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

Diarthron iranica (family: Thymelaeaceae), a medicinal plant native to Iran, contains a variety of beneficial phytochemicals, among which phenolic compounds with a spectrum of health-promoting activities hold a special place.

This study deals with the isolation and identification of the main phenolic compounds from D. iranica and investigates their inhibitory potential against α-amylase, an important enzyme in glucose metabolism, using in silico and in vitro approaches.

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 spectroscopic techniques: NMR (¹H, ¹³C, DEPT), mass spectrometry (MS), and UV-Vis spectroscopy. The in vitro α-amylase inhibition was performed in triplicate across seven concentrations (0.30 - 2.80 mg/mL) using the DNS colorimetric method. Molecular docking simulations were conducted using AutoDock 4.2, with ten conformations generated per ligand.

Several phenolic derivatives, including 5-[(β)-D-xylopyranoside-(1'''→6'')-β-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 α-amylase inhibition assay, compounds 1 and 3 exhibited moderate 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, surpassing compounds 1 and 3. Molecular docking studies revealed that compound 1 had a superior binding free energy of -7.13 kcal/mol, forming stable interactions through 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/mol with fewer stabilizing interactions. However, compound 2 demonstrated poor performance in the docking assay, despite its potent inhibitory activity in the α-amylase assay.

The phytochemical analysis carried out on the aerial parts of D. iranica yielded the identification and characterization of four phenolic compounds, including a methoxy apigenin glycoside (1), one bicoumarin (2), and two lignans (3-4). Molecular docking studies indicated that compound 1 exhibited superior inhibitory potential compared to compound 3, with stable interactions in the enzyme's binding site. In α-amylase inhibitory assays, these compounds 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.