| Background and objectiveAccording to the World Health Organization survey shows that, the prevalence of diabetes mellitus (DM) in China has grown following wih the United States, be the second in the world. The sick of adult had reached3.5percent, the total number had been over40million; and it raise0.1%annual, and90%for type II diabetes mellitus (T2DM). Because of the popular of diabetes in China, the Diabetes Guide repeatedly made it clear, to reduce the incidence of type II diabetes and early intervention are the major challenges and one of the tasks.The common characteristic of the early stage of type II diabetes is impaired fasting glucose(IFG) and/or impaired glucose tolerance(IGT). IGT/IFG, an abnormal the one glucose metabolism between normal blood sugar and diabetes, is a type II diabetes natural course stage. The normal fasting blood glucose is3.9-5.6mmol/L, diabetes’s fasting glucose≥7mmol/L. IGT performance for the oral glucose tolerance test (OGTT) is their fasting plasma glucose<7.0mmol/L. After oral glucose2h plasma glucose is7.8-11.1mmol/L, and fasting plasma glucose is5.6-7.0mmol/L when the diagnosis IFG. Insulin resistance(IR) and insulin secretion defects is the main reason of the IGT/IFG. Among them, IGT associated with defects in insulin secretion, while IFG with IR. Many studies have shown that IR and insulin secretion defects is related to the elevate of blood lipid, especially the raised of free fatty acid.Currently, the prevention of IGT/IFG advocates diet and lifestyle intervention. In recent years, some reports showed that a small dose of metformin, pioglitazone or acarbose for IGT/IFG intervention, significantly to preventive diabetes and cardiovascular complications. However, part of the IGT/IFG patients will not develop into type II diabetes and the couple of advice effect, oral hypoglycemic agents early intervention also need to conduct a risk assessment.The Chinese traditional medicine believes that IGT/IFG similar spleen pyretic abundance, the spleen pyretic abundance consequences is converted Diabetes. The primary method of Chinese medicine for the treatment of IGT/IFG is major in dialectical identifying diseases combine with treatment, syndrome type is still in the initial exploratory stage and lack of standardized clinical trials. IGT/IFG are the diseases which can representation the advantages of Chinese medicine treatment, they are the early stage of diabetes, accord with the treatment philosophy of Chinese traditional medicine, suitable for long-term treatment. Therefore, exploration and application of traditional Chinese medicine, using natural medicine to treat IGT/IFG has a broad prospects and valuable practical value.Uraria crinita is a Perennial erect shrub plant, belongs to Leguminosae Uraria Desv, mainly distributed in China’s southern coastal provinces, including Guangdong, Guangxi, Hainan, Fujian, Taiwan. The record in "Guangxizhongyaozi" shown that Uraria Desv is mild and affect in lung and stomach, can slake blood stasis and phlegm. It used to treat of gastritis, edema and cold in folk. In southern China, it is often used as a decoction or stew agent, has health care effect, enjoy the "North ginseng, South Uraria crinita" reputation.To further investigate the active fraction of Uraria crinita, a gradient extraction with ethyl acetate, ethanol and water was used to collect the Uraria crinita extraction. And then we observed the effect of different extraction of Uraria crinita on the C57BL/6J mice which was induced to be diabetes by high-fat diet and a small dose streptozotocin. The result shown that the effect of18g/kg Uraria crinita ethyl acetate, ethanol and water extraction in non-fasting blood glucose of the mice model are not observed, but significantly reduced fasting blood glucose by25%,33%and27.3%, while the control group which is given xiaokewan1500mg/kg reduced fasting blood glucose level was12%.three kinds of extractions had no significant effect on plasma insulin levels, but there are varying degrees to reduce the level of free fatty acid (FFA) and triglyceride (TG), especially ethanol extracts reduced the level of free fatty acid up to54%while the control group for33%. To sum up, there are multiple ingredients in the extraction of Uraria crinita were participated in the improvement of IGT/IFG. The improvement was relative to reduce the blood lipids, especially free fatty acid (FFA). The extraction of ethanol is the most effective active fraction in three.In2009, Tongji school of Pharmacy, Huazhong University of Science and Technology and Hubei Province Natural Medicinal Chemistry and Resource Evaluation Key Laboratory were isolation and identification of the chemical composition of Uraria crinita from the10kg Uraria crinita root ethanol extraction.14compounds were isolated and identified, weight is143mg. In the14compounds which have been reported significant hypoglycemic effect, can be used for the treatment of diabetes compounds including genistein (14mg), silymarin (8mg) and betulin(llmg), these three compounds content of total extract content23.1%. We believe that the Uraria crinita ethanol extraction had a better effect may be associated with these ingredients.The known Uraria crinita extraction, flavonoids accounted for almost40%. Flavonoids was well-known to be a strong antioxidant, order to evaluate the antioxidant activity of flavonoids in Uraria crinita, we extracted total flavonoids of Uraria crinita and studied in vitro antioxidant activity and the protective effect of oxidative stress in rat insulinoma cells (INS-1E). The results found that the total flavonoids of Uraria crinita significantly inhibited DPPH, ABTS,·OH radicals, H2O2, NO free radicals and lipid peroxidation in vitro in a concentration-dependent way. The total flavonoids of Uraria crinita can significantly improve the viability of the INS-1E cells under oxidative stress, to improve the content of glutathione (GSH) and superoxide dismutase (SOD) activity, reduced malondialdehyde (MDA) and nitric oxide (NO) content. The total flavonoids of Uraria crinite had free radical scavenging activity and protective effect on islet β-cells from oxidative stress.Above analysis shows that in the Uraria crinite extraction, genistein, silymarin and betulin are important components of IGT/IFG improvement, and there are four ways for those compounds to take effect:reducing blood liqid, increasing glucose utilization, antioxidant and promote insulin release. Moreover, these types of ingredients through the multi-target way, may play a synergistic effect. Because effective dose of single component is tens to hundreds of milligram level per kilogram of body weight (rats), while the amount of Uraria crinite which count by5-20mg/10kg herbs extraction rate is only a few10micrograms per kilogram of body weight (rats). Even if the actual content is increased by10times, the content of each component was less than it’s effective dose which is reported before10to100times, but improve effect in IGT/IFG and reduce fatty acid effect are similar, indicating that variety of ingredients in the Uraria crinite extract had a synergistic effect.Based on the above assumptions, the subject will be based on previous work, using different cells models to study the Uraria crinite extract and genistein, silymarin and betulin combination effect in the model of type II diabetes. To provide a safety and effective measures and substratum for the use of Uraria crinite and it’s active ingredients in early prevention of type II diabetes, and that had an important significance to explore the application of Chinese traditional medicine in prevent type II diabetes and reduce the incidence of this disease.Methods:1. RIN-m cells damage by oxidative stress and the protective effect of Uraria crinita total flavonoidsExponentially growing RIN-m cells were seeded in96-well plates, each hole1*104cells, the total flavonoids of Uraria crinita was0.01,0.05,0.1,0.2,0.3mg/ml, total flavonoids processing cells, and then used lOmM of SNP to building damage model, and then using the MTT assay cell viability at570nm.2. The effect of Uraria crinita total flavonoids in RIN-m cells from oxidative stress, SOD activity, MDA, and the levels of GSH and intracellular and extracellular fluid content of NOExponentially growing RIN-m cells were seeded in24well plates,1*105cells per well, the total flavonoids of Uraria crinita was0.01,0.05,0.1,0.2,0.3mg/ml, total flavonoids processing cells, and then used lOmM of SNP to building damage model, and then collect cell supernatants and cell lysates, using the appropriate reagent kit to detection MDA, GSH, the content of NO and SOD activity.3. HEPG2cells insulin resistance and the reversal effect of genistein, silymarin and betulin combinedExponential growth phase HEPG2cells were seeded in96-well plates,1*104cells per well, with a total concentration of genistein, silymarin and betulin combined is1,0.5,0.25nM, and the drup and0.25mM palmitic acid are combine to processing cell for12h, and then culture was continued with a basal medium containing1*10-7M insulin for8h, and then the supernatant glucose content was detected using glucose detection kit, and the cell viability was detected at570nm wavelength.4. INS-1E cells fatty acids injury model and the reversal effect of genistein, silymarin and betulin combinedThe exponential growth phase of INS-1E cells were seeded in96-well plates,1*104cells per well, with a total concentration of genistein, silymarin and betulin combination was2,1nM, drugs and0.5mM palmitic acid were combine to treated cells with24h, and then MTT assay was used to detect cell viability at570nm wavelength.Results:1. The protection experiments of Uraria crinita total flavonoids in RIN-m cells from oxidative stress shown that, after treated by10mM SNP for four hours, the viability of cell was significant reduces(P<0.05), and the different concentrations of Uraria crinita total flavonoids have significant improve cell viability of the model (P<0.05).2. Uraria crinita total flavonoids can improve MDA, GSH content and SOD activity in RIN-m cells from oxidative stress under conditions, but only high dose of Uraria crinita total flavonoids can decrease the content of NO intracellular and extracellular significantly. The protective effect of Uraria crinita total flavonoids in RIN-m cells from oxidative stress, the main reason may be due to its good impact on the GSH content and SOD activity.3. Genistein, the silymarin and betulin combined administration will be able to significantly improve the induction of HEPG2cell insulin resistance by palmitic acid in a lower concentration than the monomer (P<0.05), to improve utilization of glucose in the presence of insulin while the cell viability was not enhance, therefore we inferred that the.improvement of glucose utilization was due to the drugs which increase the sensibility of cell to insulin.4. Genistein, silymarin and betulin can significantly improve INS-1E cell viability damaged by palmitic acid, but the compound administered and the monomer administration were no significant difference.ConclusionsUraria crinita total flavonoids has a significant protective effect in islet cells from oxidative stress injury, can effectively improve the state of oxidative stress, the cells of MDA, GSH content and SOD activity. Genistein, silymarin and betulin which were the main ingredients of Uraria crinita total flavonoids can effectively improve the hepatocytes insulin resistance while the cell viability was not enhance, further the combined of three monomer can effectively reduce the dose. Genistein, silymarin and betulin combined can effective protective the islet cells form fatty acids injury, but the combined was not show up a better effect than the monomers. |
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