indian mustard bee pollen exhibits high antioxidant content

Epub ahead of print1Journal of Integrative Medicine

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● Research ArticleInvestigation of the nutraceutical potential of monofloral Indian mustard bee pollenSameer 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, India3. 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)60033-9Ketkar 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: [email protected]

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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 chemicalsThe 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 composition2.4.1 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 pollen2.5.1 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 examinationSensory 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 pollenA: 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 solutionHPLC-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 10-100Slope (mean±SEM) 1 153.00±18.76 2 607.00±30.44 2 514.00±40.30 2 472.00±39.25Intercept (mean±SEM) 359±1 139 -2 296±1 848 2 328±2 446 624.4±2 382.0Confidence limit of slope (95% CI) 1 100 to 1 205 2 523 to 2 692 2 402 to 2 625 2 363 to 2 581Confidence limit of intercept (95% CI) -2 802 to 3 520 -7 425 to 2 833 -4 461 to 9 117 -5 988 to 7 236r2 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 1Limit of detection (µg/mL) 1.20 0.58 0.33 0.62Limit of quantitation (µg/mL) 3.64 1.76 0.99 1.88Precision (%RSD)

Intraday (repeatability) 1.76 0.47 0.72 0.58Interday (intermediate precision) 1.23 0.39 0.22 0.89

Specificity Specific Specific Specific SpecificRecovery (%) 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 extractData 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]

1 591 to 3 485 (Sonoran Desert US)[23]

360 to 810 (Alagoas)[37]

660 to 1 090 (Parana state)[37]

850 to 1 460 (Spain)[42]

4 150 to 21 320 (Bahia, Brazil)[49]

2 190 to 2 668 (Algeria)[50]

3 046.00±8.22 (Southern Brazil)[51] 376 to 887 (Transylvania)[52]

2 167 to 2 880 (Romania)[53]

1 050 to 1 680 (Portuguese Natural-Park)[54]

Total flavonoid content (mg QE/100 g)

122.35±5.31

4 500 to 7 100 mg CE/kg (Portugal) [7]

266 to 548 (Sonoran Desert US)[23]

380 to 760 (Spain)[42] 571 to 1 457 (Algeria)[50]

892±55 (Southern Brazil)[51] 255 to 629 (Transylvania)[52]

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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indian mustard bee pollen exhibits high antioxidant content

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