| Objective:Based on the Jiuwei Tongqiao decoction and its analysis,to obtain the best prescription and the most effective extract by in vitro pharmacodynamic screening methods in vitro efficacy.Establish a method for cell membrane chromatography of brain glioma,to obtain the best chemical composition of anti brain glioma.Establishment of rat brain glioma model to explore the effects of the above mentioned components on the anti glioma effect in vivo.Methods:(1)Jiuwei Tongqiao decoction was separated to seven prescriptions based on the principles of monarch.After alcohol extract enrichment,using U251 cells of anthropogenic brain glioma,MTT was applied to determine the effect of different prescriptions for cell survival.Flow cytometry instrument was used to compare the cells apoptosis effect on optimal prescription with the whole part;Western blot was used to detect apoptosis-related protein Bcl-2 and P53 expression differences between the optimal prescription and the whole part.Alcohol extraction of the optimal prescription was concentrated with petroleum ether,chloroform and ethyl acetate.MTT was applied to determine the effect of extraction parts for cell survival.(2)The best prescription of Jiuwei Tongqiao decoction interact with U251 cells,the U251 cell membrane was used as the stationary phase and make a good combination of both.After removal of the unbound component,the cell membrane is inactivated and the active component is released from the cell membrane.The active ingredients were got by solid phase extraction,and analyzed by UPLC-TOF/MS.(3)MTT was applied to determine the effect of Radix Astragali for cell survival.Flow cytometry instrument was applied to detenine Radix Astragali effect on the cells apoptosis after 24 h.The active chemicals of Radix Astragali s were conbianed with human brain glioma membrane under imitated physical environments.The unattached ingredients were washed away.Then the attached ingredients were eluted.The released ingredients from target were collected and analyzed by UPLC-TOF/MS.Then MTT was applied to determine the effects of the active ingredients for cell survival.Flow cytometry instrument was applied to detenine the active ingredients' effects on the cells apoptosis after 24 h.(4)Rats were anesthetized and fixed on the stereotaxic apparatus,skin disinfection,determined the drilling location corresponding to the right side of the caudate nucleus,10 uL C6 cell suspension was injected in the left caudate nucleus by micro syringe,established the model of cerebral glioma in rats.Clarify the efficacy of calycosin and formononetin in the brain glioma.Results:(1)Compared with other parts,prescription ? has more obvious inhibition of U251 cells and appear a certain concentration-dependent.The apoptosis rate of formula ? treatment group and whole group are all significantly higher than blank group.The Bcl-2 protein expression level of Formula ? and the whole group are all obviously lower than the blank group.The P53 protein expression level of formula ? group and the whole part are all obviously higher than the blank group.The directive ingredients in part ? were more comprehensive.(2)Through the establishment of brain glioma cell membrane chromatography,screened 4 effects in the test solution composition,calycosin,calycosin-7-glucoside(calycosin-7-O-?-D-glucoside),ononin(formononetin-7-O-?-D-glucoside)and formononetin.(3)The results indicated that Radix Astragali has obvious inhibition of U251 cells and appear a certain concentration-dependent.The apoptosis rate of Radix Astragali is significantly higher than blank group,P<0.01.The human brain glioma membrane immobilized chromatography has binded with four active ingredients.They are calycosin,calycosin-7-glucoside(calycosin-7-O-?-D-glucoside),ononin(formononetin-7-O-?-D-glucoside)and formononetin.Calycosion and formononetin indicated obvious inhibition of U251 cells and high apoptosis rate.(4)Rat brain glioma models were successfully established after injecting rat brain glioma C6 cell line into the right caudate nucleus.Conclusion:Prescription IV is the optimal prescription.Part ? is the most effective parts of the anti-proliferative effect of U251 glioma cells.Establishment of rat brain glioma cell membrane chromatography allowed us to obtain four different active ingredients.The effects of calycosion and formononetin on cell viability were determined,and the concentration was dependent on time.The rat model of brain glioma was established successfully,which could further explore the pharmacological effects of calycosion and formononetin in vivo.. |
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