Isolation And Purification Of Fucoidan From Laminaria Japonica And Its Immunomodulatory Activities On Broiler Macrophage

1,Extracted and purified Fucoidan from Laminaria japonicaThe fucoidans was extracted and isolated from Laminaria japonica by the method of water boiling and ethanol precipitation. The optimized condition were as follows: radio of samples to water,1:25; time, 6h; temperature,100℃.Under the conditions shown above, the resulted content of Fucoidan was 5.47%. Fucoidan was further purified by ion exchange chromatography DEAE and gel filtration chromatography Sephacryl into five different Fractions, Fractionâ… to Fractionâ…¤. Their molecular weights were 40KD,74KD,163KD,240KD,360KD respectively. The polysaccharide was composed of fucose, glucose, xylose, rhamnose according to thin-layer chromatography. All five Fractions exhibited character vibration peak of polysaccharide and had no protein and nucleic acid.2,Effects of Fucoidan isolated from Laminaria japonica on growth performance and immunity of broilersThe trial was conducted to study the effects of Fucoidan extracted from Laminaria japonica on the immunity and growth performance of broilers. Total of 600 broilers were randomly allotted to one of four dietary treatments, with six replicate pens per treatment and twenty-five chickens per pens. Dietary treatments included a control corn-soybean meal (Control), a diet with 100mg/kg Fucoidan (Low level), a diet with 500mg/kg Fucoidan (Middle level), a diet with 1000mg/kg Fucoidan (High level). Birds were raised from hatch to 42 d. At the age 42d, the growth performance, the immune organ indexes, the proliferation of blood lymphocyte, the proportions of T cell subsets in blood were measured. The macrophages were harvested and the phagocytic activity, NO, iNOS and respiratory burst production of macrophages were detected. Feeding Fucoidan had no significant effects on growth performance of broilers relative to the control. Compared to the control, feeding Fucoidan significantly increased spleen index and thymus index (P<0.05), enhanced the proliferation of T lymphocyte induced by ConA (P<0.05).The percentage of CD4+ T cell was higher in 42d old chickens maintained on Fucoidan feed than in chichens receiving the control corn-soybean meal. Fucoidan had significantly increased macrophages to relesee NO, significantly promoted iNOS activity and stimulated the respiratory burst activity. The Fucoidan isolated from Laminaria japonica possess potent immunomodulatory activity on T lymphocyte and macrophages on broilers.3,Effects of Fucoidan and its Fraction isolated from Laminaria japonica on immunity of broiler macrophages in vitroThe immunomodulatory activities of Fucoidan (FLJ) and its Fraction were investigated in vitro in relation to broiler peritoneal macrophages. The broiler macrophages were cultured with Fucoidan (200μg/ml) and Fraction fromâ… toâ…¤(100μg/ml) for 24h, the cell vialibility were detected by using the mothod of MTT and the phagocytic activity were detected. The induced nitric oxide synthase (iNOS), NO, IL-1 and TNF-αin culture supernatants were measured. The respiratory burst capacity in macrophages was measured by luminal dependent chemiluminescence.Cell vialibility results showed that the proliferation of macrophages was not influenced by FLJ and its Fractions. The treatment of FLJ and its Fraction increased the phagocytic activity, enhanced the production of IL-1, NO, iNOS and the respiratory burst capacity (P<0.05). The Fucoidan and its Fraction had no significantly effect on TNF-α(P>0.05). The different Fraction had different effect on broiler macrophage immunity function. Fractionâ…¢and Fractionâ…£had the most effective promoting action. Their promotion effects were higher than Fractionâ…¤.The Fractionâ…¤had no effect on broilers macrophages immunity function. The Fucoidan isolated from Laminaria japonica had promoting effect on broiler macrophage immunity at 200μg/ml.The Fractionâ…¢and Fractionâ…£purified from Fucoidan were the effective Fraction among five Fraction.4,Effect of Fucoidan and its Fraction isolated from Laminaria japonica on immunity of broiler macrophages under oxidation stress conditionThe immunomodulatory activities of a Fucoidan (FLJ) and its Fraction were investigated in vitro under oxidation stress in broiler macrophages. After the broiler macrophages were cultured with FLJ and its Fraction for 24h, the tBOOH was added to the culture as oxygen reagent. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and induced nitric oxide synthase (iNOS) were detected. The content of lactate dehydrogenase (LDH), malonic dialdehyde (MDA), NO, IL-1 and TNF-αwere measured. The respiratory burst capacity of broiler macrophages was measured by luminal dependent chemiluminescence. The elevated MDA and LDH, decreased cell vialibility, increased SOD and GSH-Px levels indicated that adding tBOOH in culture caused the oxidation stress in broiler macrophages. The decreased of phagocytic activity, NO, iNOS, IL-1 and respiratory burst capacity indicated that the immune function of broiler macrophages were influenced by tBOOH. When the FLJ and its Fraction were present in culture, the oxidation stress were eliminated. The cell vialibility, SOD and GSH-Px were significantly increased (P<0.05), LDH and MDA were significantly decreased (P<0.05). The immune function of macrophages was enhanced. The different Fraction had different antioxidant activities. Fractionâ…¢and Fractionâ…£had the most effective antioxidant activities. These data suggest that the FLJ and its Fraction had antioxidant activities, and the antioxidant activities of FLJ and its Fraction were related to their immunomodulatory activities.5,Macrophage Immunomodulatory mechanism of Fucoidan and Fraction isolated from Laminaria japonica: Role of calciumThe effect of Fucoidan (FLJ) and its Fraction on Ca²⁺ content of broiler macrophages were investigated in vitro to conform if FLJ and its Fraction can activize Ca²⁺ signal transmit system in immune cell. Using the highly sensitive Ca²⁺ fluorescent dye, Fura-2/AM, the kinetic changes of Ca²⁺ in single intact living cells were measured, before and after FLJ and its Fraction directly treatment. Using A23187 and EGTA to change Ca²⁺ content of macrophages, the immunity of cell were detected in vitro. The intracellular Ca²⁺ content increased after FLJ treatment (P<0.05). The different Fraction had different action to the Ca²⁺ content. The Fractionâ…¢and Fractionâ…£induced intracellular Ca²⁺ content increase (P<0.05), while the Fractionâ… , Fractionâ…¡and Fractionâ…¤induced intracellular Ca²⁺ content decreased (P<0.05). Using A23187 to increase the Ca²⁺ content of lived cell, the immune function of broiler macrophages were enhanced, while using EGTA to decrease the Ca²⁺ content, the immune function of lived cell were reduced. After adding EGTA and Fraction into the culture at the same time, the phagocytic activity, the respiratory burst capacity, the production of IL-1, NO, iNOS of broiler macrophages were decreased significantly (P<0.05). The immunomodulatory activities of a Fucoidan and its Fraction had a relationship with increasing the intracellular Ca²⁺ content.