Ten grams of each honey sample were taken, diluted in tepid water and 95% ethanol, centrifuged, de-hydrated with anhydrous acetic acid, submitted to the acetolysis method with acetic anhydride and sulfuric acid (9:1) and successively centrifuged (Erdtman, 1960). After the acetolysis process, slides containing glycerinated gelatin were prepared for the mounting of the pollen grains, which were later examined and identified by optical microscopy. The frequency classes were established from counting at least 300 pollen grains for each honey sample. The classification was based upon
the following criteria: predominant pollen type (DP, >45%), secondary pollen type (SP, 16–45%), important minor pollen (IMP, 3–15%) and minor pollen (MP, <3%). The identification of pollen types found in each sample was based on selleck products pollen catalogues and comparison with the slide collection of the pollen libraries from the Federal University of the West of the Pará (PUFOPA) and the State University of the Santana
Fair (PUEFS). The determination of total phenolic content of the honey samples and the ethyl acetate fractions (EtOAct) was conducted www.selleckchem.com/products/Rapamycin.html by the colorimetric Folin–Ciocalteu method (Slinkard & Singleton, 1977). A 300-μL aliquot of methanol extract (5 mg mL−1 in MeOH) was transferred to a test tube containing 60 μL of the Folin–Ciocalteu reagent and 2.46 μL of distilled water. The mixture was stirred for 1 min before 180 μL of Na2CO3 (15%) were added. The contents were stirred for an additional 0.30 min to obtain a final extract concentration of 0.2 mg mL−1. The samples were kept in the dark for 2 h prior to analysis using a UV–Vis spectrophotometer at 760 nm. The total phenolic content (TFC) was determined Tacrolimus (FK506) by interpolation of the sample absorbance against a calibration curve built with gallic acid standards (0.001–0.015 mg mL−1
in ethanol) and expressed as milligrams of gallic acid equivalents per gram of extract (mg GAE/g). All the analyses were performed in triplicate. The ABTS test was performed according to the methodology reported by Re et al. (1999). The cation radical ABTS + was synthesised by the reaction of a 7 mM ABTS solution with a 2.45 mM potassium persulfate solution. The mixture was kept at 23 °C in the dark for 16 h. Afterwards, the ABTS + solution was diluted with ethanol until an absorbance (A) of 0.7 at 734 nm was achieved in a UV–Vis spectrophotometer. Aliquots of 2.7 mL from the ABTS + solution were added, immediately after being prepared, to the sample solutions diluted in methanol (MeOH) to reach final concentrations between 0.1 and 0.5 mg mL−1. After 10 min, the percentage inhibition of absorbance at 734 nm was calculated for each concentration, relative to the blank absorbance (ethanol).