| Diabetes is a kind of chronic progressive disease caused by sugar,protein and fat metabolism disorder in body.With the improvement of living standards,as well as the aging of the population and the increase in the incidence of obesity,the incidence of diabetes was an upward trend year after year.The World Health Organization predicts that by 2030,the number of people who have diabetes will reach 578 million all around the world.Diabetes has been a serious threat to human health.It takes the third place among all the causes of morality,just inferior to cardio-cerebral vascular diseases and cancer.Classic treatments of diabetes are mainly around the insulin secretion and sensitization,but they generally have inherent disadvantages,such as hypoglycemia,gastrointestinal tract side effects,and the decreasing hypoglycemic effect as the extension of the course of treatment.Therefore,the search for novel anti-diabetic drugs,especially the effective hypoglycemic drugs with security and ease of use from natural Chinese herbal medicine has become a hot spot.Rencent study indicated that pentacyclic triterpenes(PTs)have salient hypoglycemic activities.It is rich in resources,with a wide range of biological activities,no significant side effects,and ameliorate the clinical symptoms of patients with diabetes significantly.However,their action mechanisms are not quite clear yet.Based on the studies on the regulation of glucose and lipid metabolism of pentacyclic triterpenes and the results of its molecular mechanism study that have been reported,our study was continued on the hypoglycemic activity of PTs as GP inhibitors in vivo and in vitro.Part 1 Effects of pentacyclic triterpene natural products and derivatives on glycogen phosphorylase inhibitory activityObjective:We performed High-throughput glycogen phosphorylase inhibitory activity screening of pentacyclic triterpene derivatives was conducted,in-depth study of the mechanism of action of these compounds,and search and development of drugs with pentacyclic triterpene compounds as active substances.Methods:Caffeine,a well-known GP inhibitor,which shares the same binding site with pentacyclic triterpenes,was used as the positive control.In addition,the positive group(only adding enzyme),blank group and test compound group were set.Each test compound was dissolved in DMSO and diluted at different concentrations for IC50 determination.Rabbit muscle glycogen phosphorylase a(GPa)was added into the buffer of test compound and in 96-well microplates(Costar).After the addition of 150 ?L malachite green solution,reactions were run at 30°C for 20 min,and then the phosphate absorbance was measured at 655 nm.The IC50 values were estimated by fitting the inhibition data to a dose-dependent curve using a logistic derivative equation.Results:20 pentacyclic triterpene derivatives were tested for effects on rabbit muscle glycogen phosphorylase a.The results showed that most of the tested compounds displayed inhibitory activity against glycogen phosphorylase to some extent.Of the pentacyclic triterpene derivatives,compound13(IC50=7.76?M)showed good potential against glycogen phosphorylase.Conclusions:The results indicated that most compounds displayed inhibitory activity against glycogen phosphorylase to some extent.The study further strengthens the evident that pentacyclic triterpenes could lower blood glucose levels at least in part,through modulation of glycogen metabolism.Part 2 The effects of pentacyclic triterpenes on HepG2 liver cells' glucose consumptionObjective:To evaluate the effects of the anti-diabetes activity of the synthesized compounds,we treated HepG2 cells with different doses of synthesized compounds and measured the glucose consumption by glucose-oxidase method.Methods:HepG2 cells were cultured in vitro under insulin-resistant conditions.Changes in the glucose concentrations in aliquots from the culture media after HepG2 incubation for 24 hours were determined by glucose oxidase-peroxidase method.Results:There were 13 compounds chosen to screen the effect for 24 h incubation on glucose consumption of HepG2 cells.The results indicated that positive reference insulin(0.1 ?M)induced a significant increase in the consumption of extra-cellular glucose compared to the blank vehicle 0.1% DMSO(P<0.05).25 compounds significantly increase glucose consumption in HepG2 cells,and promote glucose uptake,in which compounds,1?5?6?7?9?11 have considerable effect as insulin,compounds10?12?13 have better effect than insulin.Conclusions:We have found that pentacyclic triterpenes displayed inhibitory activity against glycogen phosphorylase,and their anti-hyperglycemia activity could,at least in part,be due to modulation of hepatic glucose production.Moreover,pentacyclic triterpenes significantly promoted the glucose consumption in HepG2 cells.Considering the hypoglycemic phenomenon we postulate that the anti-diabetic pharmacological effect of pentacyclic triterpenes not only due to the suppression of hepatic glucose production but also maybe partly caused by the enhancement of hepatic glucose consumption.Part 3 Effect of pentacyclic triterpenes on fasted plasma glucose of hyperglycemic mice induced by adrenalineObjective:In order to provide experimental basis for the development and utilization of glycogen phosphorylase inhibitors.we observed the effects of ursolic Acid,oleanolic acid and maslinic acid on blood glucose in hyperglycemia mice induced by adrenaline.Methods:Five-to six-week old mice were randomized to five groups.Mice were then dosed p.o.daily for 7 days with glimepiride and pentacyclic triterpenes.On day 8,mice were treated p.o.with adrenaline 0.2 mg/kg to induce hyperglycemia.Then bled post-dose for plasma glucose determination by glucose oxidase-peroxidase method.Results:The preliminary animal study results showed that glimepiride significantly inhibited an increase in the fasted plasma glucose level of diabetic mice induced by adrenaline at 1 h and 2 h(p < 0.01).Ursolic Acid also significantly inhibited an increase in the fasted plasma glucose level of diabetic mice induced by adrenaline at 1 h and 2 h(p < 0.05).Oleanolic acid and maslinic acid reduced blood glucose at 4 h(p < 0.05).Conclusions:Comprehensive in vivo biological evaluations were performed on pentacyclic triterpenes.The results showed that pentacyclic triterpenes could lower blood glucose levels,whose functions were achieved,at least in part,through modulation of glycogen metabolism. |
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