Bioactive compounds, antioxidant activity and phenolic profile of stingless bee pot-pollen from Amazon, Brazil

Pot-pollen is a mix of flowers pollen, nectar and salivary substances from the bees. This product is stored in pots, made of cerumen (wax and resin), inside the beehive, where is fermented by microorganisms. Despite to be consumed by natives of the Amazon region, and its commercialization to be a good alternative to incomes generation and sustainability, the composition of Amazonian pot-pollen is still poorly known. In this context, this study aimed to investigate the bioactive compounds content, antioxidant activity (AA) and phenolic profile of Melipona seminigra pot-pollen samples from different localities in the Amazon region. The samples were collected from four localities in Amazonas state, Brazil: Manaus Urban Zone (MUZ), Manaus Rural Zone (MRZ), Iranduba (IRA) and Boa Vista do Ramos (BVR). The total carotenoid content (TCC) was extracted with ethyl acetate (80g/L), at 37°C, partitioned with hexane in a separatory funnel and determined by spectrometry, at 450nm. To determine total phenolic content (TPC), total flavonoid content (TFC), AA and phenolic profile, the pot-pollen extracts (PPEs) (80g/L in ethanol 65%) were prepared using an ultrasonic probe (400W for 30min). After to be filtered through Whatman paper n° 4, the PPEs were stored at -20ºC until analyses. TPC was determined at 725nm, using the Folin-Ciocalteu reagent method and a gallic acid standard curve. TFC was assessed by the aluminum chloride colorimetric method, using a catechin standard curve, with readings at 510nm. The AA was investigated by FRAP and ORAC assay, and the screening of phenolic compounds by High-Performance Liquid Chromatography coupled with Mass Spectrometry (HPLC-MS). All the statistical analyses (One-way ANOVA, Tukey-test and Pearson correlations) were performed using GraphPad Prism software, version 7. TCC ranged from 3.2-48.0μg/g and the highest content was found in BVR sample. TPC ranged from 7.0-15.0mg GAE/g, being the IRA and MRZ samples the ones with the highest amount. TFC ranged from 1.9-4.5mg CAE/g, where IRA sample showed the highest value. Regarding the AA assays, FRAP showed values ranging from 2.8-6.3μmol TE/g and ORAC from 224.9-1117.0μmol TE/g. The sample from IRA showed the higher AA values by both assays. Negative correlations were observed between TCC versus ORAC (r=-0.7) and FRAP (r=-0.8). On the other hand, we observed positive and significant (P<0.05) correlations between FRAP versus TPC (r=0.9) and TFC (r=0.9); as well as between ORAC versus TPC (r=0.8) and TFC (r=0.8). These data indicate that the phenolic compounds can be responsible by the AA in the samples analyzed here. A total of 21 phenolic compounds were identified in the pot-pollen samples evaluated. There were few qualitative variations despite a high quantitative variation in phenolic profile among samples. Finally, the variation observed in bioactive compounds content and AA among pot-pollen samples can be attributed to the different pollinic sources available at the different localities, due to the phenolic compounds to be products of plant metabolism. Given the known bioactive potential of polyphenols, we recommend in vivo studies to be conducted to investigate the health benefits of the stingless bee Amazonian pot-pollen.