The Study On The Anti-diabetic Effect Of Uraria Crinita Extracts

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.