Study On The Quality Control Of Pueraria Lobata In Songling Xuemaikang Capsules

Objective:Songling Xuemaikang Capsules have the function of calming the heart and calming the mind.It is mostly used to treat hyperactivity of liver yang,hypertension and hyperlipidemia.It is composed of fresh pine leaves,Pueraria lobata and pearl layer powder.The current quality standard of Songling Xuemaikang Capsules only prescribes Thin Layer Chromatography(TLC)identification method of Pueraria lobata and pine leaves and determination method of puerarin content by High Performance Liquid Chromatography(HPLC).The current standard can not guarantee the product quality and clinical efficacy.Pueraria lobata is the dried root of Pueraria lobata(Willd.)Ohwi,a leguminous plant.It is a adjuvant drug.This study focused on Pueraria lobata,to establish the fingerprint of Pueraria lobata by HPLC to control the quality of Pueraria lobata.On the basis of the fingerprint of Pueraria lobata,optimizing the chromatographic conditions and established the correlation fingerprint of Pueraria lobata-intermediate-finished product of Songling Xuemaikang Capsules by HPLC to control the stability and homogeneity of the product.To identify the chemical constituents of Pueraria lobata in vitro and the transitional constituents in blood by UHPLC-LTQ-Orbitrap MS/MS.Then establishing the zebrafish thrombosis model to study the anti-thrombotic pharmacological activity of characteristic blood-entry components so as to determine the active ingredients of Pueraria lobata in Songling Xuemaikang Capsules.After that,to establish a method for the determination of the characteristic components of Pueraria lobata into blood in Songling Xuemaikang Capsules by UPLC,which can be used to control the quality of Pueraria lobata in Songling Xuemaikang Capsules.Methods:1.To establish HPLC fingerprint of Pueraria lobata,combined with similarity evaluation and pattern recognition,and constructs the quality evaluation function to evaluate the quality of Pueraria lobata,2.To establish the fingerprint of Pueraria lobata-intermediate-finished products of Songling Xuemaikang Capsules with the same chromatographic conditions,and analyzing the sample data by similarity evaluation,principal component analysis and cluster analysis.3.To identify the chemical constituents of Pueraria lobata alcohol extract by UHPLC-LTQ-Orbitrap MS/MS,and summarize the cracking rules of them.And to infer the blood-entry components of Pueraria lobata by comparing Pueraria lobata in vitro samples,blank rat serum and administration rat serum so as to providthe basis for the basic research of the pharmacodynamics of Pueraria lobata.4.Using zebrafish thrombosis model induced by epinephrine hydrochloride to calculate the anti-thrombotic effects of Pueraria lobata in Songling Xuemaikang Capsules according to the red blood cell staining area of caudal vein,so as to confirm the antithrombotic pharmacological activity of puerarin5.To determinethe content of puerarin,3’-methoxypuerarin and puerarin apigenoside in Songling Xuemaikang Capsules by Ultra High Performance Liquid Chromatography(UPLC)with fast analysis speed and high sensitivity,which can provide reference for the quality control of Pueraria lobata in Songling Xuemaikang Capsules.Results:1.The fingerprint of 12 batches of Pueraria lobata was established by HPLC.24 common peaks were identified and 5 of them were identified as 3’-hydroxypropyl puerarin,puerarin,daidzein,genistein and daidzein.The similarity of 12 batches of Pueraria lobata was greater than 0.950.The results of cluster analysis and principal component analysis were consistent.The medicinal materials were divided into 4 categories.The quality evaluation function constructed showed that the function values of the medicinal materials clustered into the same kind were similar.2.The chromatograms of 30 batches of Pueraria lobata and 10 batches of Puerariathomsonii Benth,46 batches of intermediates and 48 batches of capsules were determined under the same chromatographic conditions.The fingerprint of Pueraria lobata-Songling Xuemaikang Capsules intermediate-finished products was established by HPLC.In the fingerprint of Pueraria lobata,15 common peaks were found and 7 common peaks were identified,respectively,3’-hydroxypropyl puerarin,puerarin,puerarinxylose,3’-methoxypuerarin,puerarin apigenin,daidzin and daidzein.The similarity of 30 batches of Pueraria lobata was greater than 0.950.The results of cluster analysis and principal component analysis were consistent.The similarity between 10 batches of Puerariathomsonii Benth and common pattern of Pueraria lobata is less than 0.850,which can be used to distinguish Puerariathomsonii Benth from Pueraria lobata.The medicinal materials were divided into 3 categories.In the fingerprint of Songling Xuemaikang Capsules and intermediate,15 common peaks were found,and 8 common peaks were identified.Among them,3’-hydroxyproperarin,puerarin,puerarinxylose,3’-methoxypuerarin,puerarin apigenin,daidzein and daidzein came from Pueraria lobata,shikimic acid from fresh pine leaves.The similarities of 48 batches of Songling Xuemaikang Capsules and 46 batches of intermediates with their common patterns were greater than 0.990,indicating that the chemical composition of Songling Xuemaikang Capsules and intermediates was in good agreement.Cluster analysis and principal component analysis showed that 48 batches of capsules could be classified into 5 categories and 46 batches of intermediates could be classified into 5 categories according to the small differences among batches of samples.3.27 chemical constituents including 26 flavonoids and 1 pueroside were identified in the ethanol extract of Pueraria lobata by UHPLC-LTQ-Orbitrap MS/MS,and 19 blood-entry constituents including 7 prototype constituents and 12 metabolites were identified.4.In the study of anti-thrombotic pharmacological effects based on zebrafish thrombosis model,compared with the model group,puerarin treatment at different concentrations(34.2,68.4,273.7 μg·mL-1 had significant antithrombotic effects(p<0.01).The antithrombotic effects of each group were 51.74%,51.94%and 52.92%respectively.The antithrombotic effects increased with the increase of the concentration and were dose-dependent.Compared with the model group,Songling Xuemaikang Capsules(250,500,2000 μg·mL-1)had significant antithrombotic effects(p<0.01).The antithrombotic effects of Songling Xuemaikang Capsules were 62.03%,79.16%and 89.07%,respectively.The antithrombotic effects increased with the increase of the concentration,and were dose-dependent.5.The contents of puerarin,3’-methoxypuerarin and puerarin apigenin in 48 batches of Songling Xuemaikang capsules are different.The total and average contents of three isoflavones is 4.59%.The precision,stability,repeatability and recovery of the method are all in accordance with the regulations,which can provide reference for the quality control of Pueraria lobata in Songling Xuemaikang Capsules.Conclusion:In this study,Pueraria lobata as the research focus,the fingerprint of Pueraria lobata was established by HPLC,which can be used to control the quality of Pueraria lobata;The correlative fingerprint of Pueraria lobata-intermediate-finished product of Songling Xuemaikang Capsule was established by using optimized HPLC chromatographic conditions,which can be used to control the stability and homogeneity of the product.The chemical constituents in vitro and the transitional constituents in blood of Pueraria lobata were studied by UHPLC-LTQ-Orbitrap MS/MS.Antithrombotic activity of puerarin,a blood-entry component of Pueraria lobata,was studied by zebrafish thrombosis model.Puerarin was identified as the active ingredient of Pueraria lobata.And a UPLC method for the determination of the characteristic constituents of Pueraria lobata into blood in Songling Xuemaikang Capsules was established,which can be used to control the quality of Pueraria lobata in Songling Xuemaikang Capsules.