Two new pregnane-type steroids from Astragalus variabilis: COX-2 inhibitory potential and structural characterization

Phytochemical investigation of Astragalus variabilis led to the isolation of three steroidal compounds, identified by spectroscopic analyses as variabilisterones A (1) and B (2), and Δ^3,5-Pregnadien-20β-ol-7-one (3). Compounds 1 and 2 were new pregnane-type steroids, while compound 3 was obtained from this plant for the first time. In vitro assays demonstrated that, compared with COX-1, compounds 1-3 exhibited a degree of selectivity toward COX-2 and showed moderate COX-2 inhibitory activities, with IC₅₀ values of 16.98 ± 2.25, 15.15 ± 0.70, and 15.16 ± 3.87 μM, respectively. To elucidate the potential mechanism, molecular docking, molecular dynamics simulations, and MM/GBSA calculations were performed for compounds 1-2, indicating stable binding within the COX-2 active site via predominantly hydrophobic interactions complemented by hydrogen bonding. Molecular dynamics results demonstrated overall structural stability of the complexes without significant perturbation of protein secondary structure, while DCCM analysis suggested minimal impact on global protein dynamics, and binding free energy calculations confirmed thermodynamically favorable interactions. ADMET predictions were further conducted to preliminarily evaluate pharmacokinetic properties. Collectively, these findings highlight the anti-inflammatory potential of pregnane-type steroids from A. variabilis and support their further investigation as potential COX-2 inhibitors.