Research On The Method For Evaluating The Quality Of Pleurotus Citrinopileatus Sing And The Effect Of It On Cyclophosphamide Hepatotoxicity In Mice

Objective:①To establish methods for the determination of Fumaric acid, Nicotinic acid and Polysaccharides in Changbai Mountain Pleurotus citrinopileatus Sing which can evaluate and control the quality of it.②To investigate the methods of P. citrinopileatus Sing polysaccharides extraction, purification and determination, and the protective effect of P. citrinopileatus Sing polysaccharides on cyclophosphamide hepatotoxicity in mice, which supplied experimental basis for clinical applications.Methods:①Reversed-phase high performance liquid chromatography (RP-HPLC) was performed using a C18(5μm,250 mm×-4.6 mm) column with methanol-0.01 mol/Lpotassium dihydrogen phosphate solution(5:95) as mobile phase detected at 216 nm to determine the content of Fumaric acid; Ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC) was performed using a C18(5μm,250 mm×4.6 mm) column with methanol-0.004mol/L heptane mahogany acid sodium-acetic acid(5:95:0.2) as mobile phase detected at 263 nm to determine the content of nicotinic acid. Polysaccharides was determined by Phenol-Sulfric acid method and Anthrone-Sulfuric acid method.②The extraction of P. citrinopileatus Sing polysaccharides was applied with water extracting-alcohol precipitating and deproteinizing with trichloroacetic acid. Polysaccharide content was detected by Phenol-Sulfric acid method and protein content was detected by Bradford method.70 mice were randomly devided into five groups:an unhandle control group, a cyclophosphamide model group and the others are P. citrinopileatus Sing polysaccharides of high-dose(0.8 g·kg-1·d-1), medium-dose(0.4 g·kg-1·d-1) and low-dose(0.2 g·kg-1·d-1) groups.10 days later, The protective effect of P. citrinopileatus Sing polysaccharides on cyclophosphamide in mice was evaluated according to the liver malondialdehyde(MDA) and glutathione(GSH), the liver microsomal Aminopyrine-N-demethylase(CYP2E1) and Erythromycin-N-demethy-lase(CYP3A) activities, and ultrastructural morphology change of the liver cells were observed.Results:①The calibration curve of fumaric acid was linear from 0.16 to 1.6μg with r=0.9999. The average recovery was 101.59% with RSD 1.28%. The calibration curve of nicotinic acid was linear from 0.08 to 0.4μg with r=0.9999. The average recovery was 103.84% with RSD 2.80%. The recovery of polysaccharides by the method of phenol-sulfuric acid was 99.85% with RSD 1.35%; the recovery of polysaccharides by the method of Anthrone-Sulfuric acid was100.07% with RSD 1.06%; The content of polysaccharides which was measured by the method of Anthrone-Sulfuric acid is higher.②Compared with the model group, drug groups decreased the hepatotoxicity induced by cyclophosphamide in mice. The liver MDA content was significantly decreased(P<0.05) and GSH content was significantly increased(P<0.01) in high-dose group. The liver microsomal CYP2E1 activity was significantly increased(P<0.05) in medium-dose group, and the CYP3A activity was significantly increased(P<0.01) in high-dose, medium-dose and low-dose groups. The cell injury of the liver cells in drug groups significantly recovered.Conclusion:①The methods were proved to be accurate and simple, and can be selected to control the quality of P. citrinopileatus Sing.②. citrinopileatus Sing polysaccharides antagonize the side effect of cyclophosphamide hepatotoxicity in mice.