Antioxidative properties of bee pollen in selected plant species
, A. Mareczek
, J. Klepacz-Baniak
, K. Czekon
Department of Plant Physiology, Faculty of Horticulture, Agricultural University, 29 Listopada 54, 31-425 Krako
Department of Pomology and Apiculture, Faculty of Horticulture, Agricultural University, 29 Listopada 54, 31-425 Krako
Received 28 March 2005; received in revised form 20 September 2005; accepted 20 September 2005
Phenolic constituents (total phenols, phenylpropanoids, ﬂavonols and anthocyanins) and antioxidant ability were determined in bee
pollen of 12 plant species. Antioxidant ability was measured as total antioxidant activity, radical-scavenging activity and activity against
free hydroxyl radical. Great variability of phenolic contents was observed in the pollen of investigated species. Total antioxidant activity
diﬀered considerably (0.8–86.4% inhibition of lipid peroxidation), however, in most of the examined pollens, it was high and corre-
sponded with the phenylpropanoid level.
Great diﬀerences in the radical-scavenging activity (8.6–91.5% of DPPH neutralization) and in the hydroxyl radical-scavenging activ-
ity (10.5–98% inhibition of deoxyribose degradation) were observed and were not correlated with the content of phenolic compounds.
In most of the investigated plant species, antioxidative capacity of bee pollen was very high.
Ó2005 Elsevier Ltd. All rights reserved.
Keywords: Pollen; Antioxidant activity; Phenolics
Recently, many investigations have been concerned with
antioxidant properties of diﬀerent nutritional products.
Antioxidant ability has usually been attributed to the activ-
ity of antioxidant enzymes (mainly superoxide dismutase,
peroxidase, catalase) as well as to the content of low-molec-
ular antioxidants such as carotenoids, tocopherols, ascor-
bic acid, phenolic substances (Bartosz, 1997; Larson,
1988). The best source of antioxidative compounds are,
undoubtedly, fruits and vegetables, recommended as health
promoting components of the human diet. Their antioxida-
tive capacity, in many cases, results from phenolic com-
pounds (Larson, 1988). The high ability of phenolic
constituents to neutralize the active oxygen species is
strongly associated with their structure, such as the conju-
gated double bonds and the number of hydroxyl groups in
the aromatic ring, mostly attributed to ﬂavonoids and cin-
namic acid derivatives (Foti, Piattelli, Baratta, & Ruberto,
1996; Natella, Nardini, Di Felice, & Saccini, 1999; Silva
et al., 2000). Interdependence between the level of diﬀerent
phenolics and antioxidant ability of fruits, vegetables and
medical plants has been studied for many years (Pietta,
Simonetti, & Mauri, 1998; Robards, Prenzler, Tucker,
Swatsitang, & Glover, 1999; Velioglu, Mazza, Gao, &
Oomah, 1998; Vinson, Hao, Su, & Zubik, 1998). Less often
the honey bee products, particularly rich in ﬂavonoids,
have been the subject of research (Nagai, Sakai, Inoue, &
Suzuki, 2001). Among them, special attention should be
paid to the ﬂoral pollen used for many years as a beneﬁcial
dietary supplement (http://www.ccpollen.com/ORAC.
The purpose of the present studies was to determine the
antioxidant activity of pollen loads of various origin in
comparison with the level of phenolic substances (total,
phenylpropanoids, ﬂavonols and anthocyanins). The
0308-8146/$ - see front matter Ó2005 Elsevier Ltd. All rights reserved.
Abbreviations: DPPH, 1,1-diphenyl-2-picrylhydrazyl; RSA, radical-s-
cavenging activity; HRSA, hydroxyl radical-scavenging activity; TAA, t-
otal antioxidant activity.
Corresponding author. Tel.: +48 12 662 52 07; fax: +48 12 662 52 66.
E-mail address: firstname.lastname@example.org (M. Leja).
Food Chemistry 100 (2007) 237–240
antioxidative properties of pollen were measured as the
radical-scavenging activity, as the inhibition of lipid perox-
idation and as the antiradical activity against free hydroxyl
radical. The simultaneous application of these three meth-
ods allows a more detailed description of the antioxidative
system of pollen.
2. Material and methods
The pollen loads were collected in 2004, in the Krako
area, from the beginning of May to the end of July by 20
honey bee colonies (Apis mellifera) settled in hives with
bottom-ﬁtted pollen traps. The fresh bee pollen was stored
at 18 °C until analysed.
The colour of the pollen was estimated according to the
tables elaborated by Hodges (1984) and Kirk (1994). Addi-
tionally, pollen loads were identiﬁed by colour and micro-
scope observations of pollen grains (Warakomska, 1962)
and compared with the pollen grains from the previously
collected ﬂowers. To describe the proper taxon, the special
key for pollen grains estimation was used (Faegri & Iver-
sen, 1978). The pollen loads of the 12 following plant spe-
cies were taken for analysis:
Aesculus hippocastanum,Chamerion angustifolium,
Lamium purpureum,Lupinus polyphyllus,Malus domestica,
Phacelia tanacetifolia,Pyrus communis,Robinia pseudoaca-
cia,Sinapis alba,Taraxacum oﬃcinale,Trifolium sp. and
For the estimation of phenolic constituents, extracts in
80% methanol were prepared. Total phenols, phenylpropa-
noids, ﬂavonols and anthocyanins were determined by the
spectrophotometric method given by Fukumoto and
Mazza (2000). Reaction mixture consisted of 0.25 cm
pollen extract with 0.25 cm
of 0.1% HCl in 96% ethanol
and 4.55 cm
of 2% HCl. The absorbance was measured
at 280 nm (chlorogenic acid as standard), 320 nm (caﬀeic
acid as standard) and 360 nm (quercetin as standard) for
total phenols, phenylpropanoids and ﬂavonols, respec-
tively. Absorbance of anthocyanin content was read at
520 nm and expressed as cyanidin, according to its molar
extinction. Radical-scavenging activity (RSA) was deter-
mined using DPPH as the stable radical and was expressed
as the percentage of its neutralization after 30 min (Pekk-
arinen, Stoeckmann, Schwarz, Heinonen, & Hopia,
1999). The ethanolic solution of DPPH (0.1 mM) was used
as a point of reference for monitoring the decrease of its
absorbance at 516 nm after addition of pollen extract.
The control was prepared with 80% methanol instead of
pollen extract. Pollen concentration in the reaction mixture
was 0.083%. Total antioxidant activity (TAA) was deter-
mined by measurements of the inhibition of linoleic acid
(LA) peroxidation, as described by Toivonen and Sweeney
(1998). The products of peroxidation of exogenous LA, ini-
tiated by ferrous-EDTA, were measured spectrophotomet-
rically at 232 nm in the solution of pollen and control. A
control consisted of the above reaction mixture with 80%
methanol in place of the pollen extract. Pollen concentra-
tion in the reaction mixture was 0.167%. TAA was
expressed as percentage inhibition of LA peroxidation by
pollen extract in comparison to the oxidation level in the
control. Hydroxyl radical-scavenging activity (HRSA) of
pollen extract, based on the inhibition of the degradation
of deoxyribose caused by the attack of hydroxyl radicals,
was evaluated using the method described by Racchi
et al. (2002). The absorbance of the reaction mixture was
read in a spectrophotometer at 532 nm against a solution
prepared without ascorbic acid. The activity was expressed
as the percentage of reaction inhibition. Pollen concentra-
tion in the reaction mixture was 0.1%.
All analyses were done in four replications (four
extracts) and the results were statistically evaluated using
DuncanÕs test for signiﬁcance P< 0.05.
3.1. Antioxidant activity
Total antioxidant activity, expressed as the percentage
of inhibition of lipid peroxidation, diﬀered considerably
in respect to the pollen species; however, most of them
(P. communis,M. domestica,T. oﬃcinale,A. hippocasta-
num,R. pseudoacacia,P. tanacetifolia and S. alba) exceeded
60% (60–90%). The medium ability of inhibition of lipid
peroxidation was associated with four species: C. angustifo-
lium,L. polyphyllus,L. purpureum and Trifolium sp. (27–
55%), while low ability was found in the Z. mays pollen
(7%) (Table 1).
Great discrepancies between radical-scavenging activity
in diﬀerent pollen species were observed. According to
the results, presented in Table 1, the pollen species can be
divided into three groups: those of high ability of DPPH
neutralization (61–91.3%, L. polyphyllus,P. tanacetifolia,
Trifolium sp., S. alba,R. pseudoacacia and A. hippocasta-
num), those of medium RSA (23.5–29.6%, Z. mays,C.
angustifolium and P. communis), and those of low RSA
(8.6–16%, L. purpureum,T. oﬃcinale and M. domestica).
According to the results obtained, hydroxyl radical-
scavenging activity was above 60% (61–98%) in eight cases
(S. alba,Trifolium sp., C. angustifolium,P. tanacetifolia,Z.
mays,L. purpureum,M. domestica and P. communis), med-
ium (15.8–24.7%) in two species (R. pseudoacacia and L.
polyphyllus), and relatively low (10.5%) in the pollen of
A. hippocastanum (Table 1).
3.2. Phenolic constituents
Great variability of phenolic content was observed in
the investigated species of pollen (Table 1). The highest
and the lowest levels of total phenols were found in pollens
from P. communis and Z. mays, respectively. Similarly, the
highest content of phenylpropanoids was observed in P.
communis pollen, followed by P. tanacetifolia, while the
lowest level was determined in Z. mays. The participation
of phenylpropanoids in total phenolics seems to be similar
238 M. Leja et al. / Food Chemistry 100 (2007) 237–240
in most of the examined pollen samples (25% on average)
excepting S. alba (38.3%).
Flavonol content showed discrepancies in the examined
pollen samples and ranged between 170 (L. purpureum) and
1349 mg 100 g
(P. communis pollen). The participation of
ﬂavonols in total phenolics diﬀered considerably, depend-
ing on the pollen species, from 4.78% (L. purpureum)to
37.3% (C. angustifolium).
The content of anthocyanins was relatively low in com-
parison with the other phenolic compounds and ranged
between 92 (Trifolium sp. and Z. mays) and 327 mg 100 g
(P. tanacetifolia). The percentage of anthocyanins in total
phenols varied in the species from 3% (L. purpureum and
P. communis)to13%(Trifolium sp.).
Variability of total antioxidant activity in the investi-
gated species seems to correspond to their phenylpropa-
noid contents (Fig. 1), being manifested by the signiﬁcant
positive correlation coeﬃcient (R
The honey bee products are considered to be abundant
sources of antioxidants. In honey, royal jelly and propolis
high antioxidant activity, expressed as the inhibition of
lipid peroxidation was found (Nagai et al., 2001). In prop-
olis water extracts high radical-scavenging activity, activity
against superoxide anion and hydroxyl radical-scavenging
activity were reported (Nagai, Inoue, Inoue, & Suzuki,
2003). Studies of Campos et al. (2000), and of Campos,
Webby, and Markham (2002), concerning antioxidant
properties of bee pollen as well as the recent reports
(http://www.ccpollen.com/ORAC.shtml), conﬁrm a very
high antiradical activity of this product. Antioxidative abil-
ity of pollen seems to be due to phenolic compounds. In
ﬂoral pollen mostly ﬂavonoids, their glycosides and deriv-
atives of cinnamic acid are present (Markham & Campos,
1996). Apart from common ﬂavonoids (quercetin, kaempf-
erol, luteolin and their derivatives), speciﬁc ﬂavonoid gly-
cosides, characteristic of some ﬂoral pollen, such as 7-a-
8-O-methylherbacetin-3-O-sophorosides (Markham &
Campos, 1996) or found in the Myrtaceae family aglycone
triacin (Campos et al., 2002) were determined.
High levels of phenolic constituents are often accompa-
nied by high antioxidative capacity of pollen; however,
according to reports of Campos, Webby, Markham, Mitc-
hall, and Cunha (2003), and Campos et al. (2000), no direct
correlation between ﬂavonoids and radical-scavenging
activity was found. The gradual decrease of RSA in the
pollen stored for 4 years was not accompanied by a parallel
reduction of ﬂavonoids (Campos et al., 2003) and some
pollens with high levels of phenolics did not present signif-
icant antiradical activity (Campos et al., 2000).
In the present investigations, great variability regarding
content of total phenols, phenylpropanoids, ﬂavonols and
antioxidant capacity in 12 examined pollens was found.
In some of them (P. tanacetifolia and S. alba), a very high
antioxidant activity, expressed as radical-scavenging activ-
ity, inhibition of lipid peroxidation and hydroxyl radical-
scavenging activity, corresponded to high levels of total
phenols, phenylpropanoids and ﬂavonols.
In pollen of R. pseudoacacia and A. hippocastanum, high
and medium levels, respectively, of phenolic compounds
(total, phenylpropanoids and ﬂavonols) were manifested
by high TAA (84.4% and 82%) and high RSA (91% and
91.3%), while the HRSA was low (15.8% and 10.5%). In
Antioxidative properties of selected bee pollens
Pollen species TAA (%) RSA (%) HRSA (%) Total phenols
(mg 100 g
(mg 100 g
(mg 100 g
(mg 100 g
Sinapis alba 86.4 g
90.0 h 61.0 d 3924 e 1503 e 914 e 236 ef
Phacelia tanacetifolia 85.9 g 66.3 e 73.5 ef 8025 i 2243 g 815 e 327 g
Robinia pseudoacacia 84.4 g 91.0 h 15.8 ab 6178 g 1875 f 1068 f 251 f
Aesculus hippocastanum 81.9 fg 91.3 h 10.5 a 3375 d 1159 d 624 cd 183 c
Taraxacum oﬃcinale 77.3 f 15.2 b 50.7 c 6307 g 1496 e 503 bc 233 ef
Malus domestica 76.5 f 16.0 b 92.7 g 7288 h 1825 f 1070 f 206 cd
Pyrus communis 66.4 e 29.6 d 98.0 g 8243 i 2307 g 1349 g 253 f
Trifolium sp. 55.1 d 82.2 g 65.1 de 1515 ab 432 ab 195 a 91.7 a
Lamium purpureum 51.1 d 8.6 a 76.1 f 3570 de 825 c 171 a 123 b
Lupinus polyphyllus 38.5 c 61.7 e 24.7 b 2836 c 741 c 595 cd 217 de
Chamerion angustifolium 27.2 b 23.7 c 67.5 def 1829b 506b 683d 147 b
Zea mays 6.8 a 23.5 c 75.8 f 1293a 308 a 378 b 92.4 a
Means followed by the same letters are not signiﬁcantly diﬀerent.
R2 = 0.6132
0 500 1000 1500 2000 2500
Fig. 1. Variability of total antioxidant activity.
M. Leja et al. / Food Chemistry 100 (2007) 237–240 239
the case of M. domestica and P. communis, high inhibition
of lipid peroxidation and very high hydroxyl radical-scav-
enging activity agreed with the high level of total phenols
and phenylpropanoids; however, radical-scavenging activ-
ity was low and medium, respectively (16.0% and 29.6%).
Special attention should be paid to L. purpureum pollen:
the relatively high (51%) TAA and high (76%) HRSA did
not reﬂect a poor (8.6%) RSA and low ﬂavonol content.
The most distinct interdependence was found between
phenylpropanoids and total antioxidant activity, resulting
in a signiﬁcant positive correlation coeﬃcient
= 0.6132). According to these results, phenylpropa-
noids might be treated as the main phenolic compounds
responsible for the protection against lipid peroxidation
in bee pollen tissue. Cinnamic acid derivatives are consid-
ered as eﬃcient antioxidants due to the substitutions on
the aromatic ring and the structure of the side chain, and
better antioxidants than their benzoic acid counterparts
(Natella et al., 1999). Very strong antioxidative properties
of caﬀeic acid and its derivatives were described by Silva
et al. (2000). However, according to Foti et al. (1996),
the best protective action against linoleic acid peroxidation
was attributed to ﬂavonoids, followed by coumarins and
In general, the direct correlation between phenolic con-
stituents and antioxidant capacity of bee pollen was ques-
tionable in some examined samples. The above results are
partially in agreement with the Campos et al. (2003,
2000) reports. However, these authors determined only
RSA activity by the DPPH method. The simultaneous esti-
mation of RSA, TAA and HRSA allowed antioxidative
properties of pollen to be characterized more precisely.
Results showed that various constituents (phenolics, and
probably, other compounds) are engaged in neutralization
of diﬀerent active oxygen species. The separation of the
individual phenolics and detection of the other antioxi-
dants will be necessary in further investigations of the pol-
len antiradical system.
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