Study On Extraction Optimization And Radiation Protection Of Polysaccharides From Poria Cocos Peels

Poria cocos peels are the dry epidermis of Poria cocos sclerotia,which are rich in active substances,but they often were discarded as by-products during the processing of Poria cocos,resulting in a serious waste of active resources.At present,studies of Poria cocos peels in the domestic and overseas mainly focus on triterpenoids,while relatively little attention is paid to Poria cocos peels polysaccharides（PCPP）.Ionizing radiation（IR）has been widely used in industrial,medical and military fields.However,long-term exposure to IR would cause adverse damages to health.Due to the disadvantages of synthetic radioprotectors such as large side effects,high cost and short effectiveness,it has gradually become a research focus to find natural radioprotectors that are safe,efficient,low-cost and suitable for long-term use.Based on this,the extraction conditions of PCPP were firstly optimized by response surface methodology（RSM）,and the biological activities of PCPP were studied in vitro,including antioxidant,immune regulation and radioprotection.Then,the radioprotection of PCPP in vivo was explored by establishing the ⁶⁰Co-γinjury model in mice.Finally,PCPP was isolated and purified,and its main radioprotective component was identified and characterized.The main results were as follows:（1）Extraction optimization of PCPP and study of antioxidant,immune regulation and radioprotection in vitroThe extraction conditions of PCPP were optimized by RSM and the antioxidant,immunomodulatory and radioprotection activities of PCPP were researched in vitro.The results showed that the optimal preparation conditions of PCPP were as follows:liquid-solid ratio 24.19 mL/g,extraction time 5.54 h and extraction temperature 100℃,with the yield of 0.71%.The biological activities of PCPP showed that it possessed significant antioxidant activity in vitro,and its scavenging abilities to DPPH free radicals and ABTS free radicals,total reducing power increased in a dose-dependent manner.In addition,PCPP could exert a good immunomodulatory effect by promoting the proliferation activity,phagocytosis function and NO secretion ability on RAW 264.7 cells.Finally,it was found that PCPP could promote the proliferation of AML-12 cells to exert a good radiation protection effect in vitro by establishing the radiation injury model of normal hepatocytes.（2）The radioprotection study of PCPP on ⁶⁰Co-γradiation-induced injury in miceThe ⁶⁰Co-γradiation injury model was established to systematically evaluate the radioprotection of PCPP in vivo.The results showed that PCPP could effectively reduce the radiation damage degree of spleens and livers in mice,and meanwhile alleviated the damage to the hematopoietic system by regulating the red blood cells,platelets and hemoglobin.Moreover,PCPP could significantly increase the activity of superoxide dismutase（SOD）and the content of glutathione（GSH）in serum,livers and spleens of irradiated mice,and correspondingly reduced the accumulation of lipid peroxide malondialdehyde（MDA）,along with the prominent reduction of alanine aminotransferase（ALT）and aspartate aminotransferase（AST）in serum and livers of mice.Above results comprehensively indicated that PCPP could effectively reduce the radiation oxidative damage in mice,suggesting that it could be used as a potential resource for the development of natural radioprotectors.（3）Purification,bioactivities determination and preliminary structural characterization of the main active components of PCPPFirstly,PCPP was purified by DEAE-52 cellulose ion-exchange column and Sephadex G-100 dexan gel-column to obtain a homogeneous polysaccharide fraction PCPP-1A,with a polysaccharide content of 91.82±2.64%.Secondly,the biological activities of PCPP-1A were evaluated.The results showed that compared with PCPP,PCPP-1A also represented good immunomodulatory activity and radioprotection effect on AML-12 cells,preliminarily indicating PCPP-1A was the main effective component of PCPP to exert radioprotection activity.Finally,the structural composition of PCPP-1A was identified using high performance liquid chromatography,spectroscopy,congo red experiment,periodic acid oxidation,nuclear magnetic resonance and electron microscope.The results showed that the molecular weight of PCPP-1A was 45.38 kDa,and it was composed of mannose,glucuronic acid,glucose,galactose and fucose with a molar ratio of 10.93:1.00:235.76:26.99:6.56.The FT-IR spectra indicated that PCPP-1A contained the characteristic absorption peaks of polysaccharides,such as O-H bonds,C-H bonds,pyran ring andα-glycoside bonds.The periodic acid oxidation and congo red experiments revealed that PCPP-1A possessed 1-2,1-4,1-2,6 or 1-4,6 glycosidic bonds without triple helix junctions.Thenuclear magnetic resonance analysis showed that PCPP-1A might have glucopyranose,which was linked byα-glycosidic bonds andβ-glycosidic bonds.In conclusion,polysaccharides from Poria cocos peels are a kind of biological macromolecules with excellent antioxidant activity,immunomodulatory function and radioprotection.Our study not only provides a new reference for comprehensive utilization of Poria cocos peels,but also lays a theoretical basis for its development as antioxidants,immunomodulators and radioprotectors.