CN116098171A - 紫苏精油或其主要成分正戊基-2-呋喃酮在蚊子防治的应用 - Google Patents
紫苏精油或其主要成分正戊基-2-呋喃酮在蚊子防治的应用 Download PDFInfo
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Abstract
本发明公开了紫苏精油或正戊基‑2‑呋喃酮作为植物源性杀蚊剂在杀灭蚊子的应用。本发明还公开了紫苏精油或正戊基‑2‑呋喃酮在制备杀蚊剂的应用。紫苏精油可使一半幼虫死亡的浓度LC50为0.0045％，正戊基2‑呋喃酮的LC50为0.0025％。紫苏精油或正戊基‑2‑呋喃酮对淡色库蚊幼虫的杀灭作用较好，在极低的浓度下就可达到100％的致死效果，可以用于杀死水体中淡色库蚊幼虫，清除孳生地，达到蚊媒防制的目的。紫苏精油与正戊基2‑呋喃酮的LC50浓度对人类胚肾细胞293T无毒害作用。说明紫苏精油与正戊基2‑呋喃酮均对人体安全无毒害。
Description
技术领域
本发明属于生物技术领域，涉及紫苏精油或其主要成分正戊基-2-呋喃酮在防治蚊子的应用，具体涉及紫苏精油或其主要成分正戊基-2-呋喃酮作为植物源性杀幼剂在杀灭幼蚊的应用。
背景技术
蚊是一种重要的媒介生物，可以携带多种病原体，引起疟疾、黄热病、登革热等多种疾病。世界卫生组织最新数据显示2020年全球疟疾病例达2.41亿例，约有62.7万人死亡[1]。据推测2050年，全球一半以上人口将面临蚊媒疾病传播的风险[2]。一直以来，主要依靠化学杀虫剂来抑制成蚊数量以控制蚊媒疾病。但随着化学杀虫剂的大量使用，蚊对拟除虫菊酯和有机磷酸盐等常见杀虫剂迅速产生抗性[3]。同时，上述杀虫剂的使用也造成了环境污染，并对生态***造成破坏，使用多年后依然有可能污染土地和水资源[4]。环境中残留的杀虫剂同样会危害人类健康，据WHO估计，每年有5000人死于有机磷农药导致的中毒。针对蚊子幼虫的控制干预措施已被证明可降低疟疾的传播率，甚至根除携带疟原虫的蚊子[5]。不同于成蚊，幼虫不能改变栖息地以逃避干预，便于统一管理施用。且同时也可配合灭成蚊等不同策略有效控制蚊的数量，减少孳生地的发生[6]。因此，迫切需要开发一种新型的安全而有效的杀幼蚊剂。
从天然植物中提取的精油是一种非常有前景的杀幼蚊剂来源，因为它通常对环境安全，对非目标生物无毒，在田间和水中的降解残留物最少，对生态环境的破坏最小[7]。此外，与化学杀虫剂不同，由于植物精油中活性化合物的多样性，可以诱发多种作用机制，蚊子很难产生抗药性[8]。唇形科植物紫苏是一种历史悠久的传统药物和功能食品，一些研究也表明它对丝状棘球菌有较好的杀伤和滋生作用，是天然杀虫剂的潜在来源。目前未见紫苏精油对蚊的杀灭作用有相关报道。
参考文献：
1.World Health,O.,World malaria report 2020:20years of globalprogress and challenges.2020,Geneva:World Health Organization.
2.Franklinos,L.H.V.,et al.,The effect of global change on mosquito-borne disease.The Lancet.Infectious Diseases,2019.19(9):p.e302-e312.
3.Namias,A.,et al.,The need for practical insecticide-resistanceguidelines to effectively inform mosquito-borne disease controlprograms.ELife,2021.10.
4.Kaushal,J.,M.Khatri,and S.K.Arya,A treatise on Organophosphatepesticide pollution:Current strategies and advancements in theirenvironmental degradation and elimination.Ecotoxicology and EnvironmentalSafety,2021.207:p.111483.
5.Fillinger,U.,et al.,Integrated malaria vector control withmicrobial larvicides and insecticide-treated nets in western Kenya:acontrolled trial.Bulletin of the World Health Organization,2009.87(9):p.655-665.
6.Derua,Y.A.,et al.,Bacterial larvicides used for malaria vectorcontrol in sub-Saharan Africa:review of their effectiveness and operationalfeasibility.Parasites&Vectors,2019.12(1):p.426.
7.Silvério,M.R.S.,et al.,Plant Natural Products for the Control of:The Main Vector of Important Arboviruses.Molecules(Basel,Switzerland),2020.25(15).
8.Senthil-Nathan,S.,A Review of Resistance Mechanisms of SyntheticInsecticides and Botanicals,Phytochemicals,and Essential Oils as AlternativeLarvicidal Agents Against Mosquitoes.Frontiers In Physiology,2019.10:p.1591.
发明内容
发明人以蛇床子、苦参、紫苏和银杏等多种材料进行提取，对提取物进行初步杀蚊幼测定，发现以水蒸气蒸馏法提取的紫苏精油对蚊幼虫致死能力最高。发明人首次对紫苏精油进行GC-MS分析，发现其主要有效成分为正戊基2-呋喃酮，并考察紫苏精油、正戊基2-呋喃酮对淡色库蚊幼蚊的杀灭效果。根据实验室环境下的生物测定结果显示，其对淡色库蚊幼虫的杀灭作用较好，在极低的浓度下就可达到100％的致死效果。紫苏精油可使一半幼虫死亡的浓度LC50为0.0045％，正戊基2-呋喃酮的LC50为0.0025％。紫苏精油和正戊基2-呋喃酮的杀灭效果均呈现为时间和剂量依赖性，处理时间越长、浓度越高，则蚊幼死亡率越高；说明紫苏精油或其主要活性成分正戊基-2-呋喃酮作为植物源性杀幼蚊剂，可以用于杀死水体中淡色库蚊幼虫，清除孳生地，达到蚊媒防制的目的。
精油有长久的应用历史，长期数据显示并无对人的明显危害。发明人采用浓度高达10％的紫苏精油与正戊基2-呋喃酮涂抹至人体皮肤上做驱蚊性测定，均未出现任何反应；紫苏精油与正戊基2-呋喃酮的LC50浓度对人类胚肾细胞293T无毒害作用。说明紫苏精油与正戊基2-呋喃酮均对人体安全无毒害。紫苏作为一种历史悠久的传统药物和功能食品，区别于其他植物源性产品的珍贵稀少，紫苏来源性广泛、原材料廉价易得、对环境无污染，易于大规模生产提取，是一种前景非常好的杀幼剂来源。
本发明的目的是提供紫苏精油或其主要活性成分正戊基-2-呋喃酮作为植物源性杀蚊剂在杀灭蚊子的应用。
优选的，所述的应用为：将紫苏精油或正戊基2-呋喃酮加入蚊幼虫孳生水体中，紫苏精油在水体中的浓度为0.004％～0.0065％，正戊基2-呋喃酮在水体中的浓度为0.0015％～0.0045％。
更优选的，紫苏精油在水体中的浓度为0.0060％～0.0065％，正戊基2-呋喃酮在水体中的浓度为0.0040％～0.0045％。
本发明的另一个目的是提供紫苏精油或正戊基-2-呋喃酮在制备杀蚊剂的应用。
本发明的另一目的是提供一种杀蚊剂，所述的杀蚊剂以紫苏精油或正戊基-2-呋喃酮为主要有效成分。
所述的紫苏精油是以紫苏叶为原料，采用Clevenger法提取得到的；包括：紫苏叶风干、压碎；将压碎的紫苏叶和5％的氯化钠溶液按照料液比1:5g/mL混合，使用Clevenger装置进行水蒸气蒸馏，取上层油状，加入Na2SO4干燥，得到紫苏精油。
所述的紫苏精油中正戊基-2-呋喃酮的含量大于49％。
所述的蚊子为淡色库蚊，优选为淡色库蚊幼虫。
附图说明
图1为紫苏精油和正戊基-2-呋喃酮的24h杀幼测定结果。
图2为紫苏精油和正戊基-2-呋喃酮的48h杀幼测定结果。
图3为紫苏精油和正戊基-2-呋喃酮对人正常细胞的毒性考察结果。
具体实施方式
下面通过具体实施方式对本发明的技术方案作进一步说明。
实施例1
Clevenger法提取紫苏精油(EO)：将风干紫苏叶压碎；采用水配制质量分数5％的氯化钠溶液，将500mL氯化钠溶液装入1L烧瓶中；把100g压碎的紫苏叶装入含有氯化钠溶液的1L烧瓶中，使用Clevenger装置进行水蒸气蒸馏约2小时，吸取上层油状，加入少量Na2SO4干燥，置于封闭玻璃瓶中4℃保存。紫苏精油的得率为1.2％。
EO的GC-MS分析：采用GC-MS03(Agilent-Technologies,Palo Alto,CA,USA)对紫苏精油进行定量和定性分析，气相色谱条件：TG-5SILMS熔融石英毛细管柱(长度30m，内径0.25mm，膜厚0.25μm)，烘箱温度程序为50℃保温3分钟，以升温速率10℃/分钟升温至100℃，再以升温速率15℃/分钟升温至280℃，280℃保温4分钟，最后以升温速率30℃/分钟升至320℃，320℃保温8分钟()。通过与计算机数据库(NIST)的质谱以及公开发表的数据来确定化合物。
表1.紫苏精油中主要化学成分
根据世界卫生组织推荐的标准方法进行杀幼虫生物测定。
杀幼虫生物测定试验：将紫苏精油、正戊基-2-呋喃酮分别溶解在1mL无水乙醇中，然后用去氯水稀释，得到100mL不同浓度的测试溶液，紫苏精油的检测浓度为35,40,45,50,55,60μg/mL，正戊基-2-呋喃酮的检测浓度15,20,25,30,35,40μg/mL，对照溶液是由1mL乙醇和99mL去氯水配成的混合溶剂；用巴斯德吸管将25批三龄或四龄淡色库蚊幼虫转移到含有100mL测试溶液的杯子中。每个浓度测试三次。在生物测定期间，不向幼虫提供食物。在幼虫分别接触测试溶液24小时、48小时后，对死亡的幼虫进行计数，以确定紫苏精油、正戊基-2-呋喃酮对幼虫的毒性。
实验结果如图1和图2所示，显示：处理24小时，在浓度0.006％(60μg/mL)下紫苏精油即可使淡色库蚊幼虫全部死亡，在浓度0.004％(40μg/mL)下正戊基2-呋喃酮可致使幼虫全部死亡。经过SPSS软件分析可使一半幼虫死亡的LC50浓度，紫苏精油的LC50为0.0045％(45μg/mL)，正戊基2-呋喃酮的LC50为0.0025％(25μg/mL)。世界卫生组织目前并没有建立统一标准的确定某一物质的杀灭幼虫活性，一般默认化合物对幼虫致死LC50<100mg/L(即0.01％)就认定其是有效的，而LC50<50mg/L(即0.005％)认定其效果是显著的。而紫苏精油与其主要活性成分测定出的LC50均小于0.005％，证明其在杀灭库蚊幼虫作用上效果显著，在极低的浓度下即可达到较好的蚊媒防治效果。
结合48h的杀灭效果，表明，紫苏精油及其主要成分正戊基2-呋喃酮对淡色库蚊幼虫的致死呈现为时间和剂量依赖性，处理时间越长、浓度越高，则库蚊幼虫死亡率越高。
以上结果均表明紫苏精油、正戊基2-呋喃酮均是很有前景的杀幼剂来源。
对人胚肾细胞的细胞毒性测定试验：使用细胞计数试剂盒-8检测(CCK-8；Beierbo，南京，中国)进行细胞毒性检测。将293T细胞悬浮液加入96孔板(每孔100μL)，在37℃和5％的二氧化碳条件下培养24小时。用0.1％(v/v)二甲亚砜(DMSO；Sigma，美国)分别溶解紫苏精油和正戊基2-呋喃酮。每孔加入紫苏精油(使最终浓度分别为45,58mg/L)或正戊基2-呋喃酮(使最终浓度分别为25和35mg/L)，以等量DMSO为对照，进一步孵育1小时；然后，每孔加入10μL CCK-8溶液，孵育1小时后在450nm处测量吸光度比较各组别差异性。每个浓度使用10个重复孔。
结果如图3所示，显示在紫苏精油与正戊基2-呋喃酮LC50和LC90浓度下，各组之间的细胞活力没有差异，无毒性显示。
Claims (7)
1.紫苏精油或正戊基-2-呋喃酮作为植物源性杀蚊剂在杀灭蚊子的应用。
2.根据权利要求1所述的应用，其特征在于：所述的应用为：将紫苏精油或正戊基2-呋喃酮加入蚊幼虫孳生水体中，紫苏精油在水体中的浓度为0.004％～0.0065％，正戊基-2-呋喃酮在水体中的浓度为0.0015％～0.0045％。
3.根据权利要求2所述的应用，其特征在于：紫苏精油在水体中的浓度为0.0060％～0.0065％，正戊基-2-呋喃酮在水体中的浓度为0.0040％～0.0045％。
4.根据权利要求1所述的应用，其特征在于：所述的紫苏精油是以紫苏叶为原料，采用Clevenger法提取得到的；包括：紫苏叶风干、压碎；将压碎的紫苏叶和5％的氯化钠溶液按照料液比1:5g/mL混合，使用Clevenger装置进行水蒸气蒸馏，取上层油状，加入Na2SO4干燥，得到紫苏精油。
5.根据权利要求1所述的应用，其特征在于：所述的蚊子为淡色库蚊。
6.紫苏精油或正戊基-2-呋喃酮在制备杀蚊剂的应用。
7.一种杀蚊剂，其特征在于：所述的杀蚊剂以紫苏精油或正戊基-2-呋喃酮为主要有效成分。
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