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Nymphaea stellata willd. flowers (NSF) are used as a traditional medicine in India and Nepal to treat diabetic disease. Different works have demonstrated that NSF extract showed antihyperglycemic effect on alloxan-induced diabetic rats. In the present work we evaluated in vitro intestinal α-glucosidase inhibition as the possible mode of action of NSF extract on suppressing postprandial hyperglycemia for curing diabetic mellitus. In addition, NSF extract was studied to assess its possible acute oral toxicity and genotoxicity. Materials and methods: Rat intestinal crude enzyme preparation and Caco-2 monolayer were used to evaluate α-glucosidase inhibitory activity of NSF extract. The main α-glucosidase inhibitors were detected by HPLC. For acute toxicity test, NSF extract was administered at doses of 2000, 5000 and 10,000mg/kg body to three groups of 10 ICR mice each, and then clinical symptoms including mortality, clinical sign and gross findings were observed once a day for 14 days. In Ames test, histidine-dependent auxotrophic mutants of Salmonella typhimurium (strains TA97, TA98, TA100, TA102 and TA1535) were used and incubated in the presence and absence of S9 metabolic activation using NSF extract with concentrations of 150–5000μg/plate. The chromosome aberration test was conducted with Chinese hamster lung (CHL) cells treated with NSF extract at doses of 150–5000μg/ml in the presence and absence of S9 metabolic activation. In the in vivo mouse micronucleus assay, 9-week-old male and female ICR mice (n =90, 25–30g) were administered daily by oral gavage at doses of 2.5, 5.0 and 10.0g/kg body for 1 or 2 days. Bone marrow smears were prepared from each treatment group 24h after last administration and then polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) were identified. Results: NSF extract showed potent rat intestinal α-glucosidase inhibitory activity for maltose hydrolysis with ED50 value of 0.1mg/ml. In Caco-2 monolayer, α-glucosidase activity for the maltose hydrolysis was down-regulated by NSF extract at a concentration of 0.05mg/well level, showing 74% inhibition compared to the saline treated control. NSF was rich in phenol contents and the main α-glucosidase inhibitor, 1,2,3,4,6-penta-O-galloyl-β-d-glucose, was identified together with two phenolic compounds of gallic acid and corilagin. In acute toxicity test, NSF extract did not produce any toxic signs or deaths and the LD50 value of this extract could be greater than 10,000mg/kg body weight. These results of genotoxicity assessment showed that NSF extract did not cause genotoxic effects in Ames test, in the in vitro chromosomal aberration assay and in the in vivo micronucleus assay. Conclusion: The current study shows that the extract from Nymphaea stellata flowers exhibits significant intestinal α-glucosidase inhibitory activity, without showing any acute toxicity or genotoxicity, which may be useful in suppressing postprandial hyperglycemia in diabetics. The results presented here suggest that the use of NSF in folk medicine as a natural antidiabetic treatment could be safe as well as beneficial. [Copyright &y& Elsevier]