CN107594132A - 可提高仔猪免疫力的饲料添加剂 - Google Patents
可提高仔猪免疫力的饲料添加剂 Download PDFInfo
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- CN107594132A CN107594132A CN201711042893.6A CN201711042893A CN107594132A CN 107594132 A CN107594132 A CN 107594132A CN 201711042893 A CN201711042893 A CN 201711042893A CN 107594132 A CN107594132 A CN 107594132A
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Abstract
本发明涉及一种可提高仔猪免疫力的饲料添加剂，其中活性成分氧化苦参碱与白芨多糖的重量比为(1:10)～(10:1)。与普通饲料添加剂相比，本发明的仔猪饲料添加剂制备方法简单，配伍合理，疗效显著，能够显著改善仔猪免疫力。由于添加剂中无西药成分，因此饲料添加剂无毒副作用，无耐药性。
Description
技术领域
本发明涉及饲料领域，具体涉及一种仔猪饲料添加剂。
背景技术
长期以来，断奶仔猪疾病高发是严重困扰我国养猪业发展的重要原因。仔猪断奶后，由于母乳被中断，环境、饲料等的突然改变，造成各系统功能紊乱、机体免疫力下降，容易感染各种免疫抑制性疾病(如蓝耳病病毒、猪瘟野毒伪狂犬病毒、圆环病毒等)，造成仔猪断奶后发病率和死亡率高等严重后果。
目前，国外主要通过调整饲粮组成(如高营养性、高适应性仔猪断奶料)、温控及其他环境的控制来防制仔猪断奶综合征。国内主要通过在饲料中添加抗生素类药物来控制继发感染，但容易造成耐药性、仔猪免疫机能下降、药物残留及污染环境等弊端。
中药制剂富含多种多糖等活性成分，在提高机体免疫力和抗病力方面有着显著的作用，现已证明数百种中草药有多方面的免疫活性，如促进免疫器官的生长发育，增强机体的细胞免疫和体液免疫，促进干扰素-γ、白细胞介素-2等细胞因子产生等作用，并且绿色、安全、不易产生耐药性。因此，中草药的开发利用已成为众望所归。
氧化苦参碱(苦参素，oxymatrine，OMT)是从中药苦参、苦豆子、广豆根中提取的生物碱，具有四环的喹嗪啶类结构，化学分子式为C15H24NO2，分子量为264，无色结晶，易溶于水。由于氧化苦参碱的广泛临床应用，对其药理作用及作用机制的研究也越来越引起相关医药学者的极大兴趣。氧化苦参碱药理作用广泛，具有抗病毒、强心、降压、抗心律失常、对心肌缺血和梗死的保护作用、抗炎、杀菌、抗肿瘤作用及镇静催眠、解热镇痛等中枢神经的抑制作用，平喘、抗过敏等作用。
现代研究表明，氧化苦参碱可通过抑制乙肝病毒(HBV)DNA的复制发挥抗HBV的作用，但其具体的作用机制尚待进一步研究。实验结果证实，氧化苦参碱能够抑制HBVDNA转染的HepG2.2.15细胞分泌HBsAg和HBeAg。在一定剂量范围内，这种抑制作用呈剂量依赖关系，对HBsAg的抑制率大于对HBeAg的抑制率；氧化苦参碱亦可通过抑制丙肝病毒(HCV)的复制发挥抗HCV的作用。氧化苦参碱还可通过影响抗柯萨奇病毒(CV)衣壳蛋白的组装抑制CV在细胞内的复制。
氧化苦参碱增强心肌收缩力，使心输出量增加，降低前负荷，纠正由于心力衰竭而造成的心室舒张末压增高，对外周血流量有降低的趋势，对总外周阻力无明显影响。因此氧化苦参碱可明显改善心脏泵血功能，产生有益的血流动力学效应。静脉注射氧化苦参碱可使麻醉大鼠和犬产生快速可靠的降压效果。氧化苦参碱能增强多沙唑嗪的降压作用，但与普萘洛尔未见明显的协同作用。氧化苦参碱降压作用强、迅速、持续时间较短，在临床上适用于短暂的血压骤升的情况。
氧化苦参碱能显著降低高脂饮食所致大鼠及家鸽的血清胆固醇及三酰甘油水平。100，200mg/kg的氧化苦参碱对75％蛋黄乳液所致小鼠高血脂症模型中血清胆固醇的抑制率分别为23.7％，28.3％。
氧化苦参碱可调节小鼠及大鼠腹腔肥大细胞释放组胺、白三烯等炎症介质，而对一些非特异性激活剂如PHA等诱导的组胺释放未见明显影响。研究证实氧化苦参碱对TNF、IL-6、NF-kB、ICAM-1等炎症细胞因子的表达具有一定的抑制作用。通过用氧化苦参碱对出血热型肾病综合征(HFRS)患者治疗1周后发现患者血清中的THP和Mβ2的强度、IL-15和细胞间黏附分子的水平较对照组明显下降。
目前国内外关于氧化苦参碱抗肿瘤作用的研究结果证实，氧化苦参碱抑制肿瘤细胞DNA的合成、抑制相关酶活性、影响肿瘤细胞的正常周期而抑制肿瘤细胞增殖；控制相关因子的表达抑制肿瘤的转移影响与肿瘤相关基因的表达、端粒酶的活性等途径，诱发细胞凋亡，诱导肿瘤细胞向正常细胞分化。
白芨为兰科植物白芨的干燥块茎，别名良姜、紫兰。李时珍释其名曰：其根白色，连及而生，故曰白芨。白芨早在《神农本草经》就有记载，1963年以后成为各版药典收载的品种之一。白芨味苦、甘、涩，性平偏凉，归肺、胃、肝经，具有补肺，止血，消肿，生肌，敛疮。白芨功能主治肺伤咳血，衄血，金疮出血，痈疽肿毒，溃疡疼痛，烫火灼伤，手足皲裂，肛裂和乳头裂等。因此素有“必涩而收，入肺止血，生肌治疮，外科最善”之称。
白芨多糖是白芨原药植物经水提醇沉提取的一种黏性多糖，其化学成分主要为葡萄糖和甘露糖(比例为1:4)，约占原药植物的55％-60％。近年来，白芨多糖作为新药开发的重要方向之一，越来越受到国内外学者的关注。
白芨多糖外用时对实质性器官(肝、脾)肌肉血管出血等有良好的收敛止血作用。研究发现，白芨多糖的止血效果优于立血止、凝血酶、医用生物蛋白胶，并能促进创面愈合。
对小给予不同剂量的白芨多糖溶液，并用断尾法测定出血时间(Bleeding time，BT)，毛细玻管法测定凝血时间(Clotting time，CT)。结果表明，白芨多糖高、中剂量组BT、CT与正常组比较都明显缩短。白芨多糖对小鼠CT的影响实验表明，其止血作用机制是增强血小板第Ⅲ因子活性、缩短凝血酶生成时间、抑制纤维蛋白酶的活性，同时促进细胞凝聚，形成人工血栓，从而达到止血之功效。
有临床研究将60例口腔溃疡患者按所用的治疗方法随机均分为传统治疗组和白芨联合治疗组，并对治疗后的临床疗效进行比较评估。结果显示，30例白芨联合治疗的患者比传统治疗组的患者治疗后评价结果更优，有效率高达80％；视觉模拟评分法(Visualanalogue scale，VAS)疼痛评分结果也显示白芨联合治疗组结果更加理想。
通过观察不同剂量的白芨多糖对免疫低下小鼠碳廓清试验结果的影响以及对小鼠脾淋巴细胞增殖的影响，发现白芨多糖能够显著提高免疫抑制小鼠的吞噬指数，并能显著增强T淋巴细胞和B淋巴细胞的增殖能力。由此证明，白芨多糖对小鼠的免疫功能有显著的增强作用。对白芨多糖作用于酪氨酸激酶信号通路的研究中，用氯化钴处理人角质形成细胞建立氧化应激炎症损伤的细胞模型，以MTT法检测细胞存活率，并检测细胞培养液中IL-6、IL-8的含量以及细胞上清液中TNF-α的分泌量。结果表明，白芨多糖提高氯化钴诱导角质形成细胞的存活率具有剂量依赖性；当白芨多糖质量浓度为0.02～0.2mg/mlTNF-α、IL-6、IL-8含量明显增加。
由白芨多糖、没食子酸和厚朴酚配伍制成的复方中药制剂对感染根管常见的黏性放线菌、具核梭杆菌、粪肠球菌都有较强的抑制作用。白芨多糖还可以抑制革兰阳性细菌活性，减少栓塞器官的感染，使药物在病变部位的血管内均匀分布，使其不良反应发生率降低，是一种理想的外围型化疗栓塞剂。白芨不同提取部位对耐甲氧西林金黄色葡萄球菌有不同程度的抑制作用，尤其是醇提水沉得到的多糖成分抑制作用最为明显。这些多糖能选择性地黏附病原体，阻断微生物病原体对靶细胞的吸附，从而发挥消炎和抗菌功效。
白芨多糖抗肿瘤功效屡有报道。研究表明中药白芨对移植性肝癌小鼠有治疗作用，可改善患癌小鼠的肝功能，延长患癌小鼠的生存时间。体外培养的人胃癌细胞，人卵巢癌细胞和肝癌细胞经过200μg/ml白芨多糖处理72h后均出现细胞生长阻滞、周期延长的现象。白芨多糖作用于U14宫颈癌模型荷瘤小鼠的实验结果显示，白芨多糖对宫颈癌荷瘤小鼠的免疫力有明显的提升作用，从而发挥其抗肿瘤作用。白芨多糖能选择性进入并聚集在肿瘤组织内，抑制肿瘤栓塞后侧支循环的再形成。研究发现，将白芨多糖作为肝动脉栓塞剂，并结合化疗药品和肝动脉结扎术能明显抑制实验性肝癌大鼠癌细胞的生长。
目前，尚未见将氧化苦参碱与白芨多糖组合作为提高仔猪免疫力的饲料添加剂的文献报道。
发明内容
本发明所要解决的技术问题是提供一种能有效提高仔猪免疫力，且毒副作用低的仔猪饲料添加剂。
本发明解决上述问题所采用的技术方案是，提供一种提高仔猪免疫力的饲料添加剂，其中活性成分氧化苦参碱与白芨多糖的重量比为(1:10)～(10:1)。
优选的，所述饲料添加剂中氧化苦参碱与白芨多糖的重量比为(1:1)～(1:9)。
更优选的，所述饲料添加剂中氧化苦参碱与白芨多糖的重量比为(1:1)～(1:4)。
最优选的，所述饲料添加剂中氧化苦参碱与白芨多糖的重量比为2:3。
优选的，所述白芨多糖的制备方法如下：白芨块茎干制品粉碎、过筛、烘干，加蒸馏水回流提取，离心后取上清液，减压浓缩后用无水乙醇沉淀，Sevag法脱蛋白，所得溶液用蒸馏水透析，真空冷冻干燥即得。
更优选的，白芨粉末中蒸馏水的加入量为15-30倍体积。
更优选的，所述回流提取温度为50-70℃，回流提取时间为2-4h，重复次数为1-3次。
更优选的，所述减压浓缩温度为60-80℃，减压后加入无水乙醇量为2-5倍体积。
更优选的，所述白芨多糖的制备方法如下：白芨块茎干制品粉碎、过筛、烘干；加15-30倍体积蒸馏水，50-70℃回流提取2-4h，重复提取1-3次，合并提取液；5000rpm离心10min，取上清抽滤，60-80℃减压浓缩至较小体积，加2-5倍体积无水乙醇沉淀，沉淀溶于最小体积水中；Sevag法脱蛋白至考马斯亮蓝G-250法检测至无蛋白质被测出；所得溶液用蒸馏水透析3-10天，真空冷冻干燥。
优选的，所述饲料添加剂进一步包括其他活性成分。
更优选的，所述其他活性成分选自氨基酸、维生素等中的一种或多种。
最优选的，所述氨基酸为甘氨酸、丙氨酸、亮氨酸、缬氨酸、半胱氨酸、甲硫氨酸、苏氨酸、丝氨酸、苯丙氨酸、酪氨酸、色氨酸、蛋氨酸、谷氨酸、精氨酸和/或组氨酸。
最优选的，所述维生素为维生素A、维生素B1、维生素B2、维生素B3、维生素B5、维生素B6、维生素B12、维生素C、维生素D、维生素E、维生素H、维生素K和/或维生素P。
本发明还提供一种上述饲料添加剂在制备改善仔猪免疫力的饲料中的应用。
优选的，所述100重量份的饲料中，含有1-5重量％的饲料添加剂。
本发明具有积极有益的效果：
令人惊奇的是，本发明意外发现将氧化苦参碱与白芨多糖组合使用，对于提高仔猪免疫力具有协同增效的效果，显著降低了活性成分的给药量，配合其他活性成分使用效果更佳。
与普通饲料添加剂相比，本发明的猪饲料添加剂制备方法简单，配伍合理，疗效显著，能够显著提高猪瘟抗体水平，实验证明对改善仔猪免疫力有特效；由于添加剂中无西药成分，因此饲料添加剂无毒副作用，无耐药性。
具体实施方式
下面结合实施例对本发明作更进一步的说明，但本发明的实施方式不限于此。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
实施例1、白芨多糖的提取
白芨块茎干制品粉碎、过筛、烘干，加20倍体积蒸馏水，60℃回流提取4h，重复提取1次，合并2次提取液。5000rpm离心10min，取上清抽滤，70℃减压浓缩至较小体积，加3倍体积无水乙醇沉淀，沉淀溶于最小体积水中，Sevag法脱蛋白至考马斯亮蓝G-250法检测至无蛋白质被测出。所得溶液用蒸馏水透析5天，透析袋截留相对分子质量为8000-10000，真空冷冻干燥。
实施例2、饲料添加剂的制备
取氧化苦参碱5重量份，实施例1制备的白芨多糖5重量份，混合后制成氧化苦参碱和白芨多糖重量比为1:1的饲料添加剂S1。
取氧化苦参碱4重量份，实施例1制备的白芨多糖6重量份，混合后制成氧化苦参碱和白芨多糖重量比为2:3的饲料添加剂S2。
取氧化苦参碱1重量份，实施例1制备的白芨多糖9重量份，混合后制成氧化苦参碱和白芨多糖重量比为1:9的饲料添加剂S3。
实施例3、本发明饲料添加剂在提高仔猪免疫力中的应用
1.试验动物及分组处理
选取临床健康、体质量相近的20日龄仔猪，采血检测猪瘟母源抗体水平，挑选出抗体水平相近的仔猪120头，随机分成6组，即本发明中药组合物组1-3(实施例2制备的饲料添加剂S1-S3)、氧化苦参碱组、白芨多糖组和对照组，每组仔猪20头。21日龄进行猪瘟兔化弱毒苗免疫。27-31日龄开始在S1-S3中药组合物组、氧化苦参碱组、白芨多糖组日粮中按照饲料总重量的1％添加上述饲料添加剂(每组加入的中药活性成分总量相同)，对照组的日粮为不添加上述饲料添加剂的相同重量的猪饲料，各组猪每日投喂3次。在55日龄对每组仔猪前腔静脉采血各5mL，分离血清用于猪瘟抗体测定，全血用于B淋巴细胞增殖的测定。
2.试验检测指标和方法
(1)血清猪瘟抗体水平
采用ELISA法测定猪瘟抗体，参照猪瘟抗体检测试剂盒说明书进行。
(2)血液B细胞增殖反应
采用MTT法进行。取各组浓度为1×106个/mL的淋巴细胞悬液加入96孔细胞培养板上，每孔加100μL，然后对照组每孔加入100μL RPMI1640(购自IBCO公司)完全培养液，试验组每孔加入100μL质量浓度为10mg/LLPS(购自sigma公司)，每个样品设置3个重复。37℃、5％CO2培养44h，每孔加入5g/L四甲基偶氮唑盐(MTT)溶液10μL，培养4h后每孔再加入DMSO溶液100μL，继续培养2h，使紫色结晶完全溶解，用酶联免疫检测仪在570nm波长下测定D值，作为淋巴细胞增殖的指标。
3.试验结果
血清猪瘟抗体水平和淋巴细胞增殖指标试验结果见表1。
表1各试验组仔猪血清猪瘟抗体水平及B淋巴细胞增殖情况
由实施例3可以看出，与对照组相比，本发明的饲料添加剂能显著提高仔猪血清中猪瘟抗体水平，改善B淋巴细胞增殖水平，是效果优异的仔猪免疫增强剂。特别是氧化苦参碱和白芨多糖的组合使用产生了协同效果，减少了药物的使用量，其中氧化苦参碱和白芨多糖重量比为2:3的饲料添加剂效果最为突出，产生了难以预期的优异效果。
Claims (8)
1.一种提高仔猪免疫力的饲料添加剂，其特征在于，其中活性成分氧化苦参碱与白芨多糖的重量比为(1:10)～(10:1)。
2.根据权利要求1所述的饲料添加剂，其特征在于，氧化苦参碱与白芨多糖的重量比为(1:1)～(1:9)。
3.根据权利要求2所述的饲料添加剂，其特征在于，氧化苦参碱与白芨多糖的重量比为(1:1)～(1:4)。
4.根据权利要求1所述的饲料添加剂，其特征在于，所述饲料添加剂进一步包括其他活性成分。
5.根据权利要求4所述的饲料添加剂，其特征在于，所述其他活性成分选自氨基酸、维生素中的一种或两种。
6.根据权利要求5所述的饲料添加剂，其特征在于，所述氨基酸选自甘氨酸、丙氨酸、亮氨酸、缬氨酸、半胱氨酸、甲硫氨酸、苏氨酸、丝氨酸、苯丙氨酸、酪氨酸、色氨酸、蛋氨酸、谷氨酸、精氨酸和/或组氨酸，所述维生素选自维生素A、维生素B1、维生素B2、维生素B3、维生素B5、维生素B6、维生素B12、维生素C、维生素D、维生素E、维生素H、维生素K和/或维生素P。
7.权利要求1-6任一项所述的饲料添加剂在制备提高仔猪免疫力的饲料中的应用。
8.根据权利要求7所述的应用，其特征在于，所述100重量份的饲料中，含有1-5重量％的饲料添加剂。
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