Phenolic Compounds from Diarthron iranica: Enzymatic and in Silico Insights Into α-Amylase Inhibitory Activity

Abstract

Background:Diarthron iranica (family: Thymelaeaceae), a medicinal plant native to Iran, contains a variety of beneficial phytochemicals, among whichphenolic compounds with a spectrum of health-promoting activities hold a special place. Objectives: This study deals with the isolation and identification of the main phenolic compounds from D. iranica and investigates their inhibitory potentialagainst alpha-amylase, an important enzyme in glucose metabolism, using in silico and in vitro approaches. Methods: The purification procedure was accomplished employing chromatographic methods, including thin-layer chromatography (TLC), medium-pressure liquid chromatography (MPLC), and high-performance liquid chromatography (HPLC). The structures were determined using spectroscopictechniques: NMR (1H, 13C, DEPT), mass spectrometry (MS), and UV-Vis spectroscopy. The in vitro alpha-amylase inhibition was performed in triplicate across sevenconcentrations (0.30 - 2.80 mg/mL) using the DNS colorimetric method. Molecular docking simulations were conducted using AutoDock 4.2, with tenconformations generated per ligand. Results: Several phenolic derivatives, including 5-(beta)-D-xylopyranoside-(1'''-> 6'')-beta-D-glucopyranoside 7-Methoxy apigenin (yuankanin, 1), 6'-Methoxy-7'-hydroxy-3'-O-7-bicoumarin (daphnoretin, 2), 4,4'-dihydroxy-3,3'-dimethoxy-7, 9':7', 9-diepoxylignan known as pinoresinol (3), and kusunokinin (4) with 3',4'-dimethoxy-3,4-methylenedioxydibenzyl butyrolactone structure were isolated and identified. In an alpha-amylase inhibition assay, compounds 1 and 3 exhibitedmoderate inhibitory activity with IC50 values of 1.32 mg/mL and 1.81 mg/mL, respectively, compared to the reference compound luteolin (IC50 = 0.63 mg/mL),indicating effective but relatively weaker inhibition. Compound 2 demonstrated the strongest inhibitory activity with an IC50 value of 0.71 mg/mL, surpassingcompounds 1 and 3. Molecular docking studies revealed that compound 1 had a superior binding free energy of -7.13 kcal/mol, forming stable interactionsthrough hydrogen bonding and van der Waals forces within the enzyme's binding site. Compound 3 showed a slightly lower binding energy of -6.43 kcal/molwith fewer stabilizing interactions. However, compound 2 demonstrated poor performance in the docking assay, despite its potent inhibitory activity in the alpha-amylase assay. Conclusions: The phytochemical analysis carried out on the aerial parts of D. iranicayielded the identification and characterization of four phenoliccompounds, including a methoxy apigenin glycoside (1), one bicoumarin (2), and two lignans (3-4). Molecular docking studies indicated that compound 1exhibited superior inhibitory potential compared to compound 3, with stable interactions in the enzyme's binding site. In alpha-amylase inhibitory assays, thesecompounds displayed varying levels of activity, with compound 2 showing the highest potency (IC50 = 0.71 mg/mL), followed by compounds 1 (IC50 = 1.32 mg/mL)and 3 (IC50 = 1.81 mg/mL). However, all were less effective than the reference compound luteolin (IC50 = 0.63 mg/mL), which demonstrated superior efficacy.