Food Chemistry 124 (2011) 387–391

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Determination of royal jelly acids in honey

V.A. Isidorov a,*, U. Czyzewska
_ a
, E. Jankowska a, S. Bakier b
a
Institute of Chemistry, Białystok University, 15-399 Białystok, Poland
b
Białystok Technical University, Department of Food Technology, 15-351 Białystok, Poland

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

Article history: In the present work we report on the compounds characteristic of larval food (royal jelly, RJ) of the hon-
Received 3 November 2009 eybee (Apis mellifera L.) that were identified in 34 different samples of genuine honey and in 3 sugar-adul-
Received in revised form 21 February 2010 terated ‘‘herbal honeys” by using solid-phase extraction and gas chromatography–mass spectrometry
Accepted 12 June 2010
(SPE/GC–MS). The unique feature of RJ is a set of C8, C10 and C12 hydroxy fatty acids. In all, ten acids char-
acteristic of this bee product were identified in different combinations in the analysed honey samples,
namely: 7- and 8-hydroxyoctanoic, 3-hydroxydecanoic, 9-hydroxydecanoic, 9-hydroxy-2-decenoic, 10-
Keywords:
hydroxydecanoic, 10-hydroxy-2-decenoic (10-HDA), 3,10-dihydroxydecanoic, 2-octene-1,8-dioic and 2-
Honey
Royal jelly
decene-1,10-dioic acids. The higher relative abundance of these compounds was determined in genuine
Hydroxy fatty acids honeydew and heather honeys, and in ‘‘herbal honeys” (23.8–40.8, 18.2–48.5, and 27.0–48.4 lg/g,
Solid-phase extraction respectively). Since RJ is known to have strong antibiotic efficacy, our results suggest that a part of the
GC–MS non-peroxide antibacterial activity of honey might be of bee origin.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Owing to its high nutritional and gustatory qualities, honey is
one of the most popular food products. It is also widely used in api-
therapy (Mizrahi & Lensky, 1996). In this connection the chemical
composition of honey is actively studied and has become the sub-
ject of numerous research works. However, the problem is far from
being solved, and new approaches to preparation of samples and
their analysis, can lead to new findings concerning the composition
of biologically active components of honey. In the present work we
announce for the first time our findings concerning the compounds
characteristic of larval food of the honeybee (Apis mellifera L.) iden-
tified in different samples of honey. Honeybee larvae are fed by bee
‘‘nurses” with a jelly-like secretion (royal jelly, RJ) of the mandibu-
lar and the hypopharyngeal glands (Winston, 1987). The chemical
composition of RJ has evoked considerable interest among
researches of different profiles in connection with both the problem
of the separation of female honeybee colony members into queen
and worker castes (Haydak, 1943; Plettner, Slessor, Winston, &
Oliver, 1996) and biological activity of the product (Lakin, 1993).
The unique feature of RJ is the set of C8–C12 hydroxy and dicarbox-
ylic fatty acids (Baker, Foster, Lamb, & Hodgson, 1959; Isidorov,
_
Czyzewska, Isidorova, & Bakier, 2009; Melliou & Chinou, 2005).
The main RJ acid, 10-hydroxy-2-decenoic acid is known to have
various pharmacological effects, including antibiotic (Blum, Novak,
& Taber, 1959; Melliou & Chinou, 2005), antitumoral (Nakaya et al.,
* Corresponding author. Tel.: +48 85 747 78 00; fax: +48 85 747 01 03.
E-mail address: isidorov@uwb.edu.pl (V.A. Isidorov).
2007; Townsend, Morgan, & Hazlett, 1959; Townsend et al., 1960),
antioxidative (Jamnik, Goranovič, & Raspor, 2007; Nagai, Inoue, Su-
zuki, & Nagashima, 2006; Nagai, Sakai, Inoue, Inoue, & Suzuki,
2001), and hypoglycaemic activities (Dixit & Patel, 1964; Kramer,
Tager, Childs, & Speirs, 1977). The pharmacological properties of
the other RJ acids are not known.
The publications emphasise the fact that RJ is the only known
source of the above component (Baker, Foster, Lamb, & Hodgson,
1959) and no other natural product containing 10-HDA has yet
been found, not even in other bee products (Melliou & Chinou,
2005). It was, therefore, no small surprise to detect this and some
other acids earlier identified in lyophilised and freshly harvested
royal jelly (Isidorov et al., 2009) during our investigations of the
composition of low-volatile polar compounds in some genuine hon-
ey samples. We expect our report to be a starting point for wider
investigations, particularly aimed at a deeper understanding of
the biology of honeybees as well as the nature of antibiotic proper-
ties of honey.
2. Experimental
2.1. Materials
The honeys investigated in this study included Polish samples
(26) of known origin from different locations of the country as well
as market samples (8) of foreign origin. Three additional Polish
samples collected from bee colonies were fed with an infusion of
young pine shoots or hawthorn flowers in ca. 500 g/L saccharose
solution (sugar-adulterated ‘‘herbal honeys”).

0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.06.044
388 V.A. Isidorov et al. / Food Chemistry 124 (2011) 387–391
Pyridine, bis(trimethylsilyl)trifluoroacetamide (BSTFA) with an
addition of 1% trimethylchlorosilane, 2-hydroxyoctanoic (98%)
and 12-hydroxydodecanoic (P98%) acids were purchased from
Sigma–Aldrich (Poznań, Poland). Extraction SpeedisksÒ C18XF for
solid-phase extraction (SPE) were acquired from Mallinckrodt Ba-
ker, Inc. (Phillipsburg, NJ, USA).
2.2. Sample preparation and analysis
Honey samples (10 g) were diluted in 50 mL of distilled water
and filtered through the previously conditioned C18XF extraction
disks. After washing the discs with 20 mL of distilled water, in or-
der to remove sugars, the adsorbed compounds of low and medium
polarity were eluted by 50 mL of diethyl ether. The solvent was re-
moved on a rotor evaporator. The dry residue was washed out by
220 lL of dry pyridine and 80 lL of BSTFA was added. The reaction
mixture was sealed and heated during 0.5 h at 60 °C. All the exper-
iments were performed in triplicates.
Solutions of trimethylsilyl (TMS) derivatives were separated on
a HP 6890 gas chromatograph with mass selective detector MSD
5973 (Agilent Technologies, USA). The device was fitted with a
HP-5MS fused silica column (30 m  0.25 mm i.d., 0.25 lm film
thickness), with electronic pressure control and split/splitless
injector. Helium flow rate through the column was 1 mL/min in a
constant flow mode. Injection of 1 lL of the sample was performed
with using HP 7673 autosampler. The injector (250 °C) worked in a
splitless mode for 5 min. The initial column temperature was 50 °C
rising to 300 °C at 5 °C/min. The MSD 5973 detector acquisition
parameters were as follows: transfer line temperature 250 °C
with the detector held at 270 °C. The EIMS spectra were obtained
at 70 eV of ionisation energy. The detection was performed in the
full scan mode from 41 to 600 a.m.u.
Hexane solution of C10–C28 n-alkanes were separated under the
above conditions. Linear temperature programmed retention indi-
ces (LTPRI) were calculated from the results of the separation of
silylated extracts.
2.3. Precision of analytical procedure
The precision of the method was estimated by six replicate SPE
extractions and analysis of a honeydew honey sample containing
higher quantities of RJ acids. The precision was expressed by rela-
tive standard deviation (RSD). The peak areas of the acids obtained
by replicate analysis were used to calculate their RSD values, which
amounted to 6%.
To determine the recovery of hydroxy acids, a special model
experiment was conducted. First, 25 or 50 lL of methanol solution
of 2-hydroxyoctanoic (0.538 mg/mL) and 12-hydroxydodecanoic
acids (0.495 mg/mL) were added to water solution of rape honey
(0.2 g/mL). Then the samples were filtered through the previously
conditioned C18XF extraction disk following the procedure de-
scribed above. The results of GC–MS analysis indicate that SPE
using C18XF extraction disks provides sufficiently good extraction
Table 1
Percentage of recovery and relative standard deviation (%) of 2-hydroxyoctanoic and
12-hydroxydodecanoic acids in a honey model solution extracted by SPE.
Acid Mean recovery (n = 3) Average recovery ± RSD (%)
2-Hydroxyoctanoic 93.7 89.7 ± 3.8
89.6
86.1
12- 76.8 82.8 ± 5.7
Hydroxydodecanoic 83.4
88.2
yield results with the recovery percentage approaching 80–90%
for C8–C12 hydroxy acids (Table 1).
To calibrate MSD, a series of five solutions of 2-hydroxyoctanoic
and 12-hydroxydodecanoic acids in methanol covering the concen-
tration range 20–2000 mg/L was prepared by successive dilutions.
To prepare TMS derivatives, 1 mL of calibration solution was trans-
ferred to a vial for HP 7673 autosampler. After the evaporation of
methanol, 220 lL of pyridine and 80 lL of BSTFA were added into
the vial and heated at 60 °C during 0.5 h. TMS derivatives were
subjected to GC–MS analysis applying the conditions described
above. Using the obtained results, a regression equation was calcu-
lated. The procedure revealed linear behaviour over the whole con-
centration range with R2 > 0.990 for both hydroxy acids. The limits
of detection were determined by comparing the signal-to-noise (S/
N) ratio of the lowest concentration to S/N = 3 and were found to
vary between 0.02 and 0.05 lg/lL. This method enables quantita-
tion of hydroxy acids in honeys at concentrations P0.05 lg/g.
3. Results and discussion
Our investigation initially aimed to determine the composition
of low-volatile polar compounds in genuine honey samples ob-
tained in the summer and autumn of 2008 and 2009 using high
performance gas chromatography coupled with mass spectrome-
try. The floral sources of Polish honeys were confirmed by GC–
MS analysis, the results of which showed the presence of charac-
teristic compounds in each of the analysed samples. For instance,
combination of vomifoliol and abscisic acid is typical for heather
honey (Guyot, Scheirman, & Collin, 1999), succinic, 4-hydroxyben-
zeneacetic and homovanillic acids are characteristic of honeydew
honey, high content of phenyllactic and 4-hydroxybenzoic acids
is characteristic of buckwheat honey, salicylic and homovanillic
acids are prevailing components in lime honey, whereas rape hon-
ey is distinguished from other types by high concentration of
methyl syringate and oleic acid (publication in preparation).
Fig. 1 shows a fragment of a typical chromatogram of TMS
derivatives of one of the samples of silver fir (Abies alba) honeydew
honey. As can be clearly seen, the chromatogram contains only
small peaks of the main components of honey, i.e. monosaccha-
rides that could hinder the identification and quantitative determi-
nation of the compounds of interest. Along with the peaks of
numerous phenol carboxylic acids, aliphatic alcohols and hydro-
carbons, the chromatograms also showed signals of hydroxy acids
and unsaturated dicarboxylic acids that were identified using the
mass spectra and chromatographic retention indices previously
obtained during the analysis of royal jelly extracts (Isidorov
et al., 2009).
Overall, 10 acids characteristic of RJ were registered in different
quantities in the genuine honey samples but also in the artificial
‘‘herbal honeys”: 7- and 8-hydroxyoctanoic, 3-hydroxydecanoic
(3-HDAA), 9-hydroxydecanoic (9-HDAA), 9-hydroxy-2-decenoic
(9-HDA), 10-hydroxydecanoic (10-HDAA), 10-HDA, 3,10-dihydr-
oxydecanoic (3,10-DDA), 2-octene-1,8-dioic (2-OenDA) and
2-decene-1,10-dioic acids (2-DecDA). In spite of the fact that as
many as 35 hydroxy and dicarboxylic C8–C14 acids have been iden-
tified in royal jelly (Isidorov et al., 2009), the 10 compounds listed
above constitute approximately 90% of acid fraction in this bee
product.
Table 2 shows the results of content analysis of eight the acids
(3-HDAA and 9-HDA were found in trace amounts only) in twelve
kinds of honey. As can be seen, the quantity of RJ acids varies lar-
gely in different honeys: the smallest quantities of acids are found
in buckwheat and rape honeys, whereas the largest ones are found
in honeydew honeys. At the same time, the latter group of honeys
appears to be very homogeneous: the coefficient of variation
 V.A. Isidorov et al. / Food Chemistry 124 (2011) 387–391 389

Fig. 1. Part of chromatogram of carboxylic acids (their TMS derivatives) extracted from honeydew honey (HDH-5). (1) Salicylic acid, (2) 7-hydroxyoctanoic acid, (3) ß-
phenyllactic acid, (4) 8-hydroxyoctanoic acid, (5) 4-hydroxybenzoic acid, (6) 4-hydroxybenzeneacetic acid, (7) 3-hydroxydecanoic acid, (8) 2-octene-1,8-dioic acid, (9) 3,4-
dihydroxyphenylethanol, (10) homovanillic acid, (11) 10-hydroxydecanoic acid, (12) protocatechuic acid, (13) 10-hydroxy-2-decenoic acid (10-HDA), (14) sebacic acid, (15)
p-coumaric acid, (16) 2-decene-1,10-dioic acid, (17) 3,10-dihydroxydecanoic acid, (18) hexadecanoic acid, (19) ferulic acid.

(CV = 100  SD/mean) for each RJ acid of seven samples (HDH-1–
HDH-7) is contained between 1% and 6%. Most likely, this homoge-
neity can be explained by their origin: all these honeys were col-
lected by different apiarists in mid-August, 2008 in the
Podkarpacki Region (south-east of Poland).
To our best knowledge, only one of the acids listed in Table 2 (2-
DecDA) was registered previously in white clover and manuka
honeys in the concentrations of 40.3 ± 57.2 and 31.1 ± 20.7 lg/g
(n = 8), respectively (Tan, Holland, Wilkins, & Molan, 1988). The
relation between the contents of 2-DecDA and 10-HDA in honey
and royal jelly should be noted here. While the latter is the main
RJ acid, 2-DecDA (with a rare exception) constituted the principal
aliphatic acid in the honeys analysed by us. It cannot be excluded
that the dicarboxylic 2-DecDA is the product of oxidation of x-hy-
droxy acid, 10-HDA, consistent with the enzymatic mechanism
postulated by Plettner et al. (1996). This assumption is supported
by a relatively strong negative correlation between concentrations
of these acids in 20 honey samples (cf. Table 2) described by the
equation: C2-DecDA = 6.8456 C10-HDA 17.776 (R2 = 0.777). How-
ever, it still remains to be seen whether the 10-HAD oxidation pro-
ceeds in the mandibular glands of workers or in honey itself.
It is interesting to note also that in some honey samples trace
quantities of 9-HDA as well as 4-hydroxy methylbenzoate (HOB)
were identified. Both compounds are ranked among queen honey
bee pheromones and were extracted from their mandibular glands
(Engels et al., 1997; Slessor, Kaminski, King, Borden, & Winston,
1988).
Royal jelly is made in the hypopharyngeal glands of bee work-
ers. Apparently the presence of RJ acids in honey is connected with
the functioning of these glands of workers, whose function is to
collect and process nectar in honey. It is known that during the
processing of nectar bees add to it different enzymes, such as
invertase, diastase and glucose oxidase. The present results show
for the first time that bees add also RJ acids to nectar. When a nec-
tar forager returns from a foraging trip, it directly transfers the col-
lected crop to two or three workers of 8–16 days old. Receiver-bees
share through subsequent trophallaxes the incoming liquid that
begins to circulate inside the hive (Wainselboim, Roces, & Farina,
2003). Hence, we can assume that the secretions produced by
the young workers’ glands get into the nectar just at the time of
its processing into honey. This assumption can be also supported
by the discovery of small but detectable amounts of 9-HDA and
HOB in some of the investigated honeys: these components of
the queen’s mandibular gland complex are licked from the queen
by a retinue of worker bees and then further propagated inside
the hive by trophallaxes (Crailsheim, 1998; Goyret & Farina,
2005; Wainselboim et al., 2003).
It is not clear, however, why the content of these compounds
varies in different kinds of honey (Table 2). Moreover, the content
of hydroxy fatty acids can be quite different in the honey collected
from different hives in the same apiary. For instance, in two sam-
ples of the analysed rape honey (i.e. RH-1 and RH-2) only trace
amounts of 10-HDA was registered. On the other hand, the third
sample (i.e. RH-3) contained rather a marked quantity (5.59 lg/g)
of this acid. Thus it can be assumed that the content of RJ acids
in honey is affected by a group of factors, such as nectar influx
and hive population (Crailsheim, 1998). It is worth noting that
the investigated sugar-adulterated ‘‘herbal honeys” (i.e. HHH,
PHH-1 and PHH-2) were rich in these acids, which can explained
by the fact that the majority of nurse bees participated in the col-
lection and processing of the syrup from the racks set directly in-
side the hives.
The content of RJ acids in honey can influence the antibiotic
activity of honey. This activity is usually connected with the
H2O2 that is formed by glucose oxidase (Molan, 1992; Molan &
Russell, 1988). However, some authors have found that the
non-peroxide activity is more important (Bogdanov, 1984, 1997;
Weston, Brocklebank, & Lu, 2000; Weston, Mitchell, & Allen,
1999) and a major part of antibacterial activity is related to yet
unidentified components of acid fraction of honey. By fractionation
of honey into different substance classes the following relative
distribution of non-peroxide antibacterial activity was
found: acids > bases = nonpolar, nonvolatiles > volatiles (Bogda-
nov, 1997). Moreover, Bogdanov also reached a conclusion that
the high antibacterial activity of honeydew honey as well as su-
gar-adulterated honey was of bee origin. Taking into account the
data given in Table 2, it is probable that RJ acids can contribute
for this specific property. Indeed, it was established that 10-HDA
exhibits antibiotic activity against many bacteria and fungi. This
fatty acid is less than one-fourth as active as penicillin against
Micrococcus pyrogenes and less than one-fifth as active as chlortet-
racycline against Escherichia coli (Blum, Novak, & Taber, 1959).
390 V.A. Isidorov et al. / Food Chemistry 124 (2011) 387–391

Table 2
Composition of ‘‘royal jelly acids” (lg/g) in the honey of different origin (n = 3), n = number of determinations with the same honey.

Sample 7-HO-C8 8-HO-C8 9-HDAA 10-HDAA 10-HDA 3,10-DDA 2-OenDA 2-DecDA

Honeydew honey (HDH), Poland
HDH-1 3.39 ± 0.02 3.68 ± 0.04 2.77 ± 0.01 3.32 ± 0.07 4.91 ± 0.26 2.60 ± 0.05 4.53 ± 0.01 12.12 ± 0.53
HDH-2 3.45 ± 0.03 3.77 ± 0.02 3.01 ± 0.01 4.20 ± 0.05 7.44 ± 0.14 2.74 ± 0.08 2.33 ± 0.01 13.83 ± 0.34
HDH-3 3.35 ± 0.03 3.44 ± 0.03 2.61 ± 0.02 3.00 ± 0.09 3.86 ± 0.23 2.42 ± 0.09 2.27 ± 0.01 6.60 ± 0.36
HDH-4 3.37 ± 0.02 3.84 ± 0.02 2.67 ± 0.01 2.95 ± 0.04 3.99 ± 0.16 2.44 ± 0.05 2.28 ± 0.01 8.13 ± 0.46
HDH-5 3.40 ± 0.03 3.59 ± 0.03 3.35 ± 0.03 3.22 ± 0.04 4.01 ± 0.14 2.60 ± 0.03 2.44 ± 0.01 11.28 ± 0.47
HDH-6 n.d.a 3.42 ± 0.01 n.d. 2.71 ± 0.01 3.26 ± 0.08 2.33 ± 0.01 2.33 ± 0.01 6.23 ± 0.33
HDH-7 3.36 ± 0.02 3.55 ± 0.02 2.58 ± 0.01 3.15 ± 0.07 4.65 ± 0.16 2.45 ± 0.04 2.32 ± 0.02 1.75 ± 0.11
Heather honey (HH), Poland
HH-1 6.60 ± 0.12 6.69 ± 0.06 6.41 ± 0.39 5.38 ± 0.05 Traceb 4.58 ± 0.08 4.89 ± 0.08 13.98 ± 1.07
HH-2 n.d. n.d. 5.72 ± 0.10 4.93 ± 0.09 n.d. 3.66 ± 0.03 4.05 ± 0.06 24.34 ± 0.64
HH-3 3.15 ± 0.05 5.39 ± 0.09 Trace 3.21 ± 0.02 n.d. 2.42 ± 0.05 2.56 ± 0.09 13.24 ± 2.03
HH-4 3.38 ± 0.01 3.38 ± 0.01 3.07 ± 0.05 2.71 ± 0.03 Trace 2.36 ± 0.07 2.50 ± 0.04 8.72 ± 0.35
HH-5 Trace 3.39 ± 0.01 Trace 2.71 ± 0.03 Trace 2.33 ± 0.02 2.30 ± 0.01 7.49 ± 0.25
HH-6 Trace 3.43 ± 0.04 Trace 2.96 ± 0.11 Trace 2.35 ± 0.03 2.44 ± 0.02 11.03 ± 0.43
Heather honey from Scotland (HHS)
HHS-1 n.d. 1.38 ± 0.01 n.d. 1.33 ± 0.01 1.35 ± 0.02 n.d. 1.31 ± 0.01 1.54 ± 0.03
HHS-2 n.d. 2.04 ± 0.03 Trace 1.93 ± 0.01 6.43 ± 0.20 Trace 1.84 ± 0.01 2.41 ± 0.14
Lime honey (LH), Poland
LH-1 4.75 ± 0.01 4.77 ± 0.02 3.65 ± 0.02 3.64 ± 0.01 4.14 ± 0.04 3.29 ± 0.05 3.29 ± 0.02 11.04 ± 0.30
LH-2 6.19 ± 0.17 Trace Trace Trace 11.36 ± 0.06 4.55 ± 0.15 4.73 ± 0.10 99.35 ± 2.77
LH-3 5.67 ± 0.17 Trace Trace 10.05 ± 0.24 16.94 ± 0.54 5.38 ± 0.15 3.75 ± 0.10 99.91 ± 3.08
LH-4 3.34 ± 0.01 3.40 ± 0.02 2.55 ± 0.06 2.66 ± 0.03 2.84 ± 0.05 2.45 ± 0.06 2.28 ± 0.02 8.50 ± 0.40
LH-5 3.08 ± 0.01 4.15 ± 0.02 1.82 ± 0.04 3.83 ± 0.03 2.67 ± 0.06 2.30 ± 0.05 3.75 ± 0.61 4.44 ± 0.06
Buckwheat honey (BH), Poland
BH-1 5.31 ± 0.01 n.d. 4.07 ± 0.01 n.d. n.d. n.d. 3.63 ± 0.04 8.40 ± 0.22
BH-2 n.d. Trace n.d. n.d. n.d. n.d. Trace 8.72 ± 0.36
BH-3 Trace n.d. n.d. n.d. n.d. n.d. 0.09 ± 0.02 2.31 ± 0.21
BH-4 7.23 ± 0.11 n.d. n.d. n.d. n.d. Trace 0.09 ± 0.02 58.22 ± 2.78
BH-5 n.d. n.d. n.d. n.d. n.d. n.d. 0.09 ± 0.02 10.75 ± 1.73
Rape honey (RH), Poland
RH-1 n.d. n.d. n.d. n.d. n.d. n.d. Trace 3.01 ± 0.13
RH-2 n.d. n.d. n.d. n.d. n.d. n.d. Trace 3.19 ± 0.02
RH-3 Trace n.d. n.d 5.05 ± 0.07 5.59 ± 0.11 Trace Trace 6.21 ± 0.64
Manuka honey (MH), New Zealand
MH-1 n.d. n.d. n.d. n.d. 3.31 ± 0.07 n.d. n.d. 3.35 ± 0.06
MH-2 n.d. n.d. n.d. n.d. 8.62 ± 0.01 n.d. n.d. 8.96 ± 0.01
Orange honey (OH), Morocco
OH n.d. n.d. n.d. n.d. Trace n.d. 2.34 ± 0.02 4.56 ± 0.23
Eucalyptus honey (EH), Morocco
EH n.d. n.d. n.d. n.d. n.d. 2.32 ± 0.01 n.d. 3.84 ± 0.07
Acacia honey (AH), Austria
AH n.d. n.d. n.d. 8.88 ± 0.05 Trace 8.84 ± 0.09 n.d. 18.10 ± 0.86
Hawthorn honey (HTH), Russia
HTH 1.54 ± 0.03 1.53 ± 0.02 n.d. 1.54 ± 0.11 1.51 ± 0.08 Trace n.d. 1.53 ± 0.10
Hawthorn herbal honey (HHH), Poland
HHH 5.03 ± 0.22 5.24 ± 0.12 4.85 ± 0.03 5.39 ± 0.14 4.69 ± 0.04 6.39 ± 0.27 5.52 ± 0.17 11.30 ± 0.69
Pine herbal honey (PHH), Poland
PHH-1 3.27 ± 0.06 3.53 ± 0.19 3.49 ± 0.14 4.56 ± 0.14 2.88 ± 0.11 3.91 ± 0.31 3.40 ± 0.18 4.08 ± 1.00
PHH-2 3.29 ± 0.09 3.68 ± 0.18 3.33 ± 0.07 3.65 ± 0.72 2.78 ± 0.05 3.56 ± 0.72 3.58 ± 0.81 3.16 ± 0.06

Abbreviations: see Section 3.

a
Not detected.
b
<0.05 lg/g.

Hence, the appearance of relatively small amounts of RJ acids with
a well documented antibacterial activity can determine additive
and synergistic effects of the hydrogen peroxide, other antibacte-
rial fatty acids (such as formic, oxalic and other low molecular
acids) as well as phenolic compounds found in honey (Boukraâ,
Niar, Benbarek, & Benhanifia, 2008).
4. Conclusion
Comparing the results of the analyses of 34 samples of honey,
we found a substantial variation in the royal jelly acid composition.
In future research it would be interesting to investigate the factors
leading to the variations. Further investigations of the content of
compounds of bee origin in different types of honey can also en-
hance our knowledge of the nature of ‘‘residual” non-peroxide
antibiotic activity of honey.
Acknowledgement
We acknowledge Prof. M. Borwaska, Bromatology Division,
Białystok Medical University for supplying the monofloral Polish
honey samples.
 V.A. Isidorov et al. / Food Chemistry 124 (2011) 387–391 391

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