two chromatographic systems, each consisting of 24
samples and 526 variables (intensities of pixels along the length line), were subjected to PCA in
order to mark compounds that are the most convenient for characterization of a particular pollen
type. Pre-treatment of the data before chemometric evaluation enabled further classification (Figure
S1, Supplementary Materials). The parameters of PCA models for six data sets revealed the best
differentiation and classification results in case of the green channel of profiles obtained with CS1.
Further, only this model will be discussed. The five-component model accounted for 93.04% of total
variance (PC1—35.61% and PC2—33.70%).
Figure 2. The line profile plots of chromatograms of (a) sample P6 with a predomination of Apiaceae
pollen type adjusted to three red/green/blue (RGB) channels, (b) samples P1 and P10, respectively,
adjusted to the green channel, (c) samples P6, P18 and P22, respectively, adjusted to the green channel.
RF : retention factor
According to the score plot (Figure 3a), two distinctive groups consisting of samples P20 and P22,
predominantly Sophora pollen, and samples P11 and P12, predominantly Rosaceae and Plantago pollen,
respectively, were classified in comparison to other samples where other pollen is more abundant.
Samples P15 (with rust spores), P18 (with a high percentage of Ranunculaceae), and P24 (with a high
amount of Chenopodiaceae), are isolated on the graph. Pollen from Brassicaceae was predominant
in five of the analyzed samples (P1, P3, P8, P9, and P13) and almost every other analyzed sample
contained some percentage of it. Going along the PC1 axis from its negative end towards the positive
values, the amount of this pollen type decreased. Samples P1, P3, P8, P9, and P13, with the highest
amount of Brassicaceae pollen, are positioned in the left upper part of the graph. Sample P3 was
slightly separated from this group, which seems to be related to the notable amount of Salix pollen
present which obviously has a high impact on the phenolic profile. Samples with predominantly
Fabaceae pollen (samples P2, P4, P5, P10, P16, P19, and P21) formed a compact cluster positioned
relatively close to the group with predominantly Brassicaceae pollen. Namely, all these samples
contain Brassicaceae pollen in an amount of 3–25%. Apiaceae pollen seems to have an important
influence on the phenolic profile, although it is present in small amounts. Samples P6, P14 and P17,
with 69%, 3–15%, and 28% of this pollen type, respectively, are grouped very close together and close
to clusters with predominantly Brassicaceae and Fabaceae pollen, and therefore probably contain some
identical phenolics. Sample P23, which contains predominantly Robinia pollen, was positioned within
the group that has predominantly Fabaceae pollen, suggesting that it has a similar phenolic content,
which is in line with the fact that false locust (Robinia) belongs to the Fabaceae plant family. There are
a certain number of scientific works that deal with determining the profile of a variety of chemical
compounds in order to define the botanical origin of bee-pollen [7,31]. Since the bee-pollen samples
investigated in this study present a mixture of more than ten different pollen types, differentiation
and classification based on chemical profile and distinct separation among the groups are difficult to
perform, particularly when observing only one class of compounds and not combining several groups
of parameters. Similar results were obtained based on NIR spectra of bee-pollen samples collected
from twelve different Brazilian states [32], while Castiglioni et al. suggest the possibility of using
IR measurements for the classification of some Italian bee-pollen samples according their botanical
origin [31]. The loading plots (Figure 3b–d) reveal that the zones with RF values of 0.04, 0.11, 0.15,
and 0.23 are parameters that showed the most positive impact on PC1 direction and can differentiate
pollen samples according to the botanical type. Zones with RF values of 0.05 and 0.15 significantly
affect the PC2 in a positive manner, while zones with a negative influence on the second component
have RF values of 0.23 and 0.63.
Figure 3. Principal component analysis (PCA) performed on data obtained from HPTLC phenolic
profiles of bee-pollen samples, (a) mutual projections of factor scores, (b) loading plot for PC1 and PC2,
(c,d) loadings for the PC1 and PC2, respectively.
3.3. Identification of Flavonoid Glycosides by UHPLC–LTQ Orbitrap MS

The qualitative profile of flavonoid glycosides with mono-, di- and trisaccharide moieties present
in bee-pollen samples was determined using an UHPLC system coupled to an LTQ Orbitrap mass
analyzer. Their characteristics (mass spectra, accurate mass, MS/MS fragmentation pattern and
characteristic retention time) were used for their identification. We also used available standards as
another confirmation for these compounds. It was found that all of the identified compounds are from
the same group of flavonols and all of them were marked as 3-O-glycosides of quercetin, isorhamnetin,
and kaempferol. The exact mass search and the study of the MS/MS fragmentation described in the
literature enabled us to identify 27 flavonol glycosides (Figure 4 and Table 1).
Figure 4. Chemical structures of different flavonol glycosides found in the bee-pollen collected in
Serbia (R1–4 = HEX—hexosyl, RHAM—rhamnosyl, PENT—pentosyl or MAL—malonyl.).
The obtained data on the retention times (tR , min) and MS parameters for the identified
glycosides are summarized in Table 1. The presence of each identified peak (glycoside) from all
bee-pollen samples is shown in Table S2 (Supplementary Material). Tentative identification of flavonol
glycosides was done using the MS fragmentation data available in the literature [5–8,33]. The nature of
interglycosidic linkages (1→2 or 1→6) for the identified flavonol glycosides was proposed according
to Ferreres et al. [11].
MS/MS fragmentation of deprotonated molecular ion [M−H]− at m/z 447 (compound 25) produced
the fragment ion Y0 − at m/z 285 ([M−H−162]− ) together with the formation of the radical anion [Y0 -H]−
at m/z 284. The observed fragment m/z 327 originates from the 0,2 X− scission of the glycoside [34],
indicating that the structure of compound 25 is kaempferol 3-O-glucoside or astragalin, which is
confirmed by the standard.
Table 1. Peak numbers, retention times (tR ), molecular formulas, calculated and exact masses, mean mass accuracy (ppm), and MS/MS fragments of flavonol glycosides
identified in bee-pollen samples.
Peak Molecular Calculated Exact Mass,

tR , min ∆, ppm MS/MS Fragmentation (%) Identification
Number Formula, [M–H]− Mass, [M–H]− [M–H]−
1 5.30 C27 H29 O17 − 625.14102 625.14233 2.10 505(5), 463(100), 462(20), 343(5), 301(30) Quercetin 3,7-di-O-hexoside
2 5.74 C33 H39 O21 − 771.19893 771.20038 1.88 609(100), 463(10), 301(5) Quercetin 3-O-(600 -O-rhamnosyl)hexoside-7-O-hexoside b
505(15), 463(15), 445(50), 343 (10), 301(50), 300(100),
3 5.85 C27 H29 O17 − 625.14102 625.14172 1.12 Quercetin 3-O-(200 -O-hexosyl)hexoside
271(20), 255(10)
4 5.89 C24 H21 O14 − 533.09368 533.09454 1.61 489(100), 285(5) Kaempferol 3-O-(600 -O-malonyl)hexoside
5 5.90 C27 H29 O16 − 609.14611 609.14636 0.41 581(10), 489(10), 447(70), 446(50), 327(5), 285(100) Kaempferol 3,7-di-O-hexoside
6 5.91 C34 H41 O21 − 785.21458 785.21509 0.65 623(100), 477(10), 315(5) Isorhamnetin 3-O-(600 -O-rhamnosyl)hexoside-7-O-hexosideb
7 5.96 C28 H31 O17 − 639.15667 639.15717 0.78 477(40), 476(20), 357(5), 315(100), 300(10) Isorhamnetin 3,7-di-O-hexoside b
753(10), 625(100), 609(30), 591(40), 573(20), 445(10),
8 5.98 C33 H39 O21 − 771.19893 771.20056 2.11 Quercetin 3-O-(200 -O-hexosyl)hexoside-7-O-rhamnosideb
427(10), 409(10), 301(45), 300(50)
475(10), 463(10), 445(15), 301(45), 300(100), 271(20),
9 6.00 C26 H27 O16 − 595.13046 595.13098 0.87 Quercetin 3-O-(200 -O-pentosyl)hexoside b
255(10)
624(20), 519(15), 491(10), 477(20), 459(65), 444(30),
10 6.02 C28 H31 O17 − 639.15667 639.15784 1.83 Isorhamnetin 3-O-(200 -O-hexosyl)hexoside
315(100), 314(50), 300(50), 299(60)
489(15), 463(10), 445(20), 429(10), 343(5), 301(20),
11 6.04 C27 H29 O16 − 609.14611 609.14642 0.51 Quercetin 3-O-(200 -O-rhamnosyl)hexoside
300(100), 285(5), 271(15), 255(10)
489(5), 447(10), 429(100), 327(10), 285(90), 284(80),
12 6.07 C27 H29 O16 − 609.14611 609.14697 1.41 Kaempferol 3-O-(200 -O-hexosyl)hexoside
255(15)
609(100), 593(90), 575(85), 429(20), 285(90), 284(70),
13 6.19 C33 H39 O20 − 755.20402 755.20471 0.91 Kaempferol 3-O-(200 -O-hexosyl)hexoside-7-O-rhamnoside b
255(25)
608(10), 503(10), 477(10), 459(30), 444(10), 327(10),
14 6.22 C28 H31 O16 − 623.16176 623.16211 0.56 Isorhamnetin 3-O-(200 -O-rhamnosyl)hexoside isomer 1
315(15), 314(100), 300(10), 299(65)
15 6.24 C27 H29 O16 − 609.14611 609.14679 1.12 343(5), 301(100), 300(20), 271(10), 255(5) Quercetin 3-O-(600 -O-rhamnosyl)glucoside (Rutin) a
− 577(15), 489(5), 477(5), 459(50), 357(10), 315(100),
16 6.26 C27 H29 O16 609.14611 609.14716 1.72 Isorhamnetin 3-O-(200 -O-pentosyl)hexoside b
314(90), 300(20), 299(30)
473(5), 447(10), 429(50), 327(5), 285(40), 284(100),
17 6.28 C27 H29 O15 − 593.15119 593.15161 0.71 Kaempferol 3-O-(200 -O-rhamnosyl)hexoside
255(20)
459(5), 447(20), 429(60), 327(10), 285(75), 284(100),
18 6.30 C26 H27 O15 − 579.13554 579.13678 2.14 Kaempferol 3-O-(200 -O-pentosyl)hexoside
255(30)
591(5), 503(10), 477(10), 459(25), 357(5), 315(35),
19 6.34 C28 H31 O16 − 623.16176 623.16248 1.16 Isorhamnetin 3-O-(200 -O-rhamnosyl)hexoside isomer 2
314(100), 300(10), 299(25)
20 6.39 C21 H19 O12 − 463.08820 463.08951 2.83 301(100), 300(20) Quercetin 3-O-galactoside (Hyperoside) a
21 6.43 C27 H29 O16 − 609.14611 609.14697 1.41 315(100), 300(20), 271(10), 255(5) Isorhamnetin 3-O-(600 -O-pentosyl)hexoside b
22 6.54 C24 H21 O15 − 549.08859 549.08923 1.17 505(100), 301(5) Quercetin 3-O-(6”-O-malonyl)hexoside
23 6.57 C28 H31 O16 − 623.16176 623.16272 1.54 315(100), 314(10), 300(20), 271(10), 255(5) Isorhamnetin 3-O-(600 -O-rhamnosyl)hexoside
24 6.65 C22 H21 O12 − 477.10385 477.10477 1.93 315(100), 314(20) Isorhamnetin 3-O-hexoside isomer 1
25 6.70 C21 H19 O11 − 447.09329 447.09424 2.12 327(20), 285(80), 284(100), 255(10) Kaempferol 3-O-glucoside (Astragalin) a
26 6.78 C22 H21 O12 − 477.10385 477.10437 1.09 449(5), 357(15), 315(30), 314(100) Isorhamnetin 3-O-hexoside isomer 2
27 6.86 C25 H23 O15 − 563.10424 563.10486 1.10 519(100), 315(5) Isorhamnetin 3-O-(600 -O-malonyl)hexoside
a Confirmed using available standards; all other compounds were identified based on MS/MS data. b Identified for the first time in bee-pollen.
Furthermore, identification of two derivatives of isorhamnetin 3-O-hexoside (compounds 24 and

26) with m/z 477 was established from the presence of the Y0 − ion and radical anion [Y0 −H]− at m/z
315 and 314, respectively. Fragment ion at m/z 449 present in compound 26 originated from loss of
small neutral molecule CO [M−H−28]− .
Compound 20 exhibited a deprotonated molecular ion [M−H]− at m/z 463. The MS/MS spectrum
in the negative ionization mode showed fragments Y0 − at m/z 301 ([M−H−162]− ) and a radical anion
[Y0 −H]−· at m/z 300, suggesting the loss of the hexosyl moiety and quercetin as aglycone. The proposed
identification of compound 20 is quercetin 3-O-galactoside or hyperoside, which was confirmed
by the standard. A similar fragmentation pattern for compounds 4 (m/z 533), 22 (m/z 549), and 27
(m/z 563) was observed with a specific fragment ion at [M−H−44]− , indicating the presence of a
carboxylic group (malonyl moiety) [33]. Further examination of mass spectra and molecular formulas
of these compounds allowed us to identify these glycosides as kaempferol 3-O-(6”-O-malonyl)hexoside,
quercetin 3-O-(6”-O-malonyl)hexoside and isorhamnetin 3-O-(6”-O-malonyl)hexoside, respectively.
In the negative ion MS/MS spectra of compounds 11 (m/z 609), 14 (m/z 623), 15 (m/z 609), 17 (m/z
593), 19 (m/z 623), and 23 (m/z 623), the Y0 − [M−H−308]− fragment originating from the losses of the
rhamnosyl-hexoside moiety was observed. Ion product spectra of these compounds also yielded
radical ions [Y0 –H]−· with higher intensity which are characteristic for 3-O-glycosides [35]. The relative
abundance of the radical [Y0 –H]−· is higher than the deprotonated Y0 − ion for compounds 11, 14,
17, and 19. In addition, fragment ions that originate from 0,2 X− scission of the terminal rhamnose
residue [M−H−120]− together with formation of the [M−H−164]− by loss of rhamnose molecule, are
also present in the mass spectra of these compounds and not observed for compounds 15 and 23.
This means that the interglycosidic linkage in compounds 11, 14, 17, and 19 was the 1→2 type, while in
compounds 15 and 23, it was the 1→6 interglycosidic linkage. [36]. Based on these considerations,
we could identify compounds 11, 14, 17, and 19 as quercetin 3-O-(2”-O-rhamnosyl)hexoside;
isorhamnetin 3-O-(2”-O-rhamnosyl)hexoside, isomer 1; kaempferol 3-O-(2”-O-rhamnosyl)hexoside;
and isorhamnetin 3-O-(2”-O-rhamnosyl)hexoside, isomer 2, respectively. The proposed fragmentation
pathways of compounds 14 and 19 are depicted in Figure S2. Compound 23 was tentatively
identified as isorhamnetin 3-O-(6”-O-rhamnosyl)hexoside and compound 15 was marked as quercetin
3-O-(6”-O-rhamnosyl)glucoside or rutin (also confirmed by the standard).
MS/MS spectrum of compounds 3 (m/z 625), 10 (m/z 639), and 12 (m/z 609) in the negative ionization
mode, showed fragment Y0 − and the radical anion [Y0 –H]− , suggesting the loss of two hexose units.
Fragments that originated by losses of the hexosyl moiety at ([M−H−162]− ) and hexosyl and H2 O
at ([M−H−162−18]− ) were characteristic for sugar units linked one to another (and not directly to
the aglycone) were also presented as well as fragment ions that originate from 0,2 X− scission of the
terminal hexose residue [M−H−120]− . This information allows us to determine that compounds 3, 10,
and 12 are 3-O-(2”-O-hexosyl)hexosides of quercetin, isorhamnetin, and kaempferol, respectively [37].
There are similar fragmentation patterns for compounds 9 (m/z 595), 16 (m/z 609), and 18 (m/z 579).
The presence of Y0 − fragment ions at m/z 301, m/z 315 and m/z 285 indicated that in these compounds,
the aglycones were quercetin, kaempferol and isorhamnetin, and it also suggested successive losses of
pentosyl (132 Da) and hexosyl (162 Da) moieties. According to presence of characteristic fragments
([M−H−120]− , [M−H−132]− , and [M−H−132−18]− ) in MS/MS spectra, these compounds were marked
as 3-O-(2”-O-pentosyl)hexosides of quercetin, isorhamnetin, and kaempferol, respectively.
Compound 21 (m/z 609) is an isomer of compound 16. In its mass spectra, only the Y0 − ion at
m/z 315 was observed, indicating a 1→6 interglycosidic linkage between sugars, so we determined
compound 21 was isorhamnetin 3-O-(6”-O-pentosyl)hexoside.
In the negative ionization mode, MS/MS spectra of compounds 1 (m/z 625), 5 (m/z 609), and 7 (m/z
639) showed high intensity of MS/MS fragments at [M−H−162]− (m/z 463, 447, and 477, respectively)
corresponding to loss of hexose in the C−3 or C−7 position. Based on these findings, the proposed
identification for these compounds was quercetin 3,7-di-O-hexoside, kaempferol 3,7-di-O-hexoside,
and isorhamnetin 3,7-di-O-hexoside, respectively [11].
Compounds 2 (m/z 771), 6 (m/z 785), 8 (m/z 771), and 13 (m/z 755) are triglycosides. MS/MS
spectra of these compounds exhibited fragment [M−H−146−162−162]− which correspond to aglycones
and the loss of two hexosyl and one deoxyhexosyl (rhamnosyl) unit. Characteristic fragments
in the MS/MS spectra of compounds 8 and 13 were at m/z 625 and 609, indicating the loss of a
rhamnosyl moiety from the hydroxyl group at the C−7 position [38]. Characteristic fragments
[M−H−146−162−18]− at m/z 445 and 429 were also present. Therefore, the proposed structures of
compounds 8 and 13 were 3-O-(2”-O-hexosyl)hexoside-7-O-rhamnosides of quercetin and kaempferol,
respectively [39,40]. Compound 2 is an isomer of compound 8, but its fragmentation pattern, with Y0 −
fragment ions at m/z 301 (deprotonated quercetin) and at m/z 609 ([M−H−162]− ), corresponds to
compound 15 with one more hexose unit. Therefore, compound 2 was marked as quercetin
3-O-(6”-O-rhamnosyl)hexoside-7-O-hexoside. The proposed structure of compound 6 was isorhamnetin
3-O-(6”-O-rhamnosyl)hexoside-7-O-hexoside, due to its similar spectral characteristics as compound
2 [41].
Analyzed bee-pollen samples are mixtures of more than ten different floral pollen types so the
twenty-seven identified flavonol glycosides was expected. Each of the analyzed bee-pollen samples
contains over fourteen flavonol glycosides, except for sample P20 (with a predomination of the Sophora
pollen type) which has twelve of these compounds. The largest number of flavonol glycosides (total
of 25) has been identified in the sample P11 (with a predomination of the Rosaceae pollen type).
Several compounds were identified in bee-pollen samples for the first time, including compounds 2, 6, 7,
8, 9, 13, 16, and 21. Compounds 3, 4, 17 and 20 are present in all bee-pollen samples. Their quantification
could help with the identification of floral pollen types by using flavonol glycosides as markers to
classify pollen from different plant species [42], but this was not conducted due to the lack of appropriate
standards. The characterization of bee-pollen, in terms of their constituent pollens, would be easier
if bee-pollen pellets were manually selected on the basis of color and size. It has been proven that
certain bee-pollen pellet types contain pollen from a single floral source [3]. However, in our research,
we opted for a mixture because the bee-pollen in this form is sent to the market. Separation into
families would otherwise be unprofitable for beekeepers.
3.4. The Total Phenolic Content of Bee-Pollen Samples

Bee-pollen samples were characterized with TPC values ranging between 5.60 and 30.24 mg of
gallic acid per gram of pollen (Table S3, Supplementary Material). The highest level of total phenolics
was found in sample P15 (with the most dominant rust spores) and the lowest level was determined in
sample P5 (with a predomination of the Fabaceae pollen type). Generally, all samples with Fabaceae as
the predominant pollen type showed lower TPC values. In addition, samples with a predomination
of the Sophora pollen type also had low levels of total phenols. The range of phenolic compound
content was wider in relation to the previously published results. Mărghitaş et al. [43] found TPC
values between 4.4 mg and 16.4 mg GAE/g in twelve bee-pollen samples from the Transylvania area of
Romania, while in ten bee-pollen samples from Turkey, TPC values ranged from 5.09 mg to 17.46 mg
GAE/g [44]. Additionally, the maximal concentration of phenolic compounds found in our study was
higher than those reported for bee-pollen from five Portuguese natural parks, which were between
10.5 mg and 16.8 mg GAE/g of pollen [16]. The results of Le Blanc et al. [17] for bee-pollen from the
Sonoran Desert, and Pascoal et al. [45], who analyzed bee-pollen from Portugal and Spain, were slightly
superior due to phenolic contents between 15.91 mg and 34.85 mg GAE/g and 18.54 mg and 32.15 mg
GAE/g, respectively. We assumed that these differences between TPC could be ascribed to different
botanical and geographical origins of the analyzed bee-pollen samples.
4. Conclusions
A comprehensive study of the phenolic compounds in bee-pollen samples from Serbia was
conducted. HPTLC fingerprinting confirmed the grouping of samples caused by specific phenolic
compositions of pollens of different botanical origin. A UHPLC system coupled to an LTQ Orbitrap
mass analyzer allowed for the identification of twenty-seven flavonol glycosides. Eight of them were
identified in bee-pollen samples for the first time. Determination of species-specific phenolic profiles is
needed for the precise characterization of samples in terms of constituent pollens. We assume that this
could be achieved by the quantification of certain flavonol glycosides and sorting bee-pollen pellets on
the basis of color and size. Analyzed bee-pollen samples have high amounts of phenolic compounds,
which may have therapeutic potential. The findings presented here corroborate the relevance of
bee-pollen from Serbia as a healthy addition to the human diet.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/9/12/783/s1,

Table S1: Frequency classes of identified pollen types and interpretation of floral origin from results of pollen
analysis published in Kostić et al., 2015 [18], Table S2: Presence of each identified glycoside in bee-pollen samples,
Table S3: Total phenolic content (TPC) of bee pollen samples, Figure S1: Raw data (a) versus preprocessed data (b)
of HPTLC profiles, Figure S2: Proposed fragmentation pathway of compounds 14 and 19 (m/z 623).
Author Contributions: Conceptualization, M.M., Ž.T., and D.M.-O.; methodology and writing—original draft,
M.M., J.T., U.G., and B.Š.; validation and investigation, M.M., J.T., and U.G.; formal analysis, M.M. and J.T.;
writing—review and editing, M.M., J.T., U.G., D.M.-O., and I.V.
Funding: This work was supported by the Ministry of Education, Science and Technological Development of
Serbia, Grant No. 172017, the Slovenian Research Agency (research core funding No. P1-0005) and RegPot project
FCUB ERA GA No. 256716 (the EC does not share responsibility for the content of the article).
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antioxidants
Review
Bee Collected Pollen and Bee Bread: Bioactive
Constituents and Health Benefits
Rodica Mărgăoan 1 , Mirela Strant, 2 , Alina Varadi 2 , Erkan Topal 3 , Banu Yücel 4 ,
Mihaiela Cornea-Cipcigan 5, * , Maria G. Campos 6,7, * and Dan C. Vodnar 8
1 Advanced Horticultural Research Institute of Transylvania, University of Agricultural Sciences and
Veterinary Medicine Cluj-Napoca, 400372 Cluj-Napoca, Romania; rodica.margaoan@usamvcluj.ro
2 Association Health with CasaBIO, 400015 Cluj-Napoca, Romania; mirela.strant@gmail.com (M.S.);
alina@casabio.ro (A.V.)
3 Apiculture Section, Aegean Agricultural Research Institute, İzmir 35661, Turkey;
topalerkan@tarimorman.gov.tr
4 Department of Animal Science, Faculty of Agriculture, Ege University, İzmir 35100, Turkey;
banu.yucel@ege.edu.tr
5 Faculty of Horticulture, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca,
400372 Cluj-Napoca, Romania
6 Observatory of Drug-Herb Interactions, Faculty of Pharmacy, University of Coimbra, Heath Sciences
Campus, Azinhaga de Santa Comba, 3000-370 Coimbra, Portugal
7 Coimbra Chemistry Centre (CQC, FCT Unit 313) (FCTUC), University of Coimbra, Rua Larga,
3000-370 Coimbra, Portugal
8 Department of Food Science, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca,
400372 Cluj-Napoca, Romania; dan.vodnar@usamvcluj.ro
* Correspondence: mihaiela.cornea@usamvcluj.ro (M.C.-C.); mgcampos@ff.uc.pt (M.G.C.)

Received: 18 October 2019; Accepted: 18 November 2019; Published: 20 November 2019
Abstract: Bee products were historically used as a therapheutic approach and in food consumption,
while more recent data include important details that could validate them as food supplements due
to their bioproperties, which support their future use as medicines. In this review data, data collected
from bee pollen (BP) and bee bread (BB) essays will be discussed and detailed for their nutritional and
health protective properties as functional foods. Dietary antioxidants intake derived from BP and BB
have been associated with the prevention and clinical treatment of multiple diseases. The beneficial
effects of BP and BB on health result from the presence of multiple polyphenols which possess
anti-inflammatory properties, phytosterols and fatty acids, which play anticancerogenic roles, as well
as polysaccharides, which stimulate immunological activity. From the main bioactivity studies with
BP and BB, in vitro studies and animal experiments, the stimulation of apoptosis and the inhibition
of cell proliferation in multiple cell lines could be one of the major therapeutic adjuvant effects to
be explored in reducing tumor growth. Tables summarizing the main data available in this field
and information about other bio-effects of BP and BB, which support the conclusions, are provided.
Additionally, a discussion about the research gaps will be presented to help further experiments that
complete the tree main World Health Organization (WHO) Directives of Efficiency, Safety and Quality
Control for these products.
Keywords: antioxidant activity; bee pollen; bee bread; cancer; diseases; health; natural product
1. Introduction
Currently, there is a change in the understanding of food production and consumption and the
development of functional foods is an important sector in the food market. Foods that are beyond their
Antioxidants 2019, 8, 568; doi:10.3390/antiox8120568 www.mdpi.com/journal/antioxidants

Antioxidants 2019, 8, 568 2 of 33
basic nutritional features, such as value-added or health-oriented products that positively influence
our well being and quality of life are known as “functional foods”. This term underlines the positive
correlation of the bioactive compounds present in these products along with health [1–3].
This new concept is assimilated to Hippocrates, the founder of medicine, with the nearly 2500
year old philosophy: “Let food be the medicine and medicine be the food”: paying more attention to a
healthy nutrition. Nowadays, due to reasons such as an increase in treatment expenses, labor loss, an
increase in life expectancy and a high percentage of populations’ aged, people desire to improve their
quality of life.
Among the 21st century diseases, one of the biggest concerns is the increase of cancer. Learning
to deal with the disease and providing better tools for health professionals that help patients during
and after treatments will be a successful way of treatment in the future. Surgeries, chemotherapy
and radiotherapy are the most used methods along with immunotherapy and molecular-targeted
therapy. The multifactor associated with these protocols and with anti-cancer agents (example:
drug-herb interactions, angiogenic and/or estrogen-like products, growth factors etc.) sometimes lead
to uncontrolled metastatic tumors as well as high rates of adverse effects, particularly among elderly
patients [4–7]. Therefore, scientists, physicians and patients with cancer make efforts and invest time
to discove and develop safer and more effective future treatment schemes. The research with bee
products has now come to a point that where a possible role in those protocols, namely efficacy, safety
and quality control, will be assured.
Bee collected pollen (BP) and bee bread (BB) have a high nutritional value and include bioactive
compounds, which have a positive effect on human health, and therefore, are regarded as “functional
foods”. These products are rich in proteins, simple sugars, essential amino acids and omega fatty acids.
These features strengthen immunity and help the body to fight bacteria, which will keep the body
healthy, provided the body can perform a quality tissue repair [8]. Bee products are in the structure
of many biochemical components found throughout functional foods, such as prebiotics, probiotics,
fibre, phytochemicals (polyphenols, phenolic acids, lignans, triterpens, steroids), bioactive peptides,
minerals, vitamins and organic acids. Furthermore, among all these compounds, phenolics, flavonoids
and carotenoids [9] have been significantly studied in people with cancer, arteriosclerosis, weakened
immune system, Parkinson’s, Alzheimer’s, cardiovascular diseases and arthritis, as well as significant
preventive and therapeutic effects on the body against premature aging [10]. The main handicap
of these activities is that what can be significant for some of them may be dangerous for others; for
instance, the angiogenic effect is beneficial for arteriosclerosis, arthritis, Parkinson’s, Alzheimer’s and
cardiovascular diseases. In cancer, they can have an anti-inflammatory effect; however, they should be
carefully evaluated for the benefit-risk ratio, as this angiogenic activity can be dangerous.
In this review, firstly BP and BB composition and bioactivities are introduced, followed by
in vivo and in vitro studies of the anti-cancer effects of BP and BB. Moreover, their effectiveness in the
prevention and treatment of diverse anti-cancer agent-induced toxicities in animal models and patients
with cancer are investigated. Additionally, we investigate the molecular mechanisms of the biological
activities of BP and BB in the treatment of cancer. Finally, based on recent publications, the present use
and therapeutic strategies in the near future of BP and BB are discussed.
2. Bee Collected Pollen (BP) Composition and Main Bioactivities

The pollen represents the male reproductive unit of flowers, and due to its high protein
content, it is necessary for the nourishment of the offspring and honey bees that serve inside the
hive [11,12]. The essential nutritional requirements of honey bees are similar to humans, namely
proteins, carbohydrates, lipids, minerals and vitamins.
Since ancient times, BP has been recognized for its nutritional values, described by the Egyptians
as “the life-giving dust”; its curative effects and its usage in human nutrition were not fully known or
discovered until the development of pollen traps after the 19th century [13]. It gained attention in
biochemical and medical fields when the discovery was made that it has anti-cancer and scavenging
activity of reactive oxygen species (ROS), due to the presence of multiple bioactive compounds [14,15].
Historically, BP was used in the treatment of different ailments, namely as an antibiotic, in liver and
kidney function or simply as a supplement of nutrients and vitamins for the human body. From all the
historically known bioactivities, the therapeutic properties of specie-specific pollens are summarized
in Table 1.
Table 1. Therapeutic properties of different pollen types in folk medicine (after [13]).
Properties Bee Pollen Type

Antibiotic Castanea spp., Eucalyptus spp., Taraxacum spp., Trifolium spp., Zea mays L.
Anti-atherogenic Aesculus hippocastanum L., Castanea sativa Mill., Prunus spp., Salix spp.
Anti-anemia Acacia spp., Citrus spp., Crataegus spp., Papaver spp., Tilia spp.
Antitussives Papaver spp.
Diuretic Centaurea cyanus L., Prunus spp., Taraxacum spp.
Digestive Acacia spp., Lavandula spp., Rosmarinus officinalis L.
Cardioprotective Crataegus spp.
Aesculus hippocastanum L., Castanea sativa Mill., Cystus incanus L., Prosopis
Hepatoprotective
juliflora (Sw.) DC., Schisandra chinensis (Turcz.) Baill., Taraxacum spp.
Brassica napus L., Phoenix dactylifera L., Schisandra chinensis Turcz.) Baill.,
Kidney function
Trifolium alexandrinum L., Zea mays L.
Immunomodulating Eucalyptus spp., Malus spp.
Ulcer healing Brassica napus L.
The above mentioned historical effects are not based on scientific or clinic studies and no connection
to specific constituents has been established until now.
Currently, BP is used as nutritious food and studied for its potential therapeutic properties.
In an overall screening of the published data, the extracts were studied in chronic prostatitis for their
anti-androgenic bioactivity [16–20], such as anti-inflammatory [20–22], antioxidants [23–25] and for
antimicrobial potential [23,25–27], as well as anti-tumor agents [28,29]. BP also shows important effects
in allergies and oral desensitization [30]. The good results in immunostimulatory activity should also
be considered in further research [31,32].
“Bee pollen” (BP) is a mix of bee-collected floral pollens that widely varies in composition and
comprises a large number of compounds, which are cited below. All of them include proteins and
free amino acids, carbohydrates, lipids including fatty acids and their esters, vitamins (as some from
B-complex and E), carotenoids, folic acid and minerals; levels might differ depending on the floral
origin. Flavonoids, phenolic acids and their derivatives are also important constituents, especially for
their bioactivities [9,33–39].
The total lipid content (g/100 g BP, dry mass) of pollen is also diverse, ranging from 1 to 13 [14,40].
The high variability depends on the type of pollen and content in fatty acids, carotenoids and lipophilic
vitamins [9]. The lipids present in pollen include high levels of long-chain essential fatty acids, the
most abundant being linoleic, γ-linolenic and palmitic acids [9,10]. In addition, different organic
acids are also found (acetic, citric, gluconic, lactic, malic, oxalic, tartaric, succinic). From these, the
gluconic acid exhibits the highest concentration. For instance, Mărgăoan et al. analyzed the fatty
acid composition of the total lipids in 16 BP samples from Romania. They identified 14 fatty acids,
from which the most abundant were α-linolenic (32.96% on average), palmitic (25.80% on average)
and linoleic (22.17% on average) acids. Based on the authors’ conclusion, the resulting percentages
are based on the samples’ various botanical origins [9]. The beneficial effect of n-3 fatty acids in the
prevention and management of cardiovascular disease, hyperinsulinemia and possibly type 2 diabetes
is well known [41]. The combination of BP with high levels of α-linolenic (n-3) acid together with a near
1:1 ratio of n-6 to n-3 polyunsaturated fatty acids (PUFAs) proves to be a balanced source of essential
PUFAs for human
aggregation, health.beThese
they could usefulacidsin theexert a variety
treatment ofof health benefits,
cardiovascular BP having
diseases. a higher
(CVD); omega-3
if PUFAs are
acid value than most vegetables. As these compounds are effective
taken as a supplement and in higher doses, they should be carefully consumed by patients with in reducing platelet aggregation,
they could
cancer be useful
in order to avoidin the treatment ofepisodes,
hemorrhagic cardiovascular
especially diseases.
if they(CVD);
will beif submitted
PUFAs areto taken as a
surgical
supplement[42].
procedures and in higher doses
Usually, doses,of they
BPshould
(15 g/day)be carefully
do not reach consumed by patients
the level with cancer
of hemorrhagia; in order to
compared to
avoid hemorrhagic episodes, especially if they will be submitted
a study which demonstrated that hemorrhagic events appear at doses of 3 g/day of omega-3 to surgical procedures [42]. Usually,
doses of BP (15 g/day)
supplementation do notwith
combined reachwarfarin
the levelor ofaspirin
hemorrhagia;
[43]. compared to a study which demonstrated
that Some
hemorrhagic
minor components from BP play key rolesofinomega-3
events appear at doses of 3 g/day nutritionsupplementation
and overall health. combined with
BP contains
warfarin or aspirin [43].
more than 100 enzymes and coenzymes, 16 fatty acids, all known vitamins and 3–8% mineral
Some minor
substances. components
Furthermore, from BP play
flavonoids, key roles inover
carotenoids, nutrition and overall
20 trace elements, health. BP contains
growth more
regulators,
than 100 enzymes and coenzymes, 16 fatty acids, all known vitamins
hormones and antioxidants are compounds that contribute to the potential bioactivities of BP in a and 3–8% mineral substances.
Furthermore, flavonoids,
broad-spectrum carotenoids,
[13]. For instance, dataover 20 trace
collected fromelements,
Colombian growth regulators,
BP include levelshormones
6.9 + 3.5 gand of
antioxidants are compounds that contribute to the potential bioactivities of
lipids, 23.8 ± 3.2 g proteins and total dietary fiber 14.5 ± 3.5 g. The moisture content was 7.7 ± 5.2 g/100 BP in a broad-spectrum [13].
gFor instance,
and data collected
dry matter-based ashfrom 2.5 Colombian
± 0.4 g. Fatty BP acids
include levels
were 6.9 + α-linolenic,
mostly 3.5 g of lipids, 23.8 ± 3.2
palmitic andg linoleic,
proteins
and total
while dietary
fructose andfiber 14.5 ± from
glucose 3.5 g. carbohydrates
The moisture content were the wasmost7.7 ±concentrated
5.2 g/100 g and dry sugars.
main matter-based
Most
ash 2.5 ± 0.4 g. Fatty acids were mostly α-linolenic, palmitic
minerals were identified, such as K, Ca and Mg [44]. In other studies, K, Ca, Na and Mg wereand linoleic, while fructose and glucose
from carbohydrates
identified as the highest were mineral
the mostcontents
concentratedin BPmain sugars.
samples, as Most
well asminerals were identified,
other metals, such as Cr,suchAl,asSr,
K,
Ca and
Sn, MgV.
Ni and [44]. In other
Among tracestudies,
minerals,K, Ca,theNa and Mg
highest were was
content identified as the highest
Mn, followed by Znmineral
and Fe, contents
Cu and Ni in
BP samples,
[39,45]. as well as other metals, such as Cr, Al, Sr, Sn, Ni and V. Among trace minerals, the highest
content
Thewas Mn, followed
relationship by Znthe
between andbotanical
Fe, Cu andoriginNi [39,45].
and chemical, antioxidant and antibacterial
The relationship between the botanical origin
properties is crucial for further investigations. In 2017, Velásquez and chemical, antioxidant andstudied
et al. antibacterial properties
the correlation
is crucial for further investigations. In 2017, Velásquez et al. studied
between the botanical origin, composition and antibacterial activity of multi-floral BP. In their the correlation between the
botanical origin,
research, Brassicacomposition
sp. and Galega and officinalis
antibacterial activity
L. BPs showed of multi-floral
antibacterial BP. activity
In their research,
against allBrassica sp.
bacteria
and Galega officinalis L. BPs showed antibacterial activity against
studied (Escherichia coli ATCC-25922, Staphylococcus aureus ATCC-25923, Pseudomonas aeruginosa all bacteria studied (Escherichia coli
ATCC-25922,
ATCC 27853 and Staphylococcus
Streptococcus aureus ATCC-25923,
pyogenes Pseudomonas
I.S.P. 364-00), and theaeruginosa ATCC 27853
extracts surpassed theand Streptococcus
effectiveness of
pyogenes I.S.P.antibiotics
conventional 364-00), and the extracts surpassed the effectiveness of conventional antibiotics [23].
[23].
As stated
As stated above,
above, the the antioxidant
antioxidant activityactivity ofof BPBP isis also
also related
related to to the
the flora
flora origin
origin [46]
[46] and
and to
to its
its
phenolic and
phenolic and polyphenolic
polyphenolic compounds,
compounds, such such as as flavonoids,
flavonoids, among among otherother constituents
constituents [24,47,48].
[24,47,48]. InIn
BP samples, from diverse flora, the flavonoids tricetin, luteolin,
BP samples, from diverse flora, the flavonoids tricetin, luteolin, selagin, myricetin, isorhamnetin selagin, myricetin, isorhamnetin
isoquercetin, quercetin
isoquercetin, quercetin and and kaempferol,
kaempferol, were were thethe most
most identified.
identified. The The latter
latter two
two andand their
their glycosidic
glycosidic
forms are
forms are the
the most
most abundant
abundant (Figure (Figure 1). 1). They
They show
show different
different ratios
ratios among
among them,them, butbut no
no distinctive
distinctive
differences are observed in the phenolic composition
differences are observed in the phenolic composition [49–53]. [49–53].
(a) (b)
Figure 1. Examples
Examples of ofFlavonoid
Flavonoidstructures
structuresfound
found in in
beebee pollen
pollen (BP)(BP)
andand bee bread
bee bread (BB),(BB),
from from [53]
[53] (with
(with permission
permission of the authors).
of the authors). Structure Structure (a): Kaempferol-3-O-[rhamnosyl
(a): Kaempferol-3-O-[rhamnosyl (1-2) glucoside]
(1-2) glucoside] (RT
(RT (retention
time) = 33.2;
(retention time)
λmax = 265,λmax
= 33.2; 290=sh,
265,
320290
sh,sh, 320
350 sh,(b):
nm); 350 Quercetin-3-O-[glucosyl
nm); (b): Quercetin-3-O-[glucosyl (RT = 30.6;
(1-2) glucoside]
(1-2) glucoside]
λmax= =30.6;
(RT 255,λ266
max =sh, 294266
255, sh,sh,
355294
nm).
sh, 355 nm).
The antioxidant power and scavenging activity of ROS are one of the most studied bioactivities
for its broad approach. Both are significant in improving clinical research approaches of diseases such
as diabetes, hypertension, obesity and cardiovascular problems, as well as in degenerative
pathologies (arthritis, Alzheimer’s, Huntington’s and Parkinson’s disease) [14,15].
The antioxidant power and scavenging activity of ROS are one of the most studied bioactivities
for its broad approach. Both are significant in improving clinical research approaches of diseases such
as diabetes, hypertension, obesity and cardiovascular problems, as well as in degenerative pathologies
(arthritis, Alzheimer’s, Huntington’s and Parkinson’s disease) [14,15].
There is evidence that oxidative stress is the result of a concentration increase of ROS in cells
that can be generated by both endogenous and exogenous factors, such as environmental factors,
as well as the superoxide anion free radical O2 – . DNA and cell membrane damage is induced by
increased levels of ROS; therefore, these effects are linked to cellular response and can induce chronic
inflammation [54,55]. If the in vivo data corroborates with the in vitro effect of the antioxidant activity
of BP substances, they may also contribute to the inhibition and removal of ROS [37], in a late sense,
contributing for the reduction of the damage caused in various diseases, even in cancer.
Pollen extracts also demonstrate significant anti-inflammatory activities. In a study from 2010,
BP (300 mg/kg) moderately suppressed the carrageenan-induced paw oedema. The water extract
(300 mg/kg) showed minor inhibitory activity, while the ethanol extract (100 and 300 mg/kg) showed
a relatively strong and significant inhibition with a mean % swelling of 48.4 and 43.5, respectively.
The authors concluded that the ethanol extract shows an effective anti-inflammatory activity through
the inhibition of NO production and cyclooxygenase-2 (COX-2) [56]. Additionally, BP affects the
release of insulin-like growth factor I (IGF-I) and steroid hormones (estradiol and progesterone), as
well as the expression of markers of apoptosis (Bcl-2, Bax and caspase-3) in rat ovarian fragments [57].
From all the cited above bioactivities below, in Table 2, the main bioactivities attributed to BP
are summarized.
Table 2. Current therapeutic properties of different bee pollen and bee bread type.
Functional
BP and BB Type Extract Type or Concentration Bioactivity References
Properties
Inhibition zone diameter: A1 (9–21 mm) against Candida albicans ATCC 14053, Bacillus cereus 7064, Enterococcus faecalis
ATCC 51299, Methicillin Resistant Staphylococcus aureus (MRSA), Micrococcus luteus, Staphylococcus aureus ATCC 6538;
A2 (9–18 mm) against Candida krusei ATCC 6258, Escherichia coli ATCC 11293, MRSA, M. luteus, S. aureus;
A3 (9–20 mm) against C. krusei, E. coli, MRSA, M. luteus, S. aureus;
10 g of Castanea sativa Mill pollen (A1–A5, E1–E4) A4 (9–21 mm) against C. parapsilosis, B. cereus, E. faecalis, MRSA, M. luteus, S. aureus;
Anti microbial Castanea sativa Mill. extracted by 100 mL of methanol from nine A5 (9–21 mm) against E. coli, MRSA, M. luteus, S. aureus; [58]
different populations E1 (9–23 mm) against C. krusei, Candida parapsilosis ATCC 22019, B. cereus, MRSA, M. luteus, S. aureus, ancomycin Resistant
Enterococcus (VRE);
E2 (9–22 mm) against C. krusei, C. albicans, B. cereus, E. coli, MRSA, M. luteus, S. aureus;
E3 (12–21 mm) against C. krusei, MRSA, M. luteus, S. aureus;
E4 (10–21 mm) C. krusei, C. albicans, C. parapsilosis, B. cereus, MRSA, M. luteus, S. aureus
Ranunculus sardous Crantz., Ulex
N/S Marked antibiotic activity against Pseudomonas aeruginosa due to herbacetin derivates [59]
europaeus L.
MEh and MEl: 2.33–3.00 mm against Listeria monocytogenes CCM 4699; 1.33–2.66 mm against Pseudomonas aeruginosa CCM
10 g of pollen extracted in 99.9% and 70% (v/v) 1960; 2.33–3.67 mm against Staphylococcus aureus CCM 3953; 2.00–3.83 mm against Salmonella enterica CCM 4420; 1.67–3.00
Brassica napus subsp. napus L. methanol (MEh and MEl) and 96% and 70% (v/v) mm against Escherichia coli CCM 3988; [60]
ethanol (Eh and El) Eh and El: 2.33–3.67 mm against L. monocytogenes; 1.67–3.67 mm against P. aeruginosa; 2.33–3.00 mm against S. aureus;
2.00–3.00 mm against S. enterica; 2.33–3.67 mm against E. coli;
MEh and MEl: 2.33–3.67 mm against L. monocytogenes; 2.00–2.67 mm against P. aeruginosa; 1.67–2.00 mm against S. aureus;
10 g of pollen extracted in 99.9% and 70% (v/v)
Helianthus annuus L. methanol (MEh and MEl) and 96% and 70% (v/v) [60]
Eh and El: 2.33–2.67 mm against L. monocytogenes; 1.00–2.67 mm against P. aeruginosa; 1.00–2.67 mm against S. aureus;
ethanol (Eh and El)
MEh and MEl: 0.67–2.67 mm against L. monocytogenes; 1.00–1.67 mm against P. aeruginosa; 1.67–2.50 mm against S. aureus;
10 g of pollen extracted in 99.9% and 70% (v/v)
Papaver somniferum L. methanol (MEh and MEl) and 96% and 70% (v/v) [60]
Eh and El: 2.00–2.33 mm against L. monocytogenes; 1.00–1.67 mm against P. aeruginosa; 1.67–3.67 mm against S. aureus;
ethanol (Eh and El)
BB—predominant Bupleurum MIC: 0.04 mg/mL against B. cereus; 0.25 mg/mL against E. coli; 0.175 mg/mL against S. aureus, L. monicytogenes, Enterobacter
spinosum Gouan.; Anethum Hydro methanolic BB extract of 20 mg/mL in water cloacae, Salmonella typhimurium; [61]
graveolens L. MBC: 0.08 mg/mL against B. cereus; 0.35 mg/mL against S. aureus, L. monicytogenes, E. coli, E. cloacae, S. typhimurium
MIC: 0.35 mg/mL against Aspergillus ochraceus; 0.50 mg/mL against Aspergillus fumigatus, 0.70 mg/mL against Penicillium
BB—predominant Bupleurum
funiculosum; 1.00 mg/mL against Aspergillus niger, Penicillium ochrochloron, Penicillium verrucosum var. cyclopium;
spinosum Gouan.; Anethum Hydro methanolic BB extract of 20 mg/mL in water [61]
MBC: 0.70 mg/mL against A. ochraceus; 1.00 mg/mL against A. fumigates, P. funiculosum; 1.40 mg/mL against A. niger, P.
graveolens L.
ochrochloron, P.v. cyclopium
DPPH value ranging between: 0.135–2.814 mmol Trolox g–1 , in Pinus spp. and Salix spp.;
Antioxidant Selected monofloral species 2 g of BP extracted in 15 mL methanol TEAC value ranging between: 0.546–6.838 mmol Trolox g–1 , in Pinus spp. and Salix spp. [47]
0.255–5.355 mmol Fe(II) g–1 , in Knautia arvensis (L.) Coulter and Matricaria chamomilla L.
Total antioxidant capacity (mg AA/g extract) 143 ± 22
BB—predominant Bupleurum 1 g of BB stirred with 30 mL methanol/water (80:20
DPPH assay (EC50 , mg/mL) 0.98 ± 0.06
spinosum Gouan.; Anethum v/v) mixture and prepared at a final concentration [61]
ABTS assay (EC50 , mg/mL) 0.50 ± 0.04
graveolens L. of 20 mg/mL in water
Reducing power (EC50 , mg/mL) 0.19 ± 0.03
Total antioxidant activity (%): 6.8–86.4 in Zea mays L. and Sinapis alba L.
Selected monofloral species 0.25 mL BP in 80% methanol DPPH value (%): 8.6–91.3 in Lamium purpureum L. and Aesculus hippocastanum L. [62]
HRSA (%): 10.5–98.0 in Aesculus hippocastanum L. and Pyrus communis L.
Methanolic extract: TPC: 816 mg/kg GAE of DW; TFC: 843 mg/kg QE DW;
0.5 g of pollen extracted with 10 mL of 80% ABTS radical scavenging activity: 95.5%;
Helianthus annus L. [63]
methanol and 50% ethanol Ethanolic extract: TPC: 2907 mg/kg GAE of DW; TFC: 865 mg/kg QE DW;
ABTS radical scavenging activity: 75%
Table 2. Cont.
Functional
Properties
1.95 g pollen fraction (chloroform extract) with Citotoxicity in MCF-7, Hela, BEL-7402, BCG-823, KB, A549 and HO8910 cells with 100 µg/mL extract
Anti-carcinogenic Brassica rapa L. 12.5, 25, 50 and 100 µg/mL of pollen extract ↑ caspase-3 enzyme activity; [64]
administered for 24 h ↓ expression of anti-apoptic proteins Bcl-2
1 g bee pollen mixed with 9 mL 70% ethanol with C. incanus extract 2 induced toxicity at 355.6 mg/mL
Cistus x incanus L., Salix alba L. final concentrations of: 1 mg/mL, 10 mg/mL and S. alba extract 2 induced toxicity at 660 mg/mL, (91.82–7.46%, [65]
100 mg/mL at 24 h until harvest (72 h) Inhibition of 17-β estradiol activity
Significant effect in formalin test of mice with pollen (100 and 200 mg/kg) at the first (0–5 min) and second phase (15–30
Three times extracted pollen with 70% min);
Anti-inflammatory Pinus densiflora Siebold & Zucc. ethanol, with orally administered dose of 100 and Dose of 200 mg/kg delayed the response of mice to hot plate [66]
200 mg/kg thermal stimulation;
↓ inflammation induced by carrageenan, formalin and arachidonic acid, due to flavonoid content in Pinus pollen
↓ inhibition of carrageenan-induced edema at 300 mg/kg water PB; ↑ inhibition of carrageenan-induced edema at 100 and
200 g of BP extracted with water or 95% ethanol;
300 mg/kg (48.4% and 43.5%)
with orally administered dose of BP (300 mg/kg),
Cistus spp. ↑ anti-inflammatory effect of ethanol extract, strong inhibition of carrageenan-induced paw edema [56]
Water BP (300 mg/kg), EtOH BP (100 and
↑ inhibition of COX-1 and COX-2 in water BP (IC50 : 150 µg/mL and 10.3 µg/mL)
300 mg/kg)
↑ inhibitionof COX-1 and COX-2 in ethanol BP (IC50 : > 150 µg/mL)
↑ calcium content (mg/g dry bone) on VD3 -induced decrease, in the femoral-diaphyseal and metaphyseal tissues by BP
dose increase
↑ calcium content (mg/g dry bone) on PGE2 -induced decrease, in the femoral-diaphyseal and metaphyseal tissues by BP
dose increase
↓ calcium content (mg/g dry bone) on PTH-induced decrease, in the femoral-diaphyseal and metaphyseal tissues by BP
5 g of BP in 20 mL distilled water, with dose increase
Anti-osteoporosis Cistus creticus L. [67]
concentrations of 10, 100, 1000 µg/mL ↓ glucose consumed (mg/g dry bone) on PTH-stimulated glucose consumption in the femoral-diaphyseal and
metaphyseal tissues by BP dose increase
↓ lactic acid production (mg/g dry bone) on PTH-stimulated lactic acid production in the femoral-diaphyseal and ↑ lactic
acid production (mg/g dry bone) in the metaphyseal tissues by BP dose increase
↓ TRACP (nmol/min/mg protein) on PTH-induced increase in TRACP activity in the femoral-diaphyseal and metaphyseal
tissues by BP dose increase
↑ calcium content (mg/g dry bone) in rat femoral-diaphyseal tissues in the presence of 50 µg/mL BP extract (< MW 1000)
5 g of BP in 20 mL distilled water, with
and moderately higher in the presence of 100 µg/mL in all BP fractioned extracts
concentrations of 10, 50 and 100 µg/mL BP extracts
↑ calcium content (mg/g dry bone) in rat femoral-diaphyseal and -methaphyseal tissues by dose increase of 25 and
Cistus creticus L. fractioned to less than MW 1000 (A), from MW [68]
50 µg/mL BP extract (< MW 1000)
1000 to MW 10,000 (B) and greater than MW 10,000
↑ osteoclast-like MNCs (number/culture) on PTH-induced osteoclastic cell formation by dose decrease (10 and 50 µg/mL)
(C)
and higer fractioned extracts
5 g of BP in 20 mL distilled water (oral ↑ calcium content (mg/g dry bone) in the femoral-diaphyseal and metaphyseal tissues by oral administration of BP water
administration) extracts (5 and 10 mg/mL/100 g body weight)
20 g of BP in 99.5% ethanol (30 mL) for use on ↑ calcium content (mg/g dry bone) in the femoral-diaphyseal and metaphyseal tissues in the presence of water-solubilized
Cistus creticus L. tissues of rats; extract (100 or 1000 µg/mL) and ethanol extract (1000 µg/mL) by dose increase [69]
Concentrations: 1, 5 or 10 mg/mL 100 g body ↑ calcium content (mg/g dry bone) in the femoral-diaphyseal tissues with water-solubilized extract (100 µg/mL)
weight orally administered to rats for 7 days; ↑ alkaline phosphatase (µmol/min/mg protein) activity and DNA (mg/g wet bone) content in the presence of
10, 100 and 1000 µg/mL water and ethanol extracts water-solubilized extract (100 or 1000 µg/mL)
5 g of BP in 20 mL distilled water (oral
administration)
Brassica napus L., Camellia 20 g of BP in 99.5% ethanol (30 mL) for use on
↑ calcium content (mg/g dry bone) in the femoral-diaphyseal or metaphyseal tissues in the presence of water-solubilized
sinensis (L.) Kuntze., Fagopyrum tissues of rats; [69]
extract (100 µg/mL), best results in C. sinensis (L.) Kuntze (> 240 mg/g dry bone calcium content)
esculentum Moench. Concentrations: 1, 5 or 10 mg/mL 100 g body
weight orally administered to rats for 7 days;
10, 100 and 1000 µg/mL water and ethanol extracts
Table 2. Cont.
Functional
Properties
↑ calcium content in the femoral-diaphyseal (5, 10 mg/100 g) or metaphyseal (5, 10 or 20 mg/100 g) tissues in the presence
of water-solubilized extract (5, 10 or 20 mg/100 g) in STZ-diabetic rats
5 g of BP in 20 mL distilled water, with final ↓ serum glucose (mg/dL) concentration by BP dose increase
concentrations of ↓ triglyceride concentration
Cistus creticus L. [70]
5, 10 or 20 mg/mL 100 g body weight orally ↑ alkaline phosphatase (µmol/min/mg protein) activity and DNA (mg/g wet bone) content in the presence of
administered to rats for 14 days water-solubilized extract (5, 10 or 20 mg/100 g) by dose increase
↓ serum calcium (mg/dL) concentration in STZ-diabetic rats by BP dose increase
↑ inorganic phosphorus (mg/dL) concentration in STZ-diabetic rats by BP dose increase
30 g of BP in 100 mL distilled water, with orally ↓ plasma and tissue (liver, kidney, brain, heart) MDA (nmol/mL-nmol/mg-prot) levels in the WSBP-treated group (G2)
administered concentrations of: G1 (control): 1mL and propoxur + WSBP (G4) compared to propoxour treated ones (G3);
of distillated water + 1 mL SO; ↑ erythrocyte and tissue (liver, kidney, brain, heart) SOD (U/mg Hb-U/mg-prot) activities in WSBP-treated group (G2) and
G2: 100 mg/kg/bw/day of WSBP +1 mL SO; Propoxur + WSBP (G4) compared to propoxour treated ones (G3);
Hepatoprotective Brasssica napus L. G3: 20 mg/kg/bw/day propoxur in 1 mL distilled ↑ erythrocyte and tissue (liver, kidney, brain, heart) GSH-Px (U/mgHb-U/mg-prot) activities in WSBP-treated group (G2) [71]
water and 1 mL SO; and propoxur + WSBP (G4) compared to propoxour treated ones (G3);
G4: 20 mg/kg/bw/day propoxur in 1 mL volume of ↑ serum T-protein and albumin (mg/dL) levels in WSBP-treated group (G2) and propoxur + WSBP (G4) compared to
soy oil and with 100mg/kg/bw/day of WSBP orally propoxour treated ones (G3);
administered for 14 days ↓ creatin (mg/dL) levels WSBP-treated group (G2) and propoxur + WSBP (G4) compared to propoxour treated ones (G3)
Protective effect of hepatocytes from oxidative stress
1 g of BP with 10 mL methanol, with orally
Healing of liver damage induced by CCI4 (carbon tetrachloride) toxicity
administered concentrations: G1 (control): 0.9%
↓ weight loss % in G6 (low pollen) and G7 (high pollen) compared to G4 (CCI4 ) G5 (silibinin) and G1-3;
NaCl (i.p.); G2 (control): 0.8 mL/kg OO (i.p.); G3
↓ plasma AST, ALT (U/L) and MDA (nmol/mL plasma) levels in G5 (silibinin), G6 (low pollen) and G7 (high pollen)
(control) 0.8 mL/kg Ethanol (i.p.); G4 (CCI4 )
compared to G4 (CCI4 ) and ↑ levels compared to control G1-3;
Castanea sativa L. 0.8 mL/kg CCI4 in OO; G5 (Silibinin) 0.8 mL/kg [72]
↑ liver MDA (nmol/g tissue) in G6 (low pollen) and G7 (high pollen) compared to G5 (silibinin) and control G1-3;
CCI4 in OO + Silibinin (50 mg/kg/day) gavage; G6
↓ SOD (U/g liver) in G6 (low pollen) and G7 (high pollen) compared to G5 (silibinin) and control G1-3
(low BP) 0,8 mL/kg CCI4 in OO (i.p.) + BP
↑ AI in G6 (low pollen) compared to G5 (silibinin)and control G1-3
(200 mg/kg/day) gavage; G7 (high BP) 0,8 mL/kg
G6 (low pollen) decreased fatty degeneration and regeneration in hepatocytes
CCI4 in OO (i.p.) + BP (400 mg/kg/day) gavage
G7 (high pollen) decreased fatty degeneration
Note: AA—allyl alcohol; ABTS—2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid; AI—apoptois index, ALT—alanine aminotransferase; AST—aspartate aminotransferase;
ATCC—American Type Culture Collection; BB—bee bread; BP—bee collected pollen; CCI4 —carbon tetrachloride; CCM—Czech Collection of Microorganisms; COX-1—cyclooxygenase-1,
COX-2—cyclooxygenase-2; DPPH—1,1-diphenyl-2-picrylhydrazyl; DW—dry weight,; Eh—96% ethanol extract; El—70% ethanol extract; GAE—gallic acid equivalents, HRSA—hydroxyl
radical-scavenging activity; i.p.—intraperitoneal, MDA—malondialdehyde; MIC—minimal inhibition concentration, MBC—minimal bactericidal concentration, Meh—99.9% methanol
extract and Mel—70% methanol extract; MNCs—multinucleated cells; MRSA—methicillin resistant Staphylococcus aureus, OO—olive oil; PGE2 —prostaglandin E2 ; PTH—synthetic human
parathyroid hormone; SOD—superoxide dismutase; TEAC—Trolox equivalent antioxidant capacity; TFC—total flavonoid content, TPC—Total phenolic content, TRACP—tartrate-resistant
acid phosphatase; SO—soy oil, STZ—streptozotocin; VD3 —1,2-dyhydroxyvitamin D3 ; QE—quercetin equivalents, WSBP—water-solubilized bee pollen extract, ↑—High; ↓—Low.
Based on multiple studies, the composition of Romanian BP varies in terms of macronutrients

and minerals. In the study conducted by Mărgăoan et al. [73], the total polyphenols, total flavonoids
and antioxidant activity were studied on six samples of fresh BP from Transylvania. Their results
showed that the highest polyphenol concentration was determined in Prunus spp. BP (8.87 mg GAE/g),
followed by Malus domestica Borkh. BP (7.74 mg gallic acid equivalents (GAE/g) and Salix spp. BP
(7.69 mg GAE/g). The lowest level of total polyphenol content was obtained for BP from Calluna
vulgaris (L.) Hull 3.76 mg GAE/g. Total flavonoids content ranged from 6.29 mg quercetin equivalents
(QE)/g (Malus domestica Borkh.) to 2.55 mg QE/g (Callendula officinalis (L.) Hull).
From Greek BP samples, three different extracts (0.5 to 10 µg/mL) showed chymotrypsin-like
(CT-L) proteasome activity in human fibroblasts. The water extract has been shown to exhibit important
antioxidant properties and create a high CT-L proteasome activity at the concentrations of 0.5 and
2 µg/mL. The microscopical analysis of the 16 different common taxa of the Greek Flora resulted in the
following species: Papaver rhoeas L., Matricaria recutita L., Sinapis arvensis L., Cistus sp., Trifolium sp.,
Dorycnium sp., Cichorium sp., Convolvulus sp., Circium sp., Malva sylvestris L., Fumaria sp., Eucalyptus
camaldulensis Dehnh., Anemone sp., Ononis sp., Asphodelus sp. and Quercus ilex L. Greek pollen, as almost
all BPs, is rich in flavonoids and phenolic acids. This composition has been reported to demonstrate
the observed free radical scavenging activity on HFL-1 human fetal lung embryonic fibroblasts along
with stimulation of cellular antioxidant mechanisms by other natural products. Additionally, these
extracts were also tested for their antimicrobial activity against gram-positive [33].
Polysaccharides are another group of major components found in BP that are investigated as
possible adjuvants for antineoplastic treatments [74–76].
Previously demonstrating that BP alleviates the distress of chemotherapy-treated patients, Wang
et al. investigated for the first time the antitumor activity of fractioned BP polysaccharides from Rosa
rugosa Thunb. The acid fractions contained rhamnogalacturonan type I (RG-I) and type II (RG-II),
homogalacturonan (HG) and arabinogalactan (AG) (Figure 2). All of them showed a potential in vitro
antitumor activity by inhibiting the proliferation of human colon carcinoma HT-29 and HCT116 cells
Antioxidants 2019, 8, x FOR PEER REVIEW 2 of 40
in a dose-dependent manner with various concentrations of BP polysaccharides for 72 h [29].
Figure 2. Rhamnogalacturonan type I (RG-I)

Figure 2. Rhamnogalacturonan (a) and
type I (RG-I) typetype
(a) and II (RG-II)
II (RG-II) (b), homogalacturonan
(b), homogalacturonan (HG) (c) and
(HG) (c)
arabinogalactan (AG)
and (d) structures.
arabinogalactan (AG) *—the compounds
(d) structures. can bind to
*—the compounds canrhamnose, galactose,
bind to rhamnose, arabinose [29].
galactose,
arabinose [29].
3. Bee Bread (BB) Composition and Main Bioactivities

“Bee Bread” (BB) is formed by adding honey and digestive enzymes to BP during its storage in
the honeycomb and by fermentation of lactic acid. The titration acidity increases during the
conversion of BP into BB, while the content in sytosterol and vitamins (ascorbic acid and pyridoxine)
decreases. The composition of BB has a major impact when it comes to the flora in the colony’s region;
3. Bee Bread (BB) Composition and Main Bioactivities

“Bee Bread” (BB) is formed by adding honey and digestive enzymes to BP during its storage in
the honeycomb and by fermentation of lactic acid. The titration acidity increases during the conversion
of BP into BB, while the content in sytosterol and vitamins (ascorbic acid and pyridoxine) decreases.
The composition of BB has a major impact when it comes to the flora in the colony’s region; it is similar
to BP and varies by botanical origin. However, the identification of the main flora can also be analyzed
using fingerprints of the phenolic and polyphenolic compounds performed by high performance liquid
chromatography with photo-diode array detection (HPLC/DAD) assays [77].
The fatty acid content of BB is very important for honeybees, whereas PUFAs are essential for a
healthy body development and productivity. Unsaturated FAs are essential for bees and for human
nutrition. Therefore, this product can be a good source of all constituents mentioned above [9,78].
Data from BB composition is scarce and difficult to compare, but as an example, in the studies
conducted by Nagai et al. [14], a content of about 20% protein, 3% lipids, 24–35% carbohydrates,
3% minerals and vitamins is shown. Fully balanced proteins containing all the necessary amino
acids, vitamins (C, B1, B2, E, K, biotin, nicotinic and folic acid), pantothenic acid, pigments and other
biologically active compounds, such as polyphenols (phenolic acid and flavonoids), carotenoids, sterols
and enzymes (saccharase, amylase, phosphatases), are also present. In addition, BB contains more
than 25 different micro- and macro- elements, such as Fe, Ca, P, K, Cu, Zn, Se and Mg.
Several recent studies complete the aforementioned research regarding the multitude of
compounds present in BB, as following: carbohydrates (glucose, fructose, sucrose, arabinose), aliphatic
acids, mainly unsaturated (α-linolenic, linoleic, oleic and 11,14,17-eicosatrienoic acids) and alkanes
(C21–C35) [79–84].
The abundant polyphenols in the structure of BB are of interest from a medicinal point of view.
Among polyphenols, flavonoids represent the most significant group of compounds present in BP
and BB. Even though the essays carried out with BB are scarce, comparatively to BP, recently, the
determination of chemical composition of ethanolic extracts (E-BB) from three different samples of BB
was performed by (gas chromatography-mass spectrometry) GC/MS and the total phenolic content
(33.43–36.52 mg GAE/g), antioxidant (0.56–1.11 mmol/L) and cytotoxic activities were also achieved.
The effects of E-BB extracts (10, 20, 30, 50, 100 µg/mL) on the viability of the glioblastoma cell line
(U87MG) were studied after 24 h, 48 h, and 72 h. A time-dependent inhibitory effect on the viability
of U87MG cells was observed after 48 h incubation, with best results of EBB1 (50 µg/mL), EBB2
(100 µg/mL), EBB3 (30 and 100 µg/mL). The main inhibitory effect was observed after 72 h; in EBB1 (10
and 30 µg/mL), EBB2 (20 and 100 µg/mL) and EBB3 (30 µg/mL) [81].
Most flavonoids known as secondary components are present in the greatest amount. It has
been reported that the total content of flavonoids in the ethanol extracts ranges from 10 to 166 mg/L.
These compounds are predominantly found in the form of glycosides [49,85] in BP samples, except, for
example, in Eucalyptus spp. Aglycones, where 3-O-methylquercetin, luteolin, tricetin and myricetin can
also be identified [49,78]. These bioactive compounds are very important due to their anti-inflammatory,
anti-allergic and anti-carcinogenic properties recognized by in vitro and in vivo studies. However,
they also cause drug-herb interactions and association with conventional therapies should be done
only when the risk is evaluated and the safety of the patient is assured [7,86].
It is well known that BB composition varies by provenance, climate conditions and seasonal
variation, as well as on the melliferous species present in the respective region. Below, we describe
multiple studies from different regions to demonstrate the above statement.
In data from Romania (Transylvania region) collected samples, the total phenolic and flavonoid
content of BB was 7.86–3.12 mg NAE g–1 (Naringin Equivalent—total flavanones), 0.696–0.168 mg
QE g–1 (Quercetin Equivalent—flavonols, flavones and isoflavones), 22.72–8.32 mg GAE g–1 (Gallic
Acid Equivalent—total phenols) [87]. For a different group of samples from Romania (Transylvania
region), the BP values for total polyphenols ranged between 20.48–10.08 mg GAE/g, 1.008–0.144 mg
QE/g (flavonol, flavone and isoflavone) and for flavanone content ranged between 16.16–2.22 mg NAE
g–1 . Regarding BB samples (triplicate analyzed) the values for total polyphenols were 13.92 mg GAE/g,
0.144 mg QE/g and 12.99 mg NAE g–1 [88]. Cocan et al. [89] performed a study with three BP extracts
and fresh BB. The content of polyphenol compounds (mg/g) in methanol extracts ranged between 25.66
(GAE) in multifloral BP and 15.33 (GAE) in BB.
In the study conducted by Baltrušaitytė et al. [90], nine BB samples, collected in Spring in Lithuania,
were assessed for their antioxidant properties by the 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic
acid (ABTS) radical cation decolourisation and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging activity. Their results showed that in the case of DPPH the values ranged between
72.5–94.0% and in the case of ABTS the values varied between 71.1–92.2%, proving to consider BB a
source of natural dietary antioxidants.
Čeksterytė et al. [91] found predominant willow pollen in Spring BB samples from Lithuania
(45.1 ± 3.0%), while in the Summer sample, rapeseed pollen was the main source (78.7 ± 4.5%).
Twenty-two FAs were identified in these samples containing five ω-3, four ω-6 and three ω-9 PUFAs.
The predominant FAs were arachidonic and oleic acids, with an average of 16.09 ± 2.38% and
15.22 ± 1.35%, respectively. The content of α-linolenic ranged mostly between 1.10% and 8.71%.
The average content of the α-linolenic acid (4.32%) in all samples was significantly similar to that
of docosahexaenoic acid (DHA) (4.24%). A significant difference was found in α-linolenic acid and
eicosapentaenoic acid (EPA) content (7.68%). The n-3 DHA also present in fish oil is known to inhibit
the development of non-small lung tumors through a ROS-mediated inactivation of the PI3K/Akt
signaling pathway [92].
Four years later the same group [93] analyzed other BB samples, from which the rapeseed pollen
varied between 54.5–80.0%, while Salix spp. was the secondary pollen source with 8.8–34.6%. In all
samples, the highest content was found to be in α-linolenic acid (27.04–43.83%), whereas ω-6 linoleic
acid content varied between 5.44% and 9.11%. Of all saturated acids, in the case of rapeseed BB,
palmitic acid content was 20.5%, while arachidic acid was 2.82%. Palmitic acid (25.02–26.21%) was
the highest in willow BB samples, which had 67.2–80.0% of this pollen. The highest reduction in
the contents of ω-6, ω-9 and saturated FAs have been detected in wet and dry BB. The research of
long-chain FAs with BB of different origin suggests that BB has more ω-3/ω-6 ratios, showing it to be
more suitable for human consumption compared to other plant-derived oils.
The Colombian samples of BB presented flavonoid and phenolic content of 3.2 ± 1.0 mg (quercetin/g)
and 8.9 ± 3.1 mg/g (gallic acid/g), respectively. In addition, the antioxidant activity of Ferric Reducing
Ability of Plasma (FRAP) and ABTS were reported to be 46.1 ± 13.0 and 61.5 ± 10.2 mmol (trolox/g),
respectively. The digestibility and bioavailability of BB were found to be significantly higher. This
suggests that the nutrient effect of BB could be higher than that of BP. This potential implies a better
profit of bioactive compounds for human use. According to these results, BB was mentioned by
Zuluaga et al. [84] as a product that should be certified as a functional food supplement, subsequently
being studied for all implementation requirements.
Ukrainian BB samples analyzed by Ivanišová et al. [83] show similar data, as presented above from
other countries, with a total polyphenol content of 12.36–18.24 GA mg/g (gallic acid equivalents/dry
weight) and flavonoids with the equivalent of 13.56–18.24 µg, QE-quercetin/dry weight).
Nagai et al. compared 1%, 10% and 100% solutions of hot water fraction (HWF-3 g of BB suspended
and extracted by boiling with 10 volumes of distilled water), water soluble fraction (WSF-3 g of BB
with 10 volumes of distilled water) and ethanol-soluble fraction (ESF-3 g of BB with 10 volumes of
ethanol). The WSF under essay has shown the highest antioxidant ability. The ESF at 10% concentration
was found to have the highest ability against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl
radicals [14]. Although there is a good correlation between the total polyphenol content and the
resistance activity of methanol extracts, no flavonoid content correlates with any of these.
Tavdidishvili et al. [94] investigated the flavonoid compounds of BB and BP Georgian samples
(Imereti region) using HPLC methods and described the naringenin, rutin and quercetin content.
The quantities were determined to constitute approximately 20% of the flavonoids full content. During
the storage of these products, the amount of flavonoid decreased to 6.17–5.03 g/kg.
In conclusion, a major percentage of the compounds found in BB is the provenance of BP and
further research should be performed to ensure a better knowledge of the product for its efficacy and
safety. Even detailed methods for quality control should be standardized.
4. Anti-Cancer Research with BP and BB and Its Bioactive Compounds
4.1. In Vitro Studies of BP and BB Correlated to the Bioactive Compounds

As described previously, the chemical composition of BP and BB may vary depending on the
botanical and geographical origin, as well as the storing conditions. Polyphenols composition present
in BP and BB determine their antioxidant activity, which tends to be species-specific [49,95].
Cancer is one of the main causes of mortality worldwide and a major health problem. Progress
in the use of anticancer drugs is often associated with adverse reactions or recurrence. Many of
these situations involve adding to the treatment of “natural products” that contribute for therapeutic
failure or toxic events [95]. Therefore, therapeutic purposes can be explored with BP and BB extracts,
depending of the taxa, to help eliminate these potential side effects. The variation on its chemical
constituents is vast, including about 200 different substances such as free amino acids, vitamins,
minerals, phenolic and polyphenolic compounds, sterols and lipids. From the latter chemical group,
a special interest in unsaturated fatty acids should be granted (linoleic, linolenic and arachidonic),
especially in α-linolenic acid (65.7%) found in the greatest amount in BP and BB [96]. The therapeutic
effects of dietary fatty acids on cancer cell progression have been verified by in vitro and in vivo
studies. PUFAs have a significant effect on the physical properties and structure of localized membrane
domains. Some isolated esters of FAs have been reported to have antitumor activity against Ehrlich
ascites tumor in mice [97]. Furthermore, in vivo studies should be improved to ensure the safety of
future approach for this type of tumors.
Phytosterols, commonly known as plant sterols, have been shown to inhibit cholesterol absorption
sites in human intestine in multiple clinical trials and also contributing to anticancer effects [98,99].
This led to researchers increased interest in phytosterols effect in human health. A fraction designated as
FV-7 in the water soluble content from the pollen extract Cernilton® (Cernitin SA, Lugano, Switzerland),
was found to inhibit the growth of prostate cancer cell line [100]. Cernilton® consists mainly of pollen
extracts, L-glutamate and Stigmasterol.
Vanderplanck et al., [101] published 5 years ago the identification of sterol compounds from
Calluna vulgaris L. Hull, Cistus spp., Cytisus scoparius (L.) Link, Salix caprea L. and Sorbus aucuparia L.
pollen. The total sterols concentration for Cistus spp./Cytisus scoparius (L.) Link and Sorbus aucuparia
L. ranged between 2.5 to 9.6 mg/g of lyophilized matter. The major phytosterols detected were
β-sitosterol (SIT) and δ5-avenasterol, but significant amounts of δ7-avenasterol (in C. vulgaris, 20.23%)
or 24-methylenecholesterol/campesterol fraction (S. aucuparia L., 84.07%) were also found in several
pollen samples.
Nine human-derived cancer and non-cancer continuous cell lines (HEP—larynx cancer,
CHANG—liver cancer, HEF—human embryo fibroblast, RT112—bladder cancer, SUZA— cancer
of the testis, DU145, 1013L, LNCaP—prostate cancer, MCF-7—breast cancer) were employed to
evaluate the relative in vitro activity of the pollen extract, Cernitin T-60. The results showed that the
androgen-insensitive 1013L and DU145 cells demonstrated significant growth inhibition, predominantly
on the 4th day. Additionally, the highest pollen concentrations (4 mg/mL) inhibited the growth of all
three prostate cell lines, while rapidly depleting the cell numbers by exposure-time. The non-prostate
derived cell lines showed no response to BP extract (1 mg/mL) even after the 4th day of exposure.
However, the highest concentration of 4 mg/mL had a small inhibition rate on HEF and RT112 cells [102].
In 2016, Mărgăoan et al., [103] demonstrated the antiproliferative effect of Filipendula ulmaria (L.)
Maxim BP methanolic extracts on C26 mice colorectal cancer cell lines. Their results showed that for
the 6 and 12 h treatment schemes, the apoptotic index was very low (<10%). After 24 and 48 h of
treatment, the index slightly increased (10–15%) for the 0.25 and 0.5 mg/mL pollen extract. The highest
apoptotic index (30%) was with 1 mg/mL extract at 24 and 48 h treatment. Additionally, the apoptosis
essay showed cellular shape modifications at the highest concentration (1 mg/mL) with longer extract
cell exposure (24 and 48 h), which led to intra-cytoplasmatic vacuolization and granulation.
Five BB samples were screened, using in vitro assays, against different human tumor cell lines,
HeLa, HepG2, MCF-7, NCI-H460, and also against non-tumor liver cells (porcine liver cells, PLP2) [104].
From all the tested samples, BB3 was the only one to inhibit the growth of all tested cell lines, solely
inhibiting the growth of HepG2. BB1 and BB2 were active against MCF-7, BB4 and BB5 against
NCI-H460, and principally BB4 along with BB1 and BB5 against HeLa. It should be noted that none of
the BB samples showed toxicity for normal cells.
The influence of the ethanolic extracts from Salix spp. BB (E-BB), with and without temozolomide
(TMZ) on diffuse astrocytoma cell lines (DASC), human glioblastoma multiform (U87MG) and normal
human astroglia (SVGp12) was investigated. The results showed that E-BB (50 mg/mL) has stronger
cytotoxic activity on U87MG cells after 72 h (26.5 of control) than TMZ alone (about 6% of control).
A higher inhibitory effect on the synthesis of DNA after 24 h was found for E-BB combined with TMZ
(56.4 ± 9.7%) than for the extracts alone. An inhibitory effect was observed in the cells incubated with
EBB (73.6 ± 6.3%) and E-BB with TMZ (67.3 ± 5.1%) after 48 h of exposure [105]. Research by Uçar
et al. [106] showed that BP shows apoptosis and affects caspase-3 activity in HL-60 cells. However,
the identification of the floral origin is crucial for a better understanding of the compounds involved
in the bioactivity; otherwise, it will be impossible to reproduce the anticancer effect. Among the BP
and BB compounds oleanolic and ursolic acids are also identified in some species showing important
antineoplastic potential bioactivities.
It is known that the vascular endothelial growth factor (VEGF) represents a key regulator of
pathogenic angiogenesis in diseases such as bronchial asthma, diabetic retinopathy [107]. It is part of
the structure that restores the oxygen supply to tissues when blood circulation is scarce as in hypoxic
conditions [108], but when over-expressed, it can induce cancer.
Some studies aimed to investigate the citotoxicity of BP including other bee products on human
umbilical vein endothelial cells (HUVECs) and cancer. In order to elucidate the mechanism of
in vitro angiogenesis, VEGF-induced HUVEC proliferation and migration were examined with or
without various concentrations of BP. Among the used bee products, BP showed limited effects against
VEGF-induced angiogenesis, while red Chinese propolis, royal jelly and Phoenix dactylifera L. pollen
extract had an important effect on it [109,110]. In Table 3, a summary of the main research regarding
this issue is provided.
Table 3. In vitro summary of the main studies regarding the effect of BP and BB on multiple cancer and non-cancer cell lines.
BP or BB Cell Lines Treatment Schemes Obtained Results References
The inhibitory patterns for both the naturally occurring
↓ Growth inhibition (1 µg/mL) of V-7 or DIBOA for day 1–6.;
fraction designated as FV-7 in the water soluble
Cernitin T-60 (water-soluble pollen extract with > ↑ Inhibitory effect (10 µg/mL) of 50% at day 1 and 80% at day 5;
Human prostate cell line DU-145 component of the pollen extract Cernilton® and an [100]
90% pollen w/w) Complete shutdown of the proliferative effects (100 µg/mL) achieved
authentic synthetic sample of DIBOA were tested at 1,
from day 1 to 6
10 and 100 g/ml
Human cancer cell lines (PC-3, lncap, CPBC remarkably induced concentration-dependent cytotoxicity in
Cell lines treated with various concentrations of CPBC
Chloroform extract from Brassica rapa L. BP (CPBC) MCF-7, Hela, BEL-7402, BCG-823, KB, PC-3 and lncap cells; 100 µg/mL CPBC could induce cytotoxicity in [64]
(12.5–100 µg/mL) for 24 h
A549 and HO8910) MCF-7, Hela, BEL7402, BCG-823, KB, A549 and HO8910 cells
Fractions and sub-fractions of WRPP showed a
Extracted and fractionated BP polysaccharides Human colon cancer HT-29 and Cells treated with varying concentrations (0, 0.5, 2,
concentration-dependent proliferation-inhibitory effect on HT-29 and [29]
from Rosa × rugosa Thunb. (WRPP) HCT116 cells 5 mg/mL) of various BP polysaccharides for 72 h
HCT116 cells
BB extract—effects of EBB1, EBB2, EBB3 (10, 20, 30, 50, time-dependent inhibitory effect on the viability of U87MG cells
BB ethanolic extracts (ebbs) obtained from three 100 µg/mL) on the viability of glioblastoma cell line treated EBB;
Glioblastoma cell line (U87MG) [81]
different samples of BB from Poland (U87MG) were studied after 24 h, 48 h and 72 h of The main inhibitory effect of EBB was observed after 72 h;
treatment. EBB treatment decreased cell viability to 49–66%.
↓ Cell viability: EBB = 62.4 ± 4.6% on U87MG after 72 h;
Cytotoxic effect using MTT assay: EBB (50 mg/mL),
↓ Cell viability: EBB + TMZ: 82.9–85.2% after 48 h and 70.7–80.0%
Salix spp. Beebread (EBB) extract DASC, U87MG, svgp12 combination with TMZ (20 mm) on cells after 24 h, 48 h [105]
after 72 h on U87MG;
and 72 h of the treatment
↓ Cell viability: EBB + TMZ: 46.2 ± 3.0% after 72 h on SVGp12
↑ Antioxidant activity of the FDPP extract of 3.16, 3.42, and 2.14 times
that of the DPP extract as determined by the ABTS, ferric reducing
Antioxidant activities were determined using DPPH
Date palm pollen (DPP) and volatile esters of antioxidant power (FRAP) and DPPH assays;
assay, the ferric reducing antioxidant power assay and
fermented and non-fermented Phoenix dactylifera L. MCF-7 cell line ↑ Anticancer activity of FDPP against the MCF-7 cell line (IC50 : [111]
ABTS assay. Anti-breast-cancer and antiviral activities
Pollens (FDPPS) 9.52 µg/mL) compared with the DPP extract (IC50 : 96.22 µg/mL);
were determined using the MTT assay
↑ Antiviral activity of FDPP (CC50 : 16.5 µg/mL) compared with DPP
(CC50 : 38.8 µg/mL).
Antioxidant activities determined with DPPH assay.
BPE EC50 : 0.5 mg/mL;
Antiproliferative activity at different concentrations of
BPE (bee pollen extract) MCF-7 and L929 cell lines BPE IC50 : 15 mg/mL on MCF-7 and 26 mg/mL in normal cell line L929; [112]
BPE and cisplatin was determined using MTT assay on
CP IC50 : 20 µmol/L on MCF-7
MCF-7 and L929 cell lines.
DMSO extracts of BP were incubated separately with ↑ Apoptosis DMSO extract of pollen (2 mg/mL): 52.2%;
Dimethyl sulfoxide (DMSO) extracts of BP HL-60 Myeloid Cancer Cell Lines [106]
HL-60 cells, and caspase-3 activity evaluated ↓ Cell viability: 62%
BB1 GI25 : 164 µg/mL on MCF-7; 345 µg/mL on Hela;
BB2 GI25 : 84 µg/mL on MCF-7; BB3 GI25 : 164 µg/mL on MCF-7;
Human tumor cell lines: MCF-7, 253 µg/mL on NCI-H460; 225 µg/mL on Hela; 67 µg/mL on HepG2;
In vitro assays—cytotoxicity (ranging from > 400 to
Six BB samples (BB1-BB5, BBC) NCI-H460, Hela, HepG2; BB4 GI25 : 85 µg/mL on NCI-H460; 209 µg/mL on Hela; [104]
68 µg/mL) on all cell lines
Non-Tumor Porcine Liver Cells: PLP2 BB5 GI25 : 68 µg/mL on NCI-H460; 276 µg/mL on Hela;
BBC GI25 : 366 µg/mL on Hela;
None of the BB samples showed toxicity for normal cells
Note: A549—lung carcinoma cell lines; BCG-823—gastric adenocarcinoma cells; BEL7402—human liver cancer cell line; CP—cisplatin; DASC—diffuse astrocytoma; DIBOA—hydroxamic
acid, 2,4-dihydroxy-1,4-benzoxazin-3-one; DU-145—human prostate cancer cell line; HCT116—human colon cancer cell line; HeLa—human cervix carcinoma cell lines; HepG2—liver
hepatocellular carcinoma; HL-60—human leukemia cell line; HO8910—ovarian carcinoma cell lines; HT-29—human colon adenocarcinoma cell line; KB—squamous carcinoma cells;
L929—mouse fibroblastic cell line; LNCaP—human prostate adenocarcinoma cells; MCF-7—human breast cancer cell line; MTT—3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; NCI-H460—human lung cancer cell line; PLP2—Non-Tumor Porcine Liver Cells; SVGp12—normal human astroglia; TMZ—temozolomide; U87MG—human glioblastoma cell
lines; ↑—increase; ↓—decrease.
4.2. In Vivo Studies in Animal Models
Hepatoprotective Effects in Animal Experiments

Cisplatin (CP) is one of the mostly used chemotherapeutic drugs, with a wide anticancer spectrum,
such as lung, prostate, testicular and ovarian cancer [113]. Indeed, CP is used as an adjuvant in radiation
or post-surgery therapy [114], even though it can induce multiple side-effects, such as hepatotoxicity,
nephrotoxicity, ototoxicity, neurotoxicity, nausea, vomiting and alopecia, among others [115]. The most
important factors in CP-induced acute renal failure are ROS and oxidative damage. In multiple studies,
CP decreased the antioxidant activity of enzymes, such as catalase (CAT), glutathione peroxidase
(GPX) and superoxide dismutase (SOD). A decrease in these enzymes could lead to an increase in
lipid peroxides, which leads to the formation of malondialdehyde (MDA), a decrease in antioxidant
status and an increase in free radical production [116]. Additionally, CP can activate mitogen-activated
protein kinase (MAPK) along with the redox-sensitive transcription factor nuclear factor kappa-B
(NF-kB), which could induce inflammation, tissue injury and cell death [117,118].
Along with multiple studies that showed the antimutagenic properties of BP, in a dose-dependent
manner, for certain types of cancer [119,120], a hepatoprotective effect from the extracts was
demonstrated to reduce liver damage and enzymatic defects.
For future studies, it is very important to clarify whether the effect is induced by a drug-interaction.
CP is metabolized by cytochrome P450 (CYP450) enzymes (mainly CYP2E1 and CYP4A11), which have
an important role in drug-induced hepatotoxicity and nephrotoxicity. Overall, it has been suggested
that “the cisplatin and CYP2E1 interaction leads to the generation of ROS and other oxidants resulting
in renal injury; and that ROS generated by both the use of cisplatin and by the CYP2E1 increases tissue
damage, induces apoptosis, and causes liver failure” [121].
The induction or inhibition of such isoenzymes by biocompounds from BP and BB as flavonoids,
among others, could change the outcomes of the treatment in both sides, improve the impact of side
effects and decrease the efficacy of the drug. It is important to be aware of, and do further research on,
the efficiacy, which likely undergoes changes, and evaluating the amount of the available drug; the
decrease implies a lower impact in the liver. Thus, this statement should be taken with care, because if
a medicated patient takes a hepatoprotective product, sometimes the outcome is unpredictable, and a
toxic event can happen; excepting studies regarding the administration of BP and BB in which adverse
effects have not been reported [122,123].
In a study carried out with male albino mice (Mus musculus L.) treated with BB and Propolis
extracts for 14 days, at doses of 140 and 8.4 mg/kg b.wt/day, combined with the intraperitoneal
administration of CP at a dose of 2.8 mg/kg b.wt, showed a significant chemoprotective activity [119].
In order to determine the effect of BP and BB as a feed additive ingredient in mice, control group
(C) and E1 were fed with 250 mg/kg pollen, while group E2 with 250 mg/kg for 21 days. The antioxidant
activity was determined with a spectrophotometer, showing no adverse effects on lipid peroxidation
(LPO), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), Glutathione-S-transferase
(GST), Glutathione peroxidase (GP), Glutathione reductase (GR) at the tested doses of these products in
mice diet. In the BP and BB administered groups, a decrease in the LPO level was observed compared to
control group. The activity of GSH, SOD, CAT, GST, GR and GP in liver increased compared to control
in BP and BB treated groups. Antioxidant potential of the groups treated with BB was determined to
be greater [124].
A decrease in the LPO levels and antioxidant enzymes was observed in another study, BP and BB
having a positive effect against bacteria when compared with antibiotics, ampicillin and amoxicillin.
Thus, it has been reported that BP and BB have protective effects on Staphylococcus aureus-induced
toxicity in the liver of mice [125].
CCI4 is a hepatotoxic agent that promotes the formation of free radicals that cause cellular LPO
and organ damage. It has been found that Castanea sativa L. BP protects hepatocytes from oxidative
stress and improves liver damage caused by CCI4 toxicity; can be safely incorporated into the daily
human diet and may help reduse the risk of diseases caused by oxidative stress. It is also stated that
C. sativa L. BP may be used as an appropriate alternative to silica in the treatment of hepatocellular
pathologies [72].
The properties of BB having antimicrobial, antioxidant, prebiotic and probiotic efficacy are very
important. The BB has likewise a high antioxidant and superoxide anion radical and a hydroxyl radical
scavenging abilities against free radicals [126].
As it was exemplified above, these activities are common to BP and BB, and further research
should be done to validate its possible use linked to certain therapies, as well as the beneficial effect of
this association. In Table 4, a summary of the main research regarding this issue is provided.
Table 4. In vivo studies regarding the effect of BP in multiple diseases which imply liver damage.
The Type of BP, Collection Site and Application
Animal Models Applied Treatment Effects of BP Administration References
Method
G1—control;
G2—allyl alcohol (AA);
G3—AA + Cernitin T60 2.5 mg/kg/day; ↑ liver cells apoptosis in G2
G4—AA + Cernitin T60 50 mg/kg/day; ↓ complete cell degeneration in G4
Eighty male Wistar rats weighing
Cernitin T60 and Cernitin GBX of specially G5—AA + Cernitin GBX 2.5 mg/kg/day; ↓ hepatotoxicity in G1 and G6
180–240 g were divided into eight [127]
selected plants, Sweden, orally administered G6—AA + Cernitin GBX 50 mg/kg/day; ↓ bilirubin level and liver weight; ↓activity ALT and AST in G3
equal groups
G7—AA + Cernitin GBX 2.5 mg/kg/day + Cernitin T60 and G4:
50 mg/kg/day; ↓ serum enzymes activity in G7 and G8
G8—AA + Cernitin GBX 50 mg/kg/day + Cernitin T60
50 mg/kg/day.
G1—control, G2—HFD, G3– HFD + pollen extracts (Cernitin The intima of the aorta of rabbits of G1 (controls) was unchanged.
Forty male mongrel rabbits with Cernitin T60 and Cernitin GBX—from six plant
T60—50 mg/kg/24 h + Cernitin GBX—10 mg/kg/24 h) orally, G2, G4 HFD fed groups— ↑ atherosclerotic plaques, the plaque
initial body weight 3.0–3.8 kg fed species: Rye grass, Maize, Timothy grass, Pine,
G4—HFD + clofibrate (Pharmaceutical Works ‘Polfa’/25 mg/kg/24 coverage averaging 83.5% compared to only 33.7% in the pollen [128]
with a standard basic diet, randomly Alder flower and Orchard grass; orally
h) orally. HFD = (g/kg/24 h) cholesterol (0.5), hydrogenated extract-treated animals.
divided into four equal groups administered
coconut oil (l.0), cholic acid (0.1). The experiment lasted 12 weeks ↑ weight of livers G2 and G4
G1—normal saline injections.
G2—control;
G3—polysaccharide LBPP (50 mg/kg body weight); ↓ growth of S180 (51.26% inhibition rate) with RPP
Brassica rapa L. pollen polysaccharide (LBPP), G4—thpolysaccharide LBPP ↓ toxicity RPP + Cy displayed synergism and reduced its toxicity
S180-bearing mice Wuhan, China; (100 mg/kg body weight); on immune organs. [28]
orally administered G5—polysaccharide LBPP ↑ antitumor activity of RPP
(200 mg/kg body weight); ↓ toxicity of RPP on liver, kidney, spleen and thymus
G6—cyclophosphamid (Cy,
20 mg/kg body weight
G1—provided with cooking oil, G2—200 mL of mesquite BP ↑ antioxidant activity in vivo on the liver of
Bee pollen from mesquite (Prosopis juliflora (Sw.) extract; G3—200 mL of extract of mesquite BP; G4—200 mL of bromobenzene-intoxicated mice;
Eighty male CF1 mice (19–21 g) DC.) collected in April in Mexico, extracts of two vitamin E (400 UI); G5 intoxicated with bromobenzene—200 mL, ↓ MDA in the in vitro biological systems, flavonols (0.07 mg/mL)
[129]
divided into 8 groups flavonol concentration (9.794 µg/mL and 94.211 mg/mL in cooking oil; G6-8 intoxicated with in mesquite pollen extract;
21.751 µg/mL), 200 µL orally bromobenzene—200 mL, 94.211 mg/mL in cooking oil after the ↓ MDA in the in vivo system, flavonols (21.751 mg/mL);
administration of vitamin E (400 UI) ↓ Liver LPO (the highest dose)
↓ TBARS in the liver, but without effect in brain;
↑ AOE in the liver, brain and lysate of erythrocytes;
Female CBA/Hr mice aged 4 months. Cystus incanus L. BP from location in Central Mice were fed 14 days before testing either with commercial food ↓ hepatic LPO;
Experimental and control group Croatia’s Dalmatia coast and offshore islands; pellets (control group) or with commercial food pellets mixed ↑ Apoptosis Hspa9a, Tnfsf6 (liver); [130]
consisted of 10 mice each orally administered to mice with bee pollen (100 mg/kg bw) ↓ Casp 1 and Cc121c;
↑ SOD, GSH-Px and CAT activity in the lysate of erythrocytes
(100 mg/kg bw)
Table 4. Cont.
The Type of BP, Collection Site and Application
Animal Models Applied Treatment Effects of BP Administration References
Method
SCPE—total phenolic content (53.74 ± 1.21 mg GAE/g), total
Schisandra chinensis (Turcz.) Baill. pollen extract
Male Kunming mice divided into In vivo study: SCPE (10, 20 and 40 g/kg) administered to flavonoid content (38.29 ± 0.91 mg Rutin/g);
(SCPE) from Xi’an, China; administered daily [131]
five groups of 12 animals each CCl4 -induced acute liver damage in mice ↓ ALT, AST in acute liver damage induced by CCl4 ,
orally for 42 days
↓ MDA formation in liver, ↑ SOD and GSH-Px
G1—control, 0.9% NaCl (i.p.); G2—control, 0.8 mL/kg olive oil
↑ AI in G4 compared to G5–G7.
(i.p.); G3—control, 0.8 mL/kg Ethanol (i.p.); G4—CCI4 , 0.8 mL/kg
BP collected during flowering season in Turkey Toxicity: ↑ plasma ALT and AST ↑ MDA in liver, RBC and plasma;
Forty-nine 12 weeks old CCI4 in olive oil; G5—Silibinin, 0.8 mL/kg CCI4 in olive oil +
(Western Black Sea region) with dominant ↓ SOD in plasma, RBC and liver
Sprague-Dawley rats divided in Silibinin (50 mg/kg/day) gavage; G6—low pollen, 0,8 mL/kg CCI4 [72]
component Castanea sativa L. (> 45%), Pollen administered groups:
seven groups in olive oil (i.p.) + Pollen (200 mg/kg/day) gavage; G7—high
200 mg/kg/day orally, 400 mg/kg/day orally, 7 days ↓ Plasma ALT: high dose
pollen, 0,8 mL/kg CCI4 in olive oil (i.p.) + Pollen (400 mg/kg/day)
↓ Plasma AST ↓ MDA in the plasma, RBC and liver
gavage
G1—negative control (0.9% NaCl solution by intraperitoneal
The treatment of mice with WEBP at a dose of 140 mg/kg b.
injection (i.p.) twice/week for 3 weeks).
wt./day, for 14 days with CDDP (2.8 mg/kg b. wt.) resulted in:
Effects of water extracts of Egyptian bee pollen G2—i.p. injection of CDDP (2.8 mg/kg b. wt.) twice/week for 3
↓ Lipid peroxidation in the liver, kidney and testis
(WEBP) from Beni-Suef, Upper Egypt, on cisplatin weeks.
Male albino mice divided in: six ↑ CAT and GSH in the liver, kidney and testis
(CDDP) induced hepatic, renal, testicular and G3—8.4 mg/kg b. wt. of propolis extract oral/day for 14 days. [132]
groups with 11 animals each. The positive control animals taken CDDP alone showed toxic
genotoxicity in male albino mice; G4—WEBP (140 mg/kg b. wt.) oral/once/day for 14 days.
histological and genetical manifestations
Orally administered G5 and G6—CDDP injected i.p. with (2.8 mg/kg b. wt.
↑ LPO in kidney, liver and testis
twice/week) alone for 1 week and for the next 2 weeks were given
↓ CAT and GSH in the kidney, liver and testis
WSDP and WEBP by oral intubation and i.p. injection of CDDP
G1—normal saline (10 mL/kg/day) for 12 days and i.p. with
saline (10 mL/kg) at the 7th day
↓ MDA in kidney and dose-dependent in liver
G2—normal saline (10 mL/kg/day) for 12 days and i.p. CP
36 adult male Sprague Dawley rats Schisandra chinensis (Turcz.) Baill. bee pollen ↓ iNOS in liver and dose-dependent in kidney
(8 mg/kg) at the 7th day
divided into six groups of six extract (SCBPE) from Jiaozhou, Shandong, China; ↑ SOD dose-dependent in liver and kidney [133]
G3—i.g., VC (400 mg/kg/day) for 12 days days and i.p. CP
animals each intragastrically administered (i.g.) in mice ↑ CAT in the liver and kidney
↑ GSH in the liver and kidney
G4-6—SCBPE (400, 800, 1200 mg/kg/day) for 12 days and i.p. CP
Note: AI—apoptosis index; ALT—alanine aminotransferase; AOE—modulated antioxidant enzymes; AST—aspartate aminotransferase; CAT—catalase; CCl4 —Carbon tetrachloride;
CDDP—cis-diamminedichloroplatinum(II), cipslatin; Cy—cyclophosphamide; GAE—gallic acid equivalent; GBX—fat-soluble (10–16% of phytosterols) substances; GSH—reduced
glutathione; GSH-Px—glutathione peroxidase; HFD—high-fat diet; iNOS—inducible nitric oxide synthase; LPO—lipid peroxides; MDA—malondialdehyde; RBC—red blood cell;
SOD—superoxide dismutase; TBARS—thiobarbituric acid reactive substances;; WSDP—water-soluble propolis derivative; ↓—decrease; ↑—increase.
4.3. Enzyme-Level Changes Induced by Bee Pollen

In this section, we have introduced the changes in enzyme levels for the prevention of these cancer
therapy-induced toxicities (Table 5).
Table 5. Enzyme-level changes induced by BB in response to anti-cancer therapies.
Molecules Organs Species Agents Change References

Oxidative stress
Liver, brain, heart rats Propoxur ↑ [71]
kidney rats Propoxur ↓ [71]
Testis rats CdCl2 ↑ [134]
CAT Plasma rats CCl4 ↓ [135]
Liver rats CCl4 ↓ [135]
Liver, kidney, testis mice Cisplatin ↑ [132]
Liver, kidney rats Cisplatin ↑ [133]
GST Liver, kidney, testis mice Cisplatin ↑ [132]
GSH Testis, prostate rats CdCl2 ↑ [134]
Brain rats F ↑ [136]
Liver, kidney, heart,
rats Propoxur ↑ [71]
brain
Testis rats CdCl2 ↑ [134]
Liver mice CCl4 ↑ [131]
SOD
Liver rats CCl4 ↑ [72]
Plasma rats CCl4 ↑ [135]
rats Propoxur ↑ [71]
GSH-Px brain
Liver mice CCl4 ↑ [131]
Liver mice Bromobenzene ↓ [129]
Brain rats F ↓ [136]
Liver, kidney rats Cisplatin ↓ [133]
Liver, kidney, testis mice Cisplatin ↓ [132]
MDA
Liver mice CCl4 ↓ [131]
rats Propoxur ↓ [71]
brain
iNOS Liver, kidney rats Cisplatin ↓ [133]
Inflammation
IL-6 Prostate, testis rats β-estradiol ↓ [137]
TNF-α Prostate, testis rats β-estradiol ↓ [137]
Note: CAT—catalase; CCl4 —carbon tetrachloride; CdCl2 —cadmium chloride; F—fluoride; GSH—reduced
glutathione; GSH-Px—glutathione peroxidase; GST—glutathione peroxidase; iNOS—inducible nitric oxide synthase;
IL-6—interleukin-6; MDA—malondialdehyde; Propoxur—2-isopropoxyphenyl methylcarbamate; TNF-α—tumor
necrosis factor-alpha; ↑—Increased molecular change by chemotherapeutic agents; ↓—Decreased molecular change
by chemotherapeutic agents.
5. Potential of BP and BB in Complementary Therapies That Can Be Associated to Antineoplasic

Treatments (Anxiety, Antinociceptive and Anti-Inflammatory Activities)
Anxiety is a common human mental illness. Medical treatment of this disease is associated
with many side effects. Some of these drugs interfere with antineoplastic drugs, causing drug-drug
interactions that will change the expected outcome for chemotherapy protocols.
For this reason, the search for new drugs with fewer side effects seems inevitable. In this study,
the potential anxiolytic effects of BP hydroalcoholic extract were carried out on 20–25 g male rats in
eight groups of three. Animals were injected intraperitoneally with BP hydroalcoholic solubles at doses
of 200, 400, 800 and 1600 mg/kg, diazepam at a dose of 1 mg/kg and saline at a dose of 10 mL/kg. Rats
receiving 800 and 1600 mg/kg hydroalcoholic BP extracts showed longer presence in the open arms of
an elevated plus maze device compared to animals receiving diazepam. As a result, BP showed an
anxiolytic effect of the hydroalcoholic BP extract in rats [138]. However, the identification of the botany
origin of the products under essay and the compounds responsible for the bioeffects are absolutely
fundamental for further investigation.
Data collected with flavonoids from medicinal plants associated to the anxiolytic effect [139,140]
explains this bio-effect. The main activity is often associated to the free amino acids as glutamate, but also
to the linkage of certain flavonoids, as luteolin and derivatives to the GABAA -benzodiazepine receptor.
The therapeutic effects of BP on glutamate excitotoxicity of the brain and glutamine-glutamate-gamma
aminobutyric acid (GABA) induced by propionic acid (PPA), a short chain fatty acid to rat pups, were
investigated. The results showed that the excitotoxicity measured by increasing PPA, glutamate and
glutamate/glutamine ratio and lowering the ratio of GABA, glutamine and GABA/glutamate leads to
multiple indications and is effective in removing the neurotoxic effects of BP to some extent [141].
Pain relief is an important part of the anticancer treatments. Nowadays, several drugs can be
given, but the resistance in the effectiveness in some patients drives to a better understanding of this
process and the importance of searching for new approaches.
For instance, pollen from pine (Pinus densiflora Siebold & Zucc.) was tested for the antinociceptive
and anti-inflammatory activity in mice using carrageenan- and formalin-induced paw oedema and
arachidonic acid-induced ear oedema. The ethanol extract of (100 and 200 mg/kg, per os (p.o.))
inhibited both tested phases of the formalin pain test in mice, reducing mouse writhing induced
by acetic acid and elevating pain threshold in the hot plate test. The P. densiflora pollen extract also
caused significant inhibition of carrageenan- and formalin-induced paw oedema, as well as arachidonic
acid-induced ear oedema, compared with the control group. The different polyphenols found in Pinus
densiflora Siebold & Zucc. pollen could account for the antinociceptive and anti-inflammatory actions.
The results obtained indicate that the extract possesses analgesic and anti-inflammatory effects [66].
Even though it is known that Pinus pollen is not usually part of the floral sources associated with
BP or BB, the data investigated until now give important information about its anti-inflamatory and
antioxidant acitivities and significant flavonoids content. Given that these compounds exist in virtually
all of the samples studied to date of BP and BB, their potential appearing to be immense, and should
be largely explored in the future.
The result of one study showed that BB supplementation diet of rabbits improved wound healing
parameters, such as wound tensile strength, neovascularization and fibroblast count in the incision
wound, but with no significant difference in the epithelization and hydroxyproline content of the
supplemented group compared to the control. This experiment indicated the possibility of using BB
in malnourished patients to improve surgical outcomes [142]. However, as highlighted above, this
bioactivity is mainly related to the angiogenic process and could be dangerous for the patient. Again,
the evaluation of the benefit/risk should be carefully performed.
Inflammation and oxidative stress are closely related with anti-cancer agent-induced toxicities [72,
132,137]. Regarding inflammation, it has been reported that BP lowered the levels of interleukin-6
(IL-6) and tumor necrosis factor-alpha (TNF-α) compared to β-estradiol-fed rats [137].
It has been established that pollen grains contain NAD(P)H oxidases that induce oxidative stress
in the airways, thus leading to the development of allergic inflammation.
GSH, glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST) and SOD are well-known
endogenous anti-oxidants and anti-oxidant parameters. Increased activities and levels of these factors
were detected in the liver, kidney and testis of rats and mice treated with CP and BP compared to
only CP-treated ones [132,133]. Moreover, increased levels of SOD were detected in the liver and
plasma of rats treated with BP compared to those administered CCl4 alone [72,131,135]. Additionally,
these studies also showed that levels of malondialdehyde (MDA), commonly used as an oxidative
stress biomarker, were significantly lowered in the kidney, liver, heart and brain tissues of rats and
mice treated with propoxour or CP and BP, compared to those administered CP alone [71,132,133].
Decreased levels of this factor were detected in the liver of rats and mice treated with BP compared to
those treated with CCl4 alone [72,131,135].
In rats and mice treated with BP, the MDA levels in the liver and kidney were significantly lowered
compared to the groups treated with CCl4 or CP alone [72,131–133,135].
The GSH-Px levels in the liver, kidney, heart and brain were significantly higher in rats administered
with propoxour and BP than those administered propoxour only [71]. Similar findings were also
reported in the liver of mice treated with CCl4 [131].
As referred above CP can induce hepato- and nephro-toxicity in patients that undergo this
treatment. Huang et al. [133] found that the GSH levels in the kidney and liver of rats treated with
BP and CP were significantly higher compared to those treated with CP alone. Similar findings were
reported in the prostate and testis of rats treated with BP.
BB in aluminium toxicity: aluminium, blood supply, transaminase, C-reactive protein and
monocytes have caused a significant rise in the number of levels, and significantly decreased
haemoglobin. These changes have improved significantly by BB administration. It has antioxidant
features and has demonstrated a protective effect on the elevation of C-reactive protein, leukocytes and
monocytes of blood and hepato-renal toxicity and inflammatory markers of aluminum-borne [143].
6. Clinical Trials
The positive effects of functional food can either maintain a welfare and health condition or reduce
the risk of pathological consequences. Adverse and unwanted side effects of foods are important for
human health, especially unprocessed functional foods. With the increase in environmental humidity
and temperature, the total number of coliform can increase due to untreated BP consumption. This
aspect may be challenging for human and animal health, due to many microorganisms that may be
harmful to body health [144]. On the other hand, BB consumption proves to be more efficient, due to
lactic fermentation that enhances storage longevity and increases digestibility.
In the samples of BP and BB collected from Transylvania, it was found out that K tested from the
BP and BB samples has the highest concentrations, followed by Ca and Mg. Noteworthy oligo-elements,
such as Fe and Zn, were also found. These results confirm that Romania’s BP and BB can be used by
people as a natural mineral source [145].
Thus, BP and BB may be excellent candidates for future studies involving BP flavonoids,
phytotherapy, molecular pharmacology, allergic and immunotherapeutic chemicals and antibacterial
and antitumor potential. Additionally, many studies suggest that phenolic compounds are the main
active agents of these products, but not the only ones [32,146].
Chronic Prostatitis
Chronic prostatitis (CP) is one of the most frequent disease in men aged over 50, with different
clinical presentations [147,148]. Class III chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is
the most frequent category in accordance to the National Institute of Health (NIH) classification [149].
Additionally, available therapies and therapeutic efficacies are scarce and require further in-depth
analysis, also due to recent discovery that CP might defect semen quality. Therefore, a noteworthy
investigation needs to be done in pathogenesis and alternate medication of CP.
Oxygen free radicals (OFR), which cause tissue damage by LPO [150], comprise mainly superoxide
free radical anion (O2 •¯), hydrogen peroxide (H2 O2 ), nitrogen monoxide (NO) and hydroxy free
radical (•OH). Destruction of cellular segment is a result of LPO yield of multiple types of secondary
free radicals and reactive compounds. With all these, cells are equipped with antioxidants, such as
vitamin C and E, CAT, GSH, SOD, which can act as defensive mechanisms against the cytotoxicity and
supernumerary of OFR [151]. Thus, OFR play an important role in pathogenesis of CP and infertility.
BP of Brassica rapa L. is widely used as a natural food supplement in China and as a herbal
medicine that strengthens the body’s resistance to diseases, including cancer. A steroid fraction of the
chloroform extract from BP of B. rapa L. could trigger cytotoxicity by inducing apoptosis. B. rapa L.
shows that the steroid fraction of chloroform extract from BP may be a promising candidate for the
treatment of advanced prostate cancer [64].
Promising results of pollen extracts in the treatment of chronic prostatitis, prostatodynia (prostate
pain) and benign prostatic hypertrophy (BPH) have been obtained. Pollen Extract Cernilton® has been
used in chronic prostate treatment for many years and has positive results. The fatty part of the pollen
extract inhibits prostaglandin synthesis by inhibiting the enzyme lipoxygenase and cyclooxygenase in
the eicosanoid chain. This results in an anti-inflammatory effect. Pollen extracts have a selective and
specific effect on prostate. Experimentally, pollen extracts inhibit the growth of prostate cells in cell
cultures. It has been established that this inhibition is related to the dose and duration of treatment.
For treatment of chronic prostatitis syndrome, 26 (36%) patients were treated with a pollen extract
Cernilton® at a dose of 1 tablet tid for 6 months, 30 (42%) had a flow rate increase, leucocytosisin
post-prostatic massage urine and ejaculate in C3/koeruloplasmin complex. Cernilton® has been
reported to be well tolerated in 97% of patients [16]. Another study evaluated the effect of pollen
extract on the lower urinary tract symptoms of the preparation Prostate/Poltit in patients with chronic
nonbacterial prostate/chronic pelvic pain syndrome. In the general clinical evaluation of the treatment
outcome, pollen extract preparation after treatment was reported to be more effective in patients treated
with prostate/poltit than placebo-treated patients [19,20].
Additionally, the efficacy of Cernilton® , a rye-grass pollen extract versus placebo in men with
category IIIA CP/CPPS was evaluated in a study consisting of 93 patients divided into two groups
(Cernilton® to Cernilton® , n = 48; placebo to Cernilton® , n = 45) up to 24 weeks. The results clearly
showed that the pain, quality of life domains and the total NIH-CPSI score improved significantly
at week 12 in the Cernilton® group versus placebo and continued to improve at week 24 in both
groups [20].
Qian et al. [152] followed the research with the evaluation of the therapeutic efficacy of Cernilton®
in BPH patients with histological prostatitis after transurethral resection of the prostate (TURP). They
concluded that Cernilton terapy may improve lower urinary tract symptoms in patients with moderate
prostatitis, as well as sexual dysfunction in patients with severe prostatitis.
Night sweats, pain, hair loss, forgetfulness, depression and sleep disorders are common problems
in breast cancer patients who are subjected to anti-hormonal treatment. Evidence has been provided
that honey and BP mixture can improve the menopausal symptoms of breast cancer patients receiving
anti-hormonal therapy [153].
In Table 6 clinical studies regarding the effect of BP and BB administration in patients with chronic
prostatitis or chronic pelvic pain syndrome are reported.
Table 6. Effects of BP extract treatmens in patients with chronic prostatitis or chronic pelvic pain syndrome.
BP Patient Disorders Treatment Schemes Obtained Results References
↑ lasting relief and symptom-free in seven patients
BP extract Cernilton® (several different 15 patients with chronic relapsing non-bacterial Cernilton® administration varied from 1 to 18
↑ improvement in six patients [154]
plants from Sweden) prostatitis or prostatodynia months
↓ response in two patients
↑ subjective improvement with Cernilton® (69%) compared
53 patients with benign prostatic hyperplasia Patients were administered Cernilton® (n = 29) with placebo (30%)
BP extract Cernilton® (several different
(BPH) entered in a double-blind and placebo (n = 24) in a dose of two capsules for 6 ↓ residual urine in Cernilton® -treated and in the [155]
plants in southern Sweden)
placebo-controlled study months antero-posterior (A-P) diameter of the prostate on
ultrasound
↑ response in the group without CFs, 56 (78%); 26 (36%)
treatment of chronic prostatitis syndrome in 90 were cured of their symptoms and signs
patients; G1—those without associated ↑ in flow rate
Cernilton® R N given in a dose of 1 tablet tid and
complicating factors (CFs) (n = 72) ↓ in leucocyturia in the post-prostate massage urine (VB3)
BP extract Cernilton® R N in most cases treatment was continued for 6 [16]
G2—those with complicating factors, i.e., urethral and
months
structures, prostatic calculi, bladder neck sclerosis ↓ complement C3/coeruloplasmin in the ejaculate in 30
(n = 18) (42%) in the patients with CFs, only one patient showed a
response.
G1: group A (n = 25): EA-10, P5 + Roxithromycin;
Pre-treatment group:
group B (n = 21): EA-10, P5; group C (n = 22):
↑ LEPS, MDA, NO
Roxithromycin;
G1—68 cases of CP ↓ Zn content and SOD;
BP extract EA-10, P5 (375 mg/pill) G2: group A (n = 20): EA-10, P5 + Roxithromycin; [156]
G2—63 cases of infertility with CP Post-treatment:
group B (n = 21): EA-10, P5; group C (n = 22):
↑ LEPS, Zn content and sperm motility and viability
Roxithromycin;
↓ MDA and NO
Administration twice daily for 4 weeks
87 patients:
↑ pain/discomfort domain score
G1: control (n = 18); Patients received two capsule daily from 4 to 6
Cernilton® /Cernitin pollen extract (G1: 0.39; G2: 9.79; G3: 1.66) [157]
G2: NIH chronic prostatitis category III (n = 34); weeks
↑ QoL (G1: 0.39; G2: 8.21; G3: 4.17)
G3: BPH (n = 35)
↓ NIH-CPSI and QoL
Each patient was given 1 tablet of prostat (70 mg
Prostat/Poltit 115 patients with chronic nonbacterial prostatitis ↓ symptom rating scores [158]
P5 + 4 mg EA10) twice a day for 8 weeks
↓ WBC counts in prostate massage fluid
Two groups: 58 patients between 20 and 55 years patients taking Prostat/Poltit:
The dose was three tablets/day. The placebo tablets
Prostat/Poltit (74 mg highly defined old with chronic nonbacterial prostatitis or chronic ↑ clinically improvement or symptom-free in 22 patients,
were identical in appearance to the active tablets, [19]
extract of BP from selected Graminae spp.) pelvic pain syndrome were randomized to receive compared to
but contained no pollen extract.
Prostat/Poltit (n = 30) or placebo (n = 28) ↑ improvement in 10 of the patients taking placebo
Participants were randomised to receive oral ↑ individual domains pain, QoL and NIH-CPSI score after
139 men randomly allocated to the pollen extract
BP extract Cernilton® capsules of the pollen extract (2 capsules q8 h) or 12 wk of treatment with pollen extract compared to placebo. [20]
(n = 70) or placebo (n = 69).
placebo for 12 wk Adverse events were minor in all patients studied.
↓ NIH-CPSI total score by ≥ 25%
Participants were randomised to receive oral ↓ Adverse events in the DEPROX 500® treatment group
87 males (25 class IIIa and 62 class IIIb) with a
DEPROX 500® (1 g pollen extract capsules of DEPROX 500® (two capsules/day; compared to ibuprofen
mean age of 33.6 ± 5.9 years with chronic [159]
(500 mg per tablet) and vitamins n = 41) or ibuprofen (600 mg, one tablet three ↑ pain relief and QoL in DEPROX 500® treatment group
prostatitis/chronic pelvic pain syndrome
times/day; n = 46) for four weeks ↑ antioxidant activity of the pollen extract and protective
effect on nerves by vitamins combination
Note: BPH—Benign Prostatic Hyperplasia; LEPS—leukocytes in expressed prostatic secretion; MDA—malondialdehyde; NIH-CPSI—The National Institutes of Health Chronic Prostatitis
Symptom Index; NIH—National Institutes of Health; NO—nitric oxide; QoL—Quality of Well-Being; SOD—superoxide dismutase; WBC—white blood cell count; ↓—decrease; ↑—increase.
In the European Association of Urology guidelines, phytotherapy, including Cernitin pollen extract
(Cernilton® ), is recommended for patients with inflammatory prostate pain syndrome, with no known
reports of severe adverse events associated with its administration. If such a non-antimicrobial drug
can effectively reduce serum prostate specific antigen (PSA) in prostate biopsy candidates with chronic
inflammation of the prostate, an optimal method including that could be developed for avoidance of an
unnecessary biopsy“ [160]. Thus, to verify this possibility, Togo et al. [160] administered Cernitin pollen
extract tablets to prostate biopsy candidates for 30 days, before carrying out a prostate biopsy procedure.
Then, they evaluated the relationship between the reduction in serum PSA levels and prostate biopsy
outcomes. The authors suggested that effective protocols using Cernitin pollen extract have potential
in avoiding unnecessary prostate biopsy procedures in patients with elevated prostate-specific antigen.
Further studies are required to confirm their findings in order to develop an extensive protocol that
can be used on a large number of participants.
7. Therapeutic Strategies for BP and BB and Future Perspectives

In today’s world, as Api-Nutrition combined with natural and healthy foods is gaining importance,
more research and innovation is required on the production, consumption and health effects of bee
products, particularly in the therapeutic field.
Compared to other natural products, BP and BB have the advantage to bring a significant amount
of nutrients that meet the human body needs, which is a premise for optimal functioning, a good
functioning of the immune system and resistance against illnesses, as well as supporting the healing
processes in the body.
BP and BB have been studied for their promising potential agents in cancer therapy. As mentioned
above, their inhibitory effect, such as the prevention of tumor growth, has been confirmed by in vitro
and in vivo studies, in animal experiments and in certain types of cancer, such as prostate cancer, but
with standardized extracts. With all these, many clinicians and researchers claim that the anti-cancer
effect exerted by BP are not satisfactory for the improvement in terms of prognosis and survival rates,
especially when it comes to the scarce publications regarding BB. In fact, a controlled clinical essay
should be performed in order to ensure the Efficacy versus the Safety, regarding other potential extracts
from more active floral sources in this area.
Other investigations showed that various fractions of BP are potential therapeutic agents for
different types of cancers, for example, a steroid fraction of chloroform extract from Brassica rapa
L. BP (CPBC) was tested on human cancer cell viability. Protein expression was detected by rabbit
polyclonal anti Bcl-2 antibody and secondary antibody (goat anti-rabbit immunoglobulin G (IgG))
conjugated with peroxidase. The study showed that among all nine cancer cell lines of different origin,
the steroid fraction displayed the strongest cytotoxicity in human prostate cancer PC-3 cells. After
the treatment, an obviously enhanced Caspase-3 activity was observed, along with a time-dependent
decrease in the expression of anti-apoptotic protein Bcl-2 [64]. In addition, this study also showed that
the cells derived from human prostate (PC-3, LNCaP) proved to be more sensitive to the treatment
than those of non-prostate origin, suggesting that CPBC may be a selective cytotoxic agent of human
prostate-derived cancer cell lines.
Plant sterols have been proven to exert anticancer effects in multiple cell lines, which inhibited the
growth of MDA-MB-231 and HT-29 cells cells [101]. Currently, brassinolide is gaining ground in the
medical field. It is a naturally occurring plant hormone that promotes growth, increases yields for grain
and fruit crops, and makes plants resistant to drought and cold conditions. Brassinolide, from Brassica
napus L. pollen, was isolated to investigate the cell viability effect on androgen-independent human
prostate cancer PC-3 cells. The cells were treated with different concentrations of brassinolide (0, 10, 20
and 40 mM) for 12, 24 and 36 h. The results clearly showed that a 12 h interval induced a concentration
dependent increase in the apoptotic rate and an increase in caspase-3 activity [64]. As human prostate
cancer was shown to be highly carcinogenic and metastatic, hormone unresponsive and resistant to
normal rates of apoptosis, brassinolide proves to be an efficient approach for future studies regarding
its effectiveness in prostate cancer and other diseases. Recently, the apoptotic effect of this plant sterol
was tested on drug-resistant (VPA17) and drug-sensitive (H69) SCLC (small-cell lung carcinoma) cells,
with high cytotoxic effect (IC50 = 2 µM) after 24 h, proving to be pharmacologically active in both
drug-sensitive and drug-resistant SCLC cells [161].
Previously, in this paper, we introduced combined therapies of BP and other anti-cancer agents
in the treatment of prostate cancer. For example, temozolomide (TMZ), an oral alkylating agent that
belongs to imidazotertrazines [162], is known to exhibit anti-cancer properties [163,164], recently tested
in human glioma cells. The combination of TMZ and BB exerted strong cytotoxic activity on human
glioblastoma multiforme cells (U87MG) than TMZ alone.
Additionally, in a recent study, the activity of Graminex pollen (assortment of standardized
pollen of Secale cereal L., Zea mays L. and Phleum pretense L.) on prostate cells (PC3) and in rat prostate
specimens along with E. coli lipopolysaccharide (LPS) was evaluated. A significant cytotoxic effect on
prostate PC3 cancer cell lines was observed at the highest tested concentration (500 µg/mL), Graminex
pollen reducing ROS production by PC3 cells and MDA, NF-κB mRNA and prostaglandin E2 (PGE2 )
levels in rat prostate specimens [165]. Other examples should be explored to amplify the possibilities
in cancer treatments.
8. Conclusions
In this review, summarized studies on the main bioactivities of BP and BB, correlated to cancer
research, were reported based on in vivo and in vitro studies.
Countless reports demonstrated that BP and BB could be explored to protect against anti-cancer
agent-induced toxicities, particularly in liver and kidney fibrosis. Furthermore, specific extracts can
do modulation of various biological activities involving protection via the anti-oxidative activity and
inflammation, which is closely associated to some constituents, for example, phenolic and polyphenolic
compounds found in BP and BB.
Therefore, a strong hypothesis for the safety incorporation of these products into the daily human
diet as a food supplement is needed. For instance, Castanea and Brassica BP would increase the inhibition
of inflammation and oxidative stress, respectively. Several clinical studies have confirmed the efficacy
of BP and BB against drug-induced toxicities; however, further research should be carried out, as most
of them show studies with small population groups, either in animal or in patients. Therefore, more
detailed examinations are vital for discussing the clinical efficacy of BB and BP in these patients, with
long term treatment, and research regarding chronic toxicity. Data for drug-herb (pollen/extracts)
are still scarce and will prove to be significantly important in future treatment protocols. Although
multiple problems remain unsolved, we consider that these bee products are useful tools for the
prognosis and improvement of patients’ quality of life with various diseases and even in anti-cancer
therapies/therapeutic protocols that should be validated for their safety.
Author Contributions: Important contributions to design and also to prepare the manuscript: R.M., M.C.-C.,
M.G.C., E.T. and M.S. The manuscript was revised by A.V., D.C.V. and B.Y. All authors helped preparing the paper
and approved the final version.
Funding: This work was supported by the National Research, Development and Innovations Programme for
2015-2020-PNII, developed with the support of UEFISCDI (Project No.PN-III-P1-1.2-PCCDI-2017-0473; 8 PCCDI,
and the publication was supported by funds from the National Research Development Projects to finance excellence
(PFE)-37/2018–2020 granted by the Romanian Ministry of Research and Innovation.
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Microbiological Research 192 (2016) 130–141
Contents lists available at ScienceDirect
Microbiological Research
journal homepage: www.elsevier.com/locate/micres
Review
Royal Jelly: An ancient remedy with remarkable antibacterial

properties
Filippo Fratini a,b,∗ , Giovanni Cilia a , Simone Mancini a , Antonio Felicioli a,b
a
Department of Veterinary Sciences, Viale delle Piagge 2, University of Pisa, Italy
b
Interdepartmental Research Center “Nutraceuticals and Food for Health”, Via del Borghetto 80, University of Pisa, Italy
a r t i c l e i n f o a b s t r a c t
Article history: Royal Jelly (RJ), a honeybee hypopharyngeal gland secretion of young nurse and an exclusive nourishment
Received 31 March 2016 for bee queen, has been used since ancient times for care and human health and it is still very important
Received in revised form 7 June 2016 in traditional and folkloristic medicine, especially in Asia within the apitherapy.
Accepted 19 June 2016
Recently, RJ and its protein and lipid components have been subjected to several investigations on their
Available online 23 June 2016
antimicrobial activity due to extensive traditional uses and for a future application in medicine.
Antimicrobial activities of crude Royal Jelly, Royalisin, 10-hydroxy-2-decenoic acid, Jelleines, Major
Keywords:
Royal Jelly Proteins against different bacteria have been reported. All these beehive products showed
Royal Jelly
Antimicrobial activity
antimicrobial activities that lead their potential employment in several fields as natural additives. RJ and
Honeybees its derived compounds show a highest activity especially against Gram positive bacteria.
Major Royal Jelly Proteins The purpose of this Review is to summarize the results of antimicrobial studies of Royal Jelly following
10-hydroxy-2-decenoic the timescale of the researches. From the first scientific applications to the isolation of the single com-
Natural peptides ponents in order to better understand its application in the past years and propose an employment in
future studies as a natural antimicrobial agent.
© 2016 Elsevier GmbH. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
1.1. Historic background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
2. Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
2.1. Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
2.2. Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
2.3. Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
2.4. Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
2.5. Minerals and other minor elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
3. Mechanisms of antimicrobial peptides action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
4. Antibacterial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
1. Introduction
Royal Jelly (RJ) is a glandular secretion white-yellowish (Fig. 1),

gelatinous-viscous sour taste, with a slight characteristic smell of
phenol (which gives it its characteristic flavour) produced from the
hypopharyngeal and mandibular salivary glands of young nurse
∗ Corresponding author at: Department of Veterinary Sciences, Viale delle Piagge (bees aged between 5 and 14 days) (Chauvin, 1968; Fujita et al.,
2, University of Pisa, Italy. 2013). RJ is the exclusive nourishment for all bee larvae, from
E-mail address: filippo.fratini@unipi.it (F. Fratini). hatching to the third day of life; those larvae which are selected
http://dx.doi.org/10.1016/j.micres.2016.06.007
0944-5013/© 2016 Elsevier GmbH. All rights reserved.
F. Fratini et al. / Microbiological Research 192 (2016) 130–141 131
to develop into queens are fed with RJ until the fifth day of larval age, of ancient dynasties of China (Cherbuliez and Domerego, 2003;
life (the time at which the cell is operculated), and then RJ remains Contessi, 2010). Jan Swammerdam (1637–1680), a Dutch natural-
a dedicated feed for the queen bee alimentation for the duration ist, microscopist and entomologist, was the first to described the
of her life. Furthermore, RJ also has a significant impact on the life compound of nourishment in the royal cell and discovered that
span: a worker bee lives around 45 days, while a queen bee could the “beehive chief” is a queen and not a king as supposed until
live up to five years during which is able to spawn in a day the the seventeenth century (Contessi, 2010; Viel and Doré, 2003). The
equivalent of her weight in eggs (approximately 2000–3000 eggs French scientist René Antoine de Réaumur (1683–1757) coined the
per day for several years). term “Royal Jelly” to name the feed of queen bee and he related
Storage conditions of RJ for its human employment is a critical the assumption of RJ with the exceptional growth of the queen
point for maintain unchanged its properties; RJ is light and heat (Cherbuliez and Domerego, 2003; Molan, 1999). In 1852 Reverend
susceptible and undergoes oxidation to a direct contact with air Langstroth, known as the father of American beekeeping, was the
(Bogdanov et al., 2004; Buttstedt et al., 2013; Kheyri et al., 2012; first to analysed chemically RJ, however he used methods did not
Sabatini et al., 2009; Scarselli et al., 2005; Zhang et al., 2012). guarantee a scientifically significant information (Domerego, 2001;
Levet, 2008). Langstroth also proposed during the fifties the use of
1.1. Historic background RJ as a commercial product, especially in areas where the produc-
tion of honey was not profitable (Contessi, 2010; Viel and Doré,
The first historical notes about human employment of RJ date 2003). The use of RJ as a functional product and health enhancer
back to ancient Greece; Greeks thought that the “ambrosia”, the was investigated since the early 60s, with the development of the
nectar which gave immortality to the gods of Olympus, was com- “Apitherapy”. From then on, particularities and properties of RJ
posed in part by RJ. At that time it was already consumed without were discovered and RJ reached a widely used in therapy for both
knowing its specific effects, and historians reported that the hon- men and bee itself (Contessi, 2010; Molan, 1999).
eycombs were shredded with inside honey, larvae, propolis, pollen
and RJ and eaten fresh (Cassaignau, 1991; Mraz, 1995). Aristotle was 2. Composition
the first to have discovered the function of RJ in the bees society and,
by studying its effects in queen bee, he attributed to the consump- RJ is an acid colloid (3.6–4.2 pH) composed mainly by water,
tion of RJ an increase of physical strength and, above all he supposed sugar, proteins, lipids, vitamins and some mineral salts (Melliou
its role in an improvement of intellectual capacity; the breakfast of and Chinou, 2005; Ramadan and Al-Ghamdi, 2012; Vecchi et al.,
his school was exclusively made with honey and RJ (Domerego, 1993).
2001; Molan, 1999). In ancient Egypt, RJ was used like a cosmetic, The major component is water, ranged from 60% to 70% (Caboni
which reached its zenith of notoriety with Cleopatra, as one of her et al., 2004; Melliou and Chinou, 2005), followed by carbohydrates
personal beauty secrets. Furthermore, in that period RJ became a from 11% to 23% (Sabatini et al., 2009; Sesta, 2006), proteins from
symbol of strength and majesty of the Pharaohs, which usually ate 9% to 18%, (Melliou and Chinou, 2005; Ramadan and Al-Ghamdi,
RJ (Emonet, 2001; Levet, 2008). In Asia, specifically in China, RJ is 2012; Simuth, 2001), lipids from 4% to 8%, (Malka et al., 2009; Nagai
used in traditional medicine since ancient time. This product of bee- and Inoue, 2005; Sabatini et al., 2009) and there are present in
keeping, which was produced exclusively in the sovereign gardens, low amount vitamins and mineral salts with other unknown sub-
was correlated with the longevity and the sexual force, even in old stances present in traces and all together could range from 0.8–3%
Fig. 1. Hive and royal cells with Royal Jelly and queen bee larvae.
A. Hive for breeding queen bees and Royal Jelly production, constitued by only royal cells. B. Queen bee larvae during development in royal cells filled with Royal Jelly. C.
Queen bee larvae removed by royal cells for the Royal Jelly collection.
132 F. Fratini et al. / Microbiological Research 192 (2016) 130–141
Fig. 2. Mean composition of Royal Jelly.
(Fig. 2) (Caboni et al., 2004; Lercker et al., 1992; Scarselli et al., 2005; 2.2. Proteins
Simúth et al., 2004; Zhang et al., 2012).
RJ composition could vary with seasonal and regional conditions Proteins can reach about the 50% of dry matter of RJ (Bilikova
of feeding (Antinelli et al., 2003; Attalla et al., 2007; Biondi et al., et al., 2002; Buttstedt et al., 2014; Furusawa et al., 2008; Scarselli
2003; Chen and Chen, 1995; Sabatini et al., 2009), with metabolites et al., 2005). During the last 20 years RJ was deeply investigated
and changes in the physiology of nurse bees as well as with the and several proteins have been identified.
larval age (Abd-alla et al., 1995; Brouwers et al., 1987; Lercker et al., Important protein components of RJ are those belonging to a
1985, 1992), with bees genetic and race (Liu et al., 2008; Malka family named Major Royal Jelly Proteins (MRJPs), or named apal-
et al., 2009; Sano et al., 2004; Zheng et al., 2011), and above all bumins, that represent the 83–90% of protein component (Scarselli
could be modified from the storage conditions postharvest (Caboni et al., 2005; Simuth, 2001). In this protein family have been identi-
et al., 2004; Li et al., 2008; Liu et al., 2008; Ragab and Ibrahim, 1999; fied eight proteins (MRJP 1–8) with molecular masses ranged from
Zheng et al., 2011). Several researches correlated these variations 49 to 87 KDa (Albert and Klaudiny, 2004; Albert et al., 1999a,b;
of the composition of RJ to its antimicrobial activity (Abd-alla et al., Hanes and Simuth, 1992; Malecova et al., 2003; Moriyama et al.,
1995; Li et al., 2008; Liu et al., 2008; Ragab and Ibrahim, 1999; 2015).
Zheng et al., 2011). The MRJPs plays an essential nutritional role in the diet of the
Pollen grains are always presents in RJ as contaminant and queen bee (Tamura et al., 2009); MRJP 1, MRJP 4 and MRJP 5 rep-
could are very useful as indicators of geographical origin and may resent the main intake of essential amino acids, as well MRJP 2,
enriched RJ with some proteins from plants origin (Biondi et al., and MRJP 5 are the most important nitrogen reserve for its growth
2003; Chen and Chen, 1995; Scarselli et al., 2005; Simúth et al., (Albert et al., 1999b; Schmitzova et al., 1998); moreover, MRJP 3 is
2004). a polymorphic protein, and this might also explain the role of MRJP
3 as nitrogen supply(Albert et al., 1999b).
2.1. Carbohydrates The MRJPs could also play an important role in the production
of other bee products, especially in formation of pollen-pellet and
Carbohydrates represent about 30% of dry matter (Sabatini pollen-bread (Simuth, 2001); and it was demonstrated that they
et al., 2009; Sesta, 2006) and they may be important indicators have a major role in the differentiation between queen bee and
of the authenticity of RJ, through the analysis of minor sug- worker (Buttstedt et al., 2013).
ars contained(Daniele and Casabianca, 2012; Lercker et al., 1981; MRJP 1 is also recovered in the honeybee neurons, this suggests
Serra Bonvehi, 1992).The most abundant sugars, as in honey, are another unknown function of the protein in addition to nurturing
fructose, glucose and sucrose (Ramadan and Al-Ghamdi, 2012; (Peixoto et al., 2009).
Wytrychowski et al., 2012), but small traces of oligosaccharides MRJP 1 and MRJP 2 have been characterized like major allergens
such as maltose, trehalose, melibiose, ribose and other sugars can in the RJ, and, in vivo, stimulate mouse macrophages TNF-alpha
also be found (Finke, 2005; Kheyri et al., 2012; Lercker et al., 1981, production (Rosmilah et al., 2008; Simúth et al., 2004).
1985, 1986; Simuth, 2001).
Other proteins, present in lower amount than MRJPs, are Royal- 2.3. Lipids
isin, Jelleines, Aspimin and Royalactina.
Several researches showed the antibacterial properties of Roy- The lipids are present from 3 to 19% of the RJ dry matter (Melliou
alisins and proposed their uses as potential antimicrobial natural and Chinou, 2005; Nabas et al., 2014; Ramadan and Al-Ghamdi,
peptides (Bilikova et al., 2001; Fujiwara et al., 1990). Royalisins are 2012).
amphipathic proteins (both hydrophobic and hydrophilic proper- Approximately 90% of lipids is constituted by fatty acids; the
ties) composed of 51 residues, with net charge +2; the origin is rest are neutral lipids, steroids, hydrocarbons and phenols (Nabas
unknown, but it is supposed that they could derive directly from et al., 2014; Ramadan and Al-Ghamdi, 2012).
honeybee. The peculiarity of its structure lies on its high con- The fatty acids of RJ have 8–10 carbon atoms, usually either
tent of cysteine (6 residues) and three intramolecular disulphide hydroxy fatty acids or dicarboxylic acids, unlike organic acids of
bridges which can give a compact structure exhibiting high sta- most animal and plant materials (Kodai et al., 2007; Noda et al.,
bility at low pH and high temperature. Royalisins has extensive 2005; Ramadan and Al-Ghamdi, 2012).
sequence homology with the sapecin (protein constituted from 40 The analysis of the lipid components can be a criterion of the
amino acids, taken from embryonic Sarcophaga peregrine cells and genuineness of the RJ, because an adulteration with honey or
phormicins from Phormia terra novae larvae (Fujiwara et al., 1990)). sugars, decrease the protein and lipid component, increase the
As Royalisins, Jelleines, showed their antimicrobial effects in in concentration of minor sugars and makes RJ insoluble in alkaline
vitro tests (Fontana et al., 2004; Romanelli et al., 2011). Jelleines medium (Boselli et al., 2003; Lercker et al., 1981; Li and Chen, 2003).
formation could be the result of tryptic digestion of MRJP 1 The main acid of fatty acid fraction is 10-hydroxy-2-decenoic
by specific proteases. The Jelleines, despite having the struc- (10-HDA) (Terada et al., 2011; Kitahara et al., 1995; Genc and Aslan,
tural base of the antimicrobial peptides and are characterized 1999), an unsaturated acid that seems to be involve in the antibac-
by hydrophobic residues, which influence the interactions with terial activity of RJ (Bloodworth et al., 1995; Nagai and Inoue, 2005).
bacterial membranes, do not show similarity with other known 10-HDA also showed to have an important biological role in the
antimicrobial peptides, including those produced by bees after a development of the colony strategies (Wu et al., 1991).Moreover,
possible infection (apidaecine, abaecine, hymenoptaecine).Jelleine the 10-HDA content has been adopted as a marker for quality and
I (PFKISIHL-NH2 ) differ to Jelleine II (TPFKISIHL-NH2 ) only for a freshness analysis of RJ (Antinelli et al., 2003; Ferioli et al., 2007).
Thr (T) residue from C-terminal portion. This modification seems Also the octanoic acid, present in lower amount than 10-HDA,
to vary the antibacterial activities of the two peptides, with a seems to cover more than only a nutritional function; recently
higher activity of Jelleine I than Jelleine II. Moreover, the removal research showed that the octanoic acid is involved in the repellence
of the residue Leu (L) at the N-terminus of Jelleine II with the action of queen cells again Varroa destructor (Nazzi et al., 2009).
formation of Jelleine IV (TPFKISIH-NH2 ) determines the complete
loss of antimicrobial activity. However, a significant decrease of
activity was showed also when a residue Thr (T) C-terminal was 2.4. Vitamins
replaced with Glu(E) as reported in the different sequence between
Jelleine II and Jelleine III (EPFKISIHL-NH2 ). Because of the pres- RJ is very abundant in B group vitamins, mainly vitamin B5
ence of an Arg (R) residue in position 373 and a Thr(T) residue followed by vitamins B1, B2, B6, B8, B9 and B12 (Li et al., 2012;
in position 374 of the primary sequence of the MRJP-1 it can be Viuda-Martos et al., 2008). Vitamin PP and vitamin C are present
supposed that the Jelleine II can be the product of digestion with only in small amounts (Liu et al., 2008; Melliou and Chinou, 2005;
trypsin of MRJP-1 (produced by the hypopharyngeal glands and Nagai et al., 2001). Liposoluble vitamins such as vitamins A, D, E
secreted in the RJ). An action of exo-proteinase both on C-terminal and K are absent (Li and Chen, 2003; Morita et al., 2012; Nagai and
to N-terminal tryptic fragment could lead, respectively, to the for- Inoue, 2005; Ramadan and Al-Ghamdi, 2012).
mation of Jelleines I and IV (Cabrera et al., 2014; Fontana et al., The vitamins content of RJ is subjected to seasonal changes as
2004). variation of the pollen of flowers collected by worker bees, as the
Apismin was also found, highly expressed, in the honey- mainly source of vitamins comes from the pollen (Biondi et al.,
bee head and it was demonstrated its capacity to strongly bind 2003; Chen and Chen, 1995; Sabatini et al., 2009).
MRJP 1 (Bilikova et al., 2002).Royalactina seems to induce the
differentiation of the queen bee as well MRJP 1 (Kamakura,
2011). 2.5. Minerals and other minor elements
Recently apolipophorin III-like protein was identified for the
first time in RJ(Han et al., 2011). ApolipophorinIII-like protein is Minerals and other elements are about 4% to 8% of RJ dry matter
a lipid binding protein that may form protein-lipid complexes in (Sabatini et al., 2009). The main elements are K, P, S, Na, Ca, Al, Mg,
order to carry lipids into aqueous environments (Fujita et al., 2012; Zn, Fe, Cu and Mn, but there are also traces of Ni, Cr, Sn, W, Sb, Bi
Kim and Jin, 2015). ApolipophorinIII-like protein may contribute and Ti (Benfenati et al., 1986; Li and Chen, 2003; Ramadan and Al-
additional to the antibacterial properties of RJ and could also play Ghamdi, 2012; Viuda-Martos et al., 2008). The presence of minerals
a significant role in the development of immune responses of hon- is related (and therefore variability) by the source of the feed, the
eybee larvae (Fujita et al., 2012; Han et al., 2011). production period, the environment and biological factors of bees
Glucose oxidase enzyme (GOx) was also detected in RJ (Li et al., (Benfenati et al., 1986; Garcia-Amoedo and de Almeida-Muradian,
2008; Sano et al., 2004). GOx that catalyses the oxidation of glucose 2007; Nation and Robinson, 1971; Sabatini et al., 2009).
to hydrogen peroxide was also detected in honey where showed a Moreover, RJ contains several minor components classified
high antibacterial activity (Sagona et al., 2015). under various chemical classes, such as heterocyclic substances,
Therefore, MRJPs, Royalisin, Jelleines, Apismin, Royalactina, biopterine and neopterine (Bogdanov, 2012). In RJ were also found
apolipophorinIII-like protein and glucose oxidase, present in RJ may low amounts of free nucleotides (adenosine, guanosine, cytidine,
contribute each other in virtue of their different chemical structure, and iridine), phosphates, ATP, ADP, AMP, acetylcholine and glu-
to the development of queen bee and to the efficient immune sys- conic, benzoic, malic, citric, and lactic acids (Bogdanov, 2012;
tems of honeybees, and provided as well an effective protective Matsuka,1993; Sabatini et al., 2009). The functions of these compo-
action of RJ both in vivo and in vitro uses. nents is still unclear, although their origin is assumed to arise from
the nurse bee.
3. Mechanisms of antimicrobial peptides action in the other models). Like the carpet model there is no formation
of pores in the cell membrane. After the formation of a binding
Antimicrobial peptides (AMPs) are fundamental defence between the peptide and the phospholipid head groups the peptide
biomolecules that could protect the host from bacteria, viruses or reaches the inner part of the cell without modified the membrane (a
fungi (Beutler, 2004; Gallo and Nizet, 2003; Zasloff, 2002).They mechanism of transport through the lipid bilayer without the for-
have been preserved evolutionarily in their innate immune mation of a stable channel). Once inside, the peptide can interact
response, which represents the first line of defence in most living with the targets (Pálffy et al., 2009; Xiao et al., 2015). Unfortunately,
organisms (Hancock and Lehrer, 1998; Brogden et al., 2003). the type of aggregates that provide the insertion of the peptide
AMPs are polypeptides of variable length containing from 10 to inside the membrane is not well defined, so it is more difficult
50 amino acids, so relatively short, and they have a positive charge to predict molecular properties who can favour this mechanism
that goes from 2 to 9 (most commonly 4 or 6), due to an excess of (Herbig et al., 2005; Li et al., 2012).
basic residues of lysine, arginine and histidine (Ebenhan et al., 2014; Besides the ability to interact with bacterial membranes, the
Splith and Neundorf, 2011).These properties allow the interaction AMPs could have other intracellular target (Ahn et al., 2006); in
between AMPs and microbial surfaces (negatively charged), and fact they can bind DNA, RNA and proteins and inhibit synthesis
to the cell membrane penetration by bilayer phospholipids head of different essential cell constituents as cell wall, DNA, RNA and
groups (Brogden et al., 2007; Pandey et al., 2011). proteins(Lan et al., 2010; Li et al., 2012). Moreover, AMPs can inter-
The sequence of amino acids of the peptide has a significant fere with bacterial cytokinesis by cell filamentation by an unique
role; in fact the presence of an amino acid or its substitution with mechanisms to translocation in side the cell in order to alter the
another one, even with similar chemical properties, may change the cytoplasmic membrane septum formation (Brown and Hancock,
effectiveness of peptide as its antimicrobial activity (Maróti et al., 2006; Lan et al., 2010; Li et al., 2012).
2011; Pandey et al., 2011). Many constituent of RJ are ascribable to the antimicrobial
The interaction between the AMPs and the surface of the peptides category such as MRJPs, Royalisin, Jelleines, Apismin,
bacterial cell membrane seems to be strictly correlated to the elec- Royalactna, and apolipophorinIII-like. The natural origin of these
trostatic interactions of the AMPs sequence and the structure of composts could be a potential added value to different products as
the bacterial membrane surface. Furthermore, even the secondary in vitro use. RJ, as well its by-products, could find a major role in the
structure of the peptide (␣-helix or ␤-sheet) plays an important control of microorganisms growth both for their proven activities
role primarily when the electrostatic interactions can not be estab- and for the low amounts needed.
lished due to the distance between the charged groups (Scott et al.,
1999; Yang et al., 2001; Zhao et al., 2001).
Several studies on the mechanism of action of AMPs have 4. Antibacterial activity
revealed different ways in which these substances exerted their
effect (Bulet et al., 1999). Although the antibacterial properties of The presence of antimicrobial properties of RJ against Gram pos-
many peptides present in RJ were demonstrated however their itive and Gram negative bacteria was scientifically showed for the
mode of actions were not been yet clarified in details. first time by McCleskey and Melampy (1939).
The classic way which AMPs exert their action is by the ability Subsequent studies of Hinglais et al. (1955), Butenandt and
to interact with cells membrane determining a permeabilization Rembold (1957), Blum et al. (1959), Iizuka and Koyama (1964) and
(Boman, 1994; Brogden, 2005; Huang et al., 2000). Muratova et al. (1967) reported the effects of RJ and 10-HDA against
There are three different models to describe possible AMPs many bacteria, including Escherichia coli and Micrococcus pyogenes.
mechanisms of action against bacteria: barrel-stave model, carpet- In 1990, Fujiwara et al. isolated and purified Royalisin from
like model, and toroidal pore model (Li et al., 2012). RJ.MIC (Minimum Inhibitory Concentration) evaluation of crude RJ
The barrel-stave model contemplates the formation of pores in showed that both Gram positive and Gram negative tested bacteria
the hydrophobic core of the membrane created by a circular assem- posses a low resistance of to this substance (Table 1).
bly of AMPs where their hydrophobic domains pointing toward the Royalisin MIC evaluation reported a strong antibacterial activ-
lipid chains of the membrane while the hydrophilics toward the ity against Gram positive bacteria, but not against Gram negative
interior of the pore (Li et al., 2012; Maróti et al., 2011; Shai, 2002). (Table 2). Bacterial strains belonging to Bifidobacterium, Clostrid-
In the carpet-like model the AMPs initially interact with the ium, Corynebacterium, Lactobacillus, Leuconostoc, Staphylococcus
external surface of the membrane, subsequently the charged region and Streptococcus genera showed an inhibitory concentration of
of the peptide interacts with the anionic phospholipids forming a Royalisin comparable with the effective concentrations of several
carpet, which extends on the surface of the target membrane. This antibiotic classes.
mode of action cause a reducing of the lipid layer surface and a The difference in antibacterial effectiveness between RJ and
consequent membrane disruption with collapse of the lipid struc- Royalisin could be explained by the presence of other compounds,
ture. The toroidal pore model also presents the formation of pores such as 10-HDA, which were completely lost during this peptide
in the membrane like barrel-stave model, but in this case the phos- splitting.
pholipids assumed a completely curvature as a double layer. In Further studies on antibacterial activity of Royalisin carried out
this process the lines of the double layer becomes a continuous by Bilikova et al. (2001). These Authors investigated the specific
structure, with the consequent formation on a pore. The toroidal action of Royalisin against Gram positive bacteria (Table 2).The aim
pore model is an intermediate case between the two previously of that study was to verify the action of the peptide against aetiolog-
described models and in some cases it is difficult to establish a ical agent of American foulbrood, Paenibacillus larvae subsp. larvae.
clear distinction. In the barrel-stave model and in the toroidal pore The results showed effective action of Royalisin against Bacillus sub-
model the peptide causes a rearrangement of the polar heads of tilis and Paenibacillus larvae subsp. larvae while no inhibition was
phospholipids by bundling the amphipathic helices and forming a determined for Micrococcus luteus (Sarcina lutea).
transmembrane pore which the hydrophilic part of the peptide fac- Shen et al. (2010, 2012) isolated and purified recombinant
ing the lumen of the pore (Li et al., 2012; Matsuzaki et al., 1996). Royalisins expressed by Escherichia coli after fusing in a vector
Currently a fourth model was described, the aggregate channel the Apis cerana cDNAs encoding for different Royalisin forms.
model. Several studies indicate that permeabilization of the cell These recombinant Royalisins showed higher antibacterial activity
membrane alone may not be enough to kill bacteria (as predict against Gram positive bacteria than Gram negative bacteria (MIC
Table 1
Antibacterial activities of Royal Jelly.
Bacterial strains MIC MBC Method Reference
Bacillus cereus 12.5 mg/ml AI Ratanavalachai and Wongchai (2002)

Bacillus subtilis RCMBA 6005 7.8–500.0 ␮g/ml AWD Moselhy et al. (2013)
Bacteroides fragilis nd BM Fujiwara et al. (1990)
Bacteroides vulgatus nd BM Fujiwara et al. (1990)
Bifidobacterium adolescentis ATCC 15703 10.0 ␮g/ml BM Fujiwara et al. (1990)
Bifidobacterium bifidum ATCC 15696 10.0 ␮g/ml BM Fujiwara et al. (1990)
Bifidobacterium breve ATCC 15700 10.0 ␮g/ml BM Fujiwara et al. (1990)
Bifidobacterium infantis ATCC 15697 10.0 ␮g/ml BM Fujiwara et al. (1990)
Bifidobacterium longum ATCC 15707 10.0 ␮g/ml BM Fujiwara et al. (1990)
Enteococcus faecium 50.0–70.0 w/wc AWD Garcia et al. (2013)
Enterococcus faecalis 40.0–100.0 w/wc AWD Garcia et al. (2013)
40.0–80.0 w/wc AWD Garcia et al. (2013)
ATCC 29212 60.0–80.0 w/wc AWD Garcia et al. (2013)
3.7–7.6 mg/ml 125.0- > 250.0 mg/ml BD Garcia et al. (2010)
ATCC 29212 5.0–13.7 mg/ml >250.0 mg/ml BD Garcia et al. (2010)
Escherichia coli 13.5 mg/ml AI Ratanavalachai and Wongchai (2002)
RCMBA 5003 500.0 ␮g/ml AWD Moselhy et al. (2013)
2.0 v/v1 BD Boukraa et al. (2009)
7.0–7.1 mg/ml > 250.0 mg/ml BD Garcia et al. (2010)
IID 861 10.0 ␮g/ml BM Fujiwara et al. (1990)
ATCC 29532 12.0 mmb DP Eshraghi (2005)
Klebsiella pneumoniae 80.0–100.0 w/wc AWD Garcia et al. (2013)
8.0–8.1 mg/ml 125.0–250.0 mg/ml BD Garcia et al. (2010)
IFO-3321 nd BM Fujiwara et al. (1990)
Lactobacillus acidophilus ATCC 314 10.0 ␮g/ml BM Fujiwara et al. (1990)
ATCC 4356 10.0 ␮g/ml BM Fujiwara et al. (1990)
Lactobacillus helveticus subsp. Jugurti 10.0 ␮g/ml BM Fujiwara et al. (1990)
Micrococcus luteus ATCC 9341 40.0–60.0 w/wc AWD Garcia et al. (2013)
ATCC 9341 7.5–11.8 mg/ml 125.0 mg/ml BD Garcia et al. (2010)
Micrococcus luteus (Sarcina lutea) 0.3 mg/ml AI Ratanavalachai and Wongchai (2002)
Proteus vulgaris 15.5 mg/ml AI Ratanavalachai and Wongchai (2002)
Pseudomonas aeruginosa 15.5 mg/ml AI Ratanavalachai and Wongchai (2002)
ATCC 27853 4.0 v/va AI Boukraa (2008)
RCMBA 1002 nd AWD Moselhy et al. (2013)
Salmonella infantis 10.0 ␮g/ml BM Fujiwara et al. (1990)
Salmonella typhi 14.5 mg/ml AI Ratanavalachai and Wongchai (2002)
Salmonella typhimurium 10.0 ␮g/ml BM Fujiwara et al. (1990)
Shigella flexneri 14.5 mg/ml AI Ratanavalachai and Wongchai (2002)
Staphylococcus aureus 12.5 mg/ml AI Ratanavalachai and Wongchai (2002)
RCMBA 2004 15.6–500.0 ␮g/ml AWD Moselhy et al. (2013)
1.7 v/va BD Boukraa et al. (2009)
ATCC 14776 15.0 mmb DP Eshraghi (2005)
Staphylococcus aureus MR 1 40.0–70.0 w/wc AWD Garcia et al. (2013)
8.0–14.5 mg/ml 125.0 mg/ml BD Garcia et al. (2010)
Staphylococcus aureus MR 2 30.0–70.0 w/wc AWD Garcia et al. (2013)
Staphylococcus aureus MS 1 ATCC 25923 20.0–80.0 w/wc AWD Garcia et al. (2013)
ATCC 25923 7.8–9.0 mg/ml 125.0 -> 250.0 mg/ml BD Garcia et al. (2010)
Staphylococcus aureus MS 2 40.0–80.0 w/wc AWD Garcia et al. (2013)
3.4–8.8 mg/ml 125.0 -> 250.0 mg/ml BD Garcia et al. (2010)
Staphylococcus epidermidis 40.0–80.0 w/wc AWD Garcia et al. (2013)
8.7–10.3 mg/ml 125.0 mg/ml BD Garcia et al. (2010)
Streptococcus agalactiae 50.0–100.0 w/wc AWD Garcia et al. (2013)
Streptococcus dysgalactiae 80.0–100.0 w/wc AWD Garcia et al. (2013)
Streptococcus uberis 60.0–70.0 w/wc AWD Garcia et al. (2013)
5.8–14.5 mg/ml 250.0 -> 250.0 mg/ml BD Garcia et al. (2010)
Streptomyces griseus ATCC 11746 14.0 mmb DP Eshraghi (2005)
nd: value not determined.

AI: Agar Infusion; AWD: Agar Well Diffusion; BM: Broth Medium; BD: Broth Dilution; DP: Drop Plate; DT.
a
Volume/volume of RJ on Muller Hinton agar medium.
b
RJ concentration 330 mg/ml.
c
Weight/weight of RJ on water.
Table 2
Antibacterial activities of Royalisin.
Bacillus subtilis 5.4–108.0 ␮g/ml DT Bilikova et al. (2001)

CMCC 63501 9.83 mma DT Shen et al. (2010)
CMCC 63501 10.53 mmb DT Shen et al. (2010)
CMCC 63501 62.5 ␮g/mlc MA Shen et al. (2012)
Bacteroides fragilis nd BM Fujiwara et al. (1990)
Bacteroides vulgatus nd BM Fujiwara et al. (1990)
Bifidobacterium adolescentis ATCC 15703 1.0 ␮M BM Fujiwara et al. (1990)
Bifidobacterium bifidum ATCC 15696 1.0 ␮M BM Fujiwara et al. (1990)
Bifidobacterium breve ATCC 15700 1.0 ␮M BM Fujiwara et al. (1990)
Bifidobacterium infantis ATCC 15697 1.0 ␮M BM Fujiwara et al. (1990)
Bifidobacterium longum ATCC 15707 1.0 ␮M BM Fujiwara et al. (1990)
Clostridium perfringens ATCC 13124 1.0 ␮M BM Fujiwara et al. (1990)
Clostridium tetani ATCC 19406 250.0 ␮g/mlc MA Shen et al. (2012)
Corynebacterium pyogenes 1.0 ␮M BM Fujiwara et al. (1990)
Escherichia coli nd DT Bilikova et al. (2001)
nd nd MA Bilikova et al. (2015)
ndd ndd MA Bilikova et al. (2015)
nde nde MA Bilikova et al. (2015)
ndf ndf MA Bilikova et al. (2015)
IID 861 nd BM Fujiwara et al. (1990)
CGMCC1.1139 >2000.0 ␮g/mlc MA Shen et al. (2012)
Klebsiella pneumoniae IFO-3321 nd BM Fujiwara et al. (1990)
Lactobacillus acidophilus ATCC 314 1.0 ␮M BM Fujiwara et al. (1990)
ATCC 4356 1.0 ␮M BM Fujiwara et al. (1990)
Lactobacillus bulgaricus ATCC 11841 1.0 ␮M BM Fujiwara et al. (1990)
Lactobacillus helveticus subsp. jugurti 1.0 ␮M BM Fujiwara et al. (1990)
Lactobacillus lactis ATCC 8000 1.0 ␮M BM Fujiwara et al. (1990)
Lactobacillus leichmannii ATCC 7830 1.0 ␮M BM Fujiwara et al. (1990)
Leuconostoc cremoris ATCC 19254 1.0 ␮M BM Fujiwara et al. (1990)
Micrococcus luteus (Sarcina lutea) nd DT Bilikova et al. (2001)
Paenibacillus larvae subsp. larvae 6.0 ␮g/ml 15.0 ␮g/ml MA Bilikova et al. (2015)
10.0 ␮g/mld ndd MA Bilikova et al. (2015)
10.0 ␮g/mle 20.0 ␮g/mle MA Bilikova et al. (2015)
50.0 ␮g/mlf ndf MA Bilikova et al. (2015)
ATCC 5084 5.4–108.0 ␮g/ml DT Bilikova et al. (2001)
Proteus vulgaris CGMCC1.1527 >2000.0 ␮g/mlc MA Shen et al. (2012)
Pseudomonas aeruginosa 10.0 ␮g/ml 15.0 ␮g/ml MA Bilikova et al. (2015)
Salmonella choleraesuis 9.0 ␮g/ml 11.0 ␮g/ml MA Bilikova et al. (2015)
Salmonella infantis nd BM Fujiwara et al. (1990)
Salmonella typhimurium nd BM Fujiwara et al. (1990)
CGMCC1.1190 >2000.0 ␮g/mlc MA Shen et al. (2012)
Serratia marcescens nd DT Bilikova et al. (2001)
Staphylococcus aureus 7.5 ␮g/ml 13.0 ␮g/ml MA Bilikova et al. (2015)
SC-D 1.0 ␮M BM Fujiwara et al. (1990)
Staphylococcus intermedius B 4.0 ␮g/ml 6.5 ␮g/ml MA Bilikova et al. (2015)
Staphylococcus xylosus 10.5 ␮g/ml 12.0 ␮g/ml MA Bilikova et al. (2015)
Table 2 (Continued)

f f
nd nd MA Bilikova et al. (2015)
Streptococcus alactolyticus 9.0 ␮g/ml 11.0 ␮g/ml MA Bilikova et al. (2015)
Streptococcus thermophilus ATCC 19258 1.0 ␮M BM Fujiwara et al. (1990)
Vibrio parahaemolyticus 4.0 ␮g/ml 6.5 ␮g/ml MA Bilikova et al. (2015)

BM: Broth Medium; DT: Diffusion Test; MA: Microplate Assay.
a
2 mg/ml of fusion protein from pre-pro-Acc-royalisin.
b
2 mg/ml of fusion protein from mature Acc-royalisin.
c
Recombinant Acc-royalisin.
d
Royalisin treated with DTT.
e
Royalisin-D.
f
Royalisin-D treated with DTT.
over 2000.0 ␮l/ml, Table 2); unfortunately no solid consideration ether-non-soluble fraction, that includes Royalisin (inhibition zone
could be formulated about the differences between the native and of 10-HDA are reported in Table 4) (Eshraghi, 2005).
recombinant Royalisins for the different method of determination Recently, Boukraa (2008) and Boukraa et al. (2009) evaluated
of the inhibitory activity. antibacterial effect of RJ against Pseudomonas aeruginosa, Staphy-
In 2002, in Thailand, Ratanavalachai and Wongchai tested the lococcus aureus and Escherichia coli. Results showed that all tested
antibacterial activities of crude RJ, and both the lipid and defat- bacterial strains were susceptible to RJ (Table 1).Antibacterial activ-
ted extracts. Authors also evaluated different storage combination ity against Pseudomonas aeruginosa is probably related to the action
time/temperature in order to enhance RJ conservation. RJ freshly of RJ demonstrated by Lerrer et al., 2007 that seems abrogate
picked was stored at room temperature (25–27 ◦ C), refrigerated lectin-dependent infection-preceding by Pseudomonas aeruginosa
temperature (2–4 ◦ C) and deep frozen (–18 ◦ C) for 12 h, 24 h and dhesion.
3 days and subsequently was tested against several bacteria. RJ is also effective against some bacteria implicated in infec-
Results showed that conservation of RJ at frozen temperature tion of skin wounds, as shown by the study conducted in Argentina
did not affect antibacterial and bacteriostatic activities (Table 1). on two different RJ samples in 2010 by Garcia et al. The MIC val-
Instead, Authors observed a decrease in RJ antibacterial activity ues of the two samples of RJ were reported in Table 1 as a range.
during storage time at all tested temperatures. Interesting results were reported as concern the inhibition and
Fontana et al. (2004) identified in RJ employing mass spectrom- the bactericidal effects: the MIC values were around twenty times
etry four antimicrobial peptides that were called Jelleines. lower than MBC values. The observed differences in the values of
The most relevant peptides with antimicrobial activity were MIC and MBC may be related to the RJ components associated to
Jelleines I and II, followed by Jelleine III, which has not demon- the geographical area or genetic variability between bee colonies.
strated activity against all the microorganisms, and finally the In 2013 Garcia et al. evaluated the antibacterial activity of four
Jelleine IV that has not given any evidence of activity (Table 3). Argentinean RJs, 10-HDA, ether-soluble fraction and fat-free RJ
Few researches were carried out on antibacterial activities of (Table 1 and Table 4).The results showed that the different samples
Jelleines and their modified forms. Romanelli et al. (2011) showed of RJ tested had a significant antibacterial activity on almost all bac-
that Jelleine I, II and III inhibit bacterial growth while the modifica- terial strains examined, with remarkable MIC values; both RJ and
tions of the structure in C and N terminals of these peptides caused 10-HDA showed lower activity against Gram negative bacteria, as
a decreasing of activity (Table 3). Results of Capparelli et al. (2012) Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa
showed a wide range of activity against Staphylococcus epidermidis than against Gram positive bacteria.
from 30 to 300 ␮g/ml for C-terminal modified peptides. In 2013, Moselhy et al. studied the antibacterial activity of Egyp-
Brudzynski and Sjaarda (2015), in a recent study about the eval- tian RJs (two samples collected in two different period, ie camphor
uation of honey glycoproteins against Escherichia coli and Bacillus and citrus seasons) and a Chinese RJ.
subtilis, attributed the antibacterial activities to the presence of Authors reported that Gram positive bacteria (Staphylococcus
MRJP-1 and likely the presence of Jelleines. aureus and Bacillus subtilis) were more sensitive to all three samples
In 2005Eshraghiinvestigated the different antibacterial prop- of RJ compared to Gram negative bacteria (Pseudomonas aeruginosa
erties of the crude RJ, the ether-non-soluble fraction and the and Escherichia coli) (Table 1).
ether-soluble fraction against different bacteria. The results In addition, the samples of Egyptian RJ were found to have a
showed a clear inhibitory effect of crude RJ; Staphylococcus aureus more effective antibacterial action than Chinese RJ. The bactericidal
strain was the most sensible followed by Streptomyces griseus and or bacteriostatic action of RJ is closely linked to the geographi-
Escherichia coli(Table 1). cal origin, the related botanical species and the genetic variability
The ether-soluble fraction of RJ showed a greater antibacterial between colonies (Boukraa and Sulaiman, 2009; Garcia et al., 2010;
action than the crude RJ, while the ether-non-soluble fraction, con- Garcia et al., 2013; Garcia-Amoedo and de Almeida-Muradian,
taining the Royalisin, was found to be less effective, even of crude 2007).
RJ. Another confirmation of the effectiveness of RJ against Staphy-
According to the experiment results, the antibacterial RJ action lococcus aureus strains was recently reported by an in vivo study
could be attributed to the ether-soluble fraction, i.e. the part con- carried out on rats by Gunaldi et al. (2014).
taining lipids and fatty acids including 10-HDA, and not to the
Table 3
The inhibitory activities of Jelleines against bacteria.
Bacterial strains MICa Reference
Jelleine I Jelleine II Jelleine III Jelleine IV
Bacillus cereus nd nd nd nd Fontana et al. (2004)

Bacillus pumilis nd nd nd nd Fontana et al. (2004)
Bacillus subtilis CCT 2471 10.0 30.0 nd nd Fontana et al. (2004)
Bacillus thuringiensis nd nd nd nd Fontana et al. (2004)
Enterobacter cloacae ATCC 23355 10.0 15.0 nd nd Fontana et al. (2004)
Escherichia coli CCT 1371 2.5 15.0 15.0 nd Fontana et al. (2004)
Klebsiella pneumoniae ATCC 13883 10.0 15.0 nd nd Fontana et al. (2004)
Listeria monocytogenes ≥200.0 200.0 Romanelli et al. (2011)
Proteus mirabilis nd nd nd nd Fontana et al. (2004)
Pseudomonas aeruginosa ATCC 27853 10.0 15.0 30.0 nd Fontana et al. (2004)
Salmonella entericaParatyphi ≥200.0 200.0 Romanelli et al. (2011)
Staphylococcus aureus ATCC 6535 10.0 15.0 30.0 nd Fontana et al. (2004)
≥200.0 200.0 Romanelli et al. (2011)
Staphylococcus saprophyticus 15.0 10.0 30.0 nd Fontana et al. (2004)

a
MIC values (␮g/ml) obtained with Microplate Assay method.
Table 4
The inhibitory activities of 10-hydroxy-2-decenoic acid against bacteria.
Bacterial strains MIC Method Reference
Streptomyces griseus ATCC 11746 29.0 mma DP Eshraghi (2005)

Staphylococcus aureus ATCC 14776 40.0 mma DP Eshraghi (2005)
Staphylococcus aureus MS 1 ATCC 25923 1.9 mg/ml AWD Garcia et al. (2013)
Staphylococcus aureus MS 2 1.9 mg/ml AWD Garcia et al. (2013)
Staphylococcus aureus MR 1 1.9 mg/ml AWD Garcia et al. (2013)
Staphylococcus aureus MR 2 2.3 mg/ml AWD Garcia et al. (2013)
Escherichia coli ATCC 29532 22.0 mma DP Eshraghi (2005)
nd AWD Garcia et al. (2013)
Enterococcus faecalis ATCC 29212 2.3 mg/ml AWD Garcia et al. (2013)
1.9 mg/ml AWD Garcia et al. (2013)
Enterococcus faecium 2.3 mg/ml AWD Garcia et al. (2013)
Streptococcus uberis 2.3 mg/ml AWD Garcia et al. (2013)
Streptococcus agalactiae ATCC 27956 nd AWD Garcia et al. (2013)
Klebsiella pneumoniae nd AWD Garcia et al. (2013)
Pseudomonas aeruginosa nd AWD Garcia et al. (2013)

DP: Drop Plate; AWD: Agar Well Diffusion.
a
Ether-soluble fraction of RJ concentration 30 mg/ml.
Rats with spinal implant inoculated with the bacteria and 5. Conclusions
treated with RJ showed a decrease in severity of the infection if
compared with the rats without RJ addition. From available literature Royal Jelly and its derivate compo-
Bilikova et al. (2015) analysed Royalisin and Royalisin-D, a nents, such as Royalisin, Jelleines and 10-hydroxy-2-decenoic acid
recombinant shortened form constructed in order to correlate the (10-HDA), showed a high activity against Gram positive bacteria
structure to the antimicrobial activity. Royalisin-D was structured while their effectiveness decrease against Gram negative. More-
as a reduced form of Royalisin that lacks of 11 amino acids at the over, several studies carried out on Royal Jelly showed that this
C-terminal (Tseng et al., 2011). product is also effective against many multidrug resistant bacteria,
In addition to investigate the importance of the disulfide bonds such as MRSA (methicillin-resistant Staphylococcus aureus). This is
in Royalisin, the two peptide were treated with dithiothreitol (DTT) particularly important since one of the major public health prob-
as a reducing agent of the disulfide bonds. The action of each pep- lems is currently represented right from the onset of an increasing
tide, crude and treated with DTT, against each microorganism was number of antibiotic resistant bacteria. The indiscriminate use of
evaluated with MIC and MBC (Table 2). antibiotics has led to the selection of resistant clones many for
All bacteria were susceptible to the peptides, with the excep- which it is often not provided an adequate therapy. Multidrug resis-
tion of Escherichia coli. Moreover, the activity of Royalisin and tant bacteria management request an increasing attention to the
Royalisin-D were very similar to each other, while a significant and antibacterial molecules/products used. For this reason researches
important difference was noted for the two peptides treated with in recent years has been directed toward the discovery of new
DTT. In fact Royalisin and Royalisin-D treated with DTT showed a antimicrobial substances, particularly natural substances such as
decreased inhibitory and bactericidal effects. These results high- plant extracts, essential oils and antimicrobial peptides isolated
light the importance of the disulfide bonds of Royalisin. from many different animals. The interest in beehive products is
also further enhanced by the fact that these products have always
represented an important resource such as functional foods, which Boselli, E., Caboni, M.F., Sabatini, A.G., Marcazzan, G.L., Lercker, G., 2003.
have not only the nutritional function, but also nutraceutical or Determination and changes of free amino acids in Royal Jelly during storage.
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Boukraa, L., 2008. Additive activity of Royal Jelly and honey against Pseudomonas
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International Centre of Traditional and Alternative Medicine "API" - the
leading apitherapeutical department.
International Centre of Traditional and Alternative Medicine "API" was founded in Chelyabinsk city in 1991.
Nowadays it is the largest apitherapeutical department in our country.
The centre's facilities are:
 hospital;
 clinical apitherapy department;
 narcological department;
 cosmetological department;
 psychoterapeutical department;
 multiple sclerosis treatment-and-rehabilitation centre;
 Parkinson's disease treatment-and-rehabilitation centre;
 manufactoring department.
All these years Igor Vladimirovich Krivopalov-Mosсvin, Doctor of Medicine and Professor, has been heading
the centre.
Igor Krivopalov-Mosсvin is the author of a new direction in apitherapy and medicine - clinical
apitherapy which let apitherapy be considered not as additional method but the basic one in the treatment of
many serious diseases.
For the first time the staff of the centre headed by Igor Krivopalov-Mosсvin carried out the research
concerning the bee venom influence on the blood system and immunity indices in the case of Multiple
sclerosis.
The original method of motion activity restoration combined with the kinesitherapy and the stress factor of
apitoxin is also of a great interest.
There were reached significant results in the treatment of narcological illnesses. In particular, we worked out
method of apinarcotherapy which allows the doctors not only to cure the alcohol addiction for total refuse but
also to normalize the process of alcohol usage.
The latest achievement is "APITOX" programme applying to the treatment of narcomania, it won EC golden
medal.
Every year Igor Vladimirovich represents Russia at the international congresses, conferences, symposiums.
Since 2000 was a chairman of the Apitherapeutical Union of 11 European countries "Apislavia". In 2001 he
became the first Russian representative at Apitherapy Committee of the World Organization "Apimondia".
Clinical apitherapy
 Health isn't provided for everyone, and that's why everybody should think about it himself;
 Apitherapy is nowadays a step towards obtaining the lost health, longer and better life;
 Bee is a single-use syringe, very effective and quite safe;
 Bee venom is a compound natural which cannot be created by a Man;
 Clinical apitherapy is a new medical direction and new possibilities of the medicine.
 Longevity should become one of the Man's conquests.
"New millennium is not only the bound of the new time calculation; it is a new
attitude towards many aspects of our life, new psychology, new people and,
of course, new medicine. A worthy place in it is taken by apitherapy, which
more and more claims the right to be called the medicine of the future".
I. V. Krivopalov-Moscvin
Clinical apitherapy
When we speak about wonder, we mean something, what we cannot explain.

Seven wonders of the world, created by a man, are widely known, but there
are almost inexplicable things, which appeared long ago before the mankind,
and which will still be unexplained for a long time. One of them is, of course,
a BEE.
It had gathered all the best features: perfect life and work organization, which
can be envied by any most highly developed country; power and courage,
which can be envied by any warrior; grace, wit and perfect bioenergetics.
And the things, produced by a bee, can always be admired. Just imagine: a spoon of pollen includes a day's
doze of vitamins and microelements; royal jelly, which is considered to be life elixir, ten times enlarges the
bee's life; propolis is the only inflammatory substance, which can resist any pathologic flora; and, of course,
honey.
Together with a bee, apitherapy (that is the bee venom and bee products treatment) came from ancient
times.
But until lately apitherapy was used mainly as an additional and secondary component of official medicine.
To make it the main method of treatment and to give it the scientific base - that is the aim of the international
medical center "API", which has worked out the new medical and apitherapeutical direction - clinical
apitherapy.
Clinical apitherapy is a kind of result. Nowadays it is not enough to speak about apitherapy only as of
something additional, as of separate cabinets, as of prophylaxis, and as of food supplement. None of the
existing alternative methods can be compared to bee venom, honey, propolis and royal jelly treatment. We
declare that, not only because we are apitherapeutists, but, because it is our reality. Reasonable attitude and
high professionalism, based on scientific research, allow us to get significant results in the treatment of many
diseases, which are slightly healed by common methods. There is a great amount of such diseases
nowadays:
 rheumatic diseases (rheumatic polyarthritis, rheumatic muscles diseases, rheumatic carditis);

 spondylartrosis deformans;
 polyarticular rheumatoid arthritis;
 peripheric nervous system diseases (lumbosacral radiculitis, sciatic nerve inflammation, and also
femoral, facial, and other nerves inflammation, intercostal neuralgia, poly neuritis, etc.);
 central nervous system diseases of organic origin;
 trophic ulcers and sluggish granulating wounds;
 vascular surgical diseases (thrombophlebitis without suppurative process, endarteritis, varicosity,
vascular atherosclerotic involvement of limbs);
 bronchial asthma and chronic bronchitis;
 migraine, neuroculatory dystonia;
 1 and 2 stage arterial hypertension;
 ischemic heart disease;
 post infarction and post stroke state;
 immunodeficiency;
 neurodermatitis, psoriasis, eczema, alopecia;
 1 and 2 stage thyrotoxicosis;
 iritis and iridocyclitis;
 radioactive influence;
 psychoactive substances addiction.
As a main medicament we use bee venom, which still cannot be created synthetically, and the effect of
which astonishes everybody.
In the treatment of psoriasis just after the first procedures psoriatic plaques disappear; in the treatment
of varicosity not only the cosmetic defects are removed, but the blood composition changes, which means
that the very predisposition to that disease is removed. Interesting is the fact of blood pressure influence.
The pressure can be either increased or lowered; i.e. it can be normalized. And the treatment of articular
pathology can be compared today to any other, even to very expensive one.
Unfortunately, because of the society's poor health, people often misuse medicine. It can give a certain
effect, but it won't be healthy, because any medicine suppresses the immune system. The man falls ill and
gets weak and has to cure all the time. But we've got only one liver and one pair of kidneys, and it is difficult
for them to stand such an enormous intoxication. Moreover, the medical addiction is formed, which is a
common thing today.
Bee venom fractions naturally fit in the cell structure, organ tissues, biochemical processes. Defensive and
inner powers are mobilized, and the organism obtains the natural activity.
Bee venom has a multicomponent structure. It includes amino acids, toxic peptides (melittine, secapine,
MSD-peptide, cardiopep, adolapin), esters, biogenic amines, proteins with enzymatic effects (phospholipase
A2, hyaluronidase), and mineral substances. The protein complex of bee venom is the main part of the
structure, and it determines the various pharmacological influences on the organism.
The venom activates a whole system of unspecific defence of the organism. Regulatory peptides' family
(apamin, MSD-peptide, melittine, etc.) influences the organism directly and indirectly, by modulating all
regulatory systems. Peptides have a number of amino acidic consequentialness coincidences with 3-10 link
peptides of the own organism, and that's why are not strange to it. It makes the various functions and
universal therapeutical activity of the bee venom more understandable. Bee venom is used in the treatment
of diseases, characterized by the presence of the focus and diffuse atrophic processes in the cerebral brain
cortex. Bee venom can influence directly its trophism, because apamin, due to the small size of its molecule,
is one of the few substances, which are able to cross the blood-brain barrier, increasing the biogenic amines
synthesis in the brain structures. Bee venom includes 18 irreplaceable amino acids, which allow it to
influence actively trophic processes. Melittine is able to activate together with the peptide (cardiopep)
biosynthesis of prostaglandins in the brain vessel walls. All that aids vasodilation and removes headache
spasms. It also influences vegetative and sympatic nervous systems, increasing the electrical activity of the
reticular and brain cells. It also has a great anticholesterol signification, anticoagulative and thrombolytic
peptide effect, which allow to restore actively the trophic processes, to increase physic and psychologic
activity. The venom improves impulse conduction in the nerve fiber; it aids the opioid peptides (endorphins,
enkephalins) production, has a great sedative effect, and causes no addiction and negative effects. The
venom activates the hypophysis-epinephros system, has an immunocorrective effect, improves the
adaptation possibility, and normalizes the sleep, appetite, mood, and the whole condition. Peptides have
prophylactic and medical anticonvulsive effect, which hinders the epileptoid syndrome development. All that
explains the high results in the bee venom treatment of such diseases as Parkinson's disease, Multiple
sclerosis, post strokes, post infarctions, Infantile Cerebral Paralysis, etc.
Bee is a single-use syringe (created by nature), which is rather effective and has, at the same time, no
danger of infection.
If there are no contraindications, bee stinging will be the most effective means of using bee venom,
because four main factors begin to work:
1) bee venom composition;,

2) reflectring;
3) stress syndrome;
4) bioenergetic factor.
All this, in connection with specially worked out programmes and complexes, reminds sometimes wonder.
And it isn't occasional that nowadays clinical apitherapy allows to widen largely the possibilities of the most
existing directions in treatment and rehabilitation.
For example, it is known that psychotherapy was weak in the treatment of heroin addiction, and bee
venom, influencing the brain cells' sensibility, allowed to increase the reception of the imposed information.
When there are motion activity disturbances, it is not enough to change and remove the physical factor;
but it is important to destroy the stereotype and to form the new reflex. It became easier to do that in
connection with kinesitherapy and the stress factor of the apitoxin influence.
Bee stinging is a reflex influence. It is well known, that the Chinese medicine and,
particularly, acupuncture, can do a lot. But acupuncture is a mechanical stimulation of the biologically active
points, and bee stinging is also the venom introduction. It contributes much to the effectiveness. However,
correct and professional attitude is important in the bee venom treatment. We shouldn't forget that it is
VENOM!
Important are a scheme and a doze, which are various for different diseases, age, sensibility and
allergization.
Venom must give a result and shouldn't be toxic.
We change the existing and outdated methods. The old principle - if there is diagnosis, there will be a
scheme of treatment, and nothing should be added - remains in the past.
Of course, our longevity and our health are important for us. But all these should become a certain conquest
of a Man. It is possible nowadays to influence the most factors, on which our longevity and health depend.
One of the main is a hormone balance.
Today a programme of the apitoxin usage for the support and necessary correction of the hormone balance
has been worked out. It opened up the new phase in the development of modern cosmetology and led to
the creation of the first professional apitherapeutical series with its quick and multiple effects (from the
youth restoration of the face to the body shape correction).
New points of view improved the quality of the dermatologic problems treatment (neurodermatitis, eczema,
psoriasis). To heal only the skin is useless. At the same time we can get a really good constant remission by
restoring the complex of disturbances, which cause the disease, by improving the connective tissue
microcirculation, by reflectring, and also giving neurotrophic and psychotrophic effects.
The apitoxins' ability to influence the opioid peptides production and disturbed catecholamine exchange
regulation allowed apitherapy to interfere into the narcologic processes (narcomania, toxicomania,
alcoholism, smoking).
A specialized in-patient department, which uses all the existing apitherapeutical methods of
treatment (from bee stinging to ultrasound influence) and a variety of apitherapeutical medicaments,
has been working for many years in Chelyabinsk. Everything here is different from the common
hospital, because of its comfort, service, method of specialists' selection, and, of course, it's
coordinated work.
А man's organism is considered to live for 150 years but we live only half that time. Let's think about
that and not miss the chance to live longer, look younger and be happier. And apitherapy, a wonder
given to us by Nature, the medicine of the Future, will help us!
The "APITOX" programme in the treatment of narcomania
 Squatted, in a hood, with a dropped head, with a cigarette, restless, often with sunglasses, with
a specific smell. If You see such a person, be sure - he is DRUG ADDICT! He is the one,
who "lives" in another world, for whom darkness is a common state, and light is left behind;
 He doesn't belong to himself; his subconciousness constantly suppresses him and makes him
provocate himself. That's why to give up using drugs independently is almost impossible. But if
it happens, he doesn't recover completely neither as a person, nor as a physically healthy man;
 " It is not enough to give up drugs. It is important to get rid of the predispositionary facts, one of
which is smoking. Almost everybody smokes. If this problem is solved, the effectiveness of
treatment will increase;
 The person, who treats, is sometimes more important for the result, than the way of treating.
Everything depends on how professionally and correctly the treatment is held;
 Sufficient physic and psychic compensation in addition to a proper mood are the guarantee of
success.
The "APITOX" programme in the treatment of narcomania
The offered treatment and rehabilitation complex is based on the influence of the apitoxin, active peptides of
animal origin (apamin, melittine, adolapin, secapine, cardiopep, MSD-peptide, etc.), and also enzymes
(phospholipase A, hyaluronidase)!
The variety of their effects is determined by their polyfunctionality and influence on different structural levels:
ultrastructural, cellular, tissue and organic.
The main apitoxine effects in the treatment of narcomania
Phase Achieved effects Assisting factors and mechanisms of the achieved effect
1 The main mechanism of the addiction  Activation of the hypophysis-epinephros
development. system;
Regulation of the disturbed catecholamine  Activation of the biogenic amines system
exchange. (dopamine, serotonin);
 Reflectring.
2 The treatment of the abstinent  Stimulation of the endogenous opioid

syndrome. production (endorphins, enkephalins);
Analgesic effect.  Ganglioblocking effect;
 Analgesic effect of adolapin, MSD-peptide.
Normalization of the physic condition  Normalization of the metabolic processes in

(somatovegetative normalization). the inner organs (including liver);
 Bloodreology improvement;
 The tissue microcirculation increase.
Normalization of the psychic condition (drug  Sedative effect of secapine;

attraction decrease, improvement of sleep,  Increase of the brain blood supply;
etc.)  Endogenous system regulation;
 Apipsychotherapy (stress and emotional
therapy).
3 Rehabilitation.  Hypophysis-epinephros system activation;

Psychological adaptation (mood, sleep,  Biogenic amines activation;
interests, etc.).  Melittine stimulating effect;
 Apipsychotherapy.
Mobilization of the defensive and  Stress-syndrome of the bee-venom

reserve powers of the organism. installation (regulative peptides activation).
Besides apitherapeutical impacts we use a great number of other procedures are used. They are aimed to
restore appearance, veins, to change the mood, to give the perspective of life, to turn to new dominants.
The treatment is carried stationary. The courses last for 10-15-20 days. A group of specialists works with
each patient; the living conditions are comfortable. It is rather important, because if there is chaos and
disorder, it will influence everything. There should be no trifles in treatment and rehabilitation of drug
addiction.
Dozed usage of alcohol
 More than one half of our countrymen have problems with alcohol, but most of them don't want
to give up drinking at all;
 The long alcohol abstinence leads to chronic stress;
 Those people who use alcohol at reasonable measures have higher intellectual capacities than
those, who don't use alcohol at all;
 Extremity is always bad. It is bad when a person has alcohol disturbances and it is bad when he
is afraid of drinkink;
 Predisposition to alcoholism doesn't mean alcoholism;
 Alcohol is the only psychoactive substance, which doesn't cause negative effects, if there is no
addiction.
A man, who can teach how to drink
Igor Vladimirovich Krivopalov-Moscvin, MD, professor, has been working with

different narcologic problems for many years. He has worked out the original
methodology of treating and rehabilitating narcomania, toxicomania,
alcoholism and smoking by using widely apitoxinotherapy in practice.
Igor Vladimirovich is the author of the programme on normalization of the

alcohol usage. He was the first to put this method into practice.
Dozed usage of alcohol

(apinarcotherapy method of Krivopalov-Moscvin)
Dosed usage is a relative term because there is no concrete doze for every day of
our life. It depends on many factors including mood, condition, wish to drink (it is a
normal thing, especially at the end of a working day) and necessity (working
situation, guests, etc.). That's why when people are speaking of treatment for a
concrete doze, for example, 200 ml, - it is nonsense and deceit.
Aims should be concrete and realistic:
 developing control during the usage;

 forming up the inhibitory effect;
 having quiet attitude towards the fact, that alcohol isn't drank up to the end;
 having quiet attitude towards the fact, that others drink;
 getting enough satisfaction with all necessary euphoria feelings, which do not lead to negative
consequences.
With the help of apitoxins (bee venom components) and apithenol, which give
certain imitation and compensation of alcohol, the addiction is lowered for the
whole period of treatment.
Apitoxins because of the activation of the hypophysis - epinephros system,

influence the main mechanism of addiction development - the catecholamine
exchange; also, influencing opioid peptides production, they eliminate
primary and secondary alcohol attraction; apitoxins also influence greatly
the alcodehydrogenase exchange. The ability of melittin to destroy the
existing old connections between neurofibers together with apamin effect, which leads to the formation of the
new control reflexes for alcohol usage, are important for new reflexes formation.
In the case of necessity outpatient and stationary treatment is carried, aimed for total refuse for a period of 6-
12 months.
Unfortunately, most treatments, which exist today, are aimed to influence only psychic or only physic
addiction; that's why the result is occasional and depends on the patient. But if to treat properly both
addictions, the result will be different.
If a man gives up drinking, the sufficient compensation is necessary, without which the organism won't feel
comfortable. In our case apitoxins give this compensation.
The treatment for total refuse is outpatient or stationary. The advantage of

stationary treatment is that apart from the inhibition of alcohol inclination, we
conduct rehabilitation after previous alcohol disturbances. Each disturbance
means earlier aging, mentality, behavior, efficiency, sleep and memory
problems. Each patient is treated by a group of specialists who let the patient
change internally and externally in a short period of time.
But this abstinence shouldn't last for a long time, considering the fact that it is
not the way to solve a problem.
Alcohol, being different from nicotine and especially drugs, is the only psychoactive substance, which doesn't
have negative effects and which has at the same time a number of positive effects, if there is no addiction.
There is also a psychological aspect. We live in such society, where everything is connected with alcohol.
We don't have any other society and we should be able to feel comfortable, to be able to control our activity
and to use alcohol if necessary, without ignoring meetings, banquets and holidays.
You decided not to drink or do you want to drink proper dozes?

We shall help you!
Information: The preparation of specialists by the method of apinarcotherapy of Krivopalov-Moscvin is

carried at the international medical center "API". The course lasts for 10 days.
The programme includes:
 clinical apitherapy;
 treatment of alcohol addiction for total refuse with the help of apitoxins;
 dozed usage of alcohol (normalization);
 treatment of tobacco smoking;
 the "APITOX" programme for the treatment of narcomania.
Address: 86 Svoboda st., Chelyabinsk, Russia, 454091.

Tel/Fax: +7(351) 2638323.
Tel:+7(351)2687252.
E-mail: api-center@chel.surnet.ru
Multiple sclerosis
 A lot of multiple sclerosis patients were left outside the medicine;

 Not the multiple sclerosis itself, but the attitude towards the patients leads them to the state of
constant chronic stress;
 Multiple sclerosis doesn't appear without the combination of three factors;
 Multiple sclerosis is the sportsmen's disease;
 Multiple sclerosis likes the weak and hates resistance;
 Today apitoxins join everything considered to be the best in the treatment of multiple sclerosis;
 We can't deny the fact that beekeepers almost do not suffer from multiple sclerosis;
 Apikinesitherapy gives new possibilities to restore motion activity;
 A proper treatment inhibits the processes and gives positive dynamics, contributes much to the
longevity.
Our point of view

Multiple sclerosis is considered to be an unusual illness, it demands unusual
approaches to its treatment. The reason is the chronic demyelinization process
of the myelin sheath with possible regenerative changes of the very nerve
tissue. Multiple sclerosis is characterized by the unpredictable clinical course.
A crisis can occur during a proper treatment and, on the contrary, the phase of
remission can develop without any healing.
There are dozens of direct and indirect causes which influence the rise and
development of multiple sclerosis, but all of them can be divided into three large groups:
1. Predisposition
а) inherited,
b) acquired;
2. Concomitant factor;
3. Provocative factors.
For multiple sclerosis to appear all three factors should be present; the absence of one factor (any)
inhibits the development of the disease (detailed classification is given in the book "Apitherapy in the
treatment of multiple sclerosis").
It is mainly the disease of young people who are in the prime of their working
life. It is an unexpected psycho-emotional stroke and after the first visits to a
doctor it seems to be a sentence. All these make the disease to develop
rapidly.
Unfortunately, not everything, what is done, is done for a patient. It also

concerns different activities (conferences, congresses, symposiums), which are held, although it is already
good, that this problem is spoken of. But everything what is going around is not interesting for the patient. He
is interested only in his concrete feelings.
Nowadays there are five main groups relating to the Multiple Sclerosis.
The 1st is the scientific research group, which possesses almost all the existing information, carries
out its own research, has a high intellectual potential and capacities. But they are far enough from a real
practical contact with a patient. That's why it doesn't always assess the recommended treatment
properly.
The 2nd group is the organizational structure, which has financial and coordination possibilities,
but doesn't have reliable information about what should be done with this category of ill people in
order to achieve results.
The 3rd group includes newly-established centers, which try "to do something", but which ignore the
fact, that multiple sclerosis is not the disease to be healed only will pills or injections. They don't
take into consideration the effect of the psycho-emotional factor, have no idea of the physical
rehabilitation of multiple sclerosis, and they don't want to use apitherapeutical methods because of their
being too conservative. For them the result is of secondary importance.
The 4th group consists of general practitioners, who work directly with such patients but have little
information and know only that multiple sclerosis is incurable, and it is no use treating it.
The 5th group includes the patients, for whom their health is still their own problem.
Of course, there should be a connection between these groups, which should aim everybody to help the
patient and not to "perform activity" and waste money. Nowadays there are more possibilities in the
treatment of multiple sclerosis than there were three-four years ago. The patients should know and feel it,
but it isn't that way.
The Multiple Sclerosis Society was organized in 2001. It seemed to be the main protector of the patients'
interests. But it didn't happen in reality. None of the patients understands, for what and for whom this
society was organized. Even the leaders of this organization don't know that.
Apitherapy in the treatment of multiple sclerosis
Multiple sclerosis grows and gets younger today. And this is because of the
confrontation, which is won not by the medicine.
Multiple sclerosis, as a disease, evidently differs from the others and that's why
it demands another approaches.
There should be a lot of specialists, who are engaged in the treatment of

multiple sclerosis: neurologist, psychologist, psychotherapeutist, urologist,
rehabilitologist and that's not all. To our point of view, the problem of multiple sclerosis will be solved quicker,
if a qualified apitherapeutist joins this list of specialists.
Since 1992 in Chelyabinsk the first Russian Treatment- and-Rehabilitation

Centre for patients with multiple sclerosis has been working. During all this
time different famous and infamous, medicaments have been used. But we
prefer apitoxins - bee venom components, which allow us a lot:
1. to inhibit the development of multiple sclerosis;

2. to decrease pathomorphologic changes in the myelin tunic;
3. to improve the remyelinization effect;
4. to influence positively the neurologic status and to improve nerve impulses conduction through
synaptic tracts;
5. to decrease the neutrophilic and monocytic leukocytosis and plasmatic reaction of the lymphoid
tissue;
6. to decrease the activity of the autoimmune inflammation;
7. to improve the metabolism and immune mechanisms;
8. to prevent infection complications;
9. to restore the lost functions;
10. to influence the coordination;
11. to influence actively the hypertonus;
12. to form the new reflex for motion activity restoration;
13. to carry on prenatal preparation and postnatal rehabilitation;
14. to change the patient's mood and his attitude towards the existing problem.
Such a variety of positive results is not occasional. Here are only some effects of the bee venom influence
on the functions of the man's body:
 stimulation of the epinephros cortex with the adequate corticosteroid

production;
 immunomodulation;
 decrease of the cholesterol level in the blood and dissolution of the
atherosclerotic plaques on the vascular walls;
 regulative effect on the blood pressure;
 anticoagulative and antiaggregant effect;
 remyelinization effect;
 regulation of the gastrointestinal tract function;
 radioprotective effect;
 reflectring.
In our clinic there were carried out the scientific research on the apitoxin
influence upon the neurological and pathomorphological disturbances of the
nerve tissue, the immune index and blood system in the case of multiple
sclerosis. We've came to a daring but a proved conclusion: "Bee venom join
everything what is considered to be the best for today in the treatment of
multiple sclerosis".
Main effects of the apitoxins in the treatment of Multiple

Sclerosis (Krivopalov-Moscvin I.V.)
Achieved effects Mechanisms and Assisting Factors of the Achieved Effect

1. Immunocorrection Specific immunity:
 phagocytosis stimulation;
 complementary activity stimulation;
 inhibition of the rosette formation;
 inhibition of the leukocyte migration speed.
Unspecific immunity:
 activation of the phagocytosis activity of leukocytes;

 increase of the bacterial activity of serum;
 increase of the properdin titer;
 increase of the lysocym and complement quantity.
2. Halt of the myelin damage  Anti-inflammatory effect of MCD-peptide, melittine,

phospholipase.
3. Halt of the nerve cells' processes  Protective effect of melittine.

degeneration  Antihypoxanth effect of bee venom combined with pollen and
royal jelly products.
4. Remyelinization  Synthesis of myelin, which is possible thanks to the

maintenance of 18 from 20 irreplaceable amino acids in bee
venom.
 "Factor of the nerves growth".
5. New physic abilities appear  Activation of the "hypothalamus - hypophysis - epinephros

cortex" system
 Influence of apamin, melittine.
 Reflectring.
 Improvement of the impulse conduction through the nerve
fiber.
6. Disseminative blood coagulation  Bee venom is a direct and indirect anticoagulant.

syndrome treatment  Fibrinolytic effect of the venom.
7. Improvement of coordination  Apitoxins penetrate through the brain-blood section and

improve functional links between the parts of spinal cord and
brain.
8. Improvement of the functions of  Restoration of the trophic processes.

the pelvic organs  Reflectring.
 Acceleration of the conduction of the nervous impulse in the
spinal cord.
 Applying of medicines on the base of propolis and royal jelly.
9. Rehabilitation of the sensibility  Improvement of the tissue microcirculation.

 Improvement of the impulse conduction through the nerve
fiber.
 Reflectring.
10. Mobilization of the protective and  Stress-syndrome on the background of the bee stinging.
reserve forces of the organism  Activation of the regulative peptides' system.
11. Improvement of the psychic  The stimulation of the production of the endogenous opioids.
status, spirit, remove of the anxiety  Sedative effect of secapine and tertiapine.
 Indirect analgesic effect of adolapin.
12. Chronic fatigue syndrome  Improvement of the production of the connections in the
treatment central nervous system.
 Stimulation of the opioid peptide production.
 Activation of the regulative peptides in the central nervous
system.
Traditionally the inflammation and myelin destruction processes' decrease, as well as the decrease of the
dropsy around the nerve fiber, are achieved by the usage of synthetic hormone medicaments -
corticosteroids. The usage of the bee venom gives a better effect because of MSD-peptide and peptide 401.
Moreover, it has a number of preeminences and the complete absence of the abstinent syndrome. The small
dozes of hormones do not give the desired result, but their regular and long usage is unfavourable, because
of a number of serious side effects. The results of the comparison are given in the scheme below:
Synthetic corticosteroids( hydrocortisone) Bee venom (MSD-peptide)

Steroid diabetes, obesity Normalization of carbohydrate, protein, fat
exchanges
Abstinent syndrome Absence of the abstinent syndrome
Increasing of arterial pressure, edemas, Itzenko- Stabilization of arterial pressure, vasodilatation,
Kushing syndrome diuretic with a potassium preserving effect
Immunodepressant Immunocorrector
Increasing of coagulability,thrombogenesis Decreasing of coagulability, thrombolysis effect
Low antiinfection resistance High antiinfection resistance
Exucleration of stomach and bowels, regeneration Antiinflammatory, regenerative effect
processes become slower
Psychic disturbances, excitement, epileptoid cramps, Antidepressant, sedative effect, anticonvulsive
insomnia, depressive state effect
The best argument for the apitherapy effectiveness in the treatment of multiple sclerosis is a fact that we
cannot deny: "Beekeepers almost never suffer multiple sclerosis".
Physical rehabilitation
Physical rehabilitation has some weak points today. Even if it is used, it usually consists of different
exercises, mainly they are difficult to perform.
Common physical exercises are almost ineffective, and, at the same time, difficult exercises are
contradicated, because they can cause hypertonus.
Much attention should be paid (especially beginning with the second disability group) to the restoration of
motion activity. To influence only the physical factor, including the demyelinization, is not enough, because
we change possibilities, but a person continues to move in a certain manner. He's got a reflex, which should
be destroyed, and a new one - formed.
The apikinesitherapy programme allows to teach how: to take care of oneself, to walk, to sit, to restore the
physiologically correct point of support.
Psychological rehabilitation
Today there is a widely spread mistake: no attention is paid to the psycho-emotional factor which is very
important for a certain category of patients.
Multiple sclerosis likes the weak and progresses more actively.
Our aim is to change a negative position of a man to an optimist one.
Information. You can find more information about the research and medical methods, which are used in our
clinics, in a book "Apitherapy in the treatment of multiple sclerosis".
One can reserve a place in our clinics by contacting the following address:
86, Svoboda st., Chelyabinsk, Russia 4540091.

Tel/Fax: +7(351)2638323.
Phone: +7(351)2687252.
Director
MD Professor
Krivolapov-Moscvin
Igor Vladimirovich
Apitoxinotherapy in the treatment of Parkinson's disease
Parkinson's disease belongs to a group of illnesses, which are hardly treated by the existing therapeutic methods.
 Tasks:
I. To do everything to stop disease development~
2. To get positive dynamics with the help of DOFA-excretion increase and catecholamine (dopamine,
adrenaline, noradrenaline );
3. Syndrome therapy;
4. Physical rehabilitation;
5. Psycho-emotional rehabilitation;
6. To change the quality of life.
No doubt, the treatment of Parkinson's disease needs strong and various influence. As a medicament,
which can influence just that way, we use apitoxins.
The aims of the bee venom treatment of Parkinson's disease are:

I. To provide necessary dopaminergic activity;
2.To inhibit the degenerative processes in the brain tissue;
3.To normalize motion activity;
4.To correct psycho-emotional state.
The table of main effects of apitoxinotherapy in the treatment of Parkinson's disease by I. V.
Krivopalov-Moscvin
Effects Assisting factors and mechanisms of the actived effect

I. Pathogenetic direction normalization -Bee venom includes necessary doses of dopamine.
of the dopamine concentration -Stimulation of the biogenic amines , system.
-Through the system of the regulative peptides.
2. Improvement of the central nervous -Direct and indirect anticoagulative action of the bee
system neurotrophy venom (particularly, of melittine ).
-Influence on the thrombocyte activity.
-Fibrinolytic action of the bee venom.
-Melittine and cardiopep widen the vascular lumen.
-Bee venom includes 18 amino acids, which take part in
the construction of the nerve fiber .
3. Improvement of the neurological -Apamin is able to penetrate through the brain-blood
status and motion activity: barrier.
a) Muscular rigidity lessens. -Improvement of the functional connections between
b) Tremor decreases. central nervous system and cervical parts of the spinal
c) Normalization of the gait. cord.
-Suppressive influence of sekapin, tertiapin.
-Inhibition effect of melittine on the subcortical structures
and brain cortex.
-Reflective stimulation of the biologically active points.
-Apikinesitherapy..
4. Normalization of the psychoemotional -Regulation of the mood through the system of
state endogenous opioids.
-Indirect effect of sekapin, tertiapin.
5. Nonspecific immunity increases -Stress-syndrome of the bee venom introduction.
-Activation of the regulatory peptides system.
6. Anticholesterol effect -The level of cholesterol in the blood decreases.
-Destruction of the atherosclerotic plaques.
On the whole positive dynamics is always gained.
The forms with low muscular tonus were more "sensible" to apitherapy.
Positive dynamics was gained not only in the clinical condition, but in the course of the disease, because
apitoxins are able to influence all the structural components of the Parkinson's disease.
Information: you can find us at the following address:
86 Svoboda st., Chelyabinsk, Russia, 454091.

Tel/Fax: +7(351) 2638323.
Tel:+7(351)2687252. E-mail: api-center@chel.surnet.ru
INFANTILE CEREBRAL PARALYSIS (ICP)
ICP (infantile cerebral paralysis) - is a disease which has characters of its own, such as diverse
abnormalities in locomotive organs' functioning, enunciation and psychic condition. ICP becomes apparent in
early childhood as a result of lesion of Central Nervous System, and does not grow worse from then on.
The reasons of contracting to ICP have not been investigated properly so far, but the risk factors that
predispose to ICP are known. They may be divided into three groups:
 Before the pregnancy (menstrual cycle disorder, short or long interval between the labour, genetic
syndromes, spontaneous abortions or mortinatuses in anamnesis, intake of thyroid hormones).
 During the pregnancy (fetal infections, small or big fetus mass, prematurity, postmaturity).
 During the labour and the infancy (lengthy oxygen starvation of the fetus during the labour, obstetrical
intervention).
There are four clinical versions of ICP based on abnormalities in locomotive organs' functioning: spastic,
dyskinetic, atactic and mixed forms.
APITHERAPY IN THE TREATMENT OF ICP
ICP might be characterized by absence of any treatment for it up till now, some methods for rehabilitation
being known just as a part of the general physical rehabilitation (physiotherapy, massage, exercise therapy)
which corrects locomotive abnormalities only.
Some years ago we developed a new approach for solving the problem within the framework of the
programme "APITOX" based on the application of bee venom fractions; it allows applying therapy and
rehabilitation simultaneously during the reasonable period of hospitalization.
We are glad to say that we can offer an absolutely new approach based on applying of apitoxinotherapy in
combination with apikinesitherapy, including medicines prepared on the basis of pollen, honey, herbs,
propolis, royal jelly along with a special rehabilitation complex.
Aims and tasks: physical and psychic medical rehabilitation of ICP patients.
Physical rehabilitation allows first of all, improving of locomotive abilities of the patient due to:
 Rehabilitation of normal muscle tone of limbs;

 Correction of contracture and deformity of limbs;
 Destruction of habitual motor skills;
 Formation of new joint range of motions;
 Fastening motor skills that have been acquired during the period of treatment.
Psychic rehabilitation improves mental power of the patient due to:
 Improvement of enunciation;
 Improvement of memory;
 Normalization of cerebral blood flow and metabolism.
Another aims of the treatment:
 Reduction of spasmodic attacks frequency;

 Removal of hyperexcitability;
 Increase of protective strength of organism;
 Normalization of metabolism.
BEE VENOME EFFECTS IN THE TREATMENT OF ICP
Effects achieved Assisting factors and mechanisms of the achieved effects

Physical rehabilitation
1. Reduction of hypertonus of limbs  Nerve impulse transmission blockade at the periphery (melittine);
(depending on ICP-forms)  Disorder of impulse conduction to central nervous system;
 Reflectionary through the biologically active points.
2. Increase in muscle tone  Stimulating action to Central Nervous System (apamin);
(depending on ICP-forms)  Improvement of functional bonds between Central Nervous
System and Peripheral Nervous System;
 Activation of biologically active points.
3. Formation of new locomotive  Improvement of nerve impulse transmission;
abilities  Activating action to Central Nervous System (apamin);
 Improvement of neural tissue trophism
4. Improvement of coordination  Improvement of functional bonds between parts of spinal marrow
and brain by permeation of apitoxins through the blood-brain barrier.
Psychic rehabilitation
5. Improvement of enunciation  Stimulation of locomotive center, situated near the speech center;
 Normalization of metabolism in the brain tissues;
 Simplification of internevronic bonds;
 Apipsychotherapy.
6. Improvement of memory  Increasing of synthesis of biogenic amines (apamin);
 Improvement of microcirculation of brain;
 Improvement of reological blood characteristics;
 Combination with api-preparations.
7. Reduce of spasmodic attacks of  Antispasmodic action of bee venom;
brain  Reflex action to biologically active points.
8. Removal of hyperexcitability  Sedative affect of secapine and tertiapine;
 Increasing of generation of endogenous peptides.
9. Normalization of metabolism  Influence through the system of regulatory peptides;
 Anticoagulant effect of bee venom;
 Vasodilating action of melittine;
 Increasing of oxygen-bonding blood functions;
 Combination with api-preparations.
10. Increase in protective strength  Stress-syndrome on the background of applying of bee venom;
of organism  Activation of regulatory peptides system.
An integral part of the treatment, which allows achieving significant results, is a new direction -
apikinesitherapy (combination of apitoxinotherapy and exercise therapy, mechanical and reflectory actions).
At the same time its opportunities extend due to:
 Muscle hypertonus reduction owing to melittine;

 Removal of contracture and increasing of joint range of motions owing to adolapine's analgetic effect;
 Fastening of new skills owing to central stimulating action of apamin;
 Improvement of metabolism in the muscular tissue due to the intensification of microcirculation and
changes of blood qualities.
This versatile and many-sided medical complex allows changing physical abilities, being used widely on the
background of therapy provided and improving significantly the quality of psychic health.

Tel/Fax: +7(351) 2638323.
Tel:+7(351)2687252.
Apitherapeutical cosmetology -
the new perspective of our life
When we have tested the API-cosmetics for the first time, everyone was
astonished, both clients and specialists. During all previous time we worked
with best foreign cosmetics (Spanish, Italian, French)... And thus appeared the
new apitherapeutical series with its quick and various effects: from the effect of
the youth restoration of the face to the body shape correction.
The attempts were made to create the cosmetics based on the royal jelly, honey, propolis, wax and pollen.
But that was only the cosmetics for home care.
The "API" cosmetological center managed to create new highly effective, professional
cosmetics.
The results were incredible: the restoration of the regeneration potential of the skin cells; skin
restructurization in the youth-like manner (the derma becomes thicker, and the corneal layer
becomes thinner); the activation of the synthetic cell activity due to the optimal balanced
composition of amino-acids, nucleotides, cofactor and ferments: production of the natural
moisturing components and hyaluronic acid, the restoration of the balance of the synthesis
processes and of the protein disintegration of the connective tissue (the strength and the
flexibility of the skin increase, the turgor is restored); the mobilization of the local defensive
factors and the delay of the skin aging. It is especially acute in our ecologically unfavourable
region, because the free-radical processes instantly run in the skin, leading to the destruction
of it in its deepest levels and simultaneous collagen destruction. As a result the skin becomes
faded and wrinkled.
Important is the fact of the MSD-peptide presence. It is included into the API-cosmetics and
is able to compensate the hormone decrease (the latter causes the loss of youth, beauty and
health).
Already after the first procedures the FACE changes quickly,

becoming younger, cleaner and more beautiful. The BODY obtains
flexibility, strength and shape and becomes light and supple.
In the case of necessity there is the combination with the traditional

professional cosmetics, computer stimulation, ultrasound influence and
a complex of various procedures.
Much attention is also paid to the psycho emotional factor. The modern rhythm of life leads to
the destruction of the natural biorhythms and the whole defensive system of the organism. All
that determines the premature aging.
Address:
Tel/Fax: +7(351) 2638323.
Tel:+7(351)2687252.
The Research Institute of Clinical Apitherapy
At the basis of the International Medical Center "API" we've opened the Research Institute of Clinical
Apitherapy.
-scientific;
-medicinal;
-educational activities gave us for an opportunity to put into practice the acquired research achievements
and gained practical experience. So we invite you to pass our training courses.
Now we offer several apitherapeutical programmes:
Programme 1
Advanced training courses on specialization -apitherapist.
1. General apitherapy.
2. Clinical apitherapy.
3. Organization of chargeable clinics including ambulatory and stationary care.
4. Narcological diseases treatment with the help of apitherapy ( alcoholism, smoking, narcomania).
The course lasts for 7-10 days, education is individual and costs 500 Euro. On course completion we give
the accepted standard certificate of the Research Institute of Clinical Apitherapy
Programme 2
Apitherapy and Treatment of Narcological Diseases

(broadened programme )
2. Clinical apitherapy.
3. Organization of chargeable clinics including ambulatory and stationary care.
4. Apinarcotherapy method:
-alcohol dependence treatment,
-nicotine dependence treatment.
5. The " APITOX" programme in the treatment of narcomania.
6. Dozed usage of alcohol (normalization of the alcohol usage).
7. Apitherapy in cosmetology.
The course lasts for 10-14 days, education is individual and costs 1500 Euro. The education is carried out in
Russian, English, French, German and Spanish. On education completion we give the accepted standard
certificate ot' the Research Institute of Clinical Apitherapy, video materials, manual for alcohol dozed usage.
Programme 3
Body Cosmetology
2. Skin and subcutaneous fat structure. Women skin and subcutaneous fat structure peculiarities.
3. Programmes devoted to body care in a beauty shop and at home. API-programmes
SPA- programmes
Wrappings, their kinds, methods, indications and contra-indications.
4. Instrumental cosmetology (myostimulation, ultrasound, thermoblanket). Principles of work, indications
and contra-indications, safety measures. Employment ofAPI-cosmetics together with instrumental
procedures.
5. Cellulitis: reasons, classification, preventive measures, treatment. Employment ofAPI-cosmetics in
cellulitis treatment.
6. Body overweight. Reasons, classification, preventive measures, methods of correction. Exclusive API-
programmes of weight correction.
7. Stretchings: reasons, preventive measures, methods of correction.
8. Body care in puerperal period.
9. Aromatherapy. Principles, indications and contra-indications, methods of oils mixing.
10.Epilation. Depilation.
11.Preparing the room for procedure.
12.Anticellulitis massage.
13.Lymphodrainage massage.
14.Honey massage.
15.Prophylactic-epidemiological regulations.
Programme 4
Face Cosmetology
1 .General apitherapy.
2. Skin structure and functions. Types of skin classification. Programmes devoted to different types of skin
care.
3. Skin appendages structure (hair, nails). Diseases of skin, hair, nails and their treatment. Exclusive
methods of treatment with API preparations.
4. Employment of API preparations in modern cosmetology. Their properties, mechanism of action and
effect.
5. Skin aging. Theories of aging. Employment of API cosmetics for aging signs prevention and correction.
API-programmes tor home care.
6. Acne. Classification. Aetiology. Pathogenesis. Clinic. Treatment. Exclusive methods of treatment with
APIcosmetics. Programmes of home care.
7. Types of skin cleaning. Mechanical cleaning.Cleaning with API cosmetics employment. APIAtraumatic
8. Instrumental cosmetology. Indications and contra-indications. Safety measures. Employment of API-
cosmetics together with instrumental methods.
9. Classical cosmetic massage.
10. Massage by Jacque methodic (medicinal).
11 .Plastic massage.
12.Desinfection. Sterilization.Norms and documents. The main prophylactic-epidemiological
The education is carried out in Russian, English, French, German and Spanish.
On education completion we give the accepted standard certificate of the Research Institute of Clinical
Apitherapy.

Tel/Fax:+7 (351)2638323.
Tel: +7(351)2687252.

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Developments in Bee Products Research & Apitherapy 2019

1. Rediscovery of Apitherapy Mechanisms: A Novel Challenge in Cardiovascular Autonomic

2. Apitherapy Have a Role in Treatment of Multiple Sclerosis

5. Comparison of Physicochemical, Microbiological Properties and Bioactive Compounds

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Keywords: Cardiovascular Autonomic Neuropharmacology; Antihypertensive Effect; Antiarrhythmic Effect;

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Miran KR. Rediscovery of Apitherapy Mechanisms: A Novel Copyright© Miran KR.

Miran KR. Rediscovery of Apitherapy Mechanisms: A Novel Copyright© Miran KR.

Miran KR. Rediscovery of Apitherapy Mechanisms: A Novel Copyright© Miran KR.

Miran KR. Rediscovery of Apitherapy Mechanisms: A Novel Copyright© Miran KR.

Miran KR. Rediscovery of Apitherapy Mechanisms: A Novel Copyright© Miran KR.

Miran KR. Rediscovery of Apitherapy Mechanisms: A Novel Copyright© Miran KR.

Miran KR. Rediscovery of Apitherapy Mechanisms: A Novel Copyright© Miran KR.

Miran KR. Rediscovery of Apitherapy Mechanisms: A Novel Copyright© Miran KR.

Miran KR. Rediscovery of Apitherapy Mechanisms: A Novel Copyright© Miran KR.

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Apitherapy Have a Role in Treatment of Multiple Sclerosis

Introduction 5]. The primary aims of therapy are returning function

OA Maced J Med Sci. 2014 Jun 15; 2(2):265-270. 265

OA Maced J Med Sci. 2014 Jun 15; 2(2):265-270. 267

Mean ± SE of signs at the end of the

Fig. 1 showed CT at the start and by the end of the study.

OA Maced J Med Sci. 2014 Jun 15; 2(2):265-270. 269

14. Ebers GC, Kukay K, Bulman DE, Sadovnick AD, Rice G,

Research Article Open Access

Novel Therapeutic Modality Employing Apitherapy for Controlling of Multiple

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J Clin Cell Immunol Volume 6 • Issue 1 • 1000299

2 Months 4 Months 6 Months 8 Months 10 Months 12 Months

J Clin Cell Immunol Volume 6 • Issue 1 • 1000299

IgE (IL) 1β (TNF) α, IL-6 IgE (IL) 1β (TNF) α, IL-6

J Clin Cell Immunol Volume 6 • Issue 1 • 1000299

J Clin Cell Immunol Volume 6 • Issue 1 • 1000299

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1. Generalities about Honeybees

Molecules 2019, 24, 2997; doi:10.3390/molecules24162997 www.mdpi.com/journal/molecules

In addition, MCD-peptide or peptide 401 is able to induce an anaphylactoid reaction by degranulating

2. Main Compounds of Bee Venom

2.3. Mast Cell Degranulating (MCD) Peptide

3.1. Anti-Inflammatory Potential

3.2. BV Application for the Treatment of Neurodegenerative Diseases

3.2.1. Parkinson’s Disease

3.2.2. Alzheimer’s Disease

3.2.3. Amyotrophic Lateral Sclerosis

3.3. BV and/or Melittin Applications in Cancer

Academic Editor: Juraj Majtan 

Keywords: grassland honey; bioactive compounds; sugar composition; phenolic compounds

Molecules 2019, 24, 2932; doi:10.3390/molecules24162932 www.mdpi.com/journal/molecules

Table 1. Physicochemical parameters of different types of honey from Romania.

Water Hue angle, Yellow Conductivity Viscosity

Table 2. Sugar content of different types of honey from North Romania.

Table 3. The results of the concentration of the twelve phenolic compounds.

SD: standard deviation; Min: minimum value; Max: maximum value.

2.1. Water Content Determination

2.2. Electrical Conductivity (EC)

2.6. Phenolic Compounds

4. Materials and Methods

4.2.1. Water Content

4.2.3. Phenolic Compounds

Academic Editor: Juraj Majtan