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复方金鹳草化学成分及抗糖尿病活性研究
蒋岚
学位类型博士
导师信学雷
2016-06-03
学位授予单位中国科学院大学
学位授予地点北京
学位专业有机化学
关键词复方金鹳草 丘陵老鹳草 腺点金丝桃 2型糖尿病 糖脂代谢
摘要

复方金鹳草来源于《阿维森纳医典》民族医传统经典验方,由两种中亚传统药用植物丘陵老鹳草(Geranium collinum Steph.ex Willd)和腺点金丝桃(Hypericum Scabrum Lnn)按7:3比例组成,民间用来治疗糖尿病,临床疗效显著。据查证,针对该复方治疗糖尿病的作用机理及两种组方药材未有深入研究,这使得该方的现代化研究和开发缺少科学依据。本论文以复方金鹳草、丘陵老鹳草和腺点金丝桃为研究对象,通过体外活性追踪,确定了复方和组方药材的有效部位及化学成分。探索性研究了复方治疗2型糖尿病的有效成分及作用靶点,阐释治疗2型糖尿病的作用机制。为复方金鹳草的进一步研究和开发提供科学依据,也为该复方抗糖尿病的作用机理提供研究思路和技术方法上的借鉴。以抗氧化、抑制PTP1B等体外活性为导向,确定了复方金鹳草有效部位的制备工艺。丘陵老鹳草和腺点金丝桃按7:3比例混合,用50%乙醇提取,提取物的AB-8大孔树脂纯化部位GC30、GC70具有良好的抗氧化活性和抑制PTP1B活性,并对GC30进行LC-MS/MS分析,鉴定了其中20个单体化合物。对组方单味药材丘陵老鹳草不同溶剂提取物测定抑制PTP1B、α-葡萄糖苷酶活性以及体外抗氧化活性,发现50%乙醇提取物具有较好抗氧化、抗糖尿病的生物活性,其总多酚和总黄酮的含量可达到350mg·g-1和96mg·g-1,并从50%乙醇提取物乙酸乙酯部位中分离鉴定了9个化合物。该研究为首次对丘陵老鹳草的化学成分及体外降糖活性进行报道。对组方单味药材腺点金丝桃进行化学成分分析以及生物活性测定。腺点金丝桃70%乙醇提取物具有最好的PTP1B抑制活性及较好的抗氧化活性,其总多酚和总黄酮的含量分别为107 mg?g?1和23mg?g?1,并从中分离得到11个化合物,除芦丁外,其他化合物均为首次在该植物中分得。在复方金鹳草抗糖尿病机理的初步探索中,发现该复方纯化部位GC30、GC70以及特征单体化合物老鹳草素、鞣花酸具有促进葡萄糖摄取、利用、合成糖原作用。试验发现,有效部位和单体化合物作用于细胞后,引起了ATP水平降低,激活了AMPK。推测该复方有效部位以AMPK及其信号通路为靶点,具有改善糖脂代谢的作用。同时,在PI3K/AKT胰岛素信号转导的经典途径中,发现复方纯化部位及特征化合物诱导成肌细胞中p-IRS-1、p-AKT、GLUT4表达量增加,可能是潜在的胰岛素增敏剂。复方金鹳草有效部位可以抑制体外脂肪肝细胞模型胞内甘油三酯的生成,说明它可能具有抑制肥胖和肝脏脂肪变性的作用;但对细胞中已经形成的甘油三酯没有促进分解的作用,推测复方金鹳草有效部位在脂代谢中,对脂类合成过程有抑制作用,而非脂类分解途径。在蛋白水平上也观察到了脂肪合成基因的主要转录调节因子SREBP的表达量降低。综上所述,复方金鹳草治疗糖尿病的依据可能是通过多组分多靶点发挥协同作用,具有多种降糖机制,从而防治糖尿病及其慢性并发症。

其他摘要

Compound “Gin Guan Cao” is ethnomedicine from Avicenna’s “Canon of Medicine”. It is used to treat diabetes mellitus and have significant effect. The compound formular is made of Geranium collinum Steph.ex Willd and Hypericum Scabrum Lnn by certain ratio 7 to 3. But the mechanism of its anti-diabetic effect was not clear and the chemical constituents of the two medical plants were not systematicly studied. For further research and application, research on chemical constituents and effective fraction of compound “Gin Guan Cao”, Geranium collinum and Hypericum scabrum and their antidiabetic biological activities are carried out. The paper aims to explore the mechanism of compound “Gin Guan Cao” in treatment of type 2 diabetes mellitus, and find out the effective components and targets. It will be helpful for clinical and experiment study on compound “Gin Guan Cao”. Guided by antioxidation activity and PTP1B inhibition in vitro, the effective fraction of compound “Gin Guan Cao” was prepared as follows. Geranium collinum Steph and Hypericum scabrum Lnn (mass ratio 7:3) were grinded into powder and extracted with 50% ethanol. The ethanol extraction was purified by AB-8 macro porous resin column, successively washed with distilled water, 30% ethanol, 70% ethanol. 30% and 70% ethanol eluants were concentrated under reduced pressure to obtain the effective fraction GC30 and GC70. Both the fractions have good antioxidation activity and PTP1B inhibition. 20 compounds in GC30 were identified by LC MS/MS. For plant Geranium collinum, the extrcation by 50% ethanol exhibit good antioxidation activity and PTP1B inhibition. The high amount of total polyphenolic compounds and total flavonoids were calculated as 350mg·g-1 gallic acid equivalents (GAE) and 96mg·g-1 quercetin equivalents (QE) in 50% ethanol extract. Nine known compounds were isolated and determined on the basis of 1D and 2D NMR and mass spectroscopic analyses as well as by comparison with literature data. To our knowledge, this is the first report on the phytochemical profile and antidiabetic activity of plant G. collinum.For plant Hypericum scabrum L., 70% ethanol extraction shows the best PTP1B inhibition and better antioxidation activity. Total polyphenolic compounds and total flavonoids contents were determined in the extract as 107 mg·g-1 and 23 mg?g?1. Eleven known compounds were isolated and determined on the basis of 1D and 2D NMR and mass spectroscopic analyses as well as by comparison with literature data. From our literature survey, it appears that the compounds except rutin were reported for the first time in plant H. scabrum.In this paper, we initially explore the anti-diabetic mechanism of compound “Gin Guan Cao”. We use different cell models to investigate the hypoglycemic and hypolipidemic effects of compound “Gin Guan Cao” effective fraction (GC30, GC70) and its characteristic compounds geraniin and ellagic acid. Both the fractions and the compounds were found to increase glucose consumption, 2-deoxy-glucose uptake and induce the glycogen accumulation. The ATP level of the cells was decreased after the treatment of the fractions and the characteristic compounds, test by the ATP Determination Kit.Through the western blot experiments, p-AMPK protein expression were increased. It is indicated that the fractions and compounds may have the effect through AMPK signaling pathways as targets to improve the glucolipid metabolism. We also checked the PI3K/AKT insulin pathway. p-IRS, p-AKT, GLUT4 protein expression were increased in the treatment with L6, which may indicated that the fractions and the compounds may have enhance the insulin sensitivity as insulin sensitizing agents. GC30 significantly reduced triglyceride content in the non-alcoholic fatty liver cells model in the induction of TG accumulation. It may indicate that GC30 is not only effective for prevention of obesity but also effective for treatment for hepatic steatosis. When GC30 was applied to the cells after establishment of TG accumulation, GC30 did not exhibit a strong activity in the reduction of TG content in cells, which suggesting that GC30 does not induce lipolysis. These data suggest that GC30 inhibits TG accumulation in hepatocytes, and the mechanism is likely inhibition of TG synthesis. It is also find that the fraction decreased the protein expression of SREBP, which is a major transcription factor in the lipogenic system. In all, the anti-diabetic mechanism of compound “Gin Guan Cao” may depend on multiple targets in regulating glucose and lipid metabolism. It may have prevention and control of diabetes and the various kinds of chronic complications.

文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/4867
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
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蒋岚. 复方金鹳草化学成分及抗糖尿病活性研究[D]. 北京. 中国科学院大学,2016.
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