NUTRITIONAL POTENTIAL

Epub ahead of print1Journal of Integrative Medicine
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● Research Article
Investigation of the nutraceutical potential of
monofloral Indian mustard bee pollen
Sameer S. Ketkar1
, Atul S. Rathore1
, Sathiyanarayanan Lohidasan1
, Lakshmi Rao2
, Anant
R. Paradkar3
, Kakasaheb R. Mahadik1
1. Centre for Advanced Research in Pharmaceutical Sciences, Poona College of Pharmacy, Bharati Vidyapeeth
University, Pune-411038, India
2. Central Bee Research and Training Institute, Pune-411016, India
3. Centre for Pharmaceutical Engineering Sciences, University of Bradford, Bradford, West Yorkshire, BD7
1DP, UK
OBJECTIVE: This study was designed to investigate the nutraceutical potential of monofloral
Indian mustard bee pollen (MIMBP).
METHODS: The nutritional value of MIMBP was examined in terms of proteins, fats, carbohydrates,
and energy value. Its chemical composition in terms of total polyphenol and flavonoid content
was determined. MIMBP was screened for free flavonoid aglycones by developing and validating
a high-performance liquid chromatography-photo diode array (HPLC-PDA) method. MIMBP
was analyzed for in vitro antioxidant effect in terms of 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical-scavenging activity.
RESULTS: MIMBP was found to be comprised of proteins ((182.2±5.9) g/kg), fats ((137.7±6.8) g/kg)
and carbohydrates ((560.6±17.4) g/kg), which result in its high energy value ((17 616.7±78.6) kJ/kg).
MIMBP was found to contain polyphenols ((18 286.1±374.0) mg gallic acid equivalent/kg) and
flavonoids ((1 223.5±53.1) mg quercetin equivalent/kg). The HPLC-PDA analysis revealed the
presence of kaempferol ((65.4±0.5) mg/kg) and quercetin ((51.4±0.4) mg/kg) in MIMBP, which
can be used as markers for determining the quality of bee pollen. The MIMBP extract showed
DPPH free radical-scavenging activity with a half maximal inhibitory concentration of 54.79 µg/mL.
CONCLUSION: The MIMBP was found to be a rich source of nutrients providing high caloric value,
which makes it a candidate for a potential nutraceutical agent. The study also illustrated the
high antioxidant content of MIMBP, especially in the principle polyphenols and flavonoids, which
suggests its potential role in the prevention of free radical-implicated diseases. The DPPH-scavenging
effect of MIMBP further confirmed its antioxidant potential. Additionally, we developed a simple,
specific and accurate HPLC-PDA method for the identification and quantification of free flavonoid
aglycones. This can be applied in future screenings of the quality of pollen collected by honeybees.
KEYWORDS: dietary supplements; bee pollen; flavonoids; mustard plants
http://dx.doi.org/10.1016/S2095-4964(14)60033-9
Ketkar SS, Rathore AS, Lohidasan S, Rao L, Paradkar AR, Mahadik KR. Investigation of the nutraceutical
potential of monofloral Indian mustard bee pollen. J Integr Med. 2014 July; Epub ahead of print.
Received March 14, 2014; accepted May 14, 2014.
Correspondence: Kakasaheb R. Mahadik, PhD, Professor; Tel: +91-2025437237; E-mail: krmahadik@
rediffmail.com

Epub ahead of print 2 Journal of Integrative Medicine
1 Introduction
Apiculture, the science of bee keeping, has contributed
to the field of nutrition and medicine by providing access
to apiproducts such as honey, propolis, and royal jelly,
which have demonstrated beneficial properties as nutraceutical
agents[1,2]
. Bee pollen is plant pollen collected from different
sources by the worker honeybee Apis melliferra feeding
its larvae in the early stages of development. Bee pollen
is known to be a rich source of polyphenols, flavonoids,
sugars, proteins, amino acids, fatty acids, minerals and
vitamins which makes it relevant and useful for humans[3-6]
.
Analyses of bee pollen’s chemical composition have reported
it to be the “only perfectly complete food”[7]
that possesses
a wide array of pharmacological activities including being
antioxidant, anti-inflammatory, antimicrobial, immuno-
modulatory, antiatherosclerotic, antianaemia, antiosteo-
porosis and antiallergic as shown in multiple studies[8-14]
.
Bee pollen has also demonstrated clinical relevancy for its
anti-prostatic effect in humans[15]
.
Studies on the palyonology, chemical composition and
benefits of bee pollen have been conducted in various regions
including Australia[16,17]
, Brazil[18]
, China[19]
, Chile[20]
,
Portugal[21]
, South Africa[22]
, and the Sonoran Desert,
USA[23]
. Bee pollen is currently used as a functional food
or supplementary nutrition in some of these countries,
however to our knowledge there are no studies examining
the composition or benefits of bee pollen from Indian
sources. Major regions for apicultural activities in India
include Punjab, Jammu-Kashmir, Himachal Pradesh,
Uttar Pradesh, Haryana, Bihar and West Bengal[24,25]
. The
forests, farms around sub Himalayan tracts, cultivated
vegetation in Madhya Pradesh, Rajasthan, Eastern Ghats
in Andhra Pradesh and Maharashtra are known to be the
major regions for honey collection[24-26]
. These regions are
home to diverse flora of nectariferous and polliniferous
plant species, which are prime conditions of apiculture
that help produce high-quality pollen. The current study
was designed with the aim of exploring the nutritional
value and chemical composition of Indian bee pollen in
order to assess its utility as a nutraceutical agent.
The composition of bee pollen varies with the plant
source and geographic origin. Standard quality pollen
with minimal variations, obtained by collecting bee pollen
from single botanical taxa, is termed monofloral pollen[27]
.
Monofloral pollen ensures uniform organoleptic and
biochemical characteristics to that of the original plant,
while heterofloral pollen exhibits variable properties[27]
.
Among various pollen-yielding sources in India, mustard
crops (Family: Brassicaceae) are one of the major sources.
Worldwide, these are used as extensive dietary crops and
possess economic significance[28,29]
. The phenolic composition
of Brassica vegetables has been well established, however
the nutritional and chemical composition of pollen from
these sources is not yet understood. Therefore, the current
study was designed to recognize floral origin and nutri-
tional value in terms of proteins, fats, carbohydrates, and
energy value; to determine chemical composition in terms
of total polyphenols and flavonoid content of monofloral
Indian mustard bee pollen (MIMBP), i.e., Brassica
juncea; and to develop a simple, specific and accurate
high-performance liquid chromatography-photo diode
array (HPLC-PDA) method for identification and quantifi-
cation of free flavonoid aglycones from the bee pollen.
2 Materials and methods
2.1 Bee pollen material and chemicals
The MIMBP pellets were collected from 24 Parganas
district of West Bengal, India during December 2012 to
January 2013. The collected fresh pollen pellets were
hand-sorted by appearance to avoid possible contamination
of pollen from other sources. The pollen samples were
identified and authenticated by Central Bee Research and
Training Institute, Pune, India (Voucher Specimen No (1/
WB/2012)). The fresh pollen was dried at temperatures
below 40 ℃, vacuum packed in food-grade polyethylene
bags and stored in a -15 ℃ freezer throughout the study.
All the analysis was performed within a period of one
month after pollen collection in order to best preserve its
nutritive value and free radical-scavenging capacity, and
avoid possible age-induced degradation of the pollen[8]
.
No signs of degradation or fermentation were observed
on the stored samples. The samples were sieved by 200 µ
mesh before analysis. Analytical standards of gallic acid,
rutin, chrysin, kaempferol and quercetin were procured
from Merck, USA. Aluminum chloride, mercuric oxide,
sodium carbonate, sodium hydroxide, and sodium sulfate
were procured from Sigma Aldrich, USA. Folin-Ciocalteu’s
phenol reagent, sulfuric acid, hydrochloric acid, O-phosphoric
acid and petroleum ether, and methanol (HPLC-grade)
were procured from Merck, USA. All reagents used during
the study were of analytical research grade. Distilled water
was used throughout the study.
2.2 Sensory analysis and microscopic examination
The MIMBP was subjected to sensory analysis in terms
of color, appearance, odor and taste. The pollen sample
was observed under a scanning electron microscope, Oxford
Instruments, Inca X Sight Model No. 6650-M. The pollen
was scattered on a 12 mm carbon grid attached to scanning
electron microscope specimen mounts and were sputter-
coated with a layer of gold/palladium. The pollen was
then subjected to standard acetolysis method[30]
followed
by microscopic examination using Nicon E800 Eclipse
compound microscope in phase contrast mode with Image

ProPlus software. Measures of polar axis (P), equatorial
diameter (E), colpus length (CL) and exine thickness were
determined for 30 pollen grains under the microscope
(×40).
2.3 Determination of nutrient composition
2.3.1 Moisture content
The moisture content of MIMBP was determined by
repeated drying of sample in an oven at about 105 ℃ until
constant weight was obtained[31]
.
2.3.2 Total protein content
The total protein content was determined by Kjeldahl’s
method[31,32]
wherein MIMBP (2 000 mg) was subjected
to digestion by heating with a mixture of sodium sulfate
(5 000 mg) and mercuric oxide (300 mg) in concentrated
sulfuric acid (25 mL) for about 6 h. The diluted sample
solution was distilled with 0.1% sulfuric acid (50 mL)
followed by addition of 8% sodium sulfate (13 mL) and
40% sodium hydroxide (50 mL). The ammonia collected
(150 mL) was titrated with hydrochloric acid (0.1 mol/L).
Protein content was estimated by multiplying the obtained
percentage of nitrogen by a conversion factor of 5.6.
2.3.3 Total fat content
Fat or lipid content of MIMBP (5 000 mg) was determined
by extracting with petroleum ether in a Soxhlet extractor
at about 100 ℃/12 h[33]
. The extraction flask was subjected
to a heating and cooling cycle to evaporate the solvent
completely followed by weighing of the mass. The difference
in weight of the flask before and after extraction was
correlated with fat content of the sample.
2.3.4 Ash content
The ash content of MIMBP was determined by drying
the sample at (550 ± 20) ℃ in a muffle furnace until constant
weight[34]
.
2.3.5 Total carbohydrate content
Total carbohydrate content of MIMBP was determined
by method based on calculating nutrient values from other
components in the sample using the following formula[35,36]
:
Carbohydrates (g) = 100 – (protein (g) + fat (g) + mois-
ture (g) + ash (g))
Total carbohydrate content of the sample estimated
includes dietary fiber, as well as other components of the
sample that are not lipid, protein, ash or water[35]
.
2.3.6 Energy value
The energy value for MIMBP was calculated based on
Atwater numbers[35,36]
using the following formula:
Energy (kJ) = 1/0.239 × [4 × (protein (g) + carbohydrate
(g)) + 9 × (fat (g))].
2.4 Determination of chemical composition
2.4.1 Preparation of bee pollen extract
Finely ground MIMBP powder (1 000 mg) was mixed
with ethanol (99%; 20 mL), vortexed for 10 min and
extracted at 70 ℃ on a rotating mechanical shaker at
33 987×g for 30 min[37]
. The MIMBP extract obtained
was centrifuged at 33 987×g. The supernatant was filtered
through a 0.45 µm membrane filter and subjected to determi-
nation of total polyphenol content and flavonoid content.
2.4.2 Total polyphenol content
Total polyphenol content of MIMBP extract was de-
termined by Folin-Ciocalteu colorimetric method[38-40]
.
Briefly, MIMBP (1 mL) was mixed with Folin-Ciocalteu’s
phenol reagent (1 mL). Sodium carbonate (7%, 10 mL)
was added to it followed by dilution to 25 mL with distilled
water. Absorbance was measured at 760 nm using Jasco
V-630 UV-Vis spectrometer after 90 min incubation of
mixture at room temperature. The total polyphenol content
was expressed in terms of mg gallic acid equivalent
(GAE)/kg of pollen.
2.4.3 Total flavonoid content
Total flavonoid content of MIMBP extract was determined
by aluminum chloride colorimetric method[39,41]
. To
MIMBP extract (1.5 mL), aluminum chloride ethanolic
solution (1.5 mL, 2%) was added. The mixture was
incubated for 1 h at room temperature and absorbance was
measured at 420 nm. Total flavonoid content of the extract
was expressed in terms of mg quercetin equivalent (QE)/kg
of pollen.
2.5 Determination of free flavonoid aglycones from
bee pollen
2.5.1 Preparation of standard solution
Standard stock solutions of rutin, chrysin, kaempferol
and quercetin (1 mg/mL) were prepared in methanol.
These were diluted with methanol to obtain mixed working
standard solutions of concentration 10, 20, 40, 60, 80 and
100 µg/mL each.
2.5.2 Preparation of MIMBP sample solution
Free flavonoid aglycones from MIMBP were determined
using a process described by Serra Bonvehí et al[42]
with
minor modifications. Finely ground MIMBP powder (2.5 g)
was mixed with ethyl acetate (25 mL). Diammonium sulfate
(40%, 12.5 mL) and meta-phosphoric acid (20%, 2.5 mL)
were added to the mixture, followed by shaking for
20 min. The extract was filtered through a 0.45 µm membrane
filter under pressure. The filtrate was transferred into a
separating funnel. The organic phase was collected and
the extraction process was repeated. The organic phase
collected was dried under reduced pressure at <40 ℃. The
residue was reconstituted in methanol (1.5 mL), filtered
through a 0.45 µm nylon syringe filter and subjected to
HPLC analysis for determination of free flavonoid aglycones.
2.5.3 HPLC-PDA analysis
HPLC analyses were performed using Jasco HPLC
system (Tokyo, Japan) on a Thermo Hypersil BDS C18
guard column (30 mm×4.6 mm, 5 μm) coupled to a
Thermo-Hypersil GOLD C18 RP column (250 mm×4.0 mm,
5 μm) using Jasco PU2089Plus quaternary gradient pump,
Jasco multiwavelength detector (PDA), ChromPass software

and a Rheodyne injector with 20 μL loop. Elution was
carried out with flow rate of 1 mL/min at ambient tem-
perature. The solvents comprised water adjusted to pH
3.0 with ortho-phosphoric acid (solvent A) and methanol
(solvent B) mixed using a linear gradient system; initial
30% B, 30%-50% B in 5 min, 50%-70% B in 10 min,
70%-75% B in 15 min followed by isocratic 75% B until
17 min. Solvent B was decreased to 30% over the next
3 min and held constant until the end of 25 min of run.
Detection was performed between 200 to 400 nm and
chromatograms were extracted at respective λmax of each
flavonoid aglycone compound for improved sensitivity.
The retention times (Rt) and UV spectra of flavonoid aglycones
from the sample solution were compared with that of the
standards and quantification was done using calibration
curves of the standard solutions.
2.5.4 Method validation
HPLC-PDA method was validated for parameters such
as linearity, limit of detection (LOD), limit of quantification
(LOQ), precision, repeatability, specificity and accuracy[43]
.
The serial dilutions of standard solutions were subjected
to HPLC analysis in triplicate. The calibration graphs
were obtained by plotting peak areas against respective
concentrations. Linear calibration curves were established
by employing least-squares linear-regression analysis and
analysis of variance (ANOVA). In order to ascertain the
linearity, residual analysis was performed. The LOD and
LOQ were determined by calibration curve method using
standard deviation (SD) and slope (S) of the calibration
curve. Intraday and interday variability was studied (n=3)
to analyze the precision at three different concentrations.
The repeatability of sample application and measurement
of peak area were expressed in terms of relative standard
deviation (%RSD). Specificity of the method was assessed
by qualitative comparison between chromatograms obtained
from sample, standard and blank solutions. Further, specificity
was ascertained by checking peak purity of standards and
sample solution. Accuracy of the HPLC method was studied
by standard addition technique to calculate percentage
recovery of all the flavonoid aglycone compounds from
the MIMBP sample solution.
2.6 In vitro antioxidant activity of MIMBP
MIMBP were screened for in vitro antioxidant activity
with respect to radical-scavenging activity against
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical as per method
described by Campos et al[8]
, Almaraz-Abarca et al[9]
, and
Leja et al[10]
. The dried MIMBP extract was reconstituted
with ethanol (99%) to prepare different concentrations in
the range of 10-100 µg/mL. The extract solution (1 mL)
was mixed with methanolic solution of DPPH (5 mL;
0.1 mmol/L) and allowed to stand for 20 min at 27 ℃
in a dark place followed by centrifugation at 3 056.1×g
for 5 min. Reduction in DPPH radical concentration was
analyzed by measuring the decrease in absorption at 517 nm
detected by a UV visible spectrometer. DPPH-scavenging
effect was calculated by the following formula:
DPPH-scavenging effect (%) = (Ac - As/Ac) × 100%
Wherein Ac is absorbance of control solution; As is
absorbance of the extract solution. The DPPH-scavenging
effect of MIMBP extract was compared with that of the
standard aqueous ascorbic acid solution.
2.7 Statistical analysis
All samples were analyzed in triplicate unless otherwise
stated and the results were expressed as mean ± SD. The
statistical analysis was carried out using Prism 5.0 version
of Graphpad software. Data for in vitro antioxidant activity
were analyzed by two way ANOVA followed by Bonferroni
post-test. P<0.05 was considered significant.
3 Results
3.1 Sensory analysis and microscopic examination
Sensory analysis revealed that MIMBP were yellowish
brown in color, and spherical to ovate in shape. They
possessed a typical odor for pollen load with a sweet taste
specific to Brassica pollen. Microscopic examination
(Figures 1A, 1B and 1C) revealed that the pollen exhibited
subprolate to suboblate shape, medium trizonocolpate
with ambtrilobed fossaperture confirming that these pollen
belongs to the Indian mustard, i.e., Brassica juncea; Family:
Brassicaceae. The size of individual pollen varies in the
range of 25-30 µm (P: 30.54 µm×E: 25.06 µm). The CL
of pollen grains was found to be 17.3-19.0 µm long, and
0.25-0.32 µm wide. The exine was found to be about
2.6 µm thick with reticulate ornamentation.
3.2 Nutrient composition
The MIMBP have been found to comprise total proteins
((182.2±5.9) g/kg), fats ((137.7±6.8) g/kg), and carbo-
hydrates ((560.6±17.4) g/kg), which together comprise a
high energy value ((17 616.7±78.6) kJ/kg). The moisture
content of MIMBP was (72.6±6.5) g/kg, and ash content
was (25.7±3.5) g/kg.
3.3 Chemical composition: total polyphenol and flavonoid
content
The MIMBP were found to have a total polyphenol content
of (18 286.1±374.0) mg GAE/kg, and a flavonoid content
of (1 223.5±53.1) mg QE/kg. Major polyphenol groups
reported in Brassica species are flavonoids and phenolic
acids, with the main flavonols being quercetin, kaempferol
and isorhamnetin with hydroxycinnamic acids[28]
. Flavonoids
are typically considered to be the hallmark constituents of
bee pollen.
3.4 Determination of free flavonoid aglycone: quantitative
analysis and method validation
Figure 2 illustrates representative chromatograms obtained
for the standard and MIMBP sample solutions. Inspection

of chromatograms (PDA extracted at 260, 268, 368
and 372 nm for rutin, chrysin, kaempferol and quercetin,
respectively) showed a well-resolved flavonoid aglycone
compound within a 25-minute run under gradient conditions.
The Rt values for standard rutin, chrysin, kaempferol and
quercetin were found to be (7.639±0.020), (15.039±0.010),
(12.106±0.010) and (10.516±0.010)min, respectively.
The chromatogram for MIMBP sample solution showed
peaks at Rt 12.097 and 10.519 min resembling the
standards kaempferol and quercetin, respectively. Figure
3 depicts UV spectra for standards recorded with PDA
multiwavelength detector. Figure 4 illustrates overlay
spectra of peaks of standards and MIMBP sample solution
at Rt 12.097 and 10.519 min, respectively. The retention
times and UV spectra of flavonoid aglycones from MIMBP
sample solution corresponding to that of the standards
confirmed presence of kaempferol and quercetin in the
MIMBP sample solution. Screening all the aglycones
detected kaempferol and quercetin in amounts of (65.4±0.5)
mg/kg and (51.4±0.4) mg/kg, respectively.
The HPLC method for quantification of flavonoid aglycones
was validated and showed good linearity (r2
> 0.998) in the
concentration range of 10-100 µg/mL, which was wide
enough to quantify constituents in the MIMBP sample
solution. Results for linearity of calibration curves, LOD,
LOQ, precision, repeatability, specificity, and accuracy
along with ANOVA and residual analysis are summarized
in Table 1. RSD values for all standards in the range of
0.22 to 1.76 indicated that the method exhibited acceptable
intraday and interday variation with respect to working
standards. The accuracy as measured by the recovery %
with small %RSD ranged from 98.52% to 100.06%. No
peak interference at the retention times for standards and
sample solution indicated specificity of method. The peak
purity factors generated using PDA detector for aglycone
peaks were within threshold values indicating no additional
co-eluting peaks in the standard and sample solutions.
3.5 In vitro antioxidant activity of MIMBP
Both the MIMBP extract solution and the standard
ascorbic acid solution exhibited a concentration-dependent
increase in free DPPH radical-scavenging effect as depicted
in Figure 5. The standard ascorbic acid solution at 40 µg/mL
showed more significant (P<0.05) scavenging effect on
free DPPH radical activity as compared to that of the
MIMBP extract solution. The IC50 values for the MIMBP
extract and the standard ascorbic acid solution were found
to be 54.79 and 18.13 µg/mL respectively.
4 Discussion
Nutraceuticals have become targets of high commercial
and research interests on account of their nutritional and
therapeutic benefits[44]
. This study explores whether the
apiproduct MIMBP is a suitable nutraceutical candidate.
The moisture content of (72.6±6.5) g/kg in dried MIMBP
falls within the acceptable range of proposed stipula-
tions[45]
. Several countries have proposed minimal require-
ments for dried bee pollen including in Brazil: maximum
4% (w/w); Poland and Switzerland: maximum 6% (w/w);
Uruguay: maximum 8% (w/w); Bulgaria: maximum 10%
(w/w)[45]
. The high protein content of (182.2±5.9) g/kg
in MIMBP is consistent with the literature, which states
that Brassicaceae plants such as Sinapis arvensis and
Sinapis alba have high protein content[6]
. MIMBP was
found to have a high fat content of (137.7±6.8) g/kg. The
pollenkitt or pollen coat is known to be a major contributor
to the fat content of bee pollen. The lipid fraction of
MIMBP is one of the major sources of energy to bees,
and plays a key role in the development, nutrition, and
reproduction of bees[46,47]
. The high carbohydrate content
of (560.6±17.4) g/kg in MIMBP makes it a rich source
of sugars, contributing to its sweet taste and high caloric
Figure 1 Light microscopic images of monofloral Indian mustard bee pollen
A: Untreated pollen without acetolysis (40×); B: Acetolyzed pollen showing reticulate ornamentation (40×); C: Scanning electron microscope image
of monofloral Indian mustard bee pollen (154×)

Figure 2 HPLC-PDA chromatogram of (A) standards, (B) flavonoid aglycone compounds from the MIMBP sample solution
HPLC-PDA: high-performance liquid chromatography-photo diode array; MIMBP: monofloral Indian mustard bee pollen.

Figure 3 UV spectra for standards (A) rutin, (B) chrysin, (C) kaempferol, and (D) quercetin
Figure 4 Overlay spectra of peaks of (A) standard kaempferol and sample solution at rentention time 12.097 min, and (B) standard
quercetin and sample solution at rentention time 10.519 min

value of about (17 616.7±78.6) kJ/kg. Several reports have
explored the antioxidant potential of bee pollen in terms
of radical-scavenging activity and total antioxidant activity,
which have been well correlated with total phenolic
content of pollen samples[4,8-11]
. Great variations in the
total phenolic content of bee pollen have been documented
depending upon the source and origin of the pollen
worldwide. Table 2 shows comparative representation of
polyphenol and flavoniod content of MIMBP with that
of different types of bee pollen worldwide. Our results
confirm the rich presence of the principle antioxidants
polyphenols and flavonoids in MIMBP. The free flavonoid
aglycone analysis has been considered an important
parameter for defining the quality of bee pollen[42,48]
. Most
pollen flavonoids exist in the form of glycosides, especially
O-glycosides. Hydrolyzing the glycosides to aglycone
provides a practical approach for effective determination
of flavonoids from samples[42]
. In case of honeybee-collected
pollen apart from honey, hypopharyngeal gland secretions
from the honeybee, along with the presence of hydrolytic
enzymes α/β glucosidase, accompany the pollen pellets,
which cause partial enzymatic hydrolysis of glycosides
to free aglycone in the free state[42]
. HPLC analysis con-
firmed the presence of flavonoids kaempferol and quercetin,
which can be considered as markers for determining the
quality of pollen. A significant reduction in DPPH concen-
tration exhibited by MIMBP confirmed their antioxidant
potential. Nutraceuticals in the form of antioxidants,
omega-3 polyunsaturated fatty acids, and certain vitamins
are often recommended to prevent conditions associated
with free radical damage, such as cardiovascular diseases,
cancer, and diabetes[44]
. This suggests possible application
of MIMBP in the prevention of such diseases. However,
despite its rich nutritional value, there is a need for further
studies on the possible allergenicity for MIMBP, as a few
reports have found the occurrence of allergies in children
Table 1 Summary of validation parameters including statistical data for calibration curves (n=3)
Parameter Rutin Chrysin Kaempferol Quercetin
Linearity range (µg/mL) 10-100 10-100 10-100 10-100
Slope (mean±SEM) 1 153.00±18.76 2 607.00±30.44 2 514.00±40.30 2 472.00±39.25
Intercept (mean±SEM) 359±1 139 -2 296±1 848 2 328±2 446 624.4±2 382.0
Confidence limit of slope(95% CI) 1 100 to 1 205 2 523 to 2 692 2 402 to 2 625 2 363 to 2 581
Confidence limit of intercept (95% CI) -2 802 to 3 520 -7 425 to 2 833 -4 461 to 9 117 -5 988 to 7 236
r2
0.998 9 0.999 5 0.999 0 0.999 0
Sy.x (standard deviation of residuals from
y (i.e., area) on x (i.e., concentration))
1 463 2 374 3 143 3 061
P value*
<0.000 1 <0.000 1 <0.000 1 <0.000 1
Limit of detection (µg/mL) 1.20 0.58 0.33 0.62
Limit of quantitation (µg/mL) 3.64 1.76 0.99 1.88
Precision (%RSD)
Intraday (repeatability) 1.76 0.47 0.72 0.58
Interday (intermediate precision) 1.23 0.39 0.22 0.89
Specificity Specific Specific Specific Specific
Recovery (%) NA NA 100.06 98.52
*
P value <0.000 1 is considered extremely significant. SEM: standard error of mean.
Figure 5 DPPH-scavenging effect of MIMBP extract
Data are expressed as mean ± standard deviation, n=3; *
P<0.05, vs
MIMBP extract solution.
MIMBP: monoflora Indian mustard bee pollen; DPPH: 2,2-diphenyl-l-
picrylhydrazyl.

and adults with the use of mustard seeds[55]
.
In conclusion, the present study is the first to find MIMBP
as an appropriate nutraceutical candidate. Its rich nutri-
tional value is comprised of more than 50% carbohydrates,
18% proteins, and over 10% fats, all by weight. Taken
together, this makes MIMBP a high caloric source. This
study also illustrated the chemical composition of MIMBP
in terms of polyphenol and flavonoid content, suggesting
its potential use in the prevention of free radical-implicated
diseases. The study further demonstrated development
of simple, specific and accurate liquid chromatographic
method for identification and quantification of free flavonoid
aglycones, which can be applied for screening the quality
of honeybee-collected pollen. The study outcome will be
useful to set national pollen standards for monofloral bee
pollen as an initial attempt in establishing quality criteria
for bee pollen worldwide.
5 Acknowledgements
Authors wish to thank the University Grants Commis-
sion, India for providing financial assistance for the work.
6 Conflict of interests
Authors have no conflict of interests.
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Table 2 Comparative representation of antioxidant principles polyphenol and flavoniod content of MIMBP with that of different
types of bee pollen worldwide
Parameter MIMBP Values documented for different types of bee pollen worldwide
Total polyphenol content
(mg GAE/100 g)
1 828.61±37.40
1 290 to 1 980 (Portugal)[7]
1 515 to 8 025 (Poland)[10]
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Total flavonoid content
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892±55 (Southern Brazil)[51]
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545 to 2 044 (Romania)[53]
MIMBP: monofloral Indian mustard bee pollen; GAE: gallic acid equivalents; QE: quercetin equivalents; CE: catechin equivalents.

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