Research On The Material Basis Of The Anti-tumor Activity Of Yu-Ping-Feng-San Based On Cell Biological Solid-phase Methods

Objective:With the establishment of cell biological chromatography of liver cancer cells(Hepal-6)and immune cells(RAW264.7 and splenocyte from C57 mouse),active ingredients of anti-hepatoma activity of Yu-Ping-Feng-San(YPFS)that exert direct killing of hepatoma cells and/or immunoenhancement effects were identified;application of cell proliferation in vitro experiment to verify the pharmacological activity of the active ingredients in order to study the material basis of anti-hepatoma activity of YPFS at the cellular level,which establishes the foundation for revealing the mechanism of anti-hepatoma activity of YPFS and extending to the application of its anti-tumor activity.Methods:The separation was carried out in Hypersil ODS column by gradient elution with acetonitrile-water mixtures used as mobile phases at a flow rate of 1.0 mL·min-1 in the wavelength of 220 nm and a column temperature of 300C.The injection volume was 5 μL.The high-performance liquid chromatography fingerprint and quality control standards of YPFS decoction were established,which is the foundation to analyze the combined ingredients between YPFS decoction and Hepal-6,RAW264.7 and splenocyte.The solid phase chromatography of Hepal-6,RAW264.7 and splenocyte was established.Sterilization of YPFS decoction was carried out by filter method,and medicine was incubated with Hepal-6,RAW264.7 and splenocyte for 1 h to make three kinds of cells inactive by eluting four times,and to make the active ingredients dissociate from cell membrane,finally to make cells lysis,and analyzed the composition of isolation solution and lysates by HPLC or HPLC/MS to verify the quasi-active ingredients.MTT method were used to study the effects of the selected quasi-active ingredients on the proliferation of three kinds of cells.Results:The high-performance liquid chromatography fingerprint and quality control standards of YPFS decoction were established,and the reference fingerprint and fifteen common peaks from the HPLC fingerprint of YPFS decoction were analyzed.The peak of 5,6,7,8,10,11,12,13,15 was prim-O-glucosylcimifugin,calycos:in-7-O-β-D-glycoside,cimifugin,4-0-β-D-glucosyl-5-O-methylvisamminol,psoralen,calycosin,sec-O-glucosylhamaudol,formononetin and atractylon,respectively.The nine components had a good linearity(r-0.9997).The average recoveries and the relative standard deviation(RSD)was 97.91%～99.81%and 0.58%～1.27%,respectively.The average content of prim-O-glucosylcimifugin,calycosin-7-O-β-D-glycoside,cimifugin,4-0-β-D-glucosyl-5-O-methylvisamminol,psoralen,calycosin,sec-O-glucosylhamaudol,formononetin,atractylon was 356.03 μg/mL,378.05 μg/mL,216.32 μg/mL,458.96 μg/mL,2.06 μg/mL,116.34 μg/mL,165.18 μg/mL,91.14 μg/mL and 51.40 μg/mL,respectively,which can be the quality control standards of YPFS decoction.With the application of Hepal-6 solid phase chromatography,we screened and analyzed eleven main quasi-active ingredients in cortex moutan water extraction.Among them,the components combined with the cell membrane were psoralen,formononetin,ononin and sec-O-glucosylhamaudol,and the components entered into the cell were 3-(2-hydroxy-3,4-dimethoxyphenyl)-3,4-dihydro-2H-chromen-7-ol,phenylalanine,atractylolide,formononetin,ononin,astragaloside Ⅳ;astragaloside Ⅱ,calycosin and atractylon.With the application of RAW264.7 solid phase chromatography,we screened and analyzed eleven main quasi-active ingredients in cortex moutan water extraction.Among them,the composition combined with the cell membrane were prim-O-glucosylcimifugin,atractylon,quercetin,atractylodes lactone Ⅲ and psoralen,and the components entered into the cell were adenosine,calycosin-7-O-β-D-glycoside,calycosin,4’-O-Glucosyl-5-O-methylvisamminol,astragaloside IV,astragaloside Ⅱ,atractylon and prim-O-glucosylcimifugin.With the application of splenocyte solid phase chromatography,we screened and analyzed ten main quasi-active ingredients in cortex moutan water extraction.Among them,the components combined with the cell membrane were prim-O-glucosylcimifugin,adenosine,calycosin-7-O-β-D-glycoside and calycosin,and the components entered into the cell were quercetin,adenosine,calycosin-7-O-β-D-glycoside,calycosin,isoastragaloside I*,cimifugin,4-0-β-D-glucosyl-5-O-methylvisamminol,formononetin and ononin.The MTT results showed that psoralen,formononetin,ononin and sec-O-glucosylhamaudol directly inhibit the proliferation of Hepal-6,and mixture constituted of these 4 components obviously inhibit the proliferation of Hepal-6,and calycosin,calycosin-7-O-β-D-glycoside,astragaloside IV,adenosine,prim-O-glucosylcimifugin and psoralen promote the proliferation of RAW264.7,and formononetin and adenosine promote the proliferation of splenocyte.Conclusion:1)The HPLC fingerprint of YPFS decoction was established,which is proved to be simple and reproducible.The quality control standards of 9 components was proved to be stable and reliable,which could be used to control YPFS’s quality.2)11,11,and 10 tested active constituents were screened respectively by establishing solid-phase medthods of Hepal-6,RAW264.7 and splenocyte.Among the tested active constituents,psoralen,formononetin,ononin and sec-O-glucosylhamaudol can inhibit the proliferation of Hepal-6 directly,and calycosin,calycosin-7-O-β-D-glycosid,astragaloside Ⅳ,adenosine,prim-O-glucosylcimifugin and psoralen can promote the proliferation of RAW264.7,and formononetin and adenosine can promote the proliferation of splenocyte.