CN110140812A - 对虾用复方中草药免疫增强剂及其制备方法 - Google Patents
对虾用复方中草药免疫增强剂及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种对虾用复方中草药免疫增强剂,主要由天门冬多糖和三七总皂苷组成。据此,发明人还建立了相应指标方法。本发明采用纯中草药提取,经合理配比,再加入适当辅料,获得了对虾用免疫增强剂,该产品具有天然、无毒副作用,免疫增强效果好。试验证明,给健康虾饲喂一定量本发明的免疫增强剂,可有效调节南美白对虾的免疫功能,提高其免疫能力。
Description
技术领域
本发明属于对虾用药技术领域,尤其涉及一种对虾用复方中草药免疫增强剂及其制备方法。
背景技术
南美白对虾(Litopenaeus vannamei),俗称白肢虾,为广温广盐性热带虾类,是世界养殖产量最高的三大优良品种之一,1988年中国科学院海洋研究所从美国夏威夷引进中国,目前在全国范围内广泛养殖。近年来,由于南美白对虾规模化养殖的扩大,养殖密度增加,水体氨氮含量超标,虾应激严重,导致对虾免疫力下降;而且,近年来各种疾病频发,如白斑病、虾白便等,以上原因导致南美白对虾养殖成功率明显降低,给养虾产业造成很大的经济损失。为提高对虾养殖成活率,一方面需要优化养殖条件,另一方面需要寻求能改善虾应激和提高南美对虾免疫力的药物。
中草药是中国传统药物,现代药理学研究表明,中草药中含有多种活性成分,其中一些具有提高免疫能力的作用,为改善南美白对虾应激及提高其免疫力提供了基础。
近几年,许多研究者将中草药或中草药有效成分饲喂南美白对虾后,均有抗应激、提高免疫力及增加存活率的报道。崔月明等饲喂马尾藻多糖脂质体7d后发现,脂质体组酚氧化酶、溶菌酶和碱性磷酸酶的活性极显著升高;随后在抗病毒保护试验中,多糖脂质体各剂量均能提高南美白对虾的存活率,高剂量组存活率最高,表明说明马尾藻多糖脂质体对南美白对虾具有免疫促进作用,并能提高其抗病力。黄辉等连续饲喂六种不同组合的黄芪多糖、绿原酸和大蒜素合剂21d后,用哈维氏弧菌(Vibrio harveyi)攻击虾发现,黄芪多糖、绿原酸和大蒜素的协同作用有助于提高虾的免疫力,抗氧化能力和抗病性,提升虾的存活率,而不会损害生物分子。Pandi Palanikumar等将蓟罂粟(Argemone mexicana)不同部位有效成分连续饲喂南美白对虾30d后攻毒白斑病毒(WSSV),发现根、茎乙酸乙酯部位可以显著降低其死亡率。王芸等连续饲喂黄芩、大黄、黄芪、鱼腥草和甘草这五种中药21d,研究其对凡纳滨对虾的非特异性免疫功能影响时发现,这5种中草药能有效提高酚氧化酶的活性,并且在14d活力达到最大值,但是并不适宜长期添加,因为后期会抑制酚氧化酶的活力,其中大黄、甘草、黄芩能够提高超氧化物歧化酶的活性,但是黄芪投药后超氧化物歧化酶的活力反而降低,分析认为,中草药既可以抑制自由基的产生,又可以直接消除自由基,还可以增强机体本身的抗氧化***的功能,从多个环节阻断自由基对机体的损伤作用。
发明内容:
本发明要解决的技术问题是提供一种纯天然提取、无毒副作用、效果良好的对虾用复方中草药免疫增强剂及其制备方法。
为解决上述技术问题,本发明采用以下技术方案:
对虾用复方中草药免疫增强剂,主要由天门冬多糖和三七总皂苷组成。
上述对虾用复方中草药免疫增强剂,由以下重量份的原料和辅料制成:天门冬多糖5~8份、三七总皂苷1~3份、水溶性二氧化硅2~4份。
上述对虾用复方中草药免疫增强剂的制备方法,按照配方称取天门冬多糖、三七总皂苷、水溶性二氧化硅,混匀,可加适当乙醇溶解,烘干,粉碎,过5号筛。
天门冬多糖采用醇碱提取法提取,再经旋转蒸发浓缩,两次醇沉,取沉淀;三七总皂苷总含量不少于75%。
天门冬多糖按以下操作制备:取天门冬适量,切成饮片,按照质量体积比1:15加入10%乙醇溶液搅匀后,用1mol/L的NaOH溶液调pH=9,浸泡1.5h后,再使用1mol/L的NaOH溶液调pH=9,80℃水浴回流2.5h,8层纱网过滤,残渣再重复提取一次,合并两次滤液,静置5h,取上清液,80℃减压浓缩至密度为1.20~1.24g/ml,加入5倍体积浓度为95%的食品级乙醇使乙醇含量达到80%,搅匀,4℃静置过夜,随后弃掉上清液,加入蒸馏水溶解沉淀至密度为1.15~1.20g/ml,搅拌充分溶解,先从中取10ml进行醇沉(方法同上),沉淀烘干至恒重,用于计算天门冬多糖提取率和剩余提取物的重量;剩下的溶液精密量取体积后醇沉(方法同上),离心,取沉淀备用。
三七总皂苷按以下操作制备:取三七粗粉适量加入10倍体积浓度70%的乙醇溶液浸泡0.5h后,90℃水浴回流提取2h,四层纱布过滤,滤渣重复提取一次,滤液静置5h,取上清液减压旋转蒸发浓缩至密度为1.10~1.13g/ml;将柱两倍体积的三七总皂苷浓缩液加入洗净的D101大孔吸附树脂至吸附平衡,蒸馏水洗去多糖,再以体积浓度80%的乙醇溶液洗脱三七总皂苷,减压浓缩,冷冻干燥得精制的三七总皂苷,备用。
三七总皂中以三七皂苷R1、人参皂苷Rg1、人参皂苷Re、人参皂苷Rb1、人参皂苷Rd各组分总含量大于75%。
上述对虾用复方中草药免疫增强剂的使用方法,将免疫增强剂按200~800mg/kg饲料拌入基础饲料投喂。
针对对虾免疫能力下降、应激严重等问题,发明人研制了一种对虾用复方中草药免疫增强剂,主要由天门冬多糖和三七总皂苷组成。据此,发明人还建立了相应指标方法。本发明采用纯中草药提取,经合理配比,再加入适当辅料,获得了对虾用免疫增强剂,该产品具有天然、无毒副作用,免疫增强效果好。试验证明,给健康虾饲喂一定量本发明的免疫增强剂,可有效调节南美白对虾的免疫功能,提高其免疫能力。
本发明对虾用复方中草药免疫增强剂(简称天冬糖苷散)组方原理如下:
天门冬为百合科天门冬属植物,在我国分布较广,其块根有养阴润燥、清肺生津等作用,常用于肺燥干咳、肠燥便秘等症,并具有抗菌、抗肿瘤、杀灭蚊蝇幼虫等生理活性,也可以作为滋补品,收敛药使用。研究表明,天门冬的不同提取方法对不同化学成分提取效果不一,如方芳报道称天门冬70%醇提液对金黄色葡萄球菌、大肠杆菌、黑曲霉有较强抑制作用,而水提液作用较差;谭娟研究发现天门冬水提液对金黄色葡萄球菌抑制效果相对较好,但是对大肠杆菌、苏云金芽孢杆菌、铜绿假单胞菌抑制效果较差,并且她认为天门冬水提液抗氧化和对自由基的清楚作用较醇提好。发明人采用10%乙醇水提方法从天门冬中提取有效成分,主要以抗氧化、提高免疫力为主,杀菌为辅;三七具有活血化瘀,补血养血,营养机体的作用,故添加一定量的三七总皂苷制成天冬糖苷散,可增加南美白对虾免疫力。
此外,天门冬多糖性粘,不好冷冻干燥,且冷冻干燥后的产物易吸潮结块,发明人通过加入适当比例的水溶性二氧化硅,将冷冻干燥改为直接加入辅料、烘干、粉碎,既解决了其干燥问题,也减少了辅料对天门冬多糖含量测定的影响。
附图说明
图1是血淋巴中酚氧化酶活力检测结果图,图中:1.空白对照组;2.黄芪多糖对照组;3.天冬糖苷散Ⅰ组;4.天冬糖苷散Ⅱ组;5.天冬糖苷散Ⅲ组;6.天冬糖苷散Ⅳ组;下图同。
图2是血淋巴中酸性磷酸酶活力检测结果图。
图3是血淋巴中碱性磷酸酶活力检测结果图。
图4是血淋巴中超氧化物歧化酶活力检测结果图。
图5是血淋巴中谷草转氨酶活力检测结果图。
图6是血淋巴中谷丙转氨酶活力检测结果图。
具体实施方式
实施例1南美白对虾免疫增强剂(天冬糖苷散)的制备
表1南美白对虾免疫增强剂配方
参考表1制备配方一至四的免疫增强剂。按照配方称取天门冬多糖、三七总皂苷、水溶性二氧化硅(上海凯茵化工有限公司、10kg/包、A200),混匀,可加适当乙醇溶解,烘干,粉碎,过5号筛。其中,
天门冬多糖按以下操作制备:取天门冬适量,切成饮片,按照质量体积比1:15加入10%乙醇溶液搅匀后,使用1mol/L的NaOH溶液调pH=9,浸泡1.5h,再使用1mol/L的NaOH溶液调pH=9,80℃水浴回流2.5h,8层纱网过滤,残渣再重复提取一次,合并两次滤液,静置5h,取上清液,80℃减压浓缩至密度为1.20~1.24g/ml,加入5倍体积浓度为95%的食品级乙醇使乙醇含量达到80%,搅匀,4℃静置过夜,随后弃掉上清液,加入蒸馏水溶解沉淀至密度为1.15~1.20g/ml,搅拌充分溶解,先从中取10ml进行醇沉(方法同上),沉淀烘干至恒重,用于计算天门冬多糖提取率和剩余提取物的重量;剩下的溶液精密量取体积后醇沉(方法同上),离心,取沉淀备用。
三七总皂苷按以下操作制备:取三七粗粉适量加入10倍体积浓度70%的乙醇溶液浸泡0.5h后,90℃水浴回流提取2h,四层纱布过滤,滤渣重复提取一次,滤液静置5h,取上清液减压旋转蒸发浓缩至密度为1.10~1.13g/ml;将柱两倍体积的三七总皂苷浓缩液加入洗净的D101大孔吸附树脂至吸附平衡,蒸馏水洗去多糖,再以体积浓度80%的乙醇溶液洗脱三七总皂苷,减压浓缩,冷冻干燥得精制的三七总皂苷,备用。经测定,三七总皂苷中三七皂苷R1、人参皂苷Rg1、人参皂苷Re、人参皂苷Rb1、人参皂苷Rd各组分总含量大于75%。
使用方法:在运输前后、疾病威胁或者虾塘水质剧烈变化导致对虾免疫能力降低时,针对性的使用天冬糖苷散以提高免疫力,防止疾病的发生。
将天冬糖苷散分别按质量饲料比100、200、400、800mg/kg,用适当的蒸馏水溶解,均匀喷洒于相应质量的基础日粮上,混匀,使其充分被饲料吸收,阴干24h备用。将含免疫增强剂的饲料每日按体重的3%~5%的量投喂,连续使用28天,各组每隔7天采血一次。结果:从第7天开始,各组血淋巴中酸性磷酸酶、碱性磷酸酶、酚氧化物酶均随用药剂量提高而提高,显著优于黄芪多糖对照组和空白对照组,且超氧化物歧化酶、谷丙转氨酶和谷草转氨酶无明显差别,这表明天冬糖苷散可以有效调节对虾免疫能力,且无明显的毒性。
实施例2天冬糖苷散对南美白对虾免疫调节作用
1材料方法
1.1材料
(1)实验用药
复方中草药免疫增强剂(实施例1配方4)。
黄芪多糖,北京生泰尔生物技术有限公司馈赠。
(2)实验用虾
南美白对虾(体重13~18g、体长10~15cm),由广西水产科学研究院提供。
(3)试剂盒
超氧化物歧化酶(20181008)、碱性磷酸酶试剂盒(20180929)、酸性磷酸酶试剂盒(20181008)、谷丙转氨酶(20180928)、谷草转氨酶(20180829)、BCA蛋白浓度测定试剂盒(20180928);均购自南京建成生物有限公司;
1ml 1mol/L的L-dopa溶液(20181030),购自索莱宝生物技术有限公司;其余试剂为分析纯。
1.2方法
(1)复方中药饲料添加剂的制备
制法同实施例1。
将药粉按照一定的药料比,加入适量蒸馏水混匀,用喷雾器将其均匀喷洒于相应质量的基础日粮上,混匀,使其充分被饲料吸收,阴干24h备用。
黄芪多糖的添加量为1g/kg饲料。
(2)实验用虾准备
南美白对虾实验前暂养于广西大学水产基地300L透明收纳箱中,每箱25只,每组两箱。
(3)实验处理
将300只体长10~15cm的虾分为6个组,分别为空白对照组、黄芪多糖组、天冬糖苷散Ⅰ组、天冬糖苷散Ⅱ组、天冬糖苷散Ⅲ组、天冬糖苷散Ⅳ组,每组25只(每组2个重复,共50只),分别于第一天开始按照体重5%投喂含不同黄芪多糖和天门冬多糖药饵,连续饲喂28天,分别于第7d、14d、21d、28d,每组取5尾采集血淋巴,经过处理,用于免疫指标检测,具体分组见表1。
表1动物分组及处理
1.3免疫因子测定
1.3.1酚氧化酶(PO)活力测定
以L-dopa为底物,参照Ashida的方法作适当修改后进行。于96孔板中,依次加入37℃预热的0.1mol/L pH=6.0的磷酸钾缓冲液100μL、待测液100μL、0.015mol/L L-dopa 100μL,置于室温下震荡混匀后,立即测反应体系490nm下OD值,每隔两个分钟测一次,连续测量20分钟,以实验条件下每分钟490nm OD值增加0.001定义为一个酶活力单位。
1.3.2其余免疫因子测定
超氧化物歧化酶、碱性磷酸酶、酸性磷酸酶、谷丙转氨酶、谷草转氨酶参考试剂盒所示方法检测。
2实验结果
2.1血淋巴中酚氧化酶(PO)活力检测结果
由表2和图1可知,连续28天饲喂南美白对虾天冬糖苷散,在第7天,各喂药组其酚氧化酶活性值均高于空白对照组,且天冬糖苷散各组在数值上大于黄芪多糖对照组;在喂药后第14天,天冬糖苷各组随着喂药量的上升,其酚氧化酶活性逐渐上升,在喂药量达到0.8%/kg饲料时,与空白对照组和黄芪多糖对照组相比差异显著(P>0.05);在喂药后第21天,天冬糖苷散各组酶活性大于黄芪多糖组和空白对照组;在喂药后第28天,黄芪多糖组和天冬糖苷散Ⅳ组与空白对照相比差异显著(P>0.05);结果表明,从喂药后第7天开始,各给药组酶活性呈上升趋势,其中天冬糖苷散组,会随着给药浓度升高而上升。
表2血淋巴中酚氧化酶活力检测结果
注:右上肩标“*”表明与空白对照组相比差异显著(P>0.05),肩标“#”表示和黄芪多糖对照组相比差异显著(P>0.05),无符号表示差异不显著,下同。
2.2血淋巴中酸性磷酸酶(ACP)活力检测结果
由表3和图2可知,连续饲喂天冬糖苷散28天,在饲喂天冬糖苷散第7天,天冬糖苷散Ⅳ组和黄芪多糖对照组相比差异显著(P>0.05);在给药后第14天,天冬糖苷散Ⅱ组和Ⅳ组与黄芪多糖对照组相比差异显著(P>0.05),天冬糖苷散Ⅳ组和空白对照组相比差异显著(P>0.05);在喂药后第21天,天冬糖苷散Ⅰ组和黄芪多糖组差异显著(P>0.05),给药后第28天,天冬糖苷散Ⅳ组与空白对照组和黄芪多糖组相比差异均显著(P>0.05);结果表明,连续饲喂天冬糖苷散28天,从第7天起,各组血清中酸性磷酸酶活性逐渐升高,表明天冬糖苷散有刺激虾血淋巴中酸性磷酸酶升高的趋势。
表3血淋巴中酸性磷酸酶活力检测结果(单位:金氏单位/100ml)
2.3血淋巴中碱性磷酸酶(AKP)活力检测结果
由表4和图3可知,在给药后第14天,天冬糖苷散Ⅰ组血淋巴中碱性磷酸酶与空白对照组相比差异显著;其余组虽差异不显著,但随着天冬糖苷散给药剂量的加大及随着给药时间的延长,虾血清中碱性磷酸酶含量数值会大于空白对照组,表明天冬糖苷散有调节碱性磷酸酶分泌的作用。
表4血淋巴中碱性磷酸酶活力检测结果(单位:金氏单位/100ml)
2.4血淋巴中超氧化物歧化酶(SOD)活力检测结果
由表5和图4可知,连续饲喂天冬糖苷散28天,各组在各采样点其血淋巴中SOD无明显改变,表明饲喂天冬糖苷散对南美白对虾血清中超氧化物歧化酶没有明显的影响。
表5血淋巴中超氧化物歧化酶活力检测结果(单位:U/ml)
2.5血淋巴中谷草转氨酶(GOT)活力检测结果
由表6和图5可知,在给药后第14天,天冬糖苷散Ⅲ组血清中谷草转氨酶含量明显低于空白对照组,在第28天,天冬糖苷散Ⅰ组、天冬糖苷散Ⅱ组及天冬糖苷散Ⅲ组血清中谷草转氨酶含量显著低于黄芪多糖组(P>0.05);表明连续饲喂天冬糖苷散28d对南美白对虾血清中谷草转氨酶的含量无明显影响。
表6血淋巴中谷草转氨酶活力检测结果(单位:卡门氏)
2.6血淋巴中谷丙转氨酶(GPT)活力检测结果
从表7和图6中可看出,连续给药28天,在第28天时天冬糖苷散各组血清中谷丙转氨酶含量显著(P>0.05)低于黄芪多糖对照组,其余无显著差异,表明天冬糖苷散对南美白对虾血液中谷丙转氨酶的分泌无显著影响。
表7血淋巴中谷丙转氨酶活力检测结果(单位:卡门氏)
3结论
本发明的虾用复方中草药免疫增强剂,取得了预期成果,值得进一步开发利用。
Claims (8)
1.一种对虾用复方中草药免疫增强剂,其特征在于主要由天门冬多糖和三七总皂苷组成。
2.根据权利要求1所述的对虾用复方中草药免疫增强剂,其特征在于由以下重量份的原料和辅料制成:天门冬多糖5~8份、三七总皂苷1~3份、水溶性二氧化硅2~4份。
3.权利要求2所述对虾用复方中草药免疫增强剂的制备方法,其特征在于按照配方称取天门冬多糖、三七总皂苷、水溶性二氧化硅,混匀,加适当乙醇溶解,烘干,粉碎。
4.根据权利要求3所述的对虾用复方中草药免疫增强剂的制备方法,其特征在于:所述天门冬多糖采用醇碱提取法提取,再经旋转蒸发浓缩,醇沉。
5.根据权利要求4所述的对虾用复方中草药免疫增强剂的制备方法,其特征在于所述天门冬多糖按以下操作制备:取天门冬适量,切成饮片,按照质量体积比1:15加入10%乙醇溶液搅匀后,使用1mol/L的NaOH溶液调pH=9,浸泡1.5h,再使用1mol/L的NaOH溶液调pH=9,80℃水浴回流2.5h,8层纱网过滤,残渣再重复提取一次,合并两次滤液,静置5h,取上清液,80℃减压浓缩至密度为1.20~1.24g/ml,加入5倍体积浓度为95%的食品级乙醇使乙醇含量达到80%,搅匀,4℃静置过夜,随后弃掉上清液,加入蒸馏水溶解沉淀至密度为1.15~1.20g/ml,搅拌充分溶解,先从中取10ml进行醇沉(方法同上),沉淀烘干至恒重,用于计算天门冬多糖提取率和剩余提取物的重量;剩下的溶液精密量取体积后醇沉,离心,取沉淀备用。
6.根据权利要5所述的对虾用复方中草药免疫增强剂的制备方法,其特征在于所述三七总皂苷按以下操作制备:取三七粗粉加入10倍体积浓度70%的乙醇溶液浸泡0.5h后,90℃水浴回流提取2h,四层纱布过滤,滤渣重复提取一次,滤液静置5h,取上清液减压旋转蒸发浓缩至密度为1.10~1.13g/ml;将柱两倍体积的三七总皂苷浓缩液加入洗净的D101大孔吸附树脂至吸附平衡,蒸馏水洗去多糖,再以体积浓度80%的乙醇溶液洗脱三七总皂苷,减压浓缩,冷冻干燥得精制的三七总皂苷,备用。
7.根据权利要6所述的对虾用复方中草药免疫增强剂的制备方法,其特征在于:所述三七总皂中以三七皂苷R1、人参皂苷Rg1、人参皂苷Re、人参皂苷Rb1、人参皂苷Rd各组分总含量大于75%。
8.权利要求2所述对虾用复方中草药免疫增强剂的使用方法,其特征在于将免疫增强剂按200~800mg/kg饲料拌入基础饲料投喂。
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