Study On HPLC Fingerprint Of Buyanghuanwu Decoction And Serum Pharmacochemistry

Buyanghuanwu decoction(BYHWD) was documented in ‘Yi Lin Gai Cuo’,which is included in Radix Astragali, Radix Angelicae Sinensis, Radix Peaoniea Rubra, Flos Carthami, Rhizoma Ligustici Chuanxiong, Lumbricus and Semen Persicae. BYHWD can reinforcing Qi, activate blood and dredge collaterals,which is a formula applicated frequently highest to treat stroke and other thrombotic diseases.It is widely used to therapy or prevent ischemic cerebrovascular disease, coronary heart disease, myocardial infarction and other thrombotic diseases. Most of the research of BYHWD were focused on its mechanism of anti cerebral ischemia, but its pharmacodynamic material basis is still unclear. In order to indicate its pharmacodynamic material basis, BYHWD concentrated solution was selected as the object of the study, used fingerprintology of traditional Chinese medicine(TCM) and serum pharmacochemistry to establish the HPLC fingerprints of BYHWD in vivo and vitro.Objective:1.To establish an HPLC fingerprint of BYHWD in vitro.2.The method of serum pharmacochemistry of traditional Chinese medicine(TCM) is used to establish the plasma dynamic chromatogram and HPLC fingerprints of BYHWD.3.To find the prototype components and metabolites in rat plasma through comparing the fingerprints of BYHWD, rat plasma-containing active components and blank plasma in order to provides a methodology basis to research the pharmacodynamic material foundation next.Methods:1.High-performance liquid chromatography with photodiode arraydetection(HPLC-DAD) and gradient elution were used to establish the HPLC fingerprints of BYHWD in vitro. Chromatographic column was C18(4.6mm ×250mm, 5 μ m), the mobile phase was acetonitrile-0.05% phosphoric acid in a gradient elution mode and the detection wavelength was 230 nm. The flow rate was0.8m L · min-1, and the column temperature was 30 ℃. Identified the components through comparing different chromatogram, besides, we had found out 5 major active ingredients by means of standard substance, and determined the content.2.SD rats were administered BYHWD and drew blood by coeliac artery after 0、15、30、45、60、90、120、150、180、240minutes, then separated and pretreatmented the plasma. Established the plasma dynamic chromatogram in the same chromatographic conditions, and pointed out the most suitable timing so as to establish the plasma HPLC fingerprints of BYHWD. The prototype components and metabolites in rat plasma were identified through comparing the fingerprints of BYHWD, rat plasma-containing active components and blank plasma.Results:1.We have established the HPLC fingerprints of BYHWD, 27 common peaks were identified from a software “Similarity Evaluation System for Chromatographic Fingerprint of TCM”, 14 came from Radix Astragali, one peak from Radix Angelicae Sinensis and Rhizoma Ligustici Chuanxiong, 2 were from Radix Peaoniea Rubra, 2were from Flos Carthami, one was from Lumbricus, one came from Radix Astragali and Radix Peaoniea Rubra,4 peaks came from all the herbs.2.The content determination of five major components was based on the chromatographic condition of BYHWD. The linear range of hydroxysafflor yellow A,paeoniflorin, ferulic acid, calycosin-7-glucoside and formononetin was 1.528~7.640μg(r1=0.9996)、0.8000~4.000μg(r2=0.9998)、0.05050~0.2525μg(r3=0.9993)、2.830~14.15μg(r4=0.9999)、1.720~8.600μg(r5=0.9999), respectively. The average recovery was 99.63%、98.87%、95.60%、96.22%、95.56% respectively.3.After the administration of BYHWD, 30 main components into blood were marked in the plasma fingerprint from 0~240 min and the content of them were different in different plasma samples.4.After the administration of BYHWD, 30 main components into blood were marked in the plasma fingerprint, 10 came from the original compound of rat plasma,one peak was the solvent peak. Other 19 were the drug-induced compositions, 4 of which were prototype constituents, other 15 were metabolites,such as 2 metabolites from Radix Astragali, 1 metabolite from Semen Persicae.Conclusions:1.The method is simple and accurate with a good reproducibility, and provides a basis for quality evaluation of BYHWD. What is more, this method also provides a methodology basis and reference to research the pharmacodynamic material foundation next, and the similarity of 10 batches of BYHWD was fine by the Similarity Evaluation System.2.This method is simple, reproducible, and can be used for determination of five components of hydroxysafflor yellow A, paeoniflorin, ferulic acid,calycosin-7-glucoside and formononetin in BYHWD, as well as a more reasonable and reliable quality control method.3.The constituents absorbed into rat blood had changed in quality and quantity over time after oral administration of BYHWD. It was the first time to establish the plasma dynamic chromatogram of BYHWD, and to further track its components into blood in different time after oral administration. The methodological evaluation showed that this method had a good feasibility.4.It was the first time to established the plasma HPLC fingerprints of BYHWD,and systematically analysed its components into blood, which showed that the pharmacodynamic material basis of BYHWD were the 19 drug-induced compositions,and also 11 common peaks were identified into blood. Furthermore, the similarity of 5batches of plasma HPLC chromatograms after BYHWD treatment was greater than0.8, which stated that this method has a good feasibility.