Chemicals and Reagents Standards gallic acid, catechin, quercetin, tannic acid, green vitriol (FeSO4, 7H2O), ascorbic acid, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), 2,4,6-tris (2-pyridyl)-1,3,5-triazine (TPTZ) were purchased from Sigma-Aldrich (St. Louis, MO). Aluminium chloride (AlCl3), sodium nitrite (NaNO2), hydrogen peroxide (H2O2), sodium hydroxide (NaOH), sodium acetate (C2H3NaO2), ammonium molybdate (NH4)2MoO4, Tri Chloro Acetic acid (TCA), sodium carbonate (Na2CO3) were purchased from Merck Co. (Darmstadt, Germany). Vitamin B1 and B2 standards (Thiamine HCl and Riboflavin) were purchased from DUCHEFA (Haarlem the Netherlands). All of the chemicals and reagents used in this study were of analytical grade.
3.1. Sample Collection and Processing Four varieties of pulse samples were purchased from the local market (also known as kacha Bazar) in Savar, Dhaka, Bangladesh in May, 2018. All the samples were washed to remove sands and dirt from them. All the samples were further dried in sunlight. The samples were powdered using a grinder machine and stored in an air tight container to be used for extraction process.
3.2. Extract Preparation Briefly, the powdered pulse of four samples (500 g) was dissolved with pure ethanol (100%) for 5 days. During this time, this mixture was shaken at regular interval for mixing the sample with solvent properly. Then the extract was filtered through cotton cloth and then through Whatman no. 1 filter paper which was fitted with a suction (Rocker 300) apparatus and the filtrate was collected.
The collected filtrate was kept in a rotary evaporator to be dried (Buchi, Tokyo, Japan) under reduced pressure (100 psi) and controlled temperature (1000C). For the complete evaporation of residual ethanol, semisolid extract was incubated overnight in an incubator at 45oC. Finally, the dried extract (lentil 11.35 g, mung bean 7.18 g, chick pea 16.25 g, and anchor 9.08 g) was preserved at −20o for subsequent analysis.
3.3. Statistical Analysis All analyses were performed in triplicate and the data were reported as the mean ± standard deviation. They were subjected to two tailed t-test (p < 0.05) to assess the variation of values given by each samples as statistical significant value. Data were analyzed using Microsoft Excel 2013 (Redmond, Washington, USA).
3.4. Nutritional Composition of Pulse Varieties Nutritional Composition of pulse samples was analyzed using following methods pH by pHPW (pH for dry powder) method, moisture by the weighed moisture box (A & D Company Ltd. N 92; P1011656, Japan), ash by AOAC Official Method 942.05 with slight modification, lipid by soxhlet extraction method, and crude fiber by AOAC, 962.09, 16th edition method with some modifications respectively.