Preparation Of Polysaccharides Isolated From Yu-ping-feng And Its Immune-enhancing Activity In Vitro And In Vivo

The application of an immunopotentiator, an agent in stimulating immune response, has a very important significance in improving the immune function, and thereby enhancing resistance for infectious diseases in animal production. However, most of the commonly used immunopotentiators are chemically synthesized, potentially having hidden dangers in the drug residues. As the food safety and security arouse public concern, the use of traditional herbal medicines has demonstrated some advantages. Many studies have shown that polysaccharide is an important component of traditional Chinese medicine, which is a class of complex natural macromolecular compounds naturally occurring in plants, microorganisms(bacteria and fungi), and seaweeds. Many pharmacological and clinical studies have shown that polysaccharides have extensive pharmacological and biological functions, such as immune-enhancing, anti-viral, anti-tumor, anti-oxidant, anti-aging, and so on.In this research the Yu-Ping-Feng Pulvis(YPFP) as a whole to pursue the active ingredients of polysaccharides and compare it to Aatractylodis polysaccharide(RAMPS). The immune-enhancing activity of Yu-Ping-Feng polysaccharide(YPF-PS) was explored in vitro and in vivo. In this research the total YPF-PS, RAMPS and three fractional RAMPSs were extracted by one-step or stepwise ethanol precipitation method respectively. Their immune-enhancing activities were compared and three polysaccharides with better efficacy were selected. We then compared the effects of lymphocyte cell cycle distribution and the percentages of CD4+ and CD8+ cells in vitro and the effect of NDV vaccine immune response in vivo. The immune-enhancing activities of these three polysaccharides were compared. The details are divided into five parts as follows:Five crude polysaccharides: crude total YPF-PS(YPF-PStc) 、 crude total RAMPS(RAMPStc) and three crude fractional Atractylodis polysaccharide RAMPS60c、RAMPS70c and RAMPS80 c were extracted by water-decocting and one-step or stepwise ethanol precipitation method. YPF-PStc and RAMPStc was purified by chromatography through DEAE sephadex A-25 to get purified YPF-PS(YPF-PStp) and purified RAMPS(RAMPStp). The carbohydrate and protein contents of all polysaccharides were determinated recpectively by phenol-sulfuric acid and coomassie brilliant blue G-250 methods. Identification of the polysaccharides structural by Fourier transforms infrared spectroscopy(FTIR). The results showed that extraction rate of YPF-PStc was 13.56 %, RAMPStc was 13.36 %, RAMPStp was 35.6 % and YPF-PStp was 16.87 %. Among three fractional atractylodis polysaccharide, extraction rate of RAMPS60 c was hightest, 14.79 %. The carbohydrate content of RAMPStp was highest to 97.14 %, was increased by 9.93 % over RAMPStc. The carbohydrate content of YPF-PStp was 85.6 %, was increased by 18.25 % over YPF-PStc. The protein contents of all polysaccharides were lower. The results indicated that the carbohydrate content of polysaccharides was increased after purified and the polysaccharides had characteristic absorption peak in infrared spectrum.Firstly the safe concentrations of YPF-PStc, YPF-PStp, RAMPStc, RAMPStp, RAMPS60 c, RAMPS70 c and RAMPS80 c on Chicken peripheral blood lymphocyte(PBL) were compared by MTT method. Then seven polysaccharides at five concentrations within safe concentration were added into the cultivating system of peripheral blood lymphocyte singly or synergistically with PHA. The peripheral blood lymphocyte proliferation was determined by MTT method(A570 value and hightest lymphocyte proliferation rate). The results showed that in single adding YPF-PStc and RAMPStp at 15.625~62.5 μg m L-1, YPF-PStp at 7.813~62.5 μg mL-1 and RAMPS70 c at 31.25 μg mL-1 could significantly stimulated lymphocyte proliferation, and the lymphocyte proliferation rate of YPF-PStp at 31.25 μg mL-1 was highest. In simultaneous adding with PHA YPF-PStc at 15.625~62.5 μg m L-1, YPF-PStp at 15.625~31.25 μg mL-1, RAMPS60 c at 0.781~1.563 μg m L-1 and RAMPStp at 3.125 μg m L-1 could significantly stimulated lymphocyte proliferation. The lymphocyte proliferation rate of RAMPS60 c at 7.813 μg m L-1 was highest. In general evaluation, YPF-PStp、RAMPS60c and RAMPStp were selected and maybe were the immune-enhancing active site of polysaccharides.The cells were treated with polysaccharide groups(RAMPS60c and RAMPStp) at 31.25 μg m L-1(selected according to the preliminary experiment results). Afterward, the cells were harvested after 24, 48, and 72 h, washed with PBS, and fixed in 70 % ethanol solution at 4 °C overnight. The samples were then analyzed by flow cytometry. The results showed that within 72 h most of the cells in G0/G1 phase within blank control group and the changes were not obvious. PHA induced a significant decrease in the percentage of G0/G1 phase(P< 0.05) and a significant increase in the percentage of S and G2/M phase(P< 0.05) compared with the normal control group. The ratio of G0/G1 phase in YPF-PStp, RAMPStp and RAMPS60 c groups decreased(P< 0.05) when the cells were stimulated with PHA for all times. By contrast, the ratio of S and G2/M phase in YPF-PStp and RAMPStp groups increased significantly(P< 0.05) compared with that of PHA group for 48 h, at the same time SPF and PI were the largest.The cells were harvested after 24, 48 and 72 h, washed twice with ice-cold PBS, and percentages of CD4+ and CD8+ cells were determined by flow cytometry. The results showed that at all time points, the percentages of CD4+ T cells in polysaccharides groups were significantly higher than those in the PHA group(P< 0.05) and in YPF-PStp and RAMPStp groups were significantly higher than those in the RAMPS60 c group in 48 h. At 48 and 72 h, the percentages of CD8+ T cells and CD4+/CD8+ in polysaccharides groups were significantly higher than those in the PHA group(P< 0.05). At 48 h, the percentages of CD8+ T cells in the YPF-PStp and RAMPStp groups were significantly higher than those in the RAMPS60 c group(P< 0.05). So, YPF-PStp and RAMPStp also effectively improved the percentages of CD4+、 CD8+ T cells and the ratio of CD4+/CD8+.In order to compare the immune-enhancing action of three polysaccharides in vivo, the effects of the polysaccharides on immune response of ND vaccine in chickens were determined. A total of 210 Hy-Line Variety Brown chickens(male) were randomly assigned into six groups, and each isolator with 30 chickens comprised one group. The 14-day-old chickens except blank control group were vaccinated with ND vaccines by nose and eye dropping method, the repeated vaccination was at 28 days old. At the same time of the first vaccination, the chickens in groups I to III were injected subcutaneously with 0.5 ml of YPF-PStp(6 mg ml-1), 0.5 ml of RAMPStp(6 mg ml-1) and 0.5 ml of RAMPS60c(6 mg ml-1), group IV with Oral 0.5 ml of levamisole hydrochloride(Lev, 10 mg kg-1), respectively, once a day for three successive days. Each chicken in group V was injected subcutaneously with 0.5 ml of physiological saline. Group VI served as the blank control group, and was not subjected to any treatment. On days 7(D7), 14(D14), 21(D21) and 28(D28) after the first vaccination, four chickens from each group were selected randomly to determine and the blood samples were collected from brachial vein for determination of the antibody titers of ND vaccine in serum, and the blood samples(2 m L per chicken) were collected from heart for determination of peripheral lymphocytes proliferation by MTT assay, cell cycle and CD4+ and CD8+ T lymphocytes. In the end the chickens were weighed and euthanized at each sampling time. Then the immune organs of thymus, spleen, and bursa of Fabricius were immediately excised surgically and weighed. The results showed that YPF-PStp and RAMPStp at most time points significantly raise antibody titer, promote T lymphocyte proliferation, promote lymphocytes into S and G2/M phases, effectively improve the percentages of CD4+ and CD8+ T cells and elevate their immune organ index. In general evaluation, YPF-PStp possessed best action and maybe was the immune-enhancing active site.