Extraction And Preparation Of Acidic Polysaccharides From Ziziphi Spinosae Semen And Its Protective Effect On Caco-2 Cells

Zizyphus jujube kernel,as one of the common medicinal and food homologues in China,is rich in saponins,phenolic acids,flavonoids,polysaccharides,amino acids and other active ingredients.It has obvious anti-oxidation,anti-inflammation,calm and sleeping effect.Main producing areas are Shaanxi,Hebei,Henan,Inner Mongolia and so on.Jujube seed polysaccharides have many biological activities.The results showed that ziziphus jujube seed polysaccharides had important significance in immunomodulatory activity.In this study,box-Behnken response surface method was used to optimize the reflux extraction process of polysaccharides from jujube seed,and macroporous resin static decolorization process and various separation and purification technologies were used to prepare polysaccharides from Jujube seed,and their physical and chemical properties and structural characteristics were detected.On this basis,the protective effects of acidic polysaccharides from ziziphus jujube kernel on colon cancer cell model Caco-2 were explored.The main results are as follows:1.The extraction process of polysaccharides from jujube seed was optimized by hot water reflux and Box-Behnken response surface methodology.The influence of various factors on the extraction rate of polysaccharides from Jujube seed was as follows:Na OH concentration（C）>solid-liquid ratio（B）>extraction time（A）.Regression analysis showed that the optimal extraction conditions were as follows:Na OH concentration 0.59 mol/L,reflux time 2.36 h,solid-liquid ratio 1:16.56（g/m L）,the predicted value was 1.98%.On this basis,the experiment was repeated for 3 times,and the measured average extraction rate of polysaccharide was 1.96%.The difference between the actual measured value and the predicted value was 0.02%,indicating that the experimental model was feasible to predict the relationship between various factors and the effect surface.2.On the basis of the screening of crude polysaccharide,the decolorization of resin was carried out,and H103 was comprehensively considered as the decolorization resin of polysaccharide.When the resin dosage was 0.3 g/m L,polysaccharide concentration was 3mg/m L and decolorization time was 4 h,the best effect was achieved.The polysaccharides were separated and purified by DEAE-Sephrose-Fast-flow column chromatography.After eluting with different concentrations of Na Cl,the fractions of polysaccharides were 0mol/L>0.2 mol/L>0.3 mol/L>0.5 mol/L.Four polysaccharide components,named ZY-1,ZY-2,ZY-3 and ZY-4,were obtained after dialysis,concentration and freeze-drying.The purity of ZY-2,ZY-3 and ZY-4 is higher.Does not contain nucleic acid,protein and other substances.3.The external morphology and microscopic observation showed that polysaccharides ZY-1,ZY-2,ZY-3 and ZY-4 were milky white,loose,porous,flocculent,and had no obvious odor.Soluble in distilled water,insoluble in organic solvents.The results of color reaction showed that the four polysaccharides were reductive polysaccharides.The molecular weights of ZY-1 were 109.39 and 8.91 k Da determined by HPLC-ELSD.The molecular weights of ZY-2,ZY-3 and ZY-4 are 7.76,10.71 and 8.31k Da.HPLC pre-column derivatization method showed that the monosaccharides were arabinose,rhamnose,glucuronic acid,xylose,galacturonic acid,glucose,mannose and galactose.Ir spectra showed that the four polysaccharides had characteristic absorption in the range of 3423～3456 cm^-1,2915～2937 cm^-1,1400～1420 cm^-1,1062-1086 cm^-1and 619～669 cm^-1.Nuclear magnetic resonance spectroscopy（~1H-NMR）showed that the four polysaccharides were mainly composed of A-pyranose.Congo red experiment results showed that the four polysaccharides did not have three helix structure.In summary,ZY-2,ZY-3 and ZY-4 are acidic polypyranose sugars.4.MTT assay showed that ZY-2,ZY-3 and ZY-4 could significantly promote the proliferation of Caco-2cells after treated with ZY-2,ZY-3 and ZY-4 in high concentration（25μg/m L）.Western Blot assay showed that the expression of tight junction protein in l PS-induced Caco-2 cells increased in a dose-dependent manner after treated with four polysaccharides at different concentrations for 24 h.RT-PCR results showed that ZY-2,ZY-3 and ZY-4 had protective effects on LPS-induced Caco-2 cells.The results showed that ZY-2,ZY-3 and ZY-4 had protective effects on LPS-induced Caco-2 cells.