| There are abundant pomegranate in China,the peel of it could be recycled but most of them were discarded at present.As one of the major active ingredient in pomegranate peel,pomegranate peel polysaccharide was less reported while the study mainly concentrated on the extract of polyphenols and its bioactivity including antioxidant and antineoplastic activities.Therefore,this paper invertigated on the extracted techniques,separation and purification methods of pomegranate peel polysaccharide,preliminarily characterize on the structure of PPP and the three fractions of isolated components and their immunoregulation activity in vitro,and finally demonstrated the immunoregulation capacity of pomegranate peel polysaccharide by the in vivo experiment which showed PPP could protect the mice from damage by CTX.The main conclusions are as following.(1)As for pectinase-assisted extraction of pomegranate polysaccharide,response surface method was used to analyse the three factors having important effect on the yield of polysaccharide in the single factor experiment.The results showed that the optimal extraction technique was:emzymolysis temperature of 55 ?,solid-liquid ratio of 20.5:1 mL/g,pH 7.5,enzymatic dosage of 0.68%,emzymolysis time of 19.7 min,the average yield of polysaccharide was 27.3±0.08%.This technique is simple and high yield.(2)The pomegranate polysaccharide obtained by pectinase-assisted extraction was processed by decolorization and deproteinization to get Pomegranate peel polysaccharide(PPP).PPP-1,PPP-2 and PPP-3 are fractions purified by Cellulose DEAE-52 and Sephadex G-100 column chromatography.The total sugar content determined by phenol-sulfuric acid method was 75.62%,82%and 90.02%,respectively.The uronic acid content determined by Sulfuric acid-carbazole method were 6.33%,5.93%and 9.53%,respectively.The infrared spectra showed that there were ?-pyranose ring in PPP,PPP-1,PPP-2 and PPP-3,which can demonstrate that they are polysaccharides.The analysis of monosaccharide components and mole percent in PPP,PPP-1,PPP-2 and PPP-3 showed that their main monosaccharide were glucuronic acid,glucose and xylose,all of which were acid heteropolysaccharide.The molecular weight analysis showed that PPP had two molecules of different weights and they were 274.86 kDa and 3.90 kDa.The molecular weight of PPP-1,PPP-2 and PPP-3 were 136 kDa,146 kDa,184 kDa,respectively.ESEM result showed that PPP-1 and PPP-2 were composed of many small fragments,PPP-3 was composed of bigger flake materials,and all of them had a smooth surface.(3)In a certain dose(4 ?g/mL,8 pg/mL,16 ?g/mL and 32 ?g/mL),PPP and its purified components(PPP-1,PPP-2 and PPP-3)acted on RAW264.7 respectively and the in vitro immunoregulation activity was identified by MTT method,neutral red test,NO release amount and cytokines content(TNF-a,IL-2,IL-6 and IFN-?).The results showed that after RAW264.7 were stimulated by PPP and its fractions,they had no poisonous effect on RAW264.7 cells and could improve the phagocytosis.Besides,it could remarkably promote the secretion of NO,TNF-?,IL-2,IL-6 and IFN-?,Therefore,the vitro test showed that both PPP and its fractions had immunological enhancement effect.(4)Mice experiment showed that PPP could improve the immunoregulation ability of the mice.The mice were immunized to different concentration of PPP,the weight of mice received CTX restrain,viscera index(spleen,thymus,liver,heart,kidney,testicle,epididymis,and epididymis fat),cytokine content in serum(Ig-A,Ig-G,Ig-M,TNF-?,IL-2 and INF-?),whole blood indicators(WBC,RBC,Hb and PLT),the rate of lymphocyte transformation and delayed type hypersensitivity can be improved in different degrees.The result showed a dose-effect relationship,which means the higher the concentration of PPP is,more obvious the effect will be. |
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