| Summary: | Diabetes mellitus (DM) is a metabolic complication and can pose a serious challenge to human health. DM is the main cause of many life-threatening diseases. Researchers of natural products have been continuously engaged in treating vital diseases in an economical and efficient way. In this research, we extensively used phytosteroids from <i>Notholirion thomsonianum</i> (Royle) Stapf for the treatment of DM. The structures of phytosteroids <b>NtSt01</b> and <b>NtSt02</b> were confirmed with gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) analyses. Through in vitro studies including α-glucosidase, α-amylase, and DPPH assays, compound <b>NtSt01</b> was found to be comparatively potent. An elevated dose of compound <b>NtSt01</b> was also found to be safe in an experimental study on rats. With a dose of 1.0 mg/kg of <b>NtSt01</b>, the effect on blood glucose levels in rats was observed to be 519 ± 3.98, 413 ± 1.87, 325 ± 1.62, 219 ± 2.87, and 116 ± 1.33 mg/dL on the 1st, 7th, 14th, 21st, and 28th, days, respectively. The in vivo results were compared with those of glibenclamide, which reduced the blood glucose level to 107 ± 2.33 mg/dL on the 28th day. On the 28th day of <b>NtSt01</b> administration, the average weights of the rats and vital organs (liver, kidney, pancreas, and heart) remained healthy, with a slight increase. The biochemical parameters of the blood, i.e., serum creatinine, blood urea, serum bilirubin, SGPT (or ALT), and serum alkaline phosphatase, of rats treated with <b>NtSt01</b> remained in the normal ranges. Similarly, the serum cholesterol, triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels also remained within the standard ranges. It is obvious from our overall results that the phytosteroids (specifically <b>NtSt01</b>) had an efficient therapeutic effect on the blood glucose level, protection of vital organs, and blood biochemistry.
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