Research On Screen Activating Blood Circultion Ingredients Of Buyang Huanwu Decoction And Injection Using Platelet Binding Assay

Buyang Huanwu Decoction,which is commonly used in the clinical treatment of ischemic stroke,is consist of astragali radix,angelicae sinensis radix,chuanxiong rhizoma,paeoniae radix rubra,persicae semen,carthami flos,pheretima.Buyang Huanwu Decoction can supply of Qi,activate blood circulation and resolve stasis.This research will screen activating blood circulation components of Buyang Huanwu Injection by high performance liquid chromatography fingerprint and platelet bingding assay.Objective:A HPLC method for Buyang Huanwu Decoction and Injection's fingerprint and simultaneous determination of amygdalin,paeoniflorin,campanulin,ferulic acid were established.At the same time,a method to analysis the antithrombotic and anti-platelet aggregation components in Buyang Huanwu Injection by platelet membrane binding assay was established.Methods:1.The Buyang Huanwu Decoction and Injection was separated on a Hyperil ODS2 column(4.6mm×250mm,5?m)by gradient elution using acetonitrile-0.1%acetic acid as the mobile phase under 250nm detection wavelength.The common mode of HPLC fingerprint for 10 batches of Buyang Huanwu Decoction and Injection was established by"Similarity evaluation system of chromatographic fingerpirnt for TCM 2004 revision A".2.Amygdalin,paeoniflorin,campanulin and ferulic acid in Buyang Huanwu Decoction were separated on a Hyperil ODS2 column by gradient elution using acetonitrile-0.1%phosphate as the mobile phase.Paeoniflorin and campanulin in Buyang Huanwu Injection were separated on the same columnby gradient elution using acetonitrile-0.1%acetic acid as the mobile phase.3.Screening activating blood circultion ingredients of Buyang Huanwu Injection by platelet membrane binding assay.Platelets were used to bind and separate the active components.4.Relative adscription analysis of chemical components between Buyang Huanwu Decoction and Injection,its single herb and its negative samples under the selected spectrum condition.Results:1.Under the selected spectrum condition,the HPLC fingerprint for 10 batches ofBuyang Huanwu Decoction and Injection were obtained.14 and 13 peaks were found in Buyang Huanwu Decoction and Injection's fingerprint The similarity of 10 batches Buyang Huanwu Decoction and Injection fingerprint chromatogram were over 0.9.The motheds were accurate,stable and reproducible.2.The content of amygdalin,paeoniflorin,campanulin and ferulic acid in Buyang Huanwu Decoction were 1.081?1.095,2.088?2.100,0.2108?0.2122,0.08672?0.09006 mg/g in 3 batches.The content of paeoniflorin and campanulin in Buyang Huanwu Injection were 793.51?868.91?g/g,43.19?50.88?g/g in 10 batches.3.The effective chemicals in Buyang Huanwu Injection were combined by the platelet membrane under imitated phsical environments.The unattached substances were washed away by the PBS ofpH 7.4.Then the attached compounds were eluted by the PBS of pH 4.0.Five active compounds were found to bind to the membrane of platelets.4.Peak No.4,10,2,13 belong to Astragali Radix;peak No.6,7,8 belong to Paeoniae Radix Rubra;peak No.3,4,9 belong to Chuanxiong Rhizoma;peak No.5,9,11 belong to Angelica sinensis Radix;peak No.6 belongs to Carthami Flos in Buyang Huanwu Decoction's common peak.Peak No.6,12 belong to Astragali Radix;peak No.8,10 Paeoniae Radix Rubra;peak No.5,9,11 belong to Chuanxiong Rhizoma;peak No.7,11 belong to Angelica sinensis Radix;peak No.8 belongs to Persicae semen in Buyang HuanwuInjection's common peak.Five characteristic chemicals in Buyang Huanwu Injection was found which bind to the membrane of platelets.Peak No.1 belongs to Persicae semen;peak No.2 belongs to Paeoniae Radix Rubra;peak No.3 belongs to Angelica sinensis Radix and Chuanxiong Rhizoma;peak No.4 belong to Astragali Radix.Peak No.2 and No.4 was indentified as paeoniflorin and campanulin.Conclusion:The establishment of HPLC fingerprint of Buyang Huanwu Decoction and Injection and the determination of amygdalin,paeoniflorin,campanulin and ferulic acid provide more reference for the quality control of Buyang Huanwu Decoction.Paeoniflorin and campanulin and the other 3 compounds,which were extracted through the platelet binding process,could be considered as the antithrombotic and anti-platelet compound of Buyang Huanwu Injection.