CN104383047B - 栀子提取物在制备抗鲍曼不动杆菌药物中的应用 - Google Patents
栀子提取物在制备抗鲍曼不动杆菌药物中的应用 Download PDFInfo
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Abstract
本发明涉及清开灵活性组分栀子提取物对鲍曼不动杆菌(Acinetobacter baumannii，A.baumannii)具有抑制作用，因此它可用于抑制该病菌并治疗和/或预防该细菌引起的疾病。
Description
本发明是中国发明专利申请(发明名称：清开灵活性组分栀子提取物在制备抗多重耐药菌的药物中的应用；申请日：2012年12月17日；申请号：201210550177.X)的分案申请。
技术领域
本发明涉及栀子的一种新的医药用途，具体地说是在栀子提取物制备抗多重耐药菌药物中的应用。
背景技术
多重耐药菌(multiple resistant bacteria)是指有多重耐药性的病原菌，即一种微生物对三类(比如氨基糖苷类、红霉素、B－内酰胺类)或三类以上抗生素同时耐药。其中最为多见的是NDM-1、MDR-TB、MDR-MRSA以及常在ICU中出现的鲍曼不动杆菌和铜绿假单胞菌。NDM-1(New Delhi metallo-β-lactamase-1，新德里金属β-内酰胺酶)，又名NDM-1细菌，是一种耐碳青霉烯类的酶。NDM-1基因常由质粒携带，但也整合在细菌的染色体上，易于在不同的菌株间传播和扩散，NDM-1编码的蛋白单体分子量为28kDa，它的序列和其他MBL的序列相似性很低。NDM-1主要分布在肠杆菌科和鲍曼不动杆菌中，携带NDM-1的细菌不但包括条件致病菌，也包括致病菌，如常见的志贺氏菌、霍乱、绿脓假单胞杆菌等。它具有高度耐药性，几乎可以水解临床常用的一线抗菌药物，包括β-内酰胺类、碳青霉烯类、氟喹诺酮类、氨基糖苷等。长期以来，抗生素药物的滥用，是催生多重耐药菌的重要原因。目前，多重耐药菌感染呈现出增长趋势，但抗菌药物选择方案却很少，因此重耐药菌感染已成为全球性棘手问题。
栀子(茜草科植物栀子Gardenia jasminoides Ellis的干燥成熟果实)为清热解毒复方中药清开灵注射液的主要原料药，具有护肝、利胆、降压、镇静、止血、消肿等作用，在中医临床常用于治疗黄疸型肝炎、扭挫伤、高血压、糖尿病等症，并且具有良好的效果。开发和研究栀子更多更好的医药功效，是许多中医药学领域的重要研究课题之一。
发明内容
本发明的目的就是要开发栀子提取物的新医药用途，同时为临床提供一种可有效拮抗多重耐药菌的药物。
本发明的发明人经长期研究发现栀子提取物对含NDM-1耐药基因细菌具有显著地抑制作用，且与药物浓度成正比，从而发明了栀子提取物的一种新的医药用途，即完成了栀子提取物在制备抗多重耐药菌药物中的应用。
本发明所述的栀子提取物可按照常规的中药提取方法进行。
本发明在此提供的方法如下：
(1)取栀子适量，加水煎煮两次，过滤，合并两次滤液；
(2)所得滤液减压浓缩，放冷后在搅拌下加乙醇使含醇量达60％，冷藏，过滤，滤液回收乙醇，加水稀释，过滤；
(3)将所得滤液上大孔吸附树脂，依次用水、乙醇洗脱，收集洗脱液，过滤，减压回收乙醇至无醇味，得栀子提取物，冷藏。
其中，步骤(1)中，加水煎煮，第一次加8倍量的水，煮沸2小时，第二次加6倍量的水，煮沸1小时；步骤(2)中，加水稀释至相对密度1.05～1.2(60℃)；步骤(3)中，洗脱剂水用量是5～7倍量柱体积、乙醇是8～10倍量柱体积的40％乙醇。
本发明所述栀子提取物，可按照现有技术所述方法制备成颗粒、冲剂、射剂、胶囊等药物制剂。
本发明中所述的多重耐药菌是指含有NMD-1基因的多重耐药菌，包括乙酸钙不动杆菌(Acinetobacter calcoaceticus)、鲍曼不动杆菌(Acinetobacter baumannii，A.baumannii)、嗜麦芽寡养窄食单胞菌(Stenotrophomonas maltophilia)或含NDM-1耐药基因大肠杆菌(Escherichia coli)。
上述含NDM-1耐药基因检测菌株为革兰氏阴性菌，具有较强的致病性和抗生素耐药性。其中，乙酸钙不动杆菌和鲍曼不动杆菌可引起肺部感染、伤口及皮肤感染、泌尿生殖系统感染、菌血症菌血症、脑膜炎等，麦芽寡养窄食单胞菌能引起严重的呼吸系统感染等，含NDM-1耐药基因大肠杆菌可引发对肠道感染且对抗生素具有抗药性。
更进一步地，本发明还提供了栀子提取物在制备抗乙酸钙不动杆菌药物中的应用，即栀子提取物在制备治疗和/或预防乙酸钙不动杆菌引起的疾病的药物中的应用。
更进一步地，本发明还提供了栀子提取物在制备抗鲍曼不动杆菌药物中的应用，即栀子提取物在制备治疗和/或预防鲍曼不动杆菌引起的疾病的药物中的应用。
更进一步地，本发明还提供了栀子提取物在制备抗嗜麦芽寡养窄食单胞菌药物中的应用，即栀子提取物在制备治疗和/或预防嗜麦芽寡养窄食单胞菌引起的疾病的药物中的应用。
更进一步地，本发明还提供了栀子提取物在制备抗NDM-1耐药基因大肠杆菌药物中的应用。
更进一步地，本发明还提供了栀子提取物在制备抗含NDM-1耐药基因大肠杆菌药物中的应用，即栀子提取物在制备治疗和/或预防含NDM-1耐药基因大肠杆菌引起的疾病的药物中的应用。
本发明所述栀子制剂可用于治疗上述由含NDM-1耐药基因多重耐药细菌引发的疾病，如含NDM-1耐药基因多重耐药细菌引发的肠道感染、呼吸系统感染、伤口及皮肤感染、泌尿生殖系统感染、菌血症菌血症、脑膜炎等，尤其是适用于治疗由乙酸钙不动杆菌、鲍曼不动杆菌、嗜麦芽寡养窄食单胞菌或含NDM-1耐药基因大肠杆菌引发的疾病。
本发明所述药物制剂在作为本发明的用途使用时，其具体用量可根据临床试验以及患者的具体情况确定。
具体实施方式
以下通过实施例和试验例来进一步阐述本发明所述药物的有益效果。
实施例1：
栀子提取物的制备方法
(1)取栀子适量，加水煎煮两次，第一次加8倍量的水，煮沸2小时，第二次加6倍量的水，煮沸1小时，过滤，合并两次滤液；(2)所得滤液减压浓缩至原药材量1.5～2倍体积时，放冷后在搅拌下加乙醇使含醇量达60％，冷藏，过滤，滤液回收乙醇，加水稀释到相对密度为1.05～1.2(60℃)，过滤；(3)将所得滤液上大孔吸附树脂，依次用5～7倍量柱体积的水、8～10倍量柱体积的40％乙醇洗脱，收集洗脱液，过滤，减压回收乙醇至无醇味，得栀子提取物，冷藏。
药效学试验
试验例1
栀子提取物对含NDM-1耐药基因细菌的抑菌效果研究
本药效学试验所采用的检测菌株：乙酸钙不动杆菌(Acinetobactercalcoaceticus，A.calcoaceticus)、鲍曼不动杆菌(Acinetobacter baumannii，A.baumannii)和嗜麦芽寡养窄食单胞菌(Stenotrophomonas maltophilia，S.maltophilia)，均含有blaNDM-1耐药基因；pGEX-4T-NDM1-DH5α是含NDM-1重组质粒的大肠杆菌(Escherichia coli，E.coli)；pGEX-4T-NDM-1-BL21(在大肠杆菌BL21中表达NDM-1)是含NDM-1重组质粒的大肠杆菌(Escherichia coli，E.coli)，其质粒上带有GST标签。上述菌株均由中国人民解放军疾病预防控制所提供。
配制LB琼脂培养基，高压冷却至60℃时，取栀子提取物，按1:9(药物：LB，V/V)比例配制不同浓度药物(0.1g/ml、0.2g/ml、0.3g/ml)平板，琼脂厚度3-4mm，制备药物平板，备用。
研究栀子对含NDM-1耐药基因细菌进行抑菌实验：
将4株含NDM-1基因耐药菌株乙酸钙不动杆菌、鲍曼不动杆菌、嗜麦芽寡养窄食单胞菌及大肠杆菌pGEX-4T-NDM1-DH5α分别接种于含16μg/ml亚胺培南的LB液体培养基中，37℃、200rpm培养过夜，次日菌液用无菌PBS缓冲液稀释10倍后，分别吸取2μl接种于药物琼脂平板表面或采用划线法接种，平板置于37℃孵育24h左右，观察各菌株生长状况，得栀子提取物的抑菌性测定结果，见表1。
采用琼脂稀释法对栀子提取物的最小抑菌浓度(MIC)进行测定，将耐药菌株接种于不同药物浓度的琼脂平板上，37℃孵育24h左右，观察各菌株生长状况，抑制菌株不能生长的最小药物浓度(MIC值)分别为乙酸钙不动杆菌0.3g/ml、鲍曼不动杆菌0.3g/ml、嗜麦芽寡养窄食单胞菌0.2g/ml、pGEX-4T-NDM-1-BL210.3g/ml。
实验结果表明：栀子对耐药菌株均有抑制作用，随着药物浓度的增加，抑制作用明显增强，说明栀子对含NDM-1耐药基因耐药菌都有抑制作用，且抑菌效果明显；药物对不同检测菌株的抗菌能力不同，MIC也有所差异，具有较强的广谱抗含NDM-1耐药基因耐药菌的能力。
表1栀子提取物浓度对菌株的影响
注：+++表示不抑制生长，++表示抑制不明显，+表示明显抑制，—表示完全抑制，不生长。
Claims (1)
1.栀子提取物在制备抗含NDM-1基因的鲍曼不动杆菌药物中的应用，其特征在于，所述栀子提取物由栀子药材，加水煎煮两次，过滤，合并滤液，所得滤液减压浓缩，放冷后在搅拌下加乙醇使含醇量达60％，冷藏，过滤，滤液回收乙醇，加水稀释到：60℃时药液相对密度为1.05～1.2，过滤，所得滤液上大孔吸附树脂，依次用5～7倍量柱体积的水、8～10倍量柱体积的40％乙醇洗脱，收集洗脱液，过滤，减压回收乙醇至无醇味制得。
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