Study On Pharmacodynamic Substances Of Shunaoxin Dropping Pills

Shunaoxin Dropping Pills,formerly known as Xionggui Dropping Pills,is a traditional Chinese medicine preparation for treating cerebrovascular diseases.It is made of Ligusticum Chuanxiong and Angelica sinensis by supercritical extraction technology and solid dispersion technology.The active ingredients were extracted and separated,and the dropping pills were prepared with polyethylene glycol.The pharmacodynamic substance basis of Shunaoxin Dropping Pills and its dynamic changes invivo are not completely clear.In this study,the content of active ingredients in Shunaoxin Dropping Pills was determined by HPLC,the fingerprint was established,and the attribution of common peaks was confirmed by comparing with the chromatogram of single herbs in compatible prescriptions.The pharmacokinetics of the main components was studied.And preliminary qualitative analysis of main componentsand blood-feeding components in Shunaoxin Dropping Pills by LC-MS.This study provides a reference for the quality control and pharmacodynamic material basis of Shunaoxin Dropping Pills.Objective:1.To establish an HPLC method for simultaneous determination of ferulic acid,senkyunolide I,senkyunolide A and ligustilide in Shunaoxin dripping pills from different manufacturers.2.The fingerprint of Shunaoxin Dropping Pills was studied to provide reference for the quality control and pharmacodynamic material basis of Shunaoxin Dropping Pills.3.A UPLC-MS/MS method was established to analyze the main chemical constituents of Shunaoxin Dropping Pills in vitro and in vivo.4.To establish an HPLC method for the determination of ferulic acid in rat plasma and apply it to the pharmacokinetic study of ferulic acid in Shunaoxin Dropping Pills.5.To establish an HPLC-PDA method for the determination of senkyunolide I and ligustilide in Shunaoxin Dropping Pills,and to investigate the pharmacokinetics of senkyunolide I and ligustilide in Shunaoxin Dropping Pills in rats after intragastric administration of Shunaoxin Dropping Pills suspension.Methods:1.HPLC:Venusil MP C18 column（250*4.6 mm,5μm American Dima Company）was used for chromatographic column.The column temperature was 30℃,the flow rate was 1 m L/min,the detection wavelength was 280 nm,the injection volume was 10μL,and the mobile phase was methanol（A）,1%acetic acid solution（B）for gradient elution.2.Shunaoxin dropping pills were extracted with 70%methanol.The fingerprint of Shunaoxin dropping pills was established by HPLC.The fingerprint of Shunaoxin dropping pills was analyzed by the software of"Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine 2012".The common peaks in the fingerprint of Shunaoxin Dropping Pills were analyzed by comparing with that of the extracts of two Chinese medicinal materials（Ligusticum Chuanxiong and Angelica sinensis）.3.Using UPLC-MS/MS analysis method,Agilent Eclipse Plus C18（2.1*50 mm,1.8μm）column was used with column temperature of 30℃,0.5 m L·min^-1,injection volume of 5μL,detection wavelength of 275 nm,using（A）Waters（containing 0.1%formic acid）and（B）acetonitrile（containing 0.1%formic acid）as mobile phase for gradient elution.ESI ion source and positive ion mode were used to collect data for mass spectrometry.Compounds were identified by comparison with standard products,mass spectrometry data analysis and literature review.4.The acidified plasma samples were treated by liquid-liquid extraction.Sym-metry C18 column（250 x 4.6 mm,5μm Waters Company）was used for chromatographic column.The flow rate was 1 m L/min,the injection volume was 10μL,and the column temperature was 30℃.The detection wavelength was 323 nm and the mobile phase was methanol（A）-1%acetic acid solution（B）=（35:65）.The plasmaconcentration of ferulic acid was determined.The data were processed by DAS2.0 software and the pharmacokinetic parameters of ferulic acid were calculated.5.After SD rats were given Shunaoxin Dropping Pills suspension,plasma samp les were collected at different time points.The plasma concentrations of senkyunoli de I and ligustilide in plasma samples at different time points were determined byh igh performance liquid chromatography.The main pharmacokinetic parameters werec alculated by DAS 2.0 pharmacokinetic software,and the pharmacokinetic-time curve s of senkyunolide I and ligustilide were established.Results:1.Ferulic acid,senkyunolide I,senkyunolide A and ligustilide showed good linear relationship in the range of 1.0-30μg·m L^-1,1.1-33μg·m L^-1,1.5-45μg·m L^-1 and2.0-60μg·m L^-1,respectively.The contents of ferulic acid,senkyunolide I,senkyunolide A and ligustilide in Shunaoxin dripping pills from manufacturers 1 were 0.15 mg/pill,0.31 mg/pi-ll,0.27 mg/pill and 0.90 mg/pill respectively.The contents of ferulic acid,senkyunolide I,senkyunolide A and ligustilide in Shunaoxin dripping pillsfrom manufacturer 2 were 0.14mg/pill,0.27 mg/pill,0.49 mg/pill and 0.57mg/pill respectively.2.The fingerprints of 10 batches of Shunaoxin Dropping Pills were analyzed,the fingerprint of Shunaoxin Dropping Pills was simulated by median method.“Thesimilarity evaluation system of chromatographic fingerprints of traditional Chinese medicine（2012edition）”was used for analysis,and eight characteristic peaks were calibrated.Four chara-cteristic peaks were identified by standard products（1-ferulic acid,2-senkyunolide I,4-senkyunolide A,5-ligustilide）,and the similarities of fingerprintsof 10 batches of Shuna-oxin dropping pills were all above 0.9.By comparing the fingerprints of Ligusticum Chua-nxiong and Angelica sinensis,the attribution of eight characteristic peaks was determined.It was found that eight characteristic peaks were found in both medicines,and other comp-onents with less content could also be found in the two medicines.3.There are nine main chemical constituents in Shunaoxin dropping pills.It’s inferr-ed that the nine compounds are ferulic acid,senkyunolide I,senkyunolide H,senkyunolide A,butylphthalide,ligustilide,butylidenphthalide and two phenylethalided-imer compoun-ds;Four blood transitional components,ferulic acid,senkyunolide I,senkyunolide A and ligustilide,were identified in the plasma of rats with Shunaoxindropping pills,which cons-titute the active components of Shunaoxin dropping pills.It is the material basis of Shuna-oxin dropping pills.4.Ferulic acid had a good linear relationship in the linear range of 0.1-10.0μg/m L（r=0.9989）,the minimum quantitative limit was 0.04μg/m L,the precision was good,RSD intra-day precision was less than 5.0%,RSD inter-day was less than 8.5%,and the recovery rate was 95.67%-98.73%.The drug-time curve of ferulic acid in Shunaoxin dropping pills in rats conforms to the two-compartment model:T_1/2αwas（0.178±0.08）h,T_1/2βwas（6.235±2.35）h,T_max was（0.33±0.02）h and C_max was（1.24±0.38）mg/L.5.The linear relationships of senkyunolide I and ligustilide were good in the range of0.11-11.00μg/m L and 0.15-10.00μg/m L,respectively.The recovery rate ofsenkyunolide I and ligustilide were over 79%,and the intra-day and inter-day precision RSD values were less than 8.0%.The main pharmacokinetic parameters of senkyunolide I after oral administration in SD rats were t_1/2α（0.307±0.07）h,t_1/2β（5.739±2.49）h,T_max（0.5±0.05）h,C_max（1.64±0.39）mg/L;The main pharmacokinetic parameters of ligustilide after oral administration in rats were t_1/2α（0.134±0.01）h,t_1/2β（18.816±1.26）h,T_max（0.33±0.03）h,C_max（0.401±0.09）mg/L.Conclusion:1.An HPLC method for the determination of multi-components in Shunaoxin dropping pills was established.Through methodological verification,it meets the require-ments.It can be used for the quantitative determination of Shunaoxin dropping pills.The contents of ferulic acid,senkyunolide I,senkyunolide A and ligustilide in Shunaoxin dripping pills from different manufacturers were different.2.The HPLC fingerprint of Shunaoxin Dropping Pills was established.Eight characteristic peaks were calibrated and four of them were identified.By comparing the fingerprints of Ligusticum Chuanxiong and Angelica sinensis,the attribution of eight characteristic peaks was determined.It was found that eight characteristic peaks were contained in both medicines,and other components with less content couldalso be attributable to the two medicines respectively,which provided experimental data for the in-depth study of Shunaoxin Dropping Pills.3.There are 9 main chemical constituents in Shunaoxin Dropping Pills and a preliminary qualitative analysis is also made.Four blood-entry transitional constituents,ferulic acid,senkyunolide I,senkyunolide A and ligustilide,are identified in the plasma of Shunaoxin Dropping Pills.These blood-entry constituents constitute the active constituents of Shunaoxin Dropping Pills,which are pharmacodynamic substance basis of Shunaoxin Dropping Pills.4.A method for the determination of ferulic acid in Shunaoxin Dropping Pillsby high performance liquid chromatography was established,which is suitable for the pharmaco-kinetic study of ferulic acid in Shunaoxin Dropping Pills.The drug-time curve of ferulic acid in Shunaoxin Dropping Pills fit to the two-compartment model.5.A high performance liquid chromatography（HPLC）method was established for the determination of senkyunolide I and ligustilide in Shunaoxin dropping pills.After intragastric administration of Shunaoxin dropping pill suspension in rats,senkyunolide I and ligustilide in rats fit to the two-compartment model.