Food and Chemical Toxicology 63 (2014) 233–239
Contents lists available at ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox
Biological activities of commercial bee pollens: Antimicrobial,
antimutagenic, antioxidant and anti-inflammatory
Ananias Pascoal a, Sandra Rodrigues a, Alfredo Teixeira b, Xesus Feás c, Leticia M. Estevinho a,⇑
a
CIMO-Mountain Research Center, Department of Biology and Biotechnology, Agricultural College of Bragança, Polytechnic Institute of Bragança, Campus Santa Apolónia,
E 5301-855 Bragança, Portugal
b
CEAV Animal and Veterinary Research Centre, University of Trás–os–Montes e Alto Douro, Vila Real, Portugal
c
Department of Organic Chemistry, Science Faculty, University of Santiago de Compostela, E-27002 Lugo, Galicia, Spain
a r t i c l e
i n f o
Article history:
Received 8 August 2013
Accepted 11 November 2013
Available online 19 November 2013
Keywords:
Antimicrobial activity
Antioxidant activity
Antimutagenicity
Bee pollen
Bioactive compounds
Inflammation
a b s t r a c t
Bee pollen is considered, since memorable times, a good source of nourishing substances and energy. The
present study aimed to evaluate the biological activities of eight commercial bee pollens purchased from
the market. The origin of sample A was not specified in the labeling; samples B, C, D and G were from
Portugal and the remaining were from Spain. The sample E presented the highest value of phenolics
(32.15 ± 2.12 mg/g) and the H the lowest (18.55 ± 095 mg/g). Sample C had the highest value of
flavonoids (10.14 ± 1.57 mg/g) and sample H the lowest (3.92 ± 0.68 mg/g). All the samples exhibited
antimicrobial activity, being Staphylococcus aureus the most sensitive and Candida glabrata the most
resistant of the microorganisms studied. All the samples exhibited antimutagenic activity, even though
some samples were more effective in decreasing the number of gene conversion colonies and mutant
colonies. Regarding the antioxidant activity, assessed using two methods, the more effective was sample
B. The anti-inflammatory activity, assessed using the hyaluronidase enzyme, was highest in samples B
and D. Pearson’s correlation coefficients between polyphenols, flavonoids, antioxidant activity and
antimicrobial activity were computed. It was also performed a discriminant analysis.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Bee pollen, commonly referred as the ‘‘life-giving dust’’,
results from the agglutination of flower pollens with nectar
and salivary substances of the honeybees and is used as food
for all the developmental stages in the hive (Almeida-Muradian
et al., 2005). The collection of this natural product is a
relatively recent development, dependent primarily on the basic
concept of scraping pollen off of the bees’ legs as they enter the
hive (Feás et al., 2012).
The major components of bee pollen are carbohydrates, crude
fibers, proteins and lipids at proportions ranging between 13%
and 55%, 0.3% and 20%, 10% and 40%, 1% and 10%, respectively
(Villanueva et al., 2002). In fact, bee pollen is referred as the ‘‘only
perfectly complete food’’, as it contains all the essential amino
acids needed for the human organism. However, the composition
of bee pollen depends strongly on plant source, together with
other factors such as climatic conditions, soil type, and beekeeper
activities (Morais et al., 2011). Generic bee pollen composition data
were considered sufficient for most purposes, but now the
⇑ Corresponding author. Tel.: +351 273 303342; fax: +351 273 325405.
E-mail address: leticia@ipb.pt (L.M. Estevinho).
0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fct.2013.11.010
usefulness of bee pollen-specific composition data is increasingly
being acknowledged (Nogueira et al., 2012; Bogdanov, 2011).
Bee pollen is considered a health food with a wide range of
therapeutic properties, among which: antimicrobial, antifungal,
antioxidant, anti-radiation, hepatoprotective, chemoprotective
and/or chemopreventive and anti-inflammatory activities (Abdella
et al., 2009; Bariliak et al., 1996; Viuda-Martos et al., 2008;
Fatrcová-Šramková et al., 2013). In addition, it has been reported
to trigger beneficial effects in the prevention of prostate problems,
arteriosclerosis, gastroenteritis, respiratory diseases, allergy desensitization, improving the cardiovascular and digestive systems,
body immunity and delaying aging (Estevinho et al., 2012). The
promotion of tissues’ repair, which results from the acceleration
on the mitotic rate, has also been lauded (Morais et al., 2011).
These therapeutic and protective effects have been related to the
content of polyphenols (Almeida-Muradian et al., 2005).
Having into account the European regulations (European Union,
2006) that state that any claims of health or nutritional benefits of
a food product must be supported by science, the purpose of the
present study was to quantify the bioactive compounds and to
determine the biological properties (antimicrobial, antioxidant,
anti-inflammatory and antimutagenic) of commercial bee pollens.
It is worth mention that, as far as we know, this is the first study on
the antigenotoxic activity of bee pollen.
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A. Pascoal et al. / Food and Chemical Toxicology 63 (2014) 233–239
2. Material and methods
2.1. Chemicals and reagents
All the reagents were of analytical grade purity. Methanol (CH3OH) and ethanol
(CH3CH2OH) were supplied by Pronolab (Lisbon, Portugal). The Folin–Ciocalteu reagent chloroform (CHCl3) sodium carbonate (Na2CO3) gentaminice and fluconazol
were obtained from Merck (Darmstadt, Germany). Gallic acid and (+)-catechin were
purchased from Sigma (St. Louis, MO, USA). The bovine testicular hyaluronidase
(350 units) and the potassium salt of human umbilical cord hyaluronic acid were
obtained from Sigma (St. Louis, MO, USA). The culture mediums were purchased
from Himedia (Mumbai, India). The TTC solution (235-Triphenyl-2H-tetrazolium
chloride) was supplied by Fluka (Buchs, Switzerland). The other chemicals were obtained from Sigma Chemical Co., (St. Louis, MO, USA). High purity water
(18 MX cm) used in all experiments was obtained from a Milli-Q purification
system (Millipore Bedford, MA, USA).
stored in Muller–Hinton medium plus 20% glycerol at 70 °C before experimental
use. The inoculum for the assays were prepared by diluting cell mass in 0.85% NaCl
solution adjusted to 0.5 MacFarland scale confirmed by spectrophotometrical reading at 580 nm for bacteria and 640 nm for yeasts. Cell suspensions were finally diluted to 104 CFU/mL in order to use them in the activity assays. Antimicrobial tests
were carried out according to Morais et al. (2011), using Nutrient Broth (NB) or
Yeasts Peptone Dextrose (YPD) on microplate (96 wells). Bee pollen extracts were
diluted in dimethylsulfoxide (DMSO) and transferred into the first well and serial
dilutions were performed. The inoculum was added to all wells and the plates were
incubated at 37 °C for 24 h (bacteria) and 25 °C for 48 h (yeast). Fluconazol and gentamicine were used as controls. In each experiment a positive control (inoculated
medium) and a negative control (medium) and DMSO control (DMSO with inoculated medium) was introduced. Antimicrobial activity was detected by adding
20 lL of 0.5% TTC solution. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of bee pollen extract that inhibited visible growth
as indicated by the TCC staining (dead cells are not stained by TTC). All the tests
were performed in triplicate (n = 3) and the results are expressed as mg/mL.
2.2. Pollen samples
2.6. Antimutagenic activity
Eight commercial pollens (A–H) of different floral sources and geographical origins were purchased from the market and left in the dark at room temperature
(±20 °C) until further analysis. The origin of sample A, in which the dominant family
was Cistaceae, was not specified in the labeling. Samples B (Fabaceae), C (Cistaceae),
D (Ericaceae) and G (Fabaceae) were from Portugal and E (Cistaceae), F (Ericaceae)
and H (Boraginaceae) were from Spain. It is worth mentioning that this botanical
classification of the samples was achieved in a previous study of the research team
(Nogueira et al., 2012). The preparation of the extracts was performed as described
in Morais et al. (2011), by mixing the bee pollen with methanol (1:2) (w/v). After
maceration, the extract was evaporated in a vacuum evaporator. The dried bee
pollen extract was kept in the dark at room temperature until further analysis.
2.3. Total phenolics and flavonoids
The total phenolic content of the extracts was recorded using the Folin–Ciocalteu method as described by Moreira et al. (2008). Briefly a dilute solution of each
bee pollen in MeOH (MeOH-bee pollen; 500 lL of 1:10 g/mL) was mixed with
500 lL of Folin–Ciocalteu reagent and 500 lL of Na2CO3 (10% w/v). After incubation
in dark at room temperature for 1 h the absorbance of the reaction mixture at
700 nm was determined against the blank (the same mixture without the
MeOH + sample) using a Unicam Helios Alpha UV–visible spectrometer (Thermo
Spectronic, Cambridge, UK). Galic Acid standard solutions (0.01 10 3 to
0.08 10 3 M) were used for constructing the calibration curve (y = 1.99813x +
0.0018; R2 = 0.9997). Total phenols content were expressed as mg of Galic Acid
equivalents per g of bee pollen (GAEs).
For flavonoids’ contents the aluminium chloride method was used. In briefly
MeOH-bee pollen (250 lL) was mixed with 1.25 mL of distilled H2O and 75 lL of
a 5% NaNO2 solution. After 5 min 150 lL of a 10% AlCl3H2O solution was added.
After 6 min 500 lL of 1 M NaOH and 275 lL of distilled H2O were added to the mixture and vortexed. The solution was well mixed and the intensity of pink colour was
measured at 510 nm. Catechin standard solutions (0.022 10 3 to 0.34 10 3 M)
were used for constructing the calibration curve (y = 1.0421x 0.0093;
R2 = 0.9918). Total flavonoids content were expressed as mg of catechin equivalents
per g of bee pollen (CAEs).
2.4. Anti-inflammatory activity – hyaluronidase assay
The anti-inflammatory activity was assessed indirectly by measuring the inhibitory effect of bee pollen on the reactions catalysed by hyaluronidase, using de
method described by Silva et al. (2012). The reaction mixture is constituted by
50 lL of bee pollens’ extract and 50 lL (350 units) of hyaluronidase enzyme (Type
IV-S: bovine testes) was incubated at 37 °C for 20 min. Then calcium chloride was
added (1.2 lL 2.5 10 3 M/L) to activate the enzyme and the mixture was incubated at 37 °C for 20 min. To start the reaction 0.5 mL of hyaluronic acid sodium salt
(0.1 M/L) were added. The mixture was incubated at 37 °C for 40 min. After this
0.1 mL of potassium tetraborate 0.8 M was added and it was incubated in waterbath at ebullition for 3 min. The mixture was placed at 10 °C and 3 mL of p-dimethylaminebenzaldehyde were added. Afterwards it was incubated at 37 °C for 20 min.
Finally the absorbance was measured at 585 nm using water as control. All the tests
were performed in triplicate.
2.5. Antimicrobial activity
In the present study it were used microorganisms isolated from biological
fluids, collected in the Hospital Centre and identified in the Microbiology Laboratory of Escola Superior Agrária de Bragança and reference strains obtained from
the authorised distributor of ATCC (LGC Standards S.L.U., Barcelona). The microorganisms were Staphylococcus aureus ATCC 6538™, S. aureus ESA 159, Pseudomonas
aeruginosa ATCC 15442™, P. aeruginosa ESA 22, Escherichia coli ATCC 25922™,
E. coli ESA37, Candida glabrata ATCC 66032™, C. glabrata ESA 123. The isolates were
The determination of the antimutagenic activity of bee pollen extracts was performed using yeast cells (D7 diploid strain of Saccharomyces cerevisiae ATCC
201137) according to the recommended by Zimmermann (1984). Prior to each
experiment the S. cerevisiae D7 strain (MATa/MATa, ade2-40/ ade 2-119, trp 5-12/
trp 5-27, ilv 1-92/ilv 1-92) was tested for the frequency of spontaneous convertants
at the tryptophan (trp) locus and revertants at the isoleucine (ilv) locus. Cells from a
culture with low spontaneous gene conversion and reverse point mutation frequencies were grown in a liquid medium at 28 °C until they reached the stationary
growth phase. The yeast cells were pelleted and re-suspended in a volume of
0.1 M sterile potassium phosphate buffer, pH 7.4, to obtain a final mixture of
2 108 cell/mL. The mixture (4 mL) contained 1 mL of cell suspension, potassium
phosphate buffer and methanolic extracts of bee pollen, in order to reach final concentrations of 0.00, 0.25, 0.50 and 0.75 mg/L. The mixture was incubated under
shaking for 2 h at 37 °C. Then the cells were plated in complete and selective media
to ascertain survival, trp convertants and ilv revertants. Ethyl methanesulfonate
(EMS), a mutagenic compound, was used as control.
2.7. Antioxidant activity
2.7.1. Inhibition of lipid peroxidation using thiobarbituric acid reactive substances
(TBARS)
Livers were obtained from pigs with an approximate body weight of 150 kg,
homogenised with a Polytron in ice-cold Tris–HCl buffer (20 mM, pH 7.4), in order
to produce a 1:2 (w/v) tissue homogenate, centrifuged at 3000g for 10 min. The bee
pollen extract (0.2 mL) was added to 0.1 mL of the supernatant, and incubated in
the presence of FeSO4 (10 lM; 0.1 mL) and ascorbic acid (0.1 mM; 0.1 mL) at 37 °C
for 1 h. The reaction was stopped by the addition of trichloroacetic acid (28% w/v,
0.5 mL), followed by thiobarbituric acid (TBA, 2% w/v, 0.38 mL). The obtained mixture was then heated at 80 °C for 20 min. After centrifugation at 3000g for 10 min to
remove the precipitated protein, the colour intensity of the malondialdehyde–TBA
complex in the supernatant was measured by its absorbance at 532 nm. The inhibition ratio (%) was calculated using the formula: Inhibition ratio (%) = [(A B)/
A] 100%, where A and B were the absorbance of the control and the sample
solution, respectively. The extract concentration providing 50% lipid peroxidation
inhibition (EC50) was calculated by interpolation from the graph of antioxidant
activity percentage against extract concentration (Ferreira et al., 2009). Butylated
hydroxyanisole was used as standard.
2.7.2. Scavenging of DPPH
The scavenging of DPPH radical was assayed following the method described by
Morais et al. (2011). Various concentrations of extracts of bee pollen (300 lL) were
mixed with 2.7 mL of a MeOH solution containing DPPH radicals (6 10 5 mol/L).
The mixture was shaken vigorously and left in the dark, until stable absorption values were obtained. The reduction of the DPPH radical was measured by continuously monitoring the decrease of absorption at 517 nm. The radical-scavenging
activity was calculated as a percentage of DPPH discoloration using the equation:
%RSA = [(ADPPH AS)/ADPPH] 100, where AS is the absorbance of the solution when
the sample extract has been added at a particular level and ADPPH is the absorbance
of the DPPH solution. The extract concentration providing 50% of radical scavenging
activity (EC50) was calculated by interpolation from the graph of RSA percentage
against extract concentration. The standards used were BHA and a-tocopherol.
2.8. Statistical analysis
Each bee pollen sample was analysed in triplicate. Results are shown as arithmetic mean values ± standard deviation. In each parameter the differences between
the samples were analysed using one-way analysis of variance (ANOVA) followed
by Tukey’s HSD. P values less than or equal to 0.05 were evaluated as statistically
significant. In addition, it were computed Pearson’s correlation coefficients between
A. Pascoal et al. / Food and Chemical Toxicology 63 (2014) 233–239
the phenolic compounds, flavonoids, antioxidant activity (assessed by TBARS and
DPPH methodologies) and the values of minimum inhibitory concentration for
the microorganisms under study. These treatments were carried out using SPSS
version 21.0. In order to determine which characteristics would be more useful to
classify the pollen samples a discriminant analysis was performed using the quadratic different covariances and the stepwise variable selection methods. The efficiency of the discriminant power of the models selected was assessed by the test
of the Wilks’ lambda value. Results were analysed in terms of the absolute assignment of individuals to the pre assigned group, the variance explained by each
canonical likelihood, and by the analysis of the standardised scoring coefficients.
Statistical analysis was performed using the statistical package JMP Pro 10.
3. Results
3.1. Total phenolics and flavonoids
In Table 1 it are presented the contents of phenolic compounds
and flavonoids of the different bee pollen samples. Highly significant differences (P < 0.001) were found using the Tukey test for
both parameters. Sample E presented the highest value of phenolic
compounds (32.15 ± 2.12 mg GAE/g pollen) and sample H the
lowest (18.55 ± 0.95 mg GAE/g pollen). Regarding the amount of
flavonoids, the highest value was obtained for sample C
(10.14 ± 1.57 mg CAE/g pollen) and the lowest for sample H
(3.92 ± 0.68 mg CAE/g pollen), even though this sample was not
significantly different from samples A, B, F and G.
3.2. Anti-inflammatory activity
Fig. 1 shows the anti-inflammatory activity of the eight bee pollen samples, where the highest was registered for sample D
(25.17 ± 3.18%), followed by sample B (23.60 ± 2.17%). It were not
Table 1
Concentration (mg/g) of phenolics and flavonoids in pollen extracts from different
samples (n = 24).
Sample
Phenolics (mg GAEs/g pollen)
Flavonoids (mg CAEs/g pollen)
A
B
C
D
E
F
G
H
23.14 ± 0.65dc
28.83 ± 2.59ab
25.31 ± 1.20bc
28.26 ± 0.77abc
32.15 ± 2.12a
24.10 ± 2.72bc
27.82 ± 2.80abc
18.55 ± 0.95d
4.36 ± 0.28c
4.63 ± 0.73c
10.14 ± 1.57a
9.25 ± 0.86ab
7.51 ± 0.96b
3.71 ± 0.25c
4.91 ± 0.83c
3.92 ± 0.68c
P-value
<0.001
<0.001
a, b, c, d – means with different superscripts are significantly different for each
attribute (P = 0.05).
235
found significant differences for these two samples (P < 0.05). The
lowest percentage of inhibition was obtained for sample H
(12.20 ± 1.40%), even though the anti-inflammatory activity of this
bee pollen did not differ significantly from the obtained for
samples A, C, E and F.
3.3. Antimicrobial activities of pollen samples
The minimum inhibitory concentrations (MIC) obtained for the
bacteria and yeasts, both isolated from biological fluids and reference strains, are presented in Table 2. It can be observed that the
antimicrobial activity varied significantly with the pollen sample,
for all the strains under study. For all the cases, bacteria were
much more sensitive to the bee pollens’ action. The most sensitive microorganisms were both strains of S. aureus, one of the
most representative bacteria involved in respiratory diseases, particularly when it was used sample H, with MIC values of
1.81 ± 0.29 mg/mL (S. aureus ATCC 6538™) and 2.58 ± 0.63 mg/
mL (S. aureus ESA 159). For this microorganism the less efficient
bee pollen was sample E. Regarding P. aeruginosa, the most efficient pollen sample was H, with MIC values of 3.71 ± 0.72 and
5.23 ± 0.37 mg/mL, for the reference and isolated strain, respectively. E. coli appeared as the most resistant of the assayed bacterial strains, for all the tested extracts. The MIC for this bacteria
ranged from 4.08 ± 0.38 to 5.65 ± 0.61 mg/mL, for the reference
strain, and from 6.19 ± 0.73 to 9.42 ± 0.52 mg/mL, for the isolated
strain. Also for this microorganism, the most efficient pollen sample was H. Concerning the assays carried out with C. glabrata the
pollen with higher inhibitory action was sample E, with values of
16.00 ± 1.32 mg/mL for the reference strain and 22.67 ± 2.25 mg/
mL for the isolated one.
In all the microorganisms under study it was observed that the
reference strains were more sensitive than the isolated from biological fluids. As expected, the controls gentamicine (antibacterial)
and fluconazol (antifungal) presented lower MIC than the pollen
extract.
3.4. Antimutagenic activity
In the present study, the anti-genotoxic effects of bee pollen
on S. cerevisiae were tested up to 0.75 mg/mL in a 0.5% fixed ethyl
methanesulfonate (EMS) concentration, which produces random
mutations in genetic material by nucleotide substitution, being
an alkylating agent. Samples D and F had the smallest value of
survivals, with percentages of 63.58 ± 1.74% and 69.76 ± 1.56%,
respectively. It was also observed that the higher the concentration of pollen, the lower the percentage of survival, what is related to its antifungal effect. The results obtained in the present
study for the antimutagenic activity reveal that all the samples
had substantial antigenotoxic activity, since they all decreased
the frequencies of gene conversion (Table 3). The highest number
of gene conversion colonies obtained was equal to
4.00 106 ± 3.17 105, corresponding to sample A, and the
lowest was obtained for sample H, with a value of
3.5 104 ± 2.0 103. However, only two samples (D and H) significantly reverted the mutations over the entire range of concentrations used.
3.5. Antioxidant activity of pollen samples
Fig. 1. Anti-inflammatory activity of the bee pollen samples.
The antioxidant activity ranged from 0.35 ± 0.02 (B) to
3.70 ± 0.00 (D) mg/mg extract, for the DPPH method, and from
2.98 ± 0.47 (B) to 6.69 ± 0.75 (D), using TBARS (Table 4). Significant
differences were obtained, for both methodologies, between the
eight analysed samples.
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A. Pascoal et al. / Food and Chemical Toxicology 63 (2014) 233–239
Table 2
Minimum inhibitory concentration (mg/mL) for the studied microorganisms.
Sample
S. aureus
ATCC 6538™
S. aureus
ESA 159
P. aeruginosa
ATCC™
P. aeruginosa
ESA 22
E. coli ATCC™
E. coli ESA 37
C. glabrata
ATCC™
C. glabrata
ESA 123
A
B
C
D
E
F
G
H
3.07 ± 0.72ab
2.06 ± 0.13ab
2.88 ± 0.42ab
2.64 ± 0.87ab
3.55 ± 0.23a
2.41 ± 0.61ab
2.84 ± 0.76ab
1.81 ± 0.29b
4.01 ± 0.44ab
3.12 ± 0.38ab
3.54 ± 0.84ab
4.28 ± 0.38a
4.19 ± 0.48a
3.74 ± 0.28ab
3.59 ± 0.64ab
2.58 ± 0.63b
5.69 ± 0.74a
3.99 ± 0.13ab
4.01 ± 1.24ab
4.78 ± 0.30ab
4.95 ± 0.28ab
4.88 ± 0.36ab
3.74 ± 0.60b
3.71 ± 0.72b
6.43 ± 1.05ab
5.04 ± 0.07b
5.62 ± 0.58ab
6.96 ± 0.17a
6.67 ± 0.39a
6.82 ± 0.40a
6.37 ± 0.69ab
5.23 ± 0.37b
5.23 ± 0.70ab
4.27 ± 0.40ab
4.73 ± 0.40ab
4.97 ± 0.41ab
4.17 ± 0.52b
5.57 ± 0.39a
5.65 ± 0.61a
4.08 ± 0.38b
6.75 ± 0.75bc
6.92 ± 1.18bc
7.50 ± 0.90abc
8.98 ± 0.78ab
7.42 ± 0.80abc
8.33 ± 0.63abc
9.42 ± 0.52a
6.19 ± 0.73c
25.23 ± 5.11ª
20.83 ± 1.44abc
24.17 ± 1.44ª
24.33 ± 1.61ª
16.67 ± 1.44bc
22.50 ± 2.50abc
16.00 ± 1.32c
23.33 ± 1.44ab
28.76 ± 1.56abc
33.92 ± 1.88ª
28.23 ± 1.95bcd
33.42 ± 1.28ad
24.38 ± 1.66cd
32.50 ± 2.50ab
22.67 ± 2.25d
32.50 ± 2.50ab
P-value
0.036
0.021
0.014
0.006
0.004
0.002
0.001
<0.001
a, b, c, d – means with different superscripts are significantly different for each attribute (P = 0.05).
Table 3
Effect of bee pollen extract on the percentage of survival of yeast cells and antigenotoxicity (mutagenesis and gene conversion).
Gene conversion colonies/105
Mutants colonies/106
Sample
Pollen concentration (mg/L)
EMS (%)
Survivals (%)
–
0.00
0
100.00 ± 0.00
0.80 ± 0.05
0.35 ± 0.04
A
0.00
0.25
0.50
0.75
0.50
0.50
0.50
0.50
88.18 ± 1.60a
81.00 ± 4.61ab
81.81 ± 2.11ab
77.64 ± 2.38b
52.55 ± 3.19b
35.71 ± 4.50a
37.33 ± 1.27a
40.01 ± 3.17a
380.56 ± 7.67b
323.60 ± 18.12a
321.58 ± 17.92a
410.66 ± 1.68b
B
0.00
0.25
0.50
0.75
0.50
0.50
0.50
0.50
85.67 ± 1.74a
84.44 ± 2.31a
81.45 ± 2.04a
80.22 ± 3.99a
53.75 ± 2.62b
33.52 ± 2.30a
35.20 ± 3.75a
34.15 ± 2.63a
369.32 ± 6.49a
390.63 ± 10.87ab
417.67 ± 5.33bc
439.15 ± 13.49c
C
0.00
0.25
0.50
0.75
0.50
0.50
0.50
0.50
88.20 ± 2.11a
88.17 ± 5.33a
84.97 ± 1.86a
85.36 ± 5.29a
51.37 ± 2.24b
32.89 ± 2.26a
34.65 ± 4.22a
39.92 ± 3.20a
379.63 ± 8.76b
308.98 ± 8.97a
314.55 ± 4.17a
403.28 ± 11.40b
D
0.00
0.25
0.50
0.75
0.50
0.50
0.50
0.50
83.07 ± 9.07b
74.85 ± 5.39ab
69.51 ± 2.70ab
63.58 ± 1.74a
57.15 ± 4.51b
25.46 ± 1.91a
24.24 ± 3.06a
16.88 ± 1.37a
408.87 ± 8.38b
68.44 ± 4.54a
68.62 ± 2.26a
67.96 ± 2.91a
E
0.00
0.25
0.50
0.75
0.50
0.50
0.50
0.50
91.04 ± 2.63a
86.57 ± 2.88a
85.14 ± 3.90a
80.77 ± 4.59a
54.74 ± 3.79b
33.97 ± 4.56a
36.62 ± 4.54a
32.04 ± 3.29a
376.51 ± 9.16a
405.91 ± 4.14ab
388.73 ± 8.30ab
418.70 ± 10.67b
F
0.00
0.25
0.50
0.75
0.50
0.50
0.50
0.50
84.22 ± 6.29b
76.56 ± 3.86ab
71.28 ± 3.10ab
69.76 ± 1.56a
56.41 ± 4.53b
0.42 ± 0.02a
0.44 ± 0.04a
0.51 ± 0.03a
384.88 ± 19.28b
328.54 ± 10.67a
380.45 ± 10.07ab
411.12 ± 16.26b
G
0.00
0.25
0.50
0.75
0.50
0.50
0.50
0.50
89.23 ± 3.11a
85.96 ± 1.95a
85.14 ± 2.37a
78.87 ± 6.19a
51.0 ± 4.02b
0.44 ± 0.03a
0.46 ± 0.04a
0.49 ± 0.03a
380.03 ± 14.63a
378.96 ± 18.27a
398.11 ± 14.08a
408.83 ± 13.24a
H
0.00
0.25
0.50
0.75
0.50
0.50
0.50
0.50
85.89 ± 4.40a
83.05 ± 5.93a
86.60 ± 4.81a
85.04 ± 3.77a
52.34 ± 3.94b
0.34 ± 0.04a
0.38 ± 0.02a
0.35 ± 0.02a
407.23 ± 16.70b
80.51 ± 5.25a
72.39 ± 5.91a
71.82 ± 2.68a
a, b, c, d – means with different superscripts are significantly different for each attribute (P = 0.05).
3.6. Correlation and discriminant analysis
4. Discussion
Pearson’s correlation coefficients between the phenolic
compounds, flavonoids, TBARS, DPPH, anti-inflammatory
activity, antioxidant activities (TBARS and DPPH) and MICs
for the microorganisms studied were computed and are presented in Table 5. The phenolic compounds had a significant
and positive correlation with the anti-inflammatory activity
and a significant and negative correlation with the antimicrobial activity obtained for C. glabrata ATTC. Scatter plot of the
first two canonical variables, of eight pollen samples considered (Fig. 2) showed that the groups were discriminated with
great accuracy.
4.1. Total phenolics and flavonoids
According to the literature, phytochemicals such as phenolic
compounds are considered beneficial for human health since they
decrease the risk of degenerative diseases by reducing oxidative
stress and inhibiting macromolecular oxidation (Silva et al.,
2004). The high ability of the phenolic compounds to neutralize
the active oxygen species is strongly associated with their
structure such as the conjugated double bonds and the number
of hydroxyl groups in the aromatic ring mostly attributed to flavonoids and cinnamic acid derivatives (Leja et al., 2007). Flavonoids
237
A. Pascoal et al. / Food and Chemical Toxicology 63 (2014) 233–239
Table 4
Antioxidant activity of pollen samples.
Sample
TBARS (mg/mg extract)
bc
DPPH (mg/mg extract)
A
B
C
D
E
F
G
H
1.59 ± 0.27
0.35 ± 0.02d
1.08 ± 0.01c
3.70 ± 0.00a
2.16 ± 0.03b
1.57 ± 0.48c
3.34 ± 0.19ª
1.01 ± 0.10c
6.63 ± 0.59ª
2.98 ± 0.47d
4.88 ± 0.50bc
6.69 ± 0.75ª
5.14 ± 0.58ª
4.44 ± 0.79bcd
6.69 ± 0.56ª
3.32 ± 0.56cd
P-values
<0.001
<0.001
a, b, c, d – means with different superscripts are significantly different for each
attribute (P = 0.05).
have a wide range of biological activities, including antibacterial,
antiviral, anti-inflammatory, antiallergic, as well as vasodilatory
actions (Abdella et al., 2009). In addition, flavonoids inhibit lipid
peroxidation, platelet aggregation, capillary permeability and fragility, and the activity of enzyme systems including cyclo-oxygenase and lipoxygenase (Estevinho et al., 2008; Viuda-Martos et al.,
2008). The results obtained in the present study were higher than
the observed by Morais et al. (2011), who obtained concentrations
of polyphenols between 10.5 and 16.8 mg GAE/g for honeybee-collected pollen from Portuguese Natural Parks. In the other hand, our
results are in agreement with the obtained by Kroyer and Hegedus
(2001) in pollen collected in Vienna; and are slightly superior to
the results of Campos et al. (2003), who analysed samples from
New Zealand and Portugal. Carpes et al. (2009) obtained higher
values in Brazilian pollens, ranging from 19.28 to 48.90 mg GAE/
g of pollen. Higher flavonoid contents were obtained by Carpes
et al. (2009). According to the later study, the discrepancies between the polyphenols’ and flavonoids concentration might be related to the differences on the botanical and geographical origins.
4.2. Anti-inflammatory activity
The inflammation process involves production and/or release of
mediators from neurons or damaged tissues, which are responsible
for different responses, including pain. Scavenging of free radicals,
generated by neutrophils, is the principal mechanism of conventional drugs (Paulino et al., 2003).
Hyaluronic acid is a polyanionic high molecular mass polysaccharide found in the extracellular matrix, sensitive to oxidantmediated fragmentation (Gao et al., 2008). The degradation of
the hyaluronic acid by the hyaluronidase enzyme may cause bone
loss, inflammation and pain. As consequence, the determination of
the hyaluronidase enzyme is an indirect way to assess the
Fig. 2. Scatter plot of the first two canonical variables, of eight groups considered.
anti-inflammatory activity (Silva et al., 2012). The lack of studies
concerning this biological activity in this specific matrix hampers
comparisons. However, the activity of bee pollen is lower than
the obtained, using the same methodology, for propolis (Silva
et al., 2012), what might be related with the lower amount of
polyphenols.
4.3. Antimicrobial activities of pollen samples
Very similar MIC values were obtained by Morais et al. (2011),
who analysed bee pollen from Portugal. In previous studies it has
been observed that the most sensitive microorganism to the action
of poppy pollen ethanolic extract was also S. aureus (FatrcováŠramková et al., 2013). The results hereby reported were slightly
superior to the obtained by Morais et al. (2011), what may be related with the different microorganism origin, since in the present
study the bacteria was isolated from biological fluids and in the
previous study it was collected from food products. As reported
in the literature it has been observed that the Gram-negative
bacteria were more resistant to the pollen action than the Grampositive bacteria, what may be related with the additional outer
layer membrane, impermeable to most molecules, that consists
of phospholipids, proteins and lipopolysaccharides (Silici and Kutluca, 2005). In all the microorganisms under study it was observed
that the reference strains were more sensitive than the isolated
from biological fluids, what has already been reported in other beehive products (Silva et al., 2012). This suggests that bee pollen
could be used combined with antibiotics, since, as far as we know;
there are not microorganisms capable of acquiring resistance to
bee pollen.
Table 5
Pearson’s correlation coefficients between phenolic compounds, flavonoids, antioxidant activity (determined using the methods TBARS and DPPH) and MIC the microorganisms
under study.
Phenolics
Flavonoids
TBARS
DPPH
Hyal. inhibition
S. aureus ATCC
S. aureus ESA
P. aeruginosa ATCC
P. aeruginosa ESA
E. coli ATCC
E. coli ESA
C. glabrata ATCC
C. glabrata ESA
*
corresponds to values <0.05 and
**
Phenolics
Flavonoids
TBARS
DPPH
1
0.369
0.338
0.083
0.477*
0.391
0.401
0.143
0.259
0.004
0.169
0.555**
0.303
1
0.302
0.286
0.205
0.333
0.263
0.033
0.203
0.177
0.179
0.144
0.143
1
0.730**
0.420*
0.303
0.486*
0.094
0.652**
0.388
0.723**
0.237
0.346
1
0.221
0.352
0.372
0.184
0.434*
0.265
0.454*
0.058
0.495*
to values <0.001.
Hyal. inhibition
1
0.149
0.232
0.112
0.047
0.002
0.126
0.221
0.151
238
A. Pascoal et al. / Food and Chemical Toxicology 63 (2014) 233–239
4.4. Antimutagenic activity
Recently, a considerable emphasis is being laid down on the use
of dietary constituents as chemoprotective measure for control of
neoplastic and genetic diseases (Abdella et al., 2009). Some studies
have lauded the antimutagenic properties of diverse beekeeping
products against the influence of some chemical and physical
mutagens (Bariliak et al., 1996). Most of the protective agents react
directly with the mutagen or interfere with the free radicals, free
oxygen species produced or inhibit cytochrome P450-mediated
metabolism. The specific mechanism of protection of the bee pollen extract is reported to involve the scavenging of potentially
toxic and mutagenic electrophiles and free radicals and the modification of antioxidant pathways (Ohta et al., 2007).
4.5. Antioxidant activity of pollen samples
The determination of the antioxidant activities is strongly influenced by the experimental conditions and heterogeneity of matrix,
reason by which it is desirable to assess this biological activity
using two methodologies (Sakanaka and Ishihara, 2008). In the
present study it were used two methods: scavenging of DPPH
and inhibition of lipid peroxidation (TBARS). Silva et al. (2006) reported that the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical is
one of the few stable organic nitrogen free radicals, being widely
used to determine the free radical scavenging ability of diverse
samples, either natural or synthetic. In the first method, results
are expressed as the ratio percentage of the absorbance decrease
of the DPPH radical solution in the presence of the extract. Regarding the second method, the reaction of malondialdehyde, which is
formed by the degradation of fatty acids by free radicals, with 2thiobarbituric acid is one of the most widely used estimators of
oxidative stress (Liu et al., 1997). The results obtained with these
methods were presented as EC50, which is the amount of antioxidant necessary to decrease by 50% the initial DPPH concentration
(Table 4). The results obtained were identical to the reported by
Morais et al. (2011). These authors found EC50 values that ranged
from 2.16 to 5.87 mg/mg extract in pollen samples collected from
five Portuguese Natural-Parks. Almaraz-Abarca et al. (2004) verified that the antioxidant activity, in vitro and in vivo, is related to
the amount of flavonoids present. However, our results, as well
as the obtained by Mărghitasß et al. (2009), revealed that there is
no strong relation between the phenolic compounds and the antioxidant activity.
4.6. Correlation and discriminant analysis
Since C. glabrata ATTC was the only microorganism whose MIC
was significantly correlated with polyphenols, while in the other
microorganisms it was related with the antioxidant activity, further studies must be conducted to elucidate the action mechanisms of bee pollen in the cells, both prokaryotic and eukaryotic.
TBARS was significantly and positively correlated with DPPH,
suggesting that both methods may be applied to determine the
antioxidant activity of bee pollen. Another interesting correlation
was found between antioxidant and anti-inflammatory activities.
Indeed, according to the literature oxidants play a significant role
in the pathogenesis of many disorders among which inflammation
(Geronikaki and Gavalas, 2006).
The first canonical dimension explained 53.85% of the total variance and the second 18.14% and a total of 72.2% of variance were
accounted for by the two canonical variables. The model accepted
one more canonical variable (P < 0.0001) and more 17.21% of total
variance were accounted reaching 89.40% of the total explained
variance for by the three canonical variables. The 98.19% of total
variance explains was obtained with a fourth canonical variable
(P < 0.0001) and all individuals of each group were assigned to
the corrected group with 100% of classified correctly. The fourth
canonical variables discriminated all 8 groups with great accuracy
and the more useful characteristics to classify the pollen samples
were TBARS, phenolics, flavonoids and anti-inflammatory activity.
5. Conclusions
All the bee pollen samples had substantial antimicrobial activity, being Gram-positive bacteria the most sensitive. In addition,
the reference strains were more sensitive than the isolated from
biological fluids. It was also demonstrated the antimutagenicity
of the eight commercial bee pollens against EMS using a S. cerevisiae D7 strain, even though only two samples significantly reverted
the mutations. Finally, the results obtained in this study demonstrated that bee pollen possesses good antioxidant activity,
suggesting that it could be useful in prevention of diseases in
which free radicals are implicated. Further in vivo studies must
be conducted in order to elucidate the action mechanisms underlying these beneficial biological properties and to determine the
functional significance of the present results.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgment
A. Pascoal would like to thank Fundação para a Ciência e Tecnologia (FCT) for is Post-doctoral Grant SFRH/BPD/91380/2012.
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