Study On The Chemical Composition And Bioactivity Of Guanxinning Injection

Ethnopharmacology:Guanxinning injection(GXNI),as atraditional Chinese medicine injection(TCMI)made from Danshen and Chuanxiong,is used for the treatment of coronary heart disease and angina pectorisin clinic for a long time.GXNIexhibited a series of pharmacological activities,such asactivating blood circulation,removing blood stasis,stimulating the pulse and nourishing the heart.GXNI isalso one of the traditional Chinese medicine injections for promoting blood circulation and removing blood stasis,which is the dominant variety in Shanxi province.There are 7manufacturers in China recently and 5 manufacturers are in Shanxi province.In addition,there areanother two TCMIs varieties for promoting blood circulation and removing blood stasis in Shanxi province,safflower injection(SI)and Shuxuening injection(SXNI),which are used for the similar clinical purpose.Moreover,the clinical application of SI and SXNI is more widely than GXNI significantly.Therefore,this study intends to conduct an in-depth study of GXNIand lay the foundation for revealing the characteristics of GXNI and expanding its clinical application.Objective:This subject takes GXNI as the research object.Firstly,chemical compositions of GXNI were quickly characterized tomake a foundation for its active ingredients research.And then the effects of low-temperature,freeze-thaw,high-temperature,and light on the main chemical components of GXNI wereevaluated,which laid the foundation for clarifying the influencing factors of GXNI during transportation and storage.Next,the anti-coagulant,inhibiting platelet aggregation and anti-oxidant activity of GXNI were evaluated and compared with that of SI and SXNI.Finally,the mechanism of GXNI on anti-thrombotic,anti-platelet aggregation,and anti-coagulation effect was revealed by network pharmacology analysis.Method:(1)The integration of UPLC-Q Orbitrap HRMS and 1H-NMR spectroscopy was used to analyze the chemical composition of GXNI and different solvent extraction fractions of GXNI.The chemical compounds were identified by analyzing the mass spectral fragmentation pattern of different structural types of compounds and molecular network.(2)To distinguish the factors influencing on the main chemical components of GXNI during its transportation and storage,the effects of low temperature,freeze-thaw,high temperature and light were studied.The contents of rosemary acid and salvianolic acid B were used as quantitative indicators,and the influence of different factors on the main components of GXNIwere evaluated by the similarity analysis of fingerprints,principal component analysis and relative content comparison.(3)The anti-coagulation,anti-platelet aggregation effected and anti-oxidant activity of GXNI were compared with other two traditional Chinese medicine injections(SI and SXNI)in vitro.In addition,network pharmacology analysis was performed to reveal themechanism of anti-thrombotic,anti-platelet aggregation,and anti-coagulation effect of GXNI.Results:(1)UPLC-Q Orbitrap HRMS and 1H-NMR were used to analyze GXNI and different extraction fraction.A total of 194 compounds were identified,including 4 sugars,11 amino acids,4nucleotides,27 organic acids,43 diterpene quinones,42 phthalides,26 salvianolic acids,1 flavonoid,2 fatty acids,2 triterpenes,1 steroid and 31 other compounds.Compared with direct analysis of injections,45 compounds in GXNIwould be further identified by analysis of different polar solvent extraction fractions.The molecular networks of diterpene quinones and phthalide compounds were constructed,which was used for speculating and predicting molecular formula.Then these predicted compounds were identified by characteristic ions and corresponding MS fragmentation pattern,such as 6 phthalide and 13 diterpene quinone.In addition,16 primary metabolites in GXNI were identified by NMR technology,and 10 compounds of them were only identified by NMR analysis.(2)Thecontent variations of main components in GXNI under the conditions of low temperature,freeze-thaw,high temperature,and strong light irradiation were clarified.The content of danshensu sodium increased in high temperature conditions,while it was relatively stable in other conditions.The content of ferulic acid,vanillic acid,chlorogenic acid,and caffeic acid generally showed the downward trend in several influencing factors.The content of senkyunolide H increased in low temperature conditions and decreased in freeze-thaw and high temperature conditions.The contents of rosemary acid and salvianolic acid B decreased in high temperature and strong light irradiation conditions,while they were relatively stable in low temperature conditions.(3)In terms of the results of in vitro anti-coagulation assay,GXNI has a significant effect on prolonging APTT,PT,and TT.The anti-coagulant effectsof GXNI and SI in vitro were similar,whichwere significantly stronger than SXNI.GXNI significantly inhibited platelet aggregation induced by PAF and ADP.For the inhibition ability against ADP induced platelet aggregation.GXNI and SI are equivalent,which was obviously stronger than SXNI.And GXNI and SI have the strongest inhibitory effect on PAFinduced platelet aggregation.In antioxidant activity,the effect of GXNI is more obvious than SXNI.In addition,71 pharmacological targets of GXNI were screened out through network pharmacology.KEGG pathway enrichment analysis showed that the activitiesof anti-coagulation,anti-thrombotic,and inhibiting platelet aggregation of GXNI might be related to complement and coagulation cascades pathway,Rap1 signal pathway,MAPKRap1 signal pathway and so on.Conclusion:In this paper,194 compounds in GXNI were identified bycombining UPLC-Q Orbitrap HRMS and 1H-NMR spectroscopy and 146 compounds were further identified based on the previous study.The influence factors of GXNI during transportation and storage were clarified,which revealed that high temperature and strong light irradiation had a greater impact on chemical components in GXNI,freezing and thawinghad a moderate impact,while low-temperature a had slight impact.Thus,thelow-temperature environment should be chosen to stored the GXNI.GXNI had the strong anti-coagulant,platelet aggregation and antioxidant effects in vitro and the anti-coagulant,thrombotic,and platelet aggregation effects might relatedwith the complement and coagulation cascades pathway and the Rap1 signaling pathway,which was revealed by network pharmacology.The study indicated that these diseases were regulated by GXNI through multi-component,multi-target,and multi-pathways.