In vitro antioxidant capacity of honeybee-collected pollen of selected floral origin harvested from Romania

Abstract

a b s t r a c t Total phenolic phytochemical concentration was measured in 12 honeybee-collected pollens of selected floral species as well as their antioxidant capacity. The content of total polyphenols was measured spec-trophotometrically using the Folin–Ciocalteu reagent with gallic acid as standard. The antioxidant prop-erties were evaluated by 2,2-diphenyl-picrylhydrazyl radical scavenging capacity (DPPH) assay, Trolox equivalent antioxidant Capacity procedure and Ferric ion reducing antioxidant power assay. A great var-iability regarding the correspondence between the antioxidant activity and the content of total polyphe-nols of honeybee-collected pollens with different botanical origin was found. Antioxidant activities were different for each floral species and were not clearly associated to their total phenolic content.

Full text

In vitro antioxidant capacity of honeybee-collected pollen of selected floral
origin harvested from Romania
Liviu A. Ma
˘rghitasß
a
, Oltica G. Stanciu
a,*
, Daniel S. Dezmirean
a
, Otilia Bobisß
a
, Olimpia Popescu
a
,
Stefan Bogdanov
b
, Maria Graca Campos
c
a
Department of Beekeeping and Sericulture, University of Agricultural Sciences and Veterinary Medicine, 3-5 Manastur Street, 400151 Cluj-Napoca, Romania
b
Swiss Bee Research Center, Forschungsanstalt fur Milchwirtschaft, CH-3003, Bern, Switzerland
c
Faculdade de Farmacia, Universitade de Coimbra, 3000-95 Coimbra, Portugal
article info
Article history:
Received 19 August 2008
Received in revised form 31 October 2008
Accepted 1 January 2009
Keywords:
Honeybee-collected pollen
Polyphenols
Antioxidant capacity
DPPH
FRAP
TEAC
abstract
Total phenolic phytochemical concentration was measured in 12 honeybee-collected pollens of selected
floral species as well as their antioxidant capacity. The content of total polyphenols was measured spec-
trophotometrically using the Folin–Ciocalteu reagent with gallic acid as standard. The antioxidant prop-
erties were evaluated by 2,2-diphenyl-picrylhydrazyl radical scavenging capacity (DPPH) assay, Trolox
equivalent antioxidant Capacity procedure and Ferric ion reducing antioxidant power assay. A great var-
iability regarding the correspondence between the antioxidant activity and the content of total polyphe-
nols of honeybee-collected pollens with different botanical origin was found. Antioxidant activities were
different for each floral species and were not clearly associated to their total phenolic content.
Ó2009 Elsevier Ltd. All rights reserved.
1. Introduction
In recent years there has been a remarkable increment in scien-
tific research dealing with natural antioxidants and their potential
health benefits. Oxidative stress, the consequence of an imbalance
between ROS (reactive oxygen species) generation and antioxi-
dants in the organism, initiates a series of harmful biochemical
events which are associated with diverse pathological processes
which can lead to various cellular damages and diseases (Sastre,
Pallardo, & Vina, 2003). Antioxidants are considered as possible
protection agents reducing oxidative damage to important biomol-
ecules, including lipoprotein and DNA (deoxyribonucleic acid)
from ROS (Gulcin, Buyukokuroglu, Oktay, & Kufrevioglu, 2003).
The growing interest in the physiological benefits of natural
antioxidants has been matched by acceleration in the development
of analytical and biological methodologies for measurement of
both the levels and antioxidant potential of these compounds.
There is increasing evidence from epidemiological, in vivo,
in vitro, and clinical trials clearly suggesting that the polyphenolic
compounds present in natural foods may reduce risk of chronic
disease such cancer, anti-inflammatory, cardiovascular and neu-
ro-degenerative diseases (Luthria, 2006).
Phenolic compounds, such as flavonoids, phenolic acid and tan-
nins, are considered to be a major contributor to the antioxidant
potential of foods. In this respect, antioxidant capacity of phenolic
compounds extracted from various foodstuffs, egg, Vegetables (Ou,
Huang, Hampsch-Woodill, Flanagan, & Deemer, 2002), honey,
propolis and royal jelly (Buratti, Benedetti, & Cosio, 2007) and wine
(Lee, Kim, Joo, & Lee, 2003), have been intensively studied using
in vitro methods.
The antioxidant activity of polyphenols is mainly due to their re-
dox properties, which can play an important role in neutralising free
radicals, quenching oxygen, or decomposing peroxides (Nijveldt
et al., 2001). In addition to their individual effects, antioxidants
interact in synergistic ways and have sparing effect in which one
may protect another against oxidative destruction (Damintoti,
Mamoudou, Simpore, & Traore, 2005). The best-described property
of almost every group of flavonoids, which are the predominant
phenolic class present in honeybee-collected pollen, is their capac-
ity to act as antioxidants (Kroyer & Hegedus, 2001). One way is the
direct scavenging of free radicals. Flavonoids are oxidised by
radicals, resulting in a more stable, less-reactive radical. In other
words, flavonoids stabilize the reactive oxygen species by reacting
with the reactive compound of the radical (Nijveldt et al., 2001).
There are various methods available in the assessment of the
antioxidant capacity of samples, they provide useful data, and
however, they are not sufficient to estimate a general antioxidant
0308-8146/$ - see front matter Ó2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.01.014
*Corresponding author. Tel.: +40 264 595825x224; fax: +40 264 430253.
E-mail address: ococan@gmail.com (O.G. Stanciu).
Food Chemistry 115 (2009) 878–883
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

ability of the sample (Filipiak, 2001). These methods differ in terms
of their assay principles and experimental conditions. Conse-
quently, in different methods, particular antioxidants have varying
contributions to total antioxidant potential (Cao & Prior, 1998).
Radical scavenging activities are very important due to the del-
eterious role of free radicals in foods and in biological systems
(Gulcin, Elias, Gepdiremen, Boyer, & Koksal, 2007). The DPPH
method (Brand-Williams, Cuvelier, & Berset, 1995) consist in the
reaction of DPPH (2,2-diphenyl-1-picrylhydrazyl) a stable free rad-
ical, which accepts an electron or hydrogen radical to become a
stable molecule, and, accordingly, is reduced in presence of an anti-
oxidant. DPPH radical are widely used for the preliminary screen-
ing of compounds capable to scavenging activated oxygen species
since they are much more stable and easier to handle than oxygen
free radical (Tominaga et al., 2005). The TEAC assay is based on the
inhibition by antioxidants of the absorbance of the radical cation of
ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonate). Due to
its operational simplicity, the TEAC assay has been used in many
research laboratories for studying antioxidant capacity, and TEAC
values of many compounds and food samples are reported. The
mechanisms of both methods are similar, in that the absorption
spectra of the stable, free radical changes when the molecule is re-
duced by an antioxidant or a free radical species. The FRAP assay
measures the ferric-to-ferrous iron reduction in the presence of
antioxidants and is very simple and convenient in terms of its
operation (Cao & Prior, 1998).
Honeybee-collected pollen is an apicultural product which is
composed of nutritionally valuable substances and contains con-
siderable amounts of polyphenolic compounds, mainly flavonoids,
which may act as potent antioxidants (Kroyer & Hegedus, 2001).
The antioxidant activity of honeybee-collected pollen has been
recognised as a free radical scavenger and as a lipid peroxidation
inhibitor as previously Almaraz-Abarca et al. (2004) reported.
In the current study, antioxidant potential of bee pollen of se-
lected floral origin was evaluated, in comparison with the level
of total phenolic and flavonoid content.
2. Materials and methods
2.1. Chemicals
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman carboxylic
acid), 2,2
0
-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
diammonium salt (ABTS), potassium persulphate (K
2
S
2
O
8
), sodium
carbonate, Folin–Ciocalteu’s phenol reagent and gallic acid were
purchased from Sigma–Aldrich (St. Louis, MO). Iron(III) chloride
6-hydrate (FeCl
3
3H
2
O), iron(II) sulfate 7-hydrate (FeSO
4
7H
2
O)
and acetic acid (CH
3
COOH) were obtained from BDH (Poole, UK).
2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) was purchased from Fluka
Chemie AG (Bushs, Switzerland). Hydrochloric acid (HCl) and
methanol were obtained from Merck (Darmstadt, Germany). All
chemicals used in the experiments were of analytical grade.
2.2. Equipments
Spectrophotometric measurements were performed by
Synergy
TM
HT Multi-Detection Microplate Reader with 96-well
plates (BioTek Instruments, Inc., P) and spectrophotometer (dou-
ble-beem) 1700 Schimadzu. In order to perform the analysis, the
following equipments were also used: Microscope Nikon Eclipse
50i 40, rotary evaporator Buchi R-215, ultrasonic bath Bandelin.
2.3. Botanical origin identification of the pollen pellets
Samples of honeybee-collected pollen were purchased from
local beekeepers from Transylvania area of Romania in 2007. The
floral origin of honeybee-collected pollen pellets was identified
by colour and light microscope examination by palynological anal-
ysis according to the acetolysis method (Erdtman, 1969). The
microscope examination was performed under normal lighting at
400magnification. Pollen types were identified by comparison
with pollen reference slides made by the authors of the present
work, and then compared with available pollen atlases (Erdtman,
1969; Ricciardelli & d’Albore, 1998; Sawyer, 1981). The pollen ref-
erence slides were prepared from anthers of flowers and the plant
taxon was identified upon the botanic atlas (Popovici, Moruzi, &
Toma, 1973).
2.4. Preparation of extracts
Each samples of beepollen (2 g) were individually extracted three
times with 15 ml of methanol solvent at the room temperature for
1 h. After sonication (15 min), maceration and filtration, the filtrate
was evaporated to dryness under vacuum. The resulting dried
extracts were dissolved in methanol and stored until analysis (4 °C).
2.5. Determination of total phenolic content
The content of total polyphenols was estimated according to the
Folin–Ciocalteu method proposed by Singleton, Orthofer, and
Lamuela-Raventos (1999) using gallic acid as reference standard
(Meda, Lamien, Romito, Millogo, & Nacoulma, 2005). The method
was adapted to the 96 well microplate reader. Briefly, the 125
l
L
Folin–Ciocalteu reagent (0.2 N) was added to 25
l
L of bee pollen
extracts and mixed for 5 min. After the addition of 100
l
L sodium
carbonate (Na
2
CO
3
) solution (75 g/L) the extracts was incubated
for 2 h. The absorbance at 760 nm was then measured against a
methanol blank. A standard curve of gallic acid was created using
an adequately range of gallic acid solutions from 0.01 to 0.25 mg/
mL. The results were expressed as Gallic Acid Equivalent (mg
GAE g
1
dry matter sample).
2.6. Determination of total flavonoid content
Total flavonoids were measured by the aluminium chloride col-
orimetric assay developed by Zhishen, Wengcheng, and Jianming
(1999) using quercetin as reference standard, as described by
(Kim, Jeong, & Lee, 2003). An aliquot (1 ml) of appropriately diluted
sample or standard solutions of quercetin (0.001–0.25 mg/mL) was
added to a 10 ml volumetric flask containing 4 ml distilled water.
At zero time, 0.3 ml 5% NaNO
2
was added to the flask. After
5 min, 0.3 ml 10% AlCl
3
was added. At 6 min, 2 ml 1 M NaOH was
added to the mixture. Immediately, the reaction flask was diluted
to volume with the addition of 2.4 ml of distilled water and thor-
oughly mixed. Absorbance of the mixture, pink in colour, was
determined at 510 nm versus prepared reagent blank. Total flavo-
noid content was expressed as mg Quercetin Equivalent (mg
QE g
1
dry matter sample).
2.7. Determination of antioxidant capacity
2.7.1. Determination of DPPH scavenging activity
The scavenging activity (H/e-transferring ability) of bee pollen
extracts against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH)
was evaluated spectrophotometrically by a slightly modified meth-
od of Brand-Williams et al. (1995) as described by Velazquez, Tour-
nier, Mordujovich de Buschiazzo, Saavedra, and Schinella (2003),
with adaptation on the micro-plate reader. Briefly, an aliquot
(40
l
L) of appropriately diluted extracts of bee pollen was mixed
with 200
l
L DPPH solution (0.02 mg/mL). Samples were kept for
15 min at room temperature and then the absorbance was mea-
sured at 517 nm. Absorbance of blank sample containing the same
L.A. Ma
˘rghitasßet al. /Food Chemistry 115 (2009) 878–883 879

amount of solvent and DPPH solution was prepared and measured
daily. The percentage of absorbance inhibition at 517 nm was cal-
culated using the equation below:
Inhibition ð%Þ¼ 1Asample
Ablank

100 ð1Þ
The extent of decolourisation is calculated as percentage reduc-
tion of absorbance, and this is determined as a function of concen-
tration and calculated relatively to the equivalent trolox
concentration (0.1–0.01 mM). The radical scavenging activity is ex-
pressed in millimol of equivalent Trolox per gram of sample
(mmol Trolox g
1
dry matter sample).
2.7.2. Determination of Trolox equivalent antioxidant capacity (TEAC)
For Trolox equivalent antioxidant Capacity assay, the procedure
followed the method of Re et al. (1999) with some modifications.
The TEAC assay is based on the scavenging of the 2,2’-azinobis-(3-
ethylbenzothiazoline- 6-sulphonic acid) (ABTS) radical (ABTS
+
)con-
verting it into a colourlessproduct. The degree of decolourisation in-
duced by a compound is related to that induced by trolox, giving the
‘‘TEAC value”. The ABTS
+
cation radical was produced by the reac-
tion between 7 mM ABTS solution and 2.45 mM potassium persul-
phate solution, stored in the dark at room temperature for 16 h.
The solution is stable for 3 days. Before usage, the ABTS
+
solution
was diluted to get an absorbance of 0.700 ± 0.025 at 734 nm with
ethanol. For the assay the resulting solution was mixed with
17
l
L of sample. The absorbance was read at 30 °C after exactly
6 min. The extent of inhibition of the sample, calculated using the
formula mentioned in the DPPH method, was then compared with
a standard curve made from the corresponding readings of Trolox
(0.4–0.04 mM). Results were expressed in millimol of equivalent
Trolox per gram of sample (mmol Trolox g
1
dry matter sample).
2.7.3. Determination of ferric reducing/antioxidant power
Total antioxidant potential of the sample was evaluated using
the ferric reducing ability (FRAP) assay as a measure of ‘‘antioxidant
power”. The ferric reducing/antioxidant power FRAP is a simple, di-
rect test of antioxidant capacity. This method was initially devel-
oped to assay plasma antioxidant capacity by Benzie and Strain
(1996) and adapted to a manual assay by Varga, Matkovics, Sasvári,
and Salgó (1998) (Szollosi & Varga, 2002). FRAP assay measures the
change in absorbance at 593 nm owing to the formation of the blue
coloured Fe
II
-tripycridyltriazine compound from the colourless oxi-
dised Fe
III
form by the action of electron donating antioxidants. The
working FRAP reagent was prepared by mixing 10 mL of 300 mmol/
L acetate buffer pH 3.6 (3.1 g sodium acetate CH
3
COONa 3H
2
Oand
16 mL acetic acid glacial CH
3
COOH per litre), with 1 mL of
10 mmol/L TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mmol/L hydro-
chloride acid and 1 mL of 20 mmol/L ferric chloride solution in
distilled water. All solutions were used on the day of preparation.
The sample consisted in 300
l
L FRAP reagent, 10
l
L of bee pollen
extract and 30
l
L deionized water, in order to obtain a final dilu-
tion of the sample in the reaction mixture of 1:34. The sample
was incubated at 37 °C throughout the monitoring period (4 min).
The antioxidant capacity of the samples under study was calculated
with reference to the reaction signal given by aqueous solutions of
Fe
II
solution of known concentration (0.1–1 mmol/L of FeS-
O
4
7H
2
O). The results were corrected for dilution and calculated
using a standard calibration curve (r
2
= 0.9965) and expressed as
FRAP value (mmol Fe
II
g
1
dry matter sample).
2.8. Statistical analysis
All determinations were performed in triplicate and results are
expressed as mean ± standard deviation calculated using spread-
sheet software Microsoft Excel. The data were analysed by an anal-
ysis of variance (p60.05) and means separated by Duncan’s
multiple range test. The relationship between antioxidant content
and the antioxidant capacity of different monofloral bee pollen
samples, as well as between different antioxidant capacity assays,
was analysed by Pearson correlation coefficients. The results were
processed by STATPlus2008 software.
3. Results and discussion
3.1. Botanical identification
The pollen loads studied here were first separated by colour
from the complex mixtures of pollen pellets from different species
of plants, resulting monochromatic pollen loads with uniform col-
our. The microscopic examination was the principal tool for the
selection of the honeybee-collected pollen pellets coming from
one species. In each microscopic preparation, pollen was deter-
mined, when possible, into genus, species or family. The selected
pollen load samples studied here presented the following colours:
grey yellow (Capsella bursa pastoris L.), red orange (Helianthus
annuus L., Matricaria chamomilla L., Taraxacum officinale Web.),
light green (Crataegus monogyna J.), light yellow (Pinus sp., Carex
sp.), violet (Carduus sp.), brown (Onobrychis viciifolia Scop.), grey
(Centaurea cyanus L.), pink (Knautia arvensis (L.) Coulter)), maroon
(Salix sp.).
3.2. Total phenol content of honeybee-collected pollen of selected floral
origin
There was a wide range of phenolic concentration in the honey-
bee-collected pollen analysed, as shown in Table 1. The highest
polyphenol concentration was determined in the methanol ex-
tracts of bee pollen from Salix sp. (16.4 mg GAE g
-1
) followed by
T. officinale Web. bee pollen (16.2 mg GAE g
-1
), C. cyanus L. bee
pollen (16.0 mg GAE g
1
), C. monogyna J. bee pollen and
(7.7 mg GAE g
1
)C. bursa pastoris L. bee pollen (15.2 mg GAE g
1
).
The lowest level of total polyphenol content was determined in
bee pollen from K. arvensis (L.) Coulter bee pollen with value of
4.4 mg GAE g
1
. Similar values were obtained for Pinus sp. and
Carex sp. bee pollen with value of 6.4 mg GAE g
1
. The total pheno-
lic content was significantly (p60.05) higher (3.7 times) in Salix
sp. than in K. arvensis (L.) Coulter bee pollen.
3.3. Total flavonoid content of honeybee-collected pollen of selected
floral origin
Total flavonoid content showed discrepancies in the examined
honeybee-collected pollen of selected floral origin. The highest lev-
els were quantified in Salix sp. bee pollen (13.6 mg QE g
1
) fol-
lowed by M. chamomilla L. (12.7 mg QE g
1
) and C. cyanus L. bee
pollen (11.8 mg QE g
1
). The bee pollen from Pinus sp. contained
the lowest total flavonoid content (0.6 mg QE g
1
). Similar lower
values were determined in K. arvensis (L.) Coulter bee pollen
(2.8 mg QE g
1
) and T. officinale Web. bee pollen (3.8 mg QE g
1
).
The flavonoid content differs significantly between samples
(p60.05), with exception of Carduus sp., O. viciifolia Scop. and C.
bursa pastoris L. bee pollens.
Depending on the pollen species, the participations of flavo-
noids in total phenols significantly differ (p60.05), the highest
participation was determinate in the case of O. viciifolia Scop. bee
pollen (91.2%), followed by M. chamomilla L. (90.7%), H. annuus L.
(89.5%) and Salix sp. bee pollens (82.9%). In comparison, the flavo-
noid fraction represents only 9.4% of total phenols in Pinus sp. bee
pollen and 23.5% in T. officinale Web. bee pollen.
880 L.A. Ma
˘rghitasßet al./ Food Chemistry 115 (2009) 878–883

3.4. Antioxidant capacity of honeybee-collected pollen of selected floral
origin
The antioxidant capacity determination results of an extract de-
pend greatly on the methodology used, that is the oxidant and the
oxidisable substrate used in the measurement. Therefore, it is
important to compare different analytical methods varying in their
oxidation initiators and targets in order to understand the biolog-
ical activity of an antioxidant and to obtain accurate data for a bet-
ter comparison with other literature (Cao & Prior, 1998;Stratil,
Klejdus, & Kuban, 2006 cited by Santas, Carbo, Gordon, & Almajano,
2008).
Recent investigations show differences between the test sys-
tems in determining antioxidant capacity. Use of at least two
methods is recommended to assess and compare the antioxidant
capacity of a sample (Sakanaka & Ishihara, 2008).
The present study present different in vitro tests based either on
the capacity to scavenge free radicals (DPPH, TEAC) or on the abil-
ity of reducing oxidants (ferric ions) the ferric-reducing ability
(FRAP) (Table 1).
In the DPPH assay, antioxidants will react with a nitrogen-
centered radical (2,2-diphenyl-1-picrylhydrazyl) which is with a
characteristic absorption at 517 nm and converted into 1,1,-diphe-
nyl-2-picryl hydrazine, at a very rapid rate. On the other hand,
antioxidants are believed to intercept the free radical chain of oxi-
dation and to donate hydrogen from the phenolic hydroxyl groups,
thereby forming stable end product, which does not initiate or
propagate further oxidation of lipid (Jayaprakasha & Patil, 2007).
Honeybee-collected pollen from different floral sources differed
significantly (p60.05) in their DPPH values. The DPPH values of
methanol extracts of the 12 monofloral bee pollens ranged from
0.135 (bee pollen from Pinus sp.) and 2.814 mmol Trolox g
1
dry
matter sample (bee pollen from Salix sp.).
TEAC method can measure the antioxidant capacity determined
by the decolourization of the ABTS
+
through measuring the reduc-
tion of the radical cation as the percentage inhibition of absorbance
at 734 nm. In ABTS
+
decolourisation method, Salix sp. bee pollen
extract possessed the highest TEAC value of 6.838 mmol
Trolox g
1
, followed by M. chamomilla L., Carex sp. and C. cyanus
L. bee pollens with 4.466 mmol Trolox g
1
, 3.770 and
3.638 mmol Trolox g
1
, respectively. In terms of ABTS decolourisa-
tion action, amongst the monofloral bee pollen extracts, the lowest
antioxidant potential was performed by Pinus sp. bee pollen
(0.546 mmol Trolox g
1
) followed by K. arvensis (L.) Coulter bee
pollen witch registered TEAC values of 0.938 mmol Trolox g
1
.
For the TEAC assay, the difference of antioxidant capacities was
very significant (p60.05).
The ferric reducing/antioxidant power (FRAP) assay, in contrast
to other tests of total antioxidant power, is a simple, speedy and ro-
bust assay (Prior, Wu, & Schaich, 2005). At low pH (optimum pH
3.6) Fe
III
–TPTZ complex is reduced by antioxidants to its intense
blue coloured form Fe
II
–TPTZ which has maximum absorbance at
593 nm. Methanol extracts of bee pollen from different floral spe-
cies differed significantly (p60.05) in their FRAP values. The FRAP
values of honeybee-collected pollens ranged from 5.355 to
0.255 mmol Fe
II
g
1
. In the present study, the highest antioxidant
potentials amongst the methanol extracts of the honeybee-
collected pollen was registered for the M. chamomilla L. bee pollen
(5.355 mmol Fe
II
g
1
), followed by the Salix sp. bee pollen
(3.760 mmol Fe
II
g
1
) and Carex sp. bee pollen (3.055 mmol
Fe
II
g
1
). The bee pollen extracts from K. arvensis (L.) Coulter, T.
officinale W. and Pinus sp. species exhibits the lower antioxidant
potentials (0.255, 0.327 and 0.697 mmol Fe
II
g
1
, respectively).
3.5. Relationship between antioxidant capacity and phenolic content
Free radical scavenging of phenolic compounds is an important
property underlying their various biological and pharmacological
activities (Damintoti et al., 2005). Total phenol content and total
antioxidant capacity differs significantly amongst 12 selected
monofloral bee pollens analysed. In the present investigation,
was found a great variability regarding the correspondence be-
tween the DPPH scavenging activity, Trolox equivalent antioxidant
capacity respectively the reducing/antioxidant power and the con-
tent of total phenolics and flavonoids.
Bee pollen of Salix alba L. contains the highest quantities of
polyphenols (16.4 mg GAE g
1
), respectively flavonoids
(13.6 mg QE g
1
) and demonstrated high antioxidant capacity in
all antioxidant systems evaluated (DPPH: 2.814 mmol Trolox g
1
,
TEAC: 6.838 mmol Trolox g
1
and FRAP: 3.670 mmol Fe
II
g
1
).
It can be observed that high antioxidant content (total phenolics
16.4 and 16.0 mg GAE g
1
; total flavonoids 13.6 and 11.8 mg
QE g
1
) are accompanied by high DPPH scavenging activity
(2.814, respectively 2.615 mmol Trolox g
1
) and TEAC values
(6.838, respectively 3.638 mmol Trolox g
1
) only in the case of
bee pollen from Salix sp. and C. cyanus L. (Table 1).
In the case of the K. arvensis L. and Pinus sp. bee pollens, the
lower antioxidant capacity (DPPH: 0.274 mmol Trolox g
1
and
0.135 mmol Trolox g
1
, respectively; TEAC: 0.938 mmol Trolox g
1
and 0.546 mmol Trolox g
1
, respectively; FRAP: 0.255 mmol
Table 1
The phenolic content and antioxidant capacity of honeybee-collected pollen of selected floral origin
*
.
Botanical name of floral
species
Total phenolics
**
(mg GAE g
1
)
Total flavonoids
**
(mg Qe g
1
)
Antioxidant capacity
**
DPPH value
(mmol Trolox g
1
)
TEAC value
(mmol Trolox g
1
)
FRAP value
(mmol Fe
II
g
1
)
Capsella bursa pastoris L. 15.2 ± 0.3 g 9.4 ± 0.2 ef 1.342 ± 0.02 g 2.365 ± 0.02 f 2.412 ± 0.02 h
Helianthus annuus L. 11.4 ± 0.2 d 10.2 ± 0.3 g 0.454 ± 0.01 d 1.860 ± 0.02 d 1.491 ± 0.02 e
Crataegus monogyna J. 15.3 ± 0.3 g 10.8 ± 0.3 h 1.313 ± 0.01 f 2.785 ± 0.03 h 2.014 ± 0.03 g
Pinus sp. 6.4 ± 0.1 b 0.6 ± 0.03 a 0.135 ± 0.01 a 0.546 ± 0.01 a 0.697 ± 0.02 c
Matricaria chamomilla L. 14.0 ± 0.3 f 12.7 ± 0.1 j 1.348 ± 0.02 g 4.466 ± 0.04 k 5.355 ± 0.04 l
Carduus sp. 12.9 ± 0.2 e 9.6 ± 0.1 f 1.432 ± 0.02 h 2.747± 0.03 g 3.382 ± 0.04 j
Taraxacum officinale Web. 16.2 ± 0.2 h 3.8 ± 0.1 c 0.348 ± 0.02 c 1.499 ± 0.02 c 0.327 ± 0.01 b
Onobrychis viciifolia Scop. 10.2 ± 0.2 c 9.3 ± 0.1 e 0.684 ± 0.01 e 1.883 ± 0.02 e 1.040 ± 0.03 d
Centaurea cyanus L. 16.0 ± 0.3 h 11.8 ± 0.2 i 2.615 ± 0.03 j 3.638 ± 0.04 i 1.980 ± 0.03 f
Knautia arvensis (L.)
Coulter
4.4 ± 0.1 a 2.8 ± 0.02 b 0.274 ± 0.01 b 0.938 ± 0.02 b 0.255 ± 0.02 a
Salix sp. 16.4 ± 0.3 i 13.6 ± 0.2 k 2.814 ± 0.03 k 6.838 ± 0.04 l 3.760 ± 0.05 k
Carex sp. 13.9 ± 0.1 f 8.8 ± 0.1 d 1.533 ± 0.02 i 3.770 ± 0.03 j 3.055 ± 0.05 i
*
Samples analysed in triplicate.
**
Means followed by the same letters are not significantly different (p60.05).
L.A. Ma
˘rghitasßet al. /Food Chemistry 115 (2009) 878–883 881

Fe
II
g
1
and 0.697 mmol Fe
II
g
1
, respectively) is reflected by the
lower antioxidant content: polyphenols (4.4 and 6.4 mg GAE g
1
)
and flavonoids (2.8 and 0.6 mg QE g
1
).
Low DPPH, TEAC and FRAP values obtained in the case of H.
annuus L. bee pollen (0,454 mmol Trolox g
1
, 1.860 mmol Trol-
ox g
1
and 1.491 mmol Fe
II
g
1
) is not reflected by their polyphenol
content (11.4 mg GAE g
1
), respectively flavonoid content
(10.2 mg QE g
1
). Similarly, high flavonoid content of bee pollen
of O. viciifolia Scop. (9.3 mg QE g
1
) is not reflected in their low
antioxidant capacities (DPPH: 0.684 mmol Trolox g
1
, TEAC:
1.883 mmol Trolox g
1
and FRAP: 1.040 mmol Fe
II
g
1
).
Bee pollen of T. officinale Web. despite the high polyphenol con-
tent (16.2 mg GAE g
1
) demonstrate low antioxidant capacity
(DPPH: 0.348 mmol Trolox g
1
and FRAP: 0.327 mmol Fe
II
g
1
)
according to their flavonoid content (3.8 mg QE g
1
).
The total polyphenol content of honeybee-collected pollen
(‘‘bee pollen”) and their extracts was previously determinate by
Kroyer and Hegedus (2001). In the bee pollen extracts (ethanol,
methanol-water 1:1 and water) the amount of total polyphenols
ranged between 21.4–24.6 mg g
1
, with the highest content in
the ethanol extract. As a result, the best antiradical activity against
the DPPH was pursued by the ethanol extract (53 %).
In a previous study, Almaraz-Abarca et al. (2004) determined
the total flavonol content in a mixture of bee pollen and their con-
stituent pollens, and the values ranged between 3.5–0.1 mg/g dry
matter of pollen. In addition, using a modified Campos method
(1997), the antiradical activity was expressed as the amount of
antioxidant needed to decrease by 50% the initial DPPH concentra-
tion (EC
50
). Comparing the antiradical activity and the flavonol
content of the samples, no correlation seems to exist between
the total extract of the mixture of bee pollen and those of its con-
stituent pollens, in terms of their flavonol content.
Leja, Mareczek, Wyzgolik, Klepacz-Baniak, and Czekonska
(2007) determined the phenolic constituents (total phenols, phe-
nylpropanoids, flavonols and anthocyanins) and antioxidant ability
in bee pollen of 12 plant species. Great variability of phenolic con-
tent was observed in the investigated species of pollen, levels of to-
tal phenols ranged between 82.4 and 12.9 mg g
1
. Flavonol content
showed discrepancies in the examined pollen samples and ranged
between 1.7 and 13.4 mg g
1
. Great differences in the radical-scav-
enging activity (8.6–91.5% of DPPH neutralisation) and in the hy-
droxyl radical-scavenging activity (10.5–98% inhibition of
deoxyribose degradation) were observed and were not direct cor-
related with the content of phenolic compounds in all examined
samples.
In the present study, antioxidant capacities were different for
each floral species and were not clearly associated to their total
phenolic content in the case of all bee pollen samples (Table 2).
The lower correlation coefficients between total phenol content
and the antioxidant capacities of the monofloral bee pollens ana-
lysed arises from the anomalous behaviour of H. annuus L., T. offi-
cinale W. and O. viciifolia Scop. bee pollens, as mentioned above.
The above results are in agreement with Leja et al. (2007), the di-
rect correlation was questionable in some species, as mentioned
above.
A better interdependence was obtained between the flavonoid
fraction of the bee pollen analysed and their antioxidant capacities
in all in vitro tests, resulting significant positive correlation coeffi-
cients (Table 2). As the previous results indicate, the flavonoid
components play a significant role in the free radical scavenging
capacity of bee pollen (Almaraz-Abarca et al., 2004). The relation-
ship is not clearly related to the high total flavonoid content in
the case of all bee pollen species. The results of the present study
supported previous conclusions, that the free radical scavenging
effectiveness is determined by its particularly phenolic or non-
phenolic constituents with their variable structure and actions
(Leja et al. 2007).
Good positive correlation was found between DPPH and ABTS
scavenging activity (r
2
= 0.880) and between the ABTS scavenging
activity and the Ferric reducing/antioxidant power (r
2
= 0.635) of
the methanol extracts of monofloral bee pollens studied (Table
2). This suggested that the compounds which could scavenge DPPH
radical in the bee pollen extracts were also able to scavenge ABTS
radical cation. The Ferric reducing/antioxidant power and the ABTS
scavenging activity were good correlated (r
2
= 0.794). Because the
redox potential of Fe
III
-TPTZ (0.7 V) is comparable with that of
ABTS
+
(0.68 V), similar compounds react in both the TEAC and
FRAP assays (Prior et al., 2005).
4. Conclusions
In the present investigations, great variability regarding content
of total phenols, total flavonoids and antioxidant capacity in exam-
ined bee pollen samples was found. Each pollen type has its own
specificity, mainly linked to the floral species or cultivars (Zaura-
low, 1983 cited by Nagai, Inoue, Suzuki, Myoda, & Nagashima,
2005).
It is known that only flavonoids of a certain structure and par-
ticularly hydroxyl position in the molecule, determine antioxidant
properties. In general, these properties depend on the ability to do-
nate hydrogen or electron to a free radical. Detailed examination of
phenolic composition in bee pollen extracts is required for the
comprehensive assessment of individual compounds exhibiting
antioxidant activity.
In addition, the redox properties of polyphenol compounds,
especially flavonoids, play an important role in absorbing and neu-
tralising free radicals, quenching oxygen and decomposing perox-
ides (Damintoti et al., 2005). This various mechanisms of
antioxidant activity permit a wide range of free radicals scavenging
and lipo-peroxidation assays in order to evaluate the complete
antioxidant potential (Sancez-Moreno, 2002). On the other hand,
different antioxidants respond differently in various measurement
methods which involve specific reaction conditions and mecha-
nisms of action. This may explain the various results for DPPH,
FRAP and TEAC assay, in regard with the antioxidant content of
bee pollen samples analysed. A specific polyphenolic compounds,
or an association of them, may have different actions as antioxi-
dant against various free radicals.
The results of this study confirm that antioxidant activities from
different assay methods strongly depend on the oxidation condi-
tions used in the particular oxidation test. Antioxidant activity is
not necessarily correlated with high amounts of phenolic com-
pounds. Total phenolic content, measured by the Folin–Ciocalteu
procedure, does not give a full idea of the nature of the phenolic
constituents in the extracts (Martha-Estrella, Niokhor, & Stevanov-
ic, 2008). In addition, it may be that antioxidant activity of specific
monofloral bee pollen extracts is not limited to phenolics.
Table 2
Pearson correlation matrix
*
.
TFC TF DSA ASA FRAP
TFC 1
TF 0.685 1
DSA 0.680 0.776 1
ASA 0.650 0.806 0.880 1
FRAP 0.502
**
0.748 0.635 0.794 1
*
TFC: Total phenolic content; TF: Total flavonoid content; DSA: DPPH scavenging
activity; ASA: ABTS radical cation scavenging activity, FRAP: Ferric reducing/anti-
oxidant power.
**
Significant at p< 0.05.
882 L.A. Ma
˘rghitasßet al./ Food Chemistry 115 (2009) 878–883

As a previous study indicate (Almaraz-Abarca et al., 2004), the
results of this preliminary study, conducted on bee pollen from dif-
ferent plant sources from Romania, demonstrated that the poly-
phenolic composition, rather that the concentration, could be the
determinant factor.
Since the previous studies on bee pollen antioxidant capacities
not included the in vitro TEAC and FRAP tests, the simultaneous
comparison between different pollen species made in the present
study may contribute to a better characterisation of their particular
antioxidant properties. Our results suggested that the antioxidant
capacity of samples might be associated with their specific
compounds.
In conclusion, future analysis is required, not only in testing
other different systems of evaluating the antioxidant activity, but
also in separation and identification the specific bioactive com-
pounds in bee pollens with different botanical origin, in order to
elucidate the differences between various samples.
Acknowledgements
The Ministry of Public Instruction and Research of Romania,
Project PN II Capacitati 105/2007 and PN II TD 352/2007 supported
this work.
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