【作者】 刘晓萍；

【导师】 庞建新；

【作者基本信息】 南方医科大学 ， 药理学， 2010， 硕士

【摘要】 研究背景随着社会的发展、生活方式的改变和人口老龄化,糖尿病(diabetes mellitus, DM)的患病率在全球范围内快速增长。在发达国家中,成为继心血管病和肿瘤之后的第三大非传染性疾病,对人类健康构成极大的威胁,给个人和社会带来极重的经济负担,已经成为严重危害人类健康的世界性公共卫生问题。据世界卫生组织估计,全球被诊断的DM人数将从2003年的1.94亿上升到2030年的3.60亿。目前我国糖尿病患者现约有4200万人,患病率也已经从80年代初的不足1%发展到3.26%,部分城市甚至超过了5%,并呈逐年增加趋势。糖尿病是遗传和环境因素共同作用而导致的慢性全身性代谢性疾病,胰岛素分泌绝对或相对不足和胰高血糖素活性增高所引起的代谢紊乱,引起体内的糖、脂肪、蛋白质及水盐代谢紊乱,表现为高血糖、糖尿等,其病情进展常伴心脑血管、肾、眼及神经病变等并发症。据文献报道,全球糖尿病的患病率在逐渐升高,在我国糖尿病患病人群中,Ⅱ型糖尿病占93.7%,Ⅰ型糖尿病占5.6%,其他类型糖尿病仅占0.7%。几乎所有的Ⅱ型糖尿病发病前都要经过糖耐量受损(impaired glucose tolerance,IGT)阶段。IGT也称为糖尿病(diabetes mellitus,DM)前期,是发展成Ⅱ型糖尿病的过渡状态。IGT的发病机制尚未完全阐明,目前普遍认为胰岛素抵抗(IR)和胰岛β细胞功能受损致胰岛素分泌不足是导致IGT发生的两个主要因素。如果不加以干预,IGT进一步发展就会导致糖尿病发病,而胰岛素抵抗贯穿于Ⅱ型糖尿病的整个发生、发展过程。当机体出现胰岛素抵抗时,会进一步促进胰岛素的分泌,内源性胰岛素水平升高。随着病程的延续,胰岛素抵抗加重,胰岛β细胞功能受损,胰岛素分泌不能代偿胰岛素的抵抗,胰岛素水平降低,同时,胰岛素刺激的葡萄糖利用减少,不能有效抑制肝糖原分解输出葡萄糖,从而导致血糖升高,最终出现糖尿病。研究认为,氧化应激时体内活性氧(reactive oxygen soecies,ROS)生成增多,损伤胰岛β细胞,引起细胞坏死或凋亡,导致胰岛素分泌缺乏,加重胰岛素抵抗；同时,脂质代谢异常也会参与ROS对胰岛β细胞的损伤,造成胰岛β细胞功能障碍。因此,通过改变机体的ROS水平,进而改善胰岛素抵抗、保护胰岛β细胞功能对于干预IGT和治疗Ⅱ型糖尿病至关重要。目前治疗Ⅱ型糖尿病的口服药物主要有促胰岛素分泌药,胰岛素增敏药、α糖苷酶抑制剂等,虽然西药治疗Ⅱ型糖尿病具有降糖快速、疗效好的特点,但对胰岛素抵抗、糖耐量受损以及以脂代谢紊乱为主的机体整体功能调节,不能令人满意,且长期应用会产生有明显的不良反应,如磺酰脲类易引起低血糖,胰岛素易导致水肿等。因此,寻找一种能有效治疗糖尿病,调节机体整体功能,毒副作用低的药物,对干预IGT和治疗Ⅱ型糖尿病具有重要意义。据文献报道,石参具有很好的抗炎作用,能有效的抑制应激性溃疡、治疗胃痛等疾病。体外研究表明,石参甲醇提取液具有清除NO、抵抗氧自由基的作用,并且在中国广东、广西省,石参作为民间药物用于治疗轻型糖尿病,但尚未有实验研究证实石参治疗糖尿病的作用。第一部分石参水煎液对STZ+高脂饮食诱导的Ⅱ型糖尿病模型小鼠的影响目的通过观察石参水煎液对STZ+高脂饮食诱导的Ⅱ型糖尿病模型小鼠的血糖、血脂以及胰岛、肝脏和附睾组织形态的影响,探讨石参水煎液的抗糖尿病作用。方法1.STZ+高脂饮食诱导的Ⅱ型糖尿病小鼠模型的建立雄性C57BL/6J小鼠,适应性喂养1周后,按完全随机方法分为正常组和造模组。正常组小鼠以普通饲料喂饲；造模组小鼠以高脂饲料喂养2周后,腹腔注射STZ(STZ现配现用,生理盐水配成10mg/ml),按100mg/kg的剂量给药,即0.1ml/10g鼠重,0.5h内注射完,每周一次,连续两周。两周后,筛选餐后血糖>200mg/dl为造模成功小鼠。2.石参水煎液的制备取干燥石参药材,加适量水煎2小时,取上清,过滤后负压旋转蒸发,浓缩至一定浓度石参水煎液,4℃低温保存备用。3.石参水煎液对STZ+高脂饮食诱导的Ⅱ型糖尿病小鼠的影响筛选餐后血糖>200mg/dl的小鼠,按完全随机方法分为：正常对照组,模型对照组,石参水煎液低(3g/kg)、高(9g/kg)剂量组,二甲双胍阳性药对照组(500mg/kg),共5组。各组小鼠连续给药21天后,检测餐后血糖的变化。治疗14天后,禁食过夜,灌胃葡萄糖,尾静脉采血后检测葡萄糖耐量。给药处理21天后,在小鼠眼眶后静脉丛采血,获得血清用于测定血清胰岛素、甘油三酯和游离脂肪酸。处死动物,取动物心脏、肝脏、双侧附睾,称重。并取胰腺,置于10%福尔马林中保存,作病理切片检查。结果1.采用STZ+高脂饮食可制备出理想的Ⅱ型糖尿病小鼠模型。2.石参水煎液可显著改善模型小鼠的葡萄糖耐量(P<0.05),调节胰岛素的分泌(P<0.05),显著降低血清甘油三酯(P<0.01)水平,抑制游离脂肪酸的产生(P<0.01),但对餐后血糖没有明显影响。3.病理切片显示,石参水煎液能减轻链脲佐菌素对胰岛β细胞的破坏,对受损伤的胰岛β细胞具有一定的修复功能。并且,石参水煎液还可以不同程度地减轻模型小鼠肝脏和附睾组织的损害。结论1.采用STZ+高脂饮食的方法成功构建了C57BL/6J小鼠Ⅱ型糖尿病模型,可作为石参抗糖尿病作用的研究。2.石参水煎液能显著改善糖尿病模型小鼠的葡萄糖耐量,调节胰岛素的分泌,降低甘油三酯和游离脂肪酸。3.石参水煎液对损伤的胰岛细胞有一定的修复作用,且能改善糖尿病小鼠肝脏及附睾组织的损伤。第二部分石参不同提取物对STZ+高脂饮食诱导的Ⅱ型糖尿病模型小鼠的影响目的通过观察石参不同提取物对STZ+高脂饮食诱导的Ⅱ型糖尿病模型小鼠的血糖、血脂的影响,探讨石参不同提取物的抗糖尿病作用。方法1.STZ+高脂饮食诱导的Ⅱ型糖尿病小鼠模型的建立适应性喂养1周后,雄性C57BL/6J小鼠再以高脂饲料喂饲2周,腹腔注射STZ(STZ现配现用,生理盐水配成10mg/ml),按100mg/kg的剂量给药,即0.1ml/10g鼠重,0.5h内注射完,每周一次,连续两周。两周后,筛选餐后血糖>200mg/dl为造模成功小鼠。2.石参所含组分分析取少量中药粗粉,分别用蒸馏水、乙醇和石油醚进行初步处理后,得到水浸液、乙醇提取液和石油醚提取液,加入不同的试剂对其进行成分预试。取干燥药材,用乙醇回流提取后滤过,药渣保留,合并滤液减压回收乙醇至100ml,平分成两份,一份蒸至稠膏状后70℃真空干燥得到提取物A。另一份浓缩到无醇味用浓氨水调pH值得到提取液。用等体积氯仿萃取,合并氯仿层,蒸干得到提取物B,水层蒸至稠膏状70℃真空干燥得到提取物C。药渣挥干乙醇后,加水煎煮后过滤,合并滤液浓缩至稠膏状,70℃真空干燥,得到提取物D。A、B、C、D加甲醇溶解后,制成供试品用于薄层色谱鉴别。3.石参不同提取物的制备取干燥药材,加乙酸乙酯,回流提取后滤过,药渣保留。滤液减压回收乙酸乙酯成稠膏状,70℃真空干燥,得到乙酸乙酯提取物。加蒸馏水制成乙酸乙酯提取物混悬液。药渣挥去乙酸乙酯后,加90%乙醇,回流提取后滤过,药渣保留。滤液减压回收乙醇成稠膏状,70℃真空干燥,得到乙醇提取物。加蒸馏水溶解制成乙醇提取液。药渣挥去乙醇后,加水回流提取,滤过,药渣弃去。滤液浓缩至稠膏状,70℃真空干燥,得到水提取物。加蒸馏水溶解制成水提取液。4.石参不同提取物对STZ+高脂饮食诱导的Ⅱ型糖尿病模型小鼠的影响筛选餐后血糖>200mg/dl的小鼠,按完全随机方法分为：模型对照组,石参乙酸乙酯提取物组(18g/kg),乙醇提取物组(18g/kg),水提取物组(18g/kg)和消渴丸阳性药对照组(500mg/kg),共5组。各组小鼠连续给药21天后,检测餐后血糖的变化。治疗14天后,禁食过夜,灌胃葡萄糖,尾静脉采血后检测葡萄糖耐量。给药处理21天后,在小鼠眼眶后静脉丛采血,获得血清用来测定血清胰岛素、甘油三酯和游离脂肪酸。结果1.石参组分分析结果证实石参含有多糖、黄酮类、皂苷及氨基酸等成分。2.石参不同提取物可降低模型小鼠的空腹血糖,改善Ⅱ型糖尿病小鼠的葡萄糖耐量,其中乙醇提取物在60min和120min时的血糖水平分别为29.66%和59.75%,血糖水平下降幅度较大。3.石参乙醇提取物可降低甘油三酯水平,抑制游离脂肪酸的分泌,改善脂质代谢,但对餐后血糖没有明显影响。结论1.石参含有多糖、黄酮类、皂苷及氨基酸等成分。2.石参不同提取物显示出一定的抗小鼠Ⅱ型糖尿病的作用,可降低Ⅱ型糖尿病小鼠的空腹血糖,对受损的糖耐量具有改善作用,并在一定程度上能降低血清甘油三酯和游离脂肪酸的水平,其中以乙醇提取物的效果较好。第三部分石参乙醇提取物对高脂饲料喂养SD大鼠血脂代谢的影响目的通过观察石参乙醇提取物对高脂饮食致肥胖性SD大鼠血脂代谢的影响,探讨石参对SD大鼠血脂代谢的调节作用。方法1.肥胖性SD大鼠模型的建立SD大鼠,适应性喂养1周后,按完全随机方法分组,正常组5只,以普通饲料喂养。造模组SD大鼠高脂饲料喂饲4周后,筛选体重>200g的SD大鼠为造模成功大鼠。2.石参乙醇提取物的制备取干燥药材,加乙酸乙酯,回流提取后滤过,药渣保留。药渣挥去乙酸乙酯后,加90%乙醇,回流提取后滤过。滤液减压回收乙醇成稠膏状,70℃真空干燥,得到乙醇提取物。加蒸馏水溶解制成乙醇提取液。3.石参乙醇提取物对SD大鼠血脂代谢的影响筛选体重>200g的SD大鼠,按完全随机方法分为：正常对照组,模型对照组,石参乙醇提取物组(18g/kg)和洛伐他汀阳性药对照组(10mg/kg),共4组。给药处理前后,均于SD大鼠眼眶后静脉丛采血,获得血清用来测定血清总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)。结果1.治疗前后比较,给予石参乙醇提取物处理的SD大鼠血清总胆固醇值下降了42.7%(P<0.05),血清高密度脂蛋白胆固醇值(HDL-C)升高8.41%(P<0.05),组间有显著差异。2.给予石参乙醇提取物处理后,SD大鼠的血清总胆固醇值有明显降低,显著低于正常大鼠TC水平(P<0.01)。3.与治疗前比较,石参乙醇提取物组大鼠的LDL-C水平下降了6.67%,但组间差异无显著性。结论石参乙醇提取物能显著降低SD大鼠血清中的总胆固醇,升高血清高密度脂蛋白胆固醇,对大鼠的血脂代谢有一定的调节作用。更多还原

【Abstract】 BackgroundThe prevalence of diabetes mellitus(DM) in the world has been increasing rapidly as the change of social development, life style and the aged tendency of population. In the developed countries, DM has become the third non-infective disease following angiocardiopathy and tumour, not only threatens the human health, but also burdens the individuals and the society greatly, which troubles the world’s social wealth problems. Statistics from the World Health Organization, the number diagnosed as DM will increase from 194 million in 2003 to 360 million in 2030 around the world. At present, the number of diabetes is approximately 420 thousand; the prevalence is up to 3.26% from 1% in 1980s, moreover, it’s over 5% in some cities, and the trend is increasing year by year.The co-interaction between inheritance and environment leads to the development of DM which results in chronic general metabolic disease, the lack of insulin secretion absolutely or relatively, and the metabolic disorder rose from the increase of glucagons activity. Furthermore, DM brought to the endosomatic disorder of glucose, fat, protein and water and salt metabolism, presents the syndromes of hyperglycaemia, carbohydraturia, etc. The evolvement of DM also associates with the complication of cerebral vessels, kidney, ophthalmos and neuropathy. The prevalence rate raised year by year. The number of typeⅡdiabetes accounts for 93.7% in the quantity of our country’s disease, among them, typeⅠdiabetes 5.6% and the remaining diabetes 0.7% only.Most parts of diabetes mellitus (DM) processes a stage of impaired glucose tolerance(IGT) before the onset of the disease. IGT is defined as the prophase of DM, which is a transient status of typeⅡdiabetes. However, the pathogenesis of IGT is remained to unknown. At present, it’s believed that IR and the function ofβcell of islet are the two main reasons caused hypoinsulinism. When insulin resistance evolved, the secretion of insulin will be promoted, and the level of endogenous insulin will increase. With the course of disease continuing, the insulin resistance will aggravate; the function ofβcell in islet will impair; the level of insulin will reduce resulted from the discompensation of insulin secretion from the insulin insistence. Meanwhile, the decrease of glucose utilization is not able to inhibit glycogenolysis from outputting glucose actively, which gives rise to hyperglycemia and turns to diabetes ultimately.It’s supposed that the ROS in the body will be increased when undergoing oxidative stress in current studies. It can impairβcell of islet that leads to necrosis and apoptosis, which decreases insulin secretion, aggravates insulin resistance. Meanwhile, the disorder of lipid metabolism anticipates the impair ofβcell of islet from ROS, which brights about the dysfunction ofβcell. Therefore, it’s essential to ameliorate insulin resistance and protect the function ofβcell in the intervention of IGT and the treatment of typeⅡdiabetes.The classification of oral medicine can be divided into the drugs of stimulating insulin secretion, insulin-sensitizing agent, glucosidase Inhibitor, etc. Western medicine has a privilege of faster and more effective towards typeⅡdiabetes, whereas, not only is it not satisfied at the function adjustment in IR, IGT and lipid metabolic disorder, but also arises obvious side effects, such as sulfonylurea which leads to hypoglycemia and insulin that brings about edama. Therefore, it’s imminent to find out a drug that more effective in curing diabetes and adjusting the whole-body functions, moreover, less toxic and side effects.According to the studies, Uraria crinita(UC) is effective in anti-inflammatory, which can inhibit the development of stress ulcer and cure stomach pain. Vitro studies indicated that UC methanol extract has the efficacy to clear up NO, resist oxygen-derived free radicals. Though UC was used as a medicine in Guangdong, Guangxi province to cure the levis diabetes, there haven’t been any studies to demonstrate the effect on diabetes treatment.Part one The effects of Uraria crinita water extract in typeⅡdiabetic mice induced by STZ and high-fat dietObjectiveTo explore the effects of UC water extract to anti-diabetes, we observed its efficacy to blood glucose, lipids and the tissue morphologies of islet, liver and epididymis in typeⅡdiabetes mice induced by STZ+ high-fat diet.Methods1.Establishment of the model of typeⅡdiabetic miceMale C57BL/6J mice were randomly assigned to normal group and model group after one-week adapting feed. Model group was received infusions of STZ (STZ was prepared when using, the concentration of normal sodium is 10mg/ml) after being fed by the high-fat diet. The dosage was according to the weight of mice, 100ml/kg. The requirement of injection was once a week, with half an hour to finish the injection. After 2 weeks, the successful model mice those postprandial blood glucose was over 200mg/dl were selected in the study. 2.Preparation of the UC water extractDry UC (500 g) was added to 4 L distilled water, boiled for 2 h, cooled to room temperature and filtered through 100 mesh sieve. The filtrate was concentrated to 75 ml in rotary evaporator at 60℃. The obtained UC water extract was stored at 4℃.3.The effects of UC water extract to the typeⅡdiabetic miceMice was selected by postprandial blood glucose over 200mg/dl were randomly assigned to five groups: normal control group, model control group, low-dose UC water extract group (3g/kg), high-dose UC water extract group (9g/kg) and metformin positive control group (500mg/kg). Postprandial blood glucose was measured after 21-day administration. After the 14-day administration, all groups were underwent overnight fasting and intragastirc administration of glucose. Glucose tolerance was detected through tail nipping. At the end of the 21-day treatment, blood saples was collected from the eyes (venous pool) to determine plasma levels of Insulin, triglyceride (TG) and free fatty acids (FFAs). The subjects were sacrificed to collect the hearts, livers and epididymises and to weight. Pancreatic glands were preserved in the 10% formalin in order to take the Pathological examination.Results1.Using STZ+ high-fat diet can establish the model of typeⅡdiabetic mice.2.UC water extract ameliorated glucose tolerance(p<0.05),regulated insulin secretion(P<0.05)and decreased the levels of TG(P<0.01)and FFAs(P<0.01),but there was no difference in postprandial blood glucose of typeⅡdiabetic mice.3.From the pathological section, UC water extract inhibited the damage ofβcells of islet from streptozotocin and repaired the damagingβcells.And UC water extract can reduced the damage to the liver and epididymises tissue to some extent. Conclusion1.The model of typeⅡdiabetic mice by STZ+ high-fat diet can be used to study the anti-diabetic effects of UC extract.2.UC water extract ameliorates glucose tolerance of the subjects, regulates insulin secretion, reduces the levels of TG and FFAs.3.UC water extract repaired the damage of islet cells,reduced the damage to the liver and epididymises tissue.Part Two The effects of Uraria crinita different extracts in type II diabetic mice induced by STZ and high-fat dietObjectiveTo explore the effects of UC different extracts to anti-diabetes, we observed the efficacy to blood glucose, lipids in typeⅡdiabetic mice induced by STZ+ high-fat diet.Methods1.Establishment of the model of typeⅡdiabetic mice Male C57BL/6J mice were assigned after one-week adapting feed. Model group was received infusions of STZ (STZ was prepared when using, the concentration of normal sodium is 10mg/ml) after being fed by the high-fat diet. The dosage was according to the weight of mice, 100ml/kg. The requirement of injection was once a week, with half an hour to finish the injection. After 2 weeks, the successful model mice those postprandial blood glucose was over 200mg/dl were selected in the study.2.The analysis to the elements of UCUC powder was treated preliminarily by distilled water, alcohol and petroleum ether respectively. Tests were followed to analyze the elements contained by various reagents. Row dry UC were extracted after recirculating with alcohol, reserving the gruffs. Decompressed the filter liquor in the concentration of 100ml alcohol and spited the liquor into two parts equally. One part of liquor was steamed into thick paste, which accepting 70℃vacuum dehydration to be extract A. The other part was adjusted the PH value by ammonia water stronger until the condensed without nonalcoholic smel. Using the same volume of chloroform to abstract the condensed, combining the abstraction, steaming to dry to acquire the extract B. Extract C was got by the procession of the steam to make thick paste and then vacuum drying at 70℃from the water layer in the extract B. Having been volatilizing the solvent fully, concentrating the gruffs into thick paste through decoction and filtration, drying at 70℃vacuum, extract D was made. Testing samples from solutions of extracts with methanol were measured by thin-layer chromatography (TLC). 3.Preparation of the UC different extractsRow dry UC were extracted after recirculating with acetoacetate, reserving the gruffs. Having been decompressed, the filter liquor was retrieved into thick paste, which was dried at 70℃in vacuum. By adding distilled water, the paste turns to be acetoacetate extract.Gruffs were extracted after recirculating with 90% alcohol and preserved. Having been decompressed, the filter liquor was retrieved into thick paste, which was dried at 70℃in vacuum. By adding distilled water, the paste turns to be alcoholic extract.Gruffs were extracted after recirculating with water and preserved. Having been decompressed, the filter liquor was retrieved into thick paste, which was dried at 70℃in vacuum. By adding distilled water, the paste turns to be water extract. 4.The effects of UC different extracts in TypeⅡDiabete Mice was selected by postprandial blood glucose over 200mg/dl were randomly assigned to five groups: model control group, UC acetoacetate extract group(18g/kg), UC alcoholic extract group (18g/kg), UC water extract group (18g/kg) and Xiaoke Wan positive control group(500mg/kg). Postprandial blood glucose was measured after 21-day administration. After the 14-day administration, all groups were underwent overnight fasting and intragastirc administration of glucose. Glucose tolerance was detected through tail nipping. At the end of the 21-day treatment, blood saple was collected from the eyes (venous pool) to determine plasma levels of Insulin, TG and FFAs.Results1.Testimony on containing elements of polycose, flavonoids, saponin, amino acid etc. by analyzing UC.2.The three kinds of UC extracts can decrease the levels of fasting blood glucose ,ameliorate glucose tolerance of the subjects in various extents. The level of blood glucose reduced to a great extent. Among them, the reducing level in alcoholic extract was 29.66% within 60 minutes, and 59.75% within 120 minutes respectively.3.Alcoholic extract decreased the level of TG more compared with the negative control group, inhibited the secretion of FFAs, ameliorated lipid metabolism,but there was no difference in postprandial blood glucose.Conclusion1.Testimony on containing elements of polycose, flavonoids, saponin, amino acid etc.2.The three kinds of UC extracts decreased the levels of fasting blood glucose,ameliorated glucose tolerance of the subjects, reduced the levels of TG and FFAs to some extent, and the alcoholic extract was the best. Part Three The effects of Uraria crinita alcoholic extract on the lipid metabolism of high-fat diet-fed ratsObjectiveThe effects of UC alcoholic extract were explored by observing its adjustment in the lipid metabolism on high-fat diet-fed SD ratsMethods1.Establishment of the model of high-fat diet-fed SD rats SD rats were divided into normal and model groups at randomly: normal group which consists of five mice was fed by usual feedstuffs. Comparably, model group was fed with high-fat diet for 4 weeks firstly, and selected the rats those weights were over 200g.2.Preparation of the UC alcoholic extractRow dry UC were extracted after recirculating with acetoacetate. Gruffs were reserved to volatilize the acetoacetate solvent, add 90% alcohol and recirculate after filtering.Having been decompressed, the filter liquor was retrieved into thick paste, which was dried at 70℃in vacuum. By adding distilled water, the paste turns to be alcoholic extract.3.The effects of UC alcoholic extract on the lipid metabolism of high-fat diet-fed rats SD rats were divided randomly into four groups selected by the weight more than 200g:normal control group, model control group, UC alcoholic extract group (18g/kg) and Lovastatin positive control group (10mg/kg). The levels of serum total cholesterol, high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were measured after administration as specimens were picked from the eyes (venous pool) of subjects. Results1.It appeared an obvious difference between the treatment before and after in the subjects:the level of total cholesterol decreased by 42.7% (P<0.05) and the level of HDL-C increased by 8.41%(P<0.05).2.There was a significant difference in the decreasing level of total cholesterol compared normal control group of UC alcoholic extract (P<0.01), with the latter having a greater effect.3.Between the treatment before and after in the subjects:the level of LDL-C decreased by 6.67%,but no obvious difference.ConclusionsUC alcoholic extract decreased the level of total cholesterol and increased the level of HDL-C evidently, which regulated the lipid metabolism.更多还原