The Research On Determination Of Effective Components And Relationship Of Compositions In Chaihushugan Powder

Chaihushugan powder is a famous prescription by Zhang Jiebin,which is composed of radix bupleuri chinensis, rhizoma chuanxiong,rhizoma cyperi, radix paeoniae alba, dried tangerine peel, auranti fructusand glycyrrhiza uralensis fisch. It can relieve qi stagnancy in liver and hasbeen widely used in clinic. The effective components were saponins,flavonoids, terpenoid, phenolic acid and so on. The aim of this study was toestablish a method for determination of saikosaponin a, paeoniflorin,hesperidin and ferulic acid in chaihushugan powder by HPLC, and theextraction process was optimized by an orthogonal test. The results of thispaper would provide a basis for further study on chaihushugan powder andoffer methods for researches on compatibility.1 Determination of effective components in chaihushugan powder byHPLC/DADThe orthogonal test design L9 (34) was used to arrange sequentexperiments to test the effect of the four factors, ethanol concentration, theamount of ethanol, times of reflux and extraction time on the dry extractrate and contents of saikosaponin a, ferulic acid, hesperidin andpaeoniflorin and to establish the optimal extraction conditions.Simultaneously the total of saponins, toatal of flavonoids and total ofphenolic content were determinated by Ultraviolet-Spectrophotometrymethods. Saponins compounds, flavonoids and organic acid inchaihushugan powder were determinated by High performance liquidchromatography . The results were as follows:1.1 According to the orthogonal test, the optimumextracting conditionwas 80% ethanol solution, 1:10 solid-liquid ratio, 2 times and 90 min for extration.1.2 Four effective Components in chaihushugan powder were determinedby HPLC. A Hypersil C18 analytical column (4.6mm×250mm, 10μm)was employed for the saparation. The mobile phase was acetonitril-water (43:57) for saikosaponin a, and acetonitrile-0.5% acetic acid (40: 60) for othercomponents. The flow rate was 0.8 ml·min^-1 and column temperature wasmaintained at 30℃. The detection wavelength was 200-400 nm.The results showed that the four components were separated wellwithin 15min. The linear ranges of Saikosaponin a, paeoniflorin, hesperidinand ferulic acid were 22.7¹67μg·ml^-1(r=0.9999), 15.9²54μg·ml^-1(r=0.9997), 22.1³53μg·ml^-1(r=0.9993), 6.30²02μg·ml^-1(r=0.9995) respectively. The average recoveries were 97.6%,97.4%,98.9% and 96.4% respectively. The RSD were 2.07%, 1.15%,1.33% and 0.71% respectively. The method was simple, convenient, andaccurate, and was appropriate to control the quality of ChaihushuganPowder.1.3 Total saponins were enriched by n-Butanol extracting. The content oftotal saponins were determinated by spectrophotometry, and the detectionwavelength was 327nm. The NaNO2-Al(NO3)3-NaOH spectrophotometrywas used to determine flavonoids. The content of the total phenolic acidwas detected by visible spectrophotometry. The reference substance wasgallic acid, the colour-developing agent was the mixture solution of 0.6%FeCl3-0.9% K3[Fe(CN)6](1:0.9), and the wavelength was 725nm.The results showed that the linear ranges of saikosaponin a was1.71⁸.00μg·ml^-1 and the calibration curve was Y=0.1503X+0.0393(r=0.999 5). The average recovery was 97.0%,and the RSD was 1.91%.The average content of total saponins in chaihushugan powder was 0.81%,and the RSD was 1.45%. The calibration curve of total flavonoids wasY=11.89X-0.0395, r=0.9996, and the linear ranges was 15.2⁴5.6μg·ml^-1.The average recovery was 96.6%,the RSD was 1.56%. The content of flavonoids in chaihushugan powder was 0.58%, (RSD=1.27%). Thecalibration curve of total phenolic acid was Y=0.1146X-0.0374, r=0.9997,and the linear range was 1.59⁴.79μg·ml^-1. The average recovery was96.8%, and the RSD was 1.53%. The content of total phenolic acid inchaihushugan powder was 0.68% (RSD=1.47%).2 Study on chemical composition and content variation of effectivecomponents between con-decoction and mon-decoction onchaihushugan powderRP-HPLC methods were developed for the determination ofsaikosaponin a, ferulic acid, hesperidin and paeoniflorin in chaihushuganpowder. The contents of saikosaponin a, paeoniflorin, and ferulic acid inwhole decoctions 17.84%, 10.82% and 10.97% respectively were higherthan dividual decoctions. The contents of hesperidin decreased afterwhole-decoction by 15.85%. The HPLC chromatogram of whole decoctionand dividual decoctions were basically similar.3 Influence of the different compatibility on content of saikosaponina in chaihushugan powderOrthogonal experimental design was used to decompose recipes ofchaihushugan powder. The reversed phased-highperformance liquidchromatography (RP-HPLC) was used to determine the decocting quantityof saikosaponin a from the different combinations of chaihushugan powder,and an analysis of the experiment results was made. The amount ofextraction was divided into 4 groups. A single-factor analysis of variancewas used to find out the influence of different combinations ofchaihushugan powder on decocting quantity of saikosaponin a.The results showed that the decocting quantity of saikosaponin a fromthe different formula combinations of chaihushugan powder has asignificant difference. The decocting quantity of saikosaponin a was higherfrom the whole formula of chaihushugan powder than from other formulacombinations. When compatibility of glycyrrhiza uralensis fisch and auranti fructus, the content of saikosaponin a were higher; but lower at thecombination of rhizome cyperi and rhizoma chuanxiong .In summary, a larger guantity of effective components inchaihushugan powder could be obtained by optimum extraction process.Determination of a variety of active ingredients of relativelycomprehensive analysis method has been established in chaihushuganpowder.The contents of the effective components have changed. Thecontent of saikosaponin a varies significantly due to the differentcompatibility.