Protective Effects And Mechanism Of Polysaccharide From Platycodon Grandiflorum On Damage Of PC12 Cells Induced By H₂O₂

Platycodon grndiflorum?Jacq?is a perennial herb of campanulaceae and is widely planted in China.Polysaccharide is one of the important nutrients in life activities and has many important and special physicochemical properties and physiological activities,such as low toxicity,safer and functional diversity,etc.It is well known that water-soluble polysaccharide extracted from plants has a lot of significant pharmacological activities,such as boosting immune function,regulating blood sugar and blood fat levels,anti-tumor,anti-oxidation,anti-aging,etc.Polysaccharide as the main component of Platycodon grandiflorum has antioxidant,anti-cancer and immune enhancement and other pharmacological effects.But There are less reports on neuroprotective effect of PGP at home and abroad.Oxidative stress-mediated cell injury via apoptosis or necrosis has long been regarded as the main factor behind neurodegenerative diseases which include Alzheimer 's disease.Peach resin was a kind of resin that made up from rosaceae plants like Prunus persica?L.?Batsch or Prunus davidiana?Carr.?Franch.The main ingredient of peach resin was polysaccharide with free radical scavenging and antioxidant effect.Object:This research will identifie the monosaccharides,molecular weight and purity of PGP,and establish the vitro cell model of PC 12 cells injured by H₂O₂ and determine the protective effects of Platycodon grandiflorum polysaccharide?PGP?on PC 12 cells injured by H₂O₂ with underlying mechanisms of PGP on neuroprotective effect,which provide the scientific basis for the treatment of PGP on neurodegenerative diseases,including AD.This study also dealt with the preparation method and optimization scheme of peach resin mask to provide the basis for the study of polysaccharide isolated from peach resin.Method:The monosaccharides,molecular weight and purity of PGP were identified by anthrone-sulfuric acid method,ultraviolet spectrophotometer,high performance liquid chromatography?HLPC?and high performance gel exclusion chromatography?HPGPC?.The cell viability was analyzed by MTT assay and the model was established by treating PC 12 cells with H₂O₂ to detect the effects of drugs on the proliferation of PC 12 cells,including the effects of H₂O₂ on the proliferation of PC 12 cells and the effect of PGP on the proliferation of PC 12 cells;The levels of LDH,MDA,SOD?WST-8?and GSH-Px were determined by Kits.ROS production was determined by DCFH-DA fluorescence with flow cytometry.mRNA and proteins of NOX2,p22phox and Racl were analyzed by Q-PCR and Western Blot methods.The original peach resin was hydrolyzed and decolorized to peach polysaccharide crude,and made into peach resin mask with accessories.Moisture retention rate was determined by single factor and orthogonal experiments to determine the formula.Results:The results of HPLC showed that PGP was mainly composed of glucose,galactose and mannose at a molar ratio of 7.0:1.1:0.6.HPGPC showed that the elution curve of PGP was a single symmetric peak with molecular weight Mw of about 5.9×103Da.MTT assay showed that the concentration of PGP at 50,100,200 ?g/mL could alleviate the oxidative damage of PC 12 cells induced by H₂O₂.Compared with the model,treatment with PGP for 24 h and 600 ?mol/L H₂O₂ for 2h significantly decrease of LDH,MDA and intracellular ROS production,as well as increase activity of SOD and GSH-px.Furthermore,PGP could down-regulate mRNA and proteins of NOX2,p22phox and Rac.Optimization of preparation program:temperature was 70?,and heating time was 20min.The optimal formulas of peach resin mask were polyvinyl alcohol 13.2g,peach resin 4g,sodium carboxymethyl cellulose 2.6g,sodium alginate 2g,glycerol 4ml,sodium benzoate 0.5g,flavor 2 drops,and pure water 100ml.Conclusion:PGP was a homogeneous polysaccharide,which exhibited protective effect on PC 12 cells injury induced by H₂O₂,it may via the suppression of NOX2 activation.Peach resin was suitable for the production of peach resin mask with good effects.Significance:The monosaccharides,molecular weight and purity of PGP were identified in this reserch,and there are few reserchs on neuroprotective effect of PGP at home and abroad.This study provides a theoretical basis and reference for PGP used on antioxidation and neuroprotective effect.This study also dealt with the preparation method and optimization scheme of peach resin mask,which provided a new direction for the use of peach resin resources,to provide the basis for the study of polysaccharide isolated from peach resin.