Screening Of IRTK Agonist From Traditional Chinese Medicine

OBJECTIVE This research was designed to screen IRTK agonist from traditional Chinese medicine with reporter gene cell strain based on a traditional Chinese medicine extract library.Then the effective traditional Chinese medicine was separated to find bioactive components.And it provides the basis for development of new drugs for treating diabetes.METHODS①Identifying and optimizing the assay condition by examined the effects of some factors such as cell number in each well,incubation time,luciferase substrate concentration,DMSO and mixed system of traditional Chinese medicine.②A traditional Chinese medicine extract library was established by petroleum benzine,acetoacetate,n-butanol,alcohol and water,on account of calculating the frequency of traditional Chinese medicine for treating diabetes.③the effect of extracts of traditional Chinese medicine on the expression of luciferase reporter gene were detected.④the effective traditional Chinese medicine was separated by SG column chromatography.⑤the effective fractions of bioactive components of traditional Chinese medicine were detected with the same method.⑥the effective fractions were expertise by TLC to infer the constituent.RESULTS①The optimal assay condition was 15,000 cell/well cultured,induced 8h,luciferase substrate to dilute 4 times,the final concentration of DMSO is 0.1%. And the mixed system of traditional Chinese medicine did not interfered the induction of Insulin and AG 1024.②A traditional Chinese medicine extract library was established as the source of screening.③The extracts of TCM001 and TCM002 with alcohol could induced remarkably the expression of luciferase,and the induction fold was 2.92 and 2.58 times.④The extract of TCM001 with alcohol was separated to 10 fractions.⑤the third and fifth fraction of TCM001 aslo could induced remarkably the expression of luciferase,and it did not effect through enhancing cell activity.⑥the third and fifth fraction of TCM001 could be constited of the compounds 2#,3# and 5# of TCM001.CONCLUSION After establishing the traditional Chinese medicine extract library and screening of IRTK agonist,the extract of TCM001 with alcohol could have the activity of agitating for IRTK,and it was intimate relation to the compounds 2#,3# and 5# of TCM001.Then it provides the basis for designing the lead compound of new drugs for treating diabetes.