CN112587557B - 一种防治拉萨裸裂尻鱼水霉病制剂 - Google Patents
一种防治拉萨裸裂尻鱼水霉病制剂 Download PDFInfo
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- CN112587557B CN112587557B CN202011601109.2A CN202011601109A CN112587557B CN 112587557 B CN112587557 B CN 112587557B CN 202011601109 A CN202011601109 A CN 202011601109A CN 112587557 B CN112587557 B CN 112587557B
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Abstract
本发明提供了一种防治西藏高原鱼类水霉病制剂,包括盐、小苏打、沙棘提取物、水杨酸和虾青素纳米颗粒,所述沙棘提取物包括重量份数比为2:1的沙棘果提取物和沙棘叶提取物。本发明所述的防治西藏高原鱼类水霉病制剂能够有效防治水霉病,降低死亡率。
Description
技术领域
本发明涉及鱼类养殖技术领域,尤其是涉及一种防治西藏高原鱼类水霉病制剂。
背景技术
水霉病又叫肤霉病,由水霉科(Saprolegniacae)中的水霉属(Saprolegnia)、绵霉属(Achlya)、丝囊霉属(Aphanomyces)和网囊霉属(Dictyuchus)的一些种类引起。水霉菌丝为管形没有横隔的多核体,一般像根样附着在水产动物的损伤处,分枝多而纤细,可深入至损伤、坏死的皮肤及肌肉称为内菌丝,具有吸收营养的功能;伸出在体外的叫外菌丝,菌丝较粗状,分枝较少,可长达3cm,形成肉眼能见的灰白色棉絮状物。水霉病的发生主要因为紧迫造成的二次性感染,鱼只因拥挤、移动或其他不良环境因素的影响,造成体表组织受伤,水中的水霉病游孢子伺机附着,于坏死组织上开始发芽形成菌丝,菌丝除寄生于坏死组织外,尚可蔓延侵入附近的正产组织,分泌消化酵素分解周围组织的覆盖物,并于末端形成孢子囊,放出游孢子到水中,经由水而传播各处。
西藏,誉为“地球的第三极”,是我国重要的生态屏障和安全屏障。文献资料显示,该地区分布的鱼类有60种和13亚种,分属于3目,5科和4亚科,22个属。伴随着生境退化或丧失,堤坝和水电站的建设,外来物种的入侵,环境的污染,加之该区域鱼类本身生长缓慢,性成熟晚等特征,受威胁鱼类(濒危、易危)有12种,近17%。分布在雅鲁藏布江7种主要鱼类,有5种处于受威胁状态,因此,亟需开展西藏受威胁鱼类抢救保护工作,从而保证这些特有鱼类的种质延续性。开展西藏高原鱼类驯养及繁殖是保存其种质资源的有效手段。西藏鱼类养殖水温:9-20℃,水温在水霉易爆发范围,西藏高原鱼类在捕捞和运输过程中易造成机械损伤,导致水霉病爆发;另外野生鱼类自身携带较多寄生虫,寄生虫滋生造成鱼体受损,导致水霉病爆发。西藏高原鱼类繁殖过程中对亲本注射催产剂、剂卵和剂精液,造成鱼体机械损伤,从而滋生水霉。在西藏鱼类养殖过程中,随着养殖鱼规格变大,需要及时分池,在转移过程中易造成机械损伤,导致水霉病爆发。因此在西藏高原鱼类驯养及繁殖过程中经常爆发水霉病,如何防治西藏高原鱼类水霉病成为了高原鱼类养护的主要难点之一。
发明内容
有鉴于此,本发明旨在提出一种防治西藏高原鱼类水霉病制剂。
为达到上述目的,本发明的技术方案是这样实现的:
一种防治西藏高原鱼类水霉病制剂,包括盐、小苏打、沙棘提取物、水杨酸和虾青素纳米颗粒,所述沙棘提取物包括重量份数比为2:1的沙棘果提取物和沙棘叶提取物。
沙棘是植物和其果实的统称,植物沙棘为胡颓子科沙棘属,是一种落叶性灌木。沙棘的根、茎、叶、花、果,特别是沙棘果实含有丰富的营养物质和生物活性物质,沙棘果富含丰富的维生素C,每百克鲜果含量达1000-1600毫克,是橘子的35倍,是梨子的250倍,苹果的400倍,有维生素之王的美称,另外还含有亚油酸、蛋白质、黄酮类物质,不仅是营养丰富的食品,同时还治疗心脏病、高血压等疾病的良药。
在沙棘提取物中含有着非常丰富的维生素e、维生素a以及sod、黄酮等活性成分,这些物质的存在能很好对抗自由基,因为自由基量少了,所以人体的衰老过程就会变得比较缓慢。沙棘果与沙棘叶中提取的总黄酮(TFH),是含有7种黄酮的一组化合物,可使心绞痛缓解,心功能及缺血性心电图好转。沙棘果的抗癌作用除去其通过免疫机制或其他途径所引起的作用外,还有直接地抑制癌细胞作用以及阻断致癌因素的作用。
沙棘果的有效成分超氧化物歧化酶(SOD)具有清除人体内自由基的作用,同时又能增强免疫系统功能,调节免疫活性细胞,是一种有效的免疫调节剂,这对于提高人体的抗病能力,延缓人体的衰老,会有极其显著的作用。沙棘果提取物中SOD含量是人参的6倍之多,它可以阻断因肌肤内物质过氧化产生的自由基,组织肌肤的过早老化,修复受损细胞组织,促进组织再生和上皮组织愈合,临床上和民间用于治疗烫伤、烧伤、刀伤和冻伤均取得极好的效果。
沙棘叶主要含有黄酮类、粗纤维、多糖类、氨基酸、维生素、微量元素、胡萝卜素、蛋白质、有机酸、叶绿素等营养及活性成分。沙棘叶黄铜99%为黄酮苷类,比沙棘果黄铜更易溶于水中,沙棘叶黄铜具有增加冠状动脉血流量,降低心肌耗氧量,抑制血小板聚集等多种药理作用,还有研究表明沙棘叶提取物具有与银杏黄酮苷类似的调节血脂的作用。沙棘叶水溶性多糖含2.3-4.5%,沙棘水溶性多糖能有效清除人体内氧自由基。沙棘叶含有多种微量元素,如Fe、Mg、Ca、Zn、Mo、K等,且含量较高,沙棘叶中镁、铁含量高于日常果蔬含量,胡萝卜素平均含量19.1mg/100g。
沙棘果提取物和沙棘叶提取物中的有效成分并不完全相同,通过将二者的提取物按照一定比例混合之后,可以弥补单独使用沙棘果提取物或沙棘叶提取物的不足,增强其效果。
水杨酸是植物柳树皮提取物,是一种天然的消炎药,在医药方面,水杨酸具有抗真菌、止痒和溶解角质作用。在鱼体表感染真菌时,水杨酸也可软化角质层,角质层脱落时,也将菌丝同时脱落,此外,水杨酸能够帮助其他抗真菌药物穿透,并抑制真菌生长。
虾青素是一种酮式类胡萝卜素,是一种断链抗氧化剂,具有极强的抗氧化能力,可以清除二氧化氮、硫化物、二硫化物等,也可降低脂质过氧化作用,有效的抑制自由基引起的脂质过氧化作用,同时,有抑制肿瘤发生,增强免疫力,清除体内自由基等多方面的生理作用,对紫外线引起的皮肤癌有很好的治疗效果,对糖尿病引起的眼病也有防治作用,在保健品、医药、化妆品、食品添加剂以及水产养殖等方面具有广阔的应用前景。虾青素强大的抗氧化活性是因为其能稳定膜的结构,降低膜通透性、限制过氧化物启动子进入细胞内,保护细胞内重要分子免受氧化损伤,同时虾青素可能具有潜在的成为促氧化剂诱导氧化应激的产生。另外,实验表明,虾青素可以增加抗氧化酶活性和蛋白质表达,不同剂量虾青素使动物细胞内过氧氢化酶和超氧化岐化酶的蛋白表达均有显著增加,其生物学活性也有明显提高,而这些物质在体内均起到较好的抗氧化作用。
虾青素纳米颗粒是利用纳米传输系统(NDS)开发的粒径小于30nm的颗粒,解决了虾青素的溶解性和稳定性的问题,同时还能更有效的渗透皮肤,将有效成分包裹在直径为纳米尺寸的胶囊中,以纳米胶囊作为载体,自动而匀速的缓释作用于皮肤组织,可以使功效成分较长时间维持在有效浓度内,起到稳定有效成分,大幅增加吸收效率和生物利用度。
进一步,所述盐、小苏打、沙棘提取物、水杨酸和虾青素纳米颗粒的重量份数比为(10-15):(10-15):(1-5):(0.5-1):(1-2)。
进一步,所述盐、小苏打、沙棘提取物、水杨酸和虾青素纳米颗粒的重量份数比为15:15:3:0.5:1。
进一步,所述沙棘提取物的颗粒直径在200-300nm。
进一步,所述沙棘提取物由包括如下步骤的方法制备得到:
1)称取新鲜沙棘果和沙棘叶,将新鲜沙棘果经真空冷冻干燥后粉碎备用,将新鲜沙棘叶粉碎并用纤维素酶预处理20-40min;
2)分别将预处理后的沙棘果粉末和沙棘叶碎加入料液比为1:10-20的低级醇溶液中常温浸泡1-2h,同时进行超声处理;
3)之后分别向浸泡有沙棘果粉末和沙棘叶碎的溶液中加入纯化水,使料液比达到1:30-40,常温下,超声处理6-12h;
4)将处理后的溶液浸提、减压浓缩后冻干,粉碎,得到沙棘果提取物和沙棘叶提取物备用;
5)将沙棘果提取物和沙棘叶提取物按照重量份数比2:1混合得到沙棘提取物。
进一步,所述沙棘果真空冷冻干燥包括预冻阶段、升华干燥阶段和解析干燥阶段;
预冻阶段:把清洗后新鲜、色泽自然、无病害的沙棘果实放入冻结盘中进行预冻,温度小于-35℃;
升华干燥阶段:对真空冷冻干燥机进行抽真空10-15min,之后控制加热搁板温度为38-45℃,物料温控制为-33--28℃,提供升华潜热,真空度在28-35pa,时间为20-40h;
解析干燥阶段:在升华干燥过程中除去了沙棘果中的自由水,为进一步除去其中的结合水,控制物料温度低于50℃;解析干燥后期,搁板温度与物料温差缩小,当板温度曲线和物料内部温度曲线趋于一致时,再持续干燥1.5-2.5h。
本发明还提供了一种如上述任一项所述的防治西藏高原鱼类水霉病制剂的制备方法,该方法包括如下步骤:
1)称取新鲜沙棘果和沙棘叶,将新鲜沙棘果经真空冷冻干燥后粉碎备用,将新鲜沙棘叶粉碎并用纤维素酶预处理20-40min;
2)分别将预处理后的沙棘果粉末和沙棘叶碎加入料液比为1:10-20的低级醇溶液中常温浸泡1-2h,同时进行200-300 W超声处理;
3)之后分别向浸泡有沙棘果粉末和沙棘叶碎的溶液中加入纯化水,使料液比达到1:30-40,常温下,200-300 W超声处理6-12h;
4)将处理后的溶液浸提、减压浓缩后冻干,粉碎,得到沙棘果提取物和沙棘叶提取物备用;
5)将沙棘果提取物和沙棘叶提取物按照重量份数比2:1混合得到沙棘提取物;
6)将配方量的盐、小苏打、沙棘提取物、水杨酸和虾青素纳米颗粒混匀得到制剂粉剂。
进一步,所述低级醇的质量浓度为90-95%。
进一步,所述低级醇为乙醇或丁二醇。
本发明还提供了上述任一项所述的防治西藏高原鱼类水霉病制剂的使用方法,将所述制剂制成粉剂后,直接撒入养鱼的鱼池中,每1L水中加入50-100g。
相对于现有技术,本发明所述的防治西藏高原鱼类水霉病制剂具有以下优势:
(1)从实验数据中可以发现,采用本发明的配方所制备的制剂对患病的拉萨裸裂尻鱼的治愈效果明显,可能是由于沙棘提取物含有较高的SOD,对受损的细胞起到促进组织再生和上皮组织愈合的效果,恢复病变细胞的活力。另外,沙棘提取物以及虾青素能够起到清除氧自由基的作用,增强机体清除氧自由基、对抗应激的能力,增强鱼体抵抗力,可以减少鱼体在应激或病害侵袭时的损伤。而且,沙棘提取物中还含有其他的有效成分,进一步提高了机体的免疫能力。再者,沙棘提取物经纳米级超微粉碎机粉碎至300nm纳米颗粒,虾青素采用的是虾青素纳米颗粒形式,纳米颗粒更有利于其有效成分穿透鱼的皮肤组织,大幅增加吸收效率和生物利用度,从而更有效的防治水霉病。水杨酸是一种天然消炎药,在鱼体表感染真菌时,水杨酸也可软化角质层,更有利于有效成分穿透皮肤组织,而且角质层脱落时,也将菌丝同时脱落,此外,水杨酸能够帮助其他抗真菌药物穿透,并抑制真菌生长。
(2)实验结果还发现同时添加沙棘果提取物和沙棘叶提取物的治疗效果比单独使用沙棘果提取物或沙棘叶提取物的效果好,说明沙棘果提取物和沙棘叶提取物之间存在互补关系,能够增强对水霉病的治疗效果;并且,当沙棘果提取物的含量大于沙棘叶提取物的含量时效果最好。
(3)通过对比组的数据可以发现,沙棘提取物、水杨酸和虾青素纳米颗粒之间存在促进作用,缺少其中任意一种成分,其防治效果都会打折扣;而且从对比四组可以发现,直接使用虾青素和使用虾青素纳米颗粒,其效果也是有显著差异的,使用虾青素纳米颗粒的效果更胜一筹,更有利于对水霉病的防治。
具体实施方式
需要说明的是,在不冲突的情况下,本发明中的实施例及实施例中的特征可以相互组合。
下面将结合实施例来详细说明本发明。
实施例1 沙棘提取物制备
1)沙棘果处理
预冻阶段:把10kg清洗后新鲜、色泽自然、无病害的沙棘果实放入冻结盘中进行预冻,温度-38℃;
升华干燥阶段:对真空冷冻干燥机进行抽真空15min,之后控制加热搁板温度为40℃,物料温控制为-30℃,提供升华潜热,真空度在30pa,时间为35h;
解析干燥阶段:在升华干燥过程中除去了沙棘果中的自由水,为进一步除去其中的结合水,控制物料温度为48±3℃;解析干燥后期,搁板温度与物料温差缩小,当板温度曲线和物料内部温度曲线趋于一致时,再持续干燥2h;
粉碎:将经真空冷冻干燥后的沙棘果粉碎成500目粉末备用;
2)沙棘叶处理
称取10kg新鲜沙棘叶粉碎并用1kg纤维素酶预处理25min;
3)分别将预处理后的沙棘果粉末和沙棘叶碎加入料液比为1:10的质量浓度为90%乙醇溶液中常温28℃浸泡1h,同时300w进行超声处理;
4)之后分别向浸泡有沙棘果粉末和沙棘叶碎的溶液中加入纯化水,使料液比达到1:40,常温28℃下,300w超声处理10h;
5)将处理后的溶液浸提、减压浓缩后冻干,之后用纳米级超微粉碎机粉碎至300nm,分别得到沙棘果提取物和沙棘叶提取物备用;
6)沙棘果提取物和沙棘叶提取物分别按照重量份数比1:0、1:1、2:1、1:2和0:1混合得到沙棘提取物A、B、C、D、E。
实施例2 制剂的制备
每1L水中加入69g本发明所述制剂,分别按照表1所示混合盐、小苏打、沙棘提取物、水杨酸和虾青素纳米颗粒得到制剂粉剂,制备制剂A、制剂B、制剂C、制剂D、制剂E。
表1 制剂成分表
实施例3 制剂对水霉病防治实验
(1)材料准备
从养殖场收购体型均匀、体重1kg左右、患有水霉病的拉萨裸裂尻鱼500条,随机平均分成10组,即制剂A组、制剂B组、制剂C组、制剂D组、制剂E组、对比一组、对比二组、对比三组、对比四组、空白对照组。
表2 对比组和对照组制剂成分表
(2)试验
将10组拉萨裸裂尻鱼分开饲养,于每天早6:00及晚7:00向养殖水中按照每1L水中加入69g制剂,饲养10天,并统计各组患病数和死亡率,结果如表3。
表3 结果对照表
从上表中可以发现,采用本发明的配方所制备的制剂对患病的拉萨裸裂尻鱼的治愈效果明显,可能是由于沙棘提取物含有较高的SOD,对受损的细胞起到促进组织再生和上皮组织愈合的效果,恢复病变细胞的活力。另外,沙棘提取物以及虾青素能够起到清除氧自由基的作用,增强机体清除氧自由基、对抗应激的能力,增强鱼体抵抗力,可以减少鱼体在应激或病害侵袭时的损伤。而且,沙棘提取物中还含有其他的有效成分,进一步提高了机体的免疫能力。再者,沙棘提取物经纳米级超微粉碎机粉碎至300nm纳米颗粒,虾青素采用的是虾青素纳米颗粒形式,纳米颗粒更有利于其有效成分穿透鱼的皮肤组织,大幅增加吸收效率和生物利用度,从而更有效的防治水霉病。水杨酸是一种天然消炎药,在鱼体表感染真菌时,水杨酸也可软化角质层,更有利于有效成分穿透皮肤组织,而且角质层脱落时,也将菌丝同时脱落,此外,水杨酸能够帮助其他抗真菌药物穿透,并抑制真菌生长。
通过制剂A-E组的数据可以发现,同时添加沙棘果提取物和沙棘叶提取物的治疗效果比单独使用沙棘果提取物或沙棘叶提取物的效果好,说明沙棘果提取物和沙棘叶提取物之间存在互补关系,能够增强对水霉病的治疗效果;并且,当沙棘果提取物的含量大于沙棘叶提取物的含量时效果最好。
通过对比一组-四组的数据可以发现,沙棘提取物、水杨酸和虾青素纳米颗粒之间存在促进作用,缺少其中任意一种成分,其防治效果都会打折扣;而且从对比四组可以发现,直接使用虾青素和使用虾青素纳米颗粒,其效果也是有显著差异的,使用虾青素纳米颗粒的效果更胜一筹,更有利于对水霉病的防治。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (7)
1.一种药物制剂用于制备防治拉萨裸裂尻鱼水霉病药物中的应用,其特征在于:所述药物由盐、小苏打、沙棘提取物、水杨酸和虾青素纳米颗粒组成,所述沙棘提取物为重量份数比为2:1的沙棘果提取物和沙棘叶提取物;所述盐、小苏打、沙棘提取物、水杨酸和虾青素纳米颗粒的重量份数比为(10-15):(10-15):(1-5):(0.5-1):(1-2);所述沙棘提取物的颗粒直径在200-300nm。
2.根据权利要求1所述的药物制剂用于制备防治拉萨裸裂尻鱼水霉病药物中的应用,其特征在于:所述盐、小苏打、沙棘提取物、水杨酸和虾青素纳米颗粒的重量份数比为15:15:3:0.5:1。
3.根据权利要求1所述的药物制剂用于制备防治拉萨裸裂尻鱼水霉病药物中的应用,其特征在于:所述沙棘提取物由包括如下步骤的方法制备得到:
1)称取新鲜沙棘果和沙棘叶,将新鲜沙棘果经真空冷冻干燥后粉碎备用,将新鲜沙棘叶粉碎并用纤维素酶预处理20-40min;
2)分别将预处理后的沙棘果粉末和沙棘叶碎加入料液比为1:10-20的低级醇溶液中常温浸泡1-2h,同时进行超声处理;
3)之后分别向浸泡有沙棘果粉末和沙棘叶碎的溶液中加入纯化水,使料液比达到1:30-40,常温下,超声处理6-12h;
4)将处理后的溶液浸提、减压浓缩后冻干,粉碎,得到沙棘果提取物和沙棘叶提取物备用;
5)将沙棘果提取物和沙棘叶提取物按照重量份数比2:1混合得到沙棘提取物。
4.根据权利要求3所述的药物制剂用于制备防治拉萨裸裂尻鱼水霉病药物中的应用,其特征在于:所述沙棘果真空冷冻干燥包括预冻阶段、升华干燥阶段和解析干燥阶段;
预冻阶段:把清洗后新鲜、色泽自然、无病害的沙棘果实放入冻结盘中进行预冻,温度小于-35℃;
升华干燥阶段:对真空冷冻干燥机进行抽真空10-15min,之后控制加热搁板温度为38-45℃,物料温控制为-33--28℃,提供升华潜热,真空度在28-35pa,时间为20-40h;
解析干燥阶段:在升华干燥过程中除去了沙棘果中的自由水,为进一步除去其中的结合水,控制物料温度低于50℃;解析干燥后期,搁板温度与物料温差缩小,当板温度曲线和物料内部温度曲线趋于一致时,再持续干燥1.5-2.5h。
5.根据权利要求1-4任一项所述的药物制剂用于制备防治拉萨裸裂尻鱼水霉病药物中的应用,其特征在于:所述药物由如下方法制备得到:
1)称取新鲜沙棘果和沙棘叶,将新鲜沙棘果经真空冷冻干燥后粉碎备用,将新鲜沙棘叶粉碎并用纤维素酶预处理20-40min;
2)分别将预处理后的沙棘果粉末和沙棘叶碎加入料液比为1:10-20的低级醇溶液中常温浸泡1-2h,同时进行200-300 W超声处理;
3)之后分别向浸泡有沙棘果粉末和沙棘叶碎的溶液中加入纯化水,使料液比达到1:30-40,常温下,200-300 W超声处理6-12h;
4)将处理后的溶液浸提、减压浓缩后冻干,粉碎,得到沙棘果提取物和沙棘叶提取物备用;
5)将沙棘果提取物和沙棘叶提取物按照重量份数比2:1混合得到沙棘提取物;
6)将配方量的盐、小苏打、沙棘提取物、水杨酸和虾青素纳米颗粒混匀得到制剂粉剂。
6.根据权利要求5所述的药物制剂用于制备防治拉萨裸裂尻鱼水霉病药物中的应用,其特征在于:所述低级醇的质量浓度为90-95%。
7.根据权利要求5所述的药物制剂用于制备防治拉萨裸裂尻鱼水霉病药物中的应用,其特征在于:所述低级醇为乙醇或丁二醇。
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