CN106138197A - 藿香挥发油在抑制耐药大肠杆菌生长中的应用 - Google Patents

藿香挥发油在抑制耐药大肠杆菌生长中的应用 Download PDF

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CN106138197A
CN106138197A CN201610553643.8A CN201610553643A CN106138197A CN 106138197 A CN106138197 A CN 106138197A CN 201610553643 A CN201610553643 A CN 201610553643A CN 106138197 A CN106138197 A CN 106138197A
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宫海燕
张晨
田树革
陈凯
李劭昱
王学涵
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Abstract

本发明公开了藿香挥发油在抑制耐药大肠杆菌生长中的应用，并从藿香挥发油提取物中获得单品化学成分胡薄荷酮，对人源耐药大肠杆菌，即人源产ESBLs大肠杆菌的生长具有较好的体外抑制作用。本发明的有益效果为：首次研究了藿香挥发油对耐药大肠杆菌的作用，发现能够抑制耐药大肠杆菌的生长，从藿香挥发油中筛选主要的成分作为耐药抑制剂进行研究，数据表明藿香挥发油的主要单体成分胡薄荷酮能够抑制产ESBLs大肠杆菌的生长，并影响其生物被膜的形成，旨在缓解或解决大肠杆菌的耐药感染，降低病死率，为解决与治疗人源耐药大肠杆菌提出来新的思路，具有重要的研究意义。

Description

藿香挥发油在抑制耐药大肠杆菌生长中的应用
技术领域
本发明涉及中药提取物应用技术领域，具体涉及藿香挥发油在抑制耐药大肠杆菌生长中的应用。
背景技术
目前，细菌耐药性已成为严重的公共卫生威胁和世界关注的临床难题，耐药菌的产生使得临床上现有的抗菌药物的疗效不断下降，药物的选择范围越来越有限。实际上，控制抗菌药物的使用，只能缓解细菌的耐药，但不能从根本上阻断或解决耐药问题。这就需要人们不断地去发展新型抗生素，但研发新型抗生素的速度远远赶不上细菌产生耐药的速度。中药作为人类与疾病斗争的经验结晶，因其成分复杂，作用机理广泛，在感染性疾病防治中发挥着重要的作用，一直被用作寻求抗生素替代药物的重要资源库。新疆中药资源丰富，中草药的种植开发具有广阔的应用与发展前景。
发明内容
本发明的目的就是针对上述现有临床耐药难题，从现存的中药资源库入手，筛选出新疆种植的藿香作为潜在的耐药抑制剂，其挥发油提取物具有较好的抑菌活性，首次从藿香中筛选出耐药抑制成分—胡薄荷酮，填补中药耐药抑制剂的筛选研究空白，为解决耐药大肠杆菌的耐药难题提供了详细的研究依据。
为了实现上述目的，本发明提供的技术方案为：藿香挥发油在抑制耐药大肠杆菌生长中的应用。
进一步的，上述的应用，所述藿香挥发油为新疆种植的藿香花部位提取得到的挥发油。
进一步的，上述的应用，所述藿香挥发油的活性成分为胡薄荷酮。
进一步的，上述的应用，所述耐药大肠杆菌为人源产ESBLs大肠杆菌。
进一步的，上述的应用，所述藿香挥发油的制备方法为：将采摘的新疆种植的藿香花部位分离并阴干后，取100g装入5000 ml的蒸馏瓶中，加水量为3500 ml, 浸泡1h后以水蒸气蒸馏法提取挥发油，提取6-8h；在所获得的馏出物中加入3g无水氯化钠进行干燥，置冰箱4℃冷藏24 h，使水、油两相分层，再用分液滤斗进行分离，滤得黄色透明的、具有浓郁芳香气味的滤液，即得到新疆种植的藿香花部位的挥发油，所述新疆种植的藿香花部位的挥发油的产率按照重量百分比计为0.29%；所述新疆种植的藿香花部位的挥发油的密度为0.84 mg/ml。
本发明的第二个目的是提供了胡薄荷酮在抑制耐药大肠杆菌生长中的应用。
进一步的，上述的应用，所述胡薄荷酮分离自权利要求2所述的新疆种植的藿香花部位的挥发油。
进一步的，上述的应用，所述耐药大肠杆菌为人源产ESBLs大肠杆菌。
本发明的有益效果为：为进一步明确藿香对人源产ESBLs大肠杆菌耐药性的作用，首次研究了藿香挥发油对耐药大肠杆菌的作用，发现能够抑制耐药大肠杆菌的生长。从藿香挥发油中筛选主要的成分作为耐药抑制剂进行研究，数据表明藿香挥发油的主要单体成分胡薄荷酮能够抑制产ESBLs大肠杆菌的生长，并影响其生物被膜的形成。
同时，结合中草药研究耐药病原菌的抑制与治疗问题，考虑新疆地区特有的中药资源藿香，获得其挥发油提取物及主要的活性单品成分胡薄荷酮，直接与临床主要的耐药病原菌产ESBLs大肠杆菌的抑制相结合，旨在缓解或解决大肠杆菌的耐药感染，降低病死率，为解决与治疗人源耐药大肠杆菌提出来新的思路，具有重要的研究意义。
附图说明
图1为新疆种植的藿香花部位挥发油未加正构烷烃谱图。
图2为新疆种植的藿香花部位挥发油加正构烷烃谱图。
图3为新疆种植的藿香花部位挥发油未加正构烷烃和加正构烷烃谱图比较（红色为加正构烷烃，黑色为未加正构烷烃）。
图4为硫酸庆大霉素对产ESBLs大肠杆菌的抑制率。
图5为胡薄荷酮对产ESBLs大肠杆菌的抑制率。
具体实施方式
实施例1：
1、新疆种植的藿香挥发油的提取与产率的测定：
将采摘的新疆种植的藿香花部位分离并阴干后, 取100 g装入5000 ml的蒸馏瓶中，加水量低于3500 ml, 浸泡1 h。按《中国药典》2005版(一部)附录XD水蒸气蒸馏法提取挥发油，提取6-8 h。由于所获得的馏出物含有少量的水分，加少许氯化钠，置冰箱冷藏24 h，使水、油两相分层，再用分液滤斗进行分离，滤得黄色透明的、具有浓郁芳香气味的液体，即新疆种植的藿香花部位的挥发油，计算挥发油产率为0.29 % (w/w)。毛细管比重法，求得新疆种植的藿香花部位的挥发油密度为0.84 mg/ml。
2、新疆种植的藿香挥发油提取物的含量测定（GC-MS）：
利用GC-MS联用技术与分析，通过与标准谱库匹配的方式，确定新疆种植的藿香挥发油提取物的化合物结构。
2.1、GC-MS联用技术与分析条件：
（1）气相条件：进样口温度 250 ℃, 柱温 40 ℃; 以5 ℃/min 升到250 ℃保持10min；
（2）流速：恒压模式 1.0 ml/min , 分流进样:分流比为100:1;
（3）质谱条件：EI源离子源温度：200 ℃；接口温度：250 ℃；溶剂切除时间：4min；检测器电压：1.08 KV。检索谱库：NIST05、NIST05s；进样量：0.5 μl（茎部位挥发油进样1.0 μl）；检索谱库：NIST05、NIST05s.
2.2、新疆种植的藿香花部位挥发油成分GC-MS分析结果：
通过GC-MS联用技术分析，与标准谱库匹配得谱图，如图1-图3所示，通过表1分析：从新疆种植的藿香挥发油提取物中共鉴定出21种化合物，根据积分面积可得其主要化学成分为pulegone (胡薄荷酮，34.10 %)、estragole (草蒿脑，29.54 %)、p-Menthan-3-one，(1R,4R)-(+)（16.70 %）。表1显示为新疆种植花部位挥发油的化学成分。
表1
。
3、藿香挥发油中耐药抑制活性成分筛选：
3.1、藿香挥发油对产ESBLs大肠杆菌的耐药抑制作用：
采用肉汤稀释法研究藿香挥发油对产ESBLs大肠杆菌的耐药抑制作用，与对照品比较，发现藿香挥发油提取物能明显的抑制耐药大肠杆菌的生长，如表2所示为藿香挥发油对产ESBLs大肠杆菌的抑制作用。
表2
。
抑菌活性单位: mg/ml。
表2中藿香挥发油对产ESBLs大肠杆菌具有一定的抑制作用，虽然与对照品比较，MIC值和MBC值存在一定的差距，但试验发现植物提取物的确能够抑制产ESBLs大肠杆菌的生长，这一点是值得去深思和深入研究的。
3.2、藿香挥发油中胡薄荷酮的耐药抑制作用：
产ESBLs大肠杆菌的抑制作用，结合其主要的化学成分胡薄荷酮 (34.10 %)、草蒿脑(29.54 %)、p-Menthan-3-one (1R,4R)-(+) (16.70 %)、柠檬油精 (8.18 %)。其中，胡薄荷酮和草蒿脑的含量较高，考虑草蒿脑疑似致癌，虽没有证实，也缺乏相关的研究资料，为了充分考虑用药安全，筛选胡薄荷酮为藿香挥发油的主要活性成分，进行耐药性抑制作用的研究。
利用肉汤稀释法研究胡薄荷酮单品成分对产ESBLs大肠杆菌的抑制作用结果：
MIC: 23.68 μg/ml。
MBC: 47.35 μg/ml。
胡薄荷酮对产ESBLs大肠杆菌的抑制作用较好。
3.3、藿香挥发油中胡薄荷酮的产ESBLs大肠杆菌生物被膜形成的影响：
通过96孔微孔板技术，在600 nm下检测胡薄荷酮对产ESBLs大肠杆菌生物被膜形成的影响，以抑制率为评价指标，结果如表3，图4-图5所示，表3为胡薄荷酮对产ESBLs大肠杆菌的抑制率。
表3
。
通过对每个药液组的不同浓度的抑制率进行组内方差分析，得硫酸庆大霉素、胡薄荷酮每个不同浓度内的抑制率的差异没有统计学意义，即浓度的变化不影响抑制率，抑制率并不随浓度的增大而产生变化。通过多重比较 (LSD法)，得硫酸庆大霉素的C1和C3的抑制率的差异具有显著性意义，P =0.026。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (8)

1.藿香挥发油在抑制耐药大肠杆菌生长中的应用。
2.根据权利要求1所述的应用，其特征在于，所述藿香挥发油为新疆种植的藿香花部位提取得到的挥发油。
3.根据权利要求1所述的应用，其特征在于，所述藿香挥发油的活性成分为胡薄荷酮。
4.根据权利要求1所述的应用，其特征在于，所述耐药大肠杆菌为人源产ESBLs大肠杆菌。
5.根据权利要求1所述的应用，其特征在于，所述藿香挥发油的制备方法为：将采摘的新疆种植的藿香花部位分离并阴干后，取100g装入5000 ml的蒸馏瓶中，加水量为3500 ml,浸泡1h后以水蒸气蒸馏法提取挥发油，提取6-8h；在所获得的馏出物中加入3g无水氯化钠进行干燥，置冰箱4℃冷藏24 h，使水、油两相分层，再用分液滤斗进行分离，滤得黄色透明的、具有浓郁芳香气味的滤液，即得到新疆种植的藿香花部位的挥发油，所述新疆种植的藿香花部位的挥发油的产率按照重量百分比计为0.29%；所述新疆种植的藿香花部位的挥发油的密度为0.84 mg/ml。
6.胡薄荷酮在抑制耐药大肠杆菌生长中的应用。
7.根据权利要求6所述的应用，其特征在于，所述胡薄荷酮分离自权利要求2所述的新疆种植的藿香花部位的挥发油。
8.根据权利要求6所述的应用，其特征在于，所述耐药大肠杆菌为人源产ESBLs大肠杆菌。
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