Classified And Integrated Pharmacokinetic Study Of Multipe Effective Components Contained In Xinfeng Capsule

Rheumatoid arthritis(RA) is a chronic, inflammatory and systemic autoimmune disease, which is characterized by synovial abnormal hyperplasia inflammation and progressive destruction of cartilage and bone. The pathological manifestation is the abnormal activetion of a variety of inflammatory cells, such as lymphocytes, neutrophils, macrophages and synovial cells. With the development of the disease, it will lead to disability. To date, the exact etiology and pathogenesis of RA are not entirely clear, but many studies have indicated that the pathogenesis of RA maybe related to heredity, infection, environment, neuroendocrine and immune system.Studies have shown that traditional Chinese medicine basic pathogenesis of RA was the deficiency of Qi and blood, damp abundance due to splenic asthenia, phlegm and blood stasis. Xinfeng Capsule(XFC), a Chinese medicine which developed by professor Liu Jian’s team in Traditional Chinese Medicine Hospital of Anhui Province who according to this method has obtained the satisfactory curative effect in clinic. XFC consists of as tragalus, coix, hook, centipede and other Chinese herbal. It has anti-inflammatory analgesic, immune regulation, acetanilide detumescence, reducing inflammation activity index, protecting erythrocyte deformation, improving local conditions of joints in patients with RA, improving blood viscosity and increasing the microcirculation perfusion, etc. Consequently, the metabolism of cells was improved and the functional recovery was promoted. In addition, some clinical studies have confirmed that the XFC has its unique advantages for the treatment of RA and has less side effects, so, its considered to be an ideal traditional Chinese medicine for the treatment of RA.XFC consists of monarch drug(Huang Qi, coix seed), official medicine(Lei Gongteng, centipede) of Chinese medicinal materials; studies have shown that the herbs of the XFC have anti-inflammatory, immunity function, which can be effective in the treatment of RA. The main active ingredient of RA therapy in XFC are Terpenes(TPS)(two terpene and three terpene) and alkaloids(ALS) components, suchasAstragaloside-IV(AGS-IV), Astragaloside-I, Astragaloside-II(AGS-II), Astragaloside-III, Astragaloside-IV(AGS-IV), Cycloastragenol, Triptolide(TPL), Tripdiolide, wilforlide A, Celastrol(CLT), Wilforgine(WFG), Wilforine(WFI), etc.In order to elucidate the mechanism of active ingredients of XFC in vivo, pharmacokinetic study is necessary. Because the efficacy of traditional Chinese medicine is based on the complex interaction between various components, the classified and integrated pharmacokinetics of Components is essential after administration of drug.An ultra-high performance liquid chromatography tandem mass spectrometric method(UHPLC-MS/MS) was established and validated to assay AGS-II, AGS-IV, TPL, CLT, WFG and WFI in plasma of adjuvant arthritis(AA) rats. And these components were classified into TPS and ALS. An approach of self-defined weighting coefficient has been created to the holistic pharmacokinetic profiles of TPS and ALS in XFC. The classified and integrated synthetic concentrations were obtained, and then the pharmacokinetic parameters of TPS and ALS of XFC in AA rats were calculated from non-compartmental model analysis. This method has been successfully applied to the pharmacokinetic study of multiple active components after oral administration of medicine.Objective: A sensitive UHPLC-MS/MS method was established and validated to assay AGS-II, AGS-IV, TPL, CLT, WFG and WFI of XFC and these components were classified into TPS and ALS. Establishing classified and integrated pharmacokinetic model, getting the integrated pharmacokinetics parameters of total TPS and ALS in XFC.Methods: AA model was induced by injecting with Freund’s complete adjuvant(FCA). Rats were randomly divided into five groups: normal group, AA model group and low, middle, high dose groups of XFC(0.3, 0.6, 1.2 g/kg, respectively), intragastric administration for 7 days, once a day. Observing the performance characteristics of AArats and evaluating the model. On day 24 after immunization, obtaining blood from canthus and getting biological samples. The following LC-MS conditions: chromatographic conditions: the column was ZORBAX Eclipse Plus C18 RRHD(Agilent, 2.1×100 mm, 1.8 μm), the column temperature keeping at 30 oC and the injection volume was 2 μL. A(methanol) and B(0.05% formic acid, V/V) were as mobile phase for gradient elution: 0.1-0.5 min, 65 % A~80 % A; 3.0-3.1 min,80% A~90 % A; 7.0-8.0 min, 90 % A~65 % A, and then quickly return to the initial ratio A-B(65:35,V/V), running 4 min. The flow rate was 0.2 m L/min, and the total running time was 12 min. Mass spectrometric conditions using multiple reaction monitoring(MRM) and under Electrospray Ionization negative mode to analyze the quantity of samples. Ion spraying voltage(ISV) was- 4000 V, the source temperature(TEM) was 350 oC, nebulizing gas(GS1, N2) was 40 psi, auxiliary gas(GS2, N2) was 45 psi and the curtain gas(CUR, N2) was 25 psi. After determining 6 components and classifing these into TPS and ALS. An approach of self-defined weighting coefficien has been created to the holistic pharmacokinetic profiles of TPS and ALS in XFC. The classified and integrated synthetic concentrations were obtained, and then the pharmacokinetic parameters of TPS and ALS were calculated from non-compartmental model analysis.Results: This method has high sensitivity and specificity and successfully applied to the simultaneous determination of the six active ingredients in XFC. Peak area and concentration of six components have good linearity in the determination range and all of correlation coefficien(r) were greater than 0.995. Moreover, the limit of detection(LOD), limit of quantification(LOQ), precision and stability were satisfactory. The intra-day and inter-day precision(RSD%) of the assay were 2.39 % and 3.47 %. The average recovery were between 96.5 %-105.1 %. The stability of components were good in 48 h. The parameters determined were satisfactory.Conclusions: We have established a new UHPLC-MS/MS method to assay AGS-II, AGS-IV, TPL, CLT, WFG and WFI in plasma of AA rats. The classified and integratedpharmacokinetic model of total TPS and ALS research was conformed to the characteristics of classical pharmacokinetic model, the parameters which were obtained from method couid maximum express the whole characteristics of Chinese herbal Component. Provided a method for the study on the holistic pharmacokinetic of traditional Chinese Component recipe. The results would help for the further study on the mechanism of XFC and provide an effective reference for the clinical application.