The Experimental Study Of Gingerols And Flfavonoids On Preparation And Anti-cerebral Ischemia In PC12 Cells

Objective:To prepare and assay the content of gingerols and flavonoids in green rhizomes, and to study the protective effects on cerebral ischemia in rats and the inhibition effects in the H2O2 induced ischemia in cultured rat pheochromocytoma cells(PC12).Methods:1. The total gingerols in green rhizomes were extracted by 75% ethanol.EtOH-H2O was used as the eluent to prepare gingerols by polyamide column. To detect the 50% and 95% ethanol dissolutions respectively in TLC with petroleum ether-ethyl acetate (7:3) as developing agent, with 1% aluminum chloride solution and 5% ferric trichloride as chromogenic reagent. Then recycled the ethanol parts respectively to get the total gingerols and total flavonoids, and then assay the ginger flavonoids by UV spectrophotometry.Petroleum ether-ethyl acetate (7:3) was used as the eluent to isolate the monomeric compounds in gingerols by column chromatograph of silicone gel. 6-gingerol and 8-gingerol were used as control, 5% ferric trichloride was used as chromogenic reagent, to detect the monomeric by TLC.Prep-HPLC chroatophic conditions for isolated total flavonoids: column: C18ODBTM10um(30mm×150mm);Mobile phase: MeOH-H2O (60:40); detection wavelength: 370nm; flow rate: 15ml/min; injection volume: 5ml.2. H2O2 model was used to induce the cytotoxicity in PC12 cells, the morphology of PC12 cells was observed by microscope, the cell damage was measured by MTT assays, to observe the protective effects of 6-gingerol,8-gingerol,main gingerols, main flavonoids and the reference substance.Results:1. 0.8kg extractum ginger was extracted from 27.06 kg green ginger by 75% ethanol, the ER was 2.96%. 126.0g total gingerols were separated from 500g extactum ginger, the ER was 25.2%. And we got two monomers of 6-gingerol and 8-gingerol. The content of flavonoids in ginger total flavonoids was 12.86% with rutin as the control, and the RSD was 1.86%.929.5mg Flavonoid-1and 40.6mg compound-2 was extracted respectively. The content of Flavonoid-1 was 0.79% in total flavonoids with Flavonoid-1 as control. 2.Compared with the model,6-gingerol 1μM,10μM;Total- ginger flavonoids (0.01—0.5μg/L )and total gingerols 10μM could significantly increased A570OD value and the survival rate of oxygen-glucose deprivation cultured PC12 cells. (P﹤0.05 or P﹤0.01).There were no significant differences of 8-gingerol and total-gingerols.Conclusions:1. The method is simple, accurate and can be used as theoretical basis for further studying in ginger flavonoids'extraction process.2.6-gingerols (1μM, 10μM), total-flavonoids0.01-0.5μg/L and 8-gingerol (10μM) can significantly reduce the apoptosis rate of induced injury PC12 cells.