Immunomodulatory and Antioxidant Effects of Stachys Japonica Miq. Ethanol Extract on Cyclophosphamide-Induced Immunosuppressed Mice and its Phytochemical Composition

Stachys japonica Miq. is an edible wild botanical drug and a traditional medicine in China. This study focused on examining the phytochemical profile and antioxidant properties of the ethanol extract of S. japonica Miq (SJEE) in vitro, as well as its immunomodulatory and antioxidant activities in cyclophosphamide (Cy)-induced immunosuppressed mice. The SJEE was analysed by UPLC-HRMS/MS. 36 Polyphenols, including flavonoids, cinnamic acid derivatives, alcohols, and polyolsphenylethaoids, and hydroxycoumarins were identified. Total phenolics (102.06 ± 2.35 mg GAE/g extract) and total flavonoids (59.77 ± 1.09 mg RE/g extract) contents were measured. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, hydroxyl radical scavenging activity, superoxide anion scavenging activity, and reducing ability tests demonstrated that SJEE possesses remarkable antioxidant properties. In Cy-treated mice, intragastric administration of SJEE (100, 200, and 400 mg/kg•d^-1) resulted in upregulation of immune organ indices, mitigation of immune organ injury, and restoration of impaired immune function. SJEE significantly enhanced splenocyte proliferation, strengthened macrophage phagocytosis, and substantially improved hemolysin levels while restoring delayed-type hypersensitivity (DTH) responses.  It also promoted body weight recovery in immunosuppressed mice.  Furthermore, the high-dose SJEE treatment resulted in increased liver enzyme activities of superoxide dismutase, glutathione peroxidase, and catalase, accompanied by reduced malondialdehyde levels, indicating potent in vivo antioxidant effects. These results suggest that SJEE has considerable immunomodulatory potential, capable of shielding the immune system from oxidative stress and helping maintain internal homeostasis. Consequently, SJEE holds promise for development as a functional food or immune adjuvant to support human health.