Acute and subchronic oral toxicity study, phytochemical profiling of a herbal formulation containing Taxus, astragalus, asparagus, and liquorice in mice

Zishangan Granule (ZSG) is a plant-derived formulation containing Taxus, Astragalus, Asparagus, and licorice. Its phytochemical composition and oral toxicological safety remain insufficiently characterized. This study profiled ZSG constituents and evaluated acute and subchronic oral toxicity in Kunming mice using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) and in vivo safety assessments. UPLC-MS/MS detected 15 candidate serum-detectable constituents in ZSG and post-administration mouse serum, including Salvianolic acid L, Lithospermic acid B, Taxine A, Liquiritin, Hyperin, and Calycosin-7-O-glucoside. In the acute study, no mortality occurred after 14 days of ZSG administration at 0.43–27.52 g/kg/day. No abnormal clinical signs were observed up to 3.44 g/kg/day; however, higher doses (6.88–27.52 g/kg/day) produced dose-related reductions in food intake, slower body-weight gain, hypoactivity, and piloerection. In the 90-day subchronic study, repeated administration at the no-observed-adverse-effect level (NOAEL; 3.44 g/kg/day) caused no treatment-related changes in body weight, food intake, organ coefficients, hematology, serum biochemistry, or histopathology. These findings indicate a wide oral safety margin for ZSG at up to eight-fold the recommended mouse dose and support further preclinical development, while additional genotoxicity, reproductive toxicity, and longer-duration studies remain needed.