CN108837029B - 一种防治水产动物水霉病的药物组合物及其制备方法 - Google Patents
一种防治水产动物水霉病的药物组合物及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种由纯中药制成用于防治水产动物水霉病的药物组合物及其制备方法,其特征在于:该组合物由川芎、蛇床子、秦艽、苏木、乌梅、厚朴、五味子、五倍子组成,经乙醇溶液回流提取,离心,浓缩,吸附,烘干,粉碎所得。其有益结果在于:本发明提供了一种防治水产动物水霉病的药物组合物,用于水产动物水霉病的防控和治疗,减少水产动物养殖过程中因水霉病害造成的损失。
Description
技术领域
本发明涉及中药领域、药剂学领域和水产疾病防控领域,具体涉及一种由纯中药制成用于防治水产动物水霉病的药物组合物及其制备方法。
背景技术
水霉病是鱼体表的一种真菌——水霉菌寄生所引起的疾病,已成为水产养殖的主要疾病之一,给渔业生产造成了重大损失。它是一类世界性分布的鱼类疾病,在鱼类体表受伤或局部皮肤坏死时,水霉菌便乘机入侵,病变部位长着大量棉絮状的菌丝,导致鱼虾游动失常,食欲减退,甚至会瘦弱而死,给渔业生产造成了重大损失。水霉病一年四季均可发生,其中冬季、初春及晚秋季节最为流行,特别在密养的越冬池,最易发生这种病。水霉病分布范围极广,几乎所有的水产养殖区域都会发病,淡水养殖区域尤其严重。水霉菌对寄主无严格选择性,各种养殖鱼类均可感染得病,从受精卵到成鱼各个阶段都可得病。水霉病为腐生性疾病,对水产动物的感染是一种继发性感染,健康的鱼由于有较强的抵抗能力,常常不易感染。
草鱼、鲢、鳙、鲤、鲫、鲂、花鲈、红点鲑、大银鱼、斑点叉尾鮰、大鲵、黄鳝、暗纹东方鲀、月鳢、鲑鱼、黑鱼、甲鱼、黄颡鱼等等主要淡水养殖品种都时常发生。其中,草鱼、鲢、鳙、鲤、鲫、鲂是我国主要的大宗淡水鱼类品种,也是淡水养殖产量的主体,产业地位十分重要。而水霉病则是造成大宗淡水鱼类养殖损失的主要病害之一。
水霉病是困扰水产养殖业的重要疾病,一直以来主要用化学药物治疗。Foster等首次应用孔雀石绿预防鱼卵水霉病,Arasaki等证实了该药物的效果,并指出0.02mg/L能够抑制菌丝的生长,且0.2mg/L能够抑制动孢子囊和厚垣孢子的萌发。自20世纪30年代,由于孔雀石绿操作方便、价格低廉和效果明显,因而被广泛使用在水产养殖上。此外,研究表明,甲醛在18.8mg/L质量浓度下即对水霉孢子表现出明显的抑制作用,在24mg/L质量浓度下浸泡2h以上对水霉菌丝开始有杀灭作用,而Gieseker等发现用37mg/L甲醛质量浓度药浴感染水霉病的虹鳟,其死亡率最低,约为29%。含有效碘质量浓度为1mg/L的碘伏对水霉的杀菌率超过99%。由于水霉绝大多数存在于淡水中,较高的盐分限制了水霉的分布,因而食盐也成为治疗水霉病的常见化学药物之一,当NaCl质量分数为1%、2%时,水霉生长被显著抑制,而NaCl质量分数大于3%时,水霉则完全不能生长。
尽管孔雀石绿能较好杀灭水霉菌,但是研究发现,孔雀石绿能在鱼体内长时间残留,并可通过食物链对哺乳动物和人类产生致畸、致癌、致突变等作用;甲醛具有辛辣强烈刺激性,并且对人体还有致突变和致癌等危害作用;而碘伏对水产动物细胞也有一定潜在的毒性,长期应用碘或碘化物也可引发一系列中毒症状。此外,这些化学药品的长期使用会污染水环境。因此,传统化学药物的危害已引起人们的高度重视,我国已禁止在食用鱼类养殖过程中使用孔雀石绿。近年来国际上一些研究者已开始研究开发新型有效药物,如腐殖酸、二氧化氯、氢过氧化物、溴硝丙二醇等,其杀虫、杀菌效果与孔雀石绿相当,但其安全性能还有待进一步确认,而且这些药物并非是防治鱼类水霉病的最佳选择。
发明一种安全有效的防治水产动物水霉病的药物是十分必要的。已有的研究表明,五倍子对水霉病有一定的防治效果,因此也不乏其它中药对水霉真菌有效。因此,我们利用水霉真菌对几千种中药进行体外抑菌筛选,最终获得了一种安全有效的防治水产动物水霉病的药物组合物。
发明内容
本发明提供了一种防治水产动物水霉病的药物组合物,用于水产动物水霉病的防控和治疗,避免因倒池、拉网等机械损伤而引发的水霉菌继发感染,减少水产动物病害的发生。同时,本发明还提供了该药物组合物的制备方法。
本发明的目的是通过下述技术方案实现的。
一种防治水产动物水霉病的中药组合物,其特征在于:该中药组合物由川芎、蛇床子、秦艽、苏木、乌梅、厚朴、五味子、五倍子8味中药组成。
一种防治水产动物水霉病的中药组合物,其特征在于:该组合物原药材组成为川芎40-60份、蛇床子20-40份、秦艽10-20份、苏木10-20份、乌梅10-20份、厚朴10-20份、五味子10-20份、五倍子10-20份。
一种防治水产动物水霉病的中药组合物的制备方法,其特征在于:称取原药材川芎40-60份、蛇床子20-40份、秦艽10-20份、苏木10-20份、乌梅10-20份、厚朴10-20份、五味子10-20份、五倍子10-20份,加入药材总质量8~12倍体积的提取溶剂,回流提取2~3次,每次1~3h,将回流提取液迅速冷却至室温后,在线高速离心,离心液低压浓缩体积至与原药材同等份量,加入1~3倍量的米糠对浓缩液进行吸附,于烘干箱内烘至干燥,粉碎后即得产品。
一种防治水产动物水霉病的中药组合物的制备方法,其特征在于:提取溶剂为50%~80%乙醇溶液。
一种防治水产动物水霉病的中药组合物的制备方法,其特征在于:回流提取的回流温度为50℃~80℃。
一种防治水产动物水霉病的中药组合物的制备方法,其特征在于:在线高速离心的转速为4000~10000rpm。
一种防治水产动物水霉病的中药组合物的制备方法,其特征在于:低压浓缩温度为50℃~80℃。
一种防治水产动物水霉病的中药组合物的制备方法,其特征在于:该工艺的烘干温度为70℃~90℃。
本发明与现有的防治水产动物水霉病的药物相比具有以下优势:
1.组方首创,首次将川芎、蛇床子、秦艽、苏木、乌梅、厚朴、五味子、五倍子混合提取物应用到水产动物水霉病的防治领域,组合后有显著增效作用,尤其是增加川芎、乌梅后对药物的防治效果更显著。
2.工艺独特,利用回流醇提技术,显著提高蛇床子素、厚朴多酚等活性成分的提取得率,保证产品在市场中的应用效果。
3.本产品能够预防水产动物水霉病的发生,显著降低水霉病暴发后的死亡率。
4.本产品为纯中药植物药物组合物,安全无毒副作用,不产生耐药性。
具体实施方式
下面结合具体的实施例对本发明作进一步说明,但并不仅局限于此。
实施例1
处方配比:川芎40份、蛇床子20份、秦艽10份、苏木10份、乌梅10份、厚朴10份、五味子10份、五倍子10份。
将配制完毕的65%的乙醇溶液按照药材质量10倍体积注入提取罐内,密封后,升温至80℃,回流提取3次,每次1h。将回流提取液迅速冷却至室温,以8000rpm在线高速离心,50℃低压浓缩体积至与原药材同等份量。利用1倍量的米糠为辅料吸附浓缩液,于混料罐中充分混合后置于70℃烘干箱内烘干,粉碎后即得产品。
实施例2
处方配比:川芎60份、蛇床子40份、秦艽20份、苏木20份、乌梅20份、厚朴20份、五味子20份、五倍子20份。
将配制完毕的65%的乙醇溶液按照药材质量10倍体积注入提取罐内,密封后,升温至80℃,回流提取3次,每次1h。将回流提取液迅速冷却至室温,以8000rpm在线高速离心,50℃低压浓缩体积至与原药材同等份量。利用1倍量的米糠为辅料吸附浓缩液,于混料罐中充分混合后置于70℃烘干箱内烘干,粉碎后即得产品。
实施例3
处方配比:蛇床子40份、秦艽20份、苏木20份、厚朴20份、五味子20份、五倍子20份。
将配制完毕的65%的乙醇溶液按照药材质量10倍体积注入提取罐内,密封后,升温至80℃,回流提取3次,每次1h。将回流提取液迅速冷却至室温,以8000rpm在线高速离心,50℃低压浓缩体积至与原药材同等份量。利用1倍量的米糠为辅料吸附浓缩液,于混料罐中充分混合后置于70℃烘干箱内烘干,粉碎后即得产品。
1.产品对水产动物水霉菌抑制杀灭试验研究
1.1试验目的
验证3个实施例中各处方配比药物对水霉菌的体外抑制及杀灭效果。
1.2材料与方法
1.2.1水霉菌株
水霉菌株来自上海海洋大学国家水生动物病原库,分离自发病草鱼,菌株编号为02。
1.2.2中药制剂的制备
将配制完毕的65%的乙醇溶液按照药材质量10倍体积注入提取罐内,密封后,升温至80℃,回流提取3次,每次1h。将回流提取液迅速冷却至室温,以8000rpm在线高速离心,50℃低压浓缩体积至与原药材同等份量。利用1倍量的米糠为辅料吸附浓缩液,于混料罐中充分混合后置于70℃烘干箱内烘干,粉碎后即得产品。
表1试验分组设计表
1.2.3培养基的制备
马铃薯葡萄糖琼脂培养基(PDA):葡萄糖20g,蛋白胨2g,马铃薯200g,琼脂粉20g,水1000mL。
含中药煎剂的PDA培养基:将药物稀释成10倍目的药物浓度的储液,与PDA培养基1:9混合均匀,分装,每试管20ml,高压蒸汽灭菌后倒板备用,一管一板。平板中的药物浓度设计为10mg/L、100mg/L、1000mg/L、10000mg/L。
1.2.4水霉菌株的培养
取保藏于试管内的水霉菌株02,挑取少量菌丝置于无菌PDA平板上,25℃下恒温培养直至水霉菌丝长满整个平板,4℃冰箱保存备用。
1.2.5水霉菌抑菌试验设计
利用打孔器在均匀长满菌丝的平板上打孔,制作直径7mm的菌饼,有菌丝的一面朝下,接种于各浓度药物平板的中心,放于25℃下培养,24h后观察菌丝生长情况,利用十字交叉法测量菌斑的直径。每个浓度的药物组有四个重复,以无药物平板作为空白对照,进行抑制率的计算。
抑制率=(阳性对照组菌斑面积-药物组菌斑面积)/(阳性对照组菌斑面积-接种菌饼的面积)×100%
1.2.6水霉菌体外杀菌试验设计
中药制剂制作方法同1.2.2,将制好的药物用蒸馏水溶解到规定浓度,灭菌后备用。
马铃薯葡萄糖琼脂培养基(PDA)配制方法同1.2.3中所述,灭菌后倒平板备用。
水霉菌株的培养方法同1.2.4中所述。
利用打孔器在均匀长满菌丝的平板上打孔,制作直径7mm的菌饼,放入上述各种浓度的药液中浸泡0.5h、1h、2h。用无菌镊子取出浸泡过的菌饼,置于马铃薯葡萄糖琼脂培养基上,25℃下培养观察24h。肉眼判定无水霉菌落生长的,表示此种药液在该浓度该作用时间下对水霉具有杀灭作用。每个浓度设5个对照,同时设经生理盐水浸浴过的菌饼作为空白对照。
1.3结果分析
1.3.1抑菌杀菌试验结果
表2实施例中各处方配比药物对水霉菌的体外抑菌及杀菌效果。
组号 | 抑菌率 | 0.5h | 1h | 2h |
空白对照组 | 0 | + | + | + |
实施例1 | 83.64% | + | - | - |
实施例2 | 89.13% | + | - | - |
实施例3 | 61.56% | + | - | - |
注:有水霉菌丝生长记为+,即水霉菌未被杀灭,没有菌丝生长记为-,即水霉菌被杀灭。
1.3.2分析
由表2的结果可知,各实施例中,实施例1、实施例2各处方配比药物对水霉菌的体外抑菌率,与空白对照组相比分别可达到83.64%和89.13%,各药物对水霉菌抑制效果良好。而去掉处方中川芎和乌梅的实施例3,其对水霉菌的体外抑制率只有61.56%,虽然效果也不错,但是远低于含有川芎和乌梅组方的抑菌率。由此可见,在水霉药物中加入川芎和乌梅,可以大大提高药物效果。各药物在0.1%浓度下,对水霉菌浸浴1小时及以上时间时可以有效杀灭水霉菌,显示出很好的杀灭水霉菌的效果。
1.4结论
通过各药物对水霉菌的体外抑制及杀灭试验结果,可得出结论,各药物可通过药浴给药的方式很好的抑制以及杀灭水霉菌,起到防治水产动物水霉疾病的效果。尤其是加入川芎和乌梅的组方,效果更好。
2.产品对水产动物水霉病防治的大区临床应用试验:
案例1
本试验在发病池塘泼洒实施例1产品,由北京生泰尔科技股份有限公司提供。
试验地点为浙江省湖州市某草鱼养殖基地,养殖面积10亩,水深2.2米。
鱼体病症:大量草鱼贴边独游,水中可发现附于鱼体上的水霉菌丝,鱼体消瘦,颜色变深,摄食量减少,甚至停食,有死亡情况出现,经鉴定是由于拉网导致鱼体出现鳞片脱落后,水霉菌继发感染引起的草鱼水霉病。
治疗方案:按照每亩米水体泼洒500g供试产品,1天1次,隔天用药1次,连用3次,第5天基本无贴边独游情况,7天之后病鱼的水霉菌丝脱落消失,基本恢复健康。
案例2
本试验在发病池塘泼洒实施例2产品,由北京生泰尔科技股份有限公司提供。
试验地点为湖南常德市安乡县官当镇某渔场,养殖面积20亩,水深2.0米。
鱼体病症:长期有零星死亡,近期死亡数量上升,取样观察,镜检体表及鳃部都附着有大量水霉菌丝,活力降低,摄食减少,游动缓慢。经鉴定为水霉菌感染。
治疗方案:按照每亩米水体泼洒500g供试产品,1天1次,连用3次,第7天基本无贴边独游情况,镜检观察,病鱼的水霉菌丝脱落消失,病鱼活力增强,摄食旺盛。
案例3
本试验在发病池塘泼洒实施例3产品,由北京生泰尔科技股份有限公司提供。
试验地点为浙江省绍兴市某罗非鱼养殖厂,养殖面积35亩,水深1.8米。
鱼体病症:病鱼食欲减退,鳃盖暗红、眼睛发白浑浊,鱼背部发黑,体表有白色棉絮状物质附着,游动缓慢,经镜检发现体表有大量水霉菌丝附着,已经深入肌肉,每天死亡十几条,大量鱼溜边。经检测为由水霉菌引起的水霉病。
治疗方案:按照每亩米水体泼洒500g供试产品,1天1次,连续用药3天,死亡情况消失,摄食情况也有明显改善,经检查大部分罗非鱼水霉病症状消失,但镜检仍有少量菌丝,第5天又增加用药一次,到第七天时镜检观察,已完全恢复健康,未检测到菌丝附着。
案例4
本试验在发病池塘泼洒实施例2产品,由北京生泰尔科技股份有限公司提供。
试验地点为广东省湛江某渔场,养殖面积24亩,水深2.5米。
鱼体病症:草鱼开口变差,摄食量明显下降,经过解剖未发现明显器官病灶,个别鱼溜边,游动缓慢,有白色絮状物附着,镜检发现鱼体鳃部有菌丝生长,判断为真菌引起的水霉病或者是鳃霉病。
治疗方案:按照每亩米水体泼洒300g供试产品,1天2次,在草鱼较集中处泼洒,连续用药3天,摄食情况也有明显改善,经检查大部分草鱼菌丝附着症状消失,活力增强,溜边现象消失,逐渐恢复。
通过上述临床试验可知,本产品在防治由真菌引起的水霉病方面能够起到良好的治疗效果,病鱼能在短时间内恢复正常,如病鱼还可以到料台集中摄食,则可以在料台附近集中泼洒药物,减少用量,节约成本。综上,本产品在防治由真菌引起的水霉病方面效果理想,可有效解决水产养殖企业及个体户的困扰,值得大力推广及应用。
应当理解的是,本领域普通技术人员可根据本说明进行改进或转换,而这些改进和转换都应该属于本发明所附权利要求的保护范围。
Claims (7)
1.一种防治水产动物水霉病的中药组合物,其特征在于:该组合物原药材组成为川芎40-60份、蛇床子20-40份、秦艽10-20份、苏木10-20份、乌梅10-20份、厚朴10-20份、五味子10-20份、五倍子10-20份。
2.一种制备权利要求1的组合物的方法,其特征在于:按照权利要求1中的组方配比称取原药材,加入药材总质量8~12倍体积的提取溶剂,回流提取2~3次,每次1~3h,将回流提取液迅速冷却至室温后,在线高速离心,低压浓缩体积至与原药材同等份量,加入1~3倍量的米糠对浓缩液进行吸附,于烘干箱内烘至干燥,粉碎后即得产品。
3.根据权利要求2所述的方法,其特征在于:提取溶剂为50%~80%乙醇溶液。
4.根据权利要求2所述的方法,其特征在于:回流提取的回流温度为50℃~80℃。
5.根据权利要求2所述的方法,其特征在于:在线高速离心的转速为4000~10000rpm。
6.根据权利要求2所述的方法,其特征在于:低压浓缩温度为50℃~80℃。
7.根据权利要求2所述的方法,其特征在于:该工艺的烘干温度为70℃~90℃。
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