| Stroke refers to a cerebrovascular disease, an acute occurrence of blood vessels or blood abnormalities result in brain blood circulation disorders, which lead to the occurrence of neural function defect syndrome sequentially. It includes ischemic stroke, which accounts for about60%-80%of the variety of stroke disease, cerebral hemorrhage and subarachnoid hemorrhage. In China, the cerebrovascular disease has become the first leading cause of disability and death in urban and rural population. With the aging population and rapid development of the economy and lifestyle changing, stroke has become one of the major diseases threatening the health of the elderly. Persimmon leaf is the fresh or dried leaf belonging to Diospyros kaki L. f. It is bitter in taste and cold in nature, the earliest use of Persimmon leaf in clinic is traced back to Dion Nan Materica Medica, which stated that the Persimmon could be applied to treat carbuncle sore. The Persimmon leaf has the effects of clearing heat and detoxifying, moistening the lung and nourishing the heart, relieving cough and stopping bleeding, and preventing and treating cancer, etc. It also physiologically characterized by protecting the cardiovascular system, anticancer, anti-oxidant, antiseptic and low toxicity. In the present study, we get the effective fraction of the treatment of ischemic stroke from preparation, separation and activity screening in Diospyros Kaki. To provide the scientific basis for developing anti-ischemic stroke drug in the future, we discuss the protective effect and mechanism of focal cerebral ischemia and reperfusion injury, explore influence of effective fraction on expression of NOS mRNA and MAPK signaling pathway. Experiment methods and results are summarized as follows:1. Investigation and screening of the crude extract process in Diospyros KakiBy using different concentrations of ethanol and pure water, crude Diospyros Kaki was extracted. The chemical composition of crude extract was analyzed by HPLC and LC-MS. Using eight different cell models and PT, APTT, TT coagulation parameters, high-throughput screening and in vitro activity screening were performed. Results showed the75%ethanol could be used to extract crude Diospyros Kaki. The crude extracts mainly include beta-sitosterol, rutin, astragalin, malic acid, kaempferol, myricetrin and other compounds.2. The preparation of different chemical fractions and effective fraction initial screening in Diospyros KakiThe75%ethanol extract was chromatograpied to macroporous resin column by gradient elution with aqueous ethanol. Using five different cell models and PT, APTT, TT coagulation parameters, high-throughput screening and in vitro activity screening were carried out. The fractions of Fr2+Fr3(10-50%ethanol elution) was initially identified as the effective fraction of Diospyros Kaki against ischemic stroke, which primarily include hyperin, isoquercetin, glycosides astragalin, quercetin, kaempferol and other compounds.3. Pharmacodynamic evaluation and acute toxicity test of effective fraction in Diospyros KakiOn MCAO model in rats, Diospyros Kaki effective fraction significantly ameliorated the neurological deficits (P<0.001) and reduced the brain water content (P<0.001), TTC staining showed infarct volume of rats was significantly smaller. The activity of SOD was decreased (P<0.001) and the contents of MDA was decreased (P<0.001). On pial microcirculation model in rats, Diospyros Kaki effective fraction significantly increased the BPU values (P<0.01) and inhibited the glutamate concentration in brain tissue, NOS activity and calcium content(P<0.01). On myocardial ischemia-reperfusion injury model in rats, the effective fraction significantly reduced the arrhythmia extent and the myocardial infarct size, inhibited the activity of AST, LDH and CK-MB, decreased the content of MDA and NO, but significantly increased SOD activity and GSH concentration in serum. On carotid artery thrombosis model in rats, the effective fraction significantly extended the electrical stimulation-induced arterial thrombosis (P<0.01), elevated serum PGI content (P<0.01),reduced the content of blood clots matrix proteins and TXA2in serum(P<0.01). On hyperviscosity model in rats, effective fraction significantly inhibited whole blood viscosity, plasma viscosity and serum viscosity (P<0.001), inhibited the increase of erythrocyte sedimentation rate and decrease of hematocrit. In addition, the acute toxicity test showed that the effective fraction had no obvious acute toxicity in mice at450times of the clinical equivalent dose.4. The study on the mechanism of anti-ischemic stroke effective fractionExpression levels of inducible nitric oxide synthase mRNA, neuronal nitric oxide synthase mRNA and endothelial nitric oxide synthase mRNA were detected by RT-PCR at reperfusion24h after cerebral ischemia. The activation extracellular-signal regulated kinasel/2and p38were investigated by Western Blot. The mechanisms the Diospyros Kaki effective fraction protect cerebral ischemia and reperfusion injury include anti-lipid peroxidation, up-regulation eNOS mRNA expression and down-regulation iNOS mRNA and nNOS mRNA expression, increasing the activation of ERK1/2and inhibiting the activation of and p38signal pathways.All in all, chemical fraction of10%-50%ethanol elution,from macroporous resin with75%ethanol extract of Diospyros Kaki, has good resistance to ischemic stroke activity and is able to regulate the expression of NOS and activation of MAPK to protect the ischemia-reperfusion injury in brain tissue. The effective fraction has a good clinical application prospect. |
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