| This thesis is composed of two parts, literatures review and experimental research.Literatures review contains two pieces, the thinking study and the method for the substancebasis of chinese traditional medicine compounds, and, brain capillary endothelial cells and hypox-ic-ischemic brain damage. Literatures research has offered clew and method for this experimentdesigning.Experimental study is according to research methods of serum pharmacochemistry, with aview to the migration compositions in blood after chinese traditional medicine oral administratio--n, according to whether drugs can permeate the blood-brain barrier or have protection on targetcell-CMEC, the two key links as cut-in drop, investigating the substance basis of the effect oftreating ischemic apoplexy and paralysis by Tongsaimai.The first part of this thesis analyzes the chemical composition inside and outside body ofTongsaimai. First, adopting technique of HPLC-UV, and carry through quite overall qualitativeand quantitative analysis for the fractions having UV absorption, and build fingerprint of 10 bat--ch samples, and deduce the medicinal materials and compounds which are attributive to all cha-racteristic peaks. With double waves method of HPLC carry through quantitative analysis to the6 upper determination components in Tongsaimai pellets. As this as Chemical basis, further ca--rry on the preliminary study on serum pharmacochemistry of Tongsaimai pellets, as rats for ex--perimental animal, and prepare serum contained bushentiaochong after the rats are dosed by irri-gating stomach, applying the method of HPLC-UV compare the Tongsaimai pellets raw liquor,The serum contained medicine after the rats and blank serum, questing chromatographic peak ofmigration from blood, and according to the relative retention time and online UV spectrogramof each peak, deduce the medicinal materials where the the migration compositions in blood co--me from and the attributive compounds, by contrast to the serum contained medicine of eachsimple preparation.The second part, carry on the study on effective component of Tongsaimai pellets enteringthe brain through the blood-brain barrier. First, research the attributive compounds of Tongsaimaipellets permeating the normal blood-brain barrier, adopting in vivo method, choice rats as anim-al pattern, anesthesia the animals after rats dosed by irrigating stomach, extracting cerebrospinalfluid in the foramen magnum from the brain of rats, after some special process, analysis in HPLC-UV and HPLC-MS. Experiments do more study on correlation components of Tongsaimaipellets permeating the ischemia damage blood-brain barrier of rats. Rats cerebral MACO modelis replicated with the suture method, shaping ischemia damage which lead to the alter of perm-eability of blood brain barrier, after rats are dosed by irrigating stomach, chafe on from cardiacperfusion to there is no blood in brain tissue, then choose the brain tissue, after some specialprocess, analysis the component which permeating the blood brain barrier by HPLC-UV andHPLC-MS.The third part, with the complex prescription of Tongsaimai pellets as research subjects, fro-m integer animal level, rats cerebral MACO model is replicated with the suture method, measu-re brain infarct size with TTC coloration, go along behavioral measurement and envalument forthe cerebral ischemia animal with neural function check method, and investigate the protectionof pharmic with the observation of pathological mechanism and ultramicro organizations.The fourth part, we study on Tongsaimai pellets and their effective components’ interventio--n effect on cerebral micrangium endothelial cells ischemic damage of rats. First, carry on thestudy on rats’ cerebral micrangium endothelial cells primary culture: 5-10 days borned rats suckl-ing mouse are used as experiment animal, acquisition brain tissue in asepsis environment, digest-ion tissues by collagenase isolate microvessels, cribration twice for extracting cerebral micrangiu-m endothelial cells, and subculture. Identify cells by cellular morphology observation, factorⅧimmunohist-ochemical detection under the invertmicroscope. Then build rats in-vitro ischemicdamage modle by the method chemical ischemic, with the alterlation of cellular morphology andthe value of MTT and LDH as the evaluating indicator of cytoactive. Experiments investigatingintervention active of Tongsaimai pellets andtheir effective components on the normal CMECand the protection for the ischemic damage CMEC differently.The fifth part, we discuss the adhesion mechanism of Tongsaimaipellets control CMEC. Ad--opting enzyme linked immune sandwich assay technique, we checkout the expression of two ad-hesion Protein selectivity, E-selection and ICAM-1.Experimental findings:Ⅰ. The analysis result of Tongsaimai pellets’ chemical component inside and outside of bod--y1. The study on HPLC-UV fingerprint of Tongsaimai pellets demarcates 18 common fingerp-rint peaks with glycyrrhiza uralensis glycoside as compared peak, get the technical parameter ofTongsaimai pellets’ HPLC fingerprint, adopting＜similarity evaluation of chromatographic finge rprint fortcm （2004A Edition）＞, we measured10 batches samples chromatography fingerprint integ-er degree of similarity is above 0.968, and elicit the contrast fingerprint. The analysis of thesource of 18 common fingerprint peaks: one peak come from angelica sinensis materia medicwhich is identified as ferulic acid; five peaks come from flos lonicerae, one of them is identifie-d as chlorogenic acid, the others can be deduced as similar chlorogenic acid components fromthe spectrogram; there peaks come from ningpo figwort, the two of them are identified as cinna-mic acid and ahbar khozaschet glycoside, the other one is unknown; 8 peaks come from glycyr-rhiza uralensis, there of them are identified as glycyrrhiza uralensis glycoside, liquiriti genin, an-d ammonium glycyrrhizinate, the other four have absorbing character of licoflavone mother nucl-eus; and there are a compound we don’t know the adscription.2. We inject once and checkout the upper 6 components chlorogenic acid, ferulic acid, cinn--amic acid, haba khozaschet glycoside, glycyrrhiza uralensis glycoside, liquiriti genin in Tongsai--mai pellets at one time by the method HPLC double waves method, the chromatographic peakof the 6 components we measured can Separate from the adjacent ones finely, the experimentalresult of methodology is fine. In allusion to butyl phthalide having the function of increasing ofcerebral blood flow to ischemic tissue, shrink cerebral infarct size, and reduction of brain edem,which has intimate correlative with complex prescription curative effect, we build the contentmeasuring method of butyl phthalide especially. The result affords the experimental foundationfor the function of anti ischemia damage with other components in Yongsaimai pellets.3.The preliminary findings of medicinal chemistry Tongsaimai pellets: The serum containedmedicine, blank serum, raw liquor of Tongsaimai pellets, and 7 known mixed reference substa-nce, they compare: there are 12 entered blood components, the 6 of them are absorbed intoblood by archetype, we find chlorogenic acid, ferulic acid, liquiriti genin, and cinnamic acid,the four known components are absorbed into blood by archetype, the other two are absorbedinto blood by archetype too, one is a unknown component of flos lonicerae, the other one is aunknown component of glycyrrhiza uralensis, by contrast to the retention time and UV actionspectrum of reference substance. The 3 of them are absorbed into blood by metabolic product,we prove one of them come from metabolic product of angelica sinensis, the other two comefrom metabolic product of glycyrrhiza uralensis, by contrast to the serum contained medicineof simple preparation. And the attribution of the 3 of components in-blood of the serum contai-ned medicine, are unknown, maybe are the metabolic product of drug interaction, and maybe are physiology stress substance brought by rats’ body after dosed of Tongsaimai complex presc-ription.Ⅱ. The findings of effective component of Tongsaimai pellets permeating the the blood-brai-n barrier of rats1.For the normal rats, we compared the cerebrospinal fluid contained medicine to the blankcerebrospinal fluid, we didn’t detect the correlation component of Tongsaimai pellets, by themethod of HPLC-UV and HPLC-MS. The peak intensity of four endogenous substance in cerebr-ospinal fluid chromatogram peak stronger obviously at retention time 51.6 min, 56.2 min, 60.1min, 61.9min （we can see they stronger 6.5, 4, 3.3,4.5 times from the specific value peak areadifferently）.2.We can detect the components of Tongsaimai pellets in groups of normal and sham oper-ation, we prove them as ferulic acid, liquiriti genin, and cinnamic acid by HPLC-UV and HPLC-MS; In conditions of brain ischemia damage brought by the MACO model of replicated by ins-erting nylon thread, the capacity into brain tissue of ferulic acid, liquiriti genin, and cinnamicacid add notability （P＜0.05）. The finding indicated ferulic acid, liquiriti genin, and cinnamic acid as the substance basis of part pharmacodynamics of Tongsaimai pellets, can permeate the blood-brain barrier when the brain is ischemia damage, can go to corresponding damage area, act on the key sites and bring into play effect.Ⅲ.The study on the protection of Yongsaimai pellets for the MACO model of ratsAll experimental animal appear differ degree nerve dysfunction. After 24h, the assign markof behavioristics of each dosage group animals appear downtrend, and lower than the model gro-w up obviously （P＜0.05）, indicating Tongsaimai pellets can improve the nerve dysfunction ofmodel animals. The brain infarction area and infarct hasty of each dosed groups rats all lowerthan the model group obviously （P＜0.05）, indicating Tongsaimai pellets can contract the braininfarction area, drop the infarct hasty. The cerebral exponential and moisture content of modelrats are upper than the sham operation group, the large dosage of Yongsaimai pellets can lowthe cerebral exponential and moisture content of model rats （P＜0.05）, indicating Tongsaimai pel—lets can improve brain edema, the action of larger dosage is more obvious. From histopathology observation, the ischemic apoplexy rat model of replicated by inserting nylon thread, becomeas brain tissue differ degree necrosis, gliocyte and seroma edema. The Tongsaimai pellets havethe action lower the brain tissue damage on different degree on apoplexy model.Ⅵ.Tongsaimai pellets and the effective components have intervention action on CMEC 1. The CMEC of rats primary cultured in this experiment are Long spindle and polygon,Signs were paving stones single convergence ;Ⅷfactor immune cytochemical staining are mascu-line. We proved it to be according to cha-racteristics of CMEC.2. We build CMEC ischemia damage models in vitro by the method of chemical lack ano--xic, the model cells have been damage of anoxic, we can see from the morphological the cellswill strut first and the diopter will be bate, and than collapse. The method of MTT determinethe OD value of cells, after we building model 6-12h, the value is at a distinct decline phase（P＜0.05）, we Ultimately determine to use the sugar Ealer’s solution and the concentration of0.85 mg·mL-1 solution Na2S2O4 role in normal cell 8h to create the model of ischemic injur--y.3. For normal brain microvascular endothelial cells: We compare Tongsaimai pellets highdose group to negative control group, we can see a significant difference （P＜0.05）, curb cell act-ivity, Performance for cell activity dose group （P＜0.05）.Low dose group compared with the con-trol group, no significant difference. The general trend of dose and the cell activity is bell-shap-ed, the cells showed inhibition of high-dose, middle dose range Promote for the activity.The serum after Tongsaimai pellets were administrated, High dose group compared with theblank group, Performance for cell activity （P＜0.01）.The effective components in Yongsaimai pe--llets, ferulic acid, liquiriti genin in the high-dose group compared with DMSO control group,shown to promote cell activity （P＜0.05）.The other components in pellets: butylphthalide, astragalus A in the high and middle dosegroup compared with DMSO control group, performance for cell activity （P＜0.05）. Chlorogenicacid in middle-dose group compared with the DMSO control group, the performance for cell act-ivity （P＜0.05）. High doses of chlorogenic acid showed inhibition （P＜0.05）4. For ischemic injury in brain microvascular endothelial cells: The dosage of Tongsaimaipellets at 1000,500μg·mL-1 of high-dose group compared with the model group, there was a sig--nificant difference （P＜0.01）, showed inhibition activity. Low-dose group compared with the mod-el group, we can see a significant difference （P＜0.05）, promote the performance of the activity.The serum after Tongsaimai pellets were administrated, High and middle dose group compa-red with the blank group, performance for cell activity （P＜0.05）. The active ingredients in Pell--ets: except cinnamic acid and low-dose licorice, ferulic acid, isoliquiritigenin, einnamie acid inall dose groups have shown activity in promoting the activity of cells, compare with the controlgroup DMSO, we can see a significant difference （P＜0.05）. The other components in pellets: butylphthalide, astragalus A in the high dose group comp--ared with the control group DMSO, have shown to promote cell activity （P＜0.05）. Chlorogenicacid in the low-dose and middle-dose group compared with the control group DMSO, to pro--mote the performance of cells （P＜0.05）. Glycyrrhiza uralensis glycoside of middle dose, harpag--oside of high dose, all have shown a protective effect （P＜0.05）.ⅤThe adhesion mechanism of Tongsaimai pellets control CMECAdopting enzyme linked immune sandwich assay technique, we checkout the expression oftwo adhesion Protein, E-selection and ICAM-1, we can see the results after CMEC 8h ischemiashowed that expression of both E-selection and ICAM-1 increased, a significant difference com-pared with normal （P＜0.01）. After Tongsaimai pellets role in ischemia, reduced E-selection andICAM-1, compare with ischemic cells, we can see a significant difference （P＜0.05）, which clos-e to the normal value of the expression （P＞0.05）.Conclusion:By this experiment, a comprehensive analysis:1.The preliminary findings of medicinal chemistry Tongsaimai pellets,pellets containing seru--m was prepared by its in vivo efficacy of the play "agents", among some of the blood compo--nents transitional chlorogenic acid, ferulic acid, liquiriti genin, cinnamic acid, it is possible toeffect a material foundation.2.Ferulic acid, liquiriti genin, cinnamic acid can be much more into the central nervous sys-tem through the blood-brain barrier when ischemic injury, as part of Pharmacodynamics materia-1 foundation of Tongsaimai pellets, to achieve the corresponding injury area, act on the key tar-get of apoplexy （nerve cell and glial celt ） and bring into play effect.3.Tongsaimai pellets and related components, and the serum Containing pellets, can promotenormal CMEC proliferation, enhance the activity of ischemic injury CMEC. We proved ferulicacid, liquiriti genin, cinnamic acid are the Pharmacodynamics material foundation of treatment ofischemic stroke, from the target cells-brain microvascular endothelial cells.4.CMEC started the adhesion mechanism when ischemia, Tongsaimai pellets intervention is--chemic CMEC, can reduce the high expression of E-selection and ICAM-1 due to ischemic inj--ury, suggest Tongsaimai pellets can inhibit the adhesion inhibited CMEC ischemic injury, byimpacting E-selection and ICAM-1 in the two channels.5.Brain microvascular endothelial cells drived from cell cultured by the modern technology,can be used as an important tool in vitro study of cerebrovascular disease and vector. |
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