Studies On Crude Polysacchairde Extracted From Abalone Viscera By PEF Combined With Enzymatic Method And Its Antioxidant Acticity

The abalone is a primitive marine shellfish. It is not only nutritious but also has a veryhigh medicinal value. The gonads and other organs, accounting for one-third of the abalone weight,are abandoned or turned into low-priced products in the process, resulting in a tremendous wasteof resources. In this study, the abalone viscera was used as experimental material to extractabalone viscera crude polysaccharide （AVCP） by pulsed electric field （PEF） combined withtraditional enzyme. The optimum extraction parameters were studied. Its antioxidant function wasstudied by conventional determination in vitro, vitro cellular level determination and vivoexperiments in mice.The study included:1. The optimum conditions of extracting crude polysaccharide from abalone visceraThe method: by single factor experiments, the key parameters of PEF treatment, and the types ofenzyme were determined. On the basis of single factor experiments, the impacts of time,the solid-liquid ratio, enzyme concentration and temperature on the extraction of AVCP, and thebest extraction conditions of crude polysaccharide from abalone viscera by PEF combined withtraditional enzyme were studied through orthogonal experiment.The results:①The electric field intensity was30kV/cm, and the number of pulses was12.②With neutral protease, the extraction time was2.5h, the solid-liquid ratio was1:6, theenzyme amount was2.4×104U/g, and the temperature was55°C.③The polysaccharide content of powder was60.39%.2.The study on the vitro antioxidant activity of AVCPThe method: the antioxidant activity of AVCP was studied by conventional methods in vitro.①The total reducing capacity was studied through potassium ferricyanide reduction method.②The effect ofAVCP on scavenging·OH were studied by1,10-Phenanthroline monohydrat-e-Fe²⁺-H₂O₂method.③The effect of AVCP on scavenging O₂^-· were studied by pyrogallol auto-oxidationmethod.The results:①The reducing capacity of AVCP was dose-effectively related with polysaccharide concent- ration. And its reducing capacity increased with the polysaccharide concentration increasing.②The clearance rate of·OH had a dose-dependent relationship with the polysaccharide conc-entration.The clearance rate of·OH was25.25%when the polysaccharide concentration was10mg/mL, IC₅0=20mg/mL, the regression equation was y=0.025x+0.000,R²=0.995.③High concentrations ofAVCP inhibited pyrogallol autoxidation. The effect of removingO₂^-·was not prominent and the clearance rate of O₂^-· was25.40%when the polysaccharide con-centration was20mg/mL,IC₅0=41.5mg/mL,the regression equation was y=0.012x+0.002,R²=0.987.However, it showed its anti-oxidation potential.3. The study on the vitro cell antioxidant activity of AVCPThe method: the antioxidant activity of AVCP was studied by using the H₂0₂-induced oxidativestress model of HepG2cells inhanced on the level of cell in virto.①The survival rate of oxidative damage cell was detected through MTT method.②The content of malondialdehyde （MDA） and superoxide dismutase （SOD） activity weredetermined.The results:①In the final concentration range of10⁸0μg/mL, cell survival rate increased from71.59%to84.32%by AVCP. So it had a protective effect as well as antioxidant potential.②The MDA content was significantly lower （P<0.01）, and had a certain therapeutic effect;the SOD activity was significantly increased （P<0.01）, showing impressive prevention.4. The study on the vivo antioxidant activity of AVCPThe method: aging model, induced by D-galactose, was used to study antioxidant activity ofAVCP in vivo.①To detect the content of malondialdehyde （MDA） in serum and liver homogenate.②To detect the content of glutathione （GSH） in serum and liver homogenate.③To detect the superoxide dismutase （SOD） activity in serum and liver homogenate.The results:High doses of AVCP significantly reduced the serum and liver MDA content of D-galactose i-nduced aging model mice （p<0.01）, and very significantly increased the SOD activity in serum and liver of mice （p<0.01）. It could significantly improve the level of GSH in the sera of mice （p<0.0 1） and significantly improved the level of GSH in the livers of mice （p<0.05）. There was agood dose-effect relationship between the effect on each indicator and the dosage of AVCP whichshowed prominent preventive effect. At the same time, high dose had a significant therapeuticeffect, which explained AVCP had antioxidant activity in vivo.