2.1. Collection and preparation of samples The fresh S. claviflorum fruits were obtained from Faridpur district of Bangladesh. All fruits were fully matured and ripen, and uniform in size and color. After collecting the sample fruits, the seeds from the pulp (with peel as edible portion) were separated manually. Later, the samples were individually vacuum packed into polythene bags and frozen at − 20 0C until further analysis.
2.2. Extraction procedure Bioactive compounds were extracted according to the method of Singh et al., with modification. At first, the seed and pulp (edible portion) of S. claviflorum fruits were dried using a freeze-dryer (Model: DC401, Yamato, Country of origin: Japan) at 0.001 mbar pressure and − 50 0C. The freeze-dried samples were pulverized by using a knife grinder to produce powder. Extraction of bioactive compounds from the freeze-dried sample was performed by adding 400 mL methanol/water (80:20, v/v) to 100 g powder and shaken continuously during the extraction using an orbital shaker (Remi, Mumbai, India) for 48 h at room temperature (25–26 0C). After that, extract was filtered by a Buchner funnel, and the residue was dissolved again in the solvent to maximize the extraction. The extraction was repeated three times with the same procedure, the filtrates were combined and evaporated in a vacuum rotary evaporator (R-205, Buchi, Switzerland) at 45 0C. Finally, the concentrated extracts were freeze-dried and preserved at − 20 0C for subsequent analysis.
2.3. Determination of total phenolics Total phenolic content (TPC) of S. claviflorum extracts was estimated, based on the Folin-Ciocalteu method with slight modifications adapted from Wootton-Beard et al.. Firstly, 50 mg of dried extract of each sample was weighed and dissolved in 50 ml of methanol in a volumetric flux. For complete mixing these samples were vortexed and sonicated for several minutes. Thus, the concentration of the solutions became 1 mg/mL (stock solutions). After that, 1 mL of each extract from stock solution was mixed with 4 mL of 7.5% (w/v) Na2CO3 solution and 5 mL Folin-Ciocalteu reagent (10 times diluted in distilled water) and mixed well with a vortex machine for 15 s. Then, the mixture was kept in an incubator for 30 min at 40 0C. A double beam scientific UV–Vis Spectrophotometer (Model: Specord-205, Analytic Jena, Germany) was used to take the absorbance at 765 nm. The concentration of TPC was determined from a gallic acid calibration curve and presented as mg gallic acid equivalent (mg GAE/g of dry extract).
2.4. Determination of total flavonoids Total flavonoid content (TFC) was measured as described by Chang et al. with slight modification. Firstly, 5 mL of methanolic extract was mixed with an aluminum trichloride reagent (2.5 mL). The mixture solution was then incubated for 15 min at room temperature (25–26 0C) and the absorbance was taken at 430 nm with a UV–Vis Spectrophotometer (Model: Specord-205, Analytic Jena, Germany). The concentration of TFC was determined from a quercetin calibration curve and presented as mg quercetin equivalents (mg QE/g dry extract).
2.5. Determination of total tannin content Total tannin content (TTC) was quantified as described by Amorim et al.. First of all, methanol extract (1 mL) was added with 0.5 mL of Folin-ciocalteu phenol reagent in 7.5 mL of demineralized water. After that, 35% Na2CO3 (1 mL) solution was added with the mixture solution and shaken well. The solution was incubated for 30 min at room temperature (25–26 0C). The absorbance was read at 725 nm, and the concentration of total tannin was calculated as mg tannic acid equivalent (TAE)/g of dry extract using a tannic acid calibration curve.
2.6. Determination of free radical-scavenging capacity 2.6.1. DPPH assay The free radical-scavenging capacity of the sample extracts was assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical following the method described by Shimada et al. with some modifications. Sample extract (2 mL) was allowed to react with 2 mL of 0.1 mM DPPH solution for 30 min at room temperature (25–26 0C) in the dark place and the absorbance of the solution was read at 517 nm. The DPPH radical scavenging capacity was calculated utilizing equation no. (1)
DPPH radical scavenging capacity (%) = Ao − As / Ao
where. A0 = Absorbance of the control solution As = Absorbance of the DPPH solution with sample extracts
2.7. Screening of phenolic compounds The methods described by Chuanphongpanich and Phanichphant [25] were used to identify and quantify the specific polyphenols in methanol extracts by using HPLC-DAD analysis. It was carried out on a Dionex UltiMate 3000 system consists of a quaternary rapid separation pump (LPG-3400RS) and a photodiode array detector (DAD-3000RS). The flow rate of 1 mL/min and injection volume of 20 μL was used to separate by Acclaim® C18 (5 μm) Dionex column (4.6 × 250 mm) at 300C. The limit of detection (LOD) and limit of quantification (LOQ) ranged from 0.01 to 0.17 μg/ml and 0.04–0.53 μg/ml, respectively. Acetonitrile (solvent A), acetic acid solution pH 3.0 (solvent B), and methanol (solvent C) were used as mobile phases with the gradient elution program of 5%A/95%B (0–5 min), 10%A/90%B (6–9), 15% A/75%B/10%C (11–15), 20%A/65%B/15%C (16–19 min), 30%A/50% B/20%C (20–29 min), 40%A/30%B/30%C (30–35) and 100%A (36–40 min). For the preparation of standard curve, a standard stock solution was prepared in methanol containing gallic acid (GA), hydroquinone (HQ), vanillic acid (VA), rosmarinic acid (RA), myricetin (MC) (4 μg/ml each), (− )-epicatechin (ECA), arbutin (AR) (5 μg/ml each), p-coumaric acid (PCA), kaempferol (KF), quercetin (QU) (2 μg/ml each), Syringic acid (SA), caffeic acid (CA), trans-ferulic acid (FA), vanillin (VL) (3 μg/ml each), ellagic acid (EA), (+)-catechin hydrate (CH) (10 μg/ml each), rutin hydrate (RH) (6 μg/ml), trans-cinnamic acid (TCA) (1 μg/ml) and benzoic acid (BA) (8 μg/ml). The concentration of extract in methanol solution was 10 mg/ml. Before HPLC analysis mixed standards, spiked solutions, and samples were filtered through a 0.20 μm syringe filter (Sartorius, Germany) and followed by degassing for 15 min in the ultrasound bath (Hwashin, Korea). Peak integration, data acquisition, and calibrations were performed by Dionex Chromeleon (Version 6.80 RS 10) software.
2.8. Statistical analysis Results were reported as the mean ± standard deviation (SD) of the three replicates for each sample. The 5% level of significance was considered to compare the quantified variables by using the one-way ANOVA. The impact of bioactive compounds on antioxidant capacity was determined by Pearson’s correlation coefficients (r). The SPSS software (version 21) was used to perform all required statistical analyses.