| Background and objectiveThe prevalence of diabetes increasing worldwide as the development of society and the living level as well as the population aging turns to be the third noninfective disease followed the cerebrovascular disease, which endangers the human health, burdens the individual and society heavily and leads to global problem of public health. Estimated from the World Health Organization, the number of diabetic patients will increase from 194 million in 2003 to 360 million in 2030. Since the large population in China, the burden of diabetes is extremely high, with the population of diabetes accounting for 1/3 in the world. Showed in the recent research, diabetic prevalence of aging standardization is 9.7%, furthermore, the rate of prediabetes is up to 15.5%, which means 1 adult in 10 is diabetic patient, and 1 adult in 4 is hyperglycemic patient.In the population of diabetic patients in China, the rate of typeⅡdiabetes is 90%. Pointed out by the typeⅡdiabetes Prevention manual, it's the prevention of typeⅡdiabetes that the main challenge and the target are:to decrease the prevalence of typeⅡdiabetes and to early intervention from getting typeⅡdiabetes. The common features of typeⅡdiabetes are impaired glucose tolerance (IGT) and impaired fasting glucose (IFG). IGT and IFG are the disorders of glycometabolism, which is a stage of pathogenesis in type II diabetes. The secrete defections of insulin resistance (Ins IR) and Ins are the main factors in IGF/IFG in association with IGF to Ins and IFG to IR.In China, the prevalence of IGT/IFG is up to 4.76%, with from 10% to 15% turning to T2DM. Though there are no DM syndrome presented in IGT/IFG patients, T2DM can be developed in those patients. Moreover, it can lead to vessel complication such as atherosclerosis, hypertension and coronary heart disease in this stage. Therefore, it's essential to strengthen the prevention of IGF/IFG on the reduction of T2DM prevalence and vessel complication.In a certain studies, oxidative stress increases vivo reactive oxygen soecies (ROS). Ros can impair beta cells of islet by various ways such as lipid peroxidation, broken DNA chain, inhibition of ATP synthesis and chemotaxis as IGT is the primary faction among the ways of impair. The removed ability of beta cells of islet to ROS is low, causing impair by ROS, cell death or apoptosis which lead to the defective secretion of insulin. Besides, the increased activity of ROS affects the combination of insulin and its receptor, which causes the abnormal of signal transduction in downstream that makes insulin resistance. Therefore, by decreasing the level of ROS in vivo, the insulin resistance can be ameliorated, and the function of beta cells of islet can be protected.Since 2008, we in our lab have made a series studies on Uraria crinita. In our studies, the distil extract of Uraria crinita shows ameliorated function in IGT and IFG by decreasing OGTT and the level of fasting plasma glucose with the association of reduction of free fatty acid (FFA), of triglyceride (TG) and the released stimulation of insulin.On the base of the prophase studies, the flavonoids of Uraria crinita are planned to extract and separation, and evaluate the total anti-oxidative ability of flavonoids by such as DPPH and ABTS. Vitro experiments will be performed to study the protection to lipid peroxidation and the removed functions of flavonoids in free radicle such as hydroxyl free radicle, superoxide anion, H2O2 and NO. Meanwhile, the protection of oxidative stress in diabetes and the relevant mechanism are explored by studying certain oxidative stress models of insuloma cells and analyzing correlated indexes.Methods:1. Analysis of chemical compositions from uraria crinitaChemical compositions from uraria crinita were extracted by soxhlet extractor with distilled water, dehydrated alcohol and acetoacetate as solvents respectively. Analysis of colour reaction was preformed on the extracts of distilled water, dehydrated alcohol and acetoacetate to test whether they contained chemical compositions such as alkaloid, saponin, flavone, anthraquinone, coumarin, sexualization and polysaccharide.2. Extraction and separation of total flavonoids from uraria crinitaThe alcohol solution of 50% was added to the comminuted power of uraria crinita followed by the ratio of 1:10 (g. mL-1). After the reflux condensation, the extract was filtered and decompressed to concentrate almost dry. Solved the concentration with distilled water and filtered it which was extracted by diethyl ether and acetoacetate followed. The total flavonoids were obtained after decompressing and freeze drying the extraction.3. Antioxidant study of total flavonoids from uraria crinita in vitroAccording to the standard curve of Rutin, solutions of total flavonoids in different concentrations were prepared 0.1,0.08,0.06,0.04 and 0.02 mg/ml. compared the solutions with Vitamin C or Vitamin E of the same concentrations of these solutions, the inhibitions of total flavonoids towards DPPH (517 nm), ABTS+ (734nm),NO (540nm),·OH (536nm) and the lipid peroxidation (500nm) by using the ultraviolet-visible light detector.4. Protection of INS-1E cells from cellular oxidation by the total flavonoids.INS-1E cells in the period of exponential growth phase were inoculated into 96-well plate (1×104/well), and disposed by extracts of different concentrations (the final concentrations of total flavonoids were 0.03,0.02,0.01,0.005 and 0.001 mg/ml). Both the model group and the administration group were treated oxidative damage by H2O2 (0.4mmol/L) or SNP (10mmol/L); the survival rate of islet cells was tested by MTT method in 570 nm after oxidative damage.5. Effects of the total flavonoids to the contents of NO, MDA and GSH and the activity of SOD under the oxidative stress status of INE-1E cellsINS-1E cells in the period of exponential growth phase were inoculated into 24-well plate (1×104/well), and disposed by extracts of different concentrations (the final concentrations of total flavonoids were 0.03,0.02,0.01,0.005 and 0.001 mg/ml). Both the model group and the administration group were treated oxidative damage by H2O2 (0.4 mmol/L) or SNP (10 mmol/L). The supernatant and the disrupted solution from cultured cells were collected and the contents of NO, MDA and GSH and the activity of SOD were detected by relevant reagent kits.6. Construction of the screening model inhibited DPP-Ⅳin vitroCaco-2 cells were cultured in vitro, and extracted the DPP-IV enzyme from the cells. The activity of DPP-Ⅳenzyme was detected by Gly-Pro-pNA (100 umol/L) as substrate since there was ultraviolet absorption in 405 nm after Gly-Pro-pNA was cut by the enzyme. The substrate was incubated 1 hour in 37℃with different concentrations of DPP-IV enzyme (10%,20%,25%,50%,75%,90% and 100%). The value of OD in 405 nm was detected and standard curve was drawn with OD as ordinate and relative concentration of enzyme as abscissa. Meanwhile, the substrate was incubate 1 hour in 37℃with DPP-IV solution of 100% relative concentration and Sitagliptin solution of different concentrations(0,1,10,20,100,500 and 1000 nM) respectively. The value of OD was obtained in 405 nm and the IC50 of Sitagliptin was calculated.7. The inhitations of Sitagliptin derivation and the total flavonoids from Uraria crinita to the activity of DPP-Ⅳ.The substrate was reacted with the mixture of the DPP-Ⅳsolution of 100% relative concentration, the different contraptions of JD-1 (0,2500,2800,3000,3200,3500,4000 nM),JD-2 (0,600,1000,1400,1800,2200,2600 nM) and the solutions of the total flavonoids from Uraria crinita (0.03,0.02,0.01, 0.005,0.001 mg/ml) respectively. The value of OD in 405 nm was tested after incubated 1 hour in 37℃, and the IC50 was calculated.Results:1. The analyzed result of compound identification indicated that acetoacetate extract of Uraria crinita contained certain active substances such as flavonoids, anthraquinones, coumarins; ethanol extract contained the active substances such as alkaloid, saponin, flavone, anthraquinone, coumarin, carbolic acid and polysaccharide; the distil extract contained active substances such as saponin, flavone, coumarin and polysaccharide.2. The vitro anti-oxidative analysis of total flavonoids extracted from Uraria crinita showed that in the range of 20mg.L-1～100mg.L-1, the total flavonoids is potent to clear free radicals such as PPH, ABTS+,-OH and NO. The maximum clearance of it can reach 60.89%±0.19%,74.87%±0.08%,41.77%±0.41%,81.01%±9.61% respectively and the maximum of inhibition to lipid peroxidation can up to 41.07%±0.10% which is concentration dependent.3. Protection Study of total flavonoids from Uraria crinita to INS-1E cells in oxidative stress presented that H2O2 (0.4mmol/L) and SNP (10mmol/L) impaired INS-1E cells significantly (P<0.01), with the cell survival rate in different concertration group of total flavonoids higher than the oxidative damage group. However, there are significant differences between H2O2 damage group and model group (P<0.01) in the total flavonoids concentration of 0.05 and 0.1 mg/ml; between the SNP damage group and the model group in the range concentration of from 0.05 to 0.3 mg/ml.4. There are significant differences in the effects between the blank group and the experimental group of the concentration of NO, MDA and GSH and the activity of SOD in the INS-1 cellular oxidation, between the blank group and the SNP damaged model group of the concentration of NO, MDA, GSH and the activity of SOD. The total flavonoids from Uraria crinita protected INS-1E cells from oxidative stress which adjusted the concentration of NO, MDA and GSH and the activity of SOD.5. The IC50 of Sitagliptin was tested; the result is 18.6594 nM which was approached to the literature already reported. Furthermore, the IC50 results of analogs JD- 1 and JD-2 are 3416.5 nM和2641nM respectively. However, the inhibition from the total flavonoids of Uraria crinita to DPP-IV was not found in this experimental study.ConclusionsUraria crinita is abundant with anti-oxidative activities and its total flavonoids is potent to clear free radicals included DPPH, ABTS,·OH and NO as well as is apparent to inhibit lipid peroxidation. Meanwhile, it can protect beta cell of islet in oxidative stress and adjust the concentrations NO, MDA and GSH, and the activity of SOD, but there is no inhibition to DPP-IV enzyme. |
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