Biological Activities And Structural Characteristics Of Polysaccharides From Abrus Cantoniensis And The Rapid Preparation For The Bioactive Polysaccharide

Abrus cantoniensis(Hance)belongs to Abrus genus in the leguminosae family and has been used widely as an edible vegetable and medicinal plant in tropical areas of Asia for thousands of years.Abrus cantoniensis is widely distributed in Guangdong,Guangxi,Fujian,Hainan and Yunnan provinces of China.Previous studies have also demonstrated the potential biological activities of this plant,including antioxidant,anti-tumour,antimicrobial,hepatoprotective,and immunomodulatory activities.In this study,the effects of different removing impurity process and purifying method on the structure and antioxidant,anti-tumor and immunomodulatory activities of water soluble polysaccharides from Abrus cantoniensis were investigated.Polysaccharide fraction AP-ACl-3 was found to be one of the bioactive polysaccharide of Abrus cantoniensis.Meanwhile the rapid preparation route for AP-ACl-3 was investigated.Based on the rapid preparation route,AP-ACl-3? was successfully extracted,isolated and purified from Abrus cantoniens and its structure was fully elucidated.Moreover,effects of ultrasonic treatment on the stucture,antioxidant,antitumor and immunomodulatory effects of AP-ACl-3 were also investigated.The results were listed as follows:(1)Removing impurity process exhibited great effects on the chemical component,antioxidant,anti-tumor and immunomodulatory activities.Abrus cantoniens pretreated with 95% ethanol and deproteinization with Sevage method can greatly avoid the presence of the pigment,polyphenol and flavonoids,and effectively protect polysaccharides with Mw larger than 10000 Da.All the crude polysaccharides were free from protein,indicating that crude polysaccharide without deproteinization treatment could not contain protein.Furthermore,polysaccharides with Mw larger than 10000 Da can be fully precipitated by 50% ethanol,and T-50 polysaccharide fractions showed better antioxidant,anti-tumor and immunomodulatory activities,followed by T-80 and T-30.(2)To fully exploit the potential of the polysaccharide in Abrus cantoniensis,nine polysaccharides(APFS)were isolated and purified.In vitro anti-tumour and immunomodulatory activities were also investigated and compared using the rank-sum ratio(RSR)method.Results demonstrated great differences existed in their structure and bioactivities among these polysaccharides.Among the nine polysaccharides,AP-ACl-3(4.4×104 Da,83.31%)had relavtively higher yield(613.5 mg/kg),sugar content(83.93±1.60%)and RSR_compr values,was considered to be one of the bioactive polysaccharide.Meanwhile the rapid preparation route for AP-ACl-3 was also established.(3)AP-ACl-3 exhibted considerable bioactivities and it could significantly(p<0.05)prevent the migration of MCF-7 cells(74.10% inhibition rate at 250 ?g/mL)and stimulate lymphocyte proliferation along with NO production of peritoneal macrophages.AP-ACl-3 could be explored as a novel potential anti-tumour and immunomodulatory agent.(4)According to the chemical component,IR spectrum,HPGPC,monosaccharide,SEM,AFM,CD,XRD and TG analysis,AP-ACl-3 and the polysaccharide showing lower RSR_compr value(AP-AOH80-1),exhibited great differences in the molecular weight,monosaccharide composition,IR spectrum,surface morphology,spatial structure,crystal structure and thermal stability,these might be resposible for the great differences in the bioactivities between AP-ACl-3 and AP-AOH80-1.(5)AP-ACl-3? was obtained from AP-ACl-3 by further purifiction using Sephadex G 100 column.AP-ACl-3?(4.4×104 Da)consisted of glucuronic acid,galacturonic acid,rhamnose,arabinose,fructose,glucose,and galactose with a molar ratio of 58.4:4.7:10.2:8.5:4.7:3.3:10.2.The main chain of AP-ACl-3? consisted of glucuronic acid,galacturonic acid,rhamnose,fructose,glucose,and galactose with a molar ratio of 67.2:16.3:12.7:1.3:1.2:1.3.Results revealed that AP-ACl-3? had a backbone consisting of two or more ?4)-GlcAp-(1?residues linked to each other and ?3)-Rhap-(1?,?4)-GalAp-(1? residues,and branching occurred at O-4 position of ?3)-Rhap-(1? residue of the backbone with 1,5-Araf,?6)-Galp-(1?,?6)-Glcp-(1? and terminated with T-Glcp,T-Galp and T-Araf.(6)HPGPC results showed that hot treatment at 80-120 °C for 5 h or at 100 °C within 1 h-12 h,and ultrasonic treatment with power under 500 W within 4 h,AP-ACl-3 showed no change in the molecular weight.After ultrasonic treatment with power of 500 W for 4 h,althought the molecular weight of AP-ACl-3 did no change,SEM,AFM,CD,XRD and TG results showned change in the surface morphology,spatial structure,crystal structure and thermal stability.Moreover,ultrasonic treatment could significantly(p<0.05)improve the antioxidant and antiproliferative activities of AP-ACl-3.Above all,using traditionally boiling water extraction and ultrasonic technique can not cause degradation of AP-ACl-3.(7)The economical,fast and effective conditions for AP-ACl-3 preparation were finally established as: Abrus cantoniensis powder was pretreated with 50% ethanol,and then extracted at 100 °C within 12 h coupled with ultrasonication-assisted extraction technique within 500 W and within 4 h,followed by concentration under reduced pressure at 60 °C.Polysacchride extraction solution was pricipitated with 50% ethanol,and the precipitate was dialyzed(cut-off: 3.5 kDa)against water for 48 h.Polysacchride solution was further purified on DEAE-Sepharose Fast Flow(Cl-)column,and collected the 0.5 mol/L NaCl elution after removing the 0.2 mol/L NaCl elution.