CN115053920A - 水黄皮提取物在制备对桔小实蝇具有杀虫活性效果的药物中的应用 - Google Patents
水黄皮提取物在制备对桔小实蝇具有杀虫活性效果的药物中的应用 Download PDFInfo
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Abstract
本发明公开了水黄皮提取物在制备对桔小实蝇具有杀虫活性效果的药物中的应用。本发明水黄皮提取物对桔小实蝇的卵孵化具有抑制作用，对其1龄幼虫有胃毒性和拒食作用，对成虫具有触杀、胃毒和产卵驱避活性。本发明所用原料水黄皮来源广、产量大，毒性低，克服了已有农药的不足之处。
Description
技术领域
本发明属于植物保护技术领域，具体涉及水黄皮提取物在制备对桔小实蝇具有杀虫活性效果药物中的应用。
背景技术
桔小实蝇(Bactrocera dorsalis)又名东方果实蝇，俗称“果蛆”。食性杂、寄主广范，繁殖能力强、世代重叠严重，传播快、防治困难。通常危害芒果、柑橘、苹果、杨桃等，是水果上一种毁灭性害虫。其雌虫将卵产于果实中，孵化的幼虫蛀食为害，造成果实表面凹凸不平，果汁外溢，严重时造成大量落果甚至绝收(M.S.Hossain et al.,2020)。目前桔小实蝇的防治大多采用化学防治、物理防治和诱捕防治，其中化学防治易产生抗药性(金梦娇等,2021；李建瑛等,2020)、物理防治作用范围有限且人力成本高(玄志友,2021；蒋小龙等,2002.)、诱捕防治操作复杂且效果有限(梁帆等,2014；赵菊鹏2017)。
植物源杀虫剂，如植物精油类香茅油、柠檬草油、百里香油、山鸡椒油、月桂油，都对桔小实蝇成虫有较好的毒杀作用，最高校正死亡率分别为89％；对桔小实蝇产卵亦有趋避作用，驱避率最高可达80.77％(郭峰等,2020)；中药提取物如紫茎泽兰、臭椿、菖蒲、青蒿、臭牡丹、樟树等对桔小实蝇成虫有较强的毒杀作用，浓度越高产卵趋避率、毒杀作用越强(郭峰等,2021)。
这些方法防治桔小实蝇绿色、低毒但仅针对桔小实蝇成虫，且价格昂贵，不适合大面积应用推广。故寻找高效、绿色、价格低廉的防治方法迫在眉睫。
发明内容
本发明的目的在于提供水黄皮提取物在制备对桔小实蝇具有杀虫活性效果的药物中的应用。
本发明的上述目的可以通过以下技术方案来实现：水黄皮提取物在制备对桔小实蝇具有杀虫活性效果的药物中的应用。
优选的，所述杀虫活性效果是指水黄皮提取物对桔小实蝇的卵孵化具有抑制作用。
优选的，所述杀虫活性效果是指水黄皮提取物对桔小实蝇的1龄幼虫具有胃毒性。
优选的，所述杀虫活性效果是指水黄皮提取物对桔小实蝇成虫具有触杀、胃毒和产卵驱避作用。
优选的，所述水黄皮提取物通过以下方法制备获得：取水黄皮荚果粉经乙酸乙酯浸泡和超声辅助提取，将滤液合并，减压浓缩至浸膏，干燥获得水黄皮提取物。
本发明水黄皮(Pongamia pinnata(L.)Merr.)属豆科水黄皮属的半红树植物，广泛分布澳大利亚、印度、马来西亚及我国南部的广东、广西、海南、台湾等省的沿海滩涂地，在民间，水黄皮的根、茎、叶、果等全株可以入药，其分布面积广，荚果产量大，且具有抗风、耐旱、耐盐、等特性。
作为本发明的一种优选的实施方式，所述水黄皮提取物采用以下方法制备：取水黄皮荚果粉500g，加入3～5倍的乙酸乙酯(v/w，g/mL)浸泡30min～60min，超声辅助提取30min，过滤，滤渣反复提取直至提取液不变色，过滤，合并滤液，减压浓缩至浸膏，真空冷冻至干燥，移至干燥器中保存。
优选的，所述水黄皮提取物使用时，将水黄皮提取物采用体积百分含量为95％的乙醇稀释，配制成浓度为0.01～0.08g/mL。
更佳的，当水黄皮提取物用于桔小实蝇的卵孵化时，所述水黄皮提取物的浓度为0.02g/mL。
更佳的，当水黄皮提取物用于桔小实蝇的1龄幼虫的胃毒性时，所述水黄皮提取物的浓度为0.04g/mL。
更佳的，当水黄皮提取物用于桔小实蝇成虫的触杀、胃毒性和产卵趋避时，所述水黄皮提取物的浓度为0.06～0.08g/mL。
优选的，所述应用包括对危害芒果、柑橘、苹果和杨桃的桔小实蝇具有杀虫活性效果的应用。
与现有技术相比，本发明具有如下优点：
(1)本发明所用原料水黄皮来源广、产量大，毒性低，克服了已有农药的不足之处；
(2)本发明水黄皮提取物对桔小实蝇的卵孵化有抑制作用，对其1龄幼虫具有胃毒性，对桔小实蝇成虫具有触杀、胃毒和产卵驱避活性。
附图说明
图1为实施例2中水黄皮提取物浓度对桔小实蝇卵孵化抑制活性的影响；
图2为实施例3中水黄皮提取物浓度对1龄桔小实蝇幼虫胃毒性的影响；
图3为实施例4中水黄皮提取物24h内对桔小实蝇成虫的胃毒活性的影响；
图4为实施例4中水黄皮提取物48h内对桔小实蝇成虫的胃毒活性的影响；
图5为实施例5中水黄皮提取物对桔小实蝇成虫触杀活性的影响；u6
图6为实施例6中水黄皮提取物对桔小实蝇成虫产卵驱避活性的影响；
图7为实施例7中水黄皮素标准品的HPLC图；
图8为实施例1中制备的水黄皮乙酸乙酯提取物HPLC图。
具体实施方式
为了便于理解本发明，下文将结合较佳的实施例对本发明做更全面、细致地描述，但本发明的保护范围并不限于以下具体实施例。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到或可通过现有方法制备得到。
实施例1水黄皮提取物的制备
称取水黄皮荚果粉500g，加入1500mL的乙酸乙酯浸泡30min，超声辅助提取30min，过滤，滤渣反复提取直至提取液不变色，过滤，合并滤液，减压浓缩至浸膏，真空冷冻干燥24h，移至干燥器中保存。
称取8g干燥浸膏，溶于100mL的95％(体积百分含量，下同)乙醇，配置成浓度为8％(g/mL)的提取物，取出部分分别稀释为1％、1.5％、2％、4％、6％(g/mL)质量体积百分含量的提取物，备用。
实施例2水黄皮提取物对桔小实蝇卵孵化抑制活性测定
将同一天产的100粒桔小实蝇的卵，浸入1％水黄皮提取物中10s，取出，放入装有饲料的培养皿中。对照组将同样数量桔小实蝇的卵，浸入95％乙醇(对照)中10s，放入装有饲料的培养皿中。5天后检查并记录孵化卵粒数，根据公式计算卵孵化抑制率和校正孵化抑制率。以此方法分别测试1.5％、2％、4％、6％、8％提取物，各个处理和对照均重复4次。
孵化抑制率(％)＝未孵化卵粒数/供试总卵粒数×100；
校正孵化抑制率(％)＝(处理组卵孵化抑制率-对照组卵孵化抑制率)/(1-对照组卵孵化抑制率)×100。
水黄皮提取物对桔小实蝇卵孵化的抑制活性的影响如图1所示，不同浓度水黄皮提取物对桔小实蝇的卵孵化有抑制作用，抑制效果随着浓度的提高呈现先上升后下降的趋势，当浓度为2％时的卵孵化校正抑制率最高，达到58.6％，显著高于浓度为1％、1.5％、8％时卵孵化抑制率，与浓度为4％、6％时卵的校正抑制率无显著性差异。故2％的水黄皮提取物对桔小实蝇卵孵化的抑制活性最强。
实施例3水黄皮提取物对桔小实蝇1龄幼虫的胃毒性测定
将苹果切成1-2mm厚，2cm×2cm的薄片，浸入2％水黄皮提取物中10s，取出，晾干，放入干净的培养皿中，接入1龄幼虫30头。对照组将同样规格的苹果片浸入95％乙醇中10s后，晾干，放入干净培养皿中，接入1龄幼虫30头。24h后更换同样处理的苹果片，记录存活幼虫数,根据公式计算死亡率和校正死亡率。以此方法分别测试4％、6％、8％提取物，各个处理和对照均重复4次。
死亡率(％)＝试虫死亡数/试虫总数×100；
校正死亡率(％)＝(处理组死亡率-对照组死亡率)/(1-对照组死亡率)×100。
结果如图2所示，24h和48h幼虫的校正死亡率随着提取液浓度的增大呈现先升高后下降的趋势，4％的水黄皮提取物对桔小实蝇1龄幼虫24h的胃毒活性最强，校正死亡率为54.1％，但和6％、8％的提取物对1龄幼虫24h的胃毒活性无显著差异，均显著高于浓度为2％的提取物的胃毒活性；4％的水黄皮提取物48h胃毒活性最强，显著高于其它浓度的胃毒活性，其校正死亡率高达95.9％。
实施例4水黄皮提取物对桔小实蝇成虫的胃毒活性
用95％乙醇为溶剂，分别配制2％、4％、6％、8％浓度的药液。取苹果片(2cm×2cm)，在药液中浸渍2s后取出，置于洁净的培养皿上，放入养虫笼中。对照组取2cm×2cm的苹果片，在95％乙醇浸渍2s后取出，置于洁净的培养皿上，放入养虫笼中。并放适量水。将桔小实蝇成虫30头(雌雄各15头)，饥饿处理24h后分别放入处理中和对照组的养虫笼中，并放入适量水。观察24h、48h以后桔小实蝇成虫死亡情况并记录。根据公式计算死亡率和校正死亡率。
死亡率(％)＝试虫死亡率/试虫总数×100
校正死亡率(％)＝(处理组死亡率-对照组死亡率)/(1-对照组死亡率)×100
水黄皮提取物对桔小实蝇成虫的胃毒活性如图3和图4所示，6％与8％浓度的水黄皮提取物的校正死亡率24h达80％以上，48h达95％以上，显著高于其它浓度的校正死亡率(P<0.05)，且两者之间没有显著性差异(P>0.05)。可见水黄皮提取物对桔小实蝇成虫有显著的胃毒活性。
实施例5水黄皮提取物对桔小实蝇成虫的触杀活性
采用药膜法，即将不同浓度提取物制成药膜。将30头(雌雄各半)羽化15～20d后完成交配的桔小实蝇成虫放入晾干的药膜瓶中，瓶口用纱布封住，接触2h后，将各处理的桔小实蝇成虫转移至养虫笼中，24h或48h后，记录死亡率。
死亡率(％)＝试虫死亡率/试虫总数×100
校正死亡率(％)＝(处理组死亡率-对照组死亡率)/(1-对照组死亡率)×100
由图5可知，桔小实蝇成虫的校正死亡率随着水黄皮提取物浓度的提高而增大，6％和8％浓度的提取物触杀效果最好，于2％和4％浓度的提取物有显著差异。
实施例6水黄皮提取物对桔小实蝇成虫的产卵驱避活性测定
采用果实喷雾法处理芒果，将水黄皮提取物处理芒果和空白对照芒果(溶剂乙醇处理)分别放置在养虫笼中对角线位置，笼中放入羽化15d已交配的桔小实蝇性成熟雌雄成虫各20头，人工饲料补充营养，24h后取出芒果，统计着卵量。
产卵驱避率(％)＝(对照组产卵量-处理组产卵量)/(对照组产卵量+处理组产卵量)×100。
水黄皮提取物对桔小实蝇的产卵驱避活性如图6所示，其产卵驱避活性随着水黄皮提取物浓度的升高而增加，8％的产卵趋避率显著高于其它浓度的产卵去趋避率(P<0.05)，高达99.62％。
实施例7水黄皮中水黄皮素的HPLC分析方法
精密称取水黄皮素对照品1.36mg于10mL量瓶中，甲醇定容，即得每1mL溶液含0.136mg水黄皮素对照品。
精密称取实施例1中制备的干燥的水黄皮素提取物3.12mg于10mL量瓶中，甲醇定容，即得每1mL溶液含0.312mg水黄皮提取物。
色谱条件：色谱柱Unitary－C18ODS2(4·6mm×250mm，5μm)；流动相：水-乙腈(梯度洗脱)：0-10min，60％-70％的乙腈，10-15min，70％-95％乙腈。流速:1mL·min-1；检测波长:258nm；柱温:30℃；进样量:10μL。
水黄皮素标准品HPLC图如图7所示。本发明实施例1中制备的水黄皮乙酸乙酯提取物HPLC图如图8所示。
图7-图8中的结果表明，本发明中水黄皮乙酸乙酯提取物的成分包括水黄皮素，除了水黄皮素之外，还含有其它成分比如黄酮类、生物碱类、二氢黄酮、三萜及氨基酸等，这些成分以及一些其它成分的组合对于桔小实蝇的卵孵化具有抑制作用，对桔小实蝇的1龄幼虫具有胃毒性，对桔小实蝇成虫具有触杀、胃毒和产卵驱避作用。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (7)
1.水黄皮提取物在制备对桔小实蝇具有杀虫活性效果的药物中的应用。
2.根据权利要求1所述的应用，其特征是：所述杀虫活性效果是指水黄皮提取物对桔小实蝇的卵孵化具有抑制作用。
3.根据权利要求1所述的应用，其特征是：所述杀虫活性效果是指水黄皮提取物对桔小实蝇的1龄幼虫具有胃毒性。
4.根据权利要求1所述的应用，其特征是：所述杀虫活性效果是指水黄皮提取物对桔小实蝇成虫具有触杀、胃毒和产卵驱避作用。
5.根据权利要求1-4任一项所述的应用，其特征是：所述水黄皮提取物通过以下方法制备获得：取水黄皮荚果粉经乙酸乙酯浸泡和超声辅助提取，将滤液合并，减压浓缩至浸膏，干燥获得水黄皮提取物。
6.根据权利要求5所述的应用，其特征是：所述水黄皮提取物使用时，将水黄皮提取物采用体积百分含量为95％的乙醇稀释，配制成浓度为0.01～0.08g/mL。
7.根据权利要求1所述的应用，其特征是：所述应用包括对危害芒果、柑橘、苹果和杨桃的桔小实蝇具有杀虫活性效果的应用。
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