2.1 Sample collection Some fresh, as well as fully mature S. cumini (Jaam), was collected from Faridpur and Manikganj (Bangladesh). Considering the absence of visible damages and infection, uniformity in size and color fruits were selected and then those were kept in frozen (-20oC) condition until laboratory analysis. 2.2 Sample extraction The Pulp of S. cumini fruits were separated from the seed and freeze-dried at - 50°C and 0.001 mbar pressure. The seeds were dried in the oven at 40oC. After the material was completely dry, it was pulverized in a knife grinder and kept at -20oC for further analysis. The dried, ground sample (200 g) was dissolved into 800 mL of methanol/water (80:20, v/v) and thoroughly mixed and shaken at room temperature (22 - 25°C) using an orbital shaker for 48 hrs and then filtered in a Buchner funnel. The thick residue was then subjected to re-extract for three times in the same way to maximize the extract. The filtrates were concentrated in a vacuum rotary-evaporator (model no. R-205, Switzerland). The concentrated filtrates were lyophilized and kept in an amber bottle at - 20oC prior to further analysis. 2.3 Analysis of proximate composition The amount of moisture in the sample was determined by AOAC (1990). The ash content was measured by Ranganna (1986). The percentage of protein in the samples was determined by Kjeldal method (AOAC, 2005). Fat content in the samples was evaluated by standard (AOAC, 2005) method. Flame Photometer was used to determine Sodium and Potassium (Ward and Johnston, 1962). Flame Atomic Absorption Spectrometric (AAS) method was used to estimate Chromium, Cadmium, Nickel, and Lead (Atomic Absorption Spectrometer Thermo-Scientific Ice 3000 series), AOAC method (AOAC, 2005) is followed for AAS.
2.4 Determination of total carotenoids and chlorophyll A and B Total carotenoids and chlorophyll A and chlorophyll B were measured by following the procedure demonstrated by Dere et al. (1998) with minor modification. Five grams of extracts were added to 30 mL 85% acetone in an amber-colored reagent bottle. Leave them at room temperature to stand for approximately 20 hours. Extracts were filtered into 100 mL volumetric flax through glass wool and then made up to the volume by 85% acetone solution. The absorbance, against a blank sample (85% acetone), of the solution was read using a UV-VIS spectrophotometer at 470, 645 and 662 nm. Following equation was followed to calculate chlorophyll A and B and total carotenoids: Chlorophyll A, CA (mg/mL) = (A662 x 11.75) – (A645 x 2.350), Chlorophyll B, CB (mg/mL) = (A645 x 18.61) – (A662 x 3.96), Total carotenoids (mg/mL) = (A470 x 1000) – (CA x 2.27) – {(81.4 x CB)/230} 2.5 Antioxidant activity determination 2.5.1 Phosphomolybdenum assay Total antioxidant capacity was assessed according to the phosphomolybdenum assay (Prieto et al., 1999). In brief, the extract reduces Molybdenum (VI) to Molybdenum (V) by forming phosphate-Molybdenum (V) complex which is green in color in acidic conditions. Reagent solution was made by mixing 28 mM trisodium phosphate with 4 mM ammonium molybdate and 0.6 M sulfuric acid at a ratio of 2:4:4. About 3 mL reagent solution was added with 0.3 mL sample (1 mg/mL) and kept in an incubator at 95°C for 90 min. After cooling at normal temperature (22 to 25°C), the absorbance was taken at 695 nm. We calculated the total antioxidant capacity in units of mg ascorbic acid equivalent (AAE). 2.5.2 Reducing power assay The method described by Oyaizu (1986) with some modification was followed to measure the reducing power of fruit extracts. Various concentrations of the sample (1 mL) was added with 2.5 mL phosphate buffer (0.2 M and pH 6.6) and 2.5 mL of 1% potassium ferricyanide and warmed in a water bath at 50°C for 20 mins. The mixture was cooled at room temperature (22– 25°C) and mixed with 2.5 mL 10% trichloroacetic acid. The subsequent solution was centrifuged at 3000 rpm for 10 mins. The supernatant (2.5 mL) was then added to 2.5 mL distilled water and 0.5 mL freshly prepared 0.1% ferric chloride solution, and the mixture was preserved for 10 min to complete the reaction properly. Reducing power was measured by taking the absorbance at 700 nm. Different concentration of ascorbic acid was taken as standard. Higher the absorbance of the solution.
2.4 Determination of total carotenoids and chlorophyll A and B Total carotenoids and chlorophyll A and chlorophyll B were measured by following the procedure demonstrated by Dere et al. (1998) with minor modification. Five grams of extracts were added to 30 mL 85% acetone in an amber-colored reagent bottle. Leave them at room temperature to stand for approximately 20 hours. Extracts were filtered into 100 mL volumetric flax through glass wool and then made up to the volume by 85% acetone solution. The absorbance, against a blank sample (85% acetone), of the solution was read using a UV-VIS spectrophotometer at 470, 645 and 662 nm. Following equation was followed to calculate chlorophyll A and B and total carotenoids: Chlorophyll A, CA (mg/mL) = (A662 x 11.75) – (A645 x 2.350), Chlorophyll B, CB (mg/mL) = (A645 x 18.61) – (A662 x 3.96), Total carotenoids (mg/mL) = (A470 x 1000) – (CA x 2.27) – {(81.4 x CB)/230} 2.5 Antioxidant activity determination 2.5.1 Phosphomolybdenum assay Total antioxidant capacity was assessed according to the phosphomolybdenum assay (Prieto et al., 1999). In brief, the extract reduces Molybdenum (VI) to Molybdenum (V) by forming phosphate-Molybdenum (V) complex which is green in color in acidic conditions. Reagent solution was made by mixing 28 mM trisodium phosphate with 4 mM ammonium molybdate and 0.6 M sulfuric acid at a ratio of 2:4:4. About 3 mL reagent solution was added with 0.3 mL sample (1 mg/mL) and kept in an incubator at 95°C for 90 min. After cooling at normal temperature (22 to 25°C), the absorbance was taken at 695 nm. We calculated the total antioxidant capacity in units of mg ascorbic acid equivalent (AAE). 2.5.2 Reducing power assay The method described by Oyaizu (1986) with some modification was followed to measure the reducing power of fruit extracts. Various concentrations of the sample (1 mL) was added with 2.5 mL phosphate buffer (0.2 M and pH 6.6) and 2.5 mL of 1% potassium ferricyanide and warmed in a water bath at 50°C for 20 mins. The mixture was cooled at room temperature (22– 25°C) and mixed with 2.5 mL 10% trichloroacetic acid. The subsequent solution was centrifuged at 3000 rpm for 10 mins. The supernatant (2.5 mL) was then added to 2.5 mL distilled water and 0.5 mL freshly prepared 0.1% ferric chloride solution, and the mixture was preserved for 10 min to complete the reaction properly. Reducing power was measured by taking the absorbance at 700 nm. Different concentration of ascorbic acid was taken as standard. Higher the absorbance of the solution.
2.6 Statistical analysis Every chemical analysis was worked out in triplicate and the findings were articulated as means± standard deviations (SD). The analysis was done with the SPSS software, version 21. One way analysis of variance (ANOVA) according to the Bonferroni test was performed to compare the quantified variables.