NMR Structure Elucidation and Molecular Modeling of Lipoxygenase and Cholinesterase Inhibiting Steroids from Hypericum oblongifolium

Abstract: Hypericum oblongifolium is a potent source of bioactive constituents. A series of pharmacological properties, ranging from wound healing and antiseptic to antiviral, anti-inflammatory, anticancer, ethanol intake inhibition, and apoptosis-inducing activities have been associated with this plant. The current research project was designed to investigate the new secondary metabolites in H. oblongifolium having excellent pharmaceutical potential. In the present investigation two new cholestane-type steroids, hyperinoate A (1) and hyperinoate B (2) were isolated from a chloroform soluble fraction of the whole plant of H. oblongifolium. Structures of isolated new compounds were elucidated based on spectroscopic data including 1D (1HNMR, 13CNMR) and 2D (HMBC, COSY, NOESY) NMR and mass spectrometry (EIMS, HREIMS) data. After structure elucidation, new compounds were named 6α-hydroxy-14α-methyl Cholestan-3-yl-acetate and 6α,25-dihydroxy-14α-methyl Cholestane-3-yl-acetate. Both steroids showed promising inhibitory activity against lipoxygenase (LOX) and acetylcholinesterase (AChE) enzymes. Especially hyperinoate A (1) inhibited the lipoxygenase (LOX) enzyme with IC50 41.7± 0.15 µM whereas Baicalein (positive control) had IC50 42.3 ± 0.20 µM. Similarly, Hyperinoate B (2) (56.3 ± 0.33 µM) showed higher inhibition than Galantamine (positive control) 53.8 ± 0.54 µM. These results were validated with in-silico molecular docking investigations. The greater binding affinity of hyperinoate A (1) (-9.2 Kcal/mol) against LOX-5(PDB ID 3V99) showed higher inhibition as compared to baicalein (positive control) (-7.7 Kcal/mol). The binding affinities of hyperinoate B (2) (-10.5 Kcal/mol) were close to galantamine (-10.6 Kcal/mol). All in-vitro and in-silico results revealed that both newly isolated compounds showed moderate inhibition against lipoxygenase (LOX) and acetylcholinesterase (AChE) enzymes.