| Both selenium and polysaccharide possess many biological activities,such as immune-enhancing activity,antioxidative activity and so on.The combination of inorganic selenium with polysaccharide(PS)into organic selenium compounds,selenizing polysaccharide(sPS),by chemical modification methods can make the pharmacological activity of selenium and polysaccharide be optimized.Therefore in this study garlic polysaccharide(GPS)and lycium barbarum polysaccharide(LBP)were chosen as the research object.extracted,purified and modified by HNO3-Na2SeO3 method under nine kinds of modification conditions according to L9(34)orthogonal design taking three factors,usage amount of sodium selenite,the reaction temperature and reaction time each at three levels as indexes.Nine selenizing GPS(sGPS,sGPS1-SGPS9)and nine selenizing LBP(sLBP,sLBP1-sLBP9)were obtained respectively,and their immune-enhancing activity and antioxidative activity were compared by the tests both in vitro and vivo.The aim of this study is to validate the probability of selenylation modification to improve the immune-enhancing activity and antioxidative activity of GPS and LBP,selected out the sPS 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 and selenizing modification of GPS and LBP Crude GPS and LBP were extracted by water extraction and ethanol precipitation method.Pure GPS and LBP were obtained by trichloroacetic acid method to eliminate protein,column chromatography of DEAE Cellulose-52 and freeze dehydration,and modified by HNO3-Na2SeO3 method under nine kinds of modification conditions according to L9(34)orthogonal design taking three factors,usage amount of sodium selenite,the reaction temperature and reaction time each at three levels.Nine sGPSs,sGPS1-sGPS9 and nine sLBPs,sLBP1-sLBP9,were obtained.Their carbohydrate and protein contents were measured respectively by phenol-sulphuric acid method and Coomassie brilliant blue G-250 method,selenium concents were determined by atomic fluorescence spectrometry,and structures were identified by infrared spectrometry.The results showed that among GPSs,GPSp1 presented the highest carbohydrate content(94.5%)and lowest protein content(1.1%).Among LBP,LBPpl presented the highest carbohydrate content(90.2%)and lowest protein content(1.7%).Among sGPSs,the yield of sGPS2 was the highest(37.64%),the selenium content of sGPS3 was the highest(12.49 mg-g-1),and the carbohydrate content of sGPS5 was the highest(56.9%).Among sLBPs,the yield of sLBP?was the highest(40.95%),the selenium content of sLBP6 was the highest(13.66 mg·g-1),and the carbohydrate content of sLBP6 was the highest(47.5%).The infrared spectra of two kinds of selenizing polysaccharide demonstrated the bond of selenite selenium ester and selenium ester,which showed that the GPS and LBP were successfully modified in selenylation.Test ?.Comparison of immune-enhancing activity in vitro of sGPSs and sLBPs The safe concentrations of nine sGPSs,nine sLBPs and non-selenizing GPS and LBP for chicken peripheral lymphocytes were determined by MTT assay.Then their effects on lymphocytes proliferation were compared at five concentrations within safe concentration scope in single or simultaneous with PHA stimulation.The results showed that in single stimulation,the A570 values of seven sGPSs and GPS in five concentrations,sGPS1 and sGPS7 in four concentrations were significantly larger than cell control group,the proliferation rate of sGPS6 group was the highest,next were sGPS5 and sGPS3 groups;the A570 values of nine sLBPs and LBP in all concentrations were significantly larger than cell control group,the proliferation rate of sLBP9 group was the highest,next were sLBP8 and sLBP5 groups.In simultaneous with PHA stimulation,the A570 values of nine sGPS and GPS in all concentrations were significantly larger than PHA control group,the proliferation rate of sGPS6 group was the highest,next were sGPS3 and sGPS5 groups;the A570 values of nine sLBPs and LBP in three concentrations were significantly larger than PHA control group,the proliferation rate of sLBP9 group was the highest,next were sLBP8 and sLBP5 groups.These results indicated that selenylation modification could significantly enhance the immune-enhancing activity in vitro of GPS and LBP,and the immune-enhancing activities of sGPS6,sGPS5,sGPS3,sLBP9,sLBP5 and sLBPg were stronger.Test ?.Comparison of antioxidative activity in vitro of sGPSs and sLBP The antioxidative activity of nine sGPSs,nine sLBPs and non-selenizing GPS and LBP were compared by free radical-scavenging test and oxidative hemolysis inhibition test.The results displayed that the hydroxy and superoxide anion scavenging rate and oxidative hemolysis inhibition rate of nine sGPSs except for sGPS7 were significantly higher or higher than those of GPS,the activities of sGPS3,sGPS5 and sGPS6 were stronger;the hydroxy and superoxide anion scavenging rate and oxidative hemolysis inhibition rate of nine sLBPs were significantly higher or higher than that of LBP,the activities of sLBP6,sLBP8 and sLBP9 were stronger.These indicated that selenylation modification could significantly enhance the antioxidative activity in vitro of GPS and LBP,and the antioxidative activities of sGPS3,sGPS5,sGPS6,sLBP6,sLBP8 and sLBP9 were stronger.Test ?.Comparasion of immune-enhancing activities in vivo of sGPSs and sLBP The immune-enhancing activities in vivo of sLBP3,sGPS5,sGPS6,sLBP5,sLBP8 and sLBP9 were compared taking unmodified GPS and LBP as control.300 14-day-old chickens were randomly assigned into 10 groups.The chickens except blank control group were vaccinated with ND vaccine,repeated vaccination at 28 days old.At the same time of the first vaccination,the chickens in 6 Sps groups were intramuscularly injected respectively with 0.5 mL of sGPS3,sGPS5,sGPS6,sLBP5,sLBP8 and sLBP9,in PS control,with 0.5 mL of GPS and LBP,in vaccination control and blank control group with equal volume of physiological saline.On days 7,14,21 and 28 after the first vaccination,the lymphocyte proliferation,serum antibody titer,IFN-y and IL-2 contents were determined.The result showed that the cellular A570 value in sGPS5,sGPS6 and sLBP9 groups at four time points,sGPS3 at three time points and sLBP8 at two time points were significantly larger than those in vaccination control,GPS and LBP groups,the lymphocyte proliferation rate in sGPS6 group was the highest,the next was sLBP9 and sGPS5 groups,in this three group were significantly larger than those in GPS and LBP group.The serum antibody titer in sGPS5,sGPS6 and sLBP9 groups at four time points,sLBP8 at latter two time points were significantly higher than those in vaccination control,GPS and LBP group,and in sGPS6 group was the highest.The serum IFN-? and IL-2 contents in 6 sPS groups were higher or significantly higher than those in vaccination control and blank control group,the serum IFN-? contents in sGPS5,sGPS6,sLBP5 and sLBP9 groups at four time points were significantly higher than those in GPS and LBP groups,the serum IL-2 contents in 6 sPS groups were significantly than those in GPS and LBP groups.These results showed that selenylation modification could significantly enhance the immune-enhancing activity in vivo of GPS and LBP,sGPS6 possessed the strongest immune-enhancing activity and its modification conditions were 400 mg of sodium selenite for 500 mg of GPS,reaction temperature of 70? and reaction time of 6 h.Test ?.Comparasion of antioxidative activity in vivo of sGPSs and sLBP The antioxidative activities in vivo of sLBP3,sGPS5,sGPS6,sLBP5,sLBP8 and sLBP9 were compared taking unmodified GPS and LBP as control.270 14-day-old chickens were randomly assigned into 9 groups.The chickens in 6 sPS groups were intramuscularly injected respectively with 0.5 mL of sLBP3,sGPS5,sGPS6,sLBP5,sLBP8 and sLBP9,in PS control groups with 0.5 mL of GPS and LBP,and in blank control group with equal volume of physiological saline.On days 7,14,21 and 28 after the first injection,the serum GSH-Px and SOD activities and MDA content were determined.The result showed that in all PS groups the serum GSH-Px and SOD activities were higher or significantly higher and MDA contents were lower or significantly lower than those in blank control group;in 6 Sps groups the serum GSH-Px and SOD activities were larger or significantly higher and MDA contents were lower or significantly lower than those in GPS and LBP groups,the differences between Slbp6 and GPS,LBP groups were most significant.These results indicated that selenylation modification could significantly enhance the antioxidative activity in vivo of GPS and LBP,sLBP6 presented the strongest antioxidative activity and its modification conditions were 400 mg of sodium selenite for 500 mg of LBP,reaction temperature of 70? and reaction time of 6 h. |
PDF Full Text Request