| Background and ObjectiveIn recent years,owing to life style changes,overtaken of high-calorie food and lack of adequate exercise result in rapid increase in obese people.Obesity and obesity-induced insulin resistance play important roles in the development of type 2 diabetes,heart diseases,vascular diseases,hypertension,stroke and other complications.Prevention of obesity reduces the occurrence of these diseases.Our previous study with ob/ob mice revealed the antiobese and antidiabetic effects of the cajanonic acid A from Cajanus cajan.For further utilization of Caianus cajan,we performed experimental studies on the antiobese and antidiabetic effects of different extracts of Cajanus cajan,as well as the combinatorial effects of the active Cajanus cajan extract with known hypolipidemic agents Panax notoginsenosides and distyrene glycoside.Methods1.In vitro experiments(1)Effects of different Cajanus cajan extracts,Panax notoginsenosides and distyrene glycoside were evaluated the effects on proliferation of 3T3-L1 preadipocytes were evaluated by cell counting kit 8.(2)Effects o f different Cajanus cajan extracts,,Panax notoginsenosides and distyrene glycoside on adipocyte differentiation of 3T3-L1 preadipocytes were investigated.Differentiated mature adipocytes were detected by Oil red 0 staining.(3)Effects of Cajanus cajan extracts extracted with dfferent concentrations of EtOH,Panax notoginsenosides and distyrene glycoside on glucose consumption of insulin-resistant adipocytes were investigated in dexamethasone(DEX)-induced 3T3-L1 adipocytes.From above tested Cajanus cajan extracts,the most active one was selected and coded as SDE.2.In vivo experiments(1)Induction of overweight and glucose metabolic disorderMale wistar rats were fed with high-fat diet for 15 weeks.Body weights were measured weekly,fasting blood glucose level and blood glucose level 2 h after oral administration of 2 g/kg glucose were measured at the end of induction.Overweight and glucose metabolic disorder were judged by comparing the corresponding values of normal rats of same age.(2)Drug interventions on bodyweight,glucose metabolism and lipid metabolismRats with fasting blood glucose level greater than 6.0 mmol/L were randomly devided into seven groups(N=6),and orally given 100 mg/kg/d of metformin,100 mg/kg/d of the 1:1 mixture of Panax notoginsenosides and distyrene glycoside(DG100 group),100 mg/kg/d of the 80%EtOH extract of Cajanus cajan(SDE100 group),100 mg/kg/d of DG in combination with 100 mg/kg/d of SDE(80)(DG100+SDE100 group),100 mg/kg/d of DG in combination with 50 mg/kg/d of SDE(80)(DG100+SDE50 group),50 mg/kg/d of DG in combination with 100 mg/kg/d of SDE(80)(DG50+SDE100 group),or 0.5%CMCNa(Control)for 8 weeks,respectively.Fasting blood glucose levels and bodyweights were recorded weekly.OGTT was performed at the start and the end of drug intervention.Rats of same age fed with standard chew were set as normal.Results1.In vitro experiments(1)3T3-L1 preadipocytes were incubated with 200,100,50 or 12.5?g/ml of the 95%EtOH extract,80%EtOH extract,60%EtOH extract,40%EtOH extract and 20%EtOH extract of Cajanus cajan,Panax notoginsenosides and distyrene glycoside for 48 h.It was found that,Cajanus cajan extracts and distyrene glycoside exhibited to be weak inhibitors to proliferation of 3T3-L1 preadipocytes,with inhibitory rates less than 20%at the concentration of 100 ?g/mL.Panax notoginsenosides showed to be more effective to inhibit cell proliferation,and the inhibition rate was more than 55%at 100 ?g/mL(2)The 95%EtOH(v:v)extract,80%EtOH extract,60%EtOH extract,40%EtOH extract and 20%EtOH extract of Cajanus cajan,Panax notoginsenosides and distyrene glycoside at the concentrations of 40?20?10 ?g/mL,the mixtures of Panax notoginsenosides and distyrene glycoside with the ratio of 40:40,20:20 and 10:10 ?g/mL,the mixture of the 80%EtOH extract of Cajannus cajan,Panax notoginsenosides and distyrene glycoside with the ratio of 40:40:40,20:20:20 and 10:10:10 ?g/mL were assessed the effects on adipocyte differentiation in 3T3-L1 preadipocytes.It was found that,? the 95%EtOH extract or the 80%EtOH extract at 40 ?g/mL effectively inhibited the differentiation of the preadipocytes into adipocytes,showing antiobese potential.Other Cajanus cajan extracts showed not inhibitory effect on adipocyte differentiation;?Panax notoginsenosides dose dependently inhibited adipocyte differentiation,with inhibition rate of 52%at the concentration of 40 ?g/mL;?the combinaton of Panax notoginsenosides and distyrene glycoside,and the combination of 80%EtOH extract,Panax notoginsenosides and distyrene glycoside effectively inhibited adipocyte differentiation in dose dependent manners,but were not more effective than Panax notoginsenosides of same concentrations.(3)Compared with 3T3-L1 adipocytes in the absence of dexamethasone(DEX-),3T3-L1 adipocytes in the presence of 1 ?M of DEX(DEX+)left much more glucose in the culture medium after 48 h of.incubation(1.53 mmol/L vs 5.42 mmol/L,DEX-vs DEX+,indicating the state of insulin resistance induced by DEX.The 95%EtOH extract,80%EtOH extract,60%EtOH extract,40%EtOH extract and 205 EtOH extract of Cajanus cajan,the water extract of Cajanus cajan,Panax notoginsenosides and distyrene glycoside were evaluated the effects on glucose consumption in DEX+ 3T3-IJ1 adipocytes within 48 h at their non-toxic concentrations to cell viability.In this study,the 80%EtOH extract of Cajanus cajan,at 20 ?g/mL,exhibited to be the most acti ve in increasing the glucose consumption of DEX-induced adipocytes.Our in vitro data showed that,the 80%EtOH extract of Cajanus cajan was active to inhibit adipogenesis and restore insulin sensitivity.2.In vivo experiments(1)High-fat diet feeding induced overweight and glucose metabolic disorder in ratsCompared with rats fed with standard chew(Normal),male Wistar rats fed with high-fat diet for 15 weeks(Control)had obviously greater bodyweight(461.50±24.08 g vs 524.18±53.72,Normal vs Control,P<0.0001),higher level of fasting blood glucose(5.18±0.41 mmol/L vs 6.10±0.66 mmol/L,Normal vs Control,p<0.001),and higher blood glucose level 2 h after oral administration of 2 g/kg of glucose(6.16 ±0.46 mmol/L vs 6.80±0.92 mmol/L,Normal vs Control,P<0.05),indicating a state of overweight and glucose metabolic disorder induced by high-fat diet.Forty-two of these rats had their fasting blood glucose level greater than 6.0 mmol/L,and the average values of fasting blood glucose,blood glucose 2 h after glucose administration and bodyweight were 6.72±0.72 mmol/L,7.09±1.14 mmol/L and 523.6±45.8 g,respectively.(2)Intervention with the extract of Cajanus cajan or its compound achieved certain regulatory effect on the bodyweight and glucose and lipid metabolismBodyweight:In general,bodyweight increases of rats treated with the test drugs including metformin were relatively slower than that of rats in control,but only rats in DG100 group and DG100+SDE50 group had significantly lighter bodyweights after 8 weeks of drug intervention(p=0.043<0.05 vs Control;p=0.036<0.05 vs Control).Fasting blood glucose:Compared to normal rats,control rats had obviously higher fasting blood glucose levels during the 8 weeks of experiment(P<0.01 vs Normal).Compared to Control,changes in fasting blood glucose level by drug treatments were not observed.OGTT:Compared to normal rats,blood glucose levels of control rats at different time-points after glucose administration as well as the area under the OGTT curve were significantly higher(P<0.05),indicating glucose metabolic disorder.Compared to Control rats,the corresponding values of rats in treatment groups were relatively smaller,especially those in DG100+SDE50 group.However,the changes were not statistical different.Fasting serum insulin level:compared to normal rats,control rats had obviously higher level of fasting serum insulin(P<0.01),suggesting a state of slight insulin resistance.Compared to control rats,the insulin levels in drug treatment groups were relatively lower.Especially,treatment with metformin,DG100 or SDE100 achieved significant reduction to the insulin level(P=0.029;P=0.013;P=0.041<0.05).Fasting plasma GLP-1 level:Compared to normal rats,rats in control group and DG100 group had unchanged GLP-1 levels,while rats in other treatment groups had relatively higher GLP-1 levels,but without statistical implication.Serum lipid level:Compared to normal rats,control rats had unchanged LDL-C level and TG level,relatively higher TC level,and significantly higher level of HDL-C(P=0.034<0.05),suggesting a state of slight lipid metabolic disorder.In this experiment,drug interventions achieved reduction in TC,HDL-C and TG to some extent,in which,treatment with metformin achieved significant reductions in TC,HDL-C and TG(P<0.05;P<0.01;P<0.05),treatment with SDE100 achieved significant reduction in HDL-C(P<0.05),and the treatment with DG100+SDE100 achieved significant reduction in TC(P<0.05).Conclusion(1)The 80%EtOH extract of Cajanus cajan,distyrene glycoside,Panax notoginsenosides,and their combination remarkably inhibited the differentiation of 3T3-L1 preadipocytes into mature adipocytes;(2)The 80%EtOH extract of Cajanus cajan significantly increased the glucose consumption of 3T3-L1 adipocytes with insulin resistance induced by dexamethasone;(3)The combination of the 80%EtOH extract of Cajanus cajan(50 mg/kg),Panax notoginsenosides(50 mg/kg)and distyrene glycoside(50 mg/kg)showed activity to significantly antagonize high-fat diet induced bodyweight increase in male Wistar rats(P<0.05);the 80%EtOH extract of Cajanus cajan(100 mg/kg)significantly reduced the serum HDL-C level and insulin level;the combination of the 80%EtOH extract of Cajanus cajan(100 mg/kg),Panax notoginsenosides(50 mg/kg)and distyrene glycoside(50 mg/kg)reduced the serum TC level significantly,while the combination of Panax notoginsenosides(50 mg/kg)and distyrene glycoside(50 mg/kg)reduced serum insulin level significantly.Our result suggested that the combination of 80%EtoH extract of Cajanus cajan,Panax notoginsenosides and distyrene glycoside could improve the lipid and glucose metabolism to some extent. |
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