| Objective and significance of the research:chickpea as a food and drug in the lives of the people of Xinjiang,has a good hypoglycemic effect.But the current research mainly concentrated in the isoflavones site and saponins site of chickpea.They have made some achievements,but the material basis of pharmacal efficacy is not explicit.And the hypoglycemic activity of the two parts is not obvious,only to repair pathological damage in a certain degree,but can not reverse the pathological damage.For the better use of national resources of chickpea,and the development of lead compounds has good hypoglycemic activity,we have synthesized a series of novel derivatives of isoflavone compounds(genistein,biochanin A,formononetin)in chickpea and the hypoglycemic activity of these compounds was screened.Research method:Based on the knowledge of medicinal chemistry and related structure activity relationship,designed the structure and synthesis route of target compounds.Derivative structures were identified through 1H-NMR,13C-NMR and high resolution mass spectrometry,elemental analysis,IR,UV and thermal analysis.The insulin resistant HepG2 cell model was selected for screening compounds which had good hypoglycemic activity.And according to the characteristics of traditional Chinese medicine of multiple composition and multiple targets,the compounds with high hypoglycemic activity were combined,and hope to further improve the hypoglycemic activity.HUVEC cell model under high glucose environment was selected to screen compounds with better protective effect on HUVEC cell under high glucose environment,and hope to obtain compounds with a better adjuvant treatment of diabetic peripheral neuropathy.It is expected that the compounds with good hypoglycemic activity and could repair the damage of diabetic complications can be obtained.And then the H4IIE cell model with recombinant resistin protein intervention was selected for screening compounds with better hypoglycemic activity.Expected to be able to obtain a better prevention and treatment of diabetes in obese patients.Finally,the compounds with good activity obtained from the above three cell models were screened on the model of insulin resistance human hepatocytes L02,which was expected to obtain compounds with good hypoglycemic activity on human hepatocyte L02.The genistein chromium element complex was tested by db/db mice,and the hypoglycemic activity of the complex was observed in vivo.Research results:114 isoflavone derivatives were obtained by structural modification and identification.Through the HepG2 cell model of insulin resistance(a total of 118 compounds were screened,and 4 compounds were synthesized in the prep work of laboratory.The 4 compounds had been marked in the list of compounds.The following different cell models were also used to screen 118 compounds too.),compounds F1?H?J1?N?C5?E5?K5?Q5 and genistein were obtained,which had better hypoglycemic activity,and the structure activity relationship was discussed,but the activity was not as positive as the positive drug metformin hydrochloride(p<0.05 or p<0.01).Through the combination of these compounds with better hypoglycemic activity and genistein and then screened by the HepG2 cell model of insulin resistance,four combinations had better hypoglycemic activity were obtained:The combination 6(genistein,compound H,compound J1)and the combination 10(compound J1,genistein,compound F1)in first batch of compounds(compounds A-T,A1-T1,A2-T2)had better hypoglycemic activity.There was no significant difference in the hypoglycemic activity of maximum concentration between the combination6,10 and the positive drug metformin hydrochloride(p>0.5).And the hypoglycemic activity of maximum concentration of combination 6 and combination 10 was much better than the other groups in the maximum concentration(p<0.05 or p<0.01).In the same dose group(500 mol/L),the combination 10 had similar hypoglycemic activity with positive group(p>0.05).The combination 3(genistein,compound E5,compound K5)and combination 6(compound C5,compound E5,compound K5)in second batch of compounds(compounds A3-T3,A4-T4,A5-T5)had better hypoglycemic activity.There was no significant difference(p>0.05)between the combination 3,6 and the positive drug metformin hydrochloride in the same dose(500?mol/L).At the dose of 1000 ?mol/L and 500 ?mol/L,the combination 3 and combination 6 had better hypoglycemic activity than the other combinations,and the difference was statistically significant(p<0.05 orp<0.01).The compounds were screened by the HUVEC cell model under high glucose environment,and the compound E,L,R2,A4,J1,E4,D5,M5 had the better protection of HUVEC cell model under high glucose environment.But the activity of these compounds was not as good as the positive drug(MiKeBao,500 ?mol/L)(p<0.05 or p<0.01).The 4 combinations were screened by HUVEC cell model in high glucose environment,we found the combination 6 and combination 10 in the first batch of compounds had a better protective effect on HUVEC cell model in high glucose environment.The difference of protective activity of the combination 6 and combination 10 for HUVEC was statistically significant(p<0.05),and the effect of combination 10 was better than combination 6.Combination 6 and combination 10 compared with the positive group,combination 10 was equivalent to the positive group at 1000 ?mol/L(p>0.05).The difference between combination 6 in the maximum dose and the positive group was statistically significant difference(p<0.05),and the activity of combination 6 was not good as positive group.The LDH,MDA,SOD activity and NO content between combination 10 and combination 6 also had significant difference(p<0.05 or p<0.01).The combination 10 significantly reduced the LDH and MDA of HUVEC secretion and the NO content and SOD activity of HUVEC were significantly increased in high glucose compared with combination 6.It was concluded that the combination 10 can better protect the HUVEC in high glucose.In the follow-up study,combination 10 may be able to play a better hypoglycemic effect,as well as better repair of diabetic complications.The compounds were screened by recombinant resistin protein intervention model of insulin resistant H4IIE cells,and compound J4,H3,B1,F2 were obtained with good activity.But the activity of these compounds was not as good as the positive drug(rosiglitazone hydrochloride)(p<0.05 or p<0.01).The above 4 combinations were screened by recombinant resistin protein intervention model of insulin resistant H4IIE cells,and we found that the combination 3 and combination 6 in the second batch had good activity.In the absence of insulin stimulation,the combination 3 had a stronger effect of stimulating H4IIE cells on glycogen synthesis than combination 6(p<0.05).And the combination 3 had the similar effect of glycogen synthesis compared with the positive drug in the same dose(500 ?mol/L)(p>0.05).In the insulin stimulated conditions,the combination 3 had a stronger effect of stimulating H4IIE cells on glycogen synthesis than combination 6(p<0.05).And the combination 3 had the similar effect of glycogen synthesis compared with the positive drug in the same dose(500 ?mol/L)(p>0.05).This showed that combination 3 had a stronger effect than combination 6 of stimulating H4IIE cells on glycogen synthesis intervened by recombinant resistin protein.Therefore,we can know that the combination 3 play a role in the prevention and treatment of the occurrence and development of diabetes in obese patients.The compounds were screened by human liver cells L02 insulin resistance model,and compound J1?D5?E5?F1?H?N?C5 were obtained with good activity.The compound F2?01?K5?L5?Q5(the initial screening used HepG2 cell insulin resistance model had good activity)did not show a good hypoglycemic activity in the L02 cell insulin resistance model.It showed that there was a certain difference between the insulin resistance model of L02 cells and the insulin resistance model of HepG2 cells.This may be related to the different expression of insulin receptors on the surface of L02 cells and HepG2 cells.Studies on the hypoglycemic activity of genistein complex in the db/db mice showed that,the complex can significantly improve the body weight of db/db mice,fasting blood glucose,random blood glucose,liver/body,renal/body(compared with the C5 7 group,the difference was not statistically significant),and the performance of OGTT(Oral Glucose Tolerance Test)and ITT(Insulin Tolerance Test)in db/db mice.The morphology of liver,kidney,pancreas and skeletal muscle also had obvious improvement and repair.Effect on serum index of db/db mice showed that,TC(Total Cholesterol),TG(Triglyceride),LDL-C(Low-Density Lipoprotein Cholesterol)had no significant improvement compared with the model group,but HDL-C(High-Density Lipoprotein Cholesterol)of genistein complex group had significant difference compared with the model group.In addition,our team also conducted a study on the toxicity of the complex,and did not find its potential toxicity.Conclusion and significance:Through structural modification of genistein,biochanin A,and formononetin from chickpea isoflavones,several derivatives with better hypoglycemic activity than the parent compound were obtained.But we did not obtain the derivative which had the same hypoglycemic activity with positive drug.By combination of compounds with better activity,we obtained combinations with considerable activity with positive drug.And some of these combinations also showed considerable activity with positive agents in other types of cell models associated with diabetes.It showed that the action of multiple components and multiple targets coud produce better effect than single compound.It also provides some reference for the development and utilization of the chemical components of traditional Chinese medicine.Through the study of the in vivo activity of the genistein chromium complex,the complex had good hypoglycemic activity in vivo,and did not find the potential toxicity,and may have a certain potential druggability. |
PDF Full Text Request