CN103393746A - 一种猪用免疫增强剂及其制备方法和应用 - Google Patents
一种猪用免疫增强剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开一种猪用免疫增强剂，按重量比3:1-3:1-3由红车轴草、甘草和绞股蓝配制而成。制备方法如下：第一步，备料：按重量比3:1-3:1-3称取红车轴草、甘草和绞股蓝，粉碎，过80目筛并混匀；第二步，醇提：上述原料用石油醚60℃脱脂6小时后，按重量体积比1：8-12的比例加入重量百分比浓度为50-70%的乙醇，在60-80℃下回流提取2-3小时，共提取1-3次；第三步，干燥：过滤上述提取液，减压浓缩滤液得浓缩液，喷雾干燥或自然干燥制得粉末，即猪用免疫增强剂。本发明按饲料重量的0.1-0.8%添加应用在饲料中。本发明可以显著增强猪体的免疫力，同时特别适用于缓解疫苗免疫应激。
Description
技术领域
本发明涉及一种猪用免疫增强剂及其制备方法和应用。
背景技术
随着畜牧业集约化和规模化的发展，健康养殖和食品安全已经引起人们的广泛关注。尤其是以抗生素为代表的化学药品的大量使用，造成细菌耐药性以及畜产品中兽药残留的问题日益严重。随着现代医学、细胞生物学及分子生物学的发展，人们逐渐认识到许多动物疾病的发生、发展与机体免疫系统功能缺陷和失调有密切的联系，治疗重点已由直接杀伤外源性病原体转向调整机体自身功能，以提高机体免疫力、抗病力，因此免疫增强剂的应用引起人们的关注。目前中草药免疫增强剂在促进畜禽健康养殖过程中的作用已得到了越来越广泛的重视。
在现代养猪业生产中，疫苗对预防、控制和消灭传染病发挥着极其重要的作用，免疫接种一方面保护了动物机体免受病原的侵害，另一方面也作为一种特殊的应激因子引起免疫应激，对机体产生一定的损害。给猪接种疫苗后，有的猪会出现减食、停食、精神沉郁或体温升高等应激现象，不仅影响猪的生长发育及生产性能，而且可能造成少数猪的死亡。猪免疫应激不但会造成经济损失，还会给猪预防免疫工作带来阻力。
目前许多猪场都是在动物发生免疫应激后用地塞米松和强的松等免疫应激药物进行治疗，但是在日粮中添加营养物质来抵抗免疫应激仍是畜牧工作者的研究重点，目前研制出的抗应激添加剂大致可分为氨基酸类添加剂、脂肪酸类添加剂、维生素类添加剂及中草药类添加剂，均收到了较好的效果。随着科技的发展，近年来利用中草药制作畜禽抗应激添加剂的研究日益增多。
在免疫应激过程中，免疫细胞（尤其单核/巨噬细胞） 过量合成和分泌的炎性细胞因子（包括IL-1、IL-6和TNF-α）起着十分重要的作用，因此许多研究围绕调节炎性细胞因子的分泌或表达来抑制免疫应激。
红车轴草提取物含有鹰嘴豆芽素A、刺芒柄花素、染料木素和大豆黄酮等多种异黄酮组分，具有抑制脂多糖诱导小鼠巨噬细胞分泌炎性细胞因子的作用，已经被报道可以缓解脂多糖诱导的仔猪免疫应激。不过目前没有关于红车轴草提取物对仔猪疫苗免疫应激的影响的报道。
甘草提取物一般包含甘草甜素、甘草酸，甘草甙、甘草类黄酮、后幕比檀素、刺芒柄花素、槲皮素等。有补脾益气，清热解毒，祛痰止咳，缓急止痛，调和诸药的功效。目前在猪生产上可被用于预防和治疗由支原体引起的猪喘气病、猪肺炎、支气管炎等疾病，能够提高仔猪断奶后的采食量,降低腹泻率。
绞股蓝提取物包含50余种绞股蓝皂甙，和人参皂甙一样，同属于四环三萜类玛皂甙。现代药理学研究证明，绞股蓝具有降血脂、降血压、增加冠状动脉和脑血流量、抗衰老、强壮、增强免疫、护肝、镇静止痛、抗溃疡等作用。绞股蓝添加剂对降低断奶仔猪的料肉比、下痢率和死亡率效果都很明显。日粮中添加0.5%、1.0%绞股蓝能够显著提高免疫球蛋白IgA、IgG、IgM以及补体C3、C4含量。
发明内容
本发明的目的在于提供一种猪用免疫增强剂及其制备方法和应用，可以显著增强猪体的免疫力，同时特别适用于缓解疫苗免疫应激。
为了达成上述目的，本发明的技术方案是：
一种猪用免疫增强剂，按重量比3:1-3:1-3由红车轴草、甘草和绞股蓝配制而成；优选配伍：红车轴草、甘草和绞股蓝的重量比为3:2:2。
一种猪用免疫增强剂的制备方法，步骤如下：
第一步，备料：按重量比3:1-3:1-3称取红车轴草、甘草和绞股蓝，粉碎，过80目筛并混匀；
第二步，醇提：上述原料用石油醚60℃脱脂6 小时后，按重量体积比1：8-12的比例加入重量百分比浓度为50-70%的乙醇，在60-80 ℃下回流提取2-3小时，共提取1-3次；
第三步，干燥：过滤上述提取液，减压浓缩滤液得浓缩液，喷雾干燥或自然干燥制得粉末，即猪用免疫增强剂。
一种猪用免疫增强剂的应用，按饲料重量的0.1-0.8%添加在饲料中。
采用上述方案后，本发明具有以下有益效果和优点：
（1）红车轴草、甘草和绞股蓝配伍的醇提取物中含有黄酮、异黄酮、皂苷、三萜酸等多种活性成分，本发明的缓解免疫应激细胞试验表明红车轴草、甘草和绞股蓝单独组分的醇提取物缓解免疫应激能力均低于配伍后的复合提取物的缓解免疫应激能力，三药配伍，互为补充，能发挥复方中草药添加剂的最大功效；
（2）本发明获得的免疫增强剂显著提高断奶仔猪免疫球蛋白IgG水平和补体C3、C4水平，显著降低断奶仔猪腹泻率，表明本免疫增强剂显著提升断奶仔猪免疫力；
（3）本发明获得的免疫增强剂显著缓解疫苗免疫应激造成的生长抑制。
附图说明
图1是本发明提取物对LPS诱导RAW264.7细胞TNF-α mRNA表达的影响；
图2是本发明提取物对LPS诱导RAW264.7细胞IL-6 mRNA表达的影响；
图3是本发明复合提取物对断奶仔猪腹泻率的影响。
具体实施方式
本发明揭示的一种猪用免疫增强剂，按重量比3:1-3:1-3由红车轴草、甘草和绞股蓝配制而成。
本发明制备方法时，先备料：按重量比3:1-3:1-3称取红车轴草、甘草和绞股蓝，粉碎，过80目筛并混匀；再醇提：上述原料用石油醚60℃脱脂6 小时后，按重量体积比1：8-12的比例加入重量百分比浓度为50-70%的乙醇，在60-80 ℃下回流提取2-3小时，共提取1-3次；然后，干燥：过滤上述提取液，减压浓缩滤液得浓缩液，喷雾干燥或自然干燥制得粉末，即猪用免疫增强剂。
本发明按饲料重量的0.1-0.8%添加在饲料中，应用于猪的喂养。
本发明的研究方法与结果：
一、复合提取物对LPS诱导炎性细胞因子表达的影响
1. 实验设计
研究各提取物对LPS诱导炎性细胞因子表达的影响来进一步比较分析各提取物缓解免疫应激的能力。将小鼠RAW264.7巨噬细胞以1*107/mL的密度铺板于6孔板，培养24 h后加入待研究各提取物孵育3 h，然后加入LPS（终浓度1 μg/mL）进行刺激，6 h后收集细胞。炎性细胞因子的表达采用实时荧光定量PCR技术分析其mRNA表达水平，即用Trizol 试剂提取细胞总RNA，利用RevertAidTM First Strand cDNA Synthesis 试剂盒(Fermentas)按厂家说明将RNA反转录成cDNA，实时荧光定量PCR扩增TNF-α、IL-6 ，以β- Actin作为内参。引物序列如下：
表1 实时荧光定量PCR的引物序列
2. 结果
如图1所示，LPS诱导RAW264.7细胞TNF-α mRNA表达水平显著上升，红车轴草提取物、甘草提取物和复合提取物显著降低LPS诱导的TNF-α mRNA表达水平的上升。如图2所示，LPS诱导RAW264.7细胞IL-6 mRNA表达水平显著上升，红车轴草提取物、甘草提取物、绞股蓝提取物和复合提取物都显著降低LPS诱导的IL-6 mRNA表达水平的上升。图1，2说明复合提取物缓解LPS诱导的炎性细胞因子表达的效果更加显著。
图1是提取物对LPS诱导RAW264.7细胞TNF-α mRNA表达的影响，注：*，与LPS组相比P＜0.05；**，与LPS组相比P＜0.01。
图2是提取物对LPS诱导RAW264.7细胞IL-6 mRNA表达的影响，注：*，与LPS组相比P＜0.05；**，与LPS组相比P＜0.01。
二、复合提取物对断奶仔猪免疫力的影响和对疫苗免疫应激的影响
1.材料与方法
1.1材料
1.1.1复合提取物
按上述方法制备所得。
1.1.2试验仔猪
福建省龙岩市龙马原种猪场21日龄杜×长×大断奶仔猪。
1.1.3试验日粮
本试验饲料采用龙马饲料厂生产的乳猪用浓缩料，日粮组成及营养水平见表2。
表2 基础日粮组成及营养水平
| 原料组成 | 所占比例(%) | 营养指标 | 营养水平(%) |
| 玉米 | 60.00 | 消化能/(MJ.kg-1) | 13.83 |
| 豆粕 | 28.00 | 粗蛋白质 | 21.60 |
| 膨化大豆 | 5.00 | 钙 | 1.10 |
| 鱼粉 | 3.00 | 总磷 | 0.83 |
| 石粉 | 1.00 | 赖氨酸 | 1.16 |
| 磷酸氢钙 | 1.70 | ||
| 食盐 | 0.30 | ||
| 添加剂 | 1.00 | ||
| 合计 | 100 |
1.2试验设计
1.2.1 复合提取物对疫苗免疫应激的影响
选择21日龄断奶仔猪120头，随机分为5组，每组24头。第1组为空白对照组，饲喂基础日粮+生理盐水，第2组为阳性对照组（疫苗组），饲喂基础日粮+猪瘟疫苗，第3组饲喂基础日粮+猪瘟疫苗+0.05%复合提取物（0.05% 提取物组），第4组饲喂基础日粮+猪瘟疫苗+0.1%复合提取物（0.1% 提取物组），第5组饲喂基础日粮+猪瘟疫苗+0.2% 复合提取物（0.2% 提取物组）。试验开始前进行7天的预饲。在28日龄时，注射猪瘟疫苗，4头份/头。正式试验期为28天。分别于试验开始、试验结束时，对试验仔猪进行空腹称重，并统计耗料量，计算平均日增重、平均日采食量和料重比。
1.2.2 复合提取物对断奶仔猪免疫力的影响
选择28日龄断奶仔猪48头，随机分为4组，每组12头。第1组为对照组，饲喂基础日粮，第2、3、4组在基础日粮中添加0.05%、0.1%、0.2% 复合提取物。试验期为28天。饲养期间，饮水中不添加任何含抗生素的药物。每日观察断奶仔猪排粪情况，记录各栏猪只腹泻个体耳牌号及腹泻发生的持续天数，统计腹泻率。在试验结束当天早晨，各组随机选择6头仔猪，前腔静脉采血5 mL，测定血清中免疫球蛋白IgG、补体C3和C4。采用猪的ELISA试剂盒检测,试剂盒购自上海亚太生物科技有限公司。
2.结果
2.1复合提取物对断奶仔猪疫苗应激后生长性能的影响
复合提取物对疫苗应激后断奶仔猪生长性能的影响见表3。由表3可知，与空白对照组相比，疫苗组的平均日增重降低了11.0%，料重比提高了5.42%；而0.05%、0.1%和0.2%复合提取物组的平均日增重分别比疫苗组提高了5.44%、15.50%和17.51%，料重比分别降低了1.71%、6.29%和7.43%。
表3复合提取物对断奶仔猪疫苗应激后生长性能的影响
| 项目 | 空白对照组 | 疫苗组 | 0.05%提取物组 | 0.1%提取物组 | 0.2%提取物组 |
| 初始体重(kg) | 8.27 | 8.30 | 8.22 | 8.28 | 8.29 |
| 结束体重(kg) | 20.66 | 19.32 | 19.84 | 21.01 | 21.24 |
| 平均日增重(g) | 442.5 | 393.6 | 415.0 | 454.6 | 462.5 |
| 平均日采食量(g) | 734.3 | 690.6 | 712.5 | 746.7 | 748.3 |
| 料重比 | 1.66:1 | 1.75:1 | 1.72:1 | 1.64:1 | 1.62:1 |
2.2复合提取物对断奶仔猪免疫性能的影响
由表4可知，在免疫球蛋白IgG方面，与对照组相比，日粮中添加0.05%提取物组差异不显著，添加0.1%提取物组、0.2%提取物组差异显著(P＜0.05)，分别提高了40.9%和38.4%。
在补体方面，与对照组相比， 0.05%提取物组的C3、C4水平差异不显著，而0.1%提取物组、0.2%提取物组的补体C3水平分别显著提高了28.0%和37.5%，补体C4水平分别显著提高了64.6%和66.2%。
表4 复合提取物对断奶仔猪免疫性能的影响
| 项目 | 对照组 | 0.05%提取物组 | 0.1%提取物组 | 0.2%提取物组 |
| IgG（μg/mL） | 81.80±8.46 a | 86.71±7.33a | 115.23±10.39b | 113.25±14.23b |
| C3（μg/mL） | 12.67±0.92a | 11.98±1.72a | 16.22±1.78b | 17.42±2.01b |
| C4（μg/mL） | 0.65±0.21a | 0.73±0.17a | 1.07±0.36b | 1.08±0.34 b |
注：同行标记相同小写字母表示差异不显著（P＞0.05）,小写字母不同表示差异显著（P＜0.05）。
复合提取物对断奶仔猪生长性能的影响见图3。由图3可知，与对照组相比，添加0.1%、0.2%复合提取物组的腹泻率较低，而添加0.05%复合提取物组的腹泻率与对照组的腹泻率接近。
Claims (4)
1.一种猪用免疫增强剂，其特征在于：按重量比3:1-3:1-3由红车轴草、甘草和绞股蓝配制而成。
2.如权利要求1所述的一种猪用免疫增强剂，其特征在于：红车轴草、甘草和绞股蓝的重量比为3:2:2。
3.如权利要求1所述的一种猪用免疫增强剂，其特征在于制备方法步骤如下：
第一步，备料：按重量比3:1-3:1-3称取红车轴草、甘草和绞股蓝，粉碎，过80目筛并混匀；
第二步，醇提：上述原料用石油醚60℃脱脂6 小时后，按重量体积比1：8-12的比例加入重量百分比浓度为50-70%的乙醇，在60-80 ℃下回流提取2-3小时，共提取1-3次；
第三步，干燥：过滤上述提取液，减压浓缩滤液得浓缩液，喷雾干燥或自然干燥制得粉末，即猪用免疫增强剂。
4.如权利要求1所述的一种猪用免疫增强剂，其特征在于：按饲料重量的0.1-0.8%添加应用在饲料中。
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