Samples and Preparation of extracts The fresh Satkara (Citrus macroptera) and Taikar (Gracinia pedunculata) fruits were collected from the Citrus Research Station, Sylhet, Bangladesh. The whole fruits were cleaned and washed with distilled water. Edible portion of fruits was separated carefully. Fruits were homogenized and extracted with 200ml of ethanol: acetone (7:3 v/v) for 1 h at 37ºC according to Lee and Wicker (1991). The extract was filtered through Whatman No.41 paper and rinsed with 50ml of ethanol: acetone (7:3 v/v). Extraction of the residue was repeated using the same conditions. The two filtrates were combined and then stored at −200C until used for analysis of the total polyphenol, total flavonoids and antioxidant capacity.
Physicochemical analysis The edible portion of these fruits was analyzed for moisture by moisture analyzer (IR-60 Denver Instrument), protein content (Micro Biuret method), total soluble solid (Hand Refractometer, ERMA 58- 92) and fat, fiber, pH contents with the following standard methods as described by AOAC (2004). The concentration of mineral and heavy metal elements were determined using Atomic Absorption Spectrophotometer (Thermo-scientific iCE 3000). Thiamine and Riboflavin were estimated by the methods of Coward (1938); Gyorgy and Pearson (1967). The Ascorbic acid (AA) content was determined using the 2, 6-dichlorophenolindophenol. It was used L-ascorbic acid to prepare a standard solution (0.5 mg/ml) and the concentration was calculated by comparison to the standard and expressed as mg/100 g fresh mass.β carotene content was estimated by the method of Holden, 1981 and values were expressed as mg/100g.
Determination of total polyphenol content (TPC) and total flavonoids (TF) The amount of total polyphenols was determined according to the Folin–Ciocalteu Spectrophotometric method (Rapisarda et al., 1999). Absorbance was measured at 765 nm. Total polyphenols was expressed as Gallic acid equivalents (mg/100g of GAE). Gallic acid (wako- 071-06095) standard solutions were prepared at a concentration ranging from 0 to 1000mgL−1. Total flavonoid content was determined using colourimetric method described by Abu Bakar et al. (2009), as adapted from Dewanto, Wu, Adom, and Liu (2002). Briefly, 0.5 ml of the extract was mixed with 2.25 ml of distilled water in a test tube followed by the addition of 0.15 ml of 5% NaNO2solution. After 6 min, 0.3 ml of a 10% AlCl3.6H20 solution was added and allowed to stand for another 5 min before 1.0 ml of 1 M NaOH was added. The mixture was mixed well by vortexing. The absorbance was measured immediately at 510 nm using a spectrophotometer. Results were expressed as mg rutin (wako-181- 00341) equivalents in 1 g of sample (mg RE/g).
Measurement of antioxidant capacity The antioxidant capacity was determined by the modified free radical DPPH method (Brand- Williams et al., 1995), based on free radical scavenging by antioxidants, and the ABTS method (Re et al., 1999), where the free radical is generated by a chemical reaction with potassium persulfate, was used to determine the antioxidant activity with modifications. The extract was obtained from about 10 g of sample in 40 ml 50% aqueous methanol and 40 ml 70% aqueous acetone centrifuged twice at 15,000 × g for 15 min (Hitachi, model Himac CR21E centrifuge, Tokyo, Japan), and three dilutions were prepared using the supernatant (1:5, 1:10 and 1:15). This procedure was adapted from Larrauri et al. (1997). For the DPPH method, a 100 μL aliquot of each dilution was added to 3.9 ml of DPPH radical (Wako, Japan), and the reading made at 515 nm in an Ultrospec 3100 pro spectrophotometer (HITACHI U-1900 Spectrophotometer) after 30 min, using methanol as the blank. For the ABTS method, an aliquot of 30 μL of each dilution was added to 3.0 ml of ABTS radical (Wako), and the reading made in the spectrophotometer at 734 nm after 6 min of reaction, using ethanol as the blank and a standard curve prepared using Trolox (Wako, Japan). The analyses of the extract were carried out in triplicate and the results presented in μM of Trolox equivalents (TE)/g.
Statistical analysis All samples were prepared and analyzed in triplicate. Statistical analysis was done by one-way analysis of variance. The Pearson correlation coefficient (R) and p-value were used to show correlations and their significance (SAS 9.0 for Windows). Probability values of p < 0.05 were considered statistically significant.