Extraction And Purification Technology Of Gekko Polysaccharide Based On The Anticancer Activities And Study Of Property

Objective:In this paper,we optimize the extraction and purification technology of Gekko polysaccharide and its anti-tumor and immune activity.We also study the physical and chemical properties,molecular weight,monosaccharide constituents,provides scientific basis for clinical application.Methods:1.Different extraction methods including water extraction,ultrasonic extraction,enzymatic hydrolysis-water extraction and enzymatic hydrolysis-ultrasonic extraction were used to determine the best extraction technologies on the content of polysaccharide.Furthermore,we tracked the anti-cancer activities of these polysaccharide.Single factor experiment and orthogonal experiment were used to investigate the optimum extracting condition.2.We optimize the purification technology including Sevage method,enzymatic hydrolysis-Sevage method,TCA method,enzymatic hydrolysis-TCA method and TCA-Sevage method of Gekko polysaccharides on the basis of the content of polysaccharides,the removal ratio of protein as well as its anti-cancer activities.DEAE-Sepharose Fast Flow(C1-)weak polar anion exchange chromatography and SephadexG-150 gel column chromatography is used to isolate and purify the Gekko polypeptides,inspect the purity of its separate components and its anti-tumor activity.3.The purified polypeptides were identified by a-naphthol,Biuret reagent,and iodide-Potassium iodide.Barium sulfate turbidity was used for determining the content of sulfate group in polypeptides.The properties and composition analysis was studied by IR spectra,GPC,paper chromatography and GC.Results:1.Gekko polysaccharide obtained from water extraction could inhibit the proliferation of HepG2 and SKBR3,as well as promote that of lymphocytes.The content of polysaccharide was 4.049%in the optimum extracting condition of extracted triple for 1 hour each time with the ratio of material to solvent(1:15).2.The deproteinization effect was optimal with papain + TCA method,which the protein extractive coefficient can be up to 70.46%,the content of polysaccharide is 14.27%and with 49.06%inhibitory ratio on human HepG2 cells.Only one component was obtained from DEAE-Sepharose Fast Flow(Cl-)weak polar anion exchange chromatography and SephadexG-150 gel column chromatography,the purity of polysaccharide is 69.73%and with 74.13%inhibitory ratio on human HepG2 cells3.Keep palace refined polysaccharide components uniform Gekko polysaccharide was light yellow powder,preliminary thought is a kind of polysaccharide,it dissolved in water easily,pH6-7,poorly dissolve in acetone,ethanol and other reagents.The content of polysaccharide was 69.73%,protein content was 18.83%,and the molecular weight was 73956 Da.IR showed the characteristic peak of polysaccharides.The monosaccharide composition of Gekko polysaccharide were mannose,galactose and glucose,which detected by GC.Mole ratio was 2:2:3.Conclusion:In the present study,water extraction was considered as the best extraction method because of relative high yield ratio and content of polysaccharides,which can be achieved with the ordinary extraction equipment and suitable for large-scale industrial production.Furthermore,Gekko sulfated polysaccharides obtained from water extraction possessed stronger inhibitory effects on the proliferation of human hepatoma and breast cancer cells and stimulative effects on the proliferation of lymphocytes compared to those obtained from other methods.We further optimized the water extraction technology of Gekko polysaccharides via single factor experiment and orthogonal experiment.In the condition of extracted triple for 1 hour each time with the ratio of material to solvent(1:15),the average content of Gekko sulfated polysaccharides was 4.49%and RSD was 1.18%.The deproteinization effect was optimal with papain + TCA method,which the protein extractive coefficient can be up to 70.46%,the content of polysaccharides is 14.27%and with 49.06%inhibitory ratio on human HepG2 cells.Only one component was obtained from DEAE-Sepharose Fast Flow(Cl-)weak polar anion exchange chromatography and SephadexG-150 gel column chromatography,the purity of polysaccharides is 69.73%and with 74.13%inhibitory ratio on human HepG2 cells.This extraction and purification technique is reasonable,feasible,and provides a scientific basis for industrial production.