2.1. Reagents Analytical-grade acetone, petroleum ether, n-hexane, dichloromethane, sodium carbonate, Folin–Ciocalteu reagent and acetic acid were purchased from Merck (Darmstadt, Germany). Gallic acid (Pub Chem CID:370) was purchased from Tokyo Chemical Industry Co., (Tokyo, Japan) and 2,20-azinobis (3-ethylbenothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). α-amylase, 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH), Tri(2-pyridyl)-s-triazine (TPTZ), trolox, potassium persulfate, and mineral standards were obtained from Sigma Aldrich (Steinheim, Germany). All chemicals used for the analysis were of analytical grade.
2.2. Sample Collection and Preparation To determine the proximate and mineral composition, total phenolic contents (TPC), total flavonoid contents (TFC), antioxidant capacities (DPPH, ferric-reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC)), and α-amylase inhibition activity, three wild plant samples were collected from different locations in Bangladesh. Two to three samples (300–600 g) were collected for each of the wild plants from every growing location. These were then mixed to make three analytes or composite test samples. The study samples were Achyranthes aspera L. (Upat Lengra), Eclipta alba L. (Kalokeshi), and Vitex negundo L. (Nirgundi). The samples were selected based on their traditional use, by interviewing local people, in treating diabetes. The identification of the samples was confirmed by a taxonomist of the Department of Botany, University of Dhaka, who accompanied the collection team, after examining the morphological characteristics. After collection, the leaves and roots of the samples were separated and gently washed with tap water immediately to remove sand and other extraneous material before being washed with distilled water and then air-dried. Then, the samples were cut into small pieces and freeze-dried (il Shin lab.Co. Ltd., Korea). The freeze-dried samples were ground and homogenized into a fine powder using a grinder. The homogenized samples were sieved to obtain an even particle size, then placed in an air-tight zipper bag and stored at −20 -Antioxidants C until further analysis.
2.3. Determination of Proximate Composition The proximate composition (moisture, total protein, total fat, total dietary fiber including soluble and insoluble, ash and total available carbohydrate content) of the selected samples was estimated according to the method described previously. Moisture and ash contents of the sample were calculated by the weight difference method, whereas the total fat content of the samples was estimated by the Association of Official Analytical Chemists (AOAC) method using petroleum ether as solvent. The total protein content was determined by using the micro-Kjeldhal method (nitrogen content of the samples × 6.25). The gravimetric method was utilized for the estimation of total dietary fiber (soluble and insoluble). Total available carbohydrate contents were calculated by difference using the formula below:
Carbohydrate content (%) = 100 − [total protein (%) + ash content (%) + total fat (%) + total fiber (%)].
2.4. Determination of Mineral Composition Mineral concentrations in the plants sample were calculated by using an atomic absorption spectrophotometric method described previously [23]. Briefly, approximately 500 mg of plant samples after drying were subjected to wet digestion with nitric acid and perchloric acid (2:1 ratio) in an auto-digestor at 325 0C to accelerate the discharge of minerals in the plant matrix. After digestion and appropriate dilution, the digested sample was aspirated into an air–acetylene flame to burn the elements into atomic components, which were then detected in a spectrophotometer at their relevant wavelengths. Proportions of calcium, magnesium, sodium, zinc, copper and iron were evaluated by atomic absorption spectrophotometry (Model-AA-7000S, Shimadzu, Tokyo, Japan). The amount of potassium was determined by flame photometry (Jenway flame photometer model PFP7, Origin UK). A standard calibration curve was plotted for each of the minerals using the respective mineral standard obtained from Sigma Chemical Co., USA.
2.7. Determination of Total Flavonoid Content The TFC was estimated by means of the colorimetric method according to Miao et al. [25] with slight modification. Briefly, 250 µL of the extract was mixed with 1.125 ml of distilled water in a test tube. To these, 75 µL of 5% NaNO2 solution was added. After 6 min, 150 µL of 10% AlCl3·6H2O solution was added. The solution was left to stand for another 5 min, and 500 µL of 1 M NaOH was added. Finally, the mixture was vortexed, and the absorbance was measured immediately at 510 nm by a UV-VIS spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). The TFC in the plant extract was calculated using a standard curve based on quercetin and results were expressed as milligrams quercetin equivalent (QE) per gram of dry weight (mg QE/g DW).
2.8.2. Ferric Reducing Antioxidant Power (FRAP) Assay This assay was carried out according to Miao et al. with little modification. Briefly, the FRAP reagent was made from by combining 10 mmol/L 2,4,6-tripyridyls-triazine (TPTZ) solution, 300 mmol/L acetate buffer (pH 3.6), and 20 mmol/L FeCl3 solution in a ratio of 1:10:1 (v/v), respectively. The FRAP reagent was freshly prepared and was incubated at 37 ?C in a water bath before use. 100 µl of plant extracts were added to 3 mL of the FRAP reagent. The mixture was vortexed, and absorbance of the solution was then measured at 593 nm (UV-1800, Shimadzu, Kyoto, Japan) after incubating at 37 0C for 30 min. Various concentrations (50–600 µmol/L) of Fe2+ solution was used to prepare the standard curve. The results were expressed as µmol Fe2+ per gram of dry weight (µmol Fe2+/g DW). 2.8.3. Trolox Equivalent Antioxidant Capacity (TEAC) Assay This assay was performed by the advanced ABTS•+ method as described by Miao et al. with little modification. ABTS•+ radical cation was produced by dissolving ABTS and potassium persulfate in distilled water to give a final concentration of 7 mmol/L and 2.45 mmol/L, respectively. The solutions were mixed, and the reaction mixture was left in the dark at room temperature for 24 h. The ABTS•+ solution was diluted with distilled water to an absorbance of 1.00 ± 0.03 at 734 nm. Then, 100 µL of plant extracts were added to 3.8 mL of diluted ABTS•+ solution and the solutions were kept in the dark for 10 min. After 10 min, the absorbance was read at 734 nm by a UV-VIS spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan) against the blank (distilled water). The trolox solution of various concentrations (0–15 µmol/L) was used to prepare the standard curve, and the results were expressed as µmol trolox per gram of dry weight extract (µmol trolox/g DW).
2.10. Statistical Analysis All experiments were carried out in three replicates and presented as mean ± standard deviation (SD) using Minitab version 18.0. (Minitab Inc., State College, PA, USA). One-way analysis of variance (ANOVA) and principal component analysis (PCA) were performed to check the differences between the nutrient contents, TPC, TFC, antioxidant activity, and α-amylase inhibition activity among the plant samples. The differences were declared significant at a level of p < 0.05. The Dunnett test to compare with control for α-amylase activity and Pearson correlation among variables were also calculated.