【作者】 姚璐；

【导师】 汪开毓；

【作者基本信息】 四川农业大学 ， 基础兽医学， 2009， 硕士

【摘要】 鱼类指环虫病是一种鱼类养殖中最常见寄生虫病害之一,流行于春末夏初。此病流行广、传播快、危害大,且其发病率和危害性正在逐年增长。国内外对于此病的防治主要是移植人药、兽药和部分农药等化学药物,但长期使用,会造成环境污染、药物残留、耐药性等一系列问题。因此,需寻找新的对环境无污染、易降解的天然植物杀虫药,以及从天然植物中分离杀虫活性成分,进而进行仿生合成,更符合人类对健康的要求。同时,也有利于植物资源的合理利用和开发。1.本研究以此为出发点开展了杀虫活性植物筛选、活性部位筛选、杀虫活性成分跟踪分离、鉴定。在20种植物中,对指环虫的最高杀灭率依次为:独活为100%:除虫菊为90%;远志、了哥王、苦树皮为80%;走马芹、半边莲、吐根、委陵菜为50%;南天竺、延胡索、藜芦、断肠草、白屈菜、血水草为20%～30%;在1000 mg.L^-1范围内,八角枫、山麻柳、千里光、水黄连、瓜蒌没有明显杀虫活性。最佳杀虫浓度从低到高依次为:了哥王为30 mg.L^-1;独活为120 mg.L^-1;白屈菜为150 mg.L^-1;走马芹为200mg.L^-1;除虫菊为240 mg.L^-1:远志为350 mg.L^-1;藜芦为400 mg.L^-1;苦树皮为500 mg.L^-1;延胡索为600 mg.L^-1:南天竺为700 mg.L^-1;血水草、断肠草、半边莲、吐根为800 mg.L^-1。在测试浓度范围内,能够导致试验金鱼在48 h死亡的如表2-2所示,其中山麻柳;瓜蒌、断肠草、半边莲、吐根、委陵菜在1000 mg.L^-1时,试验金鱼仍然未见有死亡现象。2.独活杀灭指环虫活性部位的研究:分别采用4种极性不同的溶剂对独活进行提取,并测定不同溶剂提取物的杀指环虫活性。结果显示:乙醇提取物的最佳杀虫浓度为120mg.L^-1（按生药计）,杀虫率为100%;丙酮提取物最佳杀虫浓度为70 mg.L^-1（按生药计）,但最高杀虫率为72.0%;乙酸乙酯提取物的最佳杀虫浓度为300 mg.L^-1（按生药计）,最高杀虫率只有64.8%;而石油醚提取物的最佳杀虫浓度为100 mg.L^-1（按生药计）,最高杀虫率仅为49.1%。3.独活中杀灭指环虫活性成分的跟踪分离与结构鉴定:根据2可知乙醇提取物对指环虫具有杀灭效果。采用硅胶柱层析法,以石油醚-乙酸乙酯（40:1、30:1、20:1、10:1、5:1、2:1、1:1、0:1）,乙酸乙酯-甲醇（100:1、50:1、20:1、10:1、5:1、3:1、2:1、1:1、0:1和水洗柱）作为洗脱系统,进行梯度洗脱,TLC检测,得到16个流分组A～P,经药效测定F流分和K流分组具有杀虫活性,且F流分为结晶体,TLC检测为单点。以氯仿-甲醇（50:1,25:1,10:1,9:1,8:1,7:1,5:1,3:1,2:1,1:1,甲醇和水洗柱）作为洗脱系统,对K流分组进行硅胶柱层析分离,得到11个流分组K₁～K₁₁,经药效测定K₁流分组具有杀虫活性。以石油醚-乙酸乙酯（1:0、50:1、20:1、10:1、5:1、2:1、1:1、0:1、甲醇）作为洗脱系统,对K₁流分进行硅胶柱层析分离,得到6个流分组K_1-1～K_1-6,其中经药效测定K_1-1和K_1-2具有杀虫活性,且K_1-1为油性成分。以石油醚—氯仿（6:4、6:5、6:6、6:7、氯仿）作为洗脱系统,对K_1-2进行梯度洗脱,得到4个流分组K_1-2-1～K_1-2-4,其中K_1-2-3具有杀虫活性,且为结晶物质,TLC检测为单点。F和K_1-2-3的活性测定结果显示,F 48h的100%杀指环虫浓度为1.6 mg.L^-1;K_1-2-348h在浓度为5 mg.L^-1时,对指环虫的杀灭率为74.4%;K_1-148h在浓度为20 mg.L^-1时,对指环虫的杀灭率为51.9%。通过对活性化合物进行熔点测定,并结合质谱（EI-MS）、红外光谱（IR）、质子核磁共振谱（1H-NMR）、核磁共振碳谱(¹³C-NMR)、高效液相（HPLC）、气质联用（GC-MS）等对其结构进行鉴定。F鉴定为蛇床子素(C₁₅H₁₆O₄),K_1-2-3鉴定为东莨菪内酯(C₁₀H₈O₄),K_1-1鉴定为油性成分。更多还原

【Abstract】 Dactylogyriasis is one of the serious parasitosis in aquaculture.It can occur in most of province of our nation,and its harm is becoming more and more strongly.At present, medication is the most effective way in fishery,but study and production of fish medicines mainly relay on chemotherapeutants applied in other fields such as human medicines,animal medicines and a part of pesticides.A lot of chemotherapeutants,chronically used,have caused environmental pollution,drug residue,drug fastness for our national aquatic productions. Therefore,it is necessary to find a new Botanical Insecticide which is non-pollution to environment and easy to degradation.It is a fast and economical way to optimize exploitage to plant resources to find out new drugs against aquatic bacteria and parasite from plants.In this experiment,screening of active plants,screening of active part of parasiticidally active plants,isolation and tracing of active compounds,analysis for spectroscopy,elucidation for chemical structure were conducted in this text.The results are obtained as follows:1.Screening of parasiticidally active plants:According to the pharmacodynamic test,the parasiticidal activity of extractions of 20 plants on D.intermedius was determined.From the results,the maximum killing rate of extractions of variety plants on D.intermedius in 48 hours was:Radix Angelicae Pubescentis attained100%;Pyrethrum cinerariifolium attained 90%;Radix Polygalae,Indian Stringbush Root and Celastrus angulatus attained 80%;Radix Archangeliae Decurrentis,Herba Lobeliae Chinensis,Cephaelis ipecacuanha and Herba Potentillae Chinensis attained 50%;Nandina domestica,Corydalis yanhus,Veratrumnigrum L, Gelsemium elegans,Herba Chelidonii and Eomecon chionantha Hance attained 20%-30%;the other tested samples had not shown obvious killing activities in experimental concentration of 1000mg.L^-1.The optimization killing concentration:Indian Stringbush Root was 30 mg.L^-1;Radix Angelicae Pubescentis was 120 mg.L^-1;Herba Chelidonii was 150 mg.L^-1;Radix Archangeliae Decurrentis was 200 mg.L^-1;Pyrethrum cinerariifolium was 240 mg.L^-1;Radix Polygalae was 350 mg.L^-1;Veratrumnigrum L was 400 mg.L^-1;Celastrus angulatus was 500 mg.L^-1;Corydalis yanhus was 600 mg.L^-1;Nandina domestica was 700 mg.L^-1;Eomecon chionantha Hance,Gelsemium elegans,Herba Lobeliae Chinensis and Cephaelis ipecacuanha were 800 mg.L^-1.In tested concentration,the palnts which can lead Carassius auratus to death were listed in table2-2.Among the total,Platycarya strobilacea,Fructrs Trichosanthis,Gelsemium elegans,Herba Lobeliae Chinensis,Cephaelis ipecacuanha,Herba Potentillae Chinensis can not lead Carassius auratus to death in experimental concentration of 1000mg.L^-1.2.Study of parasiticidally active part of Radix Angelicae Pubescentis:Radix Angelicae Pubescentis was respectively distilled by ethanol,acetone,petroleum ether and ethyl acetate, then activity of each extraction was measured.The results shew that when parasiticidal concentration of ethanol extraction was 100%,its optimal concentration of killing Dactylogyrus was 120 mg.L^-1（according to raw-herb count）;optimal concentration of acetone was 70 mg.L^-1（according to raw-herb count）,the killing rate was 72.0%;optimal concentration of petroleum ether was 300 mg.L^-1（according to raw-herb count）,the killing rate was 64.8%;optimal concentration of ethyl acetate was 100mg.L^-1（according to raw-herb count）,the killing rate was only 49.1%.3.Isolation,activity tracing and identification of active components from Radix Angelicae Pubescentis:through measurement of activity to different extractions,the extractions of ethanol was demonstrated to be the most effective part against Dactylogyrus. The ethanol extraction was separated into 16 parts（A-P） by silica gel column chromatography with gradually increasing polarity of petroleum ether:ethyl acetate（40:1,30:1,20:1, 10:1,5:1,2:1,1:1,0:1） and ethyl acetate:methanol（100:1,50:1,20:1,10:1,5:1,3:1,2:1,1:1,0:1 and water）.According to activity test,the part F and K were demonstrated to be the most effective part and F was pure substance and simple point.The part K was isolated by silica gel column chromatography with gradually increasing polarity of chloroform:methanol（50:1,25:1, 10:1,9:1,8:1,7:1,5:1,3:1,2:1,1:1,methanol and water）,11 parts were isolated from Part K. According to activity test,only part K₁ was effective part.The part K₁ was further isolated by silica gel column chromatography with gradually increasing polarityof petroleum ether:ethyl acetate（1:0,50:1,20:1,10:1,5:1,2:1,1:1,0:1,methanol）,6 parts were isolated from Part K₁. According to activity test,the part K_1-1and K_1-2 was effective part,and part K_1-1 was oily ingredient.The part K_1-2 was further isolated by silica gel column chromatography with gradually increasing polarityof petroleum ether:chloroform（6:4,6:5,6:6,6:7,chloroform）,4 parts were isolated from Part K_1-2.According to activity test,the part K_1-2-3 was effective part, and it was pure substance and simple point.From the results of the activity test of F and K_1-2-3, the maximum killing rate of extractions of variety plants on Dactylogyrus in 48 hours was:the concentration of F was 1.6 mg.L^-1;the killing rate of K_1-2-3 was 74.4%in the concentration of 5 mg.L^-1;the killing rate of K_1-1 was 51.9%in the concentration of 20 mg.L^-1. Based on the data of IR,EI-MS,¹H-NMR,¹³C-NMR,HPLC and GC-MS,F was identified as osthol,K_1-2-3was identified as scopletin isopimpinellin.,K_1-1 was identified as oily ingredient.更多还原