Study On The Active Components Of Longxuetongluo Capsule For The Treatment Of Ischemic Brain Injury

Longxuetongluo Capsule is a new drug,which composed of total phenol extract of Chinese dragon’s blood,and has been proved to be safe and effective for the treatment of ischemic stroke on the basis of pharmacological experiments and clinical trials.However,the active component of LTC against ischemic stroke remains unclear.The aim of the present study was to clarify the active components of LTC on the basis of composition-effect correlation,as well as drug metabolism,pharmacokinetic,metabonomics,etc.The results were included:1 Literature reviewThe substitutes for the domestic and foreign dragon’s blood were sorted.Research progress on the phenolic constituents and bioactivities of 6 spieces of the genus Dracaena has been reviewed,including Dracaena cochinchinensis,Dracaena cambodiana,Dracaena cinnabari,Dracaena draco,Dracaena loureiri,and Dracaena schizantha.These plants were rich in flavones,flavanones,homoisoflavanones,dihydrochalcones,homoisoflavanes,stilbenes,phenolic oligomers,and other phenolic compounds.A wide array of pharmacological activities including analgesic and anti-inflammatory,antibacterial,hypolipidemic,hypoglycemic,anticancer,especially the bi-directional regulation of hemorheology and the effect of cardiovascular and cerebrovascular have been reported.In addition,the analytical methods in vitro and in vivo of Chinese dragon’s blood have been reviewed,and the biomarkers relating to ischemic stroke have been summarized,which laid a foundation for the research on Chinese dragon’s blood deeply.2 Qualitative and quantitative analyses of LTCThe mass fragmentation behaviors of phenolic compounds were proposed using LCMS-IT-TOF,such as flavones,dihydroxychalcones,homoisoflavanones,chalcones,homoisoflavanes,flavanes,stilbenes,flavoid oligomers,etc.A total of 124 compounds were identified in LTC based on comparing mass spectral profiles with reference compounds as well as literature data,including 12 flavones,14 homoisoflavanones,12 flavanones,10 dihydrochalcones,10 homoisoflavanes,6 flavanes,6 anthocyanidins,3 chalcones,2 stilbenes,1 homoisoflavones,and 48 flavanoid oligomers.Among them,68 compounds were firstly identified in Chinese dragon’s blood,in particular 33 potential new compounds.Afterwords,simultaneous determination of 13 primary phenolic compounds was carried out in 8 batches of LTC using HPLC-DAD,and these compounds account for 10 percent of LTC,approximately.3 Composition-effect correlation of LTCTen core components(A-J)were prepared by separation from silica gel CC and uniform design,which have intersection and different contents for each compound in it.The ten samples were assessed using HPLC-DAD and UHPLC-MS,and the relative peak area represents the contents of each compound.Three kinds of cells which related to ischemic stroke in vitro were chosen,including BV-2 microglia,HUVEC vascular endothelial cell,and PC-12 nerve cell.The anti-inflammatory,vascular endothelial cell protective,and nerve cell protective activities of the ten core components of LTC were evaluated using corresponding cells,and the chemical composition-activity relationship of LTC was investigated by grey relation and partial least squares(PLS)analysis.Some compounds with high correlation were deemed as potential active compounds.4 Studies on the metabolism of LTCThe metabolites in rat plasma were characterized following the oral adminitration of loureirin A and loureirin C using LCMS-IT-TOF.On this basis,102 protypes and 339 metabolites were tentatively identified in rat plasma,urines,feces,and bile acids,respectively,after oral gavage of LTC at a dose of 500 mg/kg.Glucuronidation,sulfonation,methylation,etc,were common in the metabolites,and this study preliminarily clarified the form of LTC in vivo.5 Pharmacokinetics and tissue distribution studies of LTCAn UHPLC-MRM-EPI MS method to simultaneous determination of 12 phenolic compounds in rat plasma and tissues was developed,and the pharmacokinetic and tissue distribution investigations were subsequently conducted in rat after a single 500 mg/kg oral dose.Rapid absorption and elimination occurred for all analytes-of-interest.The comparative pharmacokinetics of five phenolic compounds after oral administration of LTC and monomer compounds were carried out,and the results showed that the rates for excretion of most analytes can decrease after oral administration of LTC.The developed method was also used for comparative study on the pharmacokinetics of these components in middle cerebral artery occlusion(MCAO)rats after oral dosage of LTC,and the concentration-time curves of most compounds in MCAO rat plasma exhibited double peak,and some analytes had increased Cmax in the MCAO group.Significant distributions occurred for all analytes in the liver as well as kidney,and several compounds could be found in brain,which could cross the blood-brain barrier.6 Metabolomics study of LTC on MCAO ratsThe rats were randomly divided into four goups:sham group,sham+LTC group,MCAO group,and MCAO+LTC group.After oral adimistration of LTC(300 mg/kg),the neurological scores and infarct volumes were significantly reduced in MCAO+LTC group.The rat plasma of the four groups was carried out using UHPLC-QE Orbitrap HRMS.In the principal component analysis(PCA)model,MCAO group and sham group were clearly distinguishable,while MCAO+LTC group was between the two groups,indicating that the matabolites of MCAO rats had a trend to become normal after LTC treatment.In the orthogonal partial least squares discriminant analysis(OPLS-DA)model,MCAO group was completely separated from sham group,for the considerable change of PCs,PEs,fatty acids,amino acids,and other endogenous metabolites during the ischemic injury.After administration of LTC,the PCs,phosphoenolpyruvic acid,arachidonic acid exhibited significant changes,suggested that it is likely that LTC alleviates ischemic stroke through regulating the disturbed glycerophospholipid metabolism,ether lipid metabolism,arachidonic acid metabolism,glycolysis pathway,etc.Comparision of the exogenous metabolites between MCAO+LTC group and MCAO group led to the finding that 21 metabollites were higher than sham group.In addition,comparision of the exogenous metabolites between MCAO+LTC group and sham+LTC group led to the finding that 37 metabollites were higher than sham+LTC group,and these metabolites all exhibited significant differences.UHPLC-QE Orbitrap HRMS was also applied for the left and right brain tissues of the rats from different groups.Glucose monophosphate and other endogenous metabolites were significantly perturbed in the right brain(the injured side),which may lead to disorders of starch and lactose metabolism,etc.On the other side,forty-three exogenous metabolites were identified in brain tissues,and nine compounds,which showed significant differences,had high contents in the right brain of MCAO+LTC group.The differential exogenous metabolites in plasma and brain tissues may be the potential active compounds of LTC.7 Validation of the potential active compounds in LTCValidation of the potential active compounds in LTC was carried out using 3 models,including inhibitory activity against LPS-activated NO production in BV-2 microglial cells,HUVEC cell protective and PC-12 cell protective activities against deprivation/reoxygenation(OGD/R)jury.The results indicated that 14 compounds,such as loureirin C,exhibited inhibitory activity against LPS-activated NO production in BV-2 microglial cells.Nineteen compounds,such as Loureirin D,exhibited HUVEC cell protective activity against deprivation/reoxygenation(OGD/R)jury,and 10 phenolic compunds,such as Loureirin B,showed PC-12 cell protective activity against deprivation/reoxygenation(OGD/R)jury.In addition,considering the results of activity verification and previous prediction results,3 phenolic compounds,9 phenolic compounds,and 5 phenolic compounds may be the active components towards anti-inflammatory,vascular endothelial cell protective,and nerve cell protective activities,respectively.These phenolic compounds may be the active components of LTC.This thesis preliminarily clarified the material basis of LTC,which will provide scientific basis for the clinical curative effects and rational use of LTC,and will offer scientific evidences for the popularization,application and market cultivation of innovative drugs.In addition,the research results also afford new experimental evidences for the clarification of the therapeutical principle of "promoting blood circulation to remove blood stasis" in treating ischemic stroke.At the same time,this research pattern provides the beneficial references for the study on the active components of other TCMs and compound prescriptions,which has important academic value and application prospect.