CN113262246A - 提高家畜免疫力的中药组合物及有效成分 - Google Patents
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Abstract
本发明公开了提高家畜免疫力的中药组合物及有效成分。所述中药组合物由黄芪、白术、茯苓和甘草组成。本发明中药组合物可提高哺乳母猪血清中IgA、IgG水平、IL‑2、IFN‑γ水平以及胃动素和胃泌素水平,还能提高T淋巴细胞亚群CD4+和CD8+、猪瘟及伪狂犬gB抗体水平以及哺乳母猪红细胞E‑C3bRR花环率,降低血清中IL‑10水平。本发明进一步采用LC‑MS联用技术对复方中药入乳汁中化学成分进行鉴定,确定毛蕊异黄酮、熊果酸、三表去氢茯苓酸、甘草内酯、光甘草酮、黄甘草苷、1,3二咖啡酰奎宁酸、甘草宁O、半甘草异黄酮B、甘草异黄烷酮和甘草宁H是发挥增强母猪免疫力的有效成分。
Description
技术领域
本发明涉及提高动物免疫力的药物,尤其涉及提高家畜免疫力的中药组合物及有效成分,属于提高动物免疫力的中药以及有效成分领域。
背景技术
中国是一个养猪大国,随着养猪业向规模化扩大、集约化发展,中国现有养猪模式在不断追求母猪生产性能的同时同样带来了一定问题,由于长期定位栏饲养,母猪高产等问题的存在,导致母猪抗病能力差,母猪产后不食等问题严重,从中兽医角度,中国母猪普遍存在“脾胃气虚证”,中兽药是近年来普遍认为具有开发前景的绿色药物,研究表明中草药中含有的有效成分可以有效促进畜禽采食与消化液分泌,增强养份的消化吸收与机体的合成代谢,促进生长发育,提高机体免疫力(褚秀玲,刘学彬,张晓云,等.中兽医气血理论及其在母猪生产中的应用[J].中兽医医药杂志,2016,035(005):83-85.)。
母猪群的健康状况不仅影响自身的繁殖性能,而且关系到仔猪群的健康。母猪生产性能及免疫功能的改善,可从源头上控制疾病的垂直传播并提高母猪繁殖能力。研究发现,乳汁中除母源抗体外还存在其它有防感染和增强免疫的物质并且可直接通过哺乳过程进入胎儿体内,对初生仔猪接受被动免疫、促进生长发育、提高抗病力起着重要作用。妊娠期及哺乳期母猪的健康状况、免疫功能高低、母乳的质量高低等母源性因素将直接影响仔猪的生产性能和抗病能力。因此,调节哺乳母猪免疫功能有助于提高母猪群和仔猪群的健康状况,可帮助提高养猪的经济效益。
复方中药入血化学成分研究报道较多,但对于复方中药入乳成分报道较少。复方中药中的化学成分在从血液入乳汁的过程中可能发生变化,中药复方中所含的化学成分十分复杂,但它们并不能真实的反映该方在体内药效物质的状态和数量,只有真正进入乳汁被仔猪摄取的中药才是发挥作用的中药,这会使评价药物的标准更加客观。
发明内容
本发明的目的之一是提供提高猪免疫力的中药组合物;
本发明的目的之二提供该提高猪免疫力的中药组合物入乳的有效成分;
为实现上述目的,本发明所采取的技术方案包括:
本发明首先提供了一种提高猪免疫力的中药组合物,由黄芪、白术、茯苓和甘草组成。
为了实现更好的技术效果,各组分的重量份为:黄芪9-20份,白术9-20份,茯苓9-15份,甘草5-10份。
本发明采用LC-MS联用技术对中药组合物入乳汁中化学成分进行鉴定,结果确定入乳成分为毛蕊异黄酮、熊果酸、三表去氢茯苓酸、甘草内酯、光甘草酮、黄甘草苷、1,3二咖啡酰奎宁酸、甘草宁O、半甘草异黄酮B、甘草异黄烷酮和甘草宁H,这些有效成分是该中药组合物发挥提高母猪免疫力的有效成分。
由此,本发明进一步提供了一种有效提高猪免疫力的有效成分,该有效成分选自毛蕊异黄酮、熊果酸、三表去氢茯苓酸、甘草内酯、光甘草酮、黄甘草苷、1,3二咖啡酰奎宁酸、甘草宁O、半甘草异黄酮B、甘草异黄烷酮或甘草宁H中的任何一种或一种以上按照任意比例的组合;优选的,所述提高猪免疫力的有效成分由毛蕊异黄酮、熊果酸、三表去氢茯苓酸、甘草内酯、光甘草酮、黄甘草苷、1,3二咖啡酰奎宁酸、甘草宁O、半甘草异黄酮B、甘草异黄烷酮和甘草宁H组成。
本发明还进一步提供了应用所述中药组合物提高猪免疫力的方法,包括:将所述复方中药组合物中各组分粉碎后混合均匀添加到猪的基础日粮中;作为参考,所述复合中药组合物添加到猪的基础日粮中的添加量可以为0.05-2wt%,优选为0.5wt%;
或者将毛蕊异黄酮、熊果酸、三表去氢茯苓酸、甘草内酯、光甘草酮、黄甘草苷、1,3二咖啡酰奎宁酸、甘草宁O、半甘草异黄酮B、甘草异黄烷酮或甘草宁H中的任何一种或多种按照任意比例混合得到组合物添加到猪的基础日粮中;作为参考,所述有效成分添加到猪的基础日粮中的添加量可以为0.01-0.5wt%,优选为0.05wt%。
本发明整体技术方案详述
一、在饲料中添加复方中药组合物对于母猪免疫力影响的考察
大约克猪40头,随机分为2组,每组20头。对照组饲喂基础日粮;试验组在基础日粮中添加上述复方中药0.2%,试验期为35d。在生产当天,产后7天采血测定胃动素、胃泌素,免疫球蛋白(IgG,IgM,IgA),猪瘟抗体,伪狂犬gB抗体,CD4+,CD8+淋巴细胞亚群进行测定。
饲料中添加本发明复方中药后可提高哺乳母猪血清中IgA、IgG水平与对照组相比差异显著(p<0.05);饲料中添加本发明复方中药可提高哺乳母猪血清中IL-2、IFN-γ水平,与对照组相比差异显著(p<0.05);降低哺乳母猪血清中IL-10水平,与对照组相比差异显著(p<0.05);饲料中添加复方中药可提高哺乳母猪血清中胃动素和胃泌素水平,与对照组相比差异显著(p<0.05);饲料中添加复方中药可提高哺乳母猪红细胞E-C3bRR花环率,与对照组相比差异显著(p<0.05)。降低哺乳母猪红细胞E-ICRR花环率与对照组相比差异显著(p<0.05);饲料中添加本发明复方中药可提高哺乳母猪T淋巴细胞亚群CD4+和CD8+,与对照组相比差异显著(p<0.05)。饲料中添加复方中药可提高哺乳母猪猪瘟及伪狂犬gB抗体水平,与对照组相比差异显著(p<0.05)。
二、复方中药入乳成分有效成分的确定
采用LC-MS联用技术对复方中药入乳汁中化学成分进行鉴定,确定入血成分为毛蕊异黄酮、熊果酸、三表去氢茯苓酸、甘草内酯、光甘草酮、黄甘草苷、1,3二咖啡酰奎宁酸、甘草宁O、半甘草异黄酮B、甘草异黄烷酮和甘草宁H是发挥增强母猪免疫力的有效成分。
本发明复方中药具有免疫调节作用,不仅可改善胸腺与脾脏的萎缩,提高胸腺与脾脏指数,还对机体的细胞免疫、体液免疫、神经内分泌系统有广泛的作用,对各种原因引起的免疫功能低下有明显的提升作用。
复方中药入血化学成分研究报道较多,但对于复方中药入乳成分报道较少。复方中药中的化学成分在从血液入乳汁的过程中可能发生变化,中药复方中所含的化学成分十分复杂,但它们并不能真实的反映该方在体内药效物质的状态和数量,只有真正进入乳汁被仔猪摄取的中药才是发挥作用的中药,这会使评价药物的标准更加客观。本发明系统分析了复方中药在乳汁中化学成分,这不仅对揭示复方中药真正药效物质基础具有极大的参考价值,同时对复方中药临床用药也有一定的指导意义。
附图说明
图1复方中药对母猪免疫球蛋白的影响。
图2复方中药对母猪细胞因子的影响。
图3复方中药对母猪胃泌素、胃动素的影响。
图4复方中药对母猪红细胞免疫的影响。
图5复方中药对母猪T淋巴细胞亚群的影响。
图6复方中药对猪瘟及伪狂犬疫苗抗体的影响。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但是应理解所述实施例仅是范例性的,不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改或替换均落入本发明的保护范围。
试验例1复方中药对母猪免疫力的影响及有效成分的确定
1、材料和试验方法
1.1材料
1.1.1供试中药
由黄芪、白术、茯苓和甘草组成的复方中药,其中,各组分的用量为黄芪15g,白术14g,茯苓12g,甘草6g。
1.1.2供试动物
试验在辽宁伟嘉种猪场进行,共选取3胎次健康大约克猪共40头,随机分为两组,每组20头,每头给药剂量为一天20克。试验周期为妊娠110天至仔猪断奶。
1.1.3试剂与仪器
猪血清免疫球蛋白(IgG、IgM、IgA)非特异性抗体试剂盒为北京华英生物技术研究所产品,Nexera UHPLC LC-30A超高效液相由岛津公司生产、Triple TOF 5600高分辨质谱、Mai Rui Bricyte E6流式细胞仪由迈瑞公司生产
1.2试验方法
1.2.1复方中药对母猪免疫力各项性能的考察
大约克猪40头,随机分为2组,每组20头。对照组饲喂基础日粮;试验组在基础日粮中添加上述复方中药0.2%,试验期为35d。在生产当天,产后7天采血测定胃动素、胃泌素,免疫球蛋白(IgG,IgM,IgA),猪瘟抗体,伪狂犬gB抗体,CD4+,CD8+淋巴细胞亚群进行测定。
1.2.2复方中药中乳中有效成分鉴定
将100mg样品添加到500μL含1μg/mL内标物的提取溶液中,然后将混合物涡旋1分钟,然后在10℃水中超声1h。在-40℃下放置1小时后,将样品在4℃下以12000rpm离心15分钟。最后,获得上清液,并放入新的2mL试管中进行LC-MS/MS分析。将400μL猪乳汁样品添加到40μL盐酸(2mol/L)中,然后将混合物涡旋1分钟,然后在4℃下孵育15分钟。涡旋和孵育周期重复4次。加入1.6mL乙腈,将混合物涡旋振荡5分钟,并将样品在4℃以12000rpm离心5分钟。将1600μL上清液转移至新管中并进行氮气干燥。将干燥后的样品在200μL含1μg/mL内标物的80%甲醇中复溶5分钟,然后在4℃下以12000rpm离心5分钟,然后将80μL上清液转移到新鲜的玻璃小瓶中进行LC/MS分析。
2试验结果
2.1复方中药对哺乳母猪免疫球蛋白的影响
复方中药对产后第7天哺乳母猪血清中IgA、IgG、IgM浓度的影响见图1。饲料中添加该复方中药后可提高哺乳母猪血清中IgA、IgG水平与对照组相比差异显著(p<0.05),但是对IgM的影响差异不显著。
2.2复方中药对哺乳母猪细胞因子的影响
复方中药对产后第7天哺乳母猪血清中IL-2、IL-10、TNF-α、IFN-γ浓度的影响见图2。饲料中添加复方中药可提高哺乳母猪血清中IL-2、IFN-γ水平,与对照组相比差异显著(p<0.05)。降低哺乳母猪血清中IL-10水平,与对照组相比差异显著(p<0.05)。对TNF-α的影响与对照组相比差异不显著。
2.3复方中药对哺乳母猪胃泌素、胃动素的影响
复方中药对产后第1天和第7天哺乳母猪血清中胃泌素、胃动素浓度的影响见图3。饲料中添加复方中药可提高哺乳母猪血清中胃动素和胃泌素水平,与对照组相比差异显著(p<0.05)。
2.4复方中药对哺乳母猪红细胞E-C3bRR花环率及E-ICRR花环率的影响
复方中药对产后第7天哺乳母猪红细胞E-C3bRR花环率及E-ICRR花环率的影响见图2。饲料中添加复方中药可提高哺乳母猪红细胞E-C3bRR花环率,与对照组相比差异显著(p<0.05)。降低哺乳母猪红细胞E-ICRR花环率与对照组相比差异显著(p<0.05)。
2.5复方中药对哺乳母猪T淋巴细胞亚群的影响
复方中药对产后第7天哺乳母猪T淋巴细胞亚群的影响见图2。饲料中添加复方中药可提高哺乳母猪T淋巴细胞亚群CD4+和CD8+,与对照组相比差异显著(p<0.05)。对CD4+/CD8+的影响与对照组相比差异不显著。
2.6复方中药对哺乳母猪猪瘟及伪狂犬疫苗抗体的影响
复方中药对产后第7天哺乳母猪猪瘟及伪狂犬gB抗体的影响见图2。饲料中添加复方中药可提高哺乳母猪猪瘟及伪狂犬gB抗体水平,与对照组相比差异显著(p<0.05)。
2.7复方中药母猪乳汁中有效成分的确定
采用LC-MS联用技术对复方中药入乳汁中化学成分进行鉴定,确定入血成分共个11个如表1所示。
表1复方中药在乳汁样品中的化学成分
根据试验结果可见,毛蕊异黄酮、熊果酸、三表去氢茯苓酸、甘草内酯、光甘草酮、黄甘草苷、1,3二咖啡酰奎宁酸、甘草宁O、半甘草异黄酮B、甘草异黄烷酮和甘草宁H是发挥增强母猪免疫力的有效成分。
Claims (10)
1.一种提高家畜免疫力的中药组合物,其特征在于,由黄芪、白术、茯苓和甘草组成。
2.按照权利要求1所述的中药组合物,其特征在于,各组分的重量份为:黄芪9-20份,白术9-20份,茯苓9-15份,甘草5-10份。
3.按照权利要求1所述的中药组合物,其特征在于,所述的家畜是猪。
4.一种提高家畜免疫力的有效成分,其特征在于,该有效成分选自毛蕊异黄酮、熊果酸、三表去氢茯苓酸、甘草内酯、光甘草酮、黄甘草苷、1,3二咖啡酰奎宁酸、甘草宁O、半甘草异黄酮B、甘草异黄烷酮和甘草宁H中的任何一种或多种按照任意比例的组合。
5.按照权利要求4所述的有效成分,其特征在于,所述的有效成分由毛蕊异黄酮、熊果酸、三表去氢茯苓酸、甘草内酯、光甘草酮、黄甘草苷、1,3二咖啡酰奎宁酸、甘草宁O、半甘草异黄酮B、甘草异黄烷酮和甘草宁H组成。
6.按照权利要求4所述的有效成分,其特征在于,所述的家畜是猪。
7.权利要求1或2所述的中药组合物在提高家畜免疫力中的应用,包括:将所述中药组合物中各组分粉碎后混合均匀添加到家畜的基础日粮中。
8.按照权利要求7所述的应用,其特征在于,所述中药组合物添加到家畜的基础日粮中的添加量为0.05-2wt%,优选为0.5wt%。
9.权利要求3或4所述的有效成分在提高家畜免疫力中的应用,包括:将所述中药组合物中各组分粉碎后混合均匀添加到家畜的基础日粮中。
10.按照权利要求9所述的应用,其特征在于,所述有效成分添加到家畜的基础日粮中的添加量为0.01-0.5wt%,优选为0.05wt%。
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