Food Anal.
Methods (2015) 8:1150–1163
DOI 10.1007/s12161-014-9996-2
Chemometric Analysis of Bee Pollen Based on Volatile
and Phenolic Compound Compositions and Antioxidant
Properties
Vilma Kaškonienė & Geralda Ruočkuvienė &
Paulius Kaškonas & Ieva Akuneca & Audrius Maruška
Received: 10 June 2014 / Accepted: 8 September 2014 / Published online: 18 September 2014
# Springer Science+Business Media New York 2014
Abstract The aim of this study was to determine total
amounts of phenolics and flavonoids, radical scavenging ac-
tivities, and compositions of phenolic and volatile compounds
in 14 samples of honeybee pollen collected in the Baltic
region. Radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) scav-
enging activities and total amounts of phenolics and flavo-
noids were evaluated using spectrophotometry. Volatiles from
the headspace of the samples were analyzed using solid-phase
microextraction (SPME) fiber coated with 100-μm polydi-
methylsiloxane layer, separated, and identified employing
GC-mass spectrometry (MS). Forty-two volatiles were iden-
tified in the headspace. Nonanal (1.5–20.1 %), dodecane
(1.2–-34.6 %), and tridecane (1.4–24.7 %) were found in all
samples. Screening of phenolics and flavonoids was per-
formed using high-performance liquid chromatography
(HPLC) with electrochemical detection. 2-Hydroxycinnamic
acid (43.4–179.9 μg/g), rutin (156.2–955.7 μg/g), and quer-
cetin (24.0–529.8 μg/g) were detected in all tested samples.
Total amounts of phenolic compounds and flavonoids varied
between 24.1 and 45.5 mg/g, and 6.1 and 11.6 mg/g, respec-
tively, expressed as rutin equivalents. The pollen extracts
exhibited radical scavenging activity, which scattered widely
in range of 7.1–39.2 mg/g, expressed as rutin equivalents.
Radical scavenging activity correlated with the total content
of phenolic compounds while correlation coefficient was 0.95.
Chemometric evaluation was performed to classify the sam-
ples into clusters according to the observed data.
V. Kaškonienė (*) : G. Ruočkuvienė : I. Akuneca : A. Maruška
Faculty of Natural Sciences, Vytautas Magnus University, Vileikos
st. 8, LT-40444 Kaunas, Lithuania
e-mail: v.kaskoniene@gmf.vdu.lt
P. Kaškonas
Institute of Metrology, Kaunas University of Technology, Studentų
st. 50, LT-51368 Kaunas, Lithuania
Keywords Bee pollen . Antioxidant activity .
SPME-GC-MS . HPLC with electrochemical detection .
Chemometric analysis
Introduction
Pollen collected by honeybees (Apis mellifera) is one of the
bee products, and it is valuable not less than honey, propolis,
royal jelly, or beebread. To our knowledge, there is lack of
literature data on phytochemical composition of bee pollen
collected in Lithuania and Latvia. The aim of this study was to
compare volatile compound composition and total contents of
phenolics and flavonoids together with antiradical activity and
phenolic composition of bee pollen collected in the Baltic
region.
The increasing number of publications concerning bee
pollen analysis in the last two decades demonstrates rediscov-
ery of the bee pollen. Analysis of the active ingredients is a
challenging task, because of the diversity of the botanical
composition, seasonal variation of the flavonoid content
(Freire et al. 2012), and the impact of the geographical origin.
Different biological activities of the bee pollen were
established, i.e., antimicrobial (Morais et al. 2011; Graikou
et al. 2011), anti-inflammatory (Maruyama et al. 2010; Küpeli
et al. 2010), anti-mutagenic, anti-histopathologic (Tohamy
et al. 2014), antinociceptive (Küpeli et al. 2010), and antiox-
idant (Graikou et al. 2011; Morais et al. 2011; Freire et al.
2012). The responsibility for the biological activities is attrib-
uted to phenolic compounds.
A number of methods based on thin layer chromatography
and reversed-phase high-performance liquid chromatography
(HPLC)/UV–Vis diode array detection (Campos et al. 1990),
capillary electrophoresis (Chu et al. 2007), nano-liquid chro-
matography (Fanali et al. 2013), HPLC-diode array detector
(DAD) (Freire et al. 2012), and HPLC with photodiode-array
Food Anal. Methods (2015) 8:1150–1163
detection coupled to an ion trap mass spectrometry (Ferreres
et al. 2010) have been developed and applied for separation of
bee pollen phenolic compounds. To our knowledge, this is the
first study of the bee pollen phenolic compound composition
analyzed using HPLC with electrochemical detection. HPLC
with electrochemical detection provides high sensitivity and
good selectivity for electroactive species. The oxidation reac-
tion of flavonoids is strongly related to their structure, which
contains several free phenolic hydroxyl groups, particularly
ortho-phenolic hydroxyl groups (Ghica and Brett 2005). On
the other hand, the flavonoid profile in bee pollen depends on
their botanical origin. The analysis in this study was per-
formed on natural mixtures of bee-collected pollen samples.
A mechanical separation of monofloral granules of pollen is
possible from the natural mixtures collected by bees, although
it is not the economically worthwhile for the commercial
production. However, mixed pollen samples exhibit different
results in comparison to monofloral pollen, due to the possible
synergistic/antagonistic/additive effects of the phytochemicals
present in different pollen species.
Materials and Methods
Chemicals
2,2-Diphenyl-1-picrylhydrazyl, 2N Folin–Ciocalteu’s phenol
reagent, phenolic acids (gallic, 3,4-dihydroxibenzoic,
chlorogenic, syringic, caffeic, sinapic, ferulic, and 2-
hydroxicinnamic), rutin, quercetin, and naringenin were sup-
plied by Sigma-Aldrich (St. Louis, MO, USA); acetonitrile,
methanol (both HPLC grade), and aluminum trichloride were
obtained from Carl Roth, Gmbh (Karlsruhe, Germany), and
sodium acetate was acquired from Fisher Scientific (Pitts-
burgh, PA, USA).
Bee Pollen Samples
Fourteen bee pollen samples from different geographical re-
gions were analyzed, namely, four samples from different
regions of Latvia (LV1 collected in unknown region, LV2
collected in Cesu, LV3 collected in Madona, and LV4 collect-
ed in Tukuma) and seven samples from various regions of
Lithuania (LT1 collected in Biržai, LT2 collected in Skuodas,
LT3 collected in Mažeikiai, LT4 collected in Pasvalys, LT5
collected in Telšiai, LT6 collected in Vilkaviškis, and LT7
collected in Prienai). For comparative reasons, one sample
from Spain (ESP) and two samples from China (CHN1 and
CHN2) were also analyzed. Samples CHN1, CHN2, LV1,
LT1, LT2, and LT3 were obtained from Apiproduktai, Ltd.,
while the other ones were purchased from the Lithuanian and
Latvian markets. The obtained pollen samples were stored in
the dark in the refrigerator at +4 °C. All samples were
1151
collected in 2012, except the sample from Spain, which was
collected in 2010. The Chinese sample CHN1 was of
monofloral (rape) origin, while other 13 samples were collect-
ed from multiple pollen sources. The botanical origin and
qualitative composition of polyfloral bee pollen samples were
not tested in this study.
To prepare the extracts for spectrophotometric analysis,
2.0 g of ground bee pollen was added into 22 ml vials and
extracted with 20 ml of 80 % methanol for 24 h in an orbital
shaker, “Titertek” (Flow Laboratories, Germany). The obtain-
ed extracts were filtered using a disposable membrane filter of
0.22 μm pore size (Sigma-Aldrich, Germany). The prepared
methanolic extracts of the pollen were kept in the dark at +
4 °C temperature until further analysis.
SPME-GC-MS Analysis
In order to analyze the bee pollen volatile composition in the
headspace of the samples, solid-phase microextraction
(SPME)-GC-mass spectrometry (MS) analysis was carried
out. Ground bee pollen sample (0.1 g) was placed in a 4 ml
vial, tightly capped with a PTFE/silicon septum, heated to
50 °C, and kept for 15 min to allow the sample and headspace
equilibration. Later, the 100 μm polydimethylsiloxane
(PDMS) layer-coated SPME fiber (Supelco, USA) was ex-
posed in the sample headspace for 10 min, then inserted into
the GC injection port, and heated at 230 °C for 5 min to clean
the SPME fiber and prevent cross-contamination. The extract
was directly transferred to the analytical column for analysis
using 1 min desorption time.
A gas chromatograph with mass spectrometric detector
(GC-MS) (GCMS-QP2010, Shimadzu, Tokyo, Japan)
equipped with a RTX-5MS column, 30 m×0.25 mm i.d.×
0.25 μm film thickness (Restec, Bellefonte, USA), was used.
The system was run applying a flow rate of helium of 1.6 ml/
min in splitless mode and using an injector temperature of
230 °C. The column oven temperature program was the
following:
4 -C=min 1 -C=min 4 -C=min
40 -C ð3minÞ→ 70 -C ð3minÞ→ 80 -C ð0minÞ→
10 -C=min 20 -C=min
100 -C ð0minÞ→ 180 -C ð0minÞ→ 280 -C ð2minÞ ˙
Data were acquired in electron impact (EI) mode with
ionization voltage of 70 eV and mass range m/z 33–400. The
relative amounts of volatiles, in percent, were calculated ac-
cording to the separated peak areas, dividing the area of each
component by the total area of all isolated components. The
identification of the bee pollen volatile compounds was per-
formed in respect of their mass spectra (NIST v1.7). Positive
identification was assumed when good matches (90 % and
1152
more) of mass spectra obtained with that presented in spectra
library were achieved.
Spectrophotometric Methods for Determination of Total
Amounts of Phenolic Compounds and Flavonoids and DPPH
Radical Scavenging Activity
The total amount of phenolic compounds in the bee
pollen extracts was evaluated using the Folin–Ciocalteu
method as described by Mikašauskaitė et al. (2013). The
absorbance at 760 nm was measured using a spectro-
photometer Spectronic1201 (Milton Roy, USA). If the
absorbance value exceeded the 1.000 AU limit, extracts
were additionally diluted adding 80 % methanol. The
measurements were compared to calibration curves of
gallic acid (0.01–1.00 mg/ml), rutin (0.01–1.00 mg/ml),
and quercetin (0.01–1.00 mg/ml) solutions expressed in
milligram of gallic acid equivalents (GAE), rutin equiv-
alents (RE), and quercetin equivalents (QE) per 1 g of
the bee pollen, respectively. The total amount of the
phenolic compounds was calculated according to the
given equation:
c⋅V
TPC ¼ ; ð1Þ
m dilution of the extract solution was carried out.
where TPC is the total phenolic content, mg/g; c is the
concentration of the reference compound determined from the
calibration curve, mg/ml; V is the volume of the pollen extract,
ml; and m is the weight of the pollen, g. The absorption
measurements to determine the total phenolic content were
performed five times, and the result was an average of the five
values.
The total flavonoid content of the bee pollen extracts
was determined using aluminum trichloride colorimetric
assay as described by Kaškonienė et al. (2009). The
absorbance value at 407 nm was read after 30 min
incubation at room temperature. If the value reached
more than 1.000 AU, the extracts were diluted addition-
ally with 80 % methanol. Calibration curve was con-
structed using rutin (0.01–1.0 mg/ml) and quercetin
(0.01–0.5 mg/ml) solutions measuring the absorption of
these solutions under the same conditions. Total amount
of the flavonoids was expressed in milligram of rutin
and quercetin equivalents per 1 g of the pollen sample,
RE and QE, respectively. The total amount of the fla-
vonoid was calculated according to Eq. (1). The deter-
minations were carried out five times averaging the
result.
Radical scavenging activity was determined according to
the slightly modified Kaškonienė et al. (2009) method using
2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical solution.
Food Anal. Methods (2015) 8:1150–1163
The solution of DPPH radical was prepared in 50 % (vol.) of
10 mM sodium acetate buffer, pH 5.5, 25 % (vol.) of
acetonitrile, and 25 % (vol.) of pure methanol. The final
DPPH concentration was 4×10−5 M. The absorption of
the prepared solution was adjusted diluting it with the
buffer/acetonitrile/methanol (50/25/25 %) (vol.) mixture
to achieve the absorbance value of 0.500±0.020 AU at
515 nm. Seventy-seven microliters of bee pollen extract
(or pure methanol for blank test) was added to 3.0 ml of
buffered DPPH solution and vortexed. After 15 min in-
cubation, the absorbance at 515 nm was measured using
a spectrophotometer. The radical scavenging activity was
calculated using expression (2)
AB −AA
I¼ ⋅100 %; ð2Þ
AB
where I corresponds to DPPH inhibition, %; AB is the
absorption of the blank sample (t=0 min); and AA is the
absorption of the tested bee pollen extract solution at the end
of the reaction (t=15 min).
The measurements were recorded when I value fell in the
range up to 75 %, and in other cases, preconcentration or
Rutin (0.05–0.25 mg/ml), gallic acid (0.01–0.05 mg/ml),
and quercetin (0.01–0.05 mg/ml) were used for the calibration
curves. The radical scavenging activity was expressed in
milligram of RE, GAE, and QE per 1 g of pollen. The
measurements were taken five times, and the result was an
average of five values.
HPLC with Electrochemical Detection
For separation and electrochemical detection of the
composition of the pollen samples methanolic extract,
an HPLC gradient system (ESA, USA) was used. Twen-
ty microliters of the sample was injected by means of
an autosampler, model 542, for analysis. High-pressure
gradient was formed using two Model 582 HPLC
pumps. Reversed-phase C-18 column, 80 mm×4.6 mm,
particle size 3 μm, and 120 Å pore size (ESA, USA),
was used for separations. Chromatograms were acquired
by means of CoulArray electrochemical detector, model
5600, containing an array of four cells with the poten-
tials set at 300, 500, 700, and 900 mV. Mobile phase
flow rate was set at 0.25 ml/min. The method was
described in details by Stankevičius et al. (2011).
Identification of the compounds was carried out by com-
parison of the analyte retention time and response profiles
obtained from electrochemical detector at four different po-
tentials with reference compounds. Quantitative analysis was
performed using external standard method, and the amounts
Food Anal. Methods (2015) 8:1150–1163
of determined compounds were calculated according to the
following equation:
Ax
cx ¼ cst ⋅ ; ð3Þ
Ast
where cx is the concentration of the analyte in the pollen
extract, mg/ml; cst is the concentration of the standard com-
pound, mg/ml; Ax is the peak area of the analyte in the extracts
obtained from the chromatogram; and Ast is the peak area of
the standard compound.
Chemometric Analysis
All statistical data analyses were performed using MATLAB
v7.6 software on Windows platform. In order to have repre-
sentative data sets that would let make statistically reliable
decisions, the well-known Monte Carlo data generation meth-
od assuming normal (Gaussian) distribution was employed.
The data set pretreatment stage, which included standardiza-
tion and volatile compounds having the lowest amounts of
filtering procedures, was adopted. The successive chemomet-
ric data exploration, comprised principal component analysis
(PCA), hierarchical clustering analysis (HCA), K-means clus-
tering analysis (KMCA), and discriminant analysis (DA),
followed. PCA was used as a tool for data compression by
transforming correlated initial variables to non-correlated set
of principal components. HCA and KMCA helped to classify
the analyzed bee pollen samples having similar characteristics
to classes. Linear DA was carried out in order to find the
boundaries between formed groups in the predictors’ space.
The classification techniques were employed to link the sam-
ples to clusters according to the volatile composition and total
DPPH radical scavenging activity, together with total amounts
of phenolic and flavonoid compounds and phenolic acid and
flavonoid composition.
Results and Discussion
Composition of the Bee Pollen Volatile Compounds
Determined Using SPME-GC-MS
Forty-two different compounds were detected testing bee
pollen samples. The results are reported in Table 1 and pre-
sented in Fig. 1. The identified components involve different
classes of chemical compounds including acids (2-
methylpropanoic, pentanoic, hexanoic, etc.), alcohols (1-
pentanol, 2,3-butanediol, 3-hexen-1-ol, benzyl alcohol, etc.),
ketones (6-methyl-5-hepten-2-one, 3,5-octadien-2-one, etc.),
aldehydes (hexanal, heptanal, 6-nonenal, etc.), esters (acetic
1153
acid octyl ester, benzoic acid ethyl ester, etc.), terpenes (α-
pinene, β-myrcene, eucalyptol, limonene), and saturated and
unsaturated, linear and branched hydrocarbons (decane,
undecane, 2,5-dimethyl-2-undecene, etc.). Only three com-
pounds, i.e., nonanal, dodecane and tridecane, were found in
all analyzed samples. Hexanal was identified in 13 out of the
14 samples, 12 samples contained 6-methyl-5-hepten-2-one,
11 samples contained 2-methylbutanoic acid and limonene,
pentadecane was detected in 10 samples, and butyrolactone
and 3,5-octadien-2-one were found in 9 samples. Some com-
pounds, like 4-methyl-1-pentanol, 3-hexen-1-ol, 1-heptanol,
eucalyptol, pentanoic acid, 5-ethyl-2-methyl-octane, 6-
nonenal and octanoic acid, were detected in one bee pollen
sample only (Table 1).
The literature data about the bee pollen volatiles are scarce.
Since pollen is an integral part of the honey, similar com-
pounds were detected in the headspace of honey. It is known
that phytochemical composition of pollen and honey depends
on the floral origin. Nonanal, hexanal, heptanal, octanal,
benzaldehyde, and limonene were identified in the buckwheat
honey (Pasini et al. 2013), in monofloral rape and polyfloral
honey (Kaškonienė et al. 2008) and in pine honey (except of
hexanal) (Tananaki et al. 2007). 2-Methylpropanoic acid was
found in monofloral winter rape, caraway and white clover
honey (Kaškonienė et al. 2008), lime tree, fir honeydew, and
sage honey (Lušić et al. 2007). 6-Methyl-5-hepten-2-one was
found in monofloral citrus honey (Alissandrakis et al. 2007).
However, it is obvious that the origin of this compound in
Lithuanian and Latvian bee pollen samples is not the citrus
pollen, because citrus trees do not grow in this climate.
The quantitative composition of the bee pollen volatiles
varied greatly. The amount of dodecane varied from 1.2 to
34.6 %, and the amount of nonanal scattered between 1.5 and
6.8 % in 13 samples, while sample LT6 contained 20.1 % of
nonanal. High amount of tridecane was found in six samples,
i.e., CHN1, CHN2, LV1, and LT1 to LT3, and it varied from
19.5 to 24.7 %. The Spanish sample of the pollen contained
13.4 % of this compound, while tridecane contributed from
1.3 to 2.6 % only in the rest seven samples. Found limonene
was in the range from 0.8 to 16.7 %. To our knowledge, the
amount of limonene detected in honey is less than 6.5 %
(Alissandrakis et al. 2007). Higher amounts of limonene were
reported only in some kinds of propolis: 10.5 % was found in
propolis collected in Croatia (Borčić et al. 1996), 11.2 % was
detected in propolis from Brazil (Simionatto et al. 2012), and
15.6 % was found in Uruguayan propolis (Kaškonienė et al.
2014). Limonene was the predominant constituent of the
sample LV3 from Latvia, contributing 16.7 %. The amount
of this compound also was high in other two samples from
Latvia, LV4—14.9 % and LV2—9.4 %, and two samples
from Lithuania, LT4—12.1 % and LT5—11.3 %. The evalu-
ation of antibacterial activity was not under the scope of this
study. Nevertheless, limonene could be one of the compounds
Table 1 Volatile compound composition in the headspace of the bee pollen samples analyzed using SPME with PDMS fiber
1154

Identified compounds RT Match, % Relative percentage composition (%)

ESP CHN1 CHN2 LV1 LV2 LV3 LV4 LT1 LT2 LT3 LT4 LT5 LT6 LT7

2-Methylpropanoic acid 3.922 95 1.7 2.2 9.0 1.7 2.1 5.8 4.4
1-Pentanol 4.044 94 3.2 9.7 3.0
2,3-Butanediol 4.577 98 6.6 2.5 1.5 3.4 1.7 1.5
Hexanal 4.825 97 3.1 8.0 4.6 10.3 7.0 4.8 1.7 3.1 2.8 4.9 12.9 12.1 5.7
4-Methyl-1-pentanol 5.972 98 2.3
3-Hexen-1-ol (Z) 6.575 90 2.8
2-Methylbutanoic acid 6.759 94 3.1 4.8 10.7 5.6 3.1 3.0 3.0 3.0 10.2 3.4 9.4
1-Hexanol 7.049 96 9.2 1.8
Pentanoic acid 7.764 92 2.9
Heptanal 8.156 96 1.5 3.5 2.1 1.4 3.1 2.9
Butyrolactone 8.516 96 6.4 5.0 2.1 3.2 2.5 3.0 1.4 1.8 4.5
α-Pinene 9.278 90 1.7 1.2 2.1
Benzaldehyde 10.352 98 2.1 2.8 2.5 5.5 5.0
1-Heptanol 10.868 94 2.1
6-Methyl-5-hepten-2-one 11.579 96 3.8 2.2 7.6 8.5 2.6 3.1 2.2 2.7 4.1 5.2 4.2 4.8
β-Myrcene 11.718 93 11.6 5.0 5.5
Hexanoic acid 11.800 97 7.8 5.6 3.7 1.6 2.7 6.0 7.4 9.6
Decane 12.111 97 1.5 1.5 0.8 1.4 0.7
α-Phellandrene 12.257 93 5.4 5.5 3.7
Octanal 12.299 92 1.3 2.9 2.4 1.3 0.7 4.7 4.5
D-Limonene 13.558 96 1.3 1.3 1.3 9.4 16.7 14.9 0.8 12.1 11.3 2.5 6.2
Eucalyptol 13.683 90 1.9
Benzyl alcohol 14.013 97 3.2 21.9 4.0
5-Ethyl-2-methyloctane 15.434 92 3.5
3,5-Octadien-2-one 16.365 93 1.5 1.7 2.7 4.2 1.8 2.4 9.1 3.8 5.9
Acetic acid octyl ester 17.952 90 3.9 3.5
6-Nonenal (Z) 18.263 90 4.2
Undecane 18.481 96 6.2 19.6 14.2 15.7 0.9 15.9 15.2 15.7
Nonanal 18.856 94 5.0 1.5 1.3 4.3 6.5 6.7 6.8 1.7 3.6 3.0 5.1 6.2 20.1 6.2
2,5-Dimethyl-2-undecene 19.800 90 2.1 1.6 1.8 1.5 1.5
Octanoic acid methyl ester 20.606 94 3.0 3.7
Pentyl benzene 22.995 98 1.7 11.5 8.7 6.5 10.8
Benzoic acid ethyl ester 24.319 95 3.5 1.9 20.0 16.3 12.4 17.2
Octanoic acid 25.781 96 7.8
Food Anal. Methods (2015) 8:1150–1163
Food Anal. Methods (2015) 8:1150–1163 1155

62.68
LT7
responsible for antibacterial activity of bee pollen (Van

2.1

1.2
3.3

1.3

5.1
1.5

0.7
Vuuren and Viljoen 2007).
Neither of the identified compounds was predominant in all

92.26
LT6

1.3
1.2

2.8
2.5
tested samples. However, it was found that dodecane, follow-
ed by tridecane and undecane, was dominant in 6 out of the 14

80.87
LT5

samples. These compounds composed of 61.4–79.0 % of both

1.4

2.7
3.0

0.9
samples from China, LV1 and LT1 to LT3 (Table 1).
There was not found any compound, which would be

81.06
10.8
LT4

2.1

1.4

5.1

1.2
0.6
characteristic and might be used as indicator for identifying
bee pollen of specific geographical location. However, 5-

147.29
ethyl-2-methyloctane and octanoic acid were detected in the
23.0
31.7
LT3

3.7

1.4
0.7

0.7

sample from Spain only, but as only one sample from this
region was analyzed, statistically significant and reliable con-
164.93
clusions about those compounds being specific geographical
26.6

19.5
LT2

2.5

7.0
1.5
0.7

origin indicators cannot be done.

It is known that the volatile composition of bee pollen
137.85
31.0

23.9
LT1

depends on its botanical origin (Campos et al. 2010). The

4.6

4.0
0.8

differences in quantitative and qualitative compositions of

the volatile compounds demonstrate the floral origin diversity
128.15
LV4

of the tested samples. However, geographical location, climat-

2.7

2.6

1.2
5.8

ic conditions, and bee species also play an important role in

121.04

the formation of bee pollen aroma profile.

LV3

2.7

1.5

5.0
3.6

94.62

Total Amount of Phenolic Compounds and Radical

LV2

2.4

7.3
1.4
6.0

Scavenging Activity
142.16
Relative percentage composition (%)

28.2

21.3
LV1

Total amount of phenolic compounds and radical scavenging

3.8
0.8

activity varied in the tested samples similar to the volatile

compounds. In order to compare the obtained results with
CHN2

96.90
28.1

21.6

the published data, total amount of phenolic compounds and

radical scavenging activity were expressed in equivalents of
176.51
CHN1

rutin (RE), gallic acid (GAE), and quercetin (QE), and total
The relative standard deviation did not exceed 6.5 %, with a few exceptions
34.6

24.7

0.5
1.5
0.3
1.2

0.7

amount of flavonoids was expressed in rutin (RE) and quer-

cetin (QE) equivalents as described above. The results are
158.40

listed in Table 2.
16.8

13.4
ESP

6.0
1.9

Total amount of phenolic compounds was in the range of

24.1–45.5 mg/g (RE), 14.2–26.8 mg/g (GAE), and 13.4–
Match, %

25.2 mg/g (QE). The highest content and the lowest content
were showed by samples LT6 and CHN2 from Lithuania and
96
95

96

97
97

97
96

97

China, respectively (Table 2). Our data is in accordance with

the studies of the bee pollen collected in Portugal performed
31.436
26.718

35.272

35.642
29.177

35.532
31.213

33.872

by Feás et al. (2012) and Morais et al. (2011). Total amount of

RT

phenols scattered from 12.9 to 19.8 mg/g (GAE) (Feás et al.

2-Methyl-5(1)-cyclohexen-1-one

2012) and from 10.5 to 16.8 mg/g (GAE) (Morais et al. 2011).
However, lower amounts of polyphenols were detected in the
Total GC peak area×106

bee pollen collected in Romania. They contributed 4.4–

Identified compounds

16.4 mg/g (GAE) (Mărghitaş et al. 2009). Two times higher

Table 1 (continued)

amount of the phenolic compounds was determined in the

1-Pentadecene

samples from South Brazil, 19.3 to 48.9 mg/g (GAE) (Carpes

1-Dodecanol

Pentadecane
Tetradecane
1-Tridecene
Dodecane

Tridecane

et al. 2009). According to Negri et al. (2011), the phenolic

compounds may directly contribute to the antioxidant action
due to their redox properties, which allow them to act as
1156 Food Anal. Methods (2015) 8:1150–1163

Fig. 1 GC-MS chromatograms

of SPME fraction isolated from
LT4, LV2, and CHN1 bee pollen
samples by PDMS fiber (1,
hexanal; 2, 1-hexanol; 3, 6--
methyl-5-hepten-2-one; 4, β-
myrcene; 5, limonene; 6, 3,5-
octadien-2-one; 7, undecane, 8,
nonanal; 9, pentyl benzene; 10,
benzoic acid ethyl ester; 11,
dodecane; 12, 2-methyl-5(1)-
cyclohexen-1-one; 13, tridecane;
14, 1-dodecanol; 15, 1-
pentadecene; 16, pentadecane)

Table 2 Total amount of phenolic compounds, flavonoid compounds, and radical scavenging activity of bee pollen extracts, expressed in equivalents of
rutin (RE), gallic acid (GAE), and quercetin (QE)

Sample Total phenolic amount (RSD≤6.50 %) Total flavonoid amount (RSD≤6.12 %) Antiradical activity in DPPH assay (RSD≤7.68 %)

RE (mg/g) GAE (mg/g) QE (mg/g) RE (mg/g) QE (mg/g) RE (mg/g) GAE (mg/g) QE (mg/g)

ESP 33.0 19.5 18.3 8.8 3.9 21.5 2.5 4.3

CHN1 43.9 25.9 24.3 11.6 5.2 39.2 5.4 8.4
CHN2 24.1 14.2 13.4 7.0 3.1 7.1 0.1 0.9
LV1 41.3 24.4 22.9 10.9 4.9 36.7 5.0 7.8
LV2 30.0 17.7 16.7 6.1 2.7 18.1 1.9 3.5
LV3 37.2 21.9 20.6 9.2 4.1 25.1 3.1 5.1
LV4 40.3 23.8 22.4 9.2 4.1 28.9 3.7 5.9
LT1 38.8 22.9 21.5 9.1 4.1 32.4 4.3 6.8
LT2 43.2 25.5 24.0 11.4 5.1 31.2 4.1 6.5
LT3 43.2 25.5 24.0 11.3 5.0 37.2 5.1 7.9
LT4 41.9 24.7 23.3 7.2 3.2 30.8 4.0 6.4
LT5 40.2 23.7 22.3 6.8 3.0 27.5 3.5 5.7
LT6 45.5 26.8 25.2 11.3 5.0 35.7 4.8 7.5
LT7 38.2 22.6 21.2 6.1 2.7 28.1 3.6 5.8

RSD relative standard deviation of five analyses

Food Anal. Methods (2015) 8:1150–1163
reducing agents, hydrogen donors, and singlet oxygen
quenchers.
Total amount of the flavonoids was from 3.4 to 6.3 and
from 4.2 to 7.8 times lower than the phenolic amount
expressed in RE and QE, respectively (Table 2). Mărghitaş
et al. (2009) determined 0.6–13.6 mg/g (QE) of the flavonoids
in the Romanian bee pollen study, while in present study, the
content distributed from 2.7 to 5.2 mg/g (QE). The Brazilian
bee pollen contained 2.1–28.3 mg/g (QE) of flavonoids
(Carpes et al. 2009).
Radical scavenging activity of the bee pollen extracts was
evaluated using free DPPH radical. In the DPPH assay, anti-
oxidants react with a nitrogen-centered radical (of 2,2-
diphenyl-1-picrylhydrazyl molecule), having a characteristic
absorption at 515 nm, which is converted into 1,1,-diphenyl-
2-picryl hydrazine at a very rapid rate during the reaction.
Radical scavenging activity of the tested samples spread
widely, i.e., 7.1–39.2 mg/g (RE), 0.1–5.4 mg/g (GAE), and
0.9–8.4 mg/g (QE) (Table 2). It is difficult to compare the
obtained data with other studies due to the different units used
for the expression of the results. Nonetheless, literature data
also show the variation of radical scavenging activity of the
bee pollen depending on their floral source. The extract con-
centration providing 50 % of DPPH inhibition (half maximal
inhibitory concentration (IC50)) was used for the evaluation of
the bee pollen from the Portuguese Natural Parks (Morais
et al. 2011); IC50 varied from 2.16 to 5.87 mg/ml. IC50 value
was in range of 0.015–0.145 mg/ml of Sonorar Desert bee
pollen, radical scavenging activity in the tested samples dis-
tributing in the following order: mimosa < chenopod < mes-
quite < terpentine bush < yucca < palm (LeBlanc et al. 2009).
Leja et al. (2007) evaluated radical scavenging activity of the
bee pollen of 12 plant species and classified the pollen species
into three groups according to their ability to inhibit DPPH
radical: Lupinus polyphyllus, Phacelia tanacetifolia, Trifolium
sp., Sinapis alba, Robinia pseudoacacia, and Aesculus
hippocastanum possessed the high ability of DPPH inhibition,
61.0–91.3 %; Zea mays, Chamerion angustifolium, and Pyrus
communis showed medium activity, 23.5–29.6 %, while
Lamium purpureum, Taraxacum officinale, and Malus
domestica had low activity, 8.6–16.0 %. Mărghitaş et al.
(2009) determined the difference between the DPPH radical
scavenging activities up to 20 times among 12 samples of the
monofloral bee pollen; the values were ranging from 0.135 to
2.814 mmol Trolox/g.
A strong correlation, 0.95, was observed between total
radical scavenging activity and total amount of phenolic com-
pounds, and lower correlation of 0.68 was determined be-
tween radical scavenging activity and total amount of
flavonoids.
It can be concluded from the results that bee pollen is a
source of antioxidants. The content of polyphenols in bee
pollen is higher than in many fruits or vegetables; for example,
1157
plums contain 3.69 mg/g (GAE) of polyphenols, straw-
berries—2.25 mg/g (GAE), apples—1.18 mg/g (GAE), aspar-
agus—0.64 mg/g (GAE), garlic—0.48 mg/g (GAE), toma-
toes—0.24 mg/g (GAE), and mushrooms—0.11 mg/g (GAE)
(Chun et al. 2005). The amount of phenolic compounds in
pollen is comparable to Andean blackberry, 21.67 mg/g
(GAE), capulí cherry peel, 14.94 mg/g (GAE), and banana
passion fruit, 10.10 mg/g (GAE) (Vasco et al. 2008). Howev-
er, it should be kept in mind that bee pollen cannot be used in
such amounts like fruits or vegetables due to the possible
allergic reaction to some people.
HPLC-ECD Analysis
The chromatographic system with the electrochemical
detector (HPLC-ECD system) was used for a reversed-
phase liquid chromatographic evaluation of the phenolic
compounds present in methanolic extracts of the bee
pollen samples, which revealed electrochemically active
compounds in the extracts. Polyphenolic compounds are
reducing agents, and their electrochemical responses are
due to the donation of the electrons. The chromato-
graphic profile of HPLC analysis of some bee pollen
extracts is presented in Fig. 2.
Eight phenolic acids, namely, gallic, chlorogenic,
caffeic, ferulic, syringic, synapic, 3,4-dihydrobenzoic
and 2-hydroxycinnamic, and three flavonoids, i.e., rutin,
naringenin, and quercetin, were used for identification
and quantification of the separated compounds in the
bee pollen extracts. Only 7 out of the 11 compounds
were detected in the tested samples, and only 2-
hydroxycinnamic acid, rutin, and quercetin were detect-
ed in all extracts. Naringenin was not identified in one
sample only. Gallic and caffeic acids were found in
eight samples, while ferulic acid was detected in two
samples (Table 3).
Various phenolic compounds were identified in the bee
pollen samples of the different floral and geographical origin
in the published data. Caffeic acid, kaempherol, chrysin,
pinocembrin, galangin, quercetin, and isorhamnetin were de-
tected in the hydrolyzed and/or non-hydrolyzed bee pollen
samples from Croatia (Šarić et al. 2009). Isoquercetin,
myricetin, tricetin, quercetin, luteolin, selagin, kaempferol,
and isorhamnetin were found in the pollen collected in Bahia,
Brazil (Freire et al. 2012). p-Coumaric acid, ferulic acid, o-
coumaric acid, myricetin, quercetin, cinnamic acid,
naringenin, hesperetin, and kaempferol were present in the
bee pollen sample collected in Crete, Greece (Fanali et al.
2013). Another study showed that the Greek pollen accumu-
l a t e d k ae m p h er o l , q ue r c e t i n, is o r h a m n et i n , an d
methylherbacetin glycosides (Graikou et al. 2011). The main
compounds identified in the Spanish bee pollen were rutin,
quercetin, myricetin, and trans-cinnamic acid (Bonvehí et al.
1158 Food Anal. Methods (2015) 8:1150–1163

Fig. 2 Chromatographic profile

of some tested bee pollen extracts
obtained using HPLC with an
electrochemical detector at 300,
500, and 700-mV potentials (1,
gallic acid; 2, caffeic acid; 3,
rutin; 4, ferulic acid; 5, 2-
hydroxycinnamic acid; 6,
quercetin; 7, naringenin)

2001). According to Carpes et al. (2013), the presence of rutin Quantitative analysis showed that rutin and quercetin were
in bee pollen indicates the biological and nutritional quality predominant in the tested samples, except CHN1, where the
due to its high antioxidant activity. amount of 2-hydroxycinnamic acid exceeds the amount of

Table 3 The amounts (μg/g) of

detected phenolic acids and fla- Sample Gallic Caffeic Ferulic 2-Hydroxycinnamic Rutin Naringenin Quercetin
vonoids in the bee pollen samples acid acid acid acid

ESP 15.4 17.8 68.6 179.6 955.7 28.6 215.3

CHN1 10.7 20.6 – 54.8 286.1 29.6 24.0
CHN2 3.9 13.6 – 47.0 242.3 21.7 125.6
LV1 12.5 8.5 14.6 179.9 610.0 43.6 357.8
LV2 – – – 73.4 374.6 3.1 529.8
LV3 – – – 49.9 194.0 118.0 167.1
LV4 – – – 44.1 349.3 78.3 217.2
LT1 11.2 10.6 – 75.1 428.9 27.7 127.1
LT2 32.3 14.2 – 56.1 316.9 6.4 205.9
LT3 – – – 43.4 255.2 11.7 78.8
LT4 3.6 11.2 – 45.2 241.3 16.1 311.4
LT5 3.0 – – 63.3 227.4 41.4 129.2
LT6 – 9.3 – 68.9 156.2 6.3 463.9
LT7 – – – 46.4 285.1 – 225.4
RSD relative standard deviation RSD (%) 2.6–6.7 1.6–5.0 3.9–5.7 1.8–6.8 2.0–6.9 1.0–6.6 3.0–7.1
of three analyses
Food Anal. Methods (2015) 8:1150–1163
quercetin. Total amount of rutin in the tested samples varied
between 156.2 and 955.7 μg/g, and the lowest and the highest
values were detected in LT6 and ESP samples, respectively.
Similar amounts of rutin were detected in some bee pollen
samples from Brazil (Carpes et al. 2013). The amount of
quercetin ranged from 78.8 to 529.8 μg/g in all samples,
except CHN1, which contained 24.0 μg/g of quercetin only.
The highest amount of quercetin was determined in the sam-
ple LV2 from Latvia. Not only did the Spanish sample ESP
and sample from Latvia LV1 contained the highest amount of
2-hydroxycinnamic acid, 179.6 and 179.9 μg/g, respectively,
while the values of this acid in other tested samples ranged
between 43.4 and 75.1 μg/g, but also they distinguished
themselves by the ferulic acid, 68.6 and 14.6 μg/g, respective-
ly, which was not found in the rest of the samples (Table 3).
Lower amount of 2-hydroxycinnamic acid and high amount of
ferulic acid were reported in Crete bee pollen, 36.7 and
149.1 μg/g, respectively (Fanali et al. 2013). The lowest and
the highest amount of naringenin were determined in samples
LV2 and LV3 from Latvia, 3.1 and 118.0 μg/g, respectively.
The amounts of gallic and caffeic acids in all analyzed sam-
ples were remarkably lower than the amount of 2-
hydroxycinnamic acid and distributed from 3.0 to 32.3 μg/g
and from 8.5 to 20.6 μg/g, respectively (Table 3).
In summary, it should be noted that similarly to the analysis
results of the volatiles, the quantitative analysis of bee pollen
phenolic compounds did not reveal a possibility of sample
attribution to specific geographical location according to the
identified compounds unambiguously. The botanical origin,
geographical bee pollen collection area, climatic conditions
during the collection season, and bee species are the factors
which can be responsible for the differences in qualitative and
quantitative composition of the bee pollen phenolic
compounds.
Chemometric Analysis
Chemometric analysis of the volatile compound composition
of the tested bee pollen samples was started with data matrix
building. It is very important to have a representative and large
data set to get reliable statistical analysis results. As the
number of real measurements of the samples was relatively
small, the well-known Monte Carlo method was applied to
artificially and randomly generate data points. According to
the real measurement results, there was normal (known as
Gaussian) distribution assumed having two parameters μvi
and σvi, where μvi is the mean and σvi is the standard deviation
of the ith variable, representing identified volatile compound
(Table 1), respectively, which were calculated from the real
data and i is the variable index from 1 to 42. To avoid
presenting individually the standard deviation for every vari-
able in the calculations, a standard deviation input of the
volatile compound composition measurements was chosen
1159
as the largest standard deviation value of all compounds,
which, expressed in relative form, equaled to 9.13 %. After
applying Monte Carlo method with a set of the parameters
(μvi, 9.13 %, 100), where 100 represents a chosen number of
data points being modeled, there was an initial data matrix of
size of [1,400×42] formed, corresponding to 14 analyzed bee
pollen samples and 42 identified volatile compounds.
The filtration procedure to reject the compounds contrib-
uting less than 1 % was completed on the formed initial data
array which resulted in building of a new data matrix of size
[1,400×29]. After this data pretreatment, the standardization
by subtracting the means and dividing to the standard devia-
tions followed.
As the initial variable space was composed of 29 dimen-
sions, which were possibly correlated (not orthogonal), a
technique to extract a set of linearly non-correlated variables
called principal components was employed. Although the
space of the principal components has the same size, the
elimination of the correlation allows retaining only those
components that have the highest variances. This rejection
procedure shrinks considerably the newly composed orthog-
onal principal components’ space. The number of PCA was
selected considering several statistical criteria (Cattell’s test,
Kaiser’s test, eigenvectors, etc.), which led to keep only the
first eight components, explaining 92.89 % of the total vari-
ance. Thus, 29 initial variables representing the volatile profile
were reduced to eight non-correlated principal components,
and a new data matrix of [1,400×8] was built for the succes-
sive analyses.
The data matrix corresponding to antioxidant activity of the
analyzed bee pollen samples was prepared for clustering anal-
ysis in a similar way. Again, the Monte Carlo method, assum-
ing a normal distribution function, with the following param-
eter set (μaj, 7.68 %, 100), where μaj is the mean of the jth
variable, representing antioxidant properties, namely, total
amount of phenolic compounds, total amount of flavonoids,
and DPPH radical scavenging activity (Table 2), j is the
variable index from 1 to 3, 7.68 % is the largest relative
standard deviation observed among the data of the antioxidant
properties, and 100 is the chosen number of data points being
generated, was applied. After the data standardization proce-
dure, PCA followed in order to eliminate correlation between
original data. All three computed principal components,
explaining 100 % of the total variance, were retained. The
initial data matrix of size [1,400×3] was prepared for further
statistical mining, which corresponded to 14 analyzed bee
pollen samples and 3 non-correlated principal components.
Analogous procedure of data matrix formation to those
described above was run on the phenolic acid and flavonoid
composition representing data. The Monte Carlo method with
assumed normal distribution function with parameter set (μpl,
7.12 %, 100), where μpl is the mean of the lth variable,
corresponding to determined phenolic acids and flavonoids
1160 Food Anal. Methods (2015) 8:1150–1163

(Table 3), l is the variable index from 1 to 7, 7.12 % is the

largest relative standard deviation observed among the data,
and 100 is the chosen number of data points being generated,
was applied. The built initial data matrix had dimensions
[1,400×7] corresponding to 14 analyzed bee pollen samples
and 7 phenolic acids and flavonoids. After the data standard-
ization procedure, PCA was performed. Applied principal
component rejection criteria allowed to retain four principal
components having the largest variances and explaining
91.58 % of total variance. The initial data matrix of phenolic
acid and flavonoid composition data was shrunk to size of
[1,400×4].
There were several techniques, HCA, and KMCA and
linear DA, used for the classification of the analyzed samples
into groups. HCA helped to classify the samples according to
the evaluated similarity measure (reciprocal to distance) and
calculated linkage. The distances and linkages were calculated
for the previously formed data matrices using the following
functions and rules: Spearman’s distance function and weight-
ed average distance linkage rule applied for the volatile com-
pound data [1,400×8], Euclidean distance function and fur-
thest neighbor linkage rule, described as the largest distance
between objects in the two clusters, applied for phenolic acids
and flavonoids data [1,400×4], and Spearman’s distance func-
tion and weighted average distance linkage rule applied the
antioxidant activity data [1,400×3]. The above-described
functions and rules for HCA were chosen according to calcu-
lated cophenetic correlation coefficient between calculated
distances and linkages. The cophenetic correlation coeffi-
cients were 0.93, 0.93, and 0.73 for volatile data, antioxidant
properties data, and phenolic acids and flavonoids data, re- Fig. 3 Hierarchical cluster analysis dendrograms of the tested bee pollen
samples. a Volatile compound composition data. b Antioxidant activity
spectively. The results of HCA are presented as dendrograms data. c Phenolic acid and flavonoid composition data
in Fig. 3. The dendrograms’ trees are cut at the level of 70 % of
maximum calculated distance to build clusters, as the samples
could be considered too dissimilar beyond that point. Looking samples LT1 and LT2 and Latvian sample LV1; and two non-
at the dendrograms, it could be concluded that the dependence Baltic region groups, represented by the Spanish sample ESP
of clustering results on geographical origin was not clearly together with LV1 and Chinese samples CHN1 and CHN2
exposed as expected a priori. According to the volatiles including LT1 and LT2, respectively.
(Fig. 3a), the samples clustered into three groups: the first Since HCA exposes similarities between samples
being composed of the four Lithuanian samples LT4 to LT7, linking them to clusters according to the mean values
the second group consisted of the three Latvian samples LV2 and does not take into account variances, another classi-
to LV4, and the third group included all remaining samples. fication method, K-means clustering, based on iterative
Thus, the results implied the influence of the pollen collection computation, was employed to do the classification in
location or floral source. Nevertheless, this tendency was not the predictors’ space. This tool, however, requires spec-
repeated in the dendrogram of antioxidant properties data ifying a number of the initial clusters, which can be
(Fig. 3b). All the samples were classified into two groups, reduced by the algorithm if empty cluster is created
and no information about geographical origin could be ex- during computation. This number was chosen in accor-
tracted. On the contrary, the results of phenolic acids and dance with the results of the HCA, i.e., 3, 2, and 3,
flavonoids classification (Fig. 3c) revealed the undoubted respectively. To find the global classification solution,
impact of the geographical region of the pollen collection. In i.e., to minimize the sum, over all clusters, of the
this case large Lithuanian – Latvian (i.e. Baltic Sea region) bee within-cluster sums of point-to-cluster-centroid distances,
pollen group can be distinguished, which is composed of all KMCA was replicated 200 times (for each classification
Lithuanian and Latvian analyzed samples, except Lithuanian task) retrieving the best result.
Food Anal. Methods (2015) 8:1150–1163 1161

Together with KMCA, linear DA was performed to esti-

mate the parameters of discriminant functions of the predictor
variables, which determine the boundaries between built clus-
ters in the predictors’ space. The results of the accomplished
KMCA and linear DA are presented in Fig. 4. The scatter plots
are presented in the space of the principal components,
PCv 1–PCv 2–PCv 3, in the case of the volatile compound
data (Fig. 4a), in the space of the principal components,
PCa 1–PCa 2–PCa 3, in the case of the antioxidant activity
data (Fig. 4b), and in the space in principal components,
PCp 1–PCp 2–PCp 3, in the case of the phenolic acid and
flavonoid profile data (Fig. 4c). The boundaries between
classes are planes and expressed in the following form of
linear equations:

0 ¼ g 0 þ g1 x þ g2 y þ g3 z; ð4Þ

where gk are the coefficients of the discriminant functions,

k is the index of the equation’s coefficients, k=0…3, and x, y,
and z are the predictors.
The results of KMCA together with DA shed some light on
the HCA classification. It was confirmed by statistically sig-
nificant clustering results that the samples form three groups
in the case of volatile data. However, this technique exposed
overlap of the data points between formed groups when the
data representing antioxidant activity were under classifica-
tion: The most of the samples were very similar from this data
point of view and did not expose significant difference.
Though some samples, ESP, LV2, and CHN2, clearly
corresponded to the first class, while other samples, CHN1,
LV1, LT2, LT3, and LT6 obviously fell to the second class, the
samples LV3, LV4, LT1, LT4, LT5, and LT7 were classified
on the boundary between built classes and the data points of
these samples scattered between them. The idea to classify the
data into three classes, expecting the samples “in the middle”
to form separate third cluster, did not give stable classification
results.
Comparing the HCA and KMCA technique classification
results that exhibited possible relationship to bee pollen geo-
graphical origin (Figs. 3a, c and 4a, c), the following conclu-
sions could be made: Lithuanian samples LT4 to LT7 and
Latvian samples LV2 to LV4 separated themselves from the
rest and could be attributed to the Lithuanian and Latvian bee
pollen group, respectively. It was confirmed by the classifica-
Fig. 4 K-means clustering analysis scatter plots together with segmen-
tion results of the mentioned samples according to the volatile tation of the predictors’ space computed using linear discriminant analy-
profile data. Moreover, the KMCA exposed that this grouping sis. a Volatile compound composition data. b Antioxidant activity data. c
tendency could not be rejected, analyzing the phenolic acid Phenolic acid and flavonoid composition data
and flavonoid profile data, despite the fact that these samples
fell into the same cluster. This could be explained by the
relative closeness of bee pollen collection locations with sim- samples in this group. This hypothesis was tested by trying to
ilar available floral sources. However, the Fig. 4c shows that classify the data into four groups (instead of three) using
all Latvian samples are concentrated apart from the remaining KMCA: the first two groups remained unchanged, while all
1162
Latvian samples were separated from the third group to new
cluster.
Conclusions
Forty-two different volatile compounds were identified in
tested 14 samples of bee pollen from different parts of the
Baltic Sea region, including two samples from China and a
sample from Spain for comparison. By GC-MS analysis,
nonanal, 1.5–20.1 %, dodecane, 1.2–34.6 %, and tridecane,
1.4–24.7 %, were detected in all 14 samples, while hexanal,
1.7–12.9 %, was identified in 13 samples, 6-methyl-5-hepten-
2-one, 2.2–8.5 %, was detected in 12 samples, and 2-
methylbutanoic acid, 3.0–10.7 %, and limonene, 0.8–
16.7 %, were found in 11 samples.
HPLC with electrochemical detection revealed the pres-
ence of 2-hydroxycinnamic acid, 43.4–179.9 μg/g, rutin,
156.2–955.7 μg/g, and quercetin, 24.0–529.8 μg/g, in all
tested samples. Naringenin, 3.1-118.0 μg/g, was identified in
13 out of the 14 samples. Gallic, caffeic, and ferulic acids were
detected in some samples as well. The study showed that the
bee pollen extracts exhibit radical scavenging activity, which
strongly correlates with the total amount of phenolic com-
pounds (the correlation coefficient was 0.95).
The qualitative and quantitative composition of the identi-
fied volatile and non-volatile compounds together with the
radical scavenging activity and total amount of phenolic com-
pounds and total amount of flavonoids varied depending on
the geographical collection location, likely due to the different
botanical compositions.
Performed HCA showed that the tested samples can be
grouped into three according to the volatile compound com-
position data, two groups in respect of antioxidant activity,
described by DPPH radical scavenging, total amount of phe-
nolic compounds, and total amount of flavonoids data, and
three groups corresponding to the phenolic acid and flavonoid
composition data. Another applied classification technique,
KMCA, confirmed the classification of the samples obtained
with the HCA. Despite the classification method used, the
results showed that not only the samples formed different
number of clusters, but also the observed dependence of the
samples data on the geographical origin of the pollen collec-
tion was not clearly exposed in all cases. However, the KMCA
revealed that the impact of the pollen collection region on the
classification of the pollen in regard to the volatile profile and
phenolic acid and flavonoid composition was observed. It
could be concluded that the analyzed samples formed Lithu-
anian, Latvian, and non-Baltic region groups.
The results of the linear DA performed to determine the
boundaries between clusters, built by KMCA using volatile
compound profile, antioxidant properties data, and phenolic
Food Anal. Methods (2015) 8:1150–1163
acid and flavonoid composition, respectively, allowed
segmenting the predictors’ space according to the number of
the clusters. This technique revealed the overlap of the data
points between built clusters classifying the samples accord-
ing to phenolic acid and flavonoid content and antioxidant
activity data. The boundary planes, separating different clus-
ters and expressed in the form of linear equations, could be
used for similarity analysis and prediction of the properties of
new bee pollen samples from the Baltic region.
Acknowledgments Authors would like to thank Apiproduktai, Ltd.,
for providing of the bee pollen samples.
Author Contributions V. Kaškonienė was responsible for the experi-
ments, result interpretation, and preparation of the paper concerning
chemical part. G. Ruočkuvienė performed SPME-GC-MS and spectro-
photometric and HPLC analyses. P. Kaškonas performed statistical min-
ing, interpreted the classification results, and prepared the paper part
about chemometric analysis. I. Akuneca helped in qualitative and quan-
titative analysis with HPLC. A. Maruška helped in experimental design
and interpretation of the results. All authors contributed equally.
Conflict of Interest Vilma Kaškonienė declares that she has no conflict
of interest. Geralda Ruočkuvienė declares that she has no conflict of
interest. Paulius Kaškonas declares that he has no conflict of interest.
Ieva Akuneca declares that she has no conflict of interest. Audrius
Maruška declares that he has no conflict of interest. This article does
not contain any studies with human or animal subjects.
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