The Protective Effect Of Active Flavonoids In Propolis Against Oxygen-glucose Deprivation In N2a Cells And Its Mechanisms Of Action

Bee Propolis was collected from the tree bud and bark by bees, which mixed secretion of beetongue gland and wax gland. It was processed by bees and transformed into jelly substance bybees.Propolis is rich in terpenes, phenolics, flavonoids and other bio-active substances, which areanticancer, antibacterial, anti-inflammatory, antioxidant, hepatoprotective,and protecting brain.In recentyears Propolis become the focus of research in the domestic and overseas. Nonfatal disease such asstroke and cerebral infarction which are caused by ischemia, have an elevated incidence.The studyshowed that ethanol extract from propolis could clear free radical oxidation and had high protectiveeffect against ischemia-reperfusion injury. Compounds pinocembrin apigenin and chrysin in propolisalso showed high protective effect against ischemia-reperfusion injury.In this paper, the propolis was separated and purified systematically. N2a cells injured by OGD4h,which is the model of cerebral ischemia reperfusion injury in vivo, was established. Cell viabilitymeasured by CCK-8assay was considered as the screening criterion in the experiment. After extractedand dryed under reduced pressure, the propolis was divided into five portions, including40%water-soluble portion,40%alcohol-soluble portion,70%water-soluble portion,70%alcohol-solubleportion and95%alcohol-soluble. The ethanol extract was used to be activity screened. The resultsshowed that70%ethanol extract group showed the highest protective effect.70%alcohol-solubleportion was the active site in propolis which against cerebral ischemia reperfusion injury.70%alcohol-soluble portion of propolis were successively extraced with petroleum ether, dichloromethane,ethyl acetate and acetone.The result showed that the dichloromethane extracts showed the highestprotective effect.Column chromatography used to seperating dichloromethane extracts.Then we gotFr.1～5, and Fr.3showed the highest protective effect.HPLC were usd to seperating Fr.3, then we gotpinocembrin, pinobanksin, pinobanksin-3-acetate, chrysin, galangin five components.Pinocembrin, pinobanksin, pinobanksin-3-acetate, chrysin, galangin and apigenin were used totreated with N2a cells injuryed by OGD. The results showed that pinocembrin, chrysin, pinobanksin,galangin and apigenin five monomers showed the higher protective effect. Apigenin and pinocembrinshowed higher protective effect than ligustrazine. Pinobanksin galangin and chrysin showed highlysignificant difference with model group in middle and high concentrations. We selected five higheractivity compounds to study on the mechanism of neuroprotective effect of propolis.One of the mechanism of cerebral ischemia-reperfusion injury is cell oxidative damage caused byactive oxygen free radicals. The antioxidation of Propolis experiment results showed that after treatedby pinocembrin, chrysin, pinobanksin, galangin and apigenin the five compounds, MDA content in cellsdecreased significantly, the SOD, GSH-Px activity were enhanced in cells, thereby reducing ischemiareperfusion injury induced oxidative damage to cells. Pinocembrin and galangin showed higherantioxidant effect in3concentrations(7.5μM,15μM,30μM)than ligustrazine.Pinobanksin galangin andchrysin showed high antioxidant effect in middle and high concentrations. One of the mechanism of cerebral ischemia-reperfusion injury is cell damage caused by calciumoverload.The intracellular free calcium concentration determination experiment results showed that aftertreated by pinocembrin, chrysin, pinobanksin, galangin and apigenin the five compounds, intracellularfree calcium concentration of N2a cells decreased significantly, by inhibiting intracellular calciumoverload and reduce the severity of impaired N2a cell, thereby antagonizing ischemia reperfusion cellinjury caused by OGD. Pinocembrin and galangin showed higher protective effect in3concentrationsthan ligustrazine. Pinobanksin galangin and chrysin showed highly significant difference with modelgroup in middle and high concentrations.One of the mechanism of cerebral ischemia-reperfusion injury is cell apoptosis. The anti-apoptosisof propolis experiment results showed that pinocembrin, chrysin, pinobanksin, galangin and apigeninthe five compounds can effectively reduce the apoptosis of N2a cells induced by the apoptosis rate ofOGD, and up regulate the expression of Bcl-2mRNA, and down-regulate Caspase-3Bax expression.Sopropolis can inhibit the apoptosis pathway. Pinocembrin and galangin showed higher anti-apoptosiseffect in3concentrations than ligustrazine. Pinobanksin galangin and chrysin showed highly significantdifference with model group in middle and high concentrations.