Dual COX/5-LOX inhibition by isorhamnetin-3-O-glucoside from Anthyllis cytisoides: An integrated chemical-enzymatic-computational study

Abstract

Ethnopharmacological relevanceAnthyllis cytisoides L. is a forage shrub native to the western Mediterranean basin, traditionally used in folk medicine to treat respiratory and gastrointestinal inflammation. However, its polar phytochemical profile and the underlying enzyme-level anti-inflammatory mechanisms involving COX-1/COX-2 and 5-LOX remain weakly documented. Aim of the studyTo integrate chemical, enzymatic, and computational evidence that links the constituents of A. cytisoides L to COX-1, COX-2, and 5-LOX targets, thereby contextualizing the species’ ethnopharmacological indications. Materials and methodsThe aerial parts were subjected to polarity-guided fractionation followed by LC–HRMS profiling. From the methanolic fraction, a flavonol glycoside was isolated and structurally elucidated as isorhamnetin-3-O-glucoside (I3OG) using ESI–MS, IR spectroscopy, and 1D/2D NMR analyses. Enzymatic inhibition of COX-1, COX-2, and 5-LOX was assessed (COX via PGE2 radioimmunoassay; 5-LOX via FOX assay at 560 nm), and IC50 values were calculated using four-parameter logistic regression (95 % CI). Molecular docking (COX-1: 1EQG; COX-2: 5F19; 5-LOX: 6NCF) and 100-ns molecular dynamics simulations (GROMACS) were also performed. ResultsI3OG was successfully identified and purified from A. cytisoides. It inhibited COX-1, COX-2, and 5-LOX with IC50 values of 10.65 ± 1.02, 15.09 ± 0.84, and 15.24 ± 1.18 μg/mL (≈22.3, 31.6, and 31.9 μM), respectively, surpassing the inhibitory activity of the crude extracts for all targets. Docking studies and 100-ns MD simulations confirmed stable interactions with each enzyme, in agreement with the experimental inhibition ranking (COX-1 > COX-2 ≈ 5-LOX). ConclusionI3OG emerges as a promising dual COX/5-LOX candidate from A. cytisoides, providing a coherent link between detailed phytochemical analysis, enzymatic inhibition, and atomistic binding mechanisms. Although selectivity and safety considerations fall outside the scope of this enzyme-focused study, the findings highlight A. cytisoides as a valuable source of multitarget anti-inflammatory scaffolds and support SAR-driven optimization and formulation strategies aimed at enhancing bioactivity.