海带根中α-glucosidase 和PTP-1B 靶向生物活性物质的分离纯化及其抗糖尿病作用研究

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	海带根中α-glucosidase 和PTP-1B 靶向生物活性物质的分离纯化及其抗糖尿病作用研究
其他题名	Isolation of α-glucosidase and PTP-1B Targeted Bioactive Compounds from Laminaria japonica Root and Identification of their Anti-diabetic Effects
	周通
学位类型	硕士
导师	林秀坤
	2007-06-15
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
学位专业	海洋生物学
关键词	Α-glucosidase Ptp-1b 海带根糖尿病
摘要	海带根是一种治疗糖尿病的民间中药，在沿海地区有很长的民间用药历史。食用海带根能够有效降低糖尿病患者的血糖，起到治疗作用。本文目的在于发现海带根中抗糖尿病的天然活性物质并分析它们在糖尿病治疗中的靶点；进一步开发一种低价且无毒副作用的化学类新药或中药新药。 α-glucosidase和 PTP-1B是II型糖尿病的两个重要靶点，海带根提取物能同时作用于这两个靶点。通过抑制这两种酶，降低血糖水平，85%乙醇粗提物对两种酶的IC50分别为1589ug/ml、IC50 1271ug/ml。乙酸乙酯相和石油醚相分别抑制α-glucosidase和 PTP-1B，IC50分别为380ug/ml和220ug/ml。因此以α-glucosidase和 PTP-1B的抑制活性为导向，用天然产物化学的方法对活性成分进行追踪分离，寻找单体活性物质进而鉴定其结构。由于乙酸乙酯相具有α-glucosidase抑制活性，用硅胶柱层析（石油醚：丙酮5：1、1：1），（二氯甲烷：甲醇60：1、20：1、5：1），凝胶柱层析Sephadex LH20（二氯甲烷：甲醇1：1），HPLC （80% 甲醇-水），对α-glucosidase抑制剂进行分离，得到组分IC50 为3.6ug/ml。用质谱仪和核磁共振确定结构。生物活性测定结果表明α-glucosidase和 PTP-1B是两种不同的物质，分别位于乙酸乙酯相和石油醚相。光照实验和高温实验表明抑制α-glucosidase的活性成分对光照和温度敏感。光照48h或者50℃ 12h而且对α-glucosidase的抑制活性显著降低，TLC检测并用FeCl3显色初步表明抑制α-glucosidase的活性成分可能是多数酚类物质。动物实验显示在1450ug/kg剂量下，乙酸乙酯相能够显著降低糖尿病小鼠血糖，与阴性对照组差异极显著（P<0.01）。表明，海带根提取物在体内和体外均呈现出抗糖尿病活性，是一种潜在的抗糖尿病药物。
其他摘要	According to the fact that kelp root has been a kind of civilian anti-diabetic drug for long, bioactive compounds are expected to be isolated from Laminaria japonica root, and their molecular targets to be identified in Diabetes Mellitus Ⅱ. The crude extract was discovered to dose-dependently inhibit both α-glucosidase (IC50 1589ug/ml) and PTP-1B(IC50 1271ug/ml), either of which plays important role in DMⅡ。EtOAc part and P.Ether part have inhibiting activity of α-glucosidase and PTP-1B respectively. Methodology and various techniques of natural product chemistry were subsequently employed to purify and characterize the anti-diabetic components. The purifying process was strictly guided by α-glucosidase and/or PTP-1B inhibiting bioactivity. Since α-glucosidase inhibitor was detected in EtOAc part, the bioactive compound was supposed to be purified through silica column chromatography eluted by P. ether: Actone and Dichloromethane: Methanol, and then through Sephadex LH20 filtration chromatography eluted by Dichloromethane: Methanol. Immediately after each step of chromatography enzymatic assays would locate the bioactive part and the IC50 of each step would also be calculated. HPLC was applied to further purification. Chemical structure of α-glucosidase inhibitor, with IC50 of 3.6ug/ml, was identified by MS and NMR. Bioactivity assay demonstrates that α-glucosidase inhibitor and PTP-1B inhibitor are different compounds, located themselves in EtOAc part(IC50 380ug/ml) and P.Ether part(IC50 220ug/ml) respectively. α-glucosidase inhibitor in kelp root is light sensitive and high-temperature sensitive. Treatment of 48h in light or 50℃ 12h results in remarkable reduction of bioactivity. TLC analysis, FeCl3 as chromogenic reagent, shows the labile bioactive compound seems to be polyphenol. Treatment to STZ induced diabetic mice shows that P.Ether and EtOAc part together, has remarkable anti-hyperglycemic activity, at the dose of 1450ug/kg, statistically different with standard group. In conclusion, kelp root has anti-diabetic activity both in vitro and in vivo, and could be developed as a potential new drug.
页数	56
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/421
专题	海洋环流与波动重点实验室
作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	周通. 海带根中α-glucosidase 和PTP-1B 靶向生物活性物质的分离纯化及其抗糖尿病作用研究[D]. 海洋研究所. 中国科学院海洋研究所,2007.