In vitro and In silico Studies of Plumbago zeylanica Root Fractions on Mitochondrial-Related Parameters on Male Wistar Rats

The mitochondrial permeability transition pore (mPTP) controls release of pro-apoptotic factors; its modulation is a therapeutic strategy for diseases characterized by dysregulated apoptosis. This study evaluated the effects of Plumbago zeylanica (PZ) root fractions on mitochondrial function in male Wistar rat liver and investigated molecular interactions between key phytochemicals and Cyclophilin D (CypD), a regulator protein of mPTP. Aqueous root extract was partitioned into hexane, ethyl acetate, butanol, and residual aqueous fractions. Isolated rat liver mitochondria were incubated with graded concentrations (4–20 µg/mL) of each fraction. mPTP opening was monitored spectrophotometrically at 540 nm (mitochondrial swelling assay). Mitochondrial ATPase activity and lipid peroxidation (TBARS) were measured. Phytochemical composition was profiled by HPLC/GC-FID. Selected phytochemicals were docked to human CypD using AutoDock Vina; chitranone (top hit) underwent 50 ns molecular dynamics (GROMACS). All organic fractions induced significant mPTP opening in a concentration-dependent manner (maximum fold inductions at 20 µg/mL: BRF 9.7-fold; HRF 9.4-fold; ARF 9.4-fold; ERF 9.1-fold; spermine reversed pore opening). Mitochondrial ATPase activity increased significantly (p<0.05). Lipid peroxidation inhibitory capacity decreased compared to control (p<0.05). HPLC/GC identified plumbagin and chloroplumbagin as major peaks. Docking showed stable interactions between phytochemicals and CypD; chitranone exhibited the highest binding affinity (−8.5 kcal•mol⁻¹) and formed three hydrogen bonds with Asp227, Asn141 and Gly222. MD trajectories indicated stable ligand–protein complexes. PZ root fractions modulate mPTP opening and mitochondrial function in vitro, and chitranone displays favourable binding to Cyclophilin D in silico. These data suggested a mitochondrial-mediated mechanism underlying the cytotoxic properties of PZ. Further in vitro and in vivo studies are required.