CN113402385B - 源自真菌代谢产物的抗菌化合物、制备方法及应用 - Google Patents
源自真菌代谢产物的抗菌化合物、制备方法及应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,尤其涉及源自真菌代谢产物的抗菌化合物、制备方法及应用。本发明对来源于中国传统中药贯叶连翘的内生真菌西曲霉进行培养发酵,获取其次生代谢产物。从菌株的发酵代谢产物中分离得到了新化合物1和已知化合物2‑4。选取革兰氏阴性菌(大肠杆菌、沙门氏菌)和革兰氏阳性菌(表皮葡萄球菌、金黄色葡萄球菌)为研究对象,采用微量肉汤稀释法评价化合物的抗菌活性。发现化合物1‑4均有一定的抑菌活性,可以预期发展成为抗菌药物。
Description
技术领域
本发明属于生物医药技术领域,尤其涉及源自真菌代谢产物的抗菌化合物、制备方法及应用。
背景技术
表皮葡萄球菌(Staphylococcus epidermidis)属细球菌科,革兰氏阳性球菌,存在于人体皮肤和黏膜上。过去曾经认为表皮葡萄球菌对人并不致病,现在认为它也是常见的重要致病细菌,而且发现耐药的现象也是日趋严重,造成治疗上的困难。当人体抵抗力下降或者表皮葡萄球菌离开皮肤和黏膜等正常生活的部位,到了非正常寄生的部位时就会引起许多感染,危害人体。现在,耐药的表皮葡萄球菌感染已经成为瓣膜修复术或者胸外科手术中的棘手问题。
从植物中提取化学成分在过去的几十年间一直是天然药物化学学科发现活性化合物的重要手段。贯叶连翘(Hypericum perforatum L.)是金丝桃属、藤黄科多年草本植物,光照时可抗病毒和抗肿瘤。但其在光照时可能产生光敏化副作用,使这两种生物活性的应用受到限制。近年来为扩大其临床价值,对其进行抑菌活性研究,发现不需要光照条件也可发挥作用,证明了其抑菌作用的安全性。随着人们研究的深入,可供大规模研究的野生植物逐渐变少。药用植物内生真菌资源丰富,且能产生药用植物生长调节物质及与宿主相同或类似的次生代谢产物,从而成为近年来的研究热点。这种生产天然产物的方法可无限重复,且持续获取,避免了植物资源的枯竭与浪费。
综上,现有技术的缺点:
1.从植物中提取化学成分在过去的几十年间一直是发现活性化合物的重要手段,但随着人们研究的深入,可供大规模研究的野生植物逐渐变少,容易造成资源匮乏甚至枯竭。
2.抗生素的不合理利用甚至滥用导致耐药表皮葡萄球菌的产生,耐药的表皮葡萄球菌感染已经成为棘手问题,亟需有效的药物来对抗耐药抗生素。
发明内容
针对现有技术存在的问题,本发明采用植物内生真菌进行培养发酵,药用植物内生真菌可与该植物产生相同或类似的次生代谢产物,避免了植物资源的枯竭与浪费。此外,本发明提供了一种抗菌化合物、制备方法及其应用,目的在于解决现有技术中的一部分问题或至少缓解现有技术中的一部分问题。
本发明是这样实现的,一种化合物,所述化合物的结构式如下式(I)中的化合物1-化合物4的任一结构所示,
本发明还提供了如上述的化合物在制备抗菌药物中的应用。
本发明还提供了一种抗菌药物,包含如上述的4种化合物中的至少一种。
进一步地,还包括药学上可接受的辅料。
进一步地,所述药物的剂型包括注射剂、粉针剂、口服剂、喷雾剂、胶囊、涂剂中的任一种。
进一步地,所述抗菌包括抑制大肠杆菌、沙门氏菌、表皮葡萄球菌和金黄色葡萄球菌中的至少一种。
本发明还提供了如上述的化合物的制备方法,包括以下步骤:
将西曲霉TJ23接种到大米和水混合的培养基中恒温发酵;之后用醇液浸提发酵物,浸提液浓缩得到浸膏;依次用乙酸乙酯:水和石油醚:甲醇萃取;甲醇萃取部位回收溶剂后得到浸膏;将甲醇部位浸膏经正相硅胶色谱,以二氯甲烷-甲醇体系梯度洗脱,依次得到4个流分Fr.1-Fr.4;
流分Fr.2使用正相柱以石油醚-乙酸乙酯体系进行梯度洗脱,用薄层层析硅胶板监测,依次得到组分Fr.2.1和Fr.2.2;Fr.2.2经反相高效液相色谱、手性色谱柱分离得到化合物4;
Fr.3使用凝胶柱进行分离,得到两个组分Fr.3.1和Fr.3.2;Fr.3.1用反相高效液相制备得到化合物3;
Fr.4经反相C-18柱,以甲醇-水洗脱得到70%组分,再使用正相柱层析,以石油醚-丙酮体系洗脱;然后经高效液相色谱、P4VP柱制备得到化合物1和化合物2。
进一步地,制备化合物4时,高效液相色谱条件为流动相:甲醇:水=90:10,2ml/min;手性色谱柱条件为IG柱,流动相:甲醇:水=95:5,1ml/min;
制备化合物3时,所用凝胶柱为纯甲醇Sephadex LH-20凝胶柱,反相高效液相条件为流动相:甲醇:水=75:25,2ml/min;
制备化合物1和2时,高效液相色谱条件为流动相:甲醇:水=60:40,2ml/min;P4VP柱条件为流动相:甲醇:水=70:30,1ml/min。
本发明还提供了西曲霉TJ23在制备抑制沙门氏菌的试剂中的应用。
本发明还提供了西曲霉TJ23在制备抑制沙门氏菌以及同时能够抑制大肠杆菌、表皮葡萄球菌和金黄色葡萄球菌中的至少一种的试剂中的应用。
曲霉(Aspergillus)是子囊菌门子囊菌属中最常见的一种丝状真菌,生长在富氧环境中。曲霉能够产生大量的活性次生代谢产物,如异香豆素、萜类、生物碱、细胞松弛素类、丁烯内酯、黄原酮、甾醇、二苯醚和蒽醌衍生物等,在医药和商业工业中占有重要地位。西曲霉(Aspergillius westerdijkiae)是一种产孢能力极强的真菌,含丰富的次生代谢产物,包括生物碱、萜类、聚酮、异戊二烯酮。现代药理研究表明,异香豆素类化合物通常具有抗菌、消炎、抗癌、抑制蛋白酶等多种生理和生物活性,萜类化合物通常具有抗菌、抗炎、抗肿瘤、抗氧化等多种药理活性。
本发明通过对贯叶连翘内生真菌西曲霉进行培养,分离鉴定了1个新化合物和3个已知化合物。化合物结构式如式(Ⅰ)所示。
选取革兰氏阴性菌(大肠杆菌、沙门氏菌)和革兰氏阳性菌(表皮葡萄球菌、金黄色葡萄球菌)为研究对象,采用微量肉汤稀释法评价化合物的抗菌活性。通过对抑菌试验数据分析发现,化合物1-4对四种菌均有一定的抑制效果,可剂量依赖性地抑制其生长,可以预期发展成为抗菌药物。
综上所述,本发明的优点及积极效果为:
1.通过对贯叶连翘内生真菌进行培养,期望得到与贯叶连翘相同或相似的次生代谢产物,缓解植物资源枯竭的现状;
2.药理活性实验结果表明,本发明从贯叶连翘内生真菌代谢物中分离到的化合物1-4对革兰氏阴性菌(大肠杆菌、沙门氏菌)和革兰氏阳性菌(表皮葡萄球菌、金黄色葡萄球菌)具有不同程度的抑菌效果。化合物1是一种新的化合物,可抑制表皮葡萄球菌的生长,具有抗菌活性,预期可以发展成为抗表皮葡萄球菌感染的药物;
3.本发明只需按照实验方案对菌种传代培养,即可无限放大发酵,获得具有抗菌活性的目标化合物。
附图说明
图1是化合物1的电子圆二色谱(ECD)的实验值与计算值比对图;
图2是化合物1-4对细菌生长的抑制作用。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明,各实施例及试验例中所用的设备和试剂如无特殊说明,均可从商业途径得到。此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
根据本申请包含的信息,对于本领域技术人员来说可以轻而易举地对本发明的精确描述进行各种改变,而不会偏离所附权利要求的精神和范围。应该理解,本发明的范围不局限于所限定的过程、性质或组分,因为这些实施方案以及其他的描述仅仅是为了示意性说明本发明的特定方面。实际上,本领域或相关领域的技术人员明显能够对本发明实施方式作出的各种改变都涵盖在所附权利要求的范围内。
为了更好地理解本发明而不是限制本发明的范围,在本申请中所用的表示用量、百分比的所有数字、以及其他数值,在所有情况下都应理解为以词语“大约”所修饰。因此,除非特别说明,否则在说明书和所附权利要求书中所列出的数字参数都是近似值,其可能会根据试图获得的理想性质的不同而加以改变。各个数字参数至少应被看作是根据所报告的有效数字和通过常规的四舍五入方法而获得的。本发明中,“约”指给定值或范围的10%以内,优选为5%以内。
本发明下述各实施例中未特别限定温度时,则均为常温条件。常温是指四季中自然室温条件,不进行额外的冷却或加热处理,一般常温控制在10~30℃,最好是15~25℃。
本发明披露了源自真菌代谢产物的抗菌化合物、制备方法及应用,本发明中涉及的西曲霉来源于植物贯叶连翘,拉丁文名称Aspergillus westerdijkiae,且已在申请号为CN201710951372.6的中国发明专利中公开,简称西曲霉TJ23。下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述。
实施例1抗菌化合物的制备
1、发酵与提取
将4℃保存的西曲霉菌接种到高温灭菌的马铃薯葡萄糖琼脂培养基(PDA培养基)上,于28℃培养箱中活化7d,作为种子板。10.0kg大米与水1:1混合,121℃,15psi下灭菌30分钟,冷却到室温后接入西曲霉,无菌封口膜封口,于恒温25℃发酵30d后,加入乙酸乙酯终止培养发酵。用20L 95%乙醇浸提发酵物,提取液减压浓缩得到粗提浸膏200g,然后依次用等体积乙酸乙酯:水和石油醚:95%甲醇萃取,95%甲醇萃取部位回收溶剂后得到40g浸膏。
2、分离
将40g的甲醇部位浸膏,经正相硅胶色谱,以二氯甲烷:甲醇=200:1-1:1梯度洗脱后,TLC检测,合并相同组分后,得到4个流分A-D(Fr.1-Fr.4)。
组分Fr.2使用正相柱以100:1-1:1的石油醚-乙酸乙酯体系进行梯度洗脱,用薄层层析硅胶板监测,得到组分Fr.2.1和Fr.2.2。Fr.2.2经反相高效液相色谱(流动相:甲醇:水=90:10,2ml/min)、手性色谱柱(IG柱,流动相:甲醇:水=95:5,1ml/min)分离得到化合物4。
Fr.3使用Sephadex LH-20凝胶柱(纯甲醇)进行分离,得到两个组分Fr.3.1和Fr.3.2。Fr.3.1用反相高效液相(流动相:甲醇:水=75:25,2ml/min)制备得到化合物3。
Fr.4经反相C-18柱,以甲醇-水洗脱得到70%组分,再使用正相柱层析,以石油醚-丙酮体系洗脱。然后经高效液相色谱(流动相:甲醇:水=60:40,2ml/min)、P4VP柱(流动相:甲醇:水=70:30,1ml/min)制备得到新化合物1和已知化合物2。
实施例2化合物的结构鉴定
经过核磁共振(NMR)、计算ECD方法得到了化合物1-4的结构。
1、理化数据
2′S-formoic acid A(化合物1):棕色粉末;HRESIMS[M+H]+m/z 213.0175(计算值C10H9O5,213.0763);;UV(CH3OH)λmax(logε)=225(4.726)、241(4.538)、350(4.122)nm;IR(KBr)νmax 3237,2957,2874,1649,1588,1477,1410cm–1;1H和13CNMR数据,见表1。
8-methoxymellein(化合物2):棕色针状晶体;m/z计算值C11H12O3:192.0786;1H和13C NMR数据,见表1。
Conidiogenone C(化合物3):黄色油状液体;m/z计算值C20H30O2:302.2246;1H和13CNMR数据,见表2。
n-hexacos-5,8,11-trienoic acid(化合物4):无色油状液体;m/z计算值C26H46O2:490.3498;1H和13C NMR数据,见表2。除表中数据,还有一些由于化学位移相近,未能一一进行归属:1H NMR(400MHz):δH 1.60(4H,m,2CH2),δH 1.29(24H,m,12CH2)。13C NMR(400MHz):δC34.3(CH2)、δC 32.1(CH2)、δC 31.7(CH2)、δC 30.0(CH2)、δC 29.9(CH2)、δC29.8(CH2)、δC 29.7(CH2)、δC 29.6(CH2)、δC 29.5(CH2)、δC 29.4(CH2)、δC 29.3(CH2)、δC 29.2(CH2)、δC 27.4(CH2)、δC 27.4(CH2)、δC 27.4(CH2)、δC 25.8(CH2)、δC 24.9(CH2)、δC 22.9(CH2)、δC 22.8(CH2)。
表1.1H NMR data for compounds 1,and 2(400MHz,J in Hz).
aMeasured in DMSO-d6;bMeasured in CDCl3
表2.1H NMR data for compounds 1,and 2(400MHz,J in Hz).
aMeasured in DMSO-d6;bMeasured in CDCl3
2.通过计算ECD的方法对化合物1的绝对构型进行确证。实验所测的ECD(电子圆二色谱)的Cotton效应与计算值吻合,见图1。
最终确定得到的化合物1-4的结构式如下式(Ⅰ)所示:
实施例3具有抗菌活性的化合物应用研究
选取革兰氏阴性菌(大肠杆菌、沙门氏菌)和革兰氏阳性菌(表皮葡萄球菌、金黄色葡萄球菌)为作用靶标,采用微量肉汤稀释法评价化合物的抗菌活性。
将大肠杆菌、沙门氏菌、表皮葡萄球菌和金黄色葡萄球菌接种于10ml培养液中,当细菌进入对数生长期后用NB肉汤稀释至麦氏比浊度为0.5,此时的细菌数量为108CFU/mL,然后按照1:1000稀释后分别用化合物1、2、3或4处理,放入37℃恒温培养箱培养16-24h后,观察结果。
细菌培养:大肠杆菌、沙门氏菌、表皮葡萄球菌和金黄色葡萄球菌生长于NB培养液中。培养细菌置于37℃、220rpm的恒温摇床中培养6-8h;用NB肉汤稀释至麦氏比浊度为0.5,此时的细菌数量为108CFU/mL,然后按照1:1000稀释后接种至96孔板,将其分成空白对照组、阴性对照组、给药组,每组设6个复孔。
详细实验过程如下:
实验分组及处理:
空白对照组(blank control):未加菌液及化合物;
阴性对照组(negative control):未加化合物,仅加入菌液;
给药组(compounds 1、2or 3):1:1000稀释后的菌液接种至96孔板,分别加入不同浓度的化合物。
抗菌实验:将空白对照组、阴性对照组、给药组放入37℃恒温培养箱培养16-24h后,应用酶标仪检测600nm波长处的OD值,并计算128μg/mL的抑菌率。重复3次实验后取平均值进行分析。
结果如图2所示:通过对抑菌试验的数据分析发现,化合物物1-4对四种微生物均有不同程度的抑菌效果,比较突出的包括:化合物物1对表皮葡萄球菌的抑菌浓度(MIC)为128μg/mL时的抑菌率为15.68%。化合物2对大肠杆菌的抑菌浓度(MIC)为128μg/mL时的抑菌率为45.48%、对大沙门氏菌的抑菌浓度(MIC)为128μg/mL时的抑菌率为52.83%、对金黄色葡萄球菌的抑菌浓度(MIC)为128μg/mL时的抑菌率为34.44%、对表皮葡萄球菌的抑菌浓度(MIC)为128μg/mL时的抑菌率为50.60%;化合物3对表皮葡萄球菌的抑菌浓度(MIC)为128μg/mL时的抑菌率为49.34%;化合物4对大肠杆菌的抑菌浓度(MIC)为128μg/mL时的抑菌率为43.34%、对大沙门氏菌的抑菌浓度(MIC)为128μg/mL时的抑菌率为41.16%、对金黄色葡萄球菌的抑菌浓度(MIC)为128μg/mL时的抑菌率为41.83%、对表皮葡萄球菌的抑菌浓度(MIC)为128μg/mL时的抑菌率为48.76%。化合物1-4具有一定的抑菌作用,且呈剂量依赖性,可预期发展成为抗菌药。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (9)
3.一种抗菌药物,其特征在于,包含如权利要求2中的4种化合物中的至少一种;所述抗菌包括抑制大肠杆菌、沙门氏菌、表皮葡萄球菌和金黄色葡萄球菌。
4.根据权利要求3所述的一种抗菌药物,其特征在于:还包括药学上可接受的辅料。
5.根据权利要求3所述的一种抗菌药物,其特征在于:所述药物的剂型包括注射剂、粉针剂、口服剂、喷雾剂、胶囊、涂剂中的任一种。
6.如权利要求2中的任一种化合物的制备方法,其特征在于,包括以下步骤:
将西曲霉TJ23接种到大米和水混合的培养基中恒温发酵;之后用醇液浸提发酵物,浸提液浓缩得到浸膏;依次用乙酸乙酯:水和石油醚:甲醇萃取;甲醇萃取部位回收溶剂后得到浸膏;将甲醇部位浸膏经正相硅胶色谱,以二氯甲烷-甲醇体系梯度洗脱,依次得到4个流分Fr.1-Fr.4;
流分Fr.2使用正相柱以石油醚-乙酸乙酯体系进行梯度洗脱,用薄层层析硅胶板监测,依次得到组分Fr.2.1和Fr.2.2;Fr.2.2经反相高效液相色谱、手性色谱柱分离得到化合物4;
Fr.3使用凝胶柱进行分离,得到两个组分Fr.3.1和Fr.3.2;Fr.3.1用反相高效液相制备得到化合物3;
Fr.4经反相C-18柱,以甲醇-水洗脱得到70%组分,再使用正相柱层析,以石油醚-丙酮体系洗脱;然后经高效液相色谱、P4VP柱制备得到化合物1和化合物2。
7.如权利要求6所述的化合物的制备方法,其特征在于:
制备化合物4时,高效液相色谱条件为流动相:甲醇:水=90:10,2ml/min;手性色谱柱条件为IG柱,流动相:甲醇:水=95:5,1ml/min;
制备化合物3时,所用凝胶柱为纯甲醇Sephadex LH-20凝胶柱,反相高效液相条件为流动相:甲醇:水=75:25,2ml/min;
制备化合物1和2时,高效液相色谱条件为流动相:甲醇:水=60:40,2ml/min;P4VP柱条件为流动相:甲醇:水=70:30,1ml/min。
8.西曲霉TJ23在制备抑制沙门氏菌的试剂中的应用。
9.西曲霉TJ23在制备抑制沙门氏菌以及同时能够抑制大肠杆菌、表皮葡萄球菌和金黄色葡萄球菌中的至少一种的试剂中的应用。
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