CN111303184A - 一种大环内酯类化合物及其制备方法与应用 - Google Patents
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Abstract
本发明提供了一种大环内酯类化合物及其制备方法和应用。本发明所述化合物的结构式如下式I所示，该化合物I是由冰城链霉菌突变菌株Streptomyces bingchenggensis BC‑120‑4经培养和发酵，得到含有化合物I的发酵液，然后从发酵液中分离获得。本发明还涉及含有化合物I的农药组合物，以及化合物I在制备用于防治农作物病虫害的农药组合物中的用途。
Description
技术领域
本发明属于农药技术领域，具体涉及一种具有杀虫活性的大环内酯类化合物，及其制备方法和应用。
背景技术
米尔贝霉素是由日本科学家首先从土壤放线菌Streptomyces hygroscopicussubsp.aureolacrimosus发酵培养液中分离得到的十六元大环内酯类化合物。己有数据表明，它是当今世界上最优良的杀螨剂之一。美国环保署认定其为低危险性杀虫剂，荷兰批准其为"GNO"(作物生产中的天然产物)。目前商品化最多的是米尔贝霉素A3/A4。该类药物在农牧业上对寄生虫类有较好的防治效果，且具有广谱杀虫活性，如螨类、蚜虫、黄褐天幕毛虫、肠道寄生虫及其他危害农业及畜牧业的寄生虫，其作用特点包括用量少，反应强烈且对人安全、无毒害，不污染环境，对天敌无害，也不易产生抗性。
因此，开发新型、具有更高生物活性的米尔贝霉素结构类似物具有极其重要的现实意义，其可为米尔贝霉素生物合成机制的研究提供新的视角，也为新型米尔贝霉素的药物开发提供丰富的实验材料。通过对Streptomyces bingchenggensis进行常温常压等离子诱变，我们获得了一株米尔贝霉素A3/A4高产菌株Streptomyces bingchenggensis BC-120-4。然而，与其它米尔贝霉素产生菌Streptomyces hygroscopicussubsp.aureolacrimosus和Streptomyces griseochromogenes一样，该菌的次级代谢产物是未知的，即次级代谢产物中是否含有新型较高活性的化合物是未知的，其次，即便次级代谢产物中含有符合上述条件的化合物，分离提取获得该化合物的难度也很大，本领域技术人员不可避免的分离出重复的、已知的或者活性较差的化合物，因此，如何从众多的、未知的次级代谢产物中分离提取获得新型的、具有较高生物活性的化合物是本领域技术人员亟需解决的难题。
发明内容
本申请发明人在研究冰城链霉菌突变菌株Streptomyces bingchenggensis BC-120-4(该菌株公布于Hai-Yan Wang,Ji Zhang,Yue-Jing Zhang,Bo Zhang,Chong-Xi Liu,Hai-Rong He,Xiang-Jing Wang,Wen-Sheng Xiang.Combined application of plasmamutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4 as maincomponents from Streptomyces bingchenggensis[J].Applied Microbiology andBiotechnology,2014,98:9703-9712)的次级代谢产物时，发现了一种新型的、具有显著杀虫活性的大环内酯类化合物。基于此，本发明提供了一种大环内酯类化合物，具有如下式I所示的化学结构：
本发明还提供了一种制备如前所述的化合物I的方法，包括以下步骤：
(1)将冰城链霉菌突变菌株Streptomyces bingchenggensis BC-120-4在培养基中进行发酵培养，得到含有化合物I的发酵液；
(2)将步骤(1)所得发酵液经预处理后得到粗提物，粗提物进行硅胶柱层析，得到含有化合物I的组分；将含有化合物I的组分进行反相制备柱层析，用洗脱液进行洗脱，得到化合物I。
在优选的实施方案中，步骤(1)所述培养基含有以下组分：蔗糖10-12g/L，酵母抽提物25-30g/L，麦芽抽提物25-30g/L，CaCO3 0.3-0.4g/L，pH 7.0-7.2，其余为水。
在优选的实施方案中，所述发酵的接种量为8％(v/v)，装液量为100ml/L，于250rpm，28℃下培养7-8天。
其中，在步骤(1)之前，还包括斜面培养和种子培养的步骤。
在优选的实施方案中，所述斜面培养的培养基含有：蔗糖10-12g/L，麦芽糖3-4g/L，酵母抽提粉3-4g/L，MgSO4·7H2O 0.5-0.6g/L，K2HPO4·3H2O 0.5-0.6g/L，NaCl 0.5-0.6g/L，KNO3 1.0-1.2g/L，琼脂粉20-25g/L，pH 7.0-7.2，其余为水，于121℃下灭菌20min。
在优选的实施方案中，所述斜面培养为：接菌后于28℃，培养8-9天。
在优选的实施方案中，所述种子培养为：向菌种斜面上加入无菌水制成孢子悬液，使其浓度为107-108c.f.u.ml-1，将孢子悬液接种于种子培养基中，装液量为25ml/250ml，于250rpm，28℃下培养45-50h。
其中，种子培养基含有：蔗糖5-6g/L，麦芽糊精5-6g/L，可溶性淀粉1.0-1.2g/L，酵母粉5-6g/L，牛肉膏1.0-1.2g/L，酪蛋白胨1.0-1.2g/L，pH 7.0-7.2，其余为水，于121℃下灭菌20min。
在优选的实施方案中，步骤(2)所述反相制备柱层析的填料为C18反相填料，使用的洗脱液为甲醇/乙腈/水＝42/42/16(V/V/V)，所述洗脱液流速为1-3ml/min，优选1.5mL/min。
在优选的实施方案中，步骤(1)所述预处理包括过滤步骤(1)所得发酵液中的菌丝体，用3-5倍菌丝体体积的有机溶剂浸提并浓缩至干得到粗提物，所述有机溶剂选自丙酮、甲醇、乙醇中的至少一种。
在优选的实施方案中，所述的硅胶层析所使用的硅胶粒径选自100-200目，采用石油醚:乙酸乙酯＝100:0-50:50(V/V)的混合溶剂进行梯度洗脱，流速为10-50ml/min。
本发明进一步提供了含有如前所述的化合物I的农药组合物，所述组合物还含有一种或者多种常规载体和/或稀释剂。所述农药组合物的剂型可以为水分散粒剂、乳油、水悬浮剂、油悬浮剂、微乳剂或片剂。
本发明进一步提供了如前所述的化合物I在制备用于防治农作物病虫害药物中的用途。所述农作物病虫害包括螨虫或线虫。
本发明所使用的的菌株为Streptomyces bingchenggensis BC-120-4，该菌株已在下述文献中披露，Hai-Yan Wang,Ji Zhang,Yue-Jing Zhang,Bo Zhang,Chong-Xi Liu,Hai-Rong He,Xiang-Jing Wang,Wen-Sheng Xiang.Combined application of plasmamutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4 as maincomponents from Streptomyces bingchenggensis[J].Applied Microbiology andBiotechnology,2014,98:9703-9712。
本发明得到了一个结构新颖的大环内酯类化合物，该化合物与商品化杀虫剂milbemycinA3/A4相比显示出更强的杀虫活性，可用于杀虫剂的开发，并可为米尔贝霉素生物合成机制的研究提供新的视角。
附图说明
图1是实施例2所得化合物I的1H-NMR谱图(CDCl3)。
图2是实施例2所得化合物I的13C NMR谱图(CDCl3)。
图3是实施例2所得化合物I的HSQC谱图(CDCl3)。
图4是实施例2所得化合物I的HMBC谱图(CDCl3)。
图5是实施例2所得化合物I的1H-1H COSY谱图(CDCl3)。
图6是实施例2所得化合物I的高分辨质谱图(HR-ESI-MS)。
图7是实施例2所得化合物I的红外吸收光谱图(IR)(KBr)。
图8是实施例2所得化合物I的紫外吸收光谱图(UV)(EtOH)。
具体实施方式
下面结合具体实施例对本发明作详细说明。必须指出，这些实施例是用于说明本发明，而不是对本发明的限制：
实施例1发酵生产含有化合物I的发酵液
(1)发酵菌种：发酵菌种是冰城链霉菌突变菌株，该菌株是公开于Hai-Yan Wang,Ji Zhang,Yue-Jing Zhang,Bo Zhang,Chong-Xi Liu,Hai-Rong He,Xiang-Jing Wang,Wen-Sheng Xiang.Combined application of plasma mutagenesis and geneengineering leads to 5-oxomilbemycins A3/A4 as main components fromStreptomyces bingchenggensis[J].Applied Microbiology and Biotechnology,2014,98:9703-9712的冰城链霉菌Streptomyces bingchenggensis BC-120-4。
(2)斜面培养：培养基配方(g/L)：蔗糖10，麦芽糖3，酵母抽提粉3，MgSO4·7H2O0.5，K2HPO4·3H2O 0.5，NaCl 0.5，KNO3 1.0，琼脂粉20，pH 7.0，用纯净水配制，于121℃下灭菌20min。接菌后置于28℃培养箱中，培养8d。
(3)种子培养：种子培养基配方(g/L)：蔗糖5，麦芽糊精5，可溶性淀粉1.0，酵母粉5，牛肉膏1.0，酪蛋白胨1.0，pH 7.0，用蒸馏水配制，于121℃下灭菌20min。向菌种斜面上加入10ml无菌水，用已灭菌的接种环刮下孢子制成孢子悬液，使其浓度为107c.f.u.ml-1。然后取2ml孢子悬液接种于种子培养摇瓶中，种子摇瓶装液量为25ml/250ml，置于250rpm旋转式摇床，28℃培养46h。
(4)发酵：发酵培养基配方(g/L)：蔗糖10，酵母抽提物25，麦芽抽提物25，CaCO30.3，pH 7.0，用蒸馏水配制，于121℃下灭菌20min。按8％(V/V)接种量，将种子液接入到1L发酵摇瓶中，发酵摇瓶的装液量为100ml/L。置于250rpm旋转式摇床，28℃发酵培养8d得含有化合物I的发酵液。
实施例2化合物I的提取分离
将实施例1制得的30L发酵液经200目筛网过滤得到菌丝体滤饼3L。滤饼用纯化水洗后再用9L甲醇浸提浸泡过夜，过滤得甲醇浸提液。所得浸提液在45℃减压浓缩去除甲醇相直至浓缩干，得到19g粗提物。
将所得的粗提物上硅胶柱(粒径100～200目)进行层析，用石油醚:乙酸乙酯＝100:0-50:50(V/V)进行梯度洗脱，流速30ml/min,用250ml锥形瓶依次收集洗脱液，每份250ml，经薄层层析(TLC)检测(石油醚:乙酸乙酯＝2:1)后合并相同组分，得到组分一(RF＝0.62-0.72)。
进一步将组分一进行反相制备柱层析，条件如下：
液相系统：Agilent 1260半制备高压液相色谱仪；
色谱柱:Agilent Zorbax SB C3 column(5μm,250×9.4mm)；
洗脱剂：甲醇/乙腈/水＝42/42/16(V/V/V)；流速：1.5mL/min；
检测波长：λ＝240nm；
收集保留时间为28.0min的峰得到该大环内酯类化合物I。
实施例3化合物I结构鉴定
性状：白色无定形粉末
溶解性：易溶于二氯甲烷、氯仿、乙腈、丙酮，不溶于水
分子式：C31H44O8
比旋度：
高分辨质谱(HRESI-MS):m/z 567.2903[M+Na]+(calcd for C31H44NaO8,567.2928)
紫外吸收光谱UV(EtOH)λmaxnm(logε):241(4.22)
红外吸收光谱IR(KBr)νmax:3337,2925,2869,1711,1453,1380,1269,1245,1143,1056,993cm-1
化合物I的1H和13C NMR(CDCl3)数据见表1。
表1化合物I的1H和13C NMR(CDCl3)数据
化合物I的结构式如下：
实施例4杀虫活性测定
(1)杀螨虫活性
分别将含1g/ml单个纯化合物I和1g/ml米尔贝霉素(色谱纯度为95％，米尔贝霉素A3/A4＝30：70)的甲醇溶液用含0.01％(w/v)表面活性剂烷基酚聚氧乙烯醚的水稀释10倍，制成100mg/ml的溶液，再依次稀释得到0.1,0.05,0.025,0.01和0.005mg/ml的溶液作为样品溶液。将对有机磷类杀虫药敏感的朱砂叶螨接种到豇豆的初生叶上。接种一天后，将豇豆的叶子浸泡在样品溶液中1-2s。将叶子在25℃放置3天后，用显微镜观察存活成虫的个数，计算死亡率(％)。
(2)杀线虫活性
分别将含1g/ml单个纯化合物I和1g/ml米尔贝霉素(色谱纯度为95％，米尔贝霉素A3/A4＝30：70)的甲醇溶液用水稀释10倍，制成100mg/ml的溶液，再依次稀释得到1,0.5,0.25,0.1和0.05mg/ml的溶液作为样品溶液。分别取不同浓度溶液10μl加入到90μl含活松材线虫水悬液中。混合液震荡后在25℃放置15h。在显微镜下计数不动线虫的数量和测试线虫的总数。计算测试线虫的死亡率(％)。
化合物I杀螨和杀线虫活性，见表2。
表2化合物I的杀螨和杀线虫活性
a米尔贝霉素A3和A4混合(30:70，体积比)。b三次平行实验的平均值。c与milbemycins A3/A4相比显著性差异，如果p>0.05说明无显著性差异
实验结果表明，化合物I的杀虫活性与商品化的米尔贝霉素A3/A4活性相比具有显著性差异(P<0.05)，且本发明提供的化合物I表现出了更强的杀虫活性，具有非常好的开发利用价值。
实施例5发酵生产含有化合物I的发酵液
(1)发酵菌种：发酵菌种是冰城链霉菌突变菌株，该菌株是公开于Hai-Yan Wang,Ji Zhang,Yue-Jing Zhang,Bo Zhang,Chong-Xi Liu,Hai-Rong He,Xiang-Jing Wang,Wen-Sheng Xiang.Combined application of plasma mutagenesis and geneengineering leads to 5-oxomilbemycins A3/A4 as main components fromStreptomyces bingchenggensis[J].Applied Microbiology and Biotechnology,2014,98:9703-9712的冰城链霉菌Streptomyces bingchenggensis BC-120-4。
(2)斜面培养：培养基组成(g/L)：蔗糖12，麦芽糖4，酵母抽提粉4，MgSO4·7H2O0.6，K2HPO4·3H2O 0.6，NaCl 0.6，KNO3 1.2，琼脂粉20，pH 7.2，用纯化水配制，于121℃下灭菌20min。接菌后置于28℃培养箱中，培养8d。
(3)种子培养：种子培养基组成(g/L)：蔗糖6，麦芽糊精6，可溶性淀粉1.2，酵母粉6，牛肉膏1.2，酪蛋白胨1.2，pH 7.2，用蒸馏水配制，于121℃下灭菌20min。向菌种斜面上加入10ml无菌水，用已灭菌的接种环刮下孢子制成孢子悬液，使其浓度为108c.f.u.ml-1。然后取2ml孢子悬液接种于种子培养摇瓶中，种子摇瓶装液量为25ml/250ml，置于250rpm旋转式摇床，28℃培养46h。
(4)发酵：发酵培养基组成(g/L)：蔗糖12，酵母抽提物30，麦芽抽提物30，CaCO30.4，pH 7.2，用蒸馏水配制，于121℃下灭菌20min。按8％(V/V)接种量，将种子液接入到1L发酵摇瓶中，发酵摇瓶的装液量为100ml/L。置于250rpm旋转式摇床，28℃发酵培养8d。
实施例6化合物I的提取分离
将实施例5制得的30L发酵液经200目筛网过滤得到菌丝体滤饼3L。滤饼用纯化水洗后再用15L丙酮浸提浸泡过夜，过滤得丙酮浸提液。所得浸提液在45℃减压浓缩去除丙酮直至浓缩干，得到28g粗提物。
将所得的粗提物上硅胶柱(粒径100～200目)进行层析，用石油醚:乙酸乙酯＝100:0-50:50(V/V)进行梯度洗脱，流速50ml/min,用250ml锥形瓶依次收集洗脱液，每份250ml，薄层层析(TLC)检测(石油醚:乙酸乙酯＝2:1)合并相同组分，得到组分一(RF＝0.62-0.72)。进一步将组分一进行反相制备柱层析，条件如下：
液相系统：Agilent 1260半制备高压液相色谱仪；
色谱柱:Agilent Zorbax SB C3 column(5μm,250×9.4mm)；
洗脱剂：甲醇/乙腈/水＝42/42/16(V/V/V)；流速：1.5mL/min；
检测波长：λ＝240nm；
收集保留时间为28.0min的峰得到该大环内酯类化合物I。
Claims (10)
1.一种大环内酯类化合物，其特征在于，所述化合物具有如下式I所示的化学结构：
2.一种制备权利要求1所述的化合物I的方法，其特征在于，所述方法包括以下步骤：
(1)将冰城链霉菌突变菌株Streptomyces bingchenggensis BC-120-4在培养基中进行发酵培养，得到含有化合物I的发酵液；
(2)将步骤(1)所得发酵液经预处理后得到粗提物，粗提物进行硅胶柱层析，得到含有化合物I的组分；将含有化合物I的组分进行反相制备柱层析，用洗脱液进行洗脱，得到化合物I。
3.根据权利要求2所述的方法，其特征在于，步骤(1)所述培养基含有以下组分：蔗糖10-12g/L，酵母抽提物25-30g/L，麦芽抽提物25-30g/L，CaCO3 0.3-0.4g/L，pH 7.0-7.2，其余为水。
4.根据权利要求2所述的方法，其特征在于，步骤(2)所述反相制备柱层析的填料为C18反相填料，使用的洗脱液为甲醇/乙腈/水＝42/42/16(V/V/V)，所述洗脱液流速为1-3ml/min，优选1.5mL/min。
5.根据权利要求2所述的方法，其特征在于，步骤(2)所述预处理包括过滤步骤(1)所得发酵液中的菌丝体，用3-5倍菌丝体体积的有机溶剂浸提并浓缩至干得到粗提物，所述有机溶剂选自丙酮、甲醇、乙醇中的至少一种。
6.根据权利要求2所述的方法，其特征在于，所述的硅胶层析所使用的硅胶粒径选自100-200目，采用石油醚:乙酸乙酯＝100:0-50:50(V/V)的混合溶剂进行梯度洗脱，流速为10-50ml/min。
7.根据权利要求2所述的方法，其特征在于，在步骤(1)之前，还包括斜面培养和种子培养的步骤；所述种子培养为向菌种斜面上加入无菌水制成孢子悬液，使其浓度为107-108c.f.u.ml-1，将孢子悬液接种于种子培养基中，装液量为25ml/250ml，于250rpm，28℃下培养45-50h，所述种子培养基含有：蔗糖5-6g/L，麦芽糊精5-6g/L，可溶性淀粉1.0-1.2g/L，酵母粉5-6g/L，牛肉膏1.0-1.2g/L，酪蛋白胨1.0-1.2g/L，pH 7.0-7.2，其余为水，于121℃下灭菌20min。
8.一种药物组合物，含有权利要求1所述的化合物I，以及一种或者多种常规载体和/或稀释剂。
9.权利要求1所述的化合物I在制备用于防治农作物病虫害药物中的用途。
10.根据权利要求9所述的用途，其特征在于，所述农作物病虫害包括螨虫或线虫。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101037442A (zh) * | 2007-01-17 | 2007-09-19 | 东北农业大学 | 用冰城链霉菌制备的新化合物及制备方法 |
| JP2007246462A (ja) * | 2006-03-17 | 2007-09-27 | Sankyo Agro Kk | ３，４−ジヒドロミルベマイシン誘導体及びその製造方法 |
-
2019
- 2019-12-11 CN CN201911263537.6A patent/CN111303184B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007246462A (ja) * | 2006-03-17 | 2007-09-27 | Sankyo Agro Kk | ３，４−ジヒドロミルベマイシン誘導体及びその製造方法 |
| CN101037442A (zh) * | 2007-01-17 | 2007-09-19 | 东北农业大学 | 用冰城链霉菌制备的新化合物及制备方法 |
Non-Patent Citations (1)
| Title |
|---|
| 林秀萍，等: "米尔贝霉素的产生菌、理化性质、生物学活性及应用研究进展", 《中国抗生素杂志》 * |
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|---|---|---|---|---|
| CN113248516A (zh) * | 2020-11-02 | 2021-08-13 | 台州职业技术学院 | 一种十七元大环内酯类化合物及其制备方法与应用 |
| CN113248516B (zh) * | 2020-11-02 | 2024-02-23 | 台州职业技术学院 | 一种十七元大环内酯类化合物及其制备方法与应用 |
| CN114634518A (zh) * | 2022-04-02 | 2022-06-17 | 台州职业技术学院 | 两种大环内酯类新化合物、药物组合物及其制备方法和应用 |
| CN114634518B (zh) * | 2022-04-02 | 2023-10-24 | 台州职业技术学院 | 两种大环内酯类新化合物、药物组合物及其制备方法和应用 |
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