CN106905342B - 玫瑰黄链霉菌两种活性代谢产物的结构及其制备 - Google Patents
玫瑰黄链霉菌两种活性代谢产物的结构及其制备 Download PDFInfo
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Abstract
本发明公开了玫瑰黄链霉菌（Streptomyces roseoflavus）的两种抗菌活性代谢产物—五烯大环內酯类代谢产物Roflamycoin和Menmyco‑A的结构及其制备方法。这两个具有抑菌活性的化合物是从玫瑰黄链霉菌菌株0116发酵液中分离纯化后得到的，有望用于高效农用抗生素的开发和应用。
Description
技术领域
本发明涉及玫瑰黄链霉菌(Streptomyces roseoflaver)产生的两种五烯大环內酯类抗菌活性代谢产物Roflamycoin和Menmyco-A的结构及其制备方法。
背景技术
菌种保藏编号为CGMCC N0.1471的玫瑰黄链霉菌(Streptomyces roseoflaver)菌株0116是本实验室采用自主创新的生防菌筛选方法，从马铃薯疮痂病自然衰退土壤中筛得的一株拮抗菌。该菌株及其发酵液对棉花黄萎病菌、瓜类白粉病菌、马铃薯疮痂病菌等多种重要的植物病原菌具有很强的抑制作用，有着良好的生防应用潜力。二十多年来，我们对该菌株及其发酵液抑菌谱、抑菌活性物质提取和鉴定、抗生物素合成基因簇、遗传转化体系建立、几丁质酶基因克隆、生防菌及其活性产物剂型等进行了大量研究，申请与此相关的国家发明专利6项，其中在玫瑰黄链霉菌负调控基因、发酵物水剂、发酵物菌渣方面，已分别获得了3项国家发明专利(玫瑰黄链霉菌的一个负调控基因及其制备方法和应用2012100837464；玫瑰黄链霉菌变种0116发酵液菌渣作为肥料的应用2005101174228；一种生物杀菌剂及其制备方法200510117421.3)。另外三项仍在申请中的专利有：“一种生物杀菌剂可湿性粉剂，专利申请号200910074685.3”、“一种玫瑰黄链霉菌生物杀菌剂的可湿性粉剂，专利申请号201110375945.8”和“一种生防菌营养型片剂，专利申请号201410156782.8”。以上专利申请针对的都是玫瑰黄链霉菌活菌或发酵物研发的不同制剂，并没有针对其活性代谢产物化合物结构的任何专利申请。
以往我们对玫瑰黄链霉菌菌株0116活性代谢产物结构的研究，长期没有突破性进展，直到2016年下半年，通过对玫瑰黄链霉菌代谢产物中的抗菌活性组分进行分离、提取和结构鉴定(数据未发表)，用甲醇萃取发酵液沉淀，旋转蒸发得到黄色粉末状物质，进行液相分析并纯化回收，并经上海秦珍生物科技有限公司进行结构鉴定，最终得到活性组分Roflamycoin(分子式C40H66O12，分子量738)和Menmyco-A(分子式C41H68O12，分子量752)，二者结构均为多烯大环內酯类物质。其中Roflamycoin经检索，为国外报道已知化合物，1971年由Schlegel等人首次报道，并在1981年确定其化学结构，该化合物在国内外还并没有作为医药、动物药以及其它商品登记，或者进入临床研究的任何信息，只在国外有两个作为抗菌医药和动物药的专利，WO9964040A1和WO2015164289A1。而Menmyco-A目前尚未见报道，我们将其定名为Menmyco-A。因此，本次专利申请涉及的玫瑰黄链霉菌代谢产物Roflamycoin和Menmyco-A的结构及其制备方法，具有创新性。
发明内容
本发明的目的之一在于：提供了两种五烯大环內酯抗生素Roflamycoin和Menmyco-A，结构式如式(1)、(2)所示：
(1)Roflamycoin
(2)Menmyco-A
本发明所述的Roflamycoin(曾用名flavomycoin)，为分子量738，化学式为C40H66O12，该化合物属36元五烯大环内酯类抗生素，最早从玫瑰黄链霉菌JA 5068菌丝体中分离得到，1971年初次报道，1981年确定其化学结构，目前尚未发现在中国地区的报道和应用。本发明所述的Menmyco-A，分子量为752，分子式为C41H68O12，与Roflamycoin相似，在结构上与Roflamycoin相似，除环上35号碳的取代基由异丙基变为异丁基外其他完全相同，目前该结构尚未发现报道。
本发明的另一个目的在于，提供天然来源的本发明的上述Roflamycoin和Menmyco-A的制备方法，包括如下步骤：
首先发酵培养保存编号为CGMCC No.1471的玫瑰黄链霉菌Streptomycesroseoflver菌株0116；然后从发酵液中提取、分离所述的两种抗生素Roflamycoin和Menmyco-A。
所述发酵培养步骤为：将保存编号为CGMCC No.1471的玫瑰黄链霉菌菌株0116接种于发酵培养基中培养成熟获得发酵液。
其中发酵培养基为葡萄糖2.4％，可溶性淀粉0.8％，花生饼粉1.5％，玉米浆0.8％，氯化钠0.4％，碳酸钙0.3％，磷酸二氢钾0.02％。
其中发酵培养条件为30℃，200rpm，培养5天。
所述提取、分离步骤包括：将发酵液4500rpm离心20min后，沉淀用约3倍体积甲醇浸泡并超声处理30min，滤纸过滤，得黄色甲醇溶液，在旋转蒸发仪中50℃将甲醇减压蒸发至干，再加入适量乙酸乙酯，超声振荡，使其充分混合，可见有黄色粉末状物质逐渐析出，静置2h后，抽滤，得到黄色粉末状物质，即为含有两种化合物Roflamycoin和Menmyco A的抗生素粗品。采用HPLC制备色谱对两个化合物进行制备，制备条件为色谱柱：Waters XPrep C18 5μm OBDTM 19×150mm，流动相乙腈：水＝45:55，流速10mL/min，检测器波长363nm。采用连续进样方式，在两个峰的相应保留时间内，接取检测器出口的流动相。在避光条件下，旋转蒸发仪中50℃，75r/min将乙腈最大程度的旋转蒸发掉，再在冷冻干燥机中-40℃，将含有水和少量乙腈的样品装在样品瓶内冷冻干燥，得到两个化合物纯品。
上述发酵培养和提取分离步骤为本发明物质Roflamycoin和Menmyco-A的一般制备方法，为本领域技术人员所熟知的常规手段，不应理解为对本发明的限制，在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
本发明说述的抗生素Roflamycoin和Menmyco-A是来源于分离自美国华盛顿州马铃薯疮痂病自然衰退土壤的玫瑰黄链霉菌Streptomyces roseoflaver菌株0116。所述产生菌已于2005年9月30日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市海淀区中关村北一条13号，中国科学院微生物研究所，邮编100080)，分类命名为玫瑰黄链霉菌Streptomyces roseoflaver，保藏编号为：CGMCCNo.1471。因此，本发明提供了一种用于生产Roflamycoin和Menmyco-A的玫瑰黄链霉菌Streptomycesroseoflaver菌株0116。
Roflamycoin和Menmyco-A的结构鉴定：根据HR-ESI-MS、13C-NMR、1H-NMR、1H-1HCOSY、HMBC、HSQC、NOESY的分析(见附图)，确定了Roflamycoin和Menmyco A的化学结构，二者在CD3OD中的NMR数据如表1和表2所示。
表1 Roflamycoin在CD3OD的1H-NMR(600MHz)、13C-NMR(150MHz)谱峰的结构归属
表2 Menmyco-A在CD3OD中的1H-NMR(600MHz)、13C-NMR(150MHz)谱峰的结构归属
附图说明
图1玫瑰黄链霉菌两种活性代谢产物Roflamycoin和Menmyco-A的结构。
图2化合物Roflamycoin的高分辨质谱(HR-ESI-MS)。
图3化合物Roflamycoin的1H NMR图。
图4化合物Roflamycoin的13C NMR(DEPT 135)图。
图5化合物Roflamycoin的1H-1H COSY图。
图6化合物Roflamycoin的HMBC图。
图7化合物Roflamycoin的HSQC图。
图8化合物Roflamycoin的NOESY图。
图9化合物Menmyco-A的高分辨质谱(HR-ESI-MS)。
图10化合物Menmyco-A的1H NMR图。
图11化合物Menmyco-A的13C NMR(DEPT 135)图。
图12化合物Menmyco-A的1H-1H COSY图。
图13化合物Menmyco-A的HMBC图。
图14化合物Menmyco-A的HSQC图。
图15化合物Menmyco-A的NOESY图。
Claims (5)
1.一种Menmyco-A的制备方法，其特征在于，所述Menmyco-A的结构如下所示：
所述制备方法包括如下步骤：首先发酵培养菌种保藏编号为CGMCC No.1471的玫瑰黄链霉菌菌株0116；然后从发酵液中提取、分离所述Menmyco-A。
2.如权利要求1所述的制备方法，其特征在于，发酵培养方法为：将菌种保藏编号为CGMCC No.1471的玫瑰黄链霉菌菌株0116接种于发酵培养基中培养成熟获得发酵液。
3.如权利要求1或2所述的制备方法，其特征在于，发酵培养基成分为葡萄糖2.4％，可溶性淀粉0.8％，花生饼粉1.5％，玉米浆0.8％，氯化钠0.4％，碳酸钙0.3％，磷酸二氢钾0.02％。
4.如权利要求1或2所述的制备方法，其特征在于，发酵培养条件为30℃，200rpm，培养5天。
5.如权利要求1所述制备方法，其特征在于，所述提取分离步骤包括：将发酵液4500rpm离心20mins后，沉淀用约3倍体积甲醇浸泡并超声处理30mins，滤纸过滤，得黄色甲醇溶液，在旋转蒸发仪中50℃将甲醇减压蒸发至干，再加入适量乙酸乙酯，超声振荡，使其充分混合，可见有黄色粉末状物质逐渐析出，静置2h后，抽滤，得到黄色粉末状物质，即为含有权利要求1中所述Menmyco-A的粗品；采用HPLC制备色谱对所述Menmyco-A进行制备，制备条件为色谱柱：WatersPrep C18 5μm OBDTM 19×150mm，流动相乙腈：水＝45:55，流速10mL/min，检测器波长363nm；采用连续进样方式，在所述Menmyco-A色谱峰的相应保留时间内，接取检测器出口的流动相；在避光条件下，旋转蒸发仪中50℃，75r/min将乙腈最大程度的旋转蒸发掉，再在冷冻干燥机中-40℃，将含有水和少量乙腈的样品装在样品瓶内冷冻干燥，得到所述Menmyco-A纯品。
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