Protective Effects of Moringa oleifera Aqueous Leaf Extract on TM3 Leydig Cells Against Hydrogen Peroxide‐Induced Oxidative Stress

Introduction Moringa oleifera ( M. oleifera ) is a medicinal plant rich in dietary antioxidants that may protect reproductive cells from oxidative stress‐induced damage and male fertility problems. This study investigated M. oleifera aqueous leaf extracts (ALE) effects on TM3 Leydig cells exposed to hydrogen peroxide (H 2 O 2 ). Methods M. oleifera ALE and ascorbic acid (10−1000 µg/mL) were assessed for antioxidant activity. TM3 Leydig cells were exposed to various concentrations of M. oleifera ALE (0−1000 µg/mL) or α‐tocopherol (50 µM) for 24 h, alongside 200 µM H 2 O 2 , with or without hCG (4 ng/mL). Cell viability, testosterone production, mitochondrial membrane potential (MMP), total protein and oxidative stress parameters (reactive oxygen species [ROS], superoxide dismutase [SOD], reduced glutathione [GSH], total antioxidant capacity [TAC] and lipid peroxidation [MDA]) were assessed. Results Ascorbic acid demonstrated superior DPPH radical scavenging and FRAP activity compared to M. oleifera ALE at most concentrations ( p < 0.0001). H 2 O 2 (200 µM) exposure significantly reduced cell viability, MMP, SOD, GSH and TAC levels, while increasing ROS and MDA levels ( p < 0.05). Treatment with M. oleifera ALE or α‐tocopherol significantly increased cell viability, total protein, SOD activity and GSH levels while reducing ROS and MDA compared to H 2 O 2 control ( p < 0.05). However, testosterone levels remained unchanged ( p > 0.05). Conclusions M. oleifera ALE effectively protects the TM3 Leydig cells from H 2 O 2 ‐induced oxidative stress by restoring the redox balance, though unchanged testosterone levels suggest limitations in using TM3 Leydig cells for assessing testosterone synthesis.