2.1 Sample preparation
Fresh leaves of C. alata were air-dried at room temperature of 29 ± 1 °C. The sample was authenticated by the Technologist in Charge of Biochemistry Laboratory, Faculty of Science, Kings University, Nigeria. The dried leaves were pulverized, and 10 g of each pulverized sample was extracted with 100 mL of methanol at room temperature of 29 ± 1 °C for 24 h and later filtered. Two third of the crude extract was partitioned repeatedly inside a separating funnel into an aqueous extract and n-butanol extract. The crude extract and the partitioned extracts were used as the corresponding extracts for the subsequent analyses.
2.2 Preparation of the crude α-glucosidase and sucrase solution
The preparation of the crude α-glucosidase and sucrose solution was carried out as described by Dahlqvist [6]. The animal used was handled under the approved guidelines of the Ethical Review Committee of the College of Natural and Applied Sciences, McPherson University, Seriki Sotayo, Nigeria. The mucosa of the small intestine of rats sacrificed under light anaesthesia was carefully scraped off with a glass slide, homogenized with cold sodium phosphate buffer (pH 6.8), and centrifuged at 4 °C for 20 min at 650 × g. The clear solution was used as a source of crude of α-glucosidase and sucrase solutions.
2.3 Inhibition of the α-amylase activity
The determination was carried out as described by Bernfeld [7]. In a test tube containing 1.0 mL of 2 mM phosphate buffer (pH 6.9), 0.1 mL of each extract was incubated with 0.05 mL of α-amylase solution for 20 min. Precisely 0.1 mL of 1.0% of freshly prepared starch solution was subsequently added and allowed to stand for 5 min. Next, 0.5 mL of dinitrosalicylic acid reagent held in boiling water for 5 min. The solution was subsequently cooled, and the absorption was measured at 540 nm. The result was expressed in IC50 (μg/mL) calculated as the concentration needed for inhibition of 50% of α-amylase activity.
2.4 Inhibition α-glucosidase activity
The determination was carried out as described by Kim et al. [8]. In a test tube containing 1.0 mL of 2 mM phosphate buffer (pH 6.9), 0.1 mL of each extract was incubated with 0.1 mL of mucosa solution for 20 min. Subsequently, 0.1 mL of 3 mM of para-nitrophenylglucopyranoside prepared in 20 mM phosphate buffer (pH 6.9) was added and allowed to stand for 15 min. Then, 0.5 mL of 5.0% sodium carbonate was added and incubated for 90 min. The absorbance at 450 nm was measured, and the result was expressed as the concentration of inhibition required to inhibit 50% of α-glucosidase activity (IC50 (μg/mL)).
2.5 Assay of sucrase inhibitory activity
The determination was carried out as described by Honda and Hara [9]. In a test tube containing 1.0 mL of 2 mM phosphate buffer (pH 6.9), 0.1 mL of each extract was incubated with 0.1 mL of mucosal solution for 20 min. Afterwards, 0.1 mL of 60 mM sucrose solution was added and allowed to stand for 5 min. Then, 0.5 mL of dinitrosalicylic acid reagent was transferred into the test tube and allowed to incubate in boiling water for 5 min. The test tube was cooled, and the optical density was read at 540 nm. The percentage inhibition of sucrase activity was calculated, and the result was expressed in IC50 (μg/mL) as the inhibition concentration required to inhibit 50% of sucrase activity.
2.6 In vitro glycation of albumin
The preparation of glycated albumin was carried out according to the procedure defined by Safari et al. [10] with slight modifications. The solution contained bovine serum albumin (0.1 g/mL) prepared in 0.1 M phosphate buffer (pH 7.4) containing 0.01% sodium azide and d-glucose (10 mg/mL), and the extract was combined in a ratio of 3:2:1 and incubated for 72 h.
2.7 Estimation of antiglycation capacity
The determination was carried out as described by Furth [11]. In a test tube containing 1.0 mL of glycated sample, 0.5 mL of 10% trichloroacetic acid was added. For 5 min, the solution was centrifuged at 650 × g. Then, 1.0 mL of phosphate buffer and 0.5 mL of 0.3 N oxalic acids were added to the sediment and boiled for 60 min. The solution was cooled, and 0.5 mL of 10% trichloroacetic acid solution and 0.5 mL 0.05 M thiobarbituric acid were added and boiled for 10 min. The solution was centrifuged at 650 × g and the absorbance of the supernatant was read at 443 nm. The result was reported as the percentage inhibition.
2.8 Determination of inhibition of glycation-induced oxidation of protein thiol groups
The determination was carried out as described by Ellman [12]. Accurately, 1.0 mL of 0.5 mM 5,5′-dithiobis (2-nitrobenzoic acid) in 0.1 M phosphate buffer (pH 7.4) was transferred into a test tube containing 1.0 mL of glycated sample and incubated at room temperature of 29 °C for 15 min. The absorbance at 412 nm was measured. The thiol group concentration was calculated using molar extinction = 1.34 × 104M−1cm−1. The findings were documented as a protein of nmol/mg.
2.9 Determination of inhibition of protein aggregation
The determination was carried out as described by Klunk et al. [13]. Precisely, 0.1 mL of 1% Congo red prepared in phosphate buffer with 10% ethanol was added to a test tube containing 1.0 mL of glycated sample. For 30 min, the solution was incubated and the absorption was measured at 530 nm. The percentage of the results was reported.
2.10 Statistical analysis
The findings were evaluated using one-way variance analysis (ANOVA) for the mean differences between the different extracts followed by multiple comparison tests by Duncan for post hoc correlations at p < 0.05 and reported as means ± standard deviation of three determinations.
2.11 Molecular docking and in silico evaluation of drug-likeness of the compounds
The compounds selected for docking are the major compounds that are biologically active molecules as previously reported [14, 15]. These compounds were docked with the hydrolases. The 3D SDF format structures of the compounds were obtained from the PubChem database. The compounds were emodin (PubChem CID: 3220), quercetin (PubChem CID: 5280343), chrysoeriol (PubChem CID: 5280666), and kaempferol (PubChem CID: 5280863). The target 3D structures of the enzymes were identified and retrieved from the Protein Data Bank (PDB). The enzymes were human pancreatic α-amylase (1HNY), α-glucosidase (5ZCB), and sucrase-isomaltase (3LPO). All the compounds and the enzymes were converted into AutoDock pdbqt format. The binding scores of the compounds and the target enzymes were measured using PyRx-Python Prescription 0.8 (The Scripps Research Institute), and the interactions were visualized using PyMOL ver. 1.1eval (De Lano Scientific LLC, CA, USA). The computations were compared to those generated through the virtual screening of acarbose (PubChem CID: 41774) with the hydrolases. The in silico evaluation of the drug-like nature of the selected compounds was predicted using the SwissADME server (http://www.swissadme.ch/index.php) [16].