CN101904902A - 一种以葎草为主方的免疫增强剂 - Google Patents
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Abstract
本发明公布了一种以葎草为主方的免疫增强剂。近年来，人们逐渐发现化学合成药物，特别是抗生素等有毒副残留，致使产生耐药性和“三变”(癌、畸、突变)，畜牧业生产和人类受到严重的威胁。本发明提供一种以葎草为主方的免疫增强剂。包含的有效成分按重量份葎草黄酮80份和大豆黄酮20份。按重量份5～30份的有效成分和69～94份的载体及1份表面活性剂组成。载体为淀粉、无水葡萄糖、麦芽糊精；表面活性剂为十二烷基硫酸钠。所述的一种以葎草为主方的免疫增强剂应用于动物免疫力低下、疫苗注射时配合使用，可以对病毒性传染病及细菌性传染病进行防治。本发明为首次以葎草为主方的免疫增强剂。
Description
技术领域：
本发明涉及一种动物用中药免疫增强剂的复方，特别涉及一种以葎草主方的免疫增强剂。
背景技术：
二十世纪初，随着科学技术和饲料工业的发展，欧美一些国家出现饲料添加剂及其工业，促进了饲养及饲料工业的发展，并成为一个新兴的行业。与此同时，相关技术随之渗入，尤其是化学合成物及其技术被大量采用，并在一个时期有取代天然物添加剂之势。但近几十年来，人们逐渐发现化学合成药物，特别是抗生素等饲料添加剂有毒副残留，致使产生耐药性和“三变”(癌、畸、突变)等弊端，严重影响和危害人类健康，使我们深深感到化学合成物类饲料添加剂只为增加动物生产和产品，而缺少确保人体健康的因素。因而迫使人们在认识上对天然物中草药饲料添加剂有一个辩证的复归，重新评价天然物中草药饲料添加剂，认为是理想的添加剂。
中草药是我国传统医学的重要组成部分；有2000多年的应用历史，其原料来源广泛，种类繁多。据统计，具有明显免疫增强作用的中草药将近200多种。中药免疫增强剂是利用我国丰富的天然中药，依据现代中兽医药理论科学组方。中药的有效成分，可用于提高或促进动物的免疫功能，预防继发感染。因其具有独特的免疫增强作用。中药免疫增强剂可显著提高机体的抗病能力，已成为抗病药物的研发热点。
葎草具有清热解毒、具有益气补血、扶正祛邪、滋阴助阳、润三焦、消五谷、益五脏等中药作用，能够调理胃肠道微生态平衡，尤其对胃肠道内有益微生物有利，提高机体非特异性免疫力，提高饲料报酬，降低料肉比等。如果伴随疫苗使用，可提高特异性免疫力。目前以葎草为主方的免疫增强剂却未见相关报道。
发明内容：
本发明的主要目的：
1、顺应当前世界范围内兴起的一股回归自然、崇尚自然、重视生态安全与食品安全，食用产品要求“高品质、低药物残留、绿色无公害”的热潮。
2、针对现有技术的不足之处，本发明提供一种中药免疫增强剂，因其资源丰富、作用广泛、标本兼治、安全低毒、不易产生耐药性、无污染、无残留，能提高动物免疫力、改善其生长性能，具有普遍推广使用的广阔前景。
3、本发明能够对动物胃肠道微生态平衡起到改善的功能，具有健胃消食、提高饲料利用率的作用。
4、本发明能够显著地提高动物的特异性和非特异性免疫力，增强动物的抗病能力。
本发明为达到以上目的，是通过这样的技术方案来实现的：一种以葎草为主方的免疫增强剂，按重量份包含有效成分葎草黄酮80份和大豆黄酮20份。
所述的一种以葎草为主方的免疫增强剂，其特征在于：5～30份有效成分和69～94份的载体及1份表面活性剂组成。
所述的以葎草为主方的免疫增强剂，其特征在于：载体为淀粉、无水葡萄糖、麦芽糊精。所述的以葎草为主方的免疫增强剂，其特征在于：表面活性剂为十二烷基硫钠。
所述的以葎草为主方的免疫增强剂应用于免疫力低下、疫苗注射时等使用，可以对病毒性传染病及细菌性传染病进行防治。
本发明主要从中药葎草和大豆中提取黄酮成分，并将两种提取物按一定的比例配制成复合制剂，作为免疫增强剂和抗病毒抗菌药物使用。至今，国内外应用植物黄酮作为免疫增强剂的很少，但以葎草为主方的免疫增强剂还未见报道。本发明为首次使用。
具体实施方式：
提供一种以释草为主方的免疫增强剂，该增强剂的配方组分(按重量计)为：有效成分5～30份，载体69～94份，表面活性剂1份。
1、葎草黄酮的提取工艺：(1)将新鲜葎草地上部分洗净干燥后粉碎；(2)用水提醇沉法提取得葎草提取液；(3)将上述提取液通过精密过滤装置，除去固体杂质得澄清滤液；(4)回收乙醇，浓缩提取液，70℃干燥，得到葎草浸膏；(5)通过真空微波干燥，即得葎草浸膏粉。
2、大豆黄酮的提取工艺：(1)称取定量豆粕，在80℃条件下，用60％乙醇，料液比为1∶20，提取2次，每次2h，合并提取液；(2)回收乙醇，浓缩提取液，70℃干燥，即得大豆黄酮浸提膏；(3)通过真空微波干燥，即得大豆黄酮浸膏粉。
3、所述的葎草黄酮浸膏粉和大豆黄酮浸膏粉，按以下具体配比混合均匀，得到的粉剂能够通过2号筛(24目)，烘干包装，放在阴凉干燥处保存。本发明可通过以下实例方案证实(参看表1)。
表1有效成分制成粉剂实施方案
下面试验对本发明作进一步的阐述。
1、葎草黄酮的性质鉴定(如表2)
表2葎草黄酮的性质鉴定
颜色反应与黄酮类化合物的颜色反应相似，说明葎草浸膏为黄酮类的化合物。
2、葎草黄酮和大豆黄酮复方制剂的体内药效试验
(1)葎草黄酮和大豆黄酮复方制剂对细菌腹泻的作用
大肠杆菌致腹泻模型：选用体重18～20g的SPF级小鼠50只，随机分成5组，每组10只；设空白组、模型组、复方高、中、低剂量组。除空白组外，其余组动物均按0.3ml/10g剂量腹腔注射大肠杆菌菌液(浓度为1×109CFU/ml)，造成严重腹泻模型，空白组腹腔注射等量生理盐水。3h后，空白组和模型组小鼠灌胃生理盐水(0.2ml/10g)；治疗组小鼠灌胃复方制剂高、中、低剂量(0.4ml/只，0.2ml/只，0.1ml/只，1ml相当于1g生药)，观察腹腔注射大肠杆菌注射液3h内小鼠腹泻情况及给药后3h内模型组和治疗组、对照组小鼠腹泻情况(包括稀便次数、总便次数、稀便级)，并计算出腹泻指数(腹泻指数＝稀便率×稀便级)。
数据统计与分析
应用SPSS11.0统计软件做数据处理，所有数据采用x±s表示，采用t检验，P＜0.05为有统计学意义。结果如表3
表3复方制剂对小鼠大肠杆菌腹泻的影响
注：*表示差异显著(P＜0.05)，**表示差异极显著(P＜0.01)，下表同。
(2)葎草黄酮和大豆黄酮复方制剂对小鼠小肠推进率的作用
选用体重18～20g的SPF级小鼠40只，随机分成4组，每组10只，设空白组和复方制剂高、中、低剂量组。每组小鼠空腹24h后，空白组灌胃生理盐水(0.2ml/10g)，高、中、低剂量组灌胃复方制剂((0.4ml/只，0.2ml/只，0.1ml/只，1ml相当于1g生药)，30min后，每组小鼠灌胃墨汁(用生理盐水稀释10倍，0.5ml/只)，30min后脱臼处死，取小肠(上段至幽门，下段至盲肠端)，量取小肠全长及墨汁推进长度，求出推进率。
推进率(％)＝(墨汁推进长度/小肠全长)×100％
数据统计与分析
应用SPSS11.0统计软件做数据处理，所有数据采用x±s表示，采用t检验，P＜0.05为有统计学意义。结果如表4
表4复方制剂对小鼠小肠的推进率的影响
(3)葎草黄酮和大豆黄酮复方制剂对小鼠免疫功能的影响
对小鼠免疫器官的影响取体重18～20g的小鼠40只，雌雄兼有，随机分为四组，设空白对照组和试验组，试验组按250mg/kg、500mg/kg、750mg/kg的剂量将复方制剂添加到小鼠饲料中，饲喂28d，于末次饲喂后次日，脱臼处死，剥去胸腺、脾脏天平称重，脏器重量以(mg/10g体重)χ±SD表示，随后进行组间t检验，比较组间差异的显著性。
对小鼠吞噬细胞功能的影响取体重18～20g的小鼠40只，雌雄兼有，随机分为四组，分为空白对照组和试验组，试验组按250mg/kg、500mg/kg、750mg/kg的剂量将复方制剂添加到小鼠饲料中，饲喂28d，于末次饲喂后次日，尾静脉注射墨汁(用生理盐水稀释10倍)0.05ml/10kg体重，注射后分别于2min、20min用微量采血管(先用肝素液润湿)从眶后静脉丛取血20ul，溶于2ml0.1％Na2CO3溶液中摇匀，置VIS-723G可见分光光度计在620nm处比色，测定光密度(OD)，最后将小鼠颈椎脱臼致死，分别取肝、脾称重。按以下公式计算廓清指数K或校正廓清指数α。
K＝(logOD1-logOD2)/(t2-t1)或α＝x1/3*体重/(肝重+脾重)
式中OD1、OD2为不同时间所取血样，t2-t1为取两血样的时间差。以X±SE表示各组的K值，进行组间t检验，比较差异的显著性。若肝脾重量差异不太大时，只记算K值亦可。
对小鼠溶血素的影响取体重18～20g的小鼠40只，雌雄兼有，随机分为四组，分为空白对照组和试验组，试验组按250mg/kg、500mg/kg、750mg/kg的剂量将复方制剂添加到小鼠饲料中，饲喂28d，于末次饲喂后次日，每鼠腹腔注射5％生理盐水鸡红细胞悬液0.2ml进行免疫，免疫后7天，摘眼球取血，离心，取血清，用生理盐水稀释100倍，取稀释血清1ml，与5％鸡红细胞悬液0.5ml、10％补体0.5ml混合，在37℃恒温箱中保温30min，0℃冰箱中终止反应。
离心，取上清液于VIS-723G可见分光光度计540nm处比色，测录光密度(OD)。另设不加血清的空白对照，取其上清液作为比色时调“θ”的基准。以光密度(OD)读数为判定血清溶血素的指标，比较各组的差异。
数据统计与分析
应用SPSS11.0统计软件做数据处理，所有数据采用x±s表示，采用t检验，P＜0.05为有统计学意义。结果如表5
表5复方制剂对小鼠免疫制剂的影响
(4)结果分析
有表3看出，给药组的中、高剂量对大肠杆菌在体内有极显著的抑制作用(p＜0.01)，说明该复方制剂可有效的预防细菌性腹泻。而表4结果表明：空白组与给药组比较，给药0.2mL、0.4mL能够显著(P＜0.05)抑制小肠的推进率，给药0.1mL能抑制小肠推进率，但与空白组比较差异不显著；说明该复方制剂能够抑制小肠液蠕动，从而达到止泻的作用。
表5明：饲料中添加250mg/kg、500mg/kg、750mg/kg(分别相当于1.175g、2.35g、3.53g生药)的复方制剂可使小鼠胸腺增重，但无显著差别；用鸡红细胞免疫后，给药组可极显著(p＜0.01)提高抗鸡红细胞抗体(溶血素IgM)的产生，给药组与对照组相比，能极显著提高单核吞噬细胞的吞噬功能，但给药组之间差别不显著。
(5)小结
复方制剂可有效地防治大肠杆菌引起的腹泻，也可抑制小肠蠕动，从而也可达到止泻的目的。
复方制剂可极显著地提高小鼠的免疫功能。
3、葎草黄酮和大豆黄酮复方制剂对羔羊体液免疫指标的影响
选择体重、胎次相近，健康的3-4月龄的湖羊羔羊32只(雌雄各半)，根据体重、性别随机分为4组，设对照组和试验组，每组2个重复，每重复4只，采取舍饲，自由采食、饮水，投喂基础日粮，预饲1周后进行试验，测定复方制剂对羔羊部分免疫功能的影响，试验期28天。对照组和试验组羔羊平均体重经统计分析检验，组间差异不显著(P＞0.05)。
各组羔羊试验开始前均用1％阿维菌素常规剂量皮下注射驱虫，并对羊舍进行消毒。试验羔羊舍饲，每只羔羊日均喂精料补充料350g，复方制剂添加量根据提取物中总黄酮的百分含量添加，试验组分别按5.0g/kg、7.5g/kg、10.0g/kg的量添加到精料中，为保证提取物与精料均匀地在混合，先将其预混合，而后再混至精料中，每天分两次饲喂。分别于每日8:30和16:30进行等量饲喂，早上自由采食青贮玉米秸，下午自由采食花生秧(均为少量多次添加)，自由饮水。试验期间由同一饲养员进行饲养，每天清扫圈舍。试验期间室内温度1.6～10.5℃、室外温度-2.0～12℃、相对湿度43.5％～49.7％。
数据统计与分析
应用SPSS11.0统计软件做数据处理，所有数据采用x±s表示，采用t检验，P＜0.05为有统计学意义。结果如表6
表6复方制剂对羔羊体液免疫指标的影响
结果表明，与对照组相比，添加复方制剂的低、中剂量组能显著提高IgA水平，高剂量组则极显著低于对照组，随着饲喂时间的推移，低、中剂量组的IgG、IgM水平变化趋势相同，与对照组及高剂量组相比差异性极显著，且均在第二周达到最大值，第四组则在第三周达到峰值。结果提示，复方制剂对羔羊体液免疫指标的影响差异性显著，能提高羔羊的免疫能力。
需要注意的是，上述的参照实例对一种以葎草为主方的免疫增强剂及其制备方法进行的详细描述，只是说明性的而不是限定性的，也就是说本发明不限于以上实例，还可有多种变形，因此在本发明公开的发明思想下的变形或修改，均应认为是本发明的保护范围。
本发明以葎草为主方的免疫增强剂，选用的都是最常见的原料，安全无毒、且价格低，能最大限度的控制生产成本。特别是葎草，在我国资源丰富、价格低廉，具有营养丰富、功效多，对病原菌有抑制作用，可增进动物生长，提高机体免疫力，有止泻保健功能。本发明是一种以葎草为主的免疫增强剂，不会使动物产生抗药性，可全部或大部分代替饲料中的抗生素。
Claims (5)
1.一种以葎草为主方的免疫增强剂，按重量份包含有效成分葎草黄酮80份和大豆黄酮20份。
2.如权利要求1所述的一种以葎草为主方的免疫增强剂，其特征在于：5～30份有效成分和69～94份的载体及1份表面活性剂组成。
3.如权利要求2所述的以葎草为主方的免疫增强剂，其特征在于：载体为淀粉、无水葡萄糖、麦芽糊精。
4.如权利要求2所述的以葎草为主方的免疫增强剂，其特征在于：表面活性剂为十二烷基硫酸钠。
5.如权利要求1、2、3所述的以葎草为主方的免疫增强剂应用于免疫力低下、疫苗注射时等使用，可以对病毒性传染病及细菌性传染病进行防治。
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| CN103168937A (zh) * | 2011-12-22 | 2013-06-26 | 郑州牧业工程高等专科学校 | 一种以中草药为主方的高产奶牛高峰期专用添加剂 |
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| CN102934743A (zh) * | 2012-11-27 | 2013-02-20 | 郑州牧业工程高等专科学校 | 一种用于断奶仔猪转群的中草药抗应激散及制备方法 |
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