Study On Pharmacodynamic Material Basis And Pharmacological Mechanism Of Naomaitong Against Ischemia/Reperfusion Injury

Objective: Naomaitong(NMT)prescription,which consists of 4traditional Chinese medicines: Rheum palmatum L.,Ligusticum chuanxiong Hort.,Panax ginseng C.A.Mey.and Pueraria lobata(Willd.)Ohwi.,has been applied clinically for years to treat ischemic stroke.In resent years,although researches on NMT was promising,the pharmacodynamic material basis and molecular mechanism of NMT remains unclear.As we all know,the absorption,distribution,metabolism and excretion of drugs are different between healthy condition and sick condition.In this study,we took NMT as the reseach object,aimed at exploring the pharmacodynamic material basis and molecular mechanism of NMT against ischemia/ reperfusion injury by the following methods: the comparative analysis of chemical constituents of NMT in vitro and in vivo of normal rats and middle cerebral artery occlusion(MCAO)model rats performed by ulta-perfomance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometer(UPLC-Q Exactive-Orbitrap MS),and the system pharmacology research based on the directly acting components in vivo.Methods and results:1.The qualitative analysis of NMT extract was performed by UPLC-Q-Exactive-Orbitrap MS.The chromatographic column was Waters UPLC HSS T3(2.1×100 mm,1.8 μm),and the mobile phase was acetonitrile(A)-0.1% formic acid water((B).The base peak ion chromatogram was obtained on a switched mode of positive and negative ion.Combined with XCalibur and Compound Discover 3.1(CD 3.1)software,a total of 214 compounds were identified by comparation of reference standards and literature,including 26 anthraquinones,70 flavonoids,43 ginsenosides,10 phenylpeptides,26 organic acids,2 other triterpene saponins,4 puerarin glycosides,4 stilbenes,5 phenolics and 24 other components.In this chapter,we established a database of NMT chemical composition in vitro.2.A MCAO rat model was established.After oral administration of NMT,the plasma,brain,cerebrospinal fluid,urine and fecal of normal rats and model rats were collected,and then analyzed by UPLC-Q-Exactive-Orbitrap MS.With the data batch process of CD 3.1software,analysis of prototype components of NMT in vivo were handled by comparing with the NMT database mentioned above.As a result,102 components were detected in the plasma,of which 35 components were in normal rats and 102 components were in model rats.Besides,the plasma collected after 1h of administration contains the maximum number of prototype compounds.A total of 70 components were detected in the brain,of which 9 components were in normal rats and 70 components were in model rats.Among model rats,more components were detected in the left brain than in the right brain.A total of 11 components were detected in the cerebrospinal fluid,of which 2 components were in normal rats and 11 components were in model rats.A total of 96 components were detected in the urine,of which 59 components were in normal rats and 93 components were in model rats.A total of 74 components were detected in the fecal,of which 51 components were in normal rats and 72 components were in model rats.Then the components detected in the left brain tissues of normal rats and model rats were analyzed by orthogonal partial least square-discriminate analysis(OPLS-DA).As a result,11 compounds(VIP>1)were filtered as differential components.They are: Rhein,Rhein 8-glucoside,Puerarin,3’-Methoxypuerarin,3’-Hydroxypuerarin,Mirificin,Daidzein,Formononetin,Glycitein,Catechin and Senkyunolide I.After modeling,the metabolism trend of these components in blood and their content in brain were changed.3.Based on the brain absorption components,combined with network pharmacology and molecular docking method,a system pharmacology study was conducted according to the directly acting components in vivo.The network pharmacology of 70 brain absorption components and 11 differential components between normal rats and model rats was conducted respectively.We found that NMT alleviated cerebral ischemia/ reperfusion injury by multi-component,multi-target and multi-channel.Meanwhile,Rhein,Rhein 8-glucoside,Senkyunolide I,Puerarin,Daidzein,Formononetin,Glycitein and Catechin may be related to the regulation of expression of PTGS2、AKT1、NOS3、CAPS3、VEGFA and other genes in the process of ischemic stroke.They can mediate TNF,estrogen and focal adhesion kinase signaling pathway and the downstream PI3K/Akt,MAPK,NF-κB signaling pathways.NMT alleviated cerebral ischemia/reperfusion injury by antagonizing excitatory amino acid toxicity,inhibiting inflammatory cascade reaction,anti-oxidative stress,inhibiting cell apoptosis and promoting cerebral angiogenesis.In this paper,an in vitro and in vivo analytical method and a mass spectrometry database of NMT were established.Through the comparative analysis of chemical constituents of NMT of normal rats and MCAO model rats,we showed the differences in absorption,distribution and metabolism of NMT in physiological / pathological conditions,and screened their differential components in the brain.Further more,we explained the interaction between NMT active components and protein targets via the construction of interaction network and molecular docking technology.We suggested that NMT plays an important role in treatment of cerebral ischemia / reperfusion injury by regulating energy metabolism,inflammation,oxidative stress and cerebral angiogenesis.