CN110484588A - 一种利用植物内生真菌发酵制备夫西地酸的方法 - Google Patents
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Abstract
本发明公开了一种利用植物内生真菌发酵制备夫西地酸的方法,先在菌种培养基中对十大功劳内生真菌进行菌种培养;再在发酵培养基中进行发酵培养;将发酵物用有机溶剂提取,合并提取液后,减压蒸馏浓缩得到发酵液浸膏,进行柱色谱分离得夫西地酸粗品;粗品经色谱纯化,得到夫西地酸纯品。本发明的有益效果是制备方法简便,可利用该菌株大规模生产夫西地酸。
Description
技术领域
本发明属于化学技术领域,涉及一种利用植物内生真菌发酵制备夫西地酸的方法。
背景技术
夫西地酸是一种重要的临床抗菌药物,临床上一般使用夫西地酸钠盐(注射剂或软膏),其对组织和体液具有极强的穿透力,静脉给药时血浆半衰期长,患者耐受性好。耐药性方面:夫西地酸与临床使用的其它抗菌药物之间无交叉耐药性,具有很好的安全性。夫西地酸常可以与利福平、碳霉素、氨基糖苷类、喹诺酮类、四环素类等药物联合使用,其协同用药效果显著。副作用方面:病人一般耐受性良好,主要副作用有胃肠道不适、出现恶心、消化不良、呕吐等。国内关于夫西地酸发酵与分离纯化工艺发展的报道较少。目前国内夫西地酸生产工艺产量偏低,我国临床用夫西地酸药物大多数采用外国进口,而且价格昂贵。因此,开发一种方法简便,原料易得的夫西地酸大规模制备方法显得尤为重要。
发明内容
本发明的目的在于提供一种利用植物内生真菌发酵制备夫西地酸的方法,本发明的有益效果是制备方法简便,可利用该菌株大规模生产夫西地酸。
本发明所采用的技术方案是按照以下步骤进行:
(1)先在菌种培养基中对十大功劳内生真菌Acremonium pilosumF47进行菌种培养;
(2)再在发酵培养基中进行发酵培养;
(3)将发酵物用有机溶剂提取,合并提取液后,减压蒸馏浓缩得到发酵液浸膏,进行柱色谱分离得到粗品;
(4)上述粗品经色谱分离,得到夫西地酸纯品,纯度在98%以上,产量高达87.5mg/kg。
进一步,步骤(1)中菌种培养基为马铃薯葡萄糖琼脂培养基,培养3天。
进一步,步骤(2)发酵培养基:大米培养基—80g大米,120ml双蒸水,0.3%蛋白胨,500ml锥形瓶,28℃恒温静置培养40-50天。
进一步,步骤(3)中有机溶剂采用乙酸乙酯。
进一步,步骤(3)中提取过程将粗提物过硅胶正相柱色谱,固定相为200-300目硅胶,石油醚和乙酸乙酯混合物为流动相,梯度洗脱分离得到夫西地酸混合物,收集石油醚:乙酸乙酯=2:1~1:2组分,浓缩得粗品。
进一步,步骤(4)中粗品经制备型高效液相色谱纯化,甲醇和水为流动相,得到夫西地酸纯品。
附图说明
图1是夫西地酸纯品化学式。
具体实施方式
下面结合具体实施方式对本发明进行详细说明。
实施例:内生真菌菌株:Acremonium pilosumF47;菌株来源:分离自植物阔叶十大功劳花轴;菌种培养基:马铃薯葡萄糖琼脂培养基;发酵培养基:大米培养基—80g大米,120ml双蒸水,0.3%蛋白胨,500ml锥形瓶,28℃恒温静置培养40-50天;提取:采用有机溶剂乙酸乙酯提取,获得粗提物;分离:柱层析操作,粗提物过硅胶正相柱,固定相为200-300目硅胶,石油醚和乙酸乙酯为流动相,梯度洗脱(100:0-0:100,v/v),收集石油醚:乙酸乙酯=2:1~1:2组分,得到夫西地酸粗品;纯化:将得到的夫西地酸粗品经制备型高效液相色谱纯化,甲醇和水为流动相,得到夫西地酸纯品。
产量:约87.5mg夫西地酸/1000g大米,纯度98.5%以上。
夫西地酸纯品化学式如图1所示。结构确证数据如表1所示。
表1核磁共振碳谱及氢谱数据(400,100MHz,CDCl3,TMS,δppm)
本发明以十大功劳内生真菌Acremonium pilosumF47作为工程菌株,快速大量的制备纯度较高的夫西地酸,且制备工艺简单,高效。
以上所述仅是对本发明的较佳实施方式而已,并非对本发明作任何形式上的限制,凡是依据本发明的技术实质对以上实施方式所做的任何简单修改,等同变化与修饰,均属于本发明技术方案的范围内。
Claims (6)
1.一种利用植物内生真菌发酵制备夫西地酸的方法,其特征在于:
(1)先在菌种培养基中对十大功劳内生真菌Acremonium pilosum F47进行菌种培养;
(2)再在发酵培养基中进行发酵培养;
(3)将发酵物用有机溶剂提取,合并提取液后,减压蒸馏浓缩得到发酵液浸膏,进行色谱分离得粗品;
(4)粗品经色谱纯化,得到夫西地酸纯品,纯度在98%以上,产量高达87.5mg/kg。
2.按照权利要求1所述一种利用植物内生真菌发酵制备夫西地酸的方法,其特征在于:所述步骤(1)中菌种培养基为马铃薯葡萄糖琼脂培养基,培养3天。
3.按照权利要求1所述一种利用植物内生真菌发酵制备夫西地酸的方法,其特征在于:所述步骤(2)发酵培养基:大米培养基—80g大米,120ml双蒸水,0.3%蛋白胨,500ml锥形瓶,28℃恒温静置培养40-50天。
4.按照权利要求1所述一种利用植物内生真菌发酵制备夫西地酸的方法,其特征在于:所述步骤(3)中有机溶剂采用乙酸乙酯。
5.按照权利要求1所述一种利用植物内生真菌发酵制备夫西地酸的方法,其特征在于:所述步骤(3)中提取过程将粗提物过硅胶正相柱色谱,固定相为200-300目硅胶,石油醚和乙酸乙酯混合物为流动相,梯度洗脱,收集石油醚:乙酸乙酯=2:1~1:2组分,分离得到夫西地酸粗品。
6.按照权利要求1所述一种利用植物内生真菌发酵制备夫西地酸的方法,其特征在于:所述步骤(4)中将得到的粗品经制备型高效液相色谱纯化,甲醇和水为流动相,得到夫西地酸纯品。
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US20160338360A1 (en) * | 2013-12-24 | 2016-11-24 | Indigo Ag, Inc. | Plants containing beneficial endophytes |
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US20080318267A1 (en) * | 2004-12-17 | 2008-12-25 | Cubist Pharmaceuticals, Inc. | Methods and Compositions for Ultra-High Throughput Screening of Natural Products |
US20130266645A1 (en) * | 2010-10-06 | 2013-10-10 | Justus-Liebig-Universitat Giessen | Derivatives of steroid benzylamines, having an antiparasitic antibacterial, antimycotic and/or antiviral action |
US20160338360A1 (en) * | 2013-12-24 | 2016-11-24 | Indigo Ag, Inc. | Plants containing beneficial endophytes |
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Title |
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程洁等: "顶孢霉发酵液提取物清除自由基能力的研究", 《食品工业科技》 * |
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