CN113967208A - 苯丙素类天然源化合物在作为抑菌剂在抑制人源病菌中的应用 - Google Patents
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Abstract
本发明公开了一种苯丙素类天然源化合物新的生物活性及用途——作为抗菌剂在抑制人源性病菌中的应用，属于天然药物化学领域。本发明研究表明，其中所涉及的14种苯丙素类天然源化合物对大肠杆菌、金黄色葡萄球菌、白色念珠菌均表现出优异的抑菌活性，部分高活性化合物的MIC值为400μg/mL，并展现出广谱性、高活性的特点，作为新型生物药具有进一步研究和开发的价值。
Description
技术领域
本发明涉及苯丙素类天然源化合物作为抗菌剂在抑制人源性病菌（大肠杆菌Escherichia coli ATCC 25922、金黄色葡萄球菌Staphyloccocus aureus sp. Newman和白色念珠菌Candida albicans ATCC 24433）中的新应用，属于天然药物化学领域。
背景技术
人源性病菌主要包括大肠杆菌Escherichia coli ATCC 25922、金黄色葡萄球菌Staphyloccocus aureus subsp. Newman和白色念珠菌Canidia albicans ATCC 24433三种。大肠杆菌是临床感染中最常见的革兰阴性菌之一，主要通过粪-口传播途径传播，在一定条件下可引起人和多种动物发生胃肠道、尿道等多种局部组织器官感染。金黄色葡萄球菌是临床感染中最常见的革兰阳性菌之一，常见于皮肤表面及上呼吸道黏膜，在一定条件下能够产生肠毒素，引起食物中毒，是引起食物中毒的常见致病菌。白色念珠菌是一种真菌，通常存在于正常人口腔、上呼吸道、肠道及阴道，一般在正常机体中数量少，不引起疾病。白色念珠菌在机体免疫功能、一般防御力下降或正常菌群相互制约作用失调时，才会大量繁殖并改变生长形式（芽生菌丝相）侵入细胞引起疾病。
多药耐药病原体的日益出现和传播加速了对新抗生素的需求。天然产物主导着发现抗菌剂的首选化学骨架。苯丙素是天然存在的一类苯环与三个直链碳连接（C6-C3基团）构成的化合物，一般具有苯酚结构，是酚性物质。在生物合成上，这类化合物多数由莽草酸通过苯丙氨酸和酪氨酸等芳香氨基酸，经脱氨、羟基化等一系列反应形成。CN201610153537公开了一种苯丙素类化合物在制备治疗炎症性疾病的药物中的应用，研究表明，苯丙素类化合物显示出可以抑制细胞炎症因子TNFα，IL1β，IL6的表达作用，进而具有抗炎活性，为炎症性疾病，如宫颈炎、子宫内膜炎、盆腔炎、乳腺炎、咽喉炎、关节炎等疾病的治疗药物的开发提供方向。
发明内容
本发明的目的是提供一种苯丙素类天然源化合物的新用途——作为抗菌剂在抑制人源性病菌中的应用。
本发明所涉及的苯丙素类天然源化合物包括14种不同结构的化合物，其结构及命名如下：
本发明所涉及的人源病菌包括大肠杆菌Escherichia coli ATCC 25922、金黄色葡萄球菌Staphyloccocus aureus sp. Newman和白色念珠菌Candida albicans ATCC24433。
下面通过活性实验说明苯丙素类天然源化合物对人源病菌活性的抑制作用，以期用于制备防治以其引起的人畜共患疾病的药物。
实验1：苯丙素类天然源化合物抗人源细菌活性测定
本实验中所用的人源细菌为实验室4℃保存的菌种，采用的培养基为NB固体培养基（牛肉膏：3 g，蛋白胨：10 g，氯化钠：5 g，琼脂：15 g，蒸馏水：1 L，pH7.0；121℃灭菌20min）和NB液体培养基（牛肉膏：3 g，蛋白胨：10 g，氯化钠：5 g，蒸馏水：1 L，pH7.0；121℃灭菌20 min），并于37℃下培养。
本实验中所用的人源细菌为实验室-80℃含30%甘油冻存的菌株。将冻存菌株取出，分别在人源细菌的NB固体培养基（牛肉膏：3 g，蛋白胨：5 g，酵母粉：1 g，蔗糖：10 g，琼脂：15 g，蒸馏水：1 L，pH7.0；121℃灭菌20 min）上面进行划线，在37℃下恒温培养直到长出单菌落。分别挑取NB固体培养基上单菌落至人源细菌NB液体培养基（牛肉膏：3 g，蛋白胨：5 g，酵母粉：1 g，蔗糖：10 g，蒸馏水：1 L；121℃灭菌20 min）中，在28℃、180 rpm恒温摇床振荡培养到对数生长期。将处于对数生长期的菌株用相应的液体培养基稀释至约106 CFU/mL备用。将苯丙素类化合物分别用DMSO溶解，加入NB液体培养基中，混合均匀，配制成浓度为1600 μg/mL的含药液体培养基。取50 μL含药培养基和相同体积的含约106 CFU/mL细菌培养物加入到96孔板的孔中，最终给药浓度为800 μg/mL。含等量DMSO的相同浓度100 μL菌液做对照。将96孔板在37℃恒温培养箱中培养24-48 h直至对照组菌液长出，在酶标仪上测定孔中菌液的OD值（OD600）。并且另外测定100 μL液体培养基和浓度为800 μg/mL药剂的OD值，对培养基和药剂本身造成的OD值进行矫正。校正OD值和抑制率的计算公式如下：
校正OD值 = 含菌培养基OD值-无菌培养物OD值；
抑制率 = （校正后对照培养基菌液OD值-校正后含药培养基OD值）/校正后对照培养基菌液OD值 × 100%
实验2：苯丙素类天然源化合物抗念珠菌活性测定
试验方法与实验1相同，只是将培养基更换为念珠菌的YEPD固体培养基（酵母膏：10 g，蛋白胨：20g，葡萄糖：20 g，琼脂：15 g，蒸馏水：1 L；121℃灭菌20 min）和YEPD液体培养基（酵母膏：10 g，蛋白胨：20g，葡萄糖：20 g，蒸馏水：1 L；121℃灭菌20 min），并于37℃下培养。
将高活性化合物的含药液体培养基在96孔板中通过二倍稀释法稀释得到系列浓度的50 μL含药培养基，然后根据上述相同试验方法测定系列浓度对应的抑制率。将抑制率大于90%的最低浓度定义为MIC，所有实验设置三个重复。实验结果见表2。
由表1，2显示的生测结果显示，本发明涉及的苯丙素类化合物对人源性病菌表现出较好的抑菌作用，部分高活性化合物的MIC值为400 μg/mL，展现出广谱性、高活性的特点，作为新型生物药具有进一步研究和开发的价值。
Claims (3)
1.苯丙素类天然源化合物作为抗菌剂在抑制人源性病菌中的应用。
2.根据权利要求1所述苯丙素类天然源化合物作为抗菌剂在抑制人源性病菌中的应用，其特征在于：所述苯丙素类天然源化合物的结构及命名如下：
3.如权利要求1或2所述苯丙素类天然源化合物作为抗菌剂在抑制人源性病菌中的应用，其特征在于：所述人源性病菌为大肠杆菌Escherichia coli ATCC 25922、金黄色葡萄球菌Staphyloccocus aureus sp. Newman和白色念珠菌Candida albicans ATCC 24433。
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