CN110772518A - 血根碱在抑制路邓葡萄球菌生长中的应用 - Google Patents
血根碱在抑制路邓葡萄球菌生长中的应用 Download PDFInfo
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Abstract
本发明公开了血根碱在抑制路邓葡萄球菌生长中的应用，根据血根碱对耐药路邓葡萄球菌具有较好的体外杀灭作用，能够抑制耐药路邓葡萄球菌的生长，且最低杀菌浓度为30μg/mL，最小抑菌浓度为15μg/mL，本发明提出了血根碱对路邓葡萄球菌的抑制作用，在医药等领域具有广泛的应用价值。
Description
技术领域
本发明涉及中药有效成分血根碱作为路邓葡萄球菌的抑制剂的研究开发和应用，具体涉及血根碱在抑制路邓葡萄球菌生长中的应用。
背景技术
路邓葡萄球菌(Staphylococcus lugdunensis)为毒力较强的凝固酶阴性的葡萄球菌之一，已经成为一种重要的人类病原体，它能够导致一系列的感染性疾病，例如心内膜炎、淋巴结炎、眼炎、骨关节感染，但更常见的是皮肤和软组织感染，其所引发的感染性疾病病情严重且发展迅速，并发症多、死亡率高。对路邓葡萄球菌产生的感染性疾病的治疗，根据之前学者的研究可知，其对青霉素已经产生了耐药性，对阿奇霉素、氯霉素和庆大霉素的敏感性也逐渐降低。这是由于在过去的很长一段时间里抗生素的滥用，造成了对药物具有抵抗力的耐药菌株的出现，随着细菌耐药性的增加，迫切需要开发新的抑菌剂。
中草药植物中的化学成分作为新的抑菌剂开发对象，在治疗疾病方面有毒副作用少、来源广泛，且治疗成本较低的优势。血根碱(C20H14NO4)是从白屈菜、博落回等罂粟科植物中分离得到的苯菲啶异喹啉类生物碱，具有多种药理活性，如杀虫、抗肿瘤、抑菌、抗炎、改善肝功能、增强免疫力等作用，目前报道了血根碱对白假丝酵母菌、铜绿假单胞菌、大肠杆菌和金黄色葡萄球菌等具有抑制作用，但是，路邓葡萄球菌在感染途径和治疗方案上与导致肠道、呼吸道感染的病原菌存在明显差异，而关于血根碱抑制路邓葡萄球菌生长方面也鲜有报道。
文献(刘祝英,王祥明,唐良明.血根碱的作用机制及其在动物生产上的应用效果[J].湖南饲料,2019,168(01):41-45.)从天然药用植物博落回中提取得到有效活性成分血根碱，并对血根碱的药理作用和应用现状进行了研究。结果表明，血根碱对大肠杆菌的最小抑菌浓度为640μg/mL，对四连球菌蜡样芽孢杆菌的最低抑菌浓度为80μg/mL，对金黄色葡萄球菌和枯草芽孢杆菌的最低抑菌浓度均小于40μg/mL，此外与青霉素相比，血根碱对四连球菌蜡样芽孢杆菌和枯草芽孢杆菌的抗菌活性更强。
发明内容
本发明针对目前临床医学面临的耐药难题，目的在于提供血根碱在抑制路邓葡萄球菌生长中的应用。
为了实现上述目的，本发明采用的技术方案为：
利用纸片扩散法确定路邓葡萄球菌对抗生素的耐药性，通过微量二倍稀释法确定血根碱对路邓葡萄球菌的最小抑菌浓度(MIC)和最低杀菌浓度(MBC)。结果表明：血根碱对耐药路邓葡萄球菌具有较好的抑菌作用，可以用于耐药抑制路邓葡萄球菌的生长。
优选的，所述路邓葡萄球菌为耐青霉素、克林霉素、四环素、环丙沙星、苯唑西林中多种抗生素的人源路邓葡萄球菌。
优选的，所述路邓葡萄球菌选自耐青霉素、克林霉素、四环素、环丙沙星和苯唑西林的人源路邓葡萄球菌。
优选的，所述血根碱的最低杀菌浓度为30μg/mL，最小抑菌浓度为15μg/mL。
本发明的有益效果体现在：
本发明从现存的药用植物资源库入手，挖掘潜在的耐药菌抑制剂，基于血根碱对耐药路邓葡萄球菌的作用的研究，发现非抗生素类化合物血根碱可以有效抑制路邓葡萄球菌的生长，为路邓葡萄球菌抑制剂的开发和应用提供新的思路和来源，在医药等领域具有广泛的应用价值。
进一步的，本发明明确了血根碱对耐药路邓葡萄球菌的抑制作用，将其作为抗生素替代品，能够有效缓解或解决路邓葡萄球菌的耐药及感染问题，降低病死率。
具体实施方式
下面结合实施例对本发明做进一步详细说明。所述实施例仅用于解释本发明，并非对本发明保护范围的限制。
1、人源路邓葡萄球菌的药敏实验
本发明以多株人源路邓葡萄球菌(菌株样本取自宁波市疾病预防控制中心)为出发菌株，选用青霉素、克林霉素、四环素、环丙沙星、万古霉素、苯唑西林6种常用抗生素的药敏试纸片进行药敏试验。
挑取培养18～24h的纯菌落均匀溶解于2～5mL无菌的TSB液体培养基中，调节其浊度与0.5麦氏比浊管等浊。无菌环境下吸取50～200μL菌液，均匀涂布平板，室温下放置3～5min。用无菌镊子按要求将药敏试纸片(直径为6mm)紧贴于平板表面，每种药敏试纸片做三个平行。将接种好的平板静置于36～37℃的培养箱中孵育16～24h。用0.02mm精度的游标卡尺测定抑菌圈直径，以抑菌圈的直径来判断药物的敏感性，抑菌结果根据美国临床试验室国家标准化管理委员会(CLSl2017)标准判定，判定标准见表1。实验结果见表2，结果显示，编号为3#的人源路邓葡萄球菌对青霉素、克林霉素、四环素、环丙沙星和苯唑西林这5种临床上常见的抗生素具有较强的耐受性，属于多重耐药菌株，因此将其作为下一步实验的研究对象。
表1.美国临床试验室国家标准化管理委员会(CLSl2017)标准判定结果
表2.人源路邓葡萄球菌的药敏实验中抑菌圈直径结果
注：抑菌圈直径的单位为mm，数值均为三个平行的平均值
2、血根碱对耐药路邓葡萄球菌的抑制作用
为了充分考虑用药安全，本发明以血根碱这一单一活性成分为研究对象，以ATCC700328为参考菌株(参考菌株对庆大霉素、万古霉素、利奈唑胺和莫西沙星高度敏感)，以3#人源路邓葡萄球菌作为实验菌株，进行耐药性抑制作用的研究。挑取培养过夜的单菌落接种于3mL TSB液体培养基中培养，调节其浊度与0.5麦氏比浊管等浊，并测定其OD600值。用二甲基亚砜配制浓度为10mg/mL的血根碱，利用试管二倍稀释法将药液、菌液及TSB液体培养基加入96孔培养板中过夜培养，不同浓度的药液组分别做三个平行，确保实验数据的可信度。
利用酶标仪测定血根碱对路邓葡萄球菌的最小抑菌浓度(MIC)，并将MIC浓度的前一个药物稀释浓度下的培养液转接至无菌的TSA固体培养基中继续培养24h，观察有无单菌落生成，如果无，则此浓度为血根碱对路邓葡萄球菌的最低杀菌浓度(MBC)，实验结果见表3。
表3.血根碱对耐药路邓葡萄球菌的抑制结果
由表3结果可知，血根碱对耐青霉素、克林霉素、四环素、环丙沙星和苯唑西林的路邓葡萄球菌的抑制作用较好，其MIC为15μg/mL，MBC为30μg/mL。通过与参考菌株ATCC700328的比较，结果表明血根碱不仅对路邓葡萄球菌参考菌株ATCC700328有抑菌作用，对本发明中的耐药路邓葡萄球菌(例如，3#菌株)也有抑菌作用。
根据以上实验结果，结合中草药来源广泛、不良反应少及不易产生耐药性等特点，利用中草药有效单体成分血根碱，可以直接针对临床主要的耐药路邓葡萄球菌发挥抑制作用，有效缓解或解决耐药路邓葡萄球菌的感染问题，降低病死率，为研究临床分离的路邓葡萄球菌的抑菌剂提供科学依据，为开发抑制耐药路邓葡萄球菌的药物和抗生素替代品提供新的思路和来源。
Claims (10)
1.血根碱在抑制路邓葡萄球菌生长中的应用。
2.根据权利要求1所述的应用，其特征在于：所述路邓葡萄球菌为人源路邓葡萄球菌。
3.根据权利要求1所述的应用，其特征在于：所述路邓葡萄球菌的耐药性是通过纸片扩散法确定的。
4.根据权利要求1所述的应用，其特征在于：所述路邓葡萄球菌耐受的抗生素选自青霉素、克林霉素、四环素、环丙沙星、苯唑西林中的多种。
5.根据权利要求1所述的应用，其特征在于：所述路邓葡萄球菌选自耐青霉素、克林霉素、四环素、环丙沙星和苯唑西林的路邓葡萄球菌。
6.根据权利要求5所述的应用，其特征在于：所述血根碱对路邓葡萄球菌的最低杀菌浓度为30μg/mL，最小抑菌浓度为15μg/mL。
7.根据权利要求1所述的应用，其特征在于：所述血根碱对路邓葡萄球菌具有体外杀灭作用，以及抑制路邓葡萄球菌的体外生长。
8.血根碱在制备抗路邓葡萄球菌的药物中的应用。
9.根据权利要求8所述的应用，其特征在于：所述路邓葡萄球菌为人源路邓葡萄球菌。
10.根据权利要求8所述的应用，其特征在于：所述路邓葡萄球菌选自对青霉素、克林霉素、四环素、环丙沙星、苯唑西林中多种抗生素耐药的路邓葡萄球菌。
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