Department of Public Health, Hua Xi Medicinal Center of Sichuan University, Chengdu, PR China.
J Ethnopharmacol. 2010 Sep 15;131(2):306-12. doi: 10.1016/j.jep.2010.06.035. Epub 2010 Jun 30.
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 alpha-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.
Rat intestinal crude enzyme preparation and Caco-2 monolayer were used to evaluate alpha-glucosidase inhibitory activity of NSF extract. The main alpha-glucosidase inhibitors were detected by HPLC. For acute toxicity test, NSF extract was administered at doses of 2000, 5000 and 10,000 mg/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 150-5000 microg/plate. The chromosome aberration test was conducted with Chinese hamster lung (CHL) cells treated with NSF extract at doses of 150-5000 microg/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, 25-30 g) were administered daily by oral gavage at doses of 2.5, 5.0 and 10.0 g/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.
NSF extract showed potent rat intestinal alpha-glucosidase inhibitory activity for maltose hydrolysis with ED(50) value of 0.1 mg/ml. In Caco-2 monolayer, alpha-glucosidase activity for the maltose hydrolysis was down-regulated by NSF extract at a concentration of 0.05 mg/well level, showing 74% inhibition compared to the saline treated control. NSF was rich in phenol contents and the main alpha-glucosidase inhibitor, 1,2,3,4,6-penta-O-galloyl-beta-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 LD(50) value of this extract could be greater than 10,000 mg/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.
The current study shows that the extract from Nymphaea stellata flowers exhibits significant intestinal alpha-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.
睡莲（Nymphaea stellata willd.）花在印度和尼泊尔被用作传统药物来治疗糖尿病。不同的研究已经证明，睡莲提取物对链脲佐菌素诱导的糖尿病大鼠具有降血糖作用。在本研究中，我们评估了睡莲提取物在体外抑制肠道α-葡萄糖苷酶的作用，作为其抑制餐后高血糖以治疗糖尿病的可能作用机制。此外，还研究了睡莲提取物的可能急性口服毒性和遗传毒性。
使用大鼠肠粗酶制剂和 Caco-2 单层来评估睡莲提取物的α-葡萄糖苷酶抑制活性。通过 HPLC 检测主要的α-葡萄糖苷酶抑制剂。急性毒性试验中，将睡莲提取物以 2000、5000 和 10000mg/kg 体重的剂量分别给予三组 10 只 ICR 小鼠，然后每天观察 14 天，记录临床症状，包括死亡率、临床症状和大体发现。在艾姆斯试验中，使用组氨酸依赖的营养缺陷型沙门氏菌突变体（菌株 TA97、TA98、TA100、TA102 和 TA1535），并在存在和不存在 S9 代谢激活的情况下，使用浓度为 150-5000μg/平板的睡莲提取物进行孵育。染色体畸变试验采用中国仓鼠肺（CHL）细胞进行，在存在和不存在 S9 代谢激活的情况下，用浓度为 150-5000μg/ml 的睡莲提取物处理。在体内小鼠微核试验中，9 周龄雄性和雌性 ICR 小鼠（n=90，25-30g）每天通过口服灌胃给予 2.5、5.0 和 10.0g/kg 体重的剂量，连续 1 或 2 天。末次给药后 24h 从每组处理的动物中制备骨髓涂片，然后鉴定多染红细胞（PCE）和正染红细胞（NCE）。
睡莲提取物对麦芽糖水解具有强烈的大鼠肠道α-葡萄糖苷酶抑制活性，ED（50）值为 0.1mg/ml。在 Caco-2 单层中，睡莲提取物在 0.05mg/孔的浓度下下调麦芽糖水解的α-葡萄糖苷酶活性，与生理盐水处理的对照组相比，抑制率为 74%。睡莲提取物富含酚类物质，主要的α-葡萄糖苷酶抑制剂为 1,2,3,4,6-五-O-没食子酰基-β-D-葡萄糖，同时还鉴定出两种酚类化合物没食子酸和鞣花酸。急性毒性试验中，睡莲提取物未产生任何毒性症状或死亡，该提取物的 LD（50）值可能大于 10000mg/kg 体重。遗传毒性评估的这些结果表明，睡莲提取物在艾姆斯试验、体外染色体畸变试验和体内微核试验中均未引起遗传毒性。
本研究表明，睡莲花提取物具有显著的肠道α-葡萄糖苷酶抑制活性，且无急性毒性或遗传毒性，可能有助于抑制糖尿病患者的餐后高血糖。这里提出的结果表明，在民间医学中使用睡莲作为天然抗糖尿病治疗可能既安全又有益。
