Abstract
Background: Cyclophosphamide (CP) is widely used in chemotherapy but can induce oxidative stress–mediated reproductive toxicity. This study evaluated the protective effects of an ethanolic leaf extract of Hibiscus tiliaceus against CP-induced testicular injury in male rats.
Methods: Thirty adult male Wistar rats were divided into five groups (n = 6): control, CP (150 mg/kg, intraperitoneally on day 6), CP plus H. tiliaceus extract at 250 or 500 mg/kg orally for six days, and extract alone at 500 mg/kg. Phytochemical screening, quantitative assays, and HPLC were performed. Testicular tissue levels of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and serum levels of testosterone, FSH, and LH were measured using ELISA kits, followed by histopathological examination and Johnsen scoring. The relative mRNA expression levels of TNF-α and IL-6 in testicular tissue were determined by quantitative real-time PCR and normalized to GAPDH using the 2^−ΔΔCt method.
Results: The extract contained high levels of tannins, phenolics, alkaloids, flavonoids, glycosides, and saponins, while HPLC identified quercetin, gallic acid, gossypetin, caffeic acid, kaempferol, and chlorogenic acid. Cyclophosphamide significantly increased testicular MDA and upregulated TNF-α and IL-6 expression, while reducing GSH, SOD, CAT, serum testosterone, FSH, LH, and Johnsen’s score compared with the control group. Treatment with H. tiliaceus extract significantly attenuated these biochemical, hormonal, molecular, and histopathological alterations. The 500 mg/kg dose produced greater protection than the 250 mg/kg dose, restoring FSH and LH to 46.46 ± 2.76 and 10.76 ± 2.16 mIU/mL, respectively, and reducing TNF-α and IL-6 expression to 2.30 ± 1.13-fold and 2.18 ± 0.65-fold, respectively.
Conclusion: H. tiliaceus ethanolic leaf extract exerted dose-dependent antioxidant and gonadoprotective effects against CP-induced testicular toxicity, likely through its phenolic- and flavonoid-rich composition and enhancement of endogenous antioxidant defenses.
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