| Selenium-polysaccharide possesses the function of immune enhancement,anti-oxidation,anti-aging and anti-tumor and so on.It possesses higher biological activities as compared with selenium or polysaccharide and easily absorbed by organism.In this study,astragalus polysaccharide(APS)and atractylodes macrocephala polysaccharide(AMP)were extracted purified,three APSs(APSt,APSd and APSp)and three AMPs(AMPt,AMPd and AMPp)were obtained,their immune-enhancing activity and antioxidative activity were compared by the tests in vitro,the polysaccharides,which possess the strongest activities of immune-enhancing and antioxidative,were selenizingly modified by Nitric acid-sodium selenite method,nine selenizing APS(sAPS,sAPS1?sAPS9)and nine selenizing AMP(sAMP,sAMP1?sAMP9)were obtained respectively,their immune-enhancing activity and antioxidative activity were compared by the tests in vitro and in vivo.The aim of this study is to validate the probability of selenylation modification to improve the immune-enhancing activity and antioxidative activity of APS and AMP,selected out the best selenium-polysaccharide with strongest activity and its modification conditions,and offer theoretical evidence for the development of new-type immunopotentiator and antioxidant.The tests include five parts.Test ?.Preparation of APS and AMP Total APS and AMP were extracted by water extraction and ethanol precipitation method,eliminated protein by trichloroacetic acid method,obtain APSd and AMPd,they were pured by column chromatography of DEAE Cellulose-52 and freeze dehydration,obtain APSp and AMPp.Their carbohydrate was measured by phenol-sulphuric acid method.The result is that,the yield of APSt is 6.6%,and the carbohydrate content of APSp was the highest(76.35%)among APSs;the yield of AMPt is 14.45%and its carbohydrate content was the highest(56%)among AMPs.Test ?.Selecte the strongest immune-enhancing activity polysaccaride in vitro of APS and AMP Safe concentration of chicken peripheral blood lymphocytes of APSs(APSt,APSd and APSp)and AMPs(AMPt,AMPd and AMPp)were measured by MTT method,5 concentration whitin safe concentration were chosen to test the effects on lymphocytes proliferation of each polysacchride single or simultaneous with PHA stimulation.The result showed that the lymphocytes proliferation of APSt was the highest in APSs,and the lymphocytes proliferation of AMPp was the highest in AMPs when they single stimulate cell;in simultaneous with PHA stimulation,the proliferation of APSt was the highest in APSs,and the proliferation of AMPp was the highest in AMPs.These results indicated APSs and AMPs can enhance cellular immunity when they single stimulate cell or with PHA.The effects of APSt and AMPp was strongest.They were the active area of APSs and AMPs.Test ?.Selecte the strongest antioxidant activity polysaccaride in vitro of APS and AMP The antioxidative activity of APSs and AMPs were compared by hydroxyl radical-scavenging test,DPPH radical-scavenging test and superoxide anion scavenging test.The result showed that the avarage scavenging rate of hydroxyl radical and superoxide anion of APSt were respectively 56.4%and 7.02%,they were the highest scavenging in APSs,the avarage scavenging rate of DPPH of APSd was the highest in APSs,the scavenging rate is 46.18%;the avarage scavenging rate of hydroxyl radical and superoxide anion of AMPp were the highest scavenging in AMPs,the avarage scavenging rate of DPPH of AMPt was the highest in AMPs;Test ?.Selenizing modification of APS and AMP APS and AMP were selenizingly modified by Nitric acid-sodium selenite method,under nine kinds of modification conditions according to L9(34)orthogonal design of three factors,usage amount of sodium selenite,the reaction temperature and reaction time,at three levels to obtained nine selenizing sAPSs(sAPS1?sAPS9)and sAMPs(sAMP1?sAMP9).Their carbohydrate,yield and selenium concents were measured respectively,and their structure were identified by infrared spectrometry.The result showed the carbohydrate and selenium contents of sAPS2 and sAMP4 were the highest respectively in the sAPSs and sAMPs.APS and AMP were modified successful by infrared spectrometry.Test ?.Comparison of immune-enhancing activity in vitro of sAPSs and sAMPs Safe concentration of chicken peripheral blood lymphocytes of APSs(APSt,sAPS1?sAPS9)and AMPs(AMPp,sAPS1?sAPS9)were measured by MTT method,5 concentration whitin safe concentration were chosen to test the effects on lymphocytes proliferation of each polysacchride single or simultaneous with PHA stimulation.The result showed the safe concentration of sAPSs and sAMPs were 12.5 ?g·mL-1 and 6.25 ?g·mL-1,in single stimulation,proliferation rate of all sAPSs groups and sAMPs groups were higher than APSt and AMPp group,and the value of A570 of sAPS5 at 0.781?2.50 ?g·mL-1 were significantly higher than cell control group,proliferation rate of sAPS5 was the highest in sAPSs,the value of A570 of sAMP9 at 0.390?6.250 ?g·mL-1 were significantly higher than cell control group,the proliferation rate of sAMP9 was the highest in sAMPs;In simultaneous with PHA stimulation,the A570 value of sAPSs and sAMPs groups were significantly higher than cell control group,the proliferation rate of sAPS5 and sAMP9 were respectively highest in sAPSs and sAMPs.These results indicated that selenylation modification could significantly enhance the immune-enhancing activity in vitro of APS and AMP,sAPS5 and sAMP9 were strongest.Test ?.Comparison of antioxidant activity in vitro of sAPSs and sAMPs The antioxidant activity of APSs(APSt?sAPS1?sAPS9)and AMPs(AMPp?sAMP1?sAMP9)in vitro were measured by hydroxyl radicals-scavenging test,DPPH asssy and ABTS assay.The results showed the hydroxyl radicals-scavenging rate of sAPS1?sAPS9 at 0.125?1.0 mg·mL-1,sAPS1?sAPSg at 0.0625 mg·mL-1 were significantly higher than APSt,sAMP1?sAMP9 at 0.5?1.0 mg·mL-1,sAMP3?sAMP7 at 0.0625?0.25 mg·mL-1 were significantly higher than AMPp;the DPPH radicals-scavenging rate of sAPS1?sAPS9 at 0.125?1.0 mg·mL-1,sAPS1?sAPS8 at 0.0625 mg·mL-1 were significantly higher than APSt,sAMP1?sAMP9 at 0.5?1.0 mg·mL-1,sAMP4?sAMP9 at 0.0625?0.25 mg·mL-1 were significantly higher than AMPp;the ABTS radicals-scavenging rate of sAPS1?sAPS9 at 1.0 mg·mL-1 and 0.0625 mg·mL-1,sAPS1?sAPS8 and 0.125?0.5 mg·mL-1 were significantly higher than APSt,sAMP4?sAMP8 at 0.125?1.0 mg·mL-L-1?sAMP6 at 0.0625 mg·mL-1?sAMP9 at 0.25?1.0 mg·mL-1 were significantly higher than AMPp.These results indicated that selenylation modification could enhance the antioxidant activity n vitro of APS and AMP,sAPS8 and sAMP6 were strongest.Test ?.Comparison of immune-enhancing activity in vivo of sAPS and sAMP 180 chicks of 14 days were randomly assigned into six groups and except blank control(BC)group vaccinated with Newcastle disease vaccine,repeated vaccination at 28 days old.At the same time of each vaccination,the chicks in four polysaccharide groups were intramuscularly injected with 2 mg·mL-1(0.5 ml)of sAMP9,sAPS5,AMPp and APSt,in vaccination control(VC)group and BC group with equal volume of normal saline.On days 7(D7),14(D14),21(D21),28(D28)after the first vaccination,the blood samples from each group randomly were collected for peripheral lymphocytes proliferation test,serum HI antibodytest test,serum IFN-?,IL-2 and IL-6 contents test.The results showed,the peripheral lymphocytes proliferation rate,serum HI antibody,serum IFN-?,IL-2 and IL-6 contents of sAMP9 and sAPSs groups were higher or significantly higher than AMPp and APSt group.These results indicated that selenylation modification could significantly enhance the immune-enhancing activity in vivo of APS and AMP,sAMP9 was strongest.Test ?.Comparison of antioxidant activity in vivo of sAPS and sAMP 180 chicks of 14 days were randomly assigned into six groups and except blank control(BC)group vaccinated with Newcastle disease vaccine,repeated vaccination at 28 days old.At the same time of each vaccination,the chicks in four polysaccharide groups were intramuscularly injected with 1 mg(0.5 ml)of sAMP6,sAPSg,AMPp and APSt,in vaccination control(VC)group and BC group with equal volume of normal saline.On days 7(D7),14(D14),21(321),28(328)after the first vaccination,the serum samples from each group randomly were collected to measure SOD,MDA,GSH-PX and CAT contents.The results showed the SOD,MDA,GSH-PX and CAT contents of sAMP6 and sAPSg groups were higher or significantly higher than AMPp and APSt,VC and BC groups.These results indicated that selenylation modification could significantly enhance the antioxidant activity in vivo of APS and AMP,sAMP6 was strongest. |
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