The Components Analysis Of Scallion Through Cerebral Ischemia Rats Blood Brain Barrier And Pharmacodymics Research Of Their Cerebrospinal Fluid

ObjectiveOn the basis of the existing relevant research of Tongqiaohuoxue soup, by optimizing the extraction process of scallion, the chemical composition of drugs of which scallion extract through the blood-brain barrier of Cerebral ischemia model in rats into the cerebrospinal fluid in were analyzed to determine scallion extract the component of scallion extract into the cerebrospinal fluid, as one of the bases of scallion has a protective effect of the brain. Then using the cerebrospinal fluid pharmacological methods, we study the protective effect and part of the mechanisms of the drug-containing cerebrospinal fluid on PC12 cells by glutamate induced injury.Methods1. Screening the extraction methods. The extraction methods includs supercritical fluid extraction, steam distillation, petroleum ether extraction, 80% ethanol extraction, 50% ethanol extraction and water extraction method. The extract used in PC12 cells by glutamate induced injury, by using the MTT method to analyze the cell viability and observecell morphology, to select the best extraction method.2. Using the suture method to obstruct rats middle cerebral artery, preparing cerebral ischemia model, and evaluating the model.3. Scallion extract was prepared by using extraction method has been optimized to fed the sham group and cerebral ischemia model rats respectively, and the control group fed the sham group and cerebral ischemia model rats respectively with distilled water, to collect and prepare the drug-containing cerebrospinal fluid sample and blank sample.4. Using GC-MS analyze the composition of the scallion into the cerebrospinal fluid.5. The protective effect of the drug-containing cerebrospinal fluid on PC12 cells by glutamate induced injury. Intend to use an inverted microscope to observe cell morphological changes in each group, use MTT to detect cell viability, use LDH to detect permeability of cell membranes, use flow cytometry to detect the effect of glutamate-induced apoptosis in PC12’s.6. Using western-blotting method to detect the expression of Bcl-2, Bax, caspase-3 and caspase-9.Results 1. The best extraction method for screeningThe results showed that 50% ethanol extract of scallion has the strongest protective effect on PC12 cells by glutamate induced injury. Subsequent experiments were performed using 50% ethanol to extract onions. 2. GC-MS Component analysisThe cerebrospinal fluid samples for each group and 50% ethanol extracts were analyzed comparatively by using GC-MS. The results showed that the scallion components can enter the cerebrospinal fluid of rpats in cerebral ischemia model group are Hexadecanoic acid methyl ester, Methyl cyclopentane undecane, 5,8-dimethyldodecane; The cerebrospinal fluid in sham-operated group and ischemia group all contain 5,8-dimethyl-dodecane. 3. The drug-containing cerebrospinal fluid pharmacology(1) The MTT assay test results showed that cell growth inhibition rate was 75% with a concentration of 25 mmol/L(the concentration of cells modeling) glutamate injuring PC12 cells for 24 hours; By using MTT to detect the cell viability, the results showed that, compared with the model group, 15% concentration of cerebrospinal fluid of rats were gavaged with scallion extract in sham-operated group and cerebral ischemia group have a protective effect to some extent on PC12 cells by glutamate induced injury. The protective effect of the drug-containing cerebrospinal fluid in cerebral ischemia group is better.(2) The results of LDH showed that, compared with the model group, 15% concentration of cerebrospinal fluid of rats were gavaged with scallion extract in sham-operated group and cerebral ischemia group can cause the inhibition to some extent on the leakage of lactate dehydrogenase in PC12 cells by glutamate induced injury.(3) Annexin V/PI-FITC test results showed that the drug-containing cerebrospinal fluid can significantly reduce the rate of cell apoptosis in PC12 cells by glutamate induced injury.(4) Western-Blotting protein expression results showed that, compared with the control group, the glutamate-induced injury group was significantly raised the expression of Bax, caspase-3 and caspase-9 protein and significantly reduced the expression of Bcl-2.ConclusionExperimental results show that the best extraction method of the active ingredient of scallion is 50% ethanol method. Analysis results showed that, the scallion components can can enter the cerebrospinal fluid of rats in cerebral ischemia model group are Hexadecanoic acid methyl ester, Methyl cyclopentane undecane, 5,8-dimethyldodecane; The cerebrospinal fluid in sham-operated group and ischemia group all contain 5,8-dimethyl- dodecane. These types of ingredients enter the cerebrospinal fluid through the blood-brain barrier might the active ingredient of scallion. The main anti-apoptotic mechanism is to reduce the activation of caspase-3 and caspase-9, decrease the expression of Bax and increase the expression of Bcl-2, thereby reducing the apoptosis of PC12 cells.