| Recently,many medical studies confirmed that the biological activities of Chinese herbal medicinal polysaccharides are greatly increased by molecular modification.Outstanding,the biological activity is newly generated or enhanced in some polysaccharides without or with low biological activities after they are selenizingly modified,especially the immune-enhancing and antioxidant activities possess extensive medicinal value.In this research,epimedium polysaccharide(EPS)and isatis root polysaccharide(IRPS)were chosen and selenizingly modified by Nitric acid-sodium selenite(HNO3-Na2SeO3)method to get nine selenizing EPS(sEPS)and nine selenizing IRPS(sIRPS).Their antioxidant and immune-enhancement activities were compared by the test in vitro or in vivo.The object of this study is to verify the probability that selenylation modification improve the immune-enhancing activity of two polysaccharides,select out the best selenizing polysaccharide and offer theoretical evidences for the development of new-type immunopotentiator and antioxidant.The research includes six parts as follows:Test 1.Preparation of sEPSs and sIRPSs Epimedium polysaccharide(EPS)and Isatis root polysaccharide(IRPS)were extracted by water decoction and ethanol precipitation,after eliminating protein by Sevag's method,permeating through DEAE-52 column and freeze-drying,the purified EPSs(EPS1 and EPS2)and IRPSs(IRPS1 and IRPS2)were obtained.By Nitric acid-sodium selenite(HNO3-Na2SeO3)method EPS 1 and IRPS1 were selenizingly modified according to L9(34)orthogonal design of three-factors,the usage amount of sodium selenite,reaction temperature and reaction time,each at three levels,then nine sEPSs(selenizing EPSs),sEPS1-sEPS9 and nine sIRPSs(selenizing IRPSs),sIRPS1-sIRPS9 were obtained.Their selenium contents were determined by hydride generation atomic fluorescence spectrometry(HG-AFS),and the structures were identified by Fourier transform infrared(FT-IR).The results showed that the selenium contents of sEPSs were 3.32-21.89 mg·g-1 and that of sIRPSs were 2.65-15.21 mg·g-1.There were characteristic absorptive peaks of selenite on the FT-IR spectra,which signified that the two kinds of polysaccharides were successfully modified in selenylation.Test 2.Comparison of immune-enhancing activities of sEPSs and sIRPSs in vitro In order to screening the optimum selenylation modification conditions of polysaccharides with stronger immunoenhancement action,nine sEPSs,nine sIRPSs and non-selenizing EPS and IRPS diluted with RPMI-1640 at eleven concentrations were added into the cultured chicken peripheral blood lymphocyte in single or simultaneous with PHA,respectively.After incubation for 48h,the changes of the lymphocyte proliferation(cellular A570 value and the lymphocyte proliferation rate)were determined by MTT method.The results showed that in single adding,the A570 values of all polysaccharides at 1-5 concentration groups were significantly larger than that of corresponding cell control group,the A570 values of sEPS1?sEPS4?sEPS5 and sIRPS6 at five concentration groups were significantly larger than that of corresponding cell control group,the lymphocyte proliferation rates of sEPS2?sEPS4?sEPS5 and sEPSg groups were significantly higher than that of EPS group and the lymphocyte proliferation rates of SIRPS5?sIRPS8 and sIRPS9 were significantly higher than that of IRPS.In simultaneous adding with PHA,the A570 values of all polysaccharides at 1-5 concentration groups were significantly larger than that of corresponding PHA control group,the A570 values of sEPS1-sEPS9?sIRPS2?sIRPS4?s1RPS5?sIRPSg and sIRPS9 at 5 concentration groups were significantly larger than that of corresponding PHA control group,the lymphocyte proliferation rates of sEPS2?sEPS5 and sEPSg groups were significantly higher than that of EPS group,sIRPS2 and sIRPS5 groups were significantly higher than that of IRPS group.These results indicated that selenylation modification could elevate the immune-enhancing activities of EPS and IRPS.sEPS5 and sIRPS5 were selected for experiments in vivo.Test 3.Comparison of antioxidant activities of sEPSs and sIRPSs in vitro sEPS1-sEPS9,sIRPS1-sIRPS9,non-selenizing EPS1 and IRPS1 were diluted with distilled water at five concentrations,then added into reaction system of DPPH radical,hydroxyl radical and ABTS radical,their scavenging power effects on above three kinds of radicals were determined.The results showed that in sEPS1?sEPS4 and sEPS7 groups at 2-0.5mg·mL-1 the removal rates of DPPH radical were significantly higher than that of corresponding non-selenizing EPS group;in sIRPS4?sIRPS7 and sIRPS8 groups at 2-0.125mg·mL-1 the removal rates of DPPH radical were significantly higher than that of corresponding non-selenizing IRPS group;In sEPS4 and sEPS7 groups at 1-0.0625mg-mL-1 the removal rates of hydroxyl radical were significantly higher than that of corresponding non-selenizing EPS group;in sIRPS7 group at 1-0.0625 mg·mL-1 the removal rate of hydroxyl radical was significantly higher than that of corresponding non-selenizing IRPS group;In sEPS1?sEPS4 and sEPS7 groups at 1-0.0625mg · mL-1,the removal rates of ABTS radical were significantly higher than that of corresponding non-selenizing EPS group;in sIRPS7 group at 1-0.0625mg·mL-1 the removal rate of ABTS radical was significantly higher than that of corresponding non-selenizing IRPS group.The results indicated that selenylation modification could enhance the antioxidant activities of EPS and IRPS in vitro.The antioxidant activity of sEPS7 is the best in sEPSs and that of sIRPS7 is the best in sIRPSs.Test 4.Comparison of immune-enhancing activities of sEPSs and sIRPSs in vivo The immune-enhancing activities of sEPS5 and sIRPS5 screened by the test in vitro were further compared in vivo.One hundred and eighty 14-day-old chickens were divided randomly into six groups,the chickens except blank control group were vaccinated with ND vaccine,repeated vaccination at 28 days old.At the same time of vaccinations the chickens in four polysaccharides groups were intramuscularly injected respectively with 0.5mL of sEPS5?sIRPS5?EPS and IRPS(the content of polysaccharide is 2mg·mL-1),in vaccination control and blank control groups,with equal volume of physiological saline.The changes of serum HI antibody titer,peripheral lymphocyte proliferation,the contents of the serum Interleukin(IL)-2?Interleukin(IL)-6 and Interferon(IFN)-? on days 7,14,21,and 28 after the first vaccination,and the changes of immune organ indexes on days 14 and 28 after the first vaccination were all determined.The results showed that at all time points serum HI antibody titer,peripheral lymphocyte proliferation,the contents of the serum Interleukin(IL)-2?Interleukin(IL)-6 and Interferon(IFN)-? of sEPSs and sIRPSs groups were significantly higher than that of VC and BC groups,in most cases were significantly higher than that of corresponding EPS and IRPS groups,and in most cases that of sEPS5 was the highest;On days 14 and 28 after the first vaccination the thymus index of sIRPS5 group was the highest,the spleen and the bursa of Fabricius indexes of sEPS5 group were all the highest.The above results showed that selenylation modification has improved immune-enhancing activities of two kinds of polysaccharides,sEPS5 possessed the best efficacy.Test 5.Comparison of antioxidant activities of sEPSs and sIRPSs in vivo Based on the experiments in vitro,the antioxidant activities of sEPS7 and sIRPS7 were further compared in this test.One hundred and eighty 14-day-old chickens were divided randomly into six groups,the chickens except blank control group were vaccinated with ND vaccine,repeated vaccination at 28 days old.At the same time of vaccinations the chickens in four polysaccharides groups were intramuscularly injected respectively with 0.5mL of sEPS5?sIRPS5?EPS and IRPS(the content of polysaccharide is 2mg·mL-1),in vaccination control and blank control group,with equal volume of physiological saline.On days 7,14,21,and 28 the blood was sampled after the first vaccination,the changes of serum CAT activity?SOD activity?GSH-Px activity and MDA content were determined.The results showed that the serum CAT activity of sEPS7 at every time point and that of sIRPS7 at two time points were significantly higher than that of corresponding immunization control group;the serum SOD activity of both sEPS7 and sIRPS7 groups at every time point were significantly higher than that of corresponding immunization control group;the serum GSH-Px activity of both sEPS7 and sIRPS7 groups at every time point were significantly higher than that of corresponding immunization control group;the serum MDA content of sEPS7 at every time point and that of sIRPS7 at two time points were significantly lower than that of corresponding immunization control group.These results showed that selenylation modification could enhance the antioxidant activity of EPS and IRPS,sEPS7 possessed the best efficacy.Test 6.Effects of sEPSs on mRNA expression of interleukin-2?interleukin-6 and interferon-? in chicken peripheral lymphocyte Effects of sEPS5 on mRNA expression of IL-2?IL-6 and IFN-y in chicken peripheral lymphocyte were discussed in this study.Taking EPS as control,sEPS5 at three concentrations(6.25?3.125?1.563?g·L-1)were added into the chicken peripheral blood lymphocyte and cultured for 12h,the cells were harvested and RNA was isolated,The expression of IL-2?IL-6 and IFN-y mRNA were evaluated by fluorescence quantitative RT-PCR assay.The results showed that sEPS5 at 3.125 and 1.563?g·mL-1 could promote the expression of IL-2 mRNA,sEPS5 at 1.563?g·mL-1 could promote the expression of IL-6 mRNA and IFN-? mRNA,which were all significantly stronger than PHA and unmodified polysaccharide EPS.Selenylation modification could promote the expression of IL-2?IL-6 and IFN-? mRNA,which could be one of the mechanisms of selenium polysaccharide to boost immunity. |
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