Studies On The Quality Control And Material Basis Of Ixeris Sonchifolia (Bunge) Hance Injection

Traditional Chinese medicine/herbal medicine(TCM/HM)are complex systems,which synergy with multi-component,multi-targets and multi-channel to obtain therapeutic effect with less side effects.The complex chemical constituents of TCM/HM makes them vagueness and integrity,thus adopt only one way can not control the quality of TCM/HM comprehensively.The quality evaluation and control of TCM/HM need from a single model to comprehensive and diversified ones.In this paper,a comprehensive and diversified method,including high performance liquid chromatography(HPLC)multi-wavelength fingerprint,microemulsion electrokinetic chromatography(MEEKC)fingerprint,UV spectrum fingerprint and multiple wavelength fusion fingerprint combined with multi-component quantitative simultaneously,biological activity,chemical basic research and drug metabolism study,and combined with chemometrics methods at the same time,has been established to holistic evaluation the quality of Ixeris sonchifolia(Bunge)Hance injection(ISHI):1.A RP-HPLC method of ISHI samples at five different wavelengths were evaluated,which can maximum reflect the characteristics of the HPLC fingerprint of ISHI,and avoid the one-sidedness from a single wavelength.Subsequently,a systematic quantitative fingerprint method(SQFM)was employed to holistic assess the quality grade of ISHI samples from macro qualitative and quantitative aspect.On the other hand,this method was successfully used to simultaneous quantitative of seven marker compounds,i.e.,chlorogenic acid,caffeic acid,chicoric acid,luteolin-7-β-D-glucuronide,luteolin-7-glucoside,uridine and adenosine.Therefore,the combined use of five-wavelength fingerprints,multiple marker compounds analysis,and quantitative fingerprint evaluation by SQFM,provides a valuable reference for comprehensive overall quality control of ISHI.2.The DPPH radical scavenging assay was recommended to measure the antioxidant activity of ISHI,where off line spectrophotometer method was adopted to determine the total antioxidant activity,and on line HPLC-DPPH method was employed to investigate the antioxidant activity of each chemical composition.A fingerprint-efficacy relationship linking the chemical components and in vitro antioxidant activity was established and validated using the partial least squares(PLS)and orthogonal projection to latent structures(OPLS)models.The online antioxidant activity fingerprints were evaluated by SQFM.The combined use of off line and on line DPPH radical scavenging assay in combination with fingerprint-efficacy relationship and SQFM,provides a reliable and feasible method for comprehensive overall quality control of ISHI.3.Microemulsion electrokinetic chromatography(MEEKC)fingerprints combined with quantification of seven marker compounds analysis were successfully developed to monitor the holistic quality consistency and fingerprint-efficacy relationship of ISHI samples.First of all,the aqueous buffer,pH value,voltage,injection time and range were preliminary investigated,then central composite design-response surface method was performed to optimize the microemulsions conditions of five crucial experimental parameters.Under the optimized condition,MEEKC fingerprints combined with quantification of seven marker compounds analysis were established,and SQFM was employed to holistic assess the quality grade of ISHI samples.In addition,the fingerprint-efficacy relationship between the MEEKC fingerprints and the antioxidant activities was established utilizing OPLS,which showed good prediction ability.The MEEKC fingerprints in combination with multi-marker compounds analysis and chemometrics,provides a rapid and efficient method for comprehensive overall quality control of ISHI.4.In the present study,an ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry(UHPLC/Q-TOF-MS)method was performed to investigate the chemical profiling of the ISHI sample.As a result,totally 38 compounds,including 16 organic acids,10 flavonoids,8 sesquiterpene lactones,2 nucleosides and 2 phenols,were identified or tentatively characterized.Based on the structural analysis and the maximum UV absorption obtained by UHPLC/Q-TOF-MS,the three main UV absorption bands(236 nm,286 nm and 326 nm)of the ISHI samples were preliminarily assigned from organic acids and flavonoids,which have been regarded as the major active substances in ISHI.Accordingly,multi-wavelength(236 nm,286 nm and 326 nm)fusion fingerprints and PCA were performed,and SQFM was realized the overall evaluation of the ISHI samples.In addition,the fingerprint-e fficacy relationship between HPLC fingerprints at 326 nm and antioxidant activities was established utilizing OPLS,providing important medicinal efficacy information for quality control.This study offered a scientific,rapid and effective analytical strategy for TCM/HM quality control.5.In the present study,an UHPLC/Q-TOF-MS was developed to characterize the metabolites of the main active ingredients in rat plasma following intravenous administration of ISHI.Combining information on retention behaviors,multistage mass spectra and literature data,a total of 17 prototypes and 26 metabolites were tentatively characterized,mainly organic acids,flavonoids and sesquiterpene lactones.This study revealed that glucuronidation,methylation,sulfation and methylation+sulfation were the major metabolic pathways of organic acids;methylation,sulfation,methylation+sulfation,sulfation+glucuronidation and methylation+glucuronidation were the major metabolic pathways of flavonoids;hydrogenation,hydrolysis,hydroxylation,cysteine,sulfation and glucuronidation were the major metabolic pathways of sesquiterpene lactones in ISHI in rat plasma.This study laid the foundation for the pharmacokinetic study and bioactive component screening of ISHI.