Background: Phytomedicine is becoming more acceptable as an alternative medicinal approach in the modern era. Objectives: The current study examined the antioxidant capacity and in vitro response of phytochemical constituents of Withania somnifera (Ashwagandha) on standard parameters of healthy volunteer semen. Methods: The phytochemicals and their pharmacological response in a hydroethanolic (30:70 v/v) extract of W. somnifera roots were determined using standard protocols. Results: The constituents included flavonoids, phenolic acids, alkaloids, and terpenoids were reported. High-performance liquid chromatography and Fourier-transform infrared spectroscopy determined a diverse array of biologically active chemical constituents in the extract. The extract of W. somnifera exhibits substantial antioxidant properties, including total antioxidant capacity, 2,2-diphenyl-1-picrylhydrazyl inhibition, H₂O₂ scavenging, and Fe³⁺ reducing potential (P < .05). The analysis of essential natural minerals explored adequate levels determined using atomic absorption spectrophotometer. Cytotoxic studies revealed significant thrombolytic, RBC membrane stabilization, and DNA damage protection activity (P < .05) while remaining non-mutagenic against Salmonella typhi TA98 and TA100. The best protective response of W. somnifera extract on human semen parameters (n = 30), such as total motility, progressive motility, and viability, demonstrated a significant (P < .05) improvement, particularly at the dose of 25 μg/mL and 50 μg/mL. Conclusion: The study concludes that W. somnifera possesses favorable in vitro characteristics that could aid in the preservation of sperm during intrauterine insemination and in vitro fertilization.

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