CN108835385B - 一种对虾复方中草药解毒保肝饲料添加剂 - Google Patents
一种对虾复方中草药解毒保肝饲料添加剂 Download PDFInfo
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- CN108835385B CN108835385B CN201810867168.0A CN201810867168A CN108835385B CN 108835385 B CN108835385 B CN 108835385B CN 201810867168 A CN201810867168 A CN 201810867168A CN 108835385 B CN108835385 B CN 108835385B
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Abstract
一种对虾复方中草药解毒保肝饲料添加剂,它包含柴胡、连翘、杜仲、甘草四种中草药,中草药活性成分质量比为:柴胡皂苷20%‑40%、连翘苷20%‑40%、甘草10%‑30%、绿原酸10%‑30%。该制剂生产应用后可以明显提高对虾肝胰腺解毒代谢酶活力和抗氧化水平,促进对虾的解毒能力,保护肝胰腺的正常功能。本发明通过提取四种中草药活性成分作为复方中草药解毒保肝调节剂,具有制备简单、成本低、有效成分明确、疗效高等特点,为保障对虾的生理健康提供技术保障。
Description
技术领域
本发明涉及对虾复方中草药解毒保肝饲料添加剂,属于水产配合饲料添加剂技术领域。
背景技术
随着我国对虾养殖产业的迅猛发展,由于盲目追求快速生长、高产出的养殖模式,出现了病害难于防控、渔药滥用和饲料中营养物质超量、不均衡等突出问题,对虾解毒代谢受阻,生理健康水平下降,严重制约了对虾养殖业绿色健康发展。
肝胰腺是对虾机体最重要的器官,担负着十分关键的生理功能。目前对虾配合饲料中存在蛋白质、脂肪超量添加的问题引起对虾肝胰腺脂肪堆积。同时,饲料中植物蛋白替代动物蛋白的增加会导致饲料极易滋生霉菌产生黄曲霉毒素B1等致癌、致突变物质,导致肝胰腺代谢紊乱、内毒素增加。为防止养殖池溏病害爆发,大量使用抗生素等化学物质也会给对虾肝胰腺带来沉重负担,严重损害了对虾生理健康和产业持续发展,对虾肝胰腺的养护已成为急需解决的研究课题。
目前,有关对虾饲料添加剂的研究,多集中于增强对虾机体免疫以及抗病力等方面,如通过添加免疫多糖、中草药等免疫增强剂提高对虾非特异性免疫能力,而有关对虾肝胰腺保护方面的研究尚浅。同时,传统的中草药添加剂多将药物粉碎后直接添加进饲料中,存在有效成分不明确、杂质多、适口性差等问题,易引起肝胰腺损伤和养殖环境污染。
发明内容
本发明针对现有技术存在的不足,其主要目的是提供一种对虾复方中草药解毒保肝饲料添加剂,以增强对虾肝胰腺解毒代谢能力。该制剂选用四种中草药并提取其有效成分添加进饲料中,增强对虾肝胰腺抗氧化活性和解毒代谢酶活性,并且添加成分绿色安全,不会对养殖水体造成污染。
为达到上述目的,本发明采用的具体技术方案为:
一种对虾复方中草药解毒保肝饲料添加剂,其特征在于以质量分数计,它包括柴胡皂苷20%-40%、连翘苷20%-40%、甘草10%-30%、绿原酸10%-30%。
所述的对虾复方中草药解毒保肝饲料添加剂,其特征在于柴胡皂苷:连翘苷:绿原酸:甘草酸的质量比为5:5:5:3。
所述的对虾复方中草药解毒保肝饲料添加剂,其特征在于柴胡皂苷:连翘苷:绿原酸:甘草酸的质量比为5:5:2:3。
所述的柴胡皂苷提取自柴胡,所述的连翘苷提取自连翘,所述的绿原酸提取自杜仲,所述的甘草酸提取自甘草。柴胡皂苷、连翘苷采用水提取,绿原酸采用乙酸乙酯提取,甘草酸采用稀氨水提取。
所述的柴胡皂苷、连翘苷、绿原酸、甘草酸通过以下方法制备:
所述的柴胡皂苷、连翘苷均是先将柴胡和连翘分别粉碎后,分别与蒸馏水按照质量体积比为1:10混匀,煎煮 2次,每次2小时,合并 2 次滤液,减压浓缩,冷冻干燥即得柴胡皂苷和连翘苷;
所述的绿原酸是将片状杜仲均匀粉碎后,与含0. 05 mol/L盐酸的乙酸乙酯按照质量体积比为1:10混匀,在65℃水浴上搅拌回流浸取 2小时,重复提取2次,合并滤液,旋转蒸发,即得绿原酸;
所述的甘草酸用稀氨水提取,将甘草粉碎后用0.5%稀氨水加热提取两次,每次2小时,合并两次滤液加98%浓硫酸充分搅拌调PH值到2, 静置过滤,水洗2至3次,冷冻干燥得甘草酸。
所述的对虾复方中草药解毒保肝饲料添加剂能够在制备对虾养殖饲料中的应用,能够增强对虾肝胰腺解毒代谢能力。
一种含有对虾复方中草药解毒保肝饲料添加剂的对虾饲料,其特征在于将所述的对虾复方中草药解毒保肝饲料添加剂溶于水后,均匀喷洒在基础饲料表面,再利用鱼油包裹饲料,晾干即得到含有对虾复方中草药解毒保肝饲料添加剂的对虾饲料。
每千克基础饲料中对虾复方中草药解毒保肝饲料添加剂的喷洒量为:柴胡皂苷0.1g、连翘苷0.1g、绿原酸0.1g、甘草酸0.06g;或者柴胡皂苷0.25g、连翘苷0.25g、绿原酸0.1g、甘草酸0.15g。
所述的基础饲料以质量分数计,鱼粉34%、豆粕20%、花生粕16.4%、小麦粉22%、大豆磷脂5%、复合维生素0.6%、复合矿物质2%。
本发明的优点和有益效果:
(1)提高了凡纳滨对虾总抗氧化能力。总抗氧化能力指标体现了机体包括酶类和非酶类的所有抗氧化物质的总体抗氧化活性。在投喂对虾复方中草药解毒保肝饲料添加剂的28天以及黄曲霉毒素B1毒性试验的10天内,试验组对虾T-AOC均显著高于对照组(P<0.05)。
(2)提高了凡纳滨对虾超氧化物歧化酶活性。超氧化物歧化酶是活性氧清除系统中第一个发挥作用的抗氧化酶。在投喂对对虾复方中草药解毒保肝饲料添加剂的28天以及黄曲霉毒素B1毒性试验的10天内,试验组对虾SOD活性均显著高于对照组(P<0.05)。
(3)提高了凡纳滨对虾细胞色素P450含量。细胞色素P450(CYP450)在I相代谢中催化内源性和外源性化合物的单加氧反应。在投喂对虾复方中草药解毒保肝饲料添加剂的28天以及黄曲霉毒素B1毒性试验的10天内,试验组对虾CYP450含量均显著高于对照组P<0.05)。
(4)提高了凡纳滨对虾谷胱甘肽巯基转移酶(GST)活性。谷胱甘肽巯基转移酶(GST)是体内生物转化最重要的Ⅱ相代谢酶之一,是细胞抗损伤、抗癌变的主要解毒系统。在投喂对虾复方中草药解毒保肝饲料添加剂的28天以及黄曲霉毒素B1毒性试验的10天内,试验组对虾GST活性均显著高于对照组P<0.05)。
(5)在黄曲霉毒素B1毒性试验中,降低了凡纳滨对虾血浆中谷丙转氨酶和谷草转氨酶活性。谷丙转氨酶和谷草转氨酶活性是肝细胞受损的最灵敏的指标,可以评价肝胰腺的健康程度。在投喂对虾复方中草药解毒保肝饲料添加剂的28天内,对照组和试验组谷丙转氨酶和谷草转氨酶活力无显著差异(P>0.05),在黄曲霉毒素B1毒性试验的10天内,试验组对虾谷丙转氨酶和谷草转氨酶活性均显著低于对照组(P<0.05)。
试验证明了本发明所提供的对虾复方中草药解毒保肝饲料添加剂能提高对虾胰腺解毒代谢能力和抗氧化能力,该增强剂选用柴胡、连翘、杜仲、甘草四种中草药有效进行科学配伍,本发明制备简单,成本较低,能够提高对虾养殖业的经济效益。
附图说明
图1为实施例2中对虾肝胰腺总抗氧化能力的结果图。
图2为实施例2中对虾肝胰腺超氧化物歧化酶活性的结果图。
图3为实施例2中对虾肝胰腺细胞色素P450含量的结果图。
图4为实施例2中对虾肝胰腺谷胱甘肽巯基转移酶活性的结果图。
图5为实施例2中对虾血浆谷丙转氨酶活力的结果图。
图6为实施例2中对虾血浆谷草转氨酶活力的结果图。
实施方式
实施例1:
对虾复方中草药解毒保肝饲料添加剂及其对虾饲料的制备
首先将柴胡和连翘分别粉碎后与蒸馏水按照质量体积比为1:10混匀,煎煮2次,每次2小时,合并2次滤液,减压浓缩,干燥即得柴胡皂苷和连翘苷;将片状杜仲均匀粉碎后,与乙酸乙酯(含0.05mol/L盐酸)按照质量体积比为1:10混匀,在65℃水浴上搅拌回流浸取2小时,重复提取2次,合并滤液,旋转蒸发,即得绿原酸;甘草酸用稀氨水提取,将甘草粉碎后用0.5%稀氨水加热提取两次,每次2小时,合并两次滤液加浓硫酸充分搅拌调PH值到2,水洗过滤、干燥即得甘草酸。
将中草药提取物溶于水后均匀喷洒在基础饲料表面,再利用鱼油包裹饲料,晾干即得到添加有对虾复方中草药解毒保肝饲料添加剂的基础饲料。
按照上述的制备方法分别制备两种不同浓度的增强剂及相应的养殖饲料。以质量分数计,一种对虾复方中草药解毒保肝饲料添加剂包括每千克饲料添加柴胡皂苷0.1g、连翘苷0.1g、绿原酸0.1g、甘草酸0.06g;另一种对虾复方中草药解毒保肝饲料添加剂包括每千克饲料添加柴胡皂苷0.25g、连翘苷0.25g、绿原酸0.1g、甘草酸0.15g。
上述中草药有效成分的含量可以按照现有文献所提供的方法进行测定。柴胡皂苷含量的测定方法参照:不同产地柴胡中总皂苷及柴胡皂苷a含量测定,中国药物与临床,2008年第8卷第3期;连翘苷含量的测定方法参照:连翘不同部位中连翘酯苷和连翘苷的含量分析,药物分析杂志,2008年第15卷第3期;绿原酸含量的测定方法参照:Journal ofPharmaceutical Analysis, 2011年第1卷第04期;甘草酸含量的测定方法参照:甘草及其制剂中甘草酸的定量方法研究概况,时珍国医国药,2000年第11卷第4期。
实施例
对虾复方中草药解毒保肝饲料添加剂对凡纳滨对虾抗氧化及解毒代谢能力的影响
本试验中,发明人将实施例1制备的对虾复方中草药解毒保肝饲料添加剂饲喂凡纳滨对虾,通过分析凡纳滨对虾的解毒代谢和抗氧化指标,研究本发明提高凡纳滨肝胰腺解毒代谢能力的效果。
1 材料与方法
1.1 材料
试验用凡纳滨对虾(Litopenaeus vannamei)购于青岛市崂山区沙子口对虾养殖厂,体重为3±0.32g克,试验用基础饲料的成分为:以质量分数计,鱼粉34%、豆粕20%、花生粕16.4%、小麦粉22%、大豆磷脂5%、多种维生素0.6%、复合矿物质2%。经实验计算饲料中营养成分为:以质量分数计,粗蛋白42.4%、粗脂肪7.2%、灰分8.4%。
1.2 方法
1.2.1 试验设计
将试验用对虾分为3组,每组设3个重复,每个重复组放养80尾虾。试验组分为:对照组、试验组1和试验组2,对照组投喂基础饲料(添加与试验组中的对虾等量的鱼油),试验组1、2分别投喂实施例1所制备的含有对虾复方中草药解毒保肝饲料添加剂饲料。养殖实验共持续28天。养殖实验结束后进行毒性试验,对照组和试验组均在原投喂饲料基础上添加黄曲霉毒素(2500μg/kg),毒性试验持续10天。试验期间,每日投喂2次,日投喂量为体重的5%,试验期间,水温,盐度,PH,分别为22-23℃, 34‰, 7.8-8.0。
1.2.2 测定指标和方法
分别于投喂对虾复方中草药解毒保肝饲料添加剂后第0、7、14、21、28、31、34、38天取样。每次每组随机取凡纳滨对虾6尾,使用消毒的5号针头和1mL注射器直接插入对虾头胸甲后缘围心腔内3mm左右进行采血,抽血前注射器内预先吸入了0.3mL改进的预冷凡纳滨对虾抗凝剂(0.34M NaCl, 0.01M KCl, 0.01M EDTA-Na2, 0.01 M HEPES, pH 7.45, 渗透压为780 mOsm.kg-1),最终使得抗凝剂和血淋巴的比例为1:1。血淋巴样品于4℃下800g离心10min后,取蓝色上清,即为血浆样品。解剖取肝胰腺样品,放入研钵用液氮研磨,所有样品在-80℃冰箱中保存。
T-AOC使用南京建成生物有限公司生产试剂盒测定;超氧化物歧化酶活性的测定按照现有文献的方法操作,可以参照: Involvement of the superoxide anion radicalin the autoxidation of pyrogallol and a convenient assay for superoxidedismutase. The FEBS Journal;细胞色素P450活性的测定按照现有文献的方法操作,可以参照:The carbon monoxide-binding pigment of liver microsomes I. Evidence forits hemoprotein nature. Journal of Biological Chemistry;谷胱甘肽巯基转移酶活性的测定按照现有文献的方法操作,可以参照:Glutathione S-transferases the firstenzymatic step in mercapturic acid formation. Journal of biologicalChemistry;谷丙转氨酶和谷草转氨酶使用南京建成生物有限公司生产试剂盒测定。
2 结果
2.1 总抗氧化活性
由图1可见:在投喂对虾复方中草药解毒保肝饲料添加剂的28天内,试验组对虾总抗氧化能力均显著高于对照组(P<0.05),在投喂的第7天总抗氧化能力最高,但是试验组1和试验组2之间差异不显著(P>0.05),在黄曲霉毒素B1毒性试验中,对照组总抗氧化能力下降,试验组总抗氧化能力显著高于对照组(P<0.05)。
2.2 超氧化物歧化酶活性
由图2可见:在投喂对虾复方中草药解毒保肝饲料添加剂的28天内,试验组对虾超氧化物歧化酶显著高于对照组(P<0.05),第7天和第14天的SOD活性较高,第28天试验组1和试验组2差异不显著(P>0.05)。在黄曲霉毒素B1毒性试验中,对照组总抗氧化能力大幅下降,试验组SOD活性显著高于对照组(P<0.05),试验组2的SOD活力大于试验组1,试验组间差异不显著(P>0.05)。
2.3 CYP450含量
由图3可见:在投喂对虾复方中草药解毒保肝饲料添加剂的28天内,试验组对虾肝胰腺CYP450含量显著高于对照组(P<0.05),试验组2在第7天和第21天的CYP450含量显著高于试验组1(P<0.05),,在黄曲霉毒素B1毒性试验中,对照组和试验组的CYP450含量未出现明显下降,试验组CYP450含量趋于稳定,差异不显著(P>0.05)。
2.4 谷胱甘肽巯基转移酶活性
由图4可见:在投喂对虾复方中草药解毒保肝饲料添加剂的28天内,试验组对虾肝胰腺谷胱甘肽巯基转移酶活性显著高于对照组(P<0.05),试验组GST活性在28天达到最高,其中试验组2和试验组1之间差异显著(P<0.05)。在黄曲霉毒素B1毒性试验中,对照组GST活性大幅下降,试验组GST活性显著高于对照组(P<0.05)。
2.5 谷丙转氨酶活力
由图5可见:在投喂对虾复方中草药解毒保肝饲料添加剂的28天内,试验组和对照组对虾血浆谷丙转氨酶活力无显著差异(P>0.05),表明中草药解毒保肝调节剂对凡纳滨对虾无损伤效应。在黄曲霉毒素B1毒性试验中,对照组谷丙转氨酶活力大幅上升,试验组谷丙转氨酶活力显著低于对照组(P<0.05),随着毒性试验的持续,试验组之间差异不显著(P>0.05)。
2.6 谷草转氨酶活力
由图6可见:在投喂对虾复方中草药解毒保肝饲料添加剂的28天内,试验组和对照组对虾血浆谷草转氨酶活力无显著差异(P>0.05)。在黄曲霉毒素B1毒性试验中,对照组血浆谷草转氨酶活力大幅上升,试验组谷草转氨酶活力显著低于对照组(P<0.05),随着毒性试验的持续,试验组之间差异显著(P<0.05)。
Claims (7)
1.一种对虾复方中草药解毒保肝饲料添加剂,其特征在于,以质量分数计,它包括柴胡皂苷20%-40%、连翘苷20%-40%、甘草酸10%-30%、绿原酸10%-30%,
所述的柴胡皂苷、连翘苷、绿原酸、甘草酸通过以下方法制备:
所述的柴胡皂苷、连翘苷均是先将柴胡和连翘分别粉碎后,分别与蒸馏水按照质量体积比为1:10混匀,煎煮 2次,每次2小时,合并 2 次滤液,减压浓缩,冷冻干燥即得柴胡皂苷和连翘苷;
所述的绿原酸是将片状杜仲均匀粉碎后,与含0. 05 mol/L盐酸的乙酸乙酯按照质量体积比为1:10混匀,在65℃水浴上搅拌回流浸取 2小时,重复提取2次,合并滤液,旋转蒸发,即得绿原酸;
所述的甘草酸用稀氨水提取,将甘草粉碎后用0.5%稀氨水加热提取两次,每次2小时,合并两次滤液加98%浓硫酸充分搅拌调PH值到2, 静置过滤,水洗2至3次,冷冻干燥得甘草酸。
2.如权利要求1所述的对虾复方中草药解毒保肝饲料添加剂,其特征在于,柴胡皂苷:连翘苷:绿原酸:甘草酸的质量比为5:5:5:3。
3.如权利要求1所述的对虾复方中草药解毒保肝饲料添加剂,其特征在于,柴胡皂苷:连翘苷:绿原酸:甘草酸的质量比为5:5:2:3。
4.权利要求1-3任一项所述的对虾复方中草药解毒保肝饲料添加剂在制备对虾养殖饲料中的应用。
5.一种含有对虾复方中草药解毒保肝饲料添加剂的对虾饲料,其特征在于将权利要求1所述的对虾复方中草药解毒保肝饲料添加剂溶于水后,均匀喷洒在基础饲料表面,再利用鱼油包裹饲料,晾干即得到含有对虾复方中草药解毒保肝饲料添加剂的对虾饲料。
6.如权利要求5所述的对虾饲料,其特征在于每千克基础饲料中对虾复方中草药解毒保肝饲料添加剂的喷洒量为:柴胡皂苷0.1g、连翘苷0.1g、绿原酸0.1g、甘草酸0.06g;或者柴胡皂苷0.25g、连翘苷0.25g、绿原酸0.1g、甘草酸0.15g。
7.如权利要求5或6所述的对虾饲料,其特征在于所述的基础饲料以质量分数计,鱼粉34%、豆粕20%、花生粕16.4%、小麦粉22%、大豆磷脂5%、复合维生素0.6%、复合矿物质2%。
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