2.1 Location of the study The entire research work was done at the Food Chemistry laboratory of the Department of Food Engineering and Tea Technology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh.
2.2 Chemicals and equipment The analytical grade chemicals used in this analysis were obtained from industrial suppliers. Ethanol (C2H6O), Methanol (CH3OH), Acetone (C3H6O), 2,2- diphenyl-1- picrylhydrazyl (DPPH), and Folin-Ciocalteu Phenol- Reagent were purchased from Sigma Aldrich (USA); Sodium carbonate (Na2CO3), Potassium di-hydro phosphate (KH2PO4), and Monobasic dihydrate (NaH2PO4.2H2O) were obtained from Merck (India); and Ferric Chloride (FeCl3), Trichloroacetic acid (C2HCl3O2), and Potassium ferricyanide [K3Fe(CN)6] were collected from Merc (Germany).
Oven Dryer (Model ON-01E), UV Spectrophotometer (PG Instruments Ltd., Model - T60 U), Vortex Mixer (Model - VM-2000), Incubator (AAH 26016U), Shaking incubator (Model SI-100), Blender (Panasonic, Model - MJ-M176P), and Hot water bath (NE2-9D Bennett Scientific) were used as equipment.
2.3 Preparation of samples From the regional supermarket of Sylhet, Bangladesh, Hyacinth Bean (Lablab niger Medik.), Kidney bean (Phaseolus vulgaris L.), Black Gram (Vigna mungo (L.) Hepper), Mung Bean (Vigna radiata (L.) R. Wilczek), and Green Pea (Pisum sativum L.) were purchased. Beans were then oven-dried at 60°C for 24 hrs and blended using a mechanical blender.
2.4 Extraction procedure Extraction was performed with a minor adjustment relying on Hussain et al. (2012) method. At first, 1 g of the ground sample was added separately with 15 mL of 80% (v/v) aqueous methanol, ethanol and acetone and kept at ambient temperature (25±2°C) for 2 hrs with continuous stirring using an orbital shaker (model SI100, Germany) at 150 rpm before centrifugation for 10 mins at 4000 rpm. The supernatant obtained was stored at -4°C for further experiment. Figure 1 exhibits the bird’s eye view of the entire research design.
2.5 DPPH radical scavenging activity The Brand-Williams et al. (1995) method was slightly modified for the DPPH radical scavenging assay. In a tube, 1 mL of the aliquot from each of the five extracts was added separately with 4 mL of DPPH solution. After that, the tubes were vortexed and allowed to stand in the dark for 30 mins. Then, the absorbance of the mixtures was taken using a T60 U Spectrophotometer at 517 nm. An aliquot-free DPPH solution was used as a control, and the results were expressed in percentage. The following equation was used to estimate the DPPH radical scavenging activity.
2.6 The total phenolic compounds (TPC) The total polar phenolic compound was estimated by following the Rahman et al. (2016) method with some modifications. Approximately 0.5 mL from five extracts were separately placed in a 10 mL flask. The FolinCiocalteu reagent (0.5 mL) was then applied to the solvent and shook for 3 mins. In order to prepare a 10 mL solution, one mL of saturated sodium carbonate (Na2CO3) was added, and the remaining amount was filled up with distilled water. Solutions’ absorbances were measured with a Spectrophotometer against a reagent blank at 725 nm. Pure gallic acid (GA) was used as a standard for calibration curve preparation. The results were expressed in mg GAE/ gm w/w using the equation y = 0.0004x+ 0.0013; R2 = 0.999.
2.7 Ferric reducing antioxidant power (FRAP) The modified method of Oyaizu (1986) was used to determine the total antioxidant activity of the extracts by ferric reducing antioxidant power (FRAP). An aliquot of 0.3 mL from five extracts was added separately and vortexed with 0.85 mL of 0.2 M phosphate buffer of pH 6.6 and 1%, 0.85 mL of potassium ferricyanide. After incubating the mixture at 50°C for 20 mins, 0.85 mL of trichloroacetic acid (10%) was added and vortexed well. Finally, 2.85 mL of double-distilled water and 1%, 0.57 mL of Fe2Cl3 were mixed and incubated at 25°C for half an hour. After the second incubation, absorbance was taken at 700 nm by operating a Spectrophotometer. A Blank was prepared in parallel, where distilled water was added instead of the aliquot. The standard ascorbic acid was developed by serial aqueous dilution of stock solution. The standard curve was prepared by fitting the absorbance versus its corresponding standard ascorbic solutions. The outcomes were estimated in μg ascorbic acid correspondent antioxidant capability/100 g w/w (μg AAE/100 g w/w) using the equation y = 0.176x+ 0.005; R 2 = 0.998.
2.8 Determination of total flavonoid compound The total flavonoids were determined by the aluminium chloride colourimetric assay of Pothitirat et al. (2009). Approximately 1.5 mL of the extracts were placed in a 10 mL flask. Following that, 2% AlCl3 solution and 6 mL of distilled water were added to the flask and vortexed. After half an hour, the solutions’ absorbances were measured using a T60-U Spectrophotometer (Germany) at 415nm against a blank. The equation y = 0.003x+ 0.025; R2 = 0.999 was used as a standard curve to determine the total flavonoid compounds and expressed as equivalent per gram.
2.9 Statistical analysis The result was reported as mean ± standard deviation for three replications of each treatment. The data were analyzed statistically using the Minitab-19 statistical software. Mean and Standard Deviation (SD) were measured by Tukey One Way ANOVA Test. The least significant difference was estimated at a 95% level of confidence (p < 0.05).