The Preparation Of Polysaccharides From Euphorbia Humifusa And Its Immune-enhancing Activity In Mice

Polysaccharide is an important part of traditional Chinese medicine,which has biological activities such as enhancing immunity,anti-virus,anti-tumor,anti-oxidation,anti-aging and so on.As an immunopotentiator,polysaccharide have the effect of enhancing resistance to animal diseases.Euphorbia humifusa Willd is widely distributed in China and has a wide range of pharmacological effects.It has significant antioxidant,anti-tumor,antibacterial and anti-inflammatory effects,and has important clinical application and development value.In this study,polysaccharides from Euphorbia humifusa Willd was selected as the research object,and three active sites with better effect were initially screened from the aspects of extraction and purification,structure identification and immune activity in vitro,and further study its immune enhancement activity in vitro and in vivo.Experiment 1 Extraction,decolorization and purification of Polysaccharides from Euphorbia humifusa Willd The crude total TPS(EHPS_tc)and three crude fractional EHPSs,EHPS_60c,EHPS_70cand EHPS_80c,were extracted by one-step or stepwise ethanol precipitation method,respectively.The decolorization conditions such as the amount of hydrogen peroxide,reaction temperature,p H value,and reaction time,were optimized by L₂₅(5⁶) orthogonal test taking the decolorization rate and polysaccharide retention rate as index.After decolorization,EHPS_tc was purified by DEAE-Sephadex A-25 dextran gel column chromatography,and purified EHPS_tp-1 and EHPS_tp-2 were obtained.The carbohydrate and protein contents of all polysaccharides were determinated recpectively by phenol-sulfuric acid and coomassie brilliant blue G-250 methods.The results showed that the optimal decolorization conditions of EHPS_tcwere as follows:reaction temperature 50?,reaction time 4 h,p H value 2,and hydrogen peroxide dosage 4%.The decolorization rate,retention rate and carbohydrate content were 71.10%,63.31%,and 74.52%,respectively.After purification by column chromatography,the carbohydrate content of EHPS_tp-1 and EHPS_tp-2 increased to 95.36% and92.35%,respectively.Experiment 2 Structure identification of EHPS The structures of polysaccharides were analyzed by infrared spectroscopy(IR),ultraviolet spectroscopy(UVS),PMP pre column derivatization,high performance liquid chromatography(PMP-HPLC),high performance gel permeation chromatography(HPGPC),carbazole sulfate,Congo red and nuclear magnetic resonance spectroscopy(NMR).The results showed that EHPS_tcand EHPS_tp-2 may contain a small amount of protein.EHPS_tc,EHPS_tp-1 and EHPS_tp-2 all accord with the basic characteristics of infrared spectroscopy of polysaccharides.EHPS_tp-1 mainly includes GLC(83.1%),gal(6.1%),Glc UA(1.6%).EHPS_tp-2 mainly includes gal(25.6%),galua(22.2%),Ara(16.6%);EHPS_tc mainly includes GLC(53.5%),Ara(16.8%),gal(14.3%).The weight average molecular weights of EHPS_tcand EHPS_tp-1 were 26.2 and 26.0 k Da,respectively.The weight average molecular weights of EHPS_tp-2 were 145.6 and 8.9 k Da,respectively.The uronic acid content of EHPS_tp-1 and EHPS_tp-2 were 15.9% and 27.9%,respectively.Both EHPS_tcand EHPS_tp-1 have obvious triple helix structure.EHPS_tp-1 has monosaccharide of?configuration,EHPS_tc has?and?configuration monosaccharide.Experiment 3 The effect of EHPS on immune activity of peripheral blood lymphocytes in mice EHPS_60c,EHPS_70c,EHPS_80c,EHPS_tc,EHPS_tp-1 and EHPS_tp-2 were added to the mouse peripheral blood lymphocytes,and their safe concentrations were determined by MTT method.Then,six polysaccharides within the safe concentration range were added to mouse peripheral blood lymphocytes separately or in combination with PHA.After 48 h,the changes of lymphocyte proliferation(cell A₅₇₀ value and the highest lymphocyte proliferation rate)were determined,and the better sites to enhance the immune activity were screened.The selected three polysaccharides stimulated lymphocytes at 31.25?g/mL,and the cells were collected at 24 h,48 h and 72 h,respectively.After processing,the cycle distribution at each time point and the changes of CD4⁺,CD8⁺T lymphocyte subpopulations were detected on the flow cytometer.The contents of immunoglobulin IgA,IgG and cytokines IL-2,IL-4,IL-6 and IFN-?were determined by ELISA.The results showed that the cell proliferation rate of EHPS_tp-1 was the highest at 15.625?g/mL when polysaccharide was stimulated alone,which was 15.099%,followed by EHPS_tp-2 at the same concentration,which reached 12.129%.When polysaccharides and PHA were co-stimulated,The cell proliferation rate of EHPS_tc was the highest at 15.625?g/mL,which was 23.820%;followed by EHPS_tp-1 at 31.25?g/mL,which was 20.499%..Comprehensive evaluation showed that EHPStc,EHPStp-1 and EHPStp-2 might be the better sites for enhancing the immune activity.The cell cycle distribution results showed that compared with EHPStc,the SPF and PI value increased significantly after EHPStp-1 and EHPStp-2 treated cells for 48 h and 72 h.The percentages of CD4⁺,CD8⁺T lymphocytes were significantly higher than those of the EHPS_tc group.EHPStp-1 and EHPStp-2 and EHPS_tc could significantly promote the secretion of IgA,IgG,IL-2,IL-4,IL-6 and IFN-?.Experiment 4 The effect of EHPS on the function of macrophages Take 6polysaccharides with 5 concentrations within a safe concentration range were added to the cultured mouse peritoneal macrophages individually or in combination with LPS.After 48 h of culture,the changes of macrophages proliferation were measured.Then,the phagocytic activity,NO and iNOS secretion were detected.The content of cytokines IL-2,IL-6,IFN-?,MCP-1,MIP-1?ELISA method were determined by ELISA.The expressions of CD14 and MHC-II were analyzed by flow cytometry.The results showed that EHPS_tp-1 had the highest cell proliferation rate at 31.25?g/mL when polysaccharides were stimulated alone,which was23.17%,the second was that EHPS_tc had a cell proliferation rate of 16.42%at 31.25?g/mL.When synergistic LPS stimulated macrophages,EHPS_tp-1 had the highest cell proliferation rate at 31.25?g/mL,which was 20.29%;followed by EHPS_60c at 31.25?g/mL,which had a cell proliferation rate of 17.41%.At 31.25-1.953?g/mL,EHPS_tc and EHPS_tp-1 promoted the phagocytosis of mouse peritoneal macrophages the strongest,and at 31.25-15.625?g/mL,EHPS_tc promoted phagocytosis significantly stronger than EHPS_tp-1.When EHPS_tc,EHPS_tp-1 and EHPS_tp-2 were 31.25-1.953?g/mL and 31.25-3.907?g/mL,respectively,the NO and iNOS secretion functions of mouse peritoneal macrophages were significantly stronger than that in other polysaccharide groups.EHPS_tc,EHPS_tp-1 and EHPS_tp-2 can significantly promote the secretion of cytokines.At 31.25-7.813?g/mL,the expression of CD14 and MHC-II in EHPS_tp-1 group was significantly higher than that in EHPS_tp-2 and BL groups.Experiment 5 Antioxidant effect of EHPS on mouse macrophages Five concentrations of EHPS_tc,EHPS_tp-₁ and EHPS_tp-2 within the safe concentration range were added to the cultured mouse peritoneal macrophages.After 48 h of culture,the ELSIA method was used to determine the contents of SOD,GSH-PX,MDA and MPO in murine macrophages.The results show that EHPS_tc,EHPS_tp-1 and EHPS_tp-2 could significantly increase the SOD activity of mice,and the activities of SOD and GSH-PX in EHPS_tp-1 group were significantly higher than those of other polysaccharide groups.EHPS_tc,EHPS_tp-1 and EHPS_tp-2 at 7.813-31.25?g/mL could significantly reduce MDA content and lipid peroxidation.The activity of MPO in macrophages treated with EHPS_tc,EHPS_tp-1 and EHPS_tp-2 decreased significantly,and the MPO activity of 31.25?g/mL group was significantly lower than that of other polysaccharide groups.The results indicated that EHPS_tc,EHPS_tp-1 and EHPS_tp-2 have significant antioxidant effects,which could reduce free radical damage,protect animal and enhance immune function.Experiment 6 The immunomodulatory effect of EHPS on immunosuppressive mice Establish an immunosuppressive model of cyclophosphamide(CTX)mice to study the immunomodulatory effects of EHPS_tcand EHPS_tp-1 on immunosuppressed mice.The mice were randomly divided into 5 groups(n=10).They were normal control group(NC),model control group(MC),EHPS_tc group,EHPS_tp-1 group,and positive control group(PC).The mice in the NC group were given normal saline by gavage every day,the two polysaccharide groups were given 150 mg/kg/d EHPS_tc and EHPS_tp-1 every day,and the mice in the PC group were given levamisole(100 mg/kg/D).The experiment lasted for 24 days.On the 15th,16th and 17th day,100 mg CY/kg BW was injected intraperitoneally to make immunosuppressive model.The NC group and MC group were intragastrically administered with 0.1 mL/10 g normal saline.The results showed that EHPS_tc and EHPS_tp-1 could promote T lymphocyte proliferation,promote lymphocytes to enter S phase and G2/M phase at most time points,increase the percentage of CD4⁺and CD8⁺T lymphocyte subsets,increase serum immunoglobulins and immunoglobulins,and improve the immune organ index and spleen antioxidant index in mice.The results indicated that EHPS_tc and EHPS_tp-1 can effectively improve the cellular and humoral immunity of immunosuppressed mice.Comprehensive evaluation showed that EHPS_tp-1 has the strongest immuno-enhancing activity.In this study,the total polysaccharides and various graded polysaccharides were obtained by extracting,decolorizing and purifying the polysaccharides from Humifuse Euphorbia Willd The immune activity of lymphocytes,the function of macrophages,the antioxidant capacity of macrophages and the immunomodulatory effect of polysaccharides on immunosuppressive mice were detected by using peripheral blood lymphocytes,macrophages and immunosuppressive mice.This study provides a theoretical basis for the popularization and application of polysaccharides from traditional Chinese medicine such as Humifuse Euphorbia Willd in the prevention and treatment of animal diseases.