| Objective Guided by activities for optimize the extraction process of bioactivepolysaccharides from Zhejiang zhoushan,s Mytilus coruscus originated from the sea area ofZhejiang zhoushan, extraction and active research for the mussel polysaccharide structure andactive research provided the scientific basis.Methods optimize the extraction process of bioactive polysaccharides, the hot water extra-ction and combined the papain and trypsin digestion method after water extraction for musselpolysaccharide were compared. The physicochemical properties, purification by DEAE–cellu-lose52column chromatography were get three components (MT1、MT2、MT3), after by G-100column chromatography have separation and purification the total of five components (MT1-1、MT1-2、MT2-1、MT2-2、MT3-1), the basic of system analysis physical and chemical propertiesof polysaccharides, including comparing its total sugar, Bradford method protein, uronic acidand sulfate content. The sulphation and phosphorylation structure modification of MT1, theDU-145cells and H1299cells treated with MT3-1, analyzed by FCM stained with AnnexinV-FITC/PI.Results Confirm the optimum extraction conditions of combined enzymatic hydrolysispolysaccharides of Mytilus coruscus, temperature is60℃, the optimum reaction time is3h, theratio of solid-liquid is1:20, plus the amount of enzyme papain and trypsin were1%and0.5%,the optimum pH was7,10. Proven the extract polysaccharide of Mytilus coruscus is the highestunder the condition. Factors: pH, solid-liquid ratio> temperature> amount of enzyme.Throughdata comparation, it was concluded that enzymatic extraction method is better than the hot wat-er extraction based on the yield and purity, and biological activity analysis. Yields of crude pol-ysaccharide by enzymatic extraction is23.69%. After separation andpurification, componentsanalysis showed that the combined enzyme extraction can improve glycosaminoglycanslikepolysaccharide content (MT3), which can reach13.5%. Monosaccharide composition analysisindicated that MT3mainly consisted of Man, GlcN, GlcUA, Gal and Fuc, molecular weight ofwhich was about18kd.The results of antioxidant in vitro experiment are: The concentration ofpolysaccharide was10mg/mL, DPPH scavenging rates were: MT was84.18%, RT was80.01 %. EC50were3.75mg/mL and4.97mg/mL; MT and RT superoxide anion radical scavengingrates were83.96%and76%. EC50was5.01mg/mL and6.48mg/mL; MT on lipid peroxidescavenging rate reaches82.33%, RT polysaccharide76.01%. MT and RT on lipid peroxideswere cleared EC502.41mg/mL and3.46mg/mL; MT and RT for reducing power absorbance of0.62,0.51. EC50were7.89,9.61. the most superior scavenging active component of MT.The sulfated and phosphorylated structural modification of MT1, in vitro inhibition ratesof samples were significantly increased, reached85.04%and81.67%. Compared with othercomponents, it showed good in vitro anti-tumor activity of MT3-1. in vitro prostate cancer DU-145cells and human lung cancer H1299cells inhibition rate up to88.39%and89.04%,48hIC50was2.71mg/mL,2.415mg/mL.The above two kinds of cells after HE staining, Morpholo-gical changes of apoptosis: cell shape is irregular, the increaseof megakaryocyte, chromatinagglomeration, nucleus pycnosis of etc. AO/EB double dye were studied under the fluorescentmicroscope, the control group was no obvious apoptosis cells, by24h MT3-1, with high levelsof early apoptosis cells. Effect after24h DU-145early apoptosis rate were7.49%,10.83%and26.46%, respectively, H1299early apoptosis rate were4.98%,9.86%and19.74%, significantlyhigher than the control group (P <0.05).Conclusion For mussel polysaccharide extraction process, enzymolysis technology afterhot water extraction not only can improve the purity of polysaccharide through degradation ofprotein, but also can significantly improve the the content of glycosaminoglycans. glycosamin-oglycans is the main bioactive component of mussel polysaccharide and has good antitumor ac-tivity in vitro. |
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