Study On Active Compounds Of Mai Men Dong-qian Jin Wei Jing Decoction And Chemical Constituents Of Phragmites Stem

This project was supported by the National S.& T.Supporting Program in Chinese Medicine for the 11th Five-Year Plan "Systematical biology research of TCM in regulation of lung cancer signal transduction pathway with high-capacity analysis" (2006BAI11B08-01).Mai Men Dong - Qian Jin Wei Jing decoction is an empirical formula found by a prestigious TCM specialist Zhongying Zhou after a long-term treatment of lung cancer,which is composed of two classical TCM formulae,including Mai Men Dong decoction and Qian Jin Wei Jing Fang.Mai Men Dong decoction was recorded in "Medical Treasures of the Golden Chamber" written by Zhongjing Zhang,while Qian Jin Wei Jing Fang was from Simiao Sun's "Thousand Golden Essential Prescriptions".Modern pharmacological research has shown that both Mai Men Dong decoction and Qian Jin Wei Jing Fang could inhibit tumor growth and enhance immunity.The research subject tried to study the bio-active substance of Mai Men Dong - Qian Jin Wei Jing decoction to provide the basis for the scientific content of the combined treatment of lung cancer,also provide thoughts and approaches for TCM modern research.The first part of this paper summarized the research of TCM formulae in treating lung cancer,and summed up the chemical composition,pharmalogical effects,clinical applications of Mai Men Dong decoction and Qian Jin Wei Jing Fang.The second part is experimental content,the Mai Men Dong -Qian Jin Wei Jing decoction was separated into eight chemical parts(MW-01～MW-08) through water extraction,alcohol precipitation and solvent extraction.The MTT method was used to evaluate the biological effects inhibiting the A549 lung cancer cells growth.As a result,the ethyl acetate and n-butanol fraction showed the best bio-activity,especially the ethyl acetate fraction.Using silica gel column chromatography and recrystallization,11 chemical constituents were isolated from the ethyl acetate fraction,and they were identified as p-hydroxybenzoic acid,coumaric acid,isovanillic acid,liquiritin,isoliquiritin,4'-O-[2-hydroxypropanoyl-(6)-D-glucopyranoside], formononetin,uracil,succinic acid,β-sitosterol and stigmasterol according to the physiochemical properties and spectral analysis.At the same time,succinic anhydride,uracil,ginsenoside Rg3,mannitol were isolated from n-butanol fraction.In vitro anti-lung cancer cells A549 test showed that formononetin,uracil,ginsenoside Rg3,succinic acid,β-sitosterol can inhibit the growth of A549 lung cancer cell obviously.The EtOAc fraction was analyzed by utilized the UPLC-DAD-Q-TOF-MS method.Nine compounds such as p-hydroxybenzoic acid,p-hydroxy benzaldehyde,vanillic acid, glycyrrizin,4'-O-[2-hydroxypropanoyl-(6)-D-glucopyranoside],liquiritin,isoliquiritin and formononetin were identified by comparison with the ultraviolet absorption and MS data of reference substances.Furthermore,an HPLC-DAD method was established for the first time to simultaneously determine 7 constituents,including p-hydroxybenzoic acid,p-hydroxy benzaldehyde,coumaric acid,vanillic acid,glycyrrizin,liquiritin and isoliquiritin in the EtOAc fraction.The HPLC assay was performed on a Waters sunfire C₁₈(250mm×4.6mm, 5um) column with the column temperature at 25℃.The mobile phase was composed of A (acetonitrile) and B(0.5%aqueous acetic acid,v/v).The flow rate was 1.0 mL/min and the detection wavelength was set at 277.4 nm for p-hydroxybenzoic acid and glycyrrizin,and 297.4 nm for other compounds.Fourteen compounds were isolated from the ethanol extraction of Rhizoma Phragmitis by column chromatography,their structures were identified by ¹H-NMR,¹³C-NMR,2D NMR and MS,such as aurantiamide acetate,ferulic acid,coumaric acid,syringic acid, 2,3-dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone,isovanillic acid,p-hydroxybenzoic acid,p-hydroxybenzaldehyde,palmitic acid,heptadecanoic acid,β-sitosterol, stigmasterol,α-D-glucose andβ-D-glucose.The biological activity evaluation of each ingredient in anti-lung cancer cells A549 showed that aurantiamide acetate and ferulic acid have obvious effect in inhibiting the growth of A549 lung cancer cells.Characteristics and innovation of this dissertation:1.Combination of Chemical separation and pharmacological research:Firstly,the biological fractions were certified by the biological activity evaluation of the major separation fractions of the TCM formula and major herbs;secondly,chemical constituents were isolated and analyzed by modern chromatographic technology and biologically evaluated,so as to presume the active substance and to provide substantial basis for the purpose of interpreting the treatment mechanism of TCM formula.2.Use of advanced instrumental analysis:a variety of modern chromatographic techniques, especially UPLC-Q-TOF-MS and fully automated HPLC analysis system provide a reliable technical support for the qualitative and quantitative analysis of complex chemical constituents. 3.Mai Men Dong - Qian Jin Wei Jing decoction is an empirical formula found by a prestigious TCM specialist Zhongying Zhou through a long-term clinical application,the combination research of chemical substantial basis and biological effects laid a strong foundation for the creation of new drug for preventing and treating lung cancer.