CN117917216A - 氯黄菌素及其衍生物的应用 - Google Patents

氯黄菌素及其衍生物的应用 Download PDF

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CN117917216A
CN117917216A CN202310609665.1A CN202310609665A CN117917216A CN 117917216 A CN117917216 A CN 117917216A CN 202310609665 A CN202310609665 A CN 202310609665A CN 117917216 A CN117917216 A CN 117917216A
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inhibiting
chlorofluoromycin
growth
dechlorinated
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吕雪峰
黄雪年
张伟
张璇
郭勍
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Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
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Abstract

本发明提供了氯黄菌素及其衍生物的应用，具体而言，本发明提供了氯黄菌素或去氯氯黄菌素在抑制微生物生长中的用途，或者，在制备抑制微生物生长的试剂中的用途，或者，在制备治疗由微生物所导致的疾病的药物中的用途。

Description

氯黄菌素及其衍生物的应用
技术领域
本发明属于微生物基因资源和基因工程领域，具体地，涉及氯黄菌素及其衍生物的应用。
背景技术
真菌是一类与人们的生活密切相关的真核微生物，有些真菌可以产生抑制微生物生长的抗生素，如青霉素、头孢菌素等，可有些真菌能够引起食品腐败或者导致疾病的发生，如导致食品腐败和肺曲霉病的烟曲霉，引起皮肤黏膜、内脏以及中枢神经疾病的念珠菌等。此外还有部分是农业致病菌，如齐整小核菌(Sclerotium rolfsii Sacc.)，它能够侵染多种作物导致花生、水稻、黄瓜等的白绢病。尤其花生白绢病的逐年加重，已成为制约我国花生产业健康发展的主要障碍之一。由于齐整小核菌寄主范围广，菌核抗逆性强，很难通过轮作或生防等方式来控制。目前主要依赖化学防治，如井岗霉素、嘧菌酯、噻呋酰胺等。但是化学农药的长期使用会导致病原菌产生抗药性，因此亟需开发新型的抗病原真菌生长的抗生素。
在本专利中申请人在一株亮白曲霉MEFC1001(可市售获得，保藏于中国微生物菌种保藏管理委员会普通微生物中心的真菌，登记入册编号为：CGMCC 3.15294)中分离获得了氯黄菌素(又称为氯黄酮chlorflavonin，CAS：23363-64-6)和去氯氯黄菌素(dechlorochlorflavonin，CAS:51724-52-8)，通过该化合物的抗菌活性评价，显示其对部分植物病原真菌(齐整小核菌)和人类治病真菌(白色念珠菌、烟曲霉)具有非常强的抑制活性，具有开发成为农药杀菌剂、抗生素和食品防腐剂的潜力。
发明内容
本发明提供了氯黄菌素或其衍生物的新用途。
一方面，本发明提供了氯黄菌素或去氯氯黄菌素在抑制微生物生长中的用途，或者，在制备抑制微生物生长的试剂中的用途，或者，在制备治疗由微生物所导致的疾病的药物中的用途。
在一个实施方式中，所述抑制微生物生长的试剂为抗生素。
在优选的实施方式中，所述微生物为齐整小核菌(Sclerotium rolfsii Sacc.)。
在一个实施方式中，所述疾病为植物疾病，例如，由齐整小核菌侵染作物导致花生、水稻、黄瓜等的白绢病。
本发明中的试剂和药物可以制备成不同的剂型，包括为液体制剂、固体制剂、半固体制剂或气体制剂。
本发明的试剂和药物，除了含有氯黄菌素或去氯氯黄菌素作为活性成分之外，还可以含有其他的辅助试剂。
本发明中，氯黄菌素抑制齐整小核菌的最小抑菌浓度可以低至2μg/mL以下；去氯氯黄菌素抑制齐整小核菌的最小抑菌浓度可以低至4μg/mL以下。
另一方面，本发明提供了氯黄菌素在抑制植物生长中的用途，或者，在抑制植物种子萌发和根茎发芽中的用途，或者，在制备抑制植物生长的试剂中的用途，或者，在制备除草剂中的用途；在优选的实施方式中，所述植物为拟南芥。
在制备为抑制植物生长的试剂时，优选剂型为液体剂型。
本发明中，所述氯黄菌素的结构式如下所示：
所述去氯氯黄菌素的结构式如下所示：
附图说明
图1.化合物氯黄菌素(1)和去氯氯黄菌素(2)抑制拟南芥种子的萌发；a.拟南芥种子在MS平板培养基上的萌发，阴性对照二甲基亚砜(DMSO)，阳性对照草铵膦(GA)，供试化合物氯黄菌素(1)和去氯氯黄菌素(2)。b.不同浓度下氯黄菌素(1)和去氯氯黄菌素(2)对种子萌发的生长抑制曲线。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
下列实施例中未注明具体条件的实验方法，通常按照常规条件或丝状真菌标准操作的条件或按照制造厂商所建议的条件。除非另外说明，百分比和份数按重量计。下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
酶解液：称取0.4g纤维素酶(Sigma产品，产品目录号：C1184)、0.4g裂解酶(Sigma产品，产品目录号：L1412)、0.2g蜗牛酶(生工生物工程股份有限公司产品，上海，产品目录号：SB0870)溶解于50ml的0.6M MgSO4水溶液中，经由0.22μm的无菌过滤器过滤除菌。
本发明中质粒提取采用OMEGA公司Plasmid Mini Kit I试剂盒(D6942-01)，DNA片段回收是采用OMEGA公司Cycle-Pure Kit试剂盒(D6492-01)，凝胶回收是采用OMEGA公司Gel Extraction Kit试剂盒(D2500-01)。
PDBS平板：24g/L马铃薯土豆培养基PDB干粉(BD公司产品，产品目录号：7114771)，1.2M山梨醇，4g/L琼脂糖，余量为去离子水，121℃高压灭菌20分钟后48℃保温。
PDA平板：39g/L马铃薯土豆培养基PDA干粉(BD公司产品，产品目录号：633840)，余量为去离子水，121℃高压灭菌20分钟，待冷却至约60℃制备平板。
PDAS平板：39g/L马铃薯土豆培养基PDA干粉(BD公司产品，产品目录号：633840)，1.2M山梨醇，余量为去离子水，121℃高压灭菌20分钟，待冷却至约60℃制备平板。
SGCY培养基：2％蔗糖，1％葡萄糖，0.5％酪蛋白水解物，0.5％酵母浸出粉，1％MgCl2·6H2O，余量为去离子水，121℃高压灭菌20分钟，待冷却至约30℃接种培养。
PPM培养基：15％蔗糖，2.5％豆粕，0.5％蛋白胨，0.1％NaNO3，余量为去离子水，121℃高压灭菌20分钟，待冷却至约30℃接种培养。
SM培养基:10％葡萄糖，1％蔗糖，0.5％酵母膏，0.5％蛋白胨，0.1％Na2CO3，余量为去离子水，121℃高压灭菌20分钟，待冷却至约30℃接种培养。
实施例1、氯黄菌素和去氯氯黄菌素的分离与纯化
1.1将亮白曲霉MEFC1001菌株(可市售获得，保藏于中国微生物菌种保藏管理委员会普通微生物中心的真菌，登记入册编号为：CGMCC 3.15294)置于PDA平板上静止培养，用无菌水洗涤孢子，接种于SM培养基，28℃，220rpm培养种子液，接种于PPM培养基中，28℃，220rpm发酵14天，获得发酵液。
1.2采用相同体积的乙酸乙酯对发酵液萃取三次，浓缩获得粗提物。采用干法装柱对粗提物进行柱层析，填料为十八烷基硅烷键合硅胶填料，甲醇水梯度洗脱(甲醇体积10％～100％)，每个梯度洗脱10个柱体积。目标化合物1在80％甲醇/水组分中，化合物2在60％甲醇/水组分中。将80％甲醇/水组分和60％甲醇/水组分浓缩后甲醇溶解，0.22μm滤膜过滤后半制备液相进行纯化。纯化方法：流动相A(100％水+0.05％甲酸)，流动相B(100％乙腈+0.05％甲酸)，色谱柱Waters X-bridge C18(100mm×10mm,5μm)，流速2mL/min，检测波长345nm，根据化合物1和2的极性大小，调整流动相A和B的比例，在相应保留时间收集各化合物。根据HRESI和NMR鉴定，最终确定化合物1和2分别是氯黄菌素(chlorflavonin，CAS：23363-64-6)和去氯氯黄菌素(dechlorochlorflavonin，CAS:51724-52-8)。
化合物1的结构式如下所示：
所述化合物2的结构式如下所示：
实施例2、黄酮类化合物(氯黄菌素和去氯氯黄菌素)抑制病原真菌活性评价
2.1选取两株人类致病真菌白色念珠菌(Candida albicans)、烟曲霉(Aspergillus fumigatus)，以及4株植物病原真菌齐整小核菌(Sclerotium rolfsiiSacc.)、灰霉病菌(Botrytis cinerea)、黄瓜枯萎病菌(Fusarium oxysporumf.sp.cucumerinum,FOC)和苹果炭疽病菌(Colletotrichum gloeosporioides)作为供试菌株。将白色念珠菌接种于PDB培养基中，28℃220rpm培养12小时，然后用无菌PDB培养基稀释至5×105个/mL，获得菌悬液备用。其他供试菌株接种于PDA平板上，28℃培养5-7天，待菌丝或孢子长满平板。用无菌0.85％NaCl溶液(含0.25％Tween20)洗涤并刮下菌丝，加入50mL无菌PDB培养基，得到菌悬液母液，进一步以无菌PDB培养基稀释得到5×105个/mL浓度的菌悬液备用。
2.2分别取1mg待测样品(化合物1和2)和阳性对照(两性霉素B)，溶解于100μLDMSO中，配成浓度为10mg/mL的溶液。充分混匀后，吸取50μL样品溶液到另一只离心管中，接着加入50μL DMSO，得到浓度减半的样品溶液。按照此方法，得到15组浓度依次减半的样品溶液。无菌条件下，取95μl待测的菌悬液依次加入到96孔板中，取5μL稀释后待测样品依次加入含菌悬液的96孔板中，待测化合物的终浓度依次为500、250、125、62.5、31.25、15.63、7.81、3.91、1.95、0.98、0.49、0.24、0.12、0.06和0.03μg/mL。轻轻震荡混匀后，将96孔板密封至于28℃培养72小时。使用酶标仪在600nm波长下测定每孔的吸光值(或者在明亮处肉眼观察小孔内溶液是否浑浊)，能够在小孔内完全抑制指示菌生长的最低样品浓度即为该化合物的最小抑菌浓度(MIC)。上述检测平行测定三次，检测结果如下表所示，化合物1对C.albicans、A.fumigatus和S.rolfsii Sacc.均表现出非常强的抑制活性，化合物2对C.albicans和S.rolfsii Sacc.表现出强的抑制活性。尽管黄酮类化合物抑制C.albicans和A.fumigatus已有文献报道(J.Antibiot.2001,54,1031-1035.)，但在本专利中化合物1和2对S.rolfsii Sacc.的抑制活性为首次发现。因此化合物1和2具有开发成为农药杀菌剂、抗生素和食品防腐剂的潜力。
化合物1和化合物2对病原真菌的抑制活性
a两性霉素B；NI:无抑制活性
实施例3、氯黄菌素具有抑制拟南芥种子萌发的活性
采用实施例1的方法获得化合物1(氯黄菌素)和化合物2(去氯氯黄菌素)；分别将化合物1(氯黄菌素)和化合物2(去氯氯黄菌素)溶于DMSO中，滤膜过滤除菌。加入到MS(0.216％Murashige and Skoog基础培养基+0.8％蔗糖+0.8琼脂)培养基中，稀释成不同浓度梯度(0.001μg/mL、0.02μg/mL、0.1μg/mL、0.2μg/mL、0.4μg/mL、0.6μg/mL、0.8μg/mL、1μg/mL、2μg/mL、10μg/mL)的平板。将野生型拟南芥种子用75％乙醇-10％次氯酸钠溶液消毒清洗，放置在配置好的不同浓度梯度的培养基中，4℃放置48小时。然后转移到植物培养间，20℃培养条件下，光照16小时黑暗8小时，共培养120小时，统计种子萌发情况。抑制率＝未正常萌发种子数/萌发种子数×100％。草铵膦为阳性对照，DMSO为阴性对照。实验结果显示当化合物1(氯黄菌素)在浓度为1μg/mL时可以完全抑制拟南芥种子的萌发，明显低于阳性药草铵膦的浓度(图1)。此外化合物2(去氯氯黄菌素)也具有抑制活性，其在浓度为20μg/mL时能够完全抑制拟南芥种子的萌发(图1)。因此，氯黄菌素和去氯氯黄菌素具有除草活性，具有开发成为除草剂的潜力。
虽然本发明已以较佳的实施例公开如上，但其并非用以限定本发明，在不脱离本发明精神和范围内，本领域技术人员都可以在此基础上做出各种改动与变型，因此，本发明的保护范围应该以权利要求书所界定的为准。

Claims (7)

1.氯黄菌素或去氯氯黄菌素的用途，所述用途选择以下i-iii任意一组：
i、在抑制微生物生长中的用途；
ii、在制备抑制微生物生长的试剂中的用途；
iii、在制备治疗由微生物所导致的疾病的药物中的用途；
其特征在于，所述微生物为齐整小核菌(Sclerotium rolfsii Sacc.)。
2.根据权利要求1所述的用途，其特征在于，所述抑制微生物生长的试剂为抗生素。
3.根据权利要求1所述的用途，其特征在于，所述疾病为植物疾病。
4.氯黄菌素或去氯氯黄菌素的用途，所述用途选自以下a-c任意一组：
a、在抑制植物生长中的用途；
b、在制备抑制植物生长的试剂中的用途；
c、在制备除草剂中的用途。
5.根据权利要求4所述的用途，其特征在于，所述植物为拟南芥。
6.根据权利要求1-5任一所述的用途，其特征在于，
所述氯黄菌素的结构式如下所示：
7.根据权利要求1-5任一所述的用途，其特征在于，
所述去氯氯黄菌素的结构式如下所示：
CN202310609665.1A 2022-10-21 2023-05-26 氯黄菌素及其衍生物的应用 Pending CN117917216A (zh)

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