CN101429202A - 一种顶头孢菌素及其制法和用途 - Google Patents
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Abstract
本发明提供一种新骨架顶头孢菌素,它具有如右结构式以及提供新骨架顶头孢菌素的制备方法。该新骨架顶头孢菌素具有很强的抑制大肠杆菌(Escherichia coli)、荧光假单孢菌(Pseudomonas fluorescence)、红色毛癣菌(Trichophytonrubrum)和白色念珠菌(Candida albican)作用,因此顶头孢菌素可作为具有抗菌的化合物,并有望在制备相关新型药物中得到应用。
Description
技术领域
本发明涉及从药用植物络石(Trachelospermum jasminoides(Lindl.)Lem.(Apocynaceae))根部内生菌顶头孢(Cephalosporium acremonium)固体发酵物中提取分离的一种顶头孢菌素(cephalosol)及其制法与抗菌作用。
背景技术
虽然微生物的次生代谢产物已被广泛研究,但特境微生物及其活性次生代谢产物还是没有引起人们的重视。植物内生菌是一类分布广泛、种类繁多、具有重要生理和生态功能的特境微生物,其活性次生代谢产物正成为目前天然产物化学领域的研究热点。
致病菌的扩散及其耐药性的增强严重威胁着人类的健康和生命,抗菌药物已作为常规用药广泛用于艾滋病、器官移植以及慢性消耗性疾病(如癌症、糖尿病、尿毒症等)的治疗,虽然目前临床上使用的抗菌药剂(如酮康唑、阿米卡星、庆大霉素、活力康唑、伊曲康唑、特比萘芬、二性霉素、氟康唑等)对皮肤及浅表部位感染的疗效较好,但这些抗菌药物的蓄积毒性较强,常常引起肝肾损伤、消化道刺激、头晕、过敏等,所以寻找作用机理独特的新型抗菌药物成为当今药物研发的热点之一。药用植物内生菌具有丰富的生物多样性和化学多样性,能够产生化学结构独特、活性显著的抗菌物质(参见ZhangHW,SongYC,TanRX.Nat.Prod.Rep.2006,23:753;Tan RX,Zou WX.Nat.Prod.Rep.2001,18:448)。到目前为止,尚未见从络石内生菌顶头孢(Cephalosporium acremonium)分离到抗菌物质的报道。
发明内容
本发明的目的在于:
1.提供一个具有抗菌活性的新骨架顶头孢菌素;
2.提供一种提取、分离该新骨架顶头孢菌素的方法;
3.提供一种本发明的新骨架顶头孢菌素在制备抗菌药物中的应用。
本发明的目的通过下述技术方案予以实现。
新骨架顶头孢菌素具有下述结构:
其中1,2,3,5,7,8,9,10,11,12为碳原子序数编号。
一种新骨架顶头孢菌素的制备方法,其特征是,它包括下述步骤:
步骤1.将新鲜健康的络石内生真菌顶头孢(Cephalosporium acremonium IFB-E007)菌丝体块接种到PDA培养基上,置摇床上,在140rpm、28℃条件下培养3天作为种子液;
步骤2.种子液接种于固体培养基中,固体培养基组成为每瓶中含小米7.5g,麸皮7.5g,酵母膏0.5g,FeSO4·7H2O 0.01g,酒石酸钠0.1g,谷氨酸钠0.1g,玉米油0.1mL,蒸馏水15mL,28℃条件下发酵30天;
步骤3.将步骤2所得发酵物经混合溶剂氯仿/甲醇(体积比1:1)常温浸提3次,料液比2∶5(g/ml),过滤低温真空浓缩,得到黑色粗浸膏F;
步骤4.将粗浸膏F溶于甲醇中,料液比7:10(g/mL),加热回流1h后,冷却室温并放入-18℃冰箱中,过夜,抽虑除去粗浸膏F中的蜡、油脂和盐类物质,重复三次,得到浸膏F1;
步骤5.对浸膏F1进行硅胶柱层析,依次用10倍色谱柱保留体积的氯仿、氯仿/甲醇(体积比100:1)、氯仿/甲醇(体积比100:2)、氯仿/甲醇(体积比100:4)洗脱,合并氯仿/甲醇(体积比100:4)洗脱的洗脱液,得到浸膏F2;
步骤6.将浸膏F2再上硅胶柱层析分离,用氯仿/甲醇混合溶液洗脱,合并氯仿/甲醇(体积比100:2)洗脱的洗脱液,得浸膏F3;
步骤7.利用葡聚糖凝胶Sephadex LH-20对浸膏F3进行柱层析,洗脱溶剂为氯仿/甲醇(体积比1:1),定量收集洗脱剂,TLC检测含有亮蓝色荧光的部分,合并洗脱剂后低温真空除去洗脱剂,得到顶头孢菌素。
本发明得到的新骨架顶头孢菌素对人类致病菌大肠杆菌(Escherichia coli)、荧光假单孢菌(Pseudomonas fluorescence)、红色毛癣菌(Trichophyton rubrum)和白色念珠菌(Candidaalbican)有很强的抑制作用,所以顶头孢菌素可作为具有抗菌作用的化合物,并有望在制备抗菌药物中得到应用。
与现有技术相比,本发明具有下述突出优点:
1.本发明的顶头孢菌素是全新碳骨架化合物,可以作为具有抗菌作用的新药物或先导化合物;
2.本发明的顶头孢菌素可以利用微生物进行液体发酵生产,操作工艺简便,周期短,成本低,来源有保证;
3.本发明利用生物法合成顶头孢菌素对环境无污染。
具体实施方式
通过下面给出的具体实施例可以进一步了解本发明。但它们不是对本发明的限定。
实施例1:植物内生菌顶头孢(Cephalosporium acremonium IFB-E007)的分离与纯化
取新鲜健康的络石根,装入保鲜袋中密封标记,并快速将样品拿到实验室中(20℃),5h内进行内生菌分离。络石根用清水洗净,切成1cm左右的小段后依次浸泡于75%酒精1min、1%次氯酸钠溶液(含游离氯>2.5%)10min、75%酒精1min,再用含有双抗(200IU/mL青霉素和150IU/ml链霉素)的WA培养基(20g琼脂、蒸馏水1000mL)平板培养。待菌落从切口处长出后,转接至PCA培养基(20g马玲薯、20g胡萝卜、20g琼脂、蒸馏水1000mL)进行内生真菌的分离,经菌落形态学观察、孢子形态和18S rDNA分子鉴定为顶头孢,Cephalosporiumacremonium IFB-E007,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区大屯路中国科学院微生物研究所,保藏日期:2008年7月15日,保藏号为:CGMCC NO.2595。
实施例2:植物内生菌顶头孢(Cephalosporium acremonium IFB-E007)的固体发酵
活化植物内生菌顶头孢(Cephalosporium acremonium IFB-E007),将新鲜的菌丝体块接种到1000mL锥形瓶中,每瓶含有400mL的PDA培养基,在140rpm、28℃条件下培养3天作为种子液;将20mL种子液接种于含有固体培养基的广口瓶中,28℃条件下发酵30天,其中固体培养基的组成为每瓶中含小米7.5g,麸皮7.5g,酵母膏0.5g,FeSO4·7H2O 0.01g,酒石酸钠0.1g,谷氨酸钠0.1g,玉米油0.1mL,蒸馏水15mL。
实施例3:顶头孢菌素的提取与分离
将实施例2中所得发酵物用氯仿/甲醇(体积比1:1)常温浸提3次,料液比2:5(g/ml),过滤低温真空浓缩,得到黑色粗浸膏F;将粗浸膏F溶于甲醇中,料液比7:10(g/mL),加热回流1h后,冷却室温并放入-18℃冰箱中,过夜,抽虑除去粗浸膏F中的蜡、油脂和盐类物质,重复三次,得到浸膏F1;对浸膏F1进行硅胶柱层析,依次用10倍色谱柱保留体积的氯仿、氯仿/甲醇(体积比100:1)、氯仿/甲醇(体积比100:2)、氯仿/甲醇(体积比100:4)洗脱,合并氯仿/甲醇(体积比100:4)洗脱的洗脱液,得到浸膏F2;将浸膏F2再上硅胶柱层析分离,用氯仿/甲醇混合溶液洗脱,合并氯仿/甲醇(体积比100:2)洗脱的洗脱液,得浸膏F3;利用葡聚糖凝胶Sephadex LH-20对浸膏F3进行柱层析,洗脱溶剂为氯仿/甲醇(体积比1:1),定量收集洗脱剂,TLC检测含有亮蓝色荧光的部分,合并洗脱剂后低温真空除去洗脱剂,得到顶头孢菌素。
实施例4:顶头孢菌素的结构鉴定
顶头孢菌素的结构是基于它们的质谱、核磁共振谱、红外、紫外、旋光测定和物理化学计算而确定的。
光谱学数据如下:
顶头孢菌素:浅黄色粉末,mp 206-208℃;[α]-96.2°(c0.165,CHCl3);IR(film)v:3523.2,1778.8,1727.4,1698.8,1618.0,1567.3,1513.7,1468.4,1431.2,1365.8,1224.8,1011.1cm-1;UV(CHCl3):λmax 255nm(log ε=4.9);1H NMR(500MHz,CDCl3)and13C(125MHz,CDCl3),见表1;HR-EIMS[M]+m/z 334.0690(C16H14O8,calcd 334.0689)。
表1.顶头孢菌素1H谱、13C谱和HMBC数据(溶剂:CDCl3)
S:单峰,d:双重峰,br s:宽单峰
顶头孢菌素(S型)和对应异构体(R型顶头孢菌素)的计算能量、旋光度和13C谱数据见下表2:
表2.顶头孢菌素及其对应异构体的计算能量、旋光度和13C谱数据
实施例5:顶头孢菌素抗菌活性
抗菌活性实验是采用浓度稀释的方法,每次测定重复三次,测试病原菌有大肠杆菌(Escherichia coli)、荧光假单孢菌(Pseudomonas fluorescence)、红色毛癣菌(Trichophytonrubrum)和白色念珠菌(Candida albican),菌液浓度为105个/mL。顶头孢菌素cephalosol起始浓度为500μg/mL(5%二甲基亚砜DMSO),梯度稀释至0.9μg/mL,等量体积的菌液和测试样品混合培养在96孔板中,细菌和真菌培养温度分别为37℃和28℃,培养时间24h后观察,若发现没有菌落形成时为样品最低抗菌浓度,即MIC值。该实验阳性对照为硫酸阿米卡星和酮康唑,顶头孢菌素抗菌结果见表3。
表3.顶头孢菌素抗菌MIC值(μg/mL)
以上结果提示,顶头孢菌素具有很强的抗菌活性,尤其是其抑制白色念珠菌强于阳性对照酮康唑,因此本发明的顶头孢菌素有望被用于制备新型抗菌药物。
Claims (3)
2、一种权利要求1所述的顶头孢菌素的制备方法,其特征是它包括下述步骤:
步骤1.将新鲜健康的络石内生真菌顶头孢(Cephalosporium acremonium IFB-E007)菌丝体块接种到PDA培养基上,置摇床上,在140rpm、28℃条件下培养3天作为种子液;
步骤2.种子液接种于固体培养基中,固体培养基组成为每瓶中含小米7.5g,麸皮7.5g,酵母膏0.5g,FeSO4·7H2O 0.01g,酒石酸钠0.1g,谷氨酸钠0.1g,玉米油0.1mL,蒸馏水15mL,28℃条件下发酵30天;
步骤3.将步骤2所得发酵物经混合溶剂氯仿/甲醇(体积比1:1)常温浸提3次,料液比2:5(g/ml),过滤低温真空浓缩,得到黑色粗浸膏F;
步骤4.将粗浸膏F溶于甲醇中,料液比7:10(g/mL),加热回流1h后,冷却室温并放入-18℃冰箱中,过夜,抽虑除去粗浸膏F中的蜡、油脂和盐类物质,重复三次,得到浸膏F1;
步骤5.对浸膏F1进行硅胶柱层析,依次用10倍色谱柱保留体积的氯仿、氯仿/甲醇(体积比100:1)、氯仿/甲醇(体积比100:2)、氯仿/甲醇(体积比100:4)洗脱,合并氯仿/甲醇(体积比100:4)洗脱的洗脱液,得到浸膏F2;
步骤6.将浸膏F2再上硅胶柱层析分离,用氯仿/甲醇混合溶液洗脱,合并氯仿/甲醇(体积比100:2)洗脱的洗脱液,得浸膏F3;
步骤7.利用葡聚糖凝胶Sephadex LH-20对浸膏F3进行柱层析,洗脱溶剂为氯仿/甲醇(体积比1:1),定量收集洗脱剂,TLC检测含有亮蓝色荧光的部分,合并洗脱剂后低温真空除去洗脱剂,得到顶头孢菌素。
3、根据权利要求1所述的顶头孢菌素在制备抗菌药物中的应用。
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