2.1. Materials Nine minor indigenous fruits were selected based on peoples’ choice and production catchments in Bangladesh. Analytical grade chemicals and reagents used in this study were procured from Sigma-Aldrich (Steinheim, Germany).
2.2.1. Sample Collection and Fruit Extraction Fruits were collected from the Regional Agricultural Research Station (RARS), Bangladesh Agricultural Research Institute (BARI), Akbarpur, Moulvibazar, Bangladesh. After collection, the fruits were washed with potable water and surface dried. The fruits were then freeze-dried and milled to powder using a laboratory grinder. Fruit powder of known quantity was extracted in a thermostatic water bath (Vision Scientific Co. Ltd.) at 60oC for 1 h using methanol (80%, v/v) maintaining a fruit:solvent ratio of 1:20 (w/v). The fruit extract was filtered under vacuum, centrifuged at 4000×g for 10 min and the supernatant was collected and kept at -18oC until used for analysis.
2.2.2. Determination of Physicochemical Parameters Physicochemical properties – moisture, protein, ash, total soluble solids (TSS), pH, and titrable acidity – were determined following the method of AOAC (2005). Ascorbic acid, starch, and total sugar content were determined based on the procedure of Ranganna (1995). Edible and non-edible portions of the fruits were measured using the gravimetric method.
2.2.3. Analysis of Minerals Minerals analyzed included sodium, potassium, calcium, magnesium, phosphorus, sulphur, boron, copper, manganese, iron, and zinc. Before quantification, fruits were first wet ashed and then digested in nitric and perchloric acid solution at 320oC, cooled, diluted to an appropriate concentration, and filtered. This filtrate was then used as the stock solution for further analysis. Atomic absorption spectrophotometry (Model-AA-7000S, Shimadzu, Tokyo, Japan) was used to assess levels of sodium, iron, copper, zinc, boron, manganese, calcium, and magnesium. Potassium content was measured using flame photometry, while phosphorous and sulphur were assessed by spectrophotometry. Individual minerals were quantified by comparison to the corresponding standard mineral procured from Sigma Chemical Co., USA.
2.2.4. Determination of Phytochemicals 2.2.4.1. Total Phenolic Content Twenty milligrams (0.02 g) of powder was dissolved in 1 ml of methanol to prepare a stock solution for experiments. A volume of 0.5 ml of the each extract (100 µg/ml) was mixed with 2 ml of Folin–Ciocalteu reagent (diluted 1:10 with deionized water) and neutralized with 4 ml of sodium carbonate solution (7.5%, w/v). The reaction mixture was incubated at room temperature for 30 min with intermittent shaking for color development. Absorbance of the colored solution was measured at 765 nm using double-beam UV-VIS spectrophotometry. Total phenolic content was determined from the linear equation of a standard curve prepared with gallic acid. Determination of total phenolic content in the extracts was determined according to the Folin–Ciocalteu method (Ough & Amerine, 1988), with gallic acid (GAE) as the standard and expressed (mg) as gallic acid equivalents (GAE)/g of extract (Aoshima, Hirata, & Ayabe, 2007).
2.2.4.2. Determination of Total Flavonoid Content Total flavonoid content (TFC) of the fruit powder was determined by the aluminium chloride method (Chang, Yang, Wen, & Chern, 2002) with slight modifications: 0.5 ml of sample solution was mixed with 1.5 ml of methanol. To this mixture 0.1 ml of 10% aluminium chloride and 0.1 ml of 1 M potassium acetate were added. The final volume was made up to 5 ml by adding 2.8 ml of distilled water, and the reaction was left for 30 min at room temperature. The absorbance of the solution was measured at 415 nm and expressed as mg QE/g extract. TFC was calculated from the calibration curve of quercetin plotted. The curve obtained was found to be linear with equation y = 0.0035 + 0.0056x, and the correlation coefficient was found to be R2 = 0.9993. TFC was expressed as mg quercetin equivalents per gram of extract (mg QE/g extract).
2.2.4.3. Determination of Total Anthocyanin (TA) The method was adapted from Burgos et al. (2013), the concentration of TA being calculated using the molar extinction coefficient and molecular weight of malvidin-3-p-coumaroyl-glucoside for blue-violet pigments (545 nm, 3.02×104 l/mol/cm, 718.5 g/mol), pelargonidin-3-glucoside for red pigments (515 nm, 2.73×104 l/mol/cm, 486.5 g/mol), and cyanidin-3-glucoside for purple pigments (535 nm, 3.43×104 l/mol/cm, 449.2 g/mol). Results were expressed as mg/100 g DW.
2.2.4.4. Determination of Total Carotenoid Content Total carotenoid was determined according to the method described by Thaipong, Boonprakob, Crosby, CisnerosZevallos, and Byrne (2006). Absorbance was measured at λ 470 nm. Each extract was dissolved in n-hexane proanalysis. ß-carotene solution in various concentrations was used as standard carotenoid compound and the standard curve. A linear regression equation of the standard curve was used for calculating total carotenoid content, and expressed as beta-carotene equivalents per 100 g of extract (mg/100 g).
2.2.4.5. Determination of ß-Carotene Content β-Carotene content was determined using the procedure of Holden et al. (1999), with values expressed as µg/100g of fruit extract.
2.2.5. Determination of Antioxidant Activity
2.2.5.1. Total Atioxidant Activity Total antioxidant activity was assessed using the phosphomolybdenum system according to the technique described by Prieto, Pineda, and Aguilar (1999), and results expressed as micrograms ascorbic acid (AA) per gram (µg/ml) of the sample.
2.2.5.2 Reducing Power Assay The reducing power of the fruit extract was assessed using the approach of Guo, Saravanakumar, and Wang (2018). Ascorbic acid was used as the standard for the preparation of the calibration curve.
2.2.5.3. FRAP FRAP activity was measured following the scheme outlined by Benzie and Strain (1996). A standard curve was made using ferrous sulphate aqueous solution (1–10 mM) and FRAP values were expressed as μM Fe (II)/100g of the sample.
2.2.5.4. DPPH Radical Scavenging Activity (DPPH-RSA) and IC50 DPPH-RSA was evaluated by measuring the inhibition rate following the procedure described by Brand-Williams, Cuvelier, and Berset (1995), with modification. Exactly 0.1 ml of extract was placed in a Falcon tube and 1.4 ml of methanolic solution of DPPH added. The mix was left to rest for 30 min in the dark and absorbance at 517 nm was measured against a blank. The results are expressed as percentage radical scavenging activity: