CN106810601B - 一种Destruxin类缩酚酸肽衍生物及其制备方法和应用 - Google Patents
一种Destruxin类缩酚酸肽衍生物及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及微生物来源杀虫剂,具体的说是一种Destruxin类缩酚酸肽衍生物及其制备方法和应用。所述Destruxin类缩酚酸肽衍生物如式(一)或式(二)所示。本发明所涉及的Destruxin类缩酚酸肽衍生物由菌株BeauveriafelinaAS‑70经发酵培养、提取分离制备获得,其化学结构经核磁共振和质谱等技术鉴定,具有较好杀虫活性。
Description
技术领域
本发明涉及微生物来源杀虫剂,具体的说是一种Destruxin类缩酚酸肽衍生物及其制备方法和应用。
背景技术
随着传统化学合成农药大量施用的弊端日益凸显,公众对食品安全及环境保护的关注愈加密切。一方面,植物病虫害对化学合成农药、杀虫剂等的抗药性越来越明显,另一方面,化学合成农药的大量施用也带来了环境污染、人畜中毒与三致(致癌、致畸、致突变)等突出问题。而与传统的化学合成农药相比,微生物源天然产物农药具有对人畜和非靶标生物安全性高、环境兼容性好、不易产生抗性等优点;而且微生物易于规模化发酵培养,便于产业化生产。所以微生物源农药的开发与应用对人类健康、环境保护和农业的可持续发展具有重要意义,近年来受到广泛重视。
发明内容
本发明的目的是提供一种Destruxin类缩酚酸肽衍生物及其制备方法和应用。
为实现上述目的,本发明所采用的技术方案为:
一种Destruxin类缩酚酸肽衍生物,Destruxin类缩酚酸肽衍生物如式(一)或式(二)所示;
一种Destruxin类缩酚酸肽衍生物的制备方法,
1)将菌株Beauveria feline AS-70(保藏于中国普通微生物菌种保藏管理中心,保藏日期:2012年9月29日,保藏号:CGMCC No.6643)接种于真菌固体培养基中静置发酵,经有机溶剂提取,获得提取物,待用;
2)将上述提取物进行硅胶柱层析,依次经石油醚–乙酸乙酯及氯仿–甲醇进行梯度洗脱,收集洗脱液,洗脱液再由薄层层析检测;
3)收集上述步骤2)中氯仿–甲醇体积比50~20:1梯度的洗脱组分,将收集的洗脱组分依次进行反相硅胶柱层析、Sephadex LH-20凝胶柱层析与制备高效液相色谱法分离纯化,收集保留时间tR值为12.1-18.6min的组分,即得式(一)或式(二)所示Destruxin类缩酚酸肽衍生物;
所述步骤3)收集保留时间tR值为13.5min的组分,即得式(一)所示Destruxin类缩酚酸肽衍生物;收集保留时间tR值为17.4min的组分,即得式(二)所示Destruxin类缩酚酸肽衍生物。
所述步骤1)将菌株Beauveria felineAS-70接种于真菌固体培养基中发酵30-40天,经乙酸乙酯、丙酮、氯仿、甲醇、乙醇、水中的一种或几种作为有机溶剂进行提取,获得发酵产物。
所述步骤1)中真菌固体培养基为大米培养基。
所述步骤2)中石油醚–乙酸乙酯洗脱梯度为50:1至1:1,氯仿–甲醇洗脱梯度为50:1至1:1。
所述步骤3)中反向硅胶柱层析洗脱液为体积比2:1的甲醇–水;凝胶柱层析洗脱液为丙酮;制备高效液相色谱条件为体积比1:1的乙腈–水,其中,制备高效液相色谱的流速为15mL/min,检测波长为220nm。
一种Destruxin类缩酚酸肽衍生物的应用,所述式(一)或式(二)所示Destruxin类缩酚酸肽衍生物作为制备用于杀虫活性先导化合物的应用。
本发明所具有的优点:
1)本发明所涉及的Destruxin类缩酚酸肽衍生物具有较好杀虫活性,式(一)与式(二)所示化合物的卤虫致死活性:100μg/mL时,卤虫致死率分别为54.2%与63.5%。
2)本发明所涉及的Destruxin类缩酚酸肽衍生物可以作为具有杀虫作用的先导化合物的制备。
3)本发明所涉及的Destruxin类缩酚酸肽衍生物可以利用微生物进行规模发酵,具有生产工艺简单,周期短、产品成本低等特点。
具体实施方式
为阐明对本发明特征的理解,下面结合一些非限定性的实施实例对本发明做进一步阐述。
实施例1:Destruxin类缩酚酸肽衍生物如式(一)与式(二)所示(结构中的阿拉伯数字及希腊字母为碳原子的标位)。
实施例2:Destruxin类缩酚酸肽衍生物的发酵生产及分离纯化:
1)发酵培养
菌种培养:按照微生物的常规培养方法,挑取少量保存于琼脂-麦芽膏培养基的菌株Beauveria feline AS-70,接种于PDA平板表面,28℃培养3天,作为规模发酵培养的菌种,待用。
切取上述PDA平板表面的菌种适量,接种至已灭菌的、盛有大米培养基的锥形瓶中,室温静置培养30天。加入乙酸乙酯灭菌,待用。
所述大米培养基为大米100g/瓶,蛋白胨0.6g/瓶,天然海水100mL/瓶。
2)化合物的分离纯化
将上述大米培养基用乙酸乙酯超声提取3次,合并乙酸乙酯提取液,减压蒸馏得浸膏。将其进行硅胶VLC(vacuum liquid chromatography)快速柱层析,按照洗脱液极性递增顺序,以体积比50:1至1:1的石油醚–乙酸乙酯(流速为150mL/min),体积比50:1至1:1的氯仿–甲醇(流速为150mL/min)进行梯度洗脱。收集洗脱液,并经薄层层析检测,检测时以茴香醛-浓硫酸作为显色剂,根据Rf值以及显色情况来合并相同或类似部分。收集氯仿~甲醇体积比50~20:1梯度的洗脱组分,将收集的组分进行反相硅胶柱层析,以体积比1:9至1:0的甲醇–水(流速为5mL/min)进行梯度洗脱,收集体积比为2:1的甲醇–水洗脱组分。该组分经凝胶柱层析,以丙酮为洗脱溶液(流速为1mL/min),收集洗脱液最后经制备高效液相色谱分离纯化(色谱条件为体积比1:1的乙腈–水,流速为15mL/min,检测波长为220nm),收集保留时间tR值为13.5min的组分,即得式(一)所示Destruxin类缩酚酸肽衍生物,收集保留时间tR值为17.4min的组分,即得式(二)所示Destruxin类缩酚酸肽衍生物。
式(一)所示Destruxin类缩酚酸肽衍生物,无色晶体;HRESIMS m/z580.3703[M+H]+,提示分子式为C29H49N5O7。氢谱中存在两组质子信号峰(1a与1b),比例约为1:1,表明该缩酚酸肽在溶液中存在两种构象,其1H-和13C-NMR数据见表1。
表1.式(一)所示Destruxin类缩酚酸肽的1H(500MHz)和13C NMR(125MHz)谱图数据(NMR测试所用溶剂:氘代丙酮)
式(二)所示Destruxin类缩酚酸肽衍生物,无色晶体;HRESIMS m/z594.3855[M+H]+,提示分子式为C30H51N5O7。氢谱中存在两组质子信号峰(2a与2b),比例约为3:2,表明该缩酚酸肽在溶液中也存在两种构象,其1H-和13C-NMR数据见表2。
表2.式(二)所示Destruxin类缩酚酸肽的1H(500MHz)和13C NMR(125MHz)谱图数据(NMR测试所用溶剂:氘代丙酮)
实施例3:杀虫活性试验
传统的农用杀虫活性物质的筛选方法主要依据一种或几种标准有害生物(例如粘虫、果蝇、蚜虫、玉米螟与小菜蛾等)作靶标,通过靶标生物对化合物在生长发育、形态特征、生理生化等方面的反应作为评价指标,来判断待测化合物的杀虫活性。虽然传统的筛选方法与田间条件下获得的结果接近,但是进行大规模、有目的、有步骤的筛选实验,一方面需要大量的目标昆虫作试材,另一方面试虫的培养对温度、湿度等环境因素要求非常严格,因而费时、费力、费钱,时间性和经济性不强。
应用卤虫作为杀虫活性测定的模型生物具有来源广泛、操作简单、所需化合物的量少等优势,可以显著提高筛选效率,对快速寻找杀虫活性先导化合物及后续新农药的研发具有重要意义。目前,国内外已经有应用卤虫作为模型生物快速筛选杀虫活性化合物的相关报道。王再强等[农药,2011,50(4):261–263]以卤虫作为模型生物评价了14种常见杀虫剂的杀虫活性,结果表明用卤虫筛选具有杀虫活性的化合物,方法简便,且对多种不同作用机制的杀虫剂敏感;胡志钰等[海洋通报,2000,19(4):36–41]以卤虫作为指示生物,快速筛选海洋放线菌中具有杀虫活性的化合物;Blizzard T.A.等[J.Antibiot,1989,42(8):1304–1307]报道了以卤虫为模型生物快速筛选杀虫剂阿维菌素类似物的实验结果。
1)卤虫卵的孵化
取卤虫卵100mg置于500mL烧杯中,加入人工海水400mL,用充气泵缓缓充气,室温孵化24h,除去卵壳及未孵化的卵,卤虫继续培养24h,备用。
2)样品溶液的制备
待测化合物以DMSO溶解,配制为4mg/mL溶液,并依此稀释至2,1与0.5mg/mL溶液,备用。
3)试验方法
按照Solis改良法,取96孔细胞培养板,每孔加195μL含10-15个卤虫的人工海水液,制成测试培养板。空白对照组和各浓度样品组各设三个平行孔,空白对照组加5μL人工海水,样品组加5μL所需浓度的样品液。室温培养24小时后,在双目解剖镜下检测计数卤虫死亡个体数目。
卤虫致死活性用校正死亡率表示,计算公式如下:
校正死亡率=(对照组存活率-处理组存活率)/对照组存活率×100%,并计算半数致死率LD50值。
试验结果为式(一)与式(二)所示化合物的卤虫致死活性:在浓度为100μg/mL时,卤虫致死率分别为54.2%与63.5%,具有较好杀虫活性。
上述实验结果证明本发明所涉及的化合物具有较好杀虫活性,它们可用于杀虫活性先导化合物的制备。
Claims (4)
2.一种权利要求1所述的Destruxin类缩酚酸肽衍生物的制备方法,其特征在于:
1)将菌株Beauveria feline AS-70接种于真菌固体培养基中静置发酵,经有机溶剂提取,获得提取物,待用;
2)将上述提取物进行硅胶柱层析,依次经石油醚–乙酸乙酯、氯仿–甲醇进行梯度洗脱,收集洗脱液,洗脱液再由薄层层析检测;
3)收集上述步骤2)中氯仿–甲醇体积比50~20:1梯度的洗脱组分,将收集的洗脱组分依次进行反相硅胶柱层析、Sephadex LH-20凝胶柱层析与制备高效液相色谱法分离纯化,收集保留时间tR值为12.1-18.6min的组分,即得式(一)或式(二)所示Destruxin类缩酚酸肽衍生物;
所述菌株Beauveria feline AS-70保藏于中国普通微生物菌种保藏管理中心,保藏日期:2012年9月29日,保藏号:CGMCC No.6643。
3.按权利要求2所述的Destruxin类缩酚酸肽衍生物的制备方法,其特征在于:所述步骤3)收集保留时间tR值为13.5min的组分,即得式(一)所示Destruxin类缩酚酸肽衍生物;收集保留时间tR值为17.4min的组分,即得式(二)所示Destruxin类缩酚酸肽衍生物。
4.一种权利要求1所述的Destruxin类缩酚酸肽衍生物的应用,其特征在于:所述式(一)或式(二)所示Destruxin类缩酚酸肽衍生物作为用于制备杀虫活性先导化合物的应用。
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