Research On The Preparation And Brain Protective Effect Of APG,an Active Ingredient In Clematis Tangutica

BackgroundThe plants of genus Clematis?Ranunculaceae?are widely distributed in China,which contain various types of chemical constituents.In folk,the plants were mainly used in the treatment of rheumatism,wind cold,pain and infection.As one species of the genus,Clematis tangutica is mainly distributed in the western region of China,and was used in the treatment of blood circulation and dyspepsia in Tibetan medicine.In our previous studies,saponins and flavonoids were systematically isolated and identified from the plant,and an active ingredient,apigenin-7-O-?-D-?-6''-p-coumaroyl?-glucopyranoside?APG?,was found.Up to now,progress has been made in the studies of cardiovascular protection effect and the mechanism of APG.Meanwhile,the study of brain protective effect of APG is also pushing forward.APG has the potentiality of further development as a therapeutic drug.In this study,the preparation technology of APG was explored and optimized from the extraction and synthesis aspects.And the brain protective effect of the synthetic product was verified,which provided a certain theoretical and experimental basis for the production and research of APG.Methods1.Optimization of ultrasonic extraction of total flavonoids from C.tangutica by response surface method:On the basis of three single factor experiments,the volume fraction of ethanol,the time of extraction and the liquid-material ratio,our group set up the Box-Behnken center combination experiment design on the extraction rate of total flavonoids and simulated two regression models.With the analysis of response surfaces,the total flavonoids extraction rate of C.tangutica was optimized.2.Chemical synthesis of active ingredient APG:Through consulting literature and making attempts,a synthetic route was designed,which selected apigenin,acetobromoglucose and coumaric acid vinyl ester as starting materials.And the reaction conditions were compared,such as the catalyst and solvent.On this basis,the target product APG is synthesized.3.Brain protective effect of synthetic products:A model of cerebral ischemia/reperfusion in rats was established,which was divided into sham operation group,MCAO group,vehicle group,positive control group?r-tPA?and synthetic product intervention group.Through the comparision of neurological behavioral score and cerebral infarction area,the brain protective effect of the synthetic product,apigenin-7-O-?-6''-p-coumaroyl?-glucopyranoside,was verified.Results1.Optimization of ultrasonic extraction of total flavonoids from C.tangutica by response surface method:Through model analysis and feasibility correction,it was found that ethanol volume fraction has the most significant effect on the extraction rate of total flavonoids.The best extraction condition was 80%of ethanol volume fraction,44min of extraction time,30.5:1 of liquid-material ratio,and the maximum extraction rate could reach 1.266%.2.Chemical synthesis of active ingredient APG:After the comparision experiments,the conditions are determined.Lipolase 100T was selected as the catalyst.CH₃CN was selected as the solvent.The reaction time is 76h.Synthetic results were verified by physical and chemical data,such as flavone characteristic reaction,ESI-MS,¹H-NMR and¹³C-NMR.Finally,the yield of apigenin-7-O-?-D-?-6''-p-coumaroyl?-glucopyranoside was42.27%.3.Brain protective effect of synthetic products:Compared with the MCAO group,the neurobehavioral score increased and the area of cerebral infarction decreased significantly of the rats in the synthetic product intervention group?P<0.05?.Therefore,the APG obtained through this synthetic route had brain protective effect.ConclusionsIn this study,with the aid of the methods of response surface and catalytic synthesis,the preparation technology of APG,an active ingredient from C.tangutica,was explored and optimized from two aspects,namely,extraction and synthesis.The brain protective effect of APG was verified by the model of local ischemia/reperfusion in the rat brain.It provided the experimental basis for the quantitative production of APG,and provided a theoretical basis for the development and utilization of C.tangutica,and the research and development of new cardio cerebrovascular drugs.