CN117530945A - 桉油精在制备沙门氏菌鞭毛抑制剂中的医药用途 - Google Patents
桉油精在制备沙门氏菌鞭毛抑制剂中的医药用途 Download PDFInfo
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Abstract
沙门氏菌鞭毛是沙门氏菌感染抗毒力药物研发的潜在靶标,研究发现沙门氏菌鞭毛抑制剂对于沙门氏菌的防控具有重要意义。本发明涉及桉油精在制备沙门氏菌鞭毛抑制剂中的医药用途,并通过沙门氏菌滑动运动抑制试验、最小抑菌浓度测定试验、生长曲线、沙门氏菌粘附至Caco‑2细胞抑制试验、小鼠存活率等试验,验证桉油精在不抑制细菌生长的前提下,靶向细菌毒力因子从而降低沙门氏菌的致病力。桉油精是桉树叶中的主要成分,更是传统中药艾叶的重要药效学成分,桉油精具有成本低廉、无耐药性、药物残留等特点,具有广阔的应用前景,为沙门氏菌感染防治提供了新的途径和潜在的天然化合物。
Description
技术领域
本发明涉及桉油精在制备沙门氏菌鞭毛抑制剂中的医药用途,属于医学制药技术领域。
背景技术
沙门氏菌(Salmonella typhimurium)是一种革兰氏阴性、细胞内寄生的肠杆菌,广泛存在于自然界中,不仅能引起畜禽及其它动物发生急性、慢性或隐性感染,而且还能通过污染食品导致人发生食物中毒、胃肠炎、伤寒、败血症等疾病,是一种重要的食源性病原菌,对人类和动物都有极大的危害,其中沙门氏菌相关性胃肠炎是全球所有沙门氏菌病病例中最常见的疾病,每年有近百万例患者死亡。沙门氏菌的致病机制涉及多种毒力因素,包括不同类型的分泌***、被膜、质粒和鞭毛等。其中鞭毛介导沙门氏菌的运动,细菌运动使细菌能够直接向营养物质或感染目标部位移动。鞭毛在沙门氏菌的感染过程中起着核心作用,通过帮助宿主细胞的运动,粘附和侵袭来促进病原体的毒力。在鞭毛的帮助下,病原体可以穿过粘液屏障并进入上皮层。另外鞭毛还涉及生物膜形成、免疫***调节等。鞭毛由三个主要结构组成:基体、柔性钩和细丝。鞭毛丝长10-15μm,由多达20,000鞭毛蛋白亚基构成。因此,沙门氏菌鞭毛是沙门氏菌感染抗毒力药物研发的潜在靶标,探究并发现沙门氏菌鞭毛抑制剂对于沙门氏菌的防控具有重要意义。本发明首次发现桉油精可作为沙门氏菌鞭毛抑制剂来抵抗沙门氏菌的致病力。
桉油精(Cineole),又名桉叶油醇、1,8-环氧对孟烷、桉树脑等,具有类似樟脑气味的无色液体,是来源于桉树属植物中的一种单萜烯类氧化物,主要存在于桉叶中,在桉叶油中的含量高达70%,亦存在于豆蔻、迷迭香、艾草等200余种天然植物挥发油中,是许多芳香植物精油的主要有效成分。我国传统中药艾叶成分中,桉油精是最重要的药效学成分,是艾草发挥治愈效果的关键成分。现代医学研究表明,桉油精具有广泛的生物活性,包括抑菌、抗炎、抗氧化以及促渗透作用,在医药、化妆品和香料工业领域广泛应用。近年来国内外对桉油精的抑菌、抗炎、镇痛、抗肿瘤、抗抑郁、神经保护、降血脂、防治糖尿病并发症等作用的研究日益深入,显示其尚有很大的开发潜力。近年来,大量文献对桉油精的药理活性进行了报道。但是,截止到目前国内外尚无桉油精抑制沙门氏菌鞭毛的相关研究。
发明内容
本发明提出了桉油精在制备沙门氏菌鞭毛抑制剂中的医用用途,为防治沙门氏菌感染提供了新的研究思路和候选天然化合物。
本发明通过沙门氏菌滑动运动抑制试验、最小抑菌浓度测定试验、沙门氏菌粘附细胞抑制等试验验证桉油精能够抑制细菌鞭毛介导的滑动,从而降低沙门氏菌的致病力,试验所用沙门氏菌为SL1344。
本发明所述桉油精分子式为C10H18O,分子量为154.25。桉油精的分子结构如下:
本发明的积极效果在于:
提供了桉油精在制备沙门氏菌鞭毛抑制剂中的新医用用途,公开了桉油精能够抑制鞭毛介导的细菌滑动,从而降低沙门氏菌的致病力。
附图说明
图1:桉油精抑制沙门氏菌SL1344在半固体LB平板上的滑动运动;
图2:桉油精对沙门氏菌SL1344滑动运动直径的测量结果;
图3:桉油精在有效浓度范围内不抑制沙门氏菌SL1344的生长;
图4:桉油精在有效浓度范围内对Cacao-2细胞无毒性作用;
图5:桉油精抑制沙门氏菌SL1344对Caco-2细胞的粘附;
图6:桉油精对小鼠感染沙门氏菌死亡率的影响。
具体实施方式
通过以下实施例进一步举例描述本发明,并不以任何方式限制本发明,在不背离本发明的技术解决方案的前提下,对本发明所作的本领域普通技术人员容易实现的任何改动或改变都将落入本发明的权利要求范围之内。
实施例1
桉油精可作为沙门氏菌鞭毛抑制剂用于药学上可接受的任何载体。
实施例2
桉油精可作为沙门氏菌鞭毛抑制剂用于制备治疗感染性疾病的药物。
实施例3
桉油精作为沙门氏菌鞭毛抑制剂用于治疗细菌引起的感染性疾病。
试验例1
沙门氏菌滑动运动抑制试验
过夜培养的沙门氏菌SL1344使用LB肉汤调整菌液浓度为OD600nm=0.5,并配置0.3% LB软琼脂固体平板,吸取5uL菌液滴加于桉油精处理的LB软琼脂平板中央,并置于恒温培养箱(37℃)中培养10-12h,拍照观察并测量细菌向四周运动的菌落范围大小。滑动圈越大,说明滑动能力越强;反之则说明滑动能力越弱。
结果表明:与对照组(未加入桉油精处理)比较,不同浓度桉油精(32-256μg/mL)处理能够显著抑制沙门氏菌在0.3% LB软琼脂固体平板上的滑动性(见附图1-附图2)。
试验例2
桉油精对沙门氏菌SL1344最小抑菌浓度(Minimal inhibition concentration,MIC)值测定
根据美国临床和实验室标准协会公布的最小抑菌浓度值测定试验标准稀释法进行测定桉油精对沙门氏菌SL1344的MIC值。在无菌96孔板中使用LB肉汤倍比稀释桉油精,使其终浓度为512μg/ml,256μg/ml,128μg/ml,64μg/ml,32μg/ml,16μg/ml,8μg/ml,4μg/ml,同时接种过夜培养的沙门氏菌SL1344(调整菌量为5×105CFU/ml)。振荡混匀后,置于37℃恒温培养箱内培养20h-24h,观察细菌生长情况。肉眼可见的抑制细菌生长的化合物浓度判定为桉油精对沙门氏菌的MIC值。
结果表明:桉油精对沙门氏菌SL1344的MIC值大于512μg/ml。
试验例3
沙门氏菌SL1344生长曲线
沙门氏菌SL1344接种于2mL LB培养基中过夜培养,次日按照1:100扩大培养至100mL LB培养基继续培养至OD600nm=0.3时,将菌液等量分装至5个锥形瓶内,每瓶分别加入桉油精使其终浓度分别为32、64、128、256μg/mL,同时设DMSO对照组,37℃,180rpm继续培养,每隔1h测定细菌培养物OD600nm值并记录,直至细菌生长至平台期,绘制生长曲线。
结果表明:在有效浓度范围内(32-256μg/mL),桉油精均不影响沙门氏菌SL1344的生长(见附图3)。
试验例4
桉油精对Caco-2细胞的细胞毒性
用0.25%胰酶消化Caco-2细胞,按2.0×104细胞/孔接种到96孔细胞培养板,在37℃,5% CO2的细胞培养箱中过夜培养。移去96孔板中的培养基,加入PBS轻柔清洗细胞一次。然后用DMEM培养基加入桉油精配制终浓度为32、64、128、256μg/mL的溶液,并设置等体积DMSO对照。每孔加入200μL上述所配溶液,在37℃温箱中静置孵育4h后,每孔加入10%的CCK-8溶液,加入后37℃孵育30min,在酶标仪上测定450nm处样品的吸光值。
结果表明:桉油精在有效浓度范围内(32-256μg/ml),对Caco-2细胞无明显的细胞毒性作用(见附图4)。
试验例5
桉油精抑制沙门氏菌SL1344对Caco-2细胞的粘附
用0.25%胰酶消化Caco-2细胞,按4.0×104细胞/孔接种到24孔细胞培养板,在37℃,5% CO2的细胞培养箱中过夜培养。沙门氏菌SL1344接种于2mL LB培养基中过夜培养,次日,按照1:20扩培至新鲜LB培养基,分别加入桉油精使其终浓度分别32、64、128、256μg/mL,并设置等体积DMSO对照。在37℃,180rpm继续培养4h。调整菌液的浓度至4.0×106CFUs/mL,备用。
移去24孔板中的培养基,加入上述准备好的菌液,1mL/孔。感染1h后,每孔加入1mL0.2%的TritonX-100,反复吹打。将裂解液转移至离心管,倍比稀释,涂布于链霉素抗性LB固体培养基于37℃过夜培养,次日计数。
结果表明:桉油精(128,256μg/mL)处理能够显著抑制沙门氏菌SL1344对Caco-2细胞的粘附(见附图5)。
试验例6
保护率试验
所有小鼠适应性饲养一周后,在饮水中添加5g/L链霉素连喂三天、将小鼠随机分为3组(每组n=6):PBS组、SL1344感染组、60mg/kg/天桉油精油治疗组。通过口服感染1×107CFU鼠伤寒沙门氏菌SL1344,2h后口服给药,随后每隔12h给药一次连续4天。分别在感染后不同时间点(间隔24h)统计小鼠的存活情况,最终数据汇总绘制存活率曲线。
结果表明:桉油精治疗能够显著降低沙门氏菌感染小鼠的死亡率。(见附图6)。
Claims (4)
1.桉油精在制备沙门氏菌鞭毛抑制剂中的医药用途。
2.如权利要求1所述的应用,其特征在于,所述的医药用途是指桉油精抑制沙门氏菌鞭毛介导的细菌泳动的生物学功能。
3.如权利要求1所述的应用,其特征在于,所述的沙门氏菌鞭毛抑制剂,可有效抑制沙门氏菌鞭毛介导的运动性,从而降低沙门氏菌的致病力。
4.如权利要求1所述的应用,其特征在于,所述的沙门氏菌鞭毛抑制剂桉油精在制备治疗沙门氏菌感染药物中的应用。
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