Identification of alpha-glucosidase, anti-oxidant and toxicity assessment of psychotria malayana jack leaves using metabolomics approach

Abstract

Psychotria malayana Jack belongs to Rubiaceae family and known in Malaysia as “meroyan sakat/salung”. It is widely available in Malaysia and other Southeast Asian countries where it is traditionally used to manage diabetes. However, its folk claims as an antidiabetic agent and proper metabolites profiling are yet to be carried out to further confirm its role as an efficacious antidiabetic herbal remedy. Therefore, this study was aimed to evaluate the anti-diabetic activity of this plant extracts (0, 25, 50, 75, and 100% v/v methanol–water) through α-glucosidase (AG) inhibitory assay as well as toxicity determination using zebrafish embryos/larvae (Danio rerio) model. The extracts were also assessed for antioxidant activity to further confirm its antidiabetic potential. The AG inhibitors of the plant were identified using gas and liquid chromatography fitted with mass spectrometry (GCMS and LCMS, respectively) and nuclear resonance spectroscopy (NMR) based metabolomics approach. While the ligand-protein interaction was elucidated through molecular docking study. Furthermore, a validated analytical technique was also developed applying a Fourier Transform Infrared Spectroscopy (FTIR) fingerprint and utilizing an orthogonal partial least square (OPLS). The methanol extract possessed the highest AG inhibitory activity (IC50 2.83 ± 0.32 μg/mL). In addition, methanol extract showed potent insulin sensitizing and antioxidant activities. A total of eight putative bioactive compounds were identified namely 1,3,5-benzenetriol (1); palmitic acid (2); cholesta-7,9(11)-diene-3-ol (3); 1-monopalmitin (4); β-tocopherol (5); α-tocopherol (6); 24-epicampesterol (7); and stigmast-5-ene (8) using GCMS-based metabolomics approach. Furthermore, five putative bioactive compounds, namely 1-monopalmitin (4), 4-hydroxyphenylpyruvic acid (10), 5′-hydroxymethyl-1′-(1, 2, 3, 9-tetrahydro-pyrrolo (2, 1-b) quinazolin-1-yl)-heptan-1′-one (11), α-terpinyl-β-glucoside (12), and machaeridiol-A (13) were identified through LCMS-based metabolomics approach. Additionally, using NMR-based metabolomics, two putative bioactive compounds were identified, namely 4-hydroxyphenylpyruvic acid (10) and glutamine (14). Docking results of all thirteen putative bioactive compounds showed moderate to high binding affinities (-5.5 to -10.0 kcal/mol) towards the active site of the enzymatic protein. Several residues, namely ASP352, HIE351, GLN182, ARG442, ASH215, SER311, ARG213, GLH277, GLN279, PRO312, HIE280, and GLU411 established hydrogen bond in the docked complex. The OPLS model developed by FTIR and validated using six external samples of same plant sepcies and potentially predicted the AGI activity of all extracts. Therefore, it can be suggested to be used as a tool in the plant’s quality control. The median lethal concentrations (LC50) of 0%, 25%, 50%, 75% and 100% MeOH extract were found to be toxic (252.45, 119.40, 81.28, 64.71, and 37.50 µg/mL, respectively). Conclusively, thirteen putative AG inhibitors from P. malayana were identified through metabolomics approach in this study and methanol extract is better to use based on its potency and safety. All of these findings might prove helpful for this plant to be used as a promising anti-diabetic medicine in the future.