| Polysaccharides and selenium have many bioactivities such as the immune-enhancing,antioxidant and anti-aging and others.However,the activity of polysaccharides is low and the usage of inorganic selenium can not be easy to control,and sustained intake of which will accumulate in body and cause selenium poisoning.The combition of polysaccharide with selenium will form organic selenium—selenizing polysaccharide,which could enhance the immune and antioxidant activity of polysaccharide,retain the physiological activities of selenium,even reduce the toxicity of inorganic selenium,be easily absorpted and utilizated by the body.Ophiopogon polysaccharide(OP)and lily polysaccharide(LP)were extracted and purified in this research.The active sites of immune-enhancing activity and antioxidantive activity of OP and LP were selenizingly modified by Na2SeO3-HNO3 method after the active sites were tracked.Then the active sites possessed the strongest immune-enhancing activity or strongest antioxidantive activity of selenizing polysaccharides were tracked and compared in vitro test and in vivo test.Finally,the mechanisms of immune-enhancing activity and antioxidantive activity of selenizing polysaccharide were explored.The aim of this research is to explore whether the selenylation modification can further enchan the immune activity and antioxidative activity of ophiopogon polysaccharide and lily polysaccharide,screen out the best selenizing polysaccharide and modification conditons and provide experiment basis for development of new polysaccharide immunopotentiator and new antiodidant.The tests are divided into ten parts as follows:Experiment 1.The extraction and purification of OP and LP Crude OP(OPc)and crude LP(LPc)were extracted respectively by water decoction and alcohol precipitation and hot water extraction and alcohol precipitation from traditional Chinese medicine decoction pieces of ophiopogon and lily,then by the trichloroacetic acid method to remove protein in crude polysaccharide to obtain protein-dislodging ophiopogon polysaccharide(OPd)and protein-dislodging lily polysaccharide(LPd).The purified ophiopogon polysaccharide(OPp)and purified lily polysaccharide(LPp1,LPp2)were obtained by DEAE-52 cellulose method.The extraction rate of each polysaccharide was calculated and the carbohydrate content of each polysaccharide was determined by phenol-sulfuric acid method.The results showed that the extraction rates of OPc,OPd,OPp,LPc,LPd,LPp1 and LPp2 were 33.68%,42.46%,30.42%,9.66%86.42%,62.34%,5.16%respectively,and the carbohydrate contents were 69.67%,92.20%,98.63%,35.74%,31.39%,42.04%and 15.70%respectively.Experiment 2.The tracing of immune-enhancing active site of OP and LP The immune-enhancing activity was compared of three ophiopogon polysaccharides(OPc,OPd,OPp)and three lily polysaccharides(LPc,LPd,LPp).The safe concentration of peripheral blood lymphoctyes in chickens for six polysaccharides were determined and five concentrations of every polysaccharide were choosed to stimulate lymphoctyes in single or synergetic with PHA.The results showed that the cellular A570 values of six polysaccharides were higher or significantly higher than that of cell control group(CC)at some concentration and the lymphocyte proliferation rates of OPc group was the highest in three ophiopogon polysaccharides and the lymphocyte proliferation rates of LPp group was the highest in three lily polysaccharides in single stimulation.Similarly,the cellular A570 values of six polysaccharides were higher or significantly higher than that of CC at some concentration and the lymphocyte proliferation rates of OPc group was the highest in three ophiopogon polysaccharides and the lymphocyte proliferation rates of LPp group was the highest in three lily polysaccharides in synergetic stimulation with PHA.These results indicated that OPc presented the strongest immune-enhancing activity in three ophiopogon polysaccharides,LPp presented the strongest immune-enhancing activity in three lily polysaccharides,and both of them were used as the active site of immue-enhancing activity.Experiment 3.The tracing of antioxidative active site of OP and LP The antioxidative activities were compared of three ophiopogon polysaccharides(OPc,OPd,OPp)and three lily polysaccharides(LPc,LPd,LPp).The free radical-scavenging test for OH·,DPPH·and O2-·of six polysaccharides were detected in four concentrations.The results showed that the average hydroxyl radical-scavenging rate of OPd group,the average DPPH radical-scavenging rate and the average O2-radical-scavenging rate of OPc group was the highest in three ophiopogon polysaccharides.The average hydroxyl radical-scavenging rate of LPp group,the average DPPH radical-scavenging rate and the average O2radical-scavenging rate of LPc group was the highest in three lily polysaccharides.These results indicated that OPc presented the strongest antioxidative activity in three ophiopogon polysaccharides,LPc presented the strongest antioxidative activity in three lily polysaccharides,and both of them were used as the active site of antioxidative activity.Experiment 4.The selenylation modification of OP and LP OPc,LPc and LPp screened in pre-experiment were selenizingly modified by HNO3-Na2SeO3 method.9 selenizing OPc(sOPcs),9 selenizing LPc(sLPcs)and 9 LPp(sLPps)were obtained according to orthogonal design which three factors respectively at three levels.The yields,selenium contents,carbohydrate contents,infrared spectrogram and scanning electron microscopy of selenizing polysaccharides obtained by mentioned-above method were respectively compared.The results of yields,selenium content,carbohydrate content showed that sOPc3 had the highest yield,sOPc4 had the highest selenium content and sOPc5 had the highest carbohydrate content in sOPc—sOPC9.sLPc9 had the highest yield,sLPc5 had the highest selenium content and sLPc8 had the highest carbohydrate content in sLPc1—sLPc9.sLPp6 had the highest yield and the highest selenium content,sLPp7 had the highest carbohydrate content in sLPp1—sLPp9.In the FT-IR spectra,sOPc8 had one characteristics absorption peaks at 669.22cm-1,sLPc2 at 1027.76 cm-1,sLPp6 at 669.15 cm-1 and 1025.14 cm-1.All those characteristics absorption peaks were in accordance with RSeO3R1.The surface topography of selenizing polysaccharides were more smooth compared with unmodified polysaccharide in the scanning electron microscopy.This signified that polysaccharides were successfully modified in selenylation.Experiment 5.The comparison of immune-enhancing activities of sOP and sLP in vitro sOPc1—sOPc9 and sLPp1—sLPp9 obtained in experiment 4 and its unmodified polysaccharide were used to stimulate chicken peripheral lymphocytes proliferation in single or synergetic with PHA in vitro test.The results showed that the lymphocyte proliferation rates of sOPc8 group was the highest in sOPc1—sOPC9 and significantly higher than that of OPc group.The lymphocyte proliferation rates of sLPp6 group was the highest in sLPc1—sLPc9 and significantly higher than that of LPp group.These results indicated that selenylation modification can significantly enhance the immune-enhancing activity of unmodified polysaccharide.sOPc8 presented the strongest action in sOPc1—sOPc9 and its modification conditions can be as the optimal conditions of OPc that are 300 mg of Na2SeO3 for 500 mg of OPc,the reaction temperature of 90℃ and the reaction time of 6 h.sLPp6 presented the strongest action in sLPc1—sLPc9 and its modification conditions can be as the optimal conditions of LPp that are 400 mg of Na2SeO3 for 500 mg of LPp,the reaction temperature of 70℃ and the reaction time of 6 h.Experiment 6.The comparison of antioxidative activities of sOP and sLP in vitro sOPc1—sOPc9 and sLPc1—sLPc9 obtained in experiment 4 and its unmodified polysaccharide were determinated by the free radical-scavenging test for OH·,DPPH·and ABTS·+in vitro test.The results showed that the hydroxyl radical-scavenging rates,the DPPH radical-scavenging rates and the ABTS+radical-scavenging rates of sOPc8 were the highest in sOPc1—sOPc9 and significantly higher than that of OPc group.The hydroxyl radical-scavenging rates,the DPPH radical-scavenging rates and the ABTS+radical-scavenging rates of sLPc2 were the highest in sLPc1—sLPc9 and significantly higher than that of LPc group.These results indicated that selenylation modification can significantly enhance the antioxidative activity of unmodified polysaccharide.sOPc8 presented the strongest action in sOPc1—sOPc9 and its modification conditions can be as the optimal conditions of OPc that are 300 mg of Na2SeO3 for 500 mg of OPc,the reaction temperature of 90℃ and the reaction time of 6 h.sLPc2 presented the strongest action in sLPc1—sLPc9 and its modification conditions can be as the optimal conditions of LPc that are 300 mg of Na2SeO3 for 500 mg of LPc,the reaction temperature of 50℃ and the reaction time of 8 h.Experiment 7.The comparison of immune-enhancing activities of sOP and sLP in vivo The influence of sOPc8 and sLPp6,both of which were screened in pre-experiments and presented the strongest immune-enhancing activity on chicken peripheral lymphocytes were compared taking their unmodified polysaccharide of OPc and LPp in control.The four polysaccharides were intramuscularly injected to chickens vaccinated with Newcastle disease vaccine at 14-day-old,repeated vaccination and injection at 28 days old.On days 7(D7),14(D14),21(D21)and 28(D28)after the first vaccination,the blood samples of each group were collected for the determination of peripheral lymphocytes proliferation,serum hemagglutination inhibition(HI)antibody titer,serum IL-2,IL-6 and IFN-y contents.The results showed that the A570 values,lymphocytes proliferation rates,serum antibody titer,serum IL-2,IL-6 and IFN-y contents of selenizing polysaccharides groups were higher or significantly higher than that of its unmodified polysaccharide,vaccination control or blank control at all time points.All these results indicated that selenylation modification can significantly enhance the immune-enhancing activity of unmodified polysaccharide in vivo test.sLPp6 presented the strongest immune-enhancing activity.Experiment 8.The comparison of antioxidative activities of sOP and sLP in vivo The antioxidative activities of sOPc8 and sLPc2,both of which were screened in pre-experiments and presented the strongest antioxidative activity were compared in vivo taking their unmodified polysaccharide of OPc and LPc in control.The four polysaccharides were intramuscularly injected to chickens vaccinated with Newcastle disease at 14-day-old,repeated vaccination and injection at 28 days old.On days 7(D7),14(D14),21(D21)and 28(D28)after the first vaccination,the blood samples of each group were collected for the determination of the enzyme activity of CAT,GSH-Px and T-SOD and the content of MDA in serum.The results showed that the enzyme activity of CAT,GSH-PX and T-SOD of selenizing polysaccharides groups were higher or significantly higher than that of its unmodified polysaccharide and blank control(BC)at all time points.The serum MDA content of selenizing polysaccharides groups were lower or significantly lower than that of its unmodified polysaccharide and BC at all time points.All these results indicated that selenylation modification can significantly enhance the antioxidative activity of unmodified polysaccharide in vivo test.sLPc2 presented the strongest antioxidative activity.Experiment 9.Effects of sLP on IL-2,IL-6 and IFN-γ mRNA expression of chicken peripheral lymphoctyes sLPp6,which was screened in pre-experiments and presented the strongest immune-enhancing activity in vivo test and its unmodified polysaccharide of LPp were used to stimulate chicken peripheral lymphocytes in synergetic with ConA.The total RNA was extraction after the lymphocytes cultured for 12 h.The effects of polysaccharides on IL-2,IL-6 and IFN-y mRNA relative expression of chicken lymphocytes were determined by PCR method.The results showed that with the increase of polysaccharide concentration,the IL-2,IL-6 and IFN-y mRNA relative expression increased.The IL-2,IL-6 and IFN-y mRNA relative expression of sLPp6 were higher or significantly higher than that of its unmodified polysaccharide group at the same concentration.These results indicated that selenylation modification can significantly enhance the IL-2,IL-6 and IFN-y mRNA relative expression of ummodified polysaccharide.This may be one of mechanisms by which selenizing polysaccharide enhances immunity.Experiment 10 Effects of sLP on chicken embryo hepatocyte to resist oxidative damage sLPc2,which was screened in pre-experiments and presented the strongest antioxidative activity in vivo test and its unmodified polysaccharide of LPp and NAC were used to stimulate chicken embryo hepatocyte.The activity of LDH,iNOS,AST,ALT,ROS level,SOD,GSH-PX,cells undergoing apoptosis and protein expression in mitochondrial apoptosis signaling pathways of each group were detected after H2O2-induced injury.The results showed that the activity of LDH,AST,ALT,and ROS level and cells undergoing apoptosis rates in sLPc2 were lower or significantly lower than that of LPp group at the same concentration.The activity of iNOS,SOD,GSH-PX higher or significantly higher than that of LPp group at the same concentration.The protein expression level of JNK1 and Bcl-2 were significantly higher than that of LPp group,and the protein expression level of Cytochrome c and Caspase 3 were significantly lower than that of LPp group.These results indicated that sLPc2 can significantly resist against H2O2-induced injury effects on chicken embryo hepatocyte which mechanism may be the changes of protein expression in mitochondrial apoptosis signaling pathways. |
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