Bioassay‑guided isolation, identification and molecular ligand‑target insight of lipoxygenase inhibitors from leaves of Anisomeles malabarica R.Br.

Background: Anisomeles malabarica R. Br. (Lamiaceae) is extensively used in traditional medicine in major parts of India for several medicinal purposes, including their use in rheumatism. Materials and Methods: The air‑dried leaves of A. malabarica were extracted with ethanol, defatted with n‑hexane and then successively partitioned into chloroform and n‑butanol fractions. Bioassay‑guided fractionation and purification of chloroform fraction from A. malabarica lead to the isolation of lipoxygenase (LOX) inhibitors. The structures of isolated compounds were elucidated by ultraviolet, infrared, 1H nuclear magnetic resonance (NMR), 13C NMR and mass spectrometry spectroscopic techniques and assessed further by in vitro soybean lipoxygenase (sLOX) assay. In addition, the enzyme type inhibition was evaluated through molecular docking technique as a part of computational study. Results: The bioactive compounds 3, 4 dihydroxy benzoic acid (1) and 4’, 5, 7‑trihydroxyflavone (2) were isolated from chloroform fraction of A. malabarica, whose bioactivity was observed to be dose‑dependent compared to n‑butanol fraction. Among the compounds, 3, 4 dihydroxy benzoic acid showed significant sLOX inhibitory activity with 74.04% ±2.6% followed by 4’, 5, 7‑trihydroxyflavone (34.68% ±1.9%). The computational analysis of compounds showed their molecular interaction with important amino acid residues and nonheme iron atom in the catalytic site of LOX by enlightening their potential binding mode at molecular level. Conclusions: The LOX inhibitory constituents were identified from A. malabarica by means of bioassay‑guided fractionation process. The results derived from in vitro and computational experiments confirm the potential of the isolated compounds and provide additional evidence for its traditional use in inflammatory disorders.