SCREENING OF Î±-GLUCOSIDASE INHIBITORS FROM TERMINALIA CATAPPA L. FRUITS USING MOLECULAR DOCKING METHOD AND IN VITRO TEST
Objective: Terminalia catappa L. (T. catappa L.) fruit has inhibitory activity on Î±-glucosidase, therefore, can be a potential natural source for the treatment of type II diabetes mellitus. Inhibitory activity of ethanol fruit extract with IC50 3.02 Âµg/ml was the strongest inhibition when compared with 54 medicinal plants used as an antidiabetic agent in Indonesia. This project was aimed to find the active compound from T. catappa L. fruit using molecular docking, identification ethyl acetate subfraction using TLC and GC-MC, determine in vitro test on Î±-glucosidase inhibitory activity from ethyl acetate extract and subfraction.
Methods: Molecular docking using AutoDock 4.2 was performed to predict the binding modes of Î±-glucosidase enzyme from Saccharomyces cereviciae with 13 chemical constituents of T. catappa. Î±-Glucosidase enzyme was obtained from Protein Data Bank (PDB code: 3A4A). Acarbose, voglibose and miglitol were used as standards. Docking result determines the highest binding energy (Î”G) and inhibition constants (Ki) as an active compound. Visualization of amino acid residues around the active compound was identified with PyMOL and LigPlot. Screening of active compound was carried out by T. catappa L. fruit remaceration extraction use hexane and ethyl acetate. Ethyl acetate extract was separated on silica gel column chromatography using n-hexane, ethyl acetate and methanol sequentially based on polarity of each solvent. Identification of an active compound from ethyl acetate sub fractions using TLC and GC-MS method. The inhibitory activity of the active compound of Î±-glucosidase was determined with in vitro test using Î±-glucosidase enzyme.
Results: The highest binding energy and inhibition constant is Î²â€“sitosterol with Î”G-10.61 kcal/mol and Ki 0.02 ÂµM. The ligand was situated around of 18 amino acid residues. Ethyl acetate subfractions A, B and C showed that subfraction B contains similar spot characteristic and Rf value (0.42) with Î²-Sitosterol standard. Identification with GC-MS gave Î²â€“sitosterol acetate and sitostenone. Redocking process of Î²â€“sitosterol acetate and sitostenone showed Î”G-11.14 kcal/mol and-9.79 kcal/mol with Ki 0.01 Î¼M and 0.07 Î¼M respectively. In vitro test of acarbose, ethyl acetate extract and subfraction B gave IC50 17.52; 192.51 and 296.28 Âµg/ml.
Conclusion: Three steroids that are Î²-sitosterol, Î²-sitosterol acetate and sitostenone were the active compounds responsible for Î±-glucosidase inhibitory activity of T. catappa L. fruit. According to the in vitro test, ethyl acetate extract has stronger Î±-glucosidase inhibitory activity than ethyl acetate subfraction B.
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