余甘子多糖的结构表征及免疫增强作用

本试验旨在制备余甘子多糖（Phyllanthus emblica polysaccharide，PEP），通过对其结构表征进行分析，探索PEP对鸡新城疫（Newcastle disease，ND）疫苗免疫效果的影响。利用紫外光谱（ultraviolet spectroscopy，UV）、红外光谱（Fourier transform infrared spectroscopy，FT-IR）、高效液相色谱分析法（high performance liquid chromatography，HPLC）和扫描电镜（scanning electron microscope，SEM）分析PEP结构；选取175只1日龄河田鸡，随机分成5组，每组35只，分别为空白对照组，疫苗对照组，PEP低、中、高剂量组。除空白对照组外均用ND疫苗进行免疫，7日龄首免，并于28日龄二免。在免疫的同时，PEP低、中、高剂量组分别按照10、20、30 g·L^-1的剂量给药PEP溶液0.5 mL，疫苗对照组和空白对照组给予等量生理盐水，每天1次，连续3 d。分别于7、14、21、28、35、42和49日龄时，测定血清中新城疫病毒（Newcastle disease virus，NDV）特异性抗体水平以及γ-干扰素（INF-γ）和白介素4（IL-4）的水平；于28和49日龄时测定外周血淋巴细胞增殖活性和T淋巴细胞CD4⁺/CD8⁺的比值；于49日龄剖杀后采集脾、法氏囊、胸腺和肝进行组织学观察以及检测免疫器官指数。结果表明，PEP为白色棉絮状，糖含量为（86.70±1.05）%，结构分析结果表明，PEP是含α型糖苷键的多糖，其组分均一，不含核酸和蛋白质，呈片状分布，总体结构规整且致密，单糖组成为葡萄糖醛酸（glucuronic acid，GlcA）、葡萄糖（glucose，Glu）、木糖（xylose，Xyl）和岩藻糖（fucose，Fuc），其摩尔比为0.5：8.3：1.8：0.3。动物试验结果表明，PEP能显著提高鸡血清中NDV特异性抗体、INF-γ和IL-4的水平（P<0.05），还能显著促进鸡外周血淋巴细胞增殖和T淋巴细胞CD4⁺/CD8⁺的比值（P<0.05），提高免疫器官指数（P<0.05），激活脾、法氏囊、胸腺和肝等器官的免疫功能。PEP糖含量高，结构稳定，可提高机体免疫活性，具有较良好的佐剂活性，对ND具有良好的防治效果。

Structural Characterization and Immune Enhancement of Phyllanthus emblica Polysaccharide

The purpose of this experiment was to prepare Phyllanthus emblica polysaccharide (PEP) and analyze its structural characterization and investigate the effect of PEP on immune effect of Newcastle disease (ND) vaccine. The structural characterization of PEP was analyzed by ultraviolet spectroscopy (UV), Fourier transform infrared spectroscopy (FT-IR), high performance liquid chromatography (HPLC) and scanning electron microscope (SEM). At the same time, 175 1-day-old Hetian chickens were randomly divided into 5 groups with 35 chickens in each group, including blank control group, vaccine control group, PEP low-dose group, PEP medium-dose group and PEP high-dose group. All chickens were immunized with ND vaccine at 7 days old except the blank control group, and immunized second times at 28 days old. At the same time of immunization, the low-, medium- and high-dose groups of PEP were given 0.5 mL of PEP solution according to the doses of 10, 20 and 30 g·L^-1, respectively, while vaccine control group and blank control group with physiological saline, once a day for 3 successive days. On 7, 14, 21, 28, 35, 42, and 49 days old, the levels of Newcastle disease virus (NDV) antibody, INF-γ and IL-4 were tested. On 28 and 49 days old, the proliferation of splenic lymphocytes and CD4⁺/CD8⁺ratios were measured. Chickens were slaughtered and the thymus, spleen, bursa and liver were take at 49 days old, then the organs index were calculated. The results showed that PEP was white cotton flocculent, the total carbohydrate content of PEP was (86.70±1.05)%. It was a kind of homogenous polysaccharide containing α-type glycosidic bond without nucleic acid and protein. Also, it was determined to be composed of glucuronic acid (GlcA), glucose (Glu), xylose (Xyl) and fucose (Fuc) in the molar ratio of 0.5:8.3:1.8:0.3. It was distributed in sheet shape, and its overall structure was regular and dense. The results of animal experiment found that PEP significantly increased the level of NDV specific antibody, IFN-γ and IL-4 (P<0.05), the proliferation of splenic lymphocytes, the immune organ index and CD4⁺/CD8⁺ratios (P<0.05). Also activated immune function of spleen, bursa, thymus and liver. The total carbohydrate content of PEP was high and its structure was stable. Also, PEP could improve the immune activity of the body, possess good adjuvant activity and control effect on the ND for chicken.