European Journal of Medicinal Chemistry 110 (2016) 267e279

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Review article

A pharmacological perspective on the use of Brazilian Red Propolis

and its isolated compounds against human diseases
Irlan Almeida Freires a, Severino Matias de Alencar b, Pedro Luiz Rosalen a, *
a
Department of Physiological Sciences, Piracicaba Dental School, University of Campinas, Piracicaba, SP, Brazil
b ~o Paulo, Piracicaba, SP, Brazil
Department of Agri-food Industry, Food and Nutrition, “Luiz de Queiroz” College of Agriculture, University of Sa

a r t i c l e i n f o a b s t r a c t

Article history: Propolis is a complex resinous mixture collected by bees, with high medicinal, historical and economic
Received 19 September 2015 value. The nutraceutical and pharmacological benefits of propolis have been extensively explored in
Received in revised form several fields of medicine as an important resource for prevention and treatment of oral and systemic
17 January 2016
diseases. A relatively new type of propolis, named red propolis (in Brazil, Brazilian Red Propolis - BRP),
Accepted 18 January 2016
Available online 20 January 2016
has been arousing attention for the promising pharmacological properties of some of its isolated com-
pounds (vestitol, neovestitol, quercetin, medicarpin, formononetin, etc). Due to a distinct chemical
composition, BRP and its isolated compounds (mainly isoflavones) affect a wide range of biological
Keywords:
Red propolis
targets and could have an impact against numerous diseases as an antimicrobial, anti-inflammatory and
Isoflavones immunomodulatory, antioxidant and antiproliferative agent. In this review, we comprehensively address
Antimicrobial the main aspects related to BRP bioprospection, chemistry and therapeutic potential. Further information
Anti-inflammatory is provided on mechanisms of action discovered thus far as well as clinical use in humans and regulatory
Antioxidant aspects. As of now, BRP and its isolated molecules remain a fascinating topic for further research and
Antiproliferative application in biomedical areas and dentistry.
© 2016 Elsevier Masson SAS. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
2. Botanical origin and georeferencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
3. Bioactive compounds present in Brazilian red propolis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
4. Bioactivity of Brazilian Red Propolis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
4.1. Antibacterial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
4.1.1. Anti-caries effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
4.2. Antifungal activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
4.3. Anti-inflammatory and immunomodulatory effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
4.4. Antioxidant activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
4.5. Antiproliferative activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
4.6. Other biological properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
5. Rational clinical use of BRP and regulatory aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

Abbreviations: BRP, Brazilian Red Propolis; EEP, ethanolic extract of propolis; MEP, methanolic extract of propolis; HEP, hexane extract of propolis; Chlor-fr, chloroform
fraction; Hex-fr, hexane fraction; Met-fr, methanolic fraction; GC-MS, Gas chromatography-mass spectrometry; RP-HPLC, Reversed-phase high performance liquid chro-
matography; NMR, Nuclear magnetic resonance; HPLC-PDA-ESI-MS, High-Performance Liquid Chromatography e Photodiode Array Detection/Electrospray Ionization
Tandem Mass Spectrometry; QTOF-MS/MS, Quadrupole-Time-of-Flight Mass Spectrometry; MIC/MBC/MFC, Minimum Inhibitory and Bactericidal/Fungicidal Concentration;
DPPH, 1,1-diphenyl-2-picrylhydrazyl; iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; OS, oxidative stress; ROS, reactive oxygen species;
ORAC, oxygen radical absorbance capacity; SOD, Superoxide dismutase; CAT, catalase.
* Corresponding author. Department of Physiological Sciences, Piracicaba Dental School, University of Campinas, 901 Limeira Ave., Piracicaba, SP, Brazil.
E-mail address: rosalen@fop.unicamp.br (P.L. Rosalen).

http://dx.doi.org/10.1016/j.ejmech.2016.01.033
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
268 I.A. Freires et al. / European Journal of Medicinal Chemistry 110 (2016) 267e279

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

1. Introduction low cost. The global market of functional foods, which is estimated to
over 30 billion US dollars, keeps growing every year. In this review,
Propolis is a complex resinous mixture collected by Apis melli- we discuss the most recent advances in the study of BRP as a
fera honeybees or stingless bee species from leaf buds, tree barks promising candidate for development of pharmaceutical formula-
and exudates from various plant sources [1,2]. In case of geo- tions, functional foods, and also with ethnopharmacological rele-
propolis, clay or soil is essentially the major component of this vance in folk medicine.
mixture [3]. Due to its resinous nature and mechanical properties,
bees use propolis in the assembly and repair of their hives or as a 2. Botanical origin and georeferencing
protective barrier against external invaders, thermal insulation,
humidity and wind [4]. Propolis has been used by mankind since The botanical origin of red propolis may vary in the different
the ancient times (~300 BCE) in mummification rituals, folk med- countries as a result of clime and flora diversity being specific for
icine and, more recently, in the industry of food and beverages, each region [12]. Two approaches have been commonly used to
cosmetics, mouthwashes, and toothpastes [4,5], which has sub- determine the botanical origin of propolis: the comparative
stantially increased its economic value. In this background, Brazil- chemical composition through chromatographic methods12 and
ian propolis is valued worldwide, given that from 2010 to 2012 the palynological analysis [13]. Recently, a new approach has been re-
price of a kilogram of raw Brazilian propolis increased more than ported using DNA barcoding analysis for the identification of the
50% in the international market [6] and the price of Brazilian Red botanical origin of BRP. Essentially, DNA is extracted from the
Propolis (BRP) stands currently five times more than that of other propolis resin sample, processed and then sequenced for the
types of propolis. presence of the plant source [14].
Based on physicochemical properties (color, texture, chemical Studies investigating the plant sources of BRP are relatively
composition) and geographic origin, Brazilian propolis used to be recent. The botanical origin of BRP was determined by observing
classified into 12 types [1]. In 2007, the 13th type of propolis was the collection behavior of the bees, and by comparison of the
reported in the literature as BRP, due to its intense red color [7]. Red phenolic compounds present in the plant exudate and propolis
propolis has also been found in other countries such as Cuba [8], with the use of reverse phase chromatography [2]. It was found that
Venezuela [9] and Mexico [10], with peculiarities in chemical and the botanical origin of BRP is Dalbergia ecastophyllum (L) Taud.
biological properties though. Special attention was drawn to BRP (Leguminosae), which is responsible for the red color of the prop-
due to the potential of discovering bioactive molecules hitherto olis [2,8]. Dalbergia species are known for their intensely colored
unknown in the chemical literature. heartwood pigments, due to the presence of cationic C30 isoflavans
In addition to pharmacological properties of clinical interest (retusapurpurins A and B) [8,15]. In brief, Apis mellifera bees collect
(antimicrobial, anti-inflammatory, antioxidant, antiproliferative, red exudate from the surface of the holes made by insects in the
among others), propolis in general is also considered a functional trunk of D. ecastophyllum to manufacture the propolis [2] (Fig. 1).
food, as the biologically active constituents in its extract have been BRP can be found in beehives located in the stem of mangrove
documented to provide health benefits [4,11]. The nutraceutical and bushes and sea and river coasts in the states of Alagoas, Paraíba,
biological benefits of propolis have been extensively explored in Pernambuco, Sergipe and Bahia, located in northeastern Brazil [8]
several fields of medicine as an important resource in the preven- (Fig. S1). Red propolis from Alagoas state recently obtained the
tion, management and treatment of oral and systemic diseases. In Geographical Indication (GI) by the Brazilian National Institute of
recent years, there has been an increasingly interest of the popula- Industrial Property (INPI). This state was internationally certified as
tion in holistic medicine and functional foods with easy access and the only producer of this type of propolis worldwide [16].

Fig. 1. (A) The botanical origin of Brazilian red propolis is Dalbergia ecastophyllum (L) Taud, which is a typical shrub plant belonging to the family of Leguminosae. (B) Dalbergia
species, in general, are known for their intensely colored heartwood pigments, due to the presence of isoflavans in their chemical composition. (C) Apis mellifera bees collect red
exudate from the surface of D. ecastophyllum to manufacture the propolis, which is a mixture of resin, pollen, and most importantly, plant fragments. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
 I.A. Freires et al. / European Journal of Medicinal Chemistry 110 (2016) 267e279 269

Table 1
Chemical compounds identified in Brazilian red propolis using different extraction methods.

Compound Sourcea (Extract/ Identification Geographical origin Biological properties Reference

Fraction) methodb

1,1,2-Trimethyl-3,5-bis(1-methylethenyl)- EEP GCeMS Mangrove area, Alencar et al. [7]

,(2.alpha.,3.alpha., 5.beta.)-cyclohexane Alagoas
1,2,3-Trimethoxy-5-(2-propenyl)-benzene EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
10-Octadecenoic acid, methyl ester EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
1-Methoxy-4-(1-propenyl)-benzene EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
2,2,6-Beta-trimethyl-bicyclo(4.3.0)non-9(1)- EEP GCeMS Mangrove area, Alencar et al. [7]
en-7.alpha.-ol Alagoas
2,4,6-Trimethylphenol EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
2H-1-Benzopyran-7-ol EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
40 ,7-Dimethoxy-2’-isoflavonol EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
7,40 -Dihydroxyisoflavone EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
Benzoic acid EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
Butanedioic acid, dimethyl ester EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
Hexadecanoic acid, methyl ester EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
Homopterocarpin EEP GCeMS, HPLC- Mangrove area, Piccinelli et al. [8]
PDA-ESI-MS Alagoas
Hydroxy-butanedioic acid, dimethyl ester EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
Medicarpin EEP GCeMS, NMR Mangrove area, Antibacterial, antifungal Alencar et al. [7],
n-hexane/acetone- GC-MS Alagoas Trusheva et al. [20]
fr EIS-MS Marechal Deodoro city, Silva et al. [2]
HPLC-PDA-ESI- Alagoas Frozza et al. [23]
MS Brejo Grande/Sergipe Piccinelli et al. [8]
Near Maceio city,
Alagoas

Methoxyeugenol EEP GCeMS Mangrove area, Alencar et al. [7]

Alagoas
Methyl abietate EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
Methyl o-orsellinate EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
Methyleugenol EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
m-Guaiacol EEP GCeMS Mangrove area, Alencar et al. [7]
Alagoas
Quercetin
EEP, Chlor-fr RP-HPLC Mangrove area, Anti-inflammatory Alencar et al. [7]
Alagoas

Ferulic acid
EEP, Chlor-fr RP-HPLC Mangrove area, Antioxidant Alencar et al. [7]
Alagoas

Daidzein
EEP, Chlor-fr RP-HPLC Mangrove area, Antioxidant Alencar et al. [7]
Alagoas Vasconcelos et al. [24]

(continued on next page)

270 I.A. Freires et al. / European Journal of Medicinal Chemistry 110 (2016) 267e279

Table 1 (continued )

Compound Sourcea (Extract/ Identification Geographical origin Biological properties Reference

Fraction) methodb

trans-anethol n-hexane/diethyl GCeMS Near Maceio city, Trusheva et al. [20]

ether-fr Alagoas
Methyl eugenol n-hexane/diethyl GCeMS Near Maceio city, Trusheva et al. [20]
ether-fr Alagoas
trans-methyl isoeugenol n-hexane/diethyl GCeMS Near Maceio city, Trusheva et al. [20]
ether-fr Alagoas
Elemicin n-hexane/diethyl GCeMS Near Maceio city, Trusheva et al. [20]
ether-fr Alagoas
trans-isoelemicin n-hexane/diethyl GCeMS Near Maceio city, Trusheva et al. [20]
ether-fr Alagoas
20(29)-lupen-3-one n-hexane/ethyl GCeMS, NMR Near Maceio city, Trusheva et al. [20]
methyl ketone-fr Alagoas
2,3-epoxy-2-(3-methyl-2-butenyl) n-hexane/ethyl GCeMS, NMR Near Maceio city, Trusheva et al. [20]
-1,4-naphthalenedione methyl ketone-fr Alagoas
b-amyrin n-hexane/diethyl GCeMS Near Maceio city, Trusheva et al. [20]
ether-fr Alagoas
Cycloartenol n-hexane/diethyl GCeMS Near Maceio city, Trusheva et al. [20]
ether-fr Alagoas
Lupeol n-hexane/diethyl GCeMS Near Maceio city, Trusheva et al. [20]
ether-fr Alagoas
Isosativan n-hexane/acetone- GCeMS, NMR Near Maceio city, Trusheva et al. [20]
fr Alagoas
Guttiferone E
EEP, n-hexane/ HPLC-PDA-ESI- Near Maceio city, Antibacterial, antifungal and Piccinelli et al. [8]
acetone-fr MS Alagoas antioxidant Trusheva et al. [20]
MEP GCeMS, NMR Alagoas Lotti et al. [25]
HPLC, NMR

Xanthochymol
EEP, n-hexane/ HPLC-PDA-ESI- Near Maceio city, Antibacterial, antioxidant and Piccinelli et al. [8]
acetone-fr MS Alagoas antiproliferative Trusheva et al. [20]
MEP GCeMS, NMR Alagoas Lotti et al. [25]
HPLC, NMR

Isoliquiritigenin
EEP HPLC-PDA-ESI- Alagoas Antibacterial and anti- Piccinelli et al. [8]
Chlor-fr MS Marechal Deodoro city, inflammatory Vasconcelos et al. [24]
GC-MS,NMR, Alagoas Oldoni et al. [26]
HPLC

Liquiritigenin EEP HPLC-PDA-ESI- Alagoas Piccinelli et al. [8]

MS Brejo Grande/Sergipe Vasconcelos et al. [24],
HPLC Frozza et al. [23]
ESI-MS
Formononetin
EEP HPLC-PDA-ESI- Alagoas Antiproliferative Piccinelli et al. [8]
MEP MS Brejo Grande/Sergipe Vasconcelos et al. [24]
ESI-MS Alagoas Frozza et al. [23]
HPLC, NMR Lotti et al. [25]

Biochanin A
EEP HPLC-PDA-ESI- Alagoas Antiproliferative Piccinelli et al. [8]
MEP MS Brejo Grande/Sergipe Vasconcelos et al. [24]
ESI-MS Frozza et al. [23]
HPLC, NMR Lotti et al. [25]

Vestitol
 I.A. Freires et al. / European Journal of Medicinal Chemistry 110 (2016) 267e279 271

Table 1 (continued )

Compound Sourcea (Extract/ Identification Geographical origin Biological properties Reference

Fraction) methodb

EEP HPLC-PDA-ESI- Alagoas Marechal Antibacterial, anticaries, ant- Piccinelli et al. [8]
Chlor-fr MS Deodoro city, Alagoas inflammatory and antioxidant Bueno-Silva et al. [27]
NMR, QTOF- Oldoni et al. [26]
MS/MS
GC-MS, NMR,
HPLC
Neovestitol
EEP HPLC-PDA-ESI- Alagoas Antibacterial, anticaries, ant- Piccinelli et al. [8]
Chlor-fr MS Marechal Deodoro city, inflammatory and antioxidant Bueno-Silva et al. [27]
MEP NMR, QTOF Alagoas Oldoni et al. [26]
MS/MS Alagoas Lotti et al. [25]
GC-MS,NMR,
HPLC
HPLC, NMR
7-O-methylvestitol EEP HPLC-PDA-ESI- Alagoas Piccinelli et al. [8]
MEP MS Lotti et al. [25]
HPLC, NMR
Vesticarpan EEP HPLC-PDA-ESI- Alagoas Piccinelli et al. [8]
MS
3,8-dihydroxy-9-methoxypterocarpan
EEP HPLC-PDA-ESI- Alagoas Antiproliferative Piccinelli et al. [8]
MS

3,4-dihydroxy-9-methoxypterocarpan EEP HPLC-PDA-ESI- Alagoas Piccinelli et al. [8]

MS
3-hydroxy-8,9-dimethoxypterocarpan EEP HPLC-PDA-ESI- Alagoas Piccinelli et al. [8]
MSGC-MS Marechal Deodoro city, Silva et al. [2]
Alagoas
Oblongifolin A EEP HPLC-PDA-ESI- Alagoas Piccinelli et al. [8]
MS
7,3’-dihydroxy-5’-methoxy-isoflavone EEP HPLC-PDA-ESI- Alagoas Piccinelli et al. [8]
MS
Naringenin EEP HPLC-PDA-ESI- Alagoas Piccinelli et al. [8]
MS
Mucronulatol
EEP HPLC-PDA-ESI- Alagoas Antiproliferative Piccinelli et al. [8]
MS

Retusapurpurin A EEP HPLC-PDA-ESI- Alagoas Piccinelli et al. [8]

MS
Retusapurpurin B EEP HPLC-PDA-ESI- Alagoas Piccinelli et al. [8]
MS Brejo Grande/Sergipe
EIS-MS
Frozza et al. [23]
Hesperetin 7-rhamnoglucoside EEP ESI-MS Brejo Grande/Sergipe Frozza et al. [23]
2-Hydroxy-4-methoxychalcon EEP ESI-MS Brejo Grande/Sergipe Frozza et al. [23]

Note: Compounds with any biological activity reported are in bold with their chemical structure.
a
EEP: ethanolic extract of propolis; MEP: methanolic extract of propolis; Chlor-fr: chloroform fraction of the EEP.
b
GC-MS: Gas chromatography-mass spectrometry; RP-HPLC: Reversed-phase high performance liquid chromatography; NMR: Nuclear magnetic resonance; HPLC-PDA-
ESI-MS: High-Performance Liquid ChromatographyePhotodiode Array Detection/Electrospray Ionization Tandem Mass Spectrometry; QTOF-MS/MS: Quadrupole-Time-of-
Flight Mass Spectrometry.

3. Bioactive compounds present in Brazilian red propolis
Overall, the chemical composition of propolis depends on the
biodiversity and the phytogeographic position of the beehives, with
several factors playing a role in propolis composition: seasonality,
light, altitude, flora, and type of collector bee [17e19].
A number of distinct compounds have been identified in BRP
(Table 1), including isoflavonoids, pterocarpans, chalcones, flavo-
noids, prenylated benzophenones, terpenes and tannins
[2,7,8,20,21]. The chemical markers of BRP are isoflavonoids,
including formononetin, biochanin A, pinocembrin [12] and
medicarpin [20]. The complex chemical composition of BRP ac-
counts for several biological activities (compounds with any bio-
logical activity reported in the literature are highlighted in bold in
Table 1). The structure of most of these compounds has never been
reported in other 12 types of Brazilian propolis, which makes the
BRP unique in chemical composition. However, red propolis from
different tropical zones (e.g. Cuba and Brazil) have similarities in
their chemical composition [8]. It is worth noting that BRP has a
composition similar to that of a specific type of Cuban red propolis,
produced in the province of Pinar Del Rio, which is rich in iso-
flavonoids and has several biological activities [22].
272 I.A. Freires et al. / European Journal of Medicinal Chemistry 110 (2016) 267e279

Table 2
Summary of recent studies concerning the pharmacological/functional potential of BRP and its isolated compounds in several areas that could apply to medicine and dentistry.

Pharmacological activity Tests performeda Sampleb and Reference

Geographical origin

Antibacterial
Actinomyces naeslundii ATCC 12104 MIC/MBC EEP, Chlor-fr, Hex-fr, vestitol, Oldoni et al. [26]
isoliquiritigenin/Alagoas
Actinomyces naeslundii ATCC12104 MIC/MBC EEP, vestitol, neovestitol/Alagoas Bueno-Silva et al. [27]
Aggregatibacter actinomycetemcomitans MIC/MBC, EEP/Alagoas Vasconcelos et al. [24]
ATCC 33384 Agar diffusion method,
Incorporation into bioglass
Enterococcus faecalis ATCC 12399 MIC/MBC, EEP/Alagoas Vasconcelos et al. [24]
Agar diffusion method,
Incorporation into bioglass
Enterococcus faecalis ATCC 29212 Agar diffusion method EEP/Sergipe Siqueira et al. [29]
Biofilm formation ex vivo
þ
Escherichia coli WF Agar cup method Medicarpin, guttiferone E and Trusheva et al. [20]
xanthochymol
Fusobacterium MIC/MBC, EEP/Alagoas Vasconcelos et al. [24]
nucleatum ATCC 23726 Agar diffusion method,
Incorporation into bioglass
Porphyromonas MIC/MBC, EEP/Alagoas Vasconcelos et al. [24]
gingivalis ATCC 33277 Agar diffusion method,
Incorporation into bioglass
Prevotella intermedia ATCC 25611 MIC/MBC, EEP/Alagoas Vasconcelos et al. [24]
Agar diffusion method,
Incorporation into bioglass
Staphylococcus aureus ATCC 25923 MIC/MBC EEP, Chlor-fr/Alagoas Alencar et al. [7]
Staphylococcus aureus ATCC 25923 MIC/MBC EEP, Chlor-fr, Hex-fr, vestitol, Oldoni et al. [26]
isoliquiritigenin/Alagoas
Staphylococcus aureus ATCC 25923 Propolis-containing nanosized hydroxyapatite EEP, Supplied by Pharma Ne ctar® Grenho et al. [30]
- CFU counts, Biofilm formation, (Belo Horizonte, Brazil)
confocal microscopy
Staphylococcus aureus ATCC 27664 MIC/MBC, EEP/Alagoas Vasconcelos et al. [24]
Agar diffusion method,
Incorporation into bioglass
Staphylococcus aureus ATCC25923 MIC/MBC EEP, vestitol, neovestitol/Alagoas Bueno-Silva et al. [27]
Staphylococcus aureus BTCC 209 Agar cup method Isosativan, medicarpin, guttiferone Trusheva et al. [20]
E and xanthochymol
Streptococcus mutans MIC/MBC, EEP/Alagoas Vasconcelos et al. [24]
ATCC 70069 Agar diffusion method,
Incorporation into bioglass
Streptococcus mutans Ingbritt 1600 MIC/MBC EEP, Chlor-fr, Hex-fr, vestitol, Oldini et al. [26]
isoliquiritigenin/Alagoas
Streptococcus mutans UA159 MIC/MBC EEP, vestitol, neovestitol/Alagoas Bueno-Silva et al. [27]
Streptococcus mutans UA159 MIC/MBC EEP, Chlor-fr/Alagoas Alencar et al. [7]
Streptococcus sobrinus 6715 MIC/MBC EEP, vestitol, neovestitol/Alagoas Bueno-Silva et al. [27]
Anti-caries
Reduced the incidence and severity of Topical application in a rodent model Neovestitol-vestitol (NV)/Alagoas Bueno-Silva et al. [31]
smooth and sulcal surface caries
Antifungal
Candida albicans Agar cup method Isosativan, medicarpin Trusheva et al. [20]
BTCC 562
Candida glabrata MIC/MFC, synergism with fluconazole HEP, Purchased from Natucentro® Pippi et al. [32]
(clinical isolates) (MG, Brazil)
Candida krusei MIC/MFC, synergism with fluconazole HEP, Purchased from Natucentro® Pippi et al. [32]
ATCC 6258 (MG, Brazil)
Candida parapsilosis (clinical isolates) MIC/MFC, synergism with fluconazole HEP, Purchased from Natucentro® Pippi et al. [32]
(MG, Brazil)
Candida tropicalis 72A MIC/MFC, synergism with fluconazole HEP, Purchased from Natucentro® Pippi et al. [32]
(clinical isolate) (MG, Brazil)
Saccharomyces cerevisiae 124567 CE ATPase activity assay Guttiferone E and xanthocymol Lotti et al. [25]
Rhodamine 6G transport
of Pdr5p
Trichophyton spp. MIC/MFC EEP/Paraíba Siqueira et al. [33]
Anti-inflammatory and immunomodulatory
Inhibition of neutrophil migration Experimental model in mice EEP, vestitol, neovestitol/Alagoas Bueno-Silva et al. [27]
into peritoneal cavity
Wound healing (inflammation, Topical application of 20% ointment on EEP/Alagoas Batista et al. [34]
granulation and epithelialization) induced-wounds in rats for 2, 6, 11 and 15 days
Dermal burn healing Incorporation of BRP into collagen-based dressing EEP/Sergipe Almeida et al. [35]
films used in a rodent model
Antioxidant
Activity on linoleic acid oxidation b-caroteneelinoleic acid model EEP, Chlor-fr, vestitol, Oldoni et al. [26]
neovestitol/Alagoas
Activity on linoleic acid oxidation b-caroteneelinoleic acid model MEP/Alagoas Righi et al. [36]
DPPH radical scavenging activity Guttiferone E and Xanthochymol Trusheva et al. [20]
 I.A. Freires et al. / European Journal of Medicinal Chemistry 110 (2016) 267e279 273

Table 2 (continued )

Pharmacological activity Tests performeda Sampleb and Reference

Geographical origin

DPPH radical scavenging activity
DPPH radical scavenging activity
DPPH radical scavenging activity
Oxygen radical absorbance capacity
(ORAC)
Superoxide dismutase-like (SOD) and
Catalase-like (CAT) assays
Antiproliferative
Effects on apoptosis and migration potential
in human bladder cancer cells
Effects on differential protein expression in
cancer cells
In vitro cytotoxic activity on tumor cells
In vitro cytotoxic activity on tumor cells
In vitro cytotoxic activity on tumor cells
In vitro cytotoxic activity on tumor cells
In vitro cytotoxic activity on tumor cells
In vitro cytotoxic activity on tumor cells
In vitro cytotoxic activity on tumor cells
In vitro cytotoxic activity on tumor cells
In vivo effects on the growth of melanoma
tumors in mice
In vivo modulatory activity on chemically
induced dermal carcinogenesis
Other pharmacological properties
In vivo modulatory effects on hypertension
and renal damage
Increase in ATP-binding cassette
transporter (ABCA1) expression and
cholesterol efflux from THP-1 macrophages
a
MIC/MBC/MFC: Minimum Inhibitory and Bactericidal/Fungicidal Concentration; DPPH: 1,1-diphenyl-2-picrylhydrazyl.
b
EEP: ethanolic extract of propolis; MEP: methanolic extract of propolis; HEP: hexane extract of propolis; Chlor-fr: chloroform fraction; Hex-fr: hexane fraction; Met-fr:
methanolic fraction.
Rapid DPPH
scavenging test
DPPH EEP, Chlor-fr, Hex-fr/Alagoas Alencar et al. [7]
scavenging test
DPPH EEP/Sergipe Frozza et al. [23]
scavenging test
DPPH MEP/Alagoas Righi et al. [36]
scavenging test
Lipid emulsion was prepared with the phenolic Sinapic acid and rutin from Espinosa et al. [37]
compounds. Hydroperoxide, thiobarbituric acid EEP/Alagoas
reactive substances, malondialdehyde and
phytosterol oxidation products were evaluated
as oxidative markers
SOD-like assay (inhibition of EEP/Sergipe Frozza et al. [23]
self-catalytic adrenochrome formation);
CAT-like assay (hydrogen peroxide
decomposition rate)
Inhibition of human bladder carcinoma cell line EEP/Sergipe Begnini et al. [38]
(5637) e MTT, LIVE/DEAD; Apoptosis e flow
cytometry and qRT-PCR
Proteomic analysis of Hep-2 cells treated EEP/Sergipe Frozza et al. [23]
with red propolis
Inhibition of human chronic myelogenous leukaemia EEP and Met-fr/Alagoas Novak et al. [39]
cells (K562), acute promyelocytic leukaemia (HL60),
human multiplemyeloma (RPMI 8226), and murine
melanoma (B16F10) cell lines e MTT and apoptosis assays
Inhibition of HeLa cell line (human cervical EEP/Alagoas Alencar et al. [7]
adenocarcinoma) e MTT assay
Inhibition of Hep-2 (human laryngeal epidermoid EEP/Sergipe Frozza et al. [23]
carcinoma cells) and HeLa (human cervical
adenocarcinoma) cancer cell lines e MTT assay
Inhibition of murine melanoma B16-BL6, murine Lewis 7-hydroxy-6-methoxyflavanone Li et al. [40]
lung carcinoma, human lung adenocarcinoma A549, and mucronulatol from MEP/BRP
human HT-1080 fibrosarcoma cell lines samples were provided by Nihon
Propolis Co., Ltd (Tokyo, Japan)
Inhibition of human pancreatic MEP and 3,8-dihydroxy-9- Awale et al. [41]
PANC-1 cancer cell line in nutrient-deprived conditions methoxypterocarpan/Paraíba
Inhibition of human chronic myelogenous leukaemia EEP/Northeast Franchi Jr. et al. [42]
cells (K562) and human B cell precursor leukaemia
(Nalm6) e MTT and apoptosis assays
Inhibition of human breast cancer MCF-7 EEP/BRP samples were provided Kamiya et al. [43]
cells e MTT assay, Caspase-3 activity, RT-PCR by Api Co., Ltd. (Gifu, Japan),
analysis and Western Blotting
Inhibition of human prostate cancer cells EEP/Northeast Moraes et al. [44]
(RC-58T/h/SA#4) e MTS assay
Melanoma cancer xenografts and EEP and Met-fr/Alagoas Novak et al. [39]
tumourigenesis assay in mice
Dermal carcinogenesis induced by the application EEP/Sergipe Pinheiro et al. [45]
of 9,10-dimetil-1,2-benzatraceno (DMBA) on the
backs of mice
5/6 renal ablation model in rats EEP/Alagoas Teles et al. [46]
Measurement of Liver X receptor (LXR) and EEP/BRP samples were provided Iio et al. [47]
Peroxisome proliferator-activated receptor (PPARg) by Api Co., Ltd. (Gifu, Japan)
transcriptional activity, ABCA1 promoter activity,
and cholesterol efflux

4. Bioactivity of Brazilian Red Propolis
A considerable progress has been made in the pharmacological/
functional study of BRP, which makes it a fascinating naturally-
occurring agent to be potentially used against a number of condi-
tions (Table 2). This is reflected in the increasing number of pub-
lications concerning BRP and its isolated bioactive compounds in
the last years.
Evolutionary forces usually shape the molecular complexity of
natural products in a way that chemical compounds are able to bind
to biological targets [28]. The biological properties of propolis are
attributed to a variety of major chemical constituents embedded in
the resinous mixture; in case of BRP, most of the bioactive com-
pounds have already been elucidated and are listed in Table 1. BRP
exhibits a wide range of biological properties, including
antibacterial/anti-caries, antifungal, anti-inflammatory and
immunomodulatory, antioxidant, antiproliferative, among others.
Following we discuss the most recent findings on each of these
274 I.A. Freires et al. / European Journal of Medicinal Chemistry 110 (2016) 267e279
areas in view of the prospective applicability of BRP in the man-
agement of systemic and oral diseases.
4.1. Antibacterial activity
BRP has a broad antimicrobial spectrum on gram-positive and
-negative bacteria. The ethanolic extract, chloroform fraction and/
or some isolated compounds of BRP have demonstrated a notable
antibacterial activity against oral microorganisms, such as early
colonizers of oral biofilm (Actinomyces naeslundii), cariogenic bac-
teria (Streptococcus mutans, Streptococcus sobrinus) and perio-
dontopathogens (Aggregatibacter actinomycetemcomitans,
Porphyromonas gingivalis, Prevotella intermedia), and also against
microorganisms potentially involved in systemic or focal in-
fections: Escherichia coli, Enterococcus faecalis and Staphylococcus
aureus. Overall, the EEP showed MIC values ranging between 25
and 250 mg/ml on A. naeslundii [26,27]; 50e200 mg/ml on S. mutans
[7,26,27]; <6.25 mg/ml on S. sobrinus [27]; 3.8e100 mg/ml on
S. aureus [7,24,26]; thus being considered a very strong antibacte-
rial agent based on a previous classification [48]. The effects on the
other microorganisms were observed through agar diffusion
method. The most bioactive fraction of the EEP is the chloroform
fraction (including low-polarity compounds), and isolated com-
pounds such as vestitol, neovestitol [27], isoliquiritigenin [26], are
even more active than the EEP and its subfractions.
The anti-S. mutans potential of BRP was also confirmed when
incorporated into bioactive glass particles as a drug delivery sys-
tem. Despite being a preliminary study with agar diffusion method,
the authors point out that BRP in this sustained release formulation
could be an alternative to mitigate the virulence of caries-related
oral pathogens by prolonging the substantivity of the active com-
pounds [24].
A recent study showed that BRP-containing nanosized hy-
droxyapatite (nanoHA) can prevent S. aureus growth and biofilm
formation, with little toxicity to host cells. NanoHA samples were
immersed overnight with EEP and put in contact with planktonic
and biofilm cultures. The findings indicate that it may be a prom-
ising biomaterial to be further studied with the aim of application
to orthopaedic or dental devices [49].
The mechanism of antimicrobial activity of propolis is complex
and could be attributed to the presence of several bioactive com-
pounds, particularly isoflavonoids. Cytoplasmic membrane damage
(caused by reduced membrane fluidity), inhibition of nucleic acid
synthesis (caused by topoisomerase inhibition), and inhibition of
energy metabolism (caused by NADH-cytochrome c reductase in-
hibition), and inhibition of attachment and biofilm formation have
been reported for flavonoids [50].
4.1.1. Anti-caries effects
Dental caries remains the most prevalent and costly oral infec-
tious disease worldwide, with considerable variations in preva-
lence and incidence between regions and countries [51]. The
clinical use of plant secondary metabolites has been considered as
an effective resource in the prevention of this biofilm-dependent
disease mainly caused by S. mutans [48,52].
A recent study of our research group evaluated the effects of
neovestitolevestitol (NV) containing BRP on accumulation of
S. mutans biofilm in vitro and development of dental caries in vivo.
Bueno-Silva et al. [31] showed that the topical application of
800 mg/ml of NV reduced the dry weight and biomass of soluble and
insoluble extracellular polysaccharides (EPS) in the biofilm matrix.
In addition, this same regimen was as effective as fluoride to pre-
vent the incidence and severity of smooth surface and sulcal caries
in a rodent model. Two anti-caries mechanisms were identified: (1)
inhibition of the enzymatic activity of glucosyltransferase D
(enzyme that contributes to the development of the rich biofilm
matrix of EPS), and (2) repression of specific genes associated with
stress survival/tolerance of S. mutans, such as sloA and copYAZ [31].
The in vivo anti-caries effects of other types of propolis are well
documented in the literature [53e55]. Furthermore, an experi-
mental mouthrinse containing propolis SNB-RS (from southern
Brazil) was efficient in reducing supragingival plaque formation
and insoluble polysaccharide formation under conditions of high
plaque accumulation in a double-blind, crossover, randomized
clinical trial [56]. Therefore, despite the differences in chemical
composition compared to other types of propolis, BRP has favorable
perspectives of anti-caries efficacy for clinical use. Aspects such as
non-toxicity to host cells and lasting residual activity (sub-
stantivity) should be considered though.
The study of BRP in caries research is still scarce, albeit prom-
ising. Future studies should focus on a more comprehensive
approach including other caries-related microorganisms, such as
Streptococcus sobrinus, S. salivarius, S. sanguinis (related to caries
onset) [57] and Lactobacilus casei and L. acidophilus (related to
caries progression) [58], as well as Actinomyces spp. and yeast (such
as C. albicans) in a multispecies biofilm model. The combination of
bioactive compounds of propolis (apigenin and tt-farnesol) with
fluoride has also been effective for protection from tooth decay by
combining the anti-bacterial effects of propolis constituents with
the remineralization ability of fluoride [59]. Therefore, this
combinatorial approach with fluoride could also be considered for
BRP isolated compounds (vestitol and neovestitol) in future work.
4.2. Antifungal activity
BRP and its derivatives are active against yeasts (Candida spp.
and S. cerevisiae) and dermatophytes (Trichophyton spp.). The
extract of BRP showed MIC values ranging between 3.9 and 15.6 mg/
ml on Candida spp. [32], and 64e128 mg/ml on Trichophyton spp
[33]. Isosativan and medicarpin prevented the growth of C. albicans
[20], and interestingly the isomers guttiferone E and xanthocymol
effectively inhibited multidrug efflux transporters in S. cerevisiae
such as Pdr5p and reversed multidrug resistance of cells over-
expressing these efflux pumps function [25].
A recent study evaluated the ability of C. parapsilosis and
C. glabrata to develop phenotypic resistance to a benzophenone-
enriched fraction of BRP as compared to fluconazole (FLC), and a
pharmacological synergism between them. The increase in MIC of
fluconazole for all isolates was evident and the majority developed
resistance, whereas no isolate became less susceptible to BRP. A
synergism was observed of BRP when in combination with FLC,
suggesting that BRP could be a possible therapeutic strategy for the
treatment of infections related to FLC-resistant Candida spp [32].
The antifungal mechanism of BRP on yeasts seems to be related
to action on the fungal cell wall, instead of affecting membrane
ergosterol and thus permeability [32]. Nevertheless, further
research is necessary to better elucidate the antifungal mode of
action of BRP, the effects on Candida biofilms growing on abiotic
(e.g. medical devices, dentures) or biotic surfaces (host cells), and
the effects on virulence factors (e.g. adherence, synthesis/activity of
proteases, phospholipases and hemolysins).
4.3. Anti-inflammatory and immunomodulatory effects
The anti-inflammatory properties of propolis and its derivatives
have been studied in different models of acute and chronic
inflammation [60,61], but little is known about BRP. Flavonoids, the
major compounds in BRP and other types of propolis, have recog-
nized anti-inflammatory activity [62,63]. However, the biological
properties of propolis should not be considered only as a synergic
 I.A. Freires et al. / European Journal of Medicinal Chemistry 110 (2016) 267e279
effect of the various compounds, suggesting the need for isolation
and identification of the bioactive constituents responsible for the
observed effects [60].
Red propolis from Alagoas was tested for its ability to inhibit
neutrophil migration into peritoneal cavity of mice upon induction
of inflammation with carrageenan in an experimental model. The
EEP, vestitol and neovestitol compounds administered subcutane-
ously (10 mg/kg dose) significantly inhibited neutrophil migration
at levels similar to those of dexamethasone, as seen by real-time
intra-vital microscopy [27]. Recently, it was also found that vesti-
tol and neovestitol can decrease migration and adhesion of leuko-
cytes in the inflammatory process [64]. Hence, the EEP and the
isoflavones neovestitol and vestitol may have potential therapeutic
application to modulate acute or chronic inflammatory processes in
the medical or dental settings.
The hydroalcoholic extract of BRP was also effective in
improving the biological events associated to dermal burn healing
in vivo (inflammatory infiltrate, collagen deposition, epithelializa-
tion rate) when incorporated into collagen-based dressing films,
with results better than those of green propolis [35]. In addition,
another study demonstrated the healing and anti-inflammatory
properties of the EEP of red propolis via topical application of
20% ointment on induced wounds in rats for 2, 6, 11 and 15 days. In
this study, however, the results were slightly better for green
propolis [34], likely because of the differences in the extraction
method compared to the previous study, which reflects the
chemical profile of the sample. Taken together, these findings
corroborate the potential use of BRP in wound occlusion and tissue
repair.
Compounds isolated from other types of propolis, but also
present in BRP, have shown significant anti-inflammatory activity.
Quercetin at 20 mg/kg was effective against acute inflammation
(carrageenan-induced paw edema) and chronic inflammation
(cotton pellet granuloma) in guinea pigs [63], and also inhibited
proinflammatory cytokines (TNF-a) in mice with damaged salivary
secretion [65]. Isoliquiritigenin inhibits the proinflammatory ac-
tivity of cyclooxygenase (COX) and inducible nitric oxide synthase
(iNOS), as well as modulates NF-kB and mitogen-activated protein
kinase (MAPK) signaling pathways in immune cells [66].
It is known that propolis in general acts through different
mechanisms (reviewed by Araújo et al. [60]): inhibition of cyclo-
oxygenase, inhibition of prostanoids (especially PGE2) and pro-
inflammatory cytokines (e.g. IL-6, IL-8) [67,68], effects on inflam-
matory cell activity (cell migration, macrophage activation) [27],
reduction in iNOS activity [61,66], reduced leukocyte adhesion [64],
reduced enzymatic activity during the healing process, and inhi-
bition of TNF-a [65]. Nevertheless, specific molecular mechanism(s)
underlying BRP anti-inflammatory activity have not been reported
thus far.
4.4. Antioxidant activity
Antioxidants are known to protect against oxidative stress (OS),
which is a topic of major interest in current days, because OS has
been associated with the aetiopathogenesis of several chronic
diseases, including arthritis, cancer, diabetes, atherosclerosis,
ischemia, failures in immunity and endocrine functions, among
others. Suitably, the potential beneficial effects of antioxidants in
protecting against disease have been well established [69]. An ideal
antioxidant agent should (a) prevent the formation of and scav-
enge, neutralize and remove reactive species (ROS), in addition to
(b) inhibiting oxidative chain reactions, (c) chelating reactive
metals, and (d) repairing damage to biological molecules [70]. The
studies on BRP and its isolated compounds published thus far have
focused mostly on the claims (a) and (b), showing promising results
275
with regard to scavenging properties of free radicals (e.g., DPPH)
and ability to act like antioxidant biomarkers, such as superoxide
dismutase and catalase.
The DPPH (2,20 -diphenylpicryl hydrazyl) assay is considered a
rapid, simple, inexpensive and widely used method to measure the
ability of compounds to act as free radical scavengers or hydrogen
donors [71], even though it also has some shortcomings, which are
reviewed elsewhere [72]. Flavonoids, in general, are known to have
free radical sequestering action [73]. Not surprisingly, different
extracts, fractions or isolated compounds from BRP have shown
varied antioxidant activity given as % inhibition of DPPH, as follows:
EEP (90 mg/ml: 57 ± 3.2%), Chlor-fr (90 mg/ml: 55 ± 1.3%), Hex-fr
(90 mg/ml: 78 ± 1.3%)7, MEP (25 mg/ml: 39.12%), and Guttiferone
E/Xanthochymol (3.6 mM: 49%) [20]. Furthermore, the MEP [36],
EEP, Chlor-fr, and the isoflavones vestitol and neovestitol [26] also
acted against linoleic acid oxidation, particularly Chlor-fr (57% in-
hibition at 90 mg/ml) and vestitol (39.5% inhibition at 90 mg/ml),
hence preventing the consumption of b-carotene (antioxidant) in
the system. b-carotene consumption is related to thermally induced
formation of linoleic acid hydroperoxides [26]. The b-car-
oteneelinoleic acid method has been considered an efficient
technique to detect and evaluate antioxidants from propolis sam-
ples [74,75].
The effects of eleven phenolic compounds extracted from BRP
on the oxidative stability of a functional emulsion were recently
evaluated. The emulsion consisted of Echium oil as omega 3 source
(u-3 FA); a-linolenic acid; stearidonic acid; and plant sterol esters
(PSE), without or with phenolic compounds. Hydroperoxide, thio-
barbituric acid reactive substances (TBARS), malondialdehyde and
phytosterol oxidation products (POPs) were evaluated as oxidative
markers, and the oxygen radical absorbance capacity (ORAC) was
also tested. The authors found that sinapic acid and rutin (200 ppm)
showed the same antioxidant activity as tert-butylhydroquinone
(standard antioxidant), and should be further investigated as a
potential natural antioxidant to be applied in a functional emulsion
containing u-3 FA and PSE [37]. A lipid emulsion could facilitate the
delivery of antioxidants to specific sites as well as result in pro-
phylactic or therapeutic outcomes.
Superoxide dismutase (SOD) is an important enzyme that cat-
alyzes the dismutation of reactive superoxide radical (O 2 ) into
molecular oxygen (O2) or hydrogen peroxide (H2O2) [76]. Catalase
(CAT), in turn, converts the end product of the dismutation reaction
(H2O2) into water and molecular oxygen, thus being a very
important enzyme in protecting the cell from oxidative damage by
ROS [77]. It is well known that both enzymes have an important
role in maintaining physiological redox equilibrium, decreasing
oxidative stress [23]. The hydroalcoholic extract prepared from BRP
showed important SOD-like and CAT-like activities at the concen-
tration of 100 mg/ml. SOD-like assay was done by measuring the
inhibition of self-catalytic adrenochrome formation in a medium
containing adrenaline, glycine and the BRP extract, while CAT-like
assay was performed by determining H2O2 decomposition rates
in a medium with phosphate buffer, H2O2 and the BRP extract. The
extract yielded 466.90 ± 12.40 SOD-like units, and CAT-like activity
of 13.13 ± 2.65 mmol of H2O2 decomposed/min, which are
considered promising results for antioxidant agents [23].
Altogether, the findings suggest that BRP may play a vital role in
metabolic pathways and protect cells against oxidative stress, but
further research is needed to confirm its potential, for example, as a
dietary antioxidant or ingredient of a pharmaceutical formulation.
Although there are several studies corroborating the potential
antioxidant activity of propolis, in general, there is no robust data
on the safe dose in humans [78].
Overall, several mechanisms could be involved in the protective
role of antioxidants against OS, including the catalytic systems to
276 I.A. Freires et al. / European Journal of Medicinal Chemistry 110 (2016) 267e279
neutralize or divert ROS, binding or inactivation of metal ions
(prevents generation of ROS by HabereWeiss reaction) and inhi-
bition of oxidative chain reactions [69].
4.5. Antiproliferative activity
Currently, 48.6% of novel anticancer drugs (n ¼ 85) are natural
products or compounds directly derived therefrom [79]. This
finding endorses the successful journey into natural sources for
drug discovery and fosters an active search for novel chemothera-
peutic candidates able to overcome the shortcomings (e.g. toxicity
to host cells) of synthetic monodrugs. The increasing number of
publications on the antiproliferative properties of BRP and its
constituents reveals their potential in the development of novel
anticancer agents.
Most of the studies in the literature with BRP are based on
in vitro cytotoxic assays against a wide range of tumor cells. How-
ever, effects on differential protein expression profile of cancer cell
lines and modulatory effects on carcinogenesis in vivo have also
been reported.
The EEP, MEP, Met-fr and at least three isolated compounds
showed significant antiproliferative activity (Table 2). The EEP is
cytotoxic for HeLa (human cervical adenocarcinoma) cells, with
IC50 values ranging between 7.45 mg/ml [48-h exposure]7 and
81.40 ± 6.40 mg/ml [24-h exposure] [23]. In addition, it inhibits
Hep-2 (human laryngeal epidermoid carcinoma) cells, with IC50 of
63.48 ± 3.30 mg/ml [24-h exposure] [23]; human chronic myelog-
enous leukaemia cells (K562) [IC50 42.1 ± 8.7 mg/ml], acute pro-
myelocytic leukaemia (HL60) [IC50 29.7 ± 1.5 mg/ml], human
multiplemyeloma (RPMI 8226) [IC50 36.5 ± 5.7 mg/ml], murine
melanoma (B16F10) [IC50 32.8 ± 3.8 mg/ml] cell lines [48-h expo-
sure] [39]; human chronic myelogenous leukaemia (K562) [IC50
~15 mg/ml], and human B cell precursor leukaemia (Nalm6) [IC50
~15 mg/ml] cell lines [48-h exposure] [42]; and human prostate
cancer cells (RC-58T/h/SA#4) [IC50 2.75 mg/ml] [48-h exposure]
[44]. Madjarof [80] also reported that the EEP (25 mg/ml) was
effective against a number of tumor cell lines.
Several classes of flavonoids (flavanoids, flavonol, isoflavones,
isoflavanones, isoflavans, chalcones, auronol, pterocarpans, 2-
arylbenzofuran, and neoflavonoid) and lignans isolated from the
MEP were investigated for their cytotoxic activity against murine
colon 26-L5 carcinoma, murine B16-BL6 melanoma, murine Lewis
lung carcinoma, human lung A549 adenocarcinoma, human cervix
HeLa adenocarcinoma, and human HT-1080 fibrosarcoma cell lines.
Among the tested compounds, 7-hydroxy-6-methoxyflavanone
exhibited the most potent activity against B16-BL6 (IC50,
6.66 mM), LLC (IC50, 9.29 mM), A549 (IC50, 8.63 mM), and HT-1080
(IC50, 7.94 mM), and mucronulatol against LLC (IC50, 8.38 mM) and
A549 (IC50, 9.9 mM) [40]. Other types of propolis bioprospected
worldwide have also shown cytotoxic activity against most of these
cell lines [81].
It is known that selectivity is an important parameter when
evaluating the anticancer efficacy of a given sample. In most cases,
BRP samples were selective for the tumor cells assayed. Further-
more, the antitumor efficacy of BRP was found to be comparable to
that of clinically used anticancer drugs, such as 5-fluorouracil and
doxorubicin, against the tested cell lines [40].
Two recent studies have demonstrated the modulatory effects of
EEP on experimental carcinogenesis in murine models. Novak et al.
[39] demonstrated that the administration of 10 mg/kg/day of an
active fraction of BRP containing xanthochymol and formononetin
significantly suppressed the growth (volume and weight) of mel-
anoma B16F0 tumor xenografts in C57BL/6 mice, with less general
toxicity than control group (10 mg/kg paclitaxel). In addition, the
active fraction delayed tumor growth, increasing the time
necessary for the tumor to reach a given volume. A second study by
Pinheiro et al. [45] reported the modulatory activity on chemically
induced dermal carcinogenesis in mice. The authors showed that
treatment with 100 mg/kg EEP led to a significant decrease in for-
mation, differentiation and progression of 9,10-dimetil-1,2-
benzatraceno-induced squamous cell carcinoma in the back of
mice. The average score for malignancy was significantly lower in
the EEP-treated group than the non-treated group. These findings
indicate that BRP has cytotoxic effects not only on single neoplastic
cells in vitro but also on organized and differentiated neoplastic
tissues in vivo.
The development of new blood vessels from the pre-existing
vasculature, named angiogenesis, is considered to be a critical
stage in cancer progression. A recent study investigated the anti-
angiogenic properties of quercetin and luteolin, as well as of their
derivatives. The authors observed that subtle structural modifica-
tions in these compounds led to increased antiangiogenic activity
and low cytotoxicity against host cells. These findings demonstrate
that compounds isolated from BRP, particularly quercetin, may be a
source of anticancer agents [82].
As shown in this review, the main antiproliferative mechanisms
of BRP (and propolis in general) and its isolated compounds include
cell-cycle arrest, induction of apoptosis via caspase-3, inhibition of
cell proliferation via metabolic pathways, and inhibition of tumor
development and angiogenesis. The findings reported so far sug-
gest that BRP and some of its constituents act through all of these
mechanisms and therefore could be good candidates for future
anticancer drug development.
4.6. Other biological properties
The effects of BRP on hypertension and renal damage in vivo
were recently reported. Treatment of rats with renal ablation using
EEP (150 mg/kg/day in drinking water) for 60 days significantly
reduced hypertension, proteinuria, serum creatinine retention,
glomerulosclerosis, renal macrophage infiltration and oxidative
stress, compared to age-matched untreated rats, which worsened
progressively over time. BRP renoprotective effects might be
related to the reduction of renal inflammation and oxidative stress.
However, additional studies are required to completely clarify the
mechanisms by which BRP exerts its hypotensive and renopro-
tective effects [46].
Another study demonstrated that the EEP of BRP significantly
enhanced apolipoprotein A-I-mediated cholesterol efflux in THP-1
macrophages, which was accompanied by a marked induction of
ABCA1 (ATP-binding cassette transporter) gene expression. ABCA1
is a membrane transporter that directly contributes to high-density
lipoprotein (HDL) biogenesis by regulating the cellular efflux of
cholesterol. The effect of EEP on ABCA1-dependent cholesterol
efflux was explained by the authors in part by its potency of in-
duction of PPARg (peroxisome proliferator-activated receptor) and
LXR (liver X receptor) transcriptional activity. Based on these
findings, BRP could have a potential as a diet supplement for pre-
vention and treatment of cardiovascular diseases such as athero-
sclerosis [47].
A recent study by Morsy et al. [83] demonstrated other possible
the uses of BRP in the environmental field, with sustainability
purposes. The authors compared the in vitro efficiency of EEP in
enhancing the ruminal degradability of organic matter and miti-
gating the formation of methane (CH4). However, they found that
EEP was less effective than the ionophore antibiotic monensin,
which was used as a positive control, in mitigating CH4 emissions.
An illustration of all mechanisms of action of BRP and some of its
isolated compounds in diverse metabolic pathways and patholog-
ical processes is presented in Fig. 2.
 I.A. Freires et al. / European Journal of Medicinal Chemistry 110 (2016) 267e279
Fig. 2. Mechanisms of action of Brazilian red propolis and its isolated constituents in diverse metabolic pathways and pathological processes. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
5. Rational clinical use of BRP and regulatory aspects
Currently, a number of daily products including propolis in their
composition with medicinal purposes are available in the market,
such as throat and wound sprays, toothpastes, non-alcoholic ex-
tracts, capsules or tablets as dietary supplement (antioxidant, im-
munity and energy enhancers), among others. In general, propolis
has been used by mankind for centuries and the substances iden-
tified therein are known food constituents, additives and/or sub-
stances generally regarded as safe (GRAS). However, as chemical
composition of propolis varies substantially based on geographical
location, the medicinal and nutraceutical use of propolis products
must be certified by an official regulatory agency.
Despite the advances in pharmacological research in vitro and
in vivo, no clinical trial has been found in the literature reporting
the efficacy and safety of BRP or derivatives in humans, which
suggests further avenues for the study and application of this
special type of propolis in several areas of medicine and dentistry.
More than 2880 patents have been filed worldwide related to
applications, extraction methods, therapeutic and nutritional uses
of propolis. Up to date only one patent was deposited for phar-
maceutical compositions including BRP extract (Nascimento, T.G. e
no. WO2013BR00201 20130524) [84], and another one for the
application of the isoflavones vestitol and neovestitol (Rosalen, P.L.
- BR1020120326485, INPI). However, other patents may also be
found concerning BRP isolated compounds that coexist in other
types of propolis. According to the Brazilian legislation, BRP is not
considered a native product as the collector bees (A. millifera) are
not native. However, it is worth noting that the bees are just vectors
that collect substances from the surrounding plants (in this case, D.
ecastophyllum). Therefore, whenever legislation lies upon more
precise terms, BRP should be considered part of the national genetic
resources. In Brazil, access to and study of genetic resources re-
quires authorization of the Brazilian Ministry of Environment
(Council for the Administration and Management of Genetic
Patrimony - CGEN) via the National Council for Scientific and
277
Technological Development (CNPq), so that to guarantee and
monitor the sustainable use and proper applications either for ac-
ademic or industrial purposes.
6. Concluding remarks
This review attempted to shed light on the pharmacological and
nutraceutical potential of Brazilian red propolis and its bioactive
constituents. Due to a very distinct chemical composition, BRP
exhibits a wide range of biological properties and could potentially
have an impact against a number of human diseases with antimi-
crobial, anti-inflammatory, anti-oxidant and anticancer activity.
On one hand, chemical and biological research has shown that
some of BRP compounds have high potential to become allopathic
drugs or even phytotherapics. On the other hand, the studies using
BRP extract suggest that it could also be used as a powerful and
effective functional food. Propolis is already known as such in many
countries with advanced legislation, such as Japan. In both sce-
narios (pharmaceutical and functional), BRP seems to aggregate not
only health benefits to the community, but also international eco-
nomic value.
Thus, BRP remains a fascinating topic for further research and
application in biomedical areas and dentistry. Further studies
should focus on its toxicological aspects, as well as clinical efficacy
and safety in humans.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors thank the Sa ~o Paulo Research Foundation (FAPESP,
Brazil, grants no. 2011/15984-0, no. 2013/25080-7, no. 2008/58492-
8); and the National Council for Scientific and Technological
Development (CNPq, Brazil, grant no. 308644/2011-5).
278 I.A. Freires et al. / European Journal of Medicinal Chemistry 110 (2016) 267e279
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.01.033.
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