植物多糖的制备及对肉仔鸡免疫功能影响的研究
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摘要
本研究通过5个试验研究了三种植物多糖牛膝多糖、芦荟多糖和黄芪多糖的提取、化学结构及其对肉鸡免疫功能的影响并从细胞信息传导角度探讨其作用机制。
试验一 黄芪多糖、芦荟多糖的制备及结构分析
本试验研究了黄芪多糖及芦荟多糖的提取，分别用纸层析法和气相色谱法研究了单糖的组成，硫酸苯酚法测定多糖含量，凝胶色谱法纯化多糖并测定多糖分子量，紫外和红外光谱法研究了多糖结构。结果发现芦荟粗多糖由三种组分组成，组分Ⅱ(AP2)分子量为45400，由葡萄糖、甘露糖、半乳糖、果糖及微量的岩藻糖、木糖组成，摩尔比为5.369：12.38：1：3.896：0.094：0.381。芦荟多糖的红外图谱显典型的多糖图谱。在3398.49、2934.78、1591.14、1419.22、1320.74、1093.68和879.95cm~(-1)有吸收峰，糖苷键为β-糖苷键。黄芪粗多糖由2种组分组成，组分Ⅰ(APS1)分子量为67600，纸色谱表明主要由葡萄糖组成，气相色谱显示还有甘露糖、半乳糖、果糖、木糖和岩藻糖。摩尔比为22.776：3.518：1：3.704：2.981：0.745。黄芪多糖的红外图谱显典型的多糖图谱，在3396.04、2920.28、1622.79、1418.29、1385.19、1150.93、1078.67、843.82和1023.04cm~(-1)有吸收峰，兼有α和β-糖苷键。理化性质显示芦荟多糖是酸性多糖而黄芪多糖为中性多糖。芦荟多糖旋光度为+5.56，黄芪多糖为+45.01。紫外图谱显示芦荟多糖和黄芪多糖均不含蛋白质及核酸，但蛋白质和氨基酸测定显示：芦荟粗多糖和黄芪粗多糖均是含一定蛋白质的糖蛋白。
试验二 不同植物多糖对肉仔鸡免疫功能、生产性能的影响研究
本试验首次研究了两种大分子多糖芦荟多糖和黄芪多糖及一种小分子多糖牛膝多糖在饲料中添加200mg／kg对肉仔鸡免疫功能、生产性能的影响。研究发现：三种多糖对肉鸡生产性能无显著影响，但对免疫功能有一定影响：三种多糖均显著提高了法氏囊指数(p＜0.01)，牛膝多糖还显著提高了肉鸡新城疫抗体滴度(P＜0.05)，对外周血淋巴细胞转化率也有一定影响，但差异不显著(p=0.12)，三种多糖对肉鸡ANAE阳性率及脾脏、胸腺指数均无显著影响。三种多糖对血液指标有显著影响，使血清白蛋白和钙离子浓度显著升高(p＜0.05)。以上结果提示：多糖口服有一定的免疫增强作用，但可能与分子量有关，小分子量多糖可能更容易吸收从而刺激免疫功能。
中国农业科学院博士论文摘要
试验三多糖的体外抗菌、抗病毒活性研究及攻毒保护实验研究
本试验研究了芦苔多糖、黄蔑多糖、牛膝多糖的体外抗茵、抗病毒活性以及
对新城疫强毒感染鸡的保护作用。发现在0．1％浓度下，三种多糖对金色葡萄球菌、
绿脓杆菌、沙门氏菌、大肠杆菌、禽巴氏杆菌5种细菌均无抑菌作用，黄蔑多糖
在三种浓度下对所有细菌均无抑菌作用。在0．5％和1％浓度下，芦苔多糖和牛膝
多糖对大肠杆菌和金色葡萄球菌均有一定的抑制作用。芦苔多糖在1％浓度下对绿
脓杆菌也有一定抑制作用。抗病毒活性：在低浓度下Q u g砌X各多糖对病毒
基本无作用。牛膝多糖在各浓度下对新城疫病毒均无作用。芦苔多糖在 100、500
ug加1对新城疫病毒均有显著的治疗和灭活作用，使血凝滴度显著降低。黄蔑多
糖对新城疫病毒也有显著的抑制作用，使血凝滴度降低。但只在 500 u g加l达到
了显著水平。攻毒保护实验研究：不同多糖体内注射均提高了鸡的HI抗体水平。
对于未免疫鸡，新城疫强毒攻击各处理鸡的发病率和发病死亡率均为 100兑三种
多糖对鸡起不到任何保护作用和治疗作用。对于兔疫鸡，不同多糖均在一定程度
上降低了鸡的发病率。上述结果提示：芦苔多糖和牛膝多糖在体外有一定的抗菌
作用，但需要较高的浓度。黄蔑多糖和芦苔多糖对鸡胚内新城疫病毒有一定抑制
作用。多糖能通过提高肉鸡HI抗体水平，降低新城疫强毒攻击鸡的死亡率。
试验四 不同植物多糖对鸡脾淋巴细胞免疫功能的影响
本试验研究了不同浓度黄苗多糖、芦苔多糖和牛膝多糖体外对肉鸡脾淋巴细
胞转化和细胞因子IL－1、IL－2分泌的影响。发现黄蔑多糖在三种浓度均可提高脾
淋巴细胞转化率（p＜0．05），牛膝多糖在 80、160 u g加 也促进脾淋巴转化
乃．05人 芦苔多糖对脾淋巴细胞转化无影响。三种多糖均可促进脾淋巴细胞
IL－2的分泌（P＜0．05），但作用与剂量有关：芦苔多糖在三种浓度均促进了IL－2
分泌，黄蔑多糖在 40、80 n g加 促进了 IL《的分泌，牛膝多糖在 80 u g加 促进
了IL－2的分泌。本研究首次发现除牛膝多糖外，芦苔多糖和黄芭多糖均显著抑制
了正常及地塞米松处理巨噬细胞IL－1的分泌（P（．01）。以上结果提示，三种多糖
均可促进淋巴细胞功能，并通过调节细?
A series of 5 experiments were conducted to investigate the extraction, structure, immunomodulating effects of three Chinese traditional herbal polysaccharides and approach the mechanism from view of cellular signal transduction.
Experiment 1 Studies of the extraction, purification and chemical characteristics of aloe polysaccharide and astragalan
This research was conducted to study the extraction, purification and chemical characteristics of aloe polysaccharide(AP) and astragalan polysaccharide(ASP). The results showed that crude AP has three components, The molecular weight of component 2 was 45400. It was composed of glucose, mannose, galactose and fructose, as well as trace fucose and xylose. Its mole ration was 5.369: 12.38: 1: 3.896: 0.094: 0.381. Crude ASP has two components, the molecular weight of component 1 was 67600. It was composed of glucose, mannose, galactose and fructose, as well as trace fucose and xylose. Its mole ration was 22.776: 3.518: 1: 3.704: 2.981: 0.745 respectively. The infrared spectrums of AP and ASP are typical one of polysaccharide, the peak absorption of AP was at 3398.49, 2934.78,1591.14,1419.22,1320.74,1093.68 and 879.95 cm"1, while the peak absorption of ASP was at 3396.04, 2920.28, 1622.79,1418.29, 1385.19,1150.93, 1078.67, 843.82,1023.04 cm-1. The ultraviolet spectrum revealed that both purified polysaccharide did not contain protein and nucleic acid. The chemical analysis revealed that AP is an acidic one and ASP is a neutral one.
Experiment 2 Effect of different herbal polysaccharide on immunity and growth performance of broilers
This research was conducted to study the effect of different herbal polysaccharides supplemented in the feed at the dose of 200mg/kg on immunity and growth performance of young broilers. It was found that three polysaccharides had no effect on growth performance of broilers relative to the control during the 28day trial, but affected the immunity in a different way; they all increased Bursa index significantly(p<0.01), Achyranthan(ACH) also increased HI titers significantly(p<0.05)and lymphocyte proliferation(p=0.12),three polysaccharide also
significantly affected serum index, they increased serum albumin and calcium concentration significantly. The result indicates that smaller molecular weight polysaccharide may be more effective than larger ones in stimulating immunity when administered orally.
Experiment 3 Studies on the antiviral and antibacterial activity of different herbal polysaccharides
This research was conducted to study the in vitro antiviral, antibacterial activity and protection of broilers infected with Newcastle virus of different herbal polysaccharides. It was found that at low concentrations (0.1%),three polysaccharides had no antibacterial activities to five bacteria. At the concentration of 0.5 and 1%, AP and ACH could inhibit the proliferation of Escherichia coli and staphylococcus aureus, AP had antibacterial activity to Pseudomonas aeruginosa at the concentration of 1%. No antiviral activity to Newcastle virus was observed for three polysaccharide at the concentration of 10 ug/ml, ACH showed no antiviral activity at all concentrations. AP showed significant antiviral activity to ND virus at 500 and lOO^g/ml. ASP also showed significant antiviral activity to ND virus at 50ug/ml 1. In the protection trial, all three polysaccharide significantly increased HI antibody titers when injected. Despite the polysaccharide treatment, all non-vaccined broilers were infected and died by virulent ND virus attack. But for vaccined broilers, all three polysaccharide decreased the mortality of ND virus assaulted chickens. The above results indicated that the in vitro antibacterial activity of polysaccharide is weak ASP and AP had antiviral activity to ND virus in vitro depending on the doses. Polysaccharide can decrease the mortality of ND virus infected broilers by increasing HI antibody titers, but showed no use for non-vaccined broilers.
Experiment 4 Effect of different herbal polysaccharide on splenocyte function
试验一 黄芪多糖、芦荟多糖的制备及结构分析
本试验研究了黄芪多糖及芦荟多糖的提取，分别用纸层析法和气相色谱法研究了单糖的组成，硫酸苯酚法测定多糖含量，凝胶色谱法纯化多糖并测定多糖分子量，紫外和红外光谱法研究了多糖结构。结果发现芦荟粗多糖由三种组分组成，组分Ⅱ(AP2)分子量为45400，由葡萄糖、甘露糖、半乳糖、果糖及微量的岩藻糖、木糖组成，摩尔比为5.369：12.38：1：3.896：0.094：0.381。芦荟多糖的红外图谱显典型的多糖图谱。在3398.49、2934.78、1591.14、1419.22、1320.74、1093.68和879.95cm~(-1)有吸收峰，糖苷键为β-糖苷键。黄芪粗多糖由2种组分组成，组分Ⅰ(APS1)分子量为67600，纸色谱表明主要由葡萄糖组成，气相色谱显示还有甘露糖、半乳糖、果糖、木糖和岩藻糖。摩尔比为22.776：3.518：1：3.704：2.981：0.745。黄芪多糖的红外图谱显典型的多糖图谱，在3396.04、2920.28、1622.79、1418.29、1385.19、1150.93、1078.67、843.82和1023.04cm~(-1)有吸收峰，兼有α和β-糖苷键。理化性质显示芦荟多糖是酸性多糖而黄芪多糖为中性多糖。芦荟多糖旋光度为+5.56，黄芪多糖为+45.01。紫外图谱显示芦荟多糖和黄芪多糖均不含蛋白质及核酸，但蛋白质和氨基酸测定显示：芦荟粗多糖和黄芪粗多糖均是含一定蛋白质的糖蛋白。
试验二 不同植物多糖对肉仔鸡免疫功能、生产性能的影响研究
本试验首次研究了两种大分子多糖芦荟多糖和黄芪多糖及一种小分子多糖牛膝多糖在饲料中添加200mg／kg对肉仔鸡免疫功能、生产性能的影响。研究发现：三种多糖对肉鸡生产性能无显著影响，但对免疫功能有一定影响：三种多糖均显著提高了法氏囊指数(p＜0.01)，牛膝多糖还显著提高了肉鸡新城疫抗体滴度(P＜0.05)，对外周血淋巴细胞转化率也有一定影响，但差异不显著(p=0.12)，三种多糖对肉鸡ANAE阳性率及脾脏、胸腺指数均无显著影响。三种多糖对血液指标有显著影响，使血清白蛋白和钙离子浓度显著升高(p＜0.05)。以上结果提示：多糖口服有一定的免疫增强作用，但可能与分子量有关，小分子量多糖可能更容易吸收从而刺激免疫功能。
中国农业科学院博士论文摘要
试验三多糖的体外抗菌、抗病毒活性研究及攻毒保护实验研究
本试验研究了芦苔多糖、黄蔑多糖、牛膝多糖的体外抗茵、抗病毒活性以及
对新城疫强毒感染鸡的保护作用。发现在0．1％浓度下，三种多糖对金色葡萄球菌、
绿脓杆菌、沙门氏菌、大肠杆菌、禽巴氏杆菌5种细菌均无抑菌作用，黄蔑多糖
在三种浓度下对所有细菌均无抑菌作用。在0．5％和1％浓度下，芦苔多糖和牛膝
多糖对大肠杆菌和金色葡萄球菌均有一定的抑制作用。芦苔多糖在1％浓度下对绿
脓杆菌也有一定抑制作用。抗病毒活性：在低浓度下Q u g砌X各多糖对病毒
基本无作用。牛膝多糖在各浓度下对新城疫病毒均无作用。芦苔多糖在 100、500
ug加1对新城疫病毒均有显著的治疗和灭活作用，使血凝滴度显著降低。黄蔑多
糖对新城疫病毒也有显著的抑制作用，使血凝滴度降低。但只在 500 u g加l达到
了显著水平。攻毒保护实验研究：不同多糖体内注射均提高了鸡的HI抗体水平。
对于未免疫鸡，新城疫强毒攻击各处理鸡的发病率和发病死亡率均为 100兑三种
多糖对鸡起不到任何保护作用和治疗作用。对于兔疫鸡，不同多糖均在一定程度
上降低了鸡的发病率。上述结果提示：芦苔多糖和牛膝多糖在体外有一定的抗菌
作用，但需要较高的浓度。黄蔑多糖和芦苔多糖对鸡胚内新城疫病毒有一定抑制
作用。多糖能通过提高肉鸡HI抗体水平，降低新城疫强毒攻击鸡的死亡率。
试验四 不同植物多糖对鸡脾淋巴细胞免疫功能的影响
本试验研究了不同浓度黄苗多糖、芦苔多糖和牛膝多糖体外对肉鸡脾淋巴细
胞转化和细胞因子IL－1、IL－2分泌的影响。发现黄蔑多糖在三种浓度均可提高脾
淋巴细胞转化率（p＜0．05），牛膝多糖在 80、160 u g加 也促进脾淋巴转化
乃．05人 芦苔多糖对脾淋巴细胞转化无影响。三种多糖均可促进脾淋巴细胞
IL－2的分泌（P＜0．05），但作用与剂量有关：芦苔多糖在三种浓度均促进了IL－2
分泌，黄蔑多糖在 40、80 n g加 促进了 IL《的分泌，牛膝多糖在 80 u g加 促进
了IL－2的分泌。本研究首次发现除牛膝多糖外，芦苔多糖和黄芭多糖均显著抑制
了正常及地塞米松处理巨噬细胞IL－1的分泌（P（．01）。以上结果提示，三种多糖
均可促进淋巴细胞功能，并通过调节细?
A series of 5 experiments were conducted to investigate the extraction, structure, immunomodulating effects of three Chinese traditional herbal polysaccharides and approach the mechanism from view of cellular signal transduction.
Experiment 1 Studies of the extraction, purification and chemical characteristics of aloe polysaccharide and astragalan
This research was conducted to study the extraction, purification and chemical characteristics of aloe polysaccharide(AP) and astragalan polysaccharide(ASP). The results showed that crude AP has three components, The molecular weight of component 2 was 45400. It was composed of glucose, mannose, galactose and fructose, as well as trace fucose and xylose. Its mole ration was 5.369: 12.38: 1: 3.896: 0.094: 0.381. Crude ASP has two components, the molecular weight of component 1 was 67600. It was composed of glucose, mannose, galactose and fructose, as well as trace fucose and xylose. Its mole ration was 22.776: 3.518: 1: 3.704: 2.981: 0.745 respectively. The infrared spectrums of AP and ASP are typical one of polysaccharide, the peak absorption of AP was at 3398.49, 2934.78,1591.14,1419.22,1320.74,1093.68 and 879.95 cm"1, while the peak absorption of ASP was at 3396.04, 2920.28, 1622.79,1418.29, 1385.19,1150.93, 1078.67, 843.82,1023.04 cm-1. The ultraviolet spectrum revealed that both purified polysaccharide did not contain protein and nucleic acid. The chemical analysis revealed that AP is an acidic one and ASP is a neutral one.
Experiment 2 Effect of different herbal polysaccharide on immunity and growth performance of broilers
This research was conducted to study the effect of different herbal polysaccharides supplemented in the feed at the dose of 200mg/kg on immunity and growth performance of young broilers. It was found that three polysaccharides had no effect on growth performance of broilers relative to the control during the 28day trial, but affected the immunity in a different way; they all increased Bursa index significantly(p<0.01), Achyranthan(ACH) also increased HI titers significantly(p<0.05)and lymphocyte proliferation(p=0.12),three polysaccharide also
significantly affected serum index, they increased serum albumin and calcium concentration significantly. The result indicates that smaller molecular weight polysaccharide may be more effective than larger ones in stimulating immunity when administered orally.
Experiment 3 Studies on the antiviral and antibacterial activity of different herbal polysaccharides
This research was conducted to study the in vitro antiviral, antibacterial activity and protection of broilers infected with Newcastle virus of different herbal polysaccharides. It was found that at low concentrations (0.1%),three polysaccharides had no antibacterial activities to five bacteria. At the concentration of 0.5 and 1%, AP and ACH could inhibit the proliferation of Escherichia coli and staphylococcus aureus, AP had antibacterial activity to Pseudomonas aeruginosa at the concentration of 1%. No antiviral activity to Newcastle virus was observed for three polysaccharide at the concentration of 10 ug/ml, ACH showed no antiviral activity at all concentrations. AP showed significant antiviral activity to ND virus at 500 and lOO^g/ml. ASP also showed significant antiviral activity to ND virus at 50ug/ml 1. In the protection trial, all three polysaccharide significantly increased HI antibody titers when injected. Despite the polysaccharide treatment, all non-vaccined broilers were infected and died by virulent ND virus attack. But for vaccined broilers, all three polysaccharide decreased the mortality of ND virus assaulted chickens. The above results indicated that the in vitro antibacterial activity of polysaccharide is weak ASP and AP had antiviral activity to ND virus in vitro depending on the doses. Polysaccharide can decrease the mortality of ND virus infected broilers by increasing HI antibody titers, but showed no use for non-vaccined broilers.
Experiment 4 Effect of different herbal polysaccharide on splenocyte function
引文
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