Study On The Anti Epileptic Activity Component Of Qingyangshen Based On The Process Of Digestion And Absorption Huang Cai-Shun

Screening active compounds is the core for material research of Traditional Chinese Medicine.However,due to the complexity of chemical ingredients in herbal medicine,the process is full of challenges.Although a lot of new research methods have been proposed,they are not fit for Yunnan province which is rich in Chinese herbal resources but poor in economic and technology level.We here try to establish an high efficient and economic approach to screening active components,based on the following proposition:"if focus on prototype components that are digestive-stable and absorbable from oral-administrated herbal medicine,the screen procedure will be efficient because most of them would be active".To prove this proposition,we need to prove the following assumptions:1.Some of the Chinese herbal medicine are orally effective;2.The effective Chinese herbal medicine should contain active compounds;3.Some of the active compounds are prototype components;4.Some of the prototype components are digestive-stable and able to be absorbed into blood;5.The digestive-stable and absorbable prototype components can be detected,obtained and proved to be active in a high proportion.Among them,assumption 1-3 can be considered to be established according to clinical practice and general logic,so this paper will focus on the fourth and fifth points.In this paper,we will prove the point 4-5 by literature and case research:1.The literature research:?Study on digestive stability:We reviewed the application of artificial gastrointestinal fluid model in pharmaceutical research.It is one of the main methods for digestive stability studies that can be used to guide the development of dosage forms and detect metabolism.Factors influenced the digestive stability include gastrointestinal fluid pH,digestion time,and enzymes.But current methods can not fully reflect the factors that influence the stability,so factorial design method should be applied and it can be applied in herbal medicine screen;?Serum pharmacochemistry is a method used to screen active ingredients in Chinese herbal medicine.Its core idea is that the components absorbed into blood may be effective ones.Is the method efficient and feasible?Our literature review shows that,1,There are 100 articles that directly reported and analyzed absorbable components refering to 88 herbal fonnulas;2,There are 970 absorbed components have been detected,including 582 prototype and 388 metabolites,wihich means 60%of these components are prototype composition.So,the fourth hypothesis is correct:"Some of the prototype components are digestive stable,can be absorbed into blood";3,80.37%of the absorbed prototype components have been reported about their activity(Figl),which means the fifth hypothesis is also correct,"Most of the digestive stable prototype components that absorbed into blood are active".Fig 1 The statistical chart of effective constitutes of plasma(A)and the mean activity ratio of identifiedprototype components(B)2?Experimental case:Cynanchum otophyllum is a commonly used ethnic medicine named as Qingyangshen(QYS).However,only a few monomers have been reported about its anti-seizure effect.The purpose of this paper is using the two methods,digestive stability and serum pharmacochemistry,to find the QYS's antiepileptic active components and clarify its pharmacodynamic substance,and assess the reliability of our proposed method.The content of this research are as follows:1.Simulating the gastrointestinal digestion in vivo and vitro,looking for digestive stable composition based on HPLC-UV fingerprints;2.The pharmacochemistry research of QYS,to find the absorbed constituents;Combined with the digestion and serum pharmacochemistry screen,screen out the digest-stable and absorbable compounds on printfinger,3.Isolate and identify them with the guidance of fingerprint;4.Pharmacodynamic experiment:Containing MES test and zebrafish test,to explore the anti epilepsy activity of target compounds,and their mechanism on 5-HT.5.Finally,make a preliminary evaluation,the feasibility and significance of this method.The main results are as follows:1.The result of simulated digestion:According to whether the compounds are stable or not in vivo and in vitro digestion,there are three kinds of compounds(according to the fingerprint):?:Unstable ingredients:exist in raw materials,but not in the digestion samples;?:Metabolites:Exist in the samples of large intestine,but not in raw materials,were out of the body ?:Digestive stable ingredients:existed in the raw material and the vivo and vitro digestion samples,which is the target area.2.Serum pharmacochemistry research:Only two compounds(M1,M2)were detected in plasma.They are present in raw materials and belong to the area?,which means they will be main object for the subsequent research.3.Targeted separation:based on the above research,under the guidance of fingerprints,the compounds of area ? were separated by various chromatographic techniques,such as silica gel column chromatography,ODS,MCI,and semi preparative liquid chromatography.We obtained and identified five compounds(M1,M2,D1,D2,D3).All of them are C-21 steroidal compounds with of a sugar chain with 3?4 deoxy surgars.4.Pharmacodynamic experiment:The result of MES shows that M1,M2,Dl,D2 and D3 were invalid when used along,but effective when combined with phenobarbital sodium.They have synergistic effect for anti-epilepsy in the dose of 10mg/kg and 15mg/kg.Moreover,the effect is better when M1 and M2 are combined at dose of 5mg/kg each than single used at dose of 10mg/kg,suggesting that M1+M2 is the effective group of QYS.The exploration of antiepilepsy mechanism based on 5-HT:The result shows that,the content of 5-HT in mice brain increased after administration.Therefore,the antiepileptic effect of QYS might be related to the content of 5-HT.The above research illustrated that,all of the screening compounds(M1,M2,Dl,D2,D3)are active for the treatment of epilepsy,especially the absorbed constituents(Ml,M2).We find an effective group(M1+M2),establishd the basis for clarifying the QYS's medicinal substances.It also comfirm the screen method is simple,feasible and efficient.