CN103610778B - 抗鸡热应激的中药制剂及其制备方法 - Google Patents
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Abstract
本发明涉及畜牧用药技术领域,具体为一种抗鸡热应激的中药制剂及制备方法。一种抗鸡热应激的中药制剂,包括下列重量份的各组分:党参:4份,刺五加:3份,元胡:2份,酸枣仁:2份,五味子:2份,藿香:3份,神曲:2份,甘草:2份。本发明设计合理,通过复方中药对热应激肉仔鸡HSP70mRNA表达、抗氧化作用及修复热应激受损细胞的研究,从分子水平提高对热应激的防治效果,为中药调控热应激蛋白机理及热应激的综合防制提供了理论依据。
Description
技术领域
本发明涉及畜牧用药技术领域,具体为一种抗鸡热应激的中药制剂及制备方法。
背景技术
应激是机体对应激源做出的非特异性防御反应,与传统中医学的肾虚脱症极为相似。随着全球气候温室效应的不断加剧及畜牧高度集约化生产的发展,热应激对鸡的危害日益严重。尤其肉鸡生长快、体型大、皮厚、皮下及腹部脂肪多,对热应激的抵抗力差。热应激是造成肉鸡猝死症的主要原因之一,严重制约肉鸡养殖业的健康发展,给肉鸡生产造成了严重的经济损失。
热休克蛋白(heat shock protein,HSP)是在高温和其他应激条件下动物体内合成的一系列具有内源性保护作用的蛋白质,又名热应激蛋白。HSP70是HSP家族中最重要的一员,在结构上具有高度保守性。应激状态下HSP70在细胞内迅速合成,抵抗应激造成的组织细胞损伤,提高机体的热耐受能力而起到保护机体的作用,这种热耐受能力主要与HSP70有关,而且与HSP70 的表达水平呈正相关,一些抗热应激中药可通过提高HSP的表达来改善机体抗应激的能力。HSP在应激条件下参与细胞的抗损伤、修复和热耐受过程,保护细胞生命活动。
正常生理情况下,体内自由基产生和清除处于动态平衡状态,当受到热应激时,机体自由基的产生急剧增加,清除能力不足则造成体内自由基蓄积,导致组织氧化受损。热应激诱导细胞膜内生成过量脂质超氧化物,使氧化作用加强,破坏细胞膜的完整性,引起蛋白质和脂肪分解增加,细胞膜的通透性破坏、细胞质的渗透压升高,组织液进入细胞内,使得组织细胞内水分增多,导致组织细胞发生水肿等一系列病理变化,导致细胞膜的严重损伤。SOD活性能间接地反映机体清除氧自由基的能力,而脂质过氧化产物MDA的含量则能间接反映机体细胞受自由基攻击的严重程度。因此,减少脂质过氧化物的产生,提高机体的抗氧化能力,探索清除体内过量自由基的外源植物活性成分成为当前人们研究的热点。
如何通过中药等手段提高机体抗氧化水平,抑制热应激引起的肝脏细胞凋亡,达到修复热应激受损细胞的作用,已成为分子生物学研究的重要课题。其研究成果将为抗创伤、外科手术等病理过程中缺血再灌注引起的应激反应,对细胞和器官的保护提供一个新的有效途径。
发明内容
本发明的目的在于提供一种用于抵抗鸡热应激的中药制剂及制备方法。
本发明是采用如下技术方案实现的:
一种抗鸡热应激的中药制剂,包括下列重量份的各组分:
党参:4份,刺五加:3份,元胡:2份,酸枣仁:2份,五味子:2份,藿香:3份,神曲:2份,甘草:2份。
上述抗鸡热应激的中药制剂的制备方法如下:将各规定量的各组分经水提浓缩后,去渣取汁,放置过夜使沉淀、过滤,取上清液浓缩成浸膏,然后加入糊精等载体混合后,经烘干、粉碎制成中药制剂,即成药。
优选地,所述中药制剂中,载体的含量为50%~55%;每克中药制剂中再加入30毫克的维生素C和25毫克的维生素E。
本发明以补肾健脾、益气安神、调节代谢、补充营养为治疗原则。配方中党参补中益气为君药;元胡、酸枣仁、五味子补气安神为臣药;藿香、刺五加芳香化湿、健脾利湿为佐药;甘草调和诸药为使药,诸药共用可达补气固本、卫外安神、增强免疫功能和调节代谢的作用。
本发明的试验效果如下:
1、本发明的抗热应激临床对比试验
1.1、试验动物
18日龄健康AA+肉仔鸡120只,购自山西省太原市小店区明生养殖场。
1.2、试验方法
120只18日龄AA+肉仔鸡随机分为4组,即中药复方组(本发明,即权利要求5所述方法直接获得的产品)、维生素组、纯中药组(本发明,即权利要求2所述方法直接获得的产品)和高温对照组,每组30只鸡,每组设3个重复。人工制造慢性热应激病理模型,进行本发明防治肉鸡热应激临床对比试验。试验共进行18天,包括3天预饲期和15天正式试验期。预饲期间按常规进行饲养管理和防疫,正式试验期各组基础日粮完全相同,中药制剂的添加量为0.4g /天/只,维生素的添加量按照国家标准,自由饮水。热应激环境温度、湿度的调节见表1。温湿度调节由温湿度控制器控制。
1.3、试验设计
表1 热应激模型建立的温度、湿度控制
注: 温度波动范围为±0.5℃ ,湿度波动范围为±10%。
1.4、试验结果
本发明防治肉鸡热应激试验结果见表2。
表2 抗热应激临床对比试验
注:同一列中, 右肩标字母相同表示差异不显著,相邻表示差异显著 (
P<0.05),相间表示差异极显著( P< 0.01)。
由表2可知,复方中药冲剂组平均日增重分别比维生素组、高温对照组增加7.80g(P
<0.01)和10.87g(P <0.01),差异极显著;纯中药组平均日增重分别比维生素组、高温对照组增加3.25g和6.32g(P<0.05),与高温对照组差异显著(P<0.05),其他各组间相比差异不显著。而且复方中药冲剂组和纯中药组无死淘鸡只发生,死淘率分别比维生素组和高温对照组降低10%(P<0.05)
和16.67%(P<0.01),显著差异和极显著。
2、本发明调控HSP70基因表达试验
2.1、试验动物、试验设计与试验方法同试验1。
2.2、组织处理
试验结束后每组随机选3只鸡(共12只)迅速宰杀,取出肝脏并用生理盐水冲洗干净,装于冰冻管后立即放入液氮罐中冷冻。
2.3、组织总RNA的提取及质量鉴定
将采好的肝脏组织约0.5g从液氮中取出,放入已经灼烧好的研钵中,用液氮将组织研成粉末;将组织粉末放到有1mL的Trizol的1.5 ml离心管中,剧烈混匀,静置5min;加入200μL氯仿混匀,静置3 min,12000 r/min、4℃离心15min;吸取400μL上清液加入等体积的异丙醇,室温静置10min,4℃、12000 r/min,离心10min;取出,倒掉异丙醇,再加入1mL的75%预冷的乙醇进行漂洗,4℃,7500r/min,离心5min;倒掉乙醇,自然晾干,加入20μl的ddH2O(灭菌蒸馏水),充分混匀后,进行电泳检测。
取1μL分装的RNA溶液,用核酸蛋白测定仪测定其浓度及A260、A260/280、A260/230的值。然后用常规1.2%琼脂糖凝胶电泳,检测所提取RNA的质量。
2.4、Real-Time RT-PCR反应体系建立
cDNA链的合成:10μL反应体系中含5×Prime ScriptTM
Buffer 2μL,total
RNA不多于500ng,DEPC水加至10μL。上述成分混匀后置于PCR 仪器中,反应条件为37℃15min;85℃5s。RT产物保存于-20℃备用。
扩增条件建立:每个RNA样品分别使用目的基因和内参基因引物基因进行荧光定量PCR反应。25μL反应体系如下:12.5μL的SYBR® Premix,0.5μL的PCR Forward
Primer(5μmol•L-1),0.5μL的PCR Reverse
Primer(5μmol•L-1),1μL的cDNA溶液,最后用ddH2O(灭菌蒸馏水)补充至25μL。
95℃预变性1min后,95℃10s,61℃30s,72℃30s,72℃1min;30个循环。反应结束后,由熔解曲线判定PCR反应的特异性,根据扩增动力学曲线的CT值计算定量结果。内参基因β-actin与目的基因在同一条件下不同管内扩增,每个样本设3次重复,最后取平均值。
2.5、PCR产物鉴定
根据产物片段大小,采用1%琼脂糖凝胶电泳。5μL样品与1μL溴酚蓝混合后点样,同时点DNA marker 5μL;电压120V电泳20min后,紫外灯下观察,凝胶成像系统采集图像。运用DNA
Marker 测算目的基因产物片段的大小。
2.6、结果
2.6.1、组织总RNA的提取,提取效果如图1,表示总RNA提取效果。
2.6.2、扩增产物凝胶电泳分析见图2,表示内参基因及HSP70扩增产物凝胶电泳。
2.6.3、热应激鸡肝脏组织中HSP70实时定量PCR结果见图3。
2. 7、结论
HSP70基因没有内含子,这一特异性保证了它们一旦启动转录就可产生出成熟的mRNA 以适应HSP70大量快速表达的需要,防止应激对其mRNA前体的影响。Craig等(1991)报道,高温可增强热应激转录因子的活性,加强HSP70的mRNA合成,从而增加HSP70质量浓度。本试验中复方中药冲剂组及纯中药冲剂组肉鸡肝脏HSP70基因表达量显著提高,分别比高温对照组、维生素组高2.98倍、3.28倍和4.66倍、5.13倍,说明本试验中药制剂可显著提高热应激肉鸡肝脏HSP70的mRNA的表达,增强抗热应激能力。
3、中药冲剂抗氧化作用试验
3.1、试验动物、试验设计与试验方法同试验1。
3.2、试验试剂
SOD和MDA试剂盒采用南京建成生物工程研究所产品。
3.3、组织处理
试验结束后每组随机选3只受试鸡(共12只)迅速剖杀,采集血液,分离血清,供血清SOD和MDA的含量测定。
3.4、肝脏SOD和MDA的含量测定结果
SOD测定采用黄嘌呤氧化酶法,MDA测定方法为TBA法。对热应激肉鸡的抗氧化作用见表3。
表3 肉鸡肝脏中SOD和MDA 含量检测
注:同一列中,右肩标字母相同表示差异不显著,相邻表示差异显著 ( P<0.05),相间表示差异极显著( P< 0.01)。
由表3结果可见,复方中药冲剂组与纯中药组SOD含量极显著高于维生素组和高温对照组(P<0.01),复方中药冲剂组MDA含量显著低于纯中药组(P<0.05),极显著低于维生素组和高温对照组组(P<0.01),其他各组间差异不显著。
4、中药冲剂修复热应激受损细胞试验
4.1、试验动物、试验设计与试验方法同试验1。
4.2、组织处理
试验结束后每组随机选3只鸡(共12只)迅速宰杀,取肝脏组织并用生理盐水冲洗干净,修整为1cm3大小的组织块,以中性甲醛固定,置于-70℃冰箱中保存,备用。
4.3、肝组织超薄切片制备
将肝组织从培养液中转移到预冷的戊二醛液滴中固定10min,再转移到新鲜的戊二醛液滴中,在4℃冰箱中放置2h,经0.01mol/LPBS清洗3次,用10g.L-1饿酸室温避光固定1.5h,PBS清洗3次,将组织转移到PCR反应管内,加入50ulPBS,2000r.min-1离心1~2min,吸弃PBS后缓慢加20g.L-1琼脂糖溶液100ul,立即以2000r.min-1离心1~2min,待琼脂糖凝固取出,琼脂糖块分别经300、500、700和800ml.L-1丙酮脱水2次各10min,再经2种Epon812包埋剂渗透4~6h,并于37℃温箱内过夜,45℃聚合12h,60℃聚合24~36h,聚合完毕后将样品保存于干燥器中,先将样品切成0.5um的半薄切片,经10g.L-1甲苯胺蓝液染色1min,光镜下定位后再切出50~100nm的超薄切片,用醋酸双氧铀和硝酸铅对其进行双重染色蒸馏水冲洗后于电镜下观察。
4.4、结果
中药冲剂对热应激受损细胞修复结果见图4,表示中药冲剂对热应激受损细胞修复超微结构分析。
由图4可知:复方中药组肝细胞核物质排列均匀,细胞核双层膜结构较明显,细胞内溶酶体数量较多,内质网一粗面内质网为主,线粒体数量较多,嵴排列整齐;维生素组肝细胞核膜微扩,核膜肿胀、内陷或破裂,内质网和线粒体扩张,细胞核内异染色质增多,核仁被挤向一侧,有脂滴出现;纯中药组肝细胞出现不正常的双核现象,内质网肿胀,线粒体略肿胀、嵴断裂或消失;高温对照组肉鸡肝细胞核偏向一侧、双层膜结构破裂、内质网肿胀、染色质聚集,糖原异常聚集、线粒体数量显著减少、肿胀、破裂、嵴紊乱、缺少、内容物外陷、有的空泡化。说明热应激条件下肉鸡肝脏会出现超微结构的病理变化,中药对热应激受损肝细胞有一定的修复作用,经中药加维生素干预修复后细胞形态基本恢复正常。
4.5、结论
肝脏是热应激时容易损伤的组织之一,热应激可损伤肝细胞的细胞膜结构,导致肝细胞的肿胀、坏死。还诱导细胞膜内蛋白质和脂肪分解增加,细胞膜通透性破坏、细胞质的渗透压升高,组织液进入细胞内导致细胞内水分增多、发生水肿等一系列病理变化,导致细胞膜的严重损伤。HSP70在应激条件下参与细胞的抗损伤、修复和热耐受过程,保护细胞生命活动。
正常状况下,线粒体是有明显双层单位膜套叠而成的封闭囊状结构。这种结构为各类参与氧化磷酸化过程及其他生物化学过程的酶提供了附着位点。细胞内线粒体的存在状况往往反映了细胞对能量的要求。因此,结构框架的完整性与功能的合理表达之间存在着密切的关系。
在温度等应激条件下,组织细胞会发生一系列病理变化,细胞核核膜结构出现皱缩、扭曲、内陷,表面形成许多浅沟等现象;细胞核内异染色质增多,其主要成分糖原颗粒分布呈现出斑块化状态;内质网结构呈现破碎化状态;线粒体双层膜结构严重扩张、破裂或溶解,结构坍塌,嵴脱落或消失,线粒体数量减少,结构严重破碎化;线粒体肿胀、破裂或溶解后,结构的异常导致功能异常,糖原不能被线粒体充分利用而造成堆积。肝脏糖原颗粒的异常增多,推测与线粒体功能的改变密切相关。
本发明设计合理,通过复方中药对热应激肉仔鸡HSP70mRNA表达、抗氧化作用及修复热应激受损细胞的研究,从分子水平提高对热应激的防治效果,为中药调控热应激蛋白机理及热应激的综合防制提供了理论依据。
附图说明
图1表示组织中总RNA提取效果。
图2表示内参基因及HSP70扩增产物凝胶电泳。
图3表示HSP70实时定量PCR结果对比。
图4表示中药冲剂对热应激受损细胞修复超微结构分析对比。
具体实施方式
下面对本发明的具体实施例进行详细说明。
实施例
1
一种抗鸡热应激的中药制剂,包括下列重量份的各组分:(每份10g)
党参:4份,刺五加:3份,元胡:2份,酸枣仁:2份,五味子:2份,藿香:3份,神曲:2份,甘草:2份。
制备方法如下:
将党参40g、刺五加30g、元胡20g、酸枣仁20g、五味子20g、藿香30g、神曲20g、甘草20g等8味药材经水提(煎煮)浓缩后,去渣取汁,放置过夜使沉淀、过滤,取上清液浓缩成浸膏,然后加入糊精等载体混合后,经烘干、粉碎制成中药制剂,即成药,每克成药中含0.5克的载体和0.5克的原生药。
实施例
2
一种抗鸡热应激的中药制剂,包括下列重量份的各组分:(每份50g)
党参:4份,刺五加:3份,元胡:2份,酸枣仁:2份,五味子:2份,藿香:3份,神曲:2份,甘草:2份。
制备方法如下:
将党参200g、刺五加150g、元胡100g、酸枣仁100g、五味子100g、藿香150g、神曲100g、甘草100g等8味药材经水提(煎煮)浓缩后,去渣取汁,放置过夜使沉淀、过滤,取上清液浓缩成浸膏,然后加入糊精等载体混合后,经烘干、粉碎制成中药制剂,即成药,每克成药中含0.55克的载体和0.45克的原生药;然后,每克中药制剂中加入30毫克的维生素C和25毫克的维生素E,即得到冲剂。
实施例
3
一种抗鸡热应激的中药制剂,包括下列重量份的各组分:(每份100g)
党参:4份,刺五加:3份,元胡:2份,酸枣仁:2份,五味子:2份,藿香:3份,神曲:2份,甘草:2份。
制备方法如下:
将党参400g、刺五加300g、元胡200g、酸枣仁200g、五味子200g、藿香300g、神曲200g、甘草200g等8味药材经水提(煎煮)浓缩后,去渣取汁,放置过夜使沉淀、过滤,取上清液浓缩成浸膏,然后加入糊精等载体混合后,经烘干、粉碎制成中药制剂,即成药,每克成药中含0.52克的载体和0.48克的原生药;然后,每克中药制剂中加入30毫克的维生素C和25毫克的维生素E,即得到冲剂。
Claims (4)
1.一种抗鸡热应激的中药制剂,其特征在于:
配方由下列重量份的各组分构成:党参:4份,刺五加:3份,元胡:2份,酸枣仁:2份,五味子:2份,藿香:3份,神曲:2份,甘草:2份;
通过如下步骤制备:将各规定量的各组分经水提浓缩后,去渣取汁,放置过夜使沉淀、过滤,取上清液浓缩成浸膏,然后加入载体混合后,经烘干、粉碎制成中药制剂,即成药。
2.根据权利要求1所述的抗鸡热应激的中药制剂,其特征在于:所述载体为糊精。
3.根据权利要求1或2所述的抗鸡热应激的中药制剂,其特征在于:所述中药制剂中,载体的含量为50%~55%。
4.一种中药组合物,其特征在于:每克权利要求3所述的中药制剂中加入30毫克的维生素C和25毫克的维生素E。
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