Chemoprotective action of Dactylorhiza hatagirea (D. Don) Soo ethanol extract against cyclophosphamide induced reproductive toxicity and oxidative stress on TM3 cells

Cyclophosphamide (CP) is one of the common therapeutic agents which possesses anti-cancer potential, however it causes neuronal and reproductive toxicity due to rise in oxidative stress which is a major limitation to its therapeutic use. Despite of this, several research findings suggest that phytocompounds possess antioxidant potential and thus can be utilized as potential therapeutic agent for reactive oxygen species (ROS) induced damage to the cells. So as to explore the effect of root of Dactylorhiza hatagirea (D. Don) Soo belonging to the family Orchidaceae towards reproductive toxicity induced by cyclophosphamide the current study was conducted towards TM3:mouse leyding cells. In this study, ethanolic extraction roots was performed by using soxhlet and compound identification was done through GC-MS. Further, cytotoxicity and protection against reproductive toxicity effect of ethanolic root extract was analysed by using MTT assay followed by evaluation of ROS scavenging effect of extract post-treatment with CP in TM3 cells. In order to elucidate the molecular mechanism behind the protective effect of the extract, qRT-PCR was performed to analyse the expression of antioxidant and steroidogenic gene markers. The results from GC-MS analysis showed presence of two major compounds namely Benzenemethanol, 3fluoro (C1) and 2-Mercaptophenol (C2). It was also identified that the extract was non-toxic to TM3 cells and pre-treatment with enhanced cell viability in CP-treated TM3 cells. Apart from this, reduction in ROS generation was observed in pre-treated D. hatagirea CP induced cells. Additionally, the extract has enhanced the antioxidant response (via CAT, GPx1, SOD1 and SOD2) to combat the oxidative stress caused by CP, which could help in reducing the side effects of the treatment. Moreover, the extract seems to modulate the steroidogenic gene response induced by cyclophosphamide via upregulation of AR, 3βHSD, Cyp11a1, Cyp19 and StAR. These findings suggest that DH has potential therapeutic value in mitigating the adverse effects of CP through antioxidant and steroidogenic pathways.