2.1. Materials Fresh vegetables, namely ash gourd (BINA CHALKUMRA 1), bitter gourd (BARI KOROLA 1), brinjal (BARI BEGUN 8), Indian spinach (BARI PUISHAK 1), kangkong (BARI GIMAKOLMI 1), okra (BARI DHEROSH 2), ridge gourd (BARI JHINGA 1), snake gourd (BARI CHICHINGA 1), and stem amaranth (BARI DATA 1), were collected from the Bangladesh Agricultural Research Institute (BARI), Gazipur, Bangladesh. The vegetables were dried in the oven at 40 0C, ground into powders, and then stored at −20 0 C. Porcine pancreatic α-amylase, porcine PL, rat intestinal acetone powder, acarbose, orlistat, 4-nitrophenyl α-D-glucopyranoside (PNP), N-(3-[2-furyl]acryloyl)-phenylalanyl glycyl glycine (FAPGG), and rabbit lung ACE were purchased from Sigma-Aldrich (St. Louis, MO, USA). A human recombinant renin inhibitor screening assay kit was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Other analytical grade chemical reagents were obtained from Fisher Scientific Company (Oakville, ON, Canada).
2.2. Extraction of Polyphenolic Compounds Water-soluble free (unbound) polyphenols were extracted using a previous method with minor modifications as follows. Briefly, dried vegetable powders were weighed and one part mixed with 20 parts of distilled water (1:20) in a 500 mL beaker, which was then adjusted to 60 0C and stirred for 2 h. After cooling to ambient temperature, the mixture was centrifuged for 30 min at 5600× g, and the supernatant passed through a muslin cloth to collect filtrate #1. The residue was mixed with water (1:20) and the extraction, centrifugation, and filtration processes repeated to collect filtrate #2, which was then combined with filtrate #1. The combined filtrate was partially evaporated in a rotary evaporator (Heidolph Instruments GmbH & CO., Schwabach, Germany) under vacuum (~24 mm Hg) at 60 0C, and the aqueous residue freeze-dried and stored at −20 0C.
2.3. UHPLC MS/MS Analysis of Polyphenolic Compounds An Agilent 1290 UHPLC system (Santa Clara, CA, USA) coupled with an HSS T3 2.1 × 100 mm 1.7 µm column from Waters Corp (Milford, MA, USA) was used to perform UHPLC analysis of the vegetable extracts. Samples were mixed with distilled water, vortexed, and passed through a 0.2 µm filter. Then, a 5 µL portion of the filtrate was injected onto the column followed by elution at a flow rate of 0.5 mL/min using mobile phases A and B (0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively) at 40 0C. The following gradients were used: initial holding time 0.5 min, mobile phase B ramped up to 50% after 5 min, 95% after 6 min, held for 1 min, and re-equilibrated for 1.5 min. Compounds were identified using a diode array detector at a wavelength range of 230–640 nm in 2 nm increments and a frequency of 5 Hz. The mass spec was carried out in an Agilent 6550 QTOF (Santa Clara, CA, USA) at 200 0C, using a drying gas pressure of 18 psi, 40 psi nebulizer and 350 0C sheath gas, a pressure of 12 psi, and 3500 V capillary with a 1000 V nozzle, and ran in positive ion electrospray at a frequency of 3Hz and acquisition from 30–1700 m/z. The MS/MS was performed at a narrow quadruple setting (1.3 atomic mass units), using 10, 20, and 40 eV collision energy and 30–1700 m/z. The compounds were identified using MS/MS fragmentation patterns and quantified based on the MS peak area.
2.4. Inhibition of α-Amylase Activity α-amylase inhibition assay was carried out using a previous method with slight modifications. Plant extracts were dissolved in 1 mL of 0.2 mM sodium phosphate buffer, pH 6.9 containing 6 mM NaCl. Then, 100 µL of sample aliquot (0.03–10 mg/mL final concentration) and 100 µL of α-amylase solution were added together in a test tube and incubated for 10 min at 25 0C. A 100 µL amount of 1% starch (previously dissolved in the same buffer, heated, and cooled) was added to the mixture and incubated again at 25 0C for 10 min. Then, 200 µL of 96 mM dinitrosalicylic acid (DNSA), prepared in 2 M sodium potassium tartrate tetrahydrate, were added to terminate the reaction and heated in a water bath at 100 0C for 15 min. Subsequently, a 3 mL amount of double-distilled water was added after the reaction mixture was cooled down to room temperature. From this reaction mixture, a 200 µL aliquot was transferred to a 96-well microplate and absorbance read at 540 nm using a Synergy™ H4 microplate reader (Biotek™, Winooski, VT, USA) at 25 0C. The phosphate buffer was used as a blank and its absorbance subtracted from each well to calculate enzyme activity. Acarbose, a known α-amylase inhibitor, was used as standard and assayed concomitantly with the samples. The inhibitory activity of α-amylase was calculated using the following equation:
Inhibition (%) = (Ac − As/Ac) × 100)
where Ac = Absorbance of the control (no inhibitor) and As = Absorbance of the sample.
2.5. Inhibition of α-Glucosidase Activity α-glucosidase inhibitory activity of the samples was determined according to a previously described method with the following modifications. First, a 300 mg portion of rat intestinal powder was mixed with 9 mL of 0.9% (w/v) NaCl solution and centrifuged at 5600× g for 30 min, and the supernatant was used as the source of α-glucosidase activity. Plant extracts were dissolved (final concentration of 0.03–10 mg/mL) in 0.1 M sodium phosphate buffer pH 6.9 and 50 µL mixed with 50 µL of the α-glucosidase solution in a 96-well microplate followed by incubation for 10 min at 37 0C. Then, 100 µL 5 mM (PNP) also dissolved in the phosphate buffer were added to each well and the absorbance read at 405 nm in 30 s intervals for 30 min using the Synergy™ H4 microplate reader with temperature maintained at 37 0C. A blank measurement was taken without the addition of the enzyme, and its absorbance was subtracted from each well. Acarbose, an α-glucosidase inhibitor, was used as standard and assayed using the same protocol. The following equation was used to determine the α-glucosidase inhibitory activity of the samples: Inhibition (%) = (Ac − As)/(Ac) × 100 where Ac = Absorbance of the control (no inhibitor) and As = Absorbance of the sample.
2.6. Inhibition of Lipase Activity The method described by Tang et al. with slight modifications was used to determine PL inhibition by measuring the release of 4-methyl umbelliferone (4MU) from 4-methyl umbelliferyl oleate (4MUO). PL solution (final concentration of 3.125 U/mL) was prepared in 13 mM Tris-HCl buffer, pH 8.0 containing 1.3 mM CaCl2 and 25 µL added to the mixture containing 225 µL of a 0.5 mM 4-MUO solution and 25 µL of sample (different concentrations) to start the enzyme reaction, followed by incubation for 1 h at 37 0C. The Synergy™ H4 microplate reader was set at 400 nm and used to measure the amount of 4MU released during the reaction. Orlistat, a commonly used pharmacological agent against PL, was used as the standard. The following equation was used to calculate PL inhibition: Inhibition (%) = (Ac − As)/(Ac) × 100 where Ac = Absorbance of control (no inhibitor) and As = Absorbance of the sample.