The Analysis Of Chemical Constituents And Anti-glioma Effect Of Jiuwei-Tongqiao Soup In Vitro

Objective:Jiuwei-Tongqiao soup has a good and exact effect on the prevention,treatment and improvement of symptoms of stroke,migraine,tinnitus and deafness resulting from cerebral trauma in clinical.A series of experimental studies have shown that several single medicinal materials and single active ingredients of Jiuwei-Tongqiao soup have aiti-glioma effects.The prescription probably acts on the multiple aspects of the production and development of tumor through the anti-tumor effect multi-target,multi-link and multi-effect and as well as has the characteristics of low toxic side effects,low probability of drug resistance and effect of improve immunity.The prescription of Jiuwei-Tongqiao soup has no clear and reliable indicatos of quality control.The fingerprints of Jiuwei-Tongqiao soup and its single medicinal materials were detected by LC-MS and the structures of major components were confirmed in comparison with the reference substances and the content of the representative active chemical constituent was measured by HPLC.The methods of molecular biology were applied to the preliminary exploration of the anti-tumor pharmacological effects and mechanism of Jiuwei-Tongqiao soup and quercetin.The paper reviewed the study progress of pharmacological effects against glioma of herbal extracts and the single active ingredients.The major contents were achieved as follows.Methods:1)LC-MS was applied to detect qualitatively the representative active chemical constituents of Jiuwei-Tongqiao soup,Astragalus membranaceus and Oldenlandia diffusa.2)The content of quercetin in Jiuwei-Tongqiao soup and Oldenlandia diffusa were measured by HPLC.3)The contents of calycosin,calycosin-7-glucoside,formononetin and ononin in Jiuwei-Tongqiao soup,its extractive fractions and Astragalus membranaceus were detected by HPLC.4)MTT colorimetry assay was performed to evaluate the proliferation inhibition activity of the ethanol extract from Jiuwei-Tongqiao soup and quercetin on human glioma cells U251 in vitro and flow cytometry was applied to detect the apoptosis rate of U251 cells.Results:1)There are totally 28 peaks in the fingerprints of the ethanol extract from Jiuwei-Tongqiao soup.The 15th peak represents quercetin whose tR is 31.55min,maximum UV absorption wavelength is 209,256 and 263nm,molecular ion peaks are[M+H]+on 303,[M-H]-on 301 and[2M-H]-on 603 and is confirmed as a representative active chemical constituent of Oldenlandia diffusa.The 14th peak represents calycosin whose tR is 31.33min,maximum UV absorption wavelength is 219,249 and 290nm,molecular ion peaks are[M+H]+on 285,[2M+H]+on 569,[M-H]" on 283 and[2M-H]" on 567.The 5th peak represents calycosin-7-glucoside whose tR is 20.50min,maximum UV absorption wavelength is 220 and 258nm,molecular ion peaks is[M+H]+ on 447.The 18th peak represents formononetin whose tR is 40.90min,maximum UV absorption wavelength is 249nm,molecular ion peaks is[M+H]+on 269.The 12th peak represents ononin whose tR is 28.02min,maximum UV absorption wavelength is 250nm,molecular ion peaks is[M+H]+ on 431.Calycosin,calycosin-7-glucoside,formononetin and ononin are confirmed as representative active chemical constituents of Astragalus membranaceus.2)The content of quercetin in the ethanol extract from 3 lots of Jiuwei-Tongqiao soup and Oldenlandia diffusa are 33.11μpg/g of Lot.20131202,38.29μg/g of Lot.20131203 and 24.25μg/g of Lot.20131204.The place of production of Oldenlandia diffusa of Lot.20131202 is Jiangsu province.The content of quercetin in the ethanol extract from Oldenlandia diffusa is 115.96μg/g.3)The analysis results show that the contents of representative constituents in ethyl acetate fraction is close to that in ethanol extract from Astragalus membranaceus though the ingredient formononetin is absent in the fraction.The contents of representative constituents in chloroform fraction is close to that in ethanol extract from Jiuwei-Tongqiao soup and the 4 kinds of representative constituents can be measured in the fraction.4)The results show that the ethanol extract from Jiuwei-Tongqiao soup and quercetin have the proliferation inhibition activity of on human glioma cells U251 with the increase of time and concentration in vitro via inducing apoptosis.Conclusion:The representative active chemical constituents of Jiuwei-Tongqiao soup were detected and analyzed qualitatively and quantificationally and its pharmacological effects and mechanism against glioma were explored in this paper.The results show that Jiuwei-Tongqiao soup and its active consitituent quercetin have the anti-glioma effect and induce the tumor cell apoptosis.The mechanism of induction of apoptosis need further study.