The Pharmacokinetic Study And Proteomics Of The Active Ingredient Group Of Xiaoxuan Decoction

Xiao-Xu-Ming Decoction(XXMD),a traditional Chinese medicine,is composed of Saposhnikovia Radix,Aconiti Lateralis Radix Praeparata,ginger,almond,ephedra,ginseng,Cinnamomi Ramulus,Paeonia lactiflora Pall,Ligusticum chuanxiong,Stephania Tetrandrae Radix,Scutellaria baicalensis,Glycyrrhizae Radix et Rhizoma.XXMD is mainly used in the treatment of the apoplexy and sequela of apoplexy.XXMD has a good clinical efficacy and long-term application history and is one of the most representative prescriptions in Chinese herbal medicines.Recently studies have shown that in addition to the apoplexy and sequela of apoplexy,XXMD has an obvious therapeutic effect on Alzheimer's disease,diabetic peripheral neuropathy,hypertension and other diseases.The Chinese medicine compound(Fang Ji)is the most basic form of Chinese medicine treatment and its composition can be divided into active ingredients,toxic constituents and independent component that can be proved by the high throughput screening technology and the evaluation method of effective ingredients of the Chinese medicine compound.The effective component of the Chinese medicine compound is the basis for the therapeutic effects of Chinese medicine compound,that is neither simple effective parts nor disordered groups of active site,but a organic combination of active ingredients that can reflect the TCM Prescription Theory,known as the effective components group.In this study,the material base,plasma multicomponent methodology establishment,pharmacokinetic studies and proteomics of effective components groups of XXMD were studied to provide an effective reference for the study of material basis and biological basis of XXMD.In order to clarify the chemical composition and the source of active component Xiao-Xu-Ming decoction(AF-XXMD)quickly and comprehensively,whole and individual herbs of AF-Xiao-Xu-Ming decoction were analyzed by Ultra-Performance Liquid Chromatography with Quadrupole-Time-of-Flight Mass Spectrometry(UPLC/Q-TOF-MS).Based on comparison with standard samples,database matching analysis and reviewing relevant literature,42 compounds were identified from AF-Xiao-Xu-Ming decoction.A LC-MS/MS method for the quantitation of flavonoids,alkaloids and triterpenoid saponins of AF-XXMD have been developed and applied to the pharmacokinetic study after oral administration of AF-XXMD.The method provide a simple,reliable,sensitive and specific assay for the preclinical pharmacokinetic study of XXMD in plasma.The interaction between the two is bound to affect the absorption,metabolism and other processes.In order to explore the effect and biological basis of active components of Xiao-Xu-Ming decoction on cerebral ischemia rat,proteomics method baesd on UPLC-Q-Exactive was applied to explore the biological basis.Compared with model group,702 significant difference proteins were found in XXMD group,and 789 significant difference proteins were found in the blank group.Through the biological function analysis,the found proteins could be classified into proteins involved in cellular process,biological regulation,metabolic process,response to stimulus,cellular component organization or biogenesis,immune system process.The above target proteins and their regulation pathways may be the biological basis which XXMD played a role of cerebral ischemia.1.Xiao-Xu-Ming decoction(XXMD)has been a traditional Chinese prescription of treating stroke since Tang and Song Dynasty.However,it was silence for hundred of years due to some reasons.Now in recent years,it has been gradually regain.The chemical composition of XXMD is complex,and the studies on its effective substance basis and pharmacological mechanism of anti-stroke are still in the preliminary stage of development.It is highly necessary to study and explore how to monitor the complex changes,confirm the action target and the biological effect of each component on the basis of screening and analysis of the effective components in XXMD.This article review and summarize the research progress of the material basis and pharmacological research on XXMD in recent years,and provide the references for the modernization prescription studies of XXMD.Rats were intragastric administration of Xiaoxuming Decoction group of effective components in plasma by HPLC-MS/MS,wogonin and oroxylin A glycosides,baicalin,glycyrrhizin,oroxylin A,baicalin,baicalein,isoliquiritin,ephedrine,cimifugin,concentration of 5-O methyl glycosides by using WinNonLin software,and the pharmacokinetic parameters were calculated.2.In order to clarify the chemical composition and the source of active component Xiao-Xu-Ming decoction(AF-XXMD)quickly and comprehensively,whole and individual herbs of AF-Xiao-Xu-Ming decoction were analyzed by Ultra-Performance Liquid Chromatography with Quadrupole-Time-of-Flight Mass Spectrometry(UPLC/Q-TOF-MS).Under identical experiment conditions,chromatography results were compared between experiment groups.A Waters ACQUITY UPLC HSS T3(2.1 mm×100 mm,1.8?m)was used with 0.1%formic acid solution(A)-acetonitrile(B)as the mobile phase for gradient elution.The flow rate was set to 0.35mL·min-1 and the column temperature was maintained at 40 ?.Electrospray ion(ESI)source was applied for the qualitative analysis under the positive and negative ion mode.Finally,based on comparison with standard samples,database matching analysis and reviewing relevant literature,42 compounds were identified from AF-Xiao-Xu-Ming decoction.The results implied that flavonoids,alkaloids and triterpenoid saponins were the main components in the effective part of AF-Xiao-Xu-Ming decoction.The herbal sources of these peaks were assigned.This method established is simple and rapid for elucidation the constituents of AF-Xiao-Xu-Ming decoction and the results could be used for the quality control and the further material basis research for AF-Xiao-Xu-Ming decoction.3.A LC-MS/MS method for the quantitation of flavonoids,alkaloids and triterpenoid saponins of AF-XXMD have been developed and applied to the pharmacokinetic study after oral administration of AF-XXMD.The method provide a simple,reliable,sensitive and specific assay for the preclinical pharmacokinetic study of XXMD in plasma.An Agilent Poroshell 120 SB-C18(2.1×100mm,2.7?m)reversed-phase column was used for chromatography,the mobile phase consisted of acetonitrile and 0.1%formic acid in water,gradient elution,flow rate was 0.35 mL·min-1,injection volume was 10?L.The analyte was detected using electrospray ionization(ESI)in multiple reaction monitor-ing(MRM)modes.This part established the LC-MSn detection method of 37 compounds of AF-XXMD in plasma.The method is simple,sensitive and stable,and can be applied to the detection of AF-XXMD in plasma.4.A fully validated LC-MS/MS method was developed and has been successful applied to the pharmacokinetic study of XXMD when given in rat after oral administration.The content of Wogonin,Oroxylin A,Wogonoside,OroxylinA7-O-glucuronide,Liquritigen,Isoliquiritoside,Baicalin,Baicalein,Methylephedrine Hydrochloride,Cimifugin,5-O-methylvisammioside were 0.31%,0.69%,0.93%,0.04%,0.17%,4.28%,0.98%,0.15%,0.45%,0.12%,0.31%respectively.Rats were intragastric administration of Xiaoxuming Decoction group of effective components in plasma by HPLC-MS/MS,wogonin and oroxylin A glycosides,baicalin,glycyrrhizin,oroxylin A,baicalin,baicalein,isoliquiritin,ephedrine,cimifugin,concentration of 5-O methyl glycosides by using WinNonLin software,and the pharmacokinetic parameters were calculated.From the pharmacokinetic curve,wogonoside rats,oroxylin A glycosides,oroxylin A,baicalin and isoliquiritin existed in double peak significantly.These compounds will undoubtedly increase and maintain the plasma concentration of each component in the body is high,and thus contribute to the effective components of Xiaoxuming Decoction group.Therapeutic effect of Chinese compound into complex,various components.The interaction between the two is bound to affect the absorption,metabolism and other processes5.In order to explore the effect and biological basis of active components of Xiao-Xu-Ming decoction on cerebral ischemia rat,proteomics method baesd on UPLC-Q-Exactive was applied to explore the biological basis.The cerebral ischemia rat model was established by nylon wire with 2 hours ischemic time on healthy male SD rats.Proteins of pallium from the blank group,the model group and the XXMD group were detected by UPLC-Q-Exactive systerm.The data were imported into Proteome Discoverer and Mascot software to identify and analyzed all the proteins.Compared with model group,702 significant difference proteins were found in XXMD group,and 789 significant difference proteins were found in the blank group.Through the biological function analysis,the found proteins could be classified into proteins involved in cellular process,biological regulation,metabolic process,response to stimulus,cellular component organization or biogenesis,immune system process.The above target proteins and their regulation pathways may be the biological basis which XXMD played a role of cerebral ischemia.