【作者】 傅佳；

【导师】 王静凤；

【作者基本信息】 中国海洋大学 ， 食品科学， 2010， 硕士

【摘要】 海参(sea cucumber)是具有较高食用和药用价值的海洋生物资源。化学分析及药理学研究表明,海参体壁富含海参多糖、海参皂苷和海参蛋白多肽等多种活性物质,具有抗肿瘤、抗氧化、降血压和提高免疫力等生物活性。蛹虫草(Cordyceps militaris)是目前冬虫夏草的最佳替代品,其含有的活性成分包括虫草多糖、虫草酸和虫草素等,并证实具有增强免疫力、降血脂、抗氧化和降血糖等多种活性。目前已被列为我国的新资源食品。中医理论认为,海参性温补,蛹虫草味甘性平,二者组方合用,可增强生津养阴,益气温阳的功效。本论文利用腹腔注射链脲佐菌素(streptozotocin, STZ)的方法建立糖尿病大鼠模型,灌胃由日本刺参(Apostichopus japonicas)与蛹虫草配成的复剂,研究海参虫草复剂的降血糖活性,并对其作用机制进行初步探讨。主要研究结果如下：海参虫草复剂具有显著的降血糖作用。糖尿病大鼠灌胃给予海参虫草复剂35d后,大鼠空腹血糖含量显著降低(P<0.01)。海参虫草复剂不但能显著提高大鼠的葡萄糖耐量(P<0.01),提高机体对葡萄糖的耐受力,还能显著提高大鼠血清胰岛素含量(P<0.01)、胰岛素分泌指数(P<0.01)和p细胞功能指数(P<0.05)；增加肌、肝糖元的含量(P<0.01)。提示海参虫草复剂对糖尿病大鼠体内葡萄糖代谢具有较好的调控作用。海参虫草复剂显著地降低了糖化血红蛋白和糖化血清蛋白含量(P<0.01),表明海参虫草复剂能较好的控制糖尿病病情的发展。结果表明,海参虫草复剂良好的降血糖效果与其有效改善体内的葡萄糖代谢水平、控制病情恶化有关,并且海参虫草复剂的降血糖效果明显好于海参、蛹虫草单剂作用。海参虫草复剂具有提高抗氧化能力和保护胰岛p细胞的作用。海参虫草复剂能显著提高肝脏中SOD、CAT和GSH-Px酶的活力(P<0.01)；高剂量海参虫草复剂能显著提高胰腺中CAT酶活力(P<0.01)。采用HE染色和透射电镜技术研究了海参虫草复剂对糖尿病大鼠胰腺组织的显微和亚显微结构的影响。结果表明：灌胃海参虫草复剂的糖尿病大鼠,其胰岛破坏程度减轻,形态恢复正常,胰岛p细胞的数量明显增多。电镜下可观察到β细胞结构与正常对照组接近,细胞界限清晰。细胞核呈圆形,核膜光滑；核仁清晰且染色质分布均匀；胞质内的细胞器结构肿胀程度较模型对照组明显减轻。提示海参虫草复剂能提高机体和胰腺的抗氧化能力,较好的保护了胰岛β细胞结构,使其结构完整,提高其合成和分泌胰岛素的功能。利用RT-PCR和Western Blot的方法检测了糖尿病大鼠外周组织中胰岛素信号通路关键蛋白激酶和葡萄糖转运蛋白4(Glucose transporter4, Glut4)的mRNA及蛋白表达,并对其降血糖机制进行了探讨。结果表明：海参虫草复剂能提高肌肉和脂肪组织中磷脂酰肌醇-3-激酶(Phosphatidylinositol 3-kinase, PI3K)、蛋白激酶B(Protein kinase B, PKB/Akt)和Glut4的mRNA表达。肌肉组织中,海参虫草复剂低、高剂量组PI3K mRNA的表达量与模型对照组比较具有显著性差异(P<0.01)；高剂量能明显提高Glut4 mRNA的表达。脂肪组织中,复剂高剂量组的PI3K mRNA表达量提高了42.86%,而复剂低、高剂量组Glut4的表达量平均提高了32.63%,相比于模型对照组均具有显著性差异(P<0.05,P<0.01)。另外,海参虫草复剂能增强糖尿病大鼠脂肪组织中Glut4的蛋白表达,呈现剂量效应关系。提示海参虫草复剂通过调节胰岛素信号通路,增强葡萄糖转运蛋白Glut4的表达,促进外周组织对葡萄糖的利用,达到降血糖的目的。本实验较为系统全面的评价了海参虫草复剂的降血糖活性,并对其机制进行了初步探讨。为海参虫草复剂的高值化开发利用提供了理论依据。更多还原

【Abstract】 Sea cucumber belongs to the Holothuroidea in biological classification and is distributed throughout the world seas. It has been a traditional tonic food in China and other Asian countries due to its antitumor, immunoregulatory, antiatherosclerotic, and anti-aging properties. Nowadays, many studies show that the body wall of sea cucumber contains many bioactive substances, such as polysaccharides, collagen, triterpene glycosides and so on, which exhibit excellent effect of immunomodulatory, anti-oxidation, anti-tumor, and anti-hypertension. Cordyceps militaris is known as the Chinese rare caterpillar fungus, and it has been listed as a new source food in China since 2008. Its chemical components, including cordycepin, D-mannitol, and various polysaccharides are similar to the substances contained in famous Chinese traditional medicine C. sinensis. The efficacy of C. militaris was confirmed in the treatment of cardiovascular and renal dysfunction, in immune enhancement and in the anti-hyperlipidemia effect. Use the mixture of sea cucumber and C. militaris may enhance the effect on some diseases.The aim of this study is to investigate the hypoglycemia effect of the mixture of Apostichopus japonicas and C. sinensis on diabetic rats, which were established by intraperitoneal injection of streptozotocin (STZ), and to discuss the mechanism involved in this effect. The major results are as follows:The mixture exhibit a significant hypoglycemia effect on STZ-induced diabetic rats. With mixture treated 35 days, a dramatic decrease in fasting blood level was observed in both low and high dosage groups (P<0.01), and the glucose tolerance was improved. The mixture can increase serum insulin contents (P<0.01), insulin secretion index (P<0.01) andβ-cell function index (P<0.01); moreover, it can improve the contents of muscle glycogen and liver glycogen notably (P<0.01). Meanwhile, the contents of glycosylated hemoglobin and glycosylated serum protein were significantly decreased by this mixture (P<0.05, P<0.01). These results suggested that the mixture of A. japonicus and C. militaris could improve the glucose metabolism level and have a better hypoglycemic effect on diabetic rats, and the effect in mixture groups are more better than that in A. japonicas or C. militaris.The mixture can prevent the damage of oxidation stress by enhance the ability of antioxidation. The results showed that the antioxidase activities of Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GSH-Px) in liver were improved obviously in both low and high dosage groups (P<0.01). The mixture can also increase the activity of CAT in pancreas (P<0.01). The protect effect of the mixture on islet of diabetic rats was observed using the method of hematoxylin-eosin stain and transmission electron microscope. In the groups treated with the mixture, the number ofβcells of islet was increased in comparision to the model group, and the degree of oxidation damage was reverted to a normal level. The observation of transmission electron microscope showed that, the nucleus architecture in mixture group are remarkably reverted to a normal status, and the membrane of nucleus was smooth, nucleolus and chromatins were clear. Only a slightly swelling in mitochondrion and granular endoplasmic was observed in the group of the mixture. All the results indicated that mixture of A. japonicas and C.militaris can enhance the ability of anti-oxidation of the body and the pancreas to protect theβcells, keep the normal morphology of cells, and improve the function of synthesis and secret insulin.The investigation of hypoglycemia mechanisms of the mixture were conducted by RT-PCR and Western Blot. The mRNA and protein expression levels of phosphatidylinositol 3-kinase (PI3K), serine-threonine kinases (Akt) and glucose transporter 4 (Glut4) in skeletal muscle and adipose tissues were examined. The results implied that this mixture could significantly increase the mRNA expressions of PI3K and Glut4 in skeletal muscle (P<0.01). In adipose tissues, PI3K expression levels of high dosage group was increased by 42.86%, and that of Glut4 in low and high group was average increased by 32.63% (P<0.05, P<0.01). The protein expression levels of Glut4 were also enhanced by the mixture in adipose tissue of low and high dosage group (P<0.01) and there exist a dosage-dependent relationship. Results suggest that the mixture of A. japonicas and C. militaris exhibits significant hypoglycemia activity through improvement of insulin signaling pathway by which enhance the glucose uptake and utilization in peripheral tissues and finally decreased the high glucose level in blood.In conclusion, the hypoglycemia effect of the mixture of A. japonicas and C. militaris may be related to its antioxidant bioactivity, and the mixture can upregulat the gene and protein expression of key protein kinases and Glut4 in skeletal muscle and adipose tissues, that may be one of the most important mechanism involved in this effect.更多还原