CN100586477C青蒿素及其衍生物与抗菌药物---预防及治疗细菌感染性疾病｜CN100586477C: Artemisinin and Its Derivatives Combined with Antibacterial Drugs for the Prevention and Treatment of Bacterial Infectious Diseases
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CN100586477C青蒿素及其衍生物与抗菌药物---预防及治疗细菌感染性疾病
CN100586477C: Artemisinin and Its Derivatives Combined with Antibacterial Drugs for the Prevention and Treatment of Bacterial Infectious Diseases
青蒿素及其衍生物与抗菌药物的联合应用
本发明公开了一种现有的抗疟药青蒿素及其衍生物二氢青蒿素、蒿甲醚、蒿乙醚、青蒿琥酯新的用途，即，与抗菌药物联合应用抑制细菌生长，增强抗菌药物抗菌效力。尤其是使得青蒿琥酯除作为治疗疟疾药物外，能够在预防及治疗细菌感染性疾病中得到应用。
青蒿素及其衍生物与抗菌药物的联合应用
技术领域
本发明涉及药物应用领域，尤其涉及青蒿素及其衍生物的抗菌应用。
背景技术
感染性疾病是由于病原微生物侵入体内，在体内大量地繁殖所引起的疾病，是临床病人死亡的重要原因之一。造成患者死亡的原因其一是细菌大量繁殖释放毒素导致组织器官的损伤；其二是细菌成分通过激活免疫细胞，诱导TNF-α、IL-1、IL-6、NO等致炎细胞因子释放，引起多种炎症介质所形成的瀑布效应，可使炎症反应扩大甚至失去控制，最终导致以细胞自身性破坏为特征的全身性炎症即脓毒症的发生。细菌感染曾是人类第一死因，抗生素的发明带给了人类希望之光。但抗生素的普遍使用使越来越多的细菌产生了耐药性，如果不解决抗生素滥用的问题，等待人类的将是下一次黑暗。
青蒿提取物用来治疗疟疾相关的发热已经有一千多年的历史，除传统的抗疟作用外，青蒿类物质还具有其它方面的作用，如平喘、抗癌、抗血吸虫及对免疫系统的调节等。国内外有关青蒿素及其衍生物的研究很多，但研究范围主要集中在抗疟、抗癌、抗血吸虫的作用及其机制上。
青蒿琥酯(Artesunate)，化学名为二氢青蒿素-1，2-α-琥珀酸单酯，分子式为C19O8 H28，分子量384，是具有倍半萜结构的抗疟药青蒿素的衍生物。青蒿琥酯是新型的抗疟药，较之青蒿素，青蒿琥酯可配制成任何常规的制剂形式，包括固体和液体形式，给药非常方便。青蒿琥酯作为抗疟药，不但效价高，而且不易产生耐受性。
对青蒿素及其衍生物的抗炎作用已有报道，研究发现青蒿素可抑制LPS/TNF-α诱导性NO合酶的合成及NF-κB的激活；青蒿琥酯对LPS及合并干扰素刺激小鼠腹腔巨噬细胞NO的合成有明显的抑制作用；青蒿琥酯对LPS刺激的小鼠巨噬细胞RAW264.7也具有相似的保护作用；谭余庆等还发现青蒿提取物、青蒿素可降低内毒素休克小鼠LPO、ACP、内毒素、TNF-α、P450浓度，升高SOD活性，降低小鼠死亡率，延长小鼠的平均生存时间，对小鼠肝、肺组织形态也有一定的保护作用；王俊等发现青蒿素能抑制CpG ODN、LPS、热灭活的EC诱导细胞释放炎症因子，并对脓毒症模型小鼠具有显著保护作用。但是青蒿琥酯是否具有抗菌作用，是否能改善细菌对抗生素的耐药，增强抗生素的抗菌效力和对细菌导致的感染是否有效，未见报告。我国是青蒿素和其衍生物的主要生产地，由于该药物疗效高、毒性低，WHO已经将青蒿素列为世界治疗疟疾的主要药物，我国正在扩大青蒿种植和青蒿素生产，因此扩宽青蒿素类衍生物的适应症，将其应用于细菌感染疾病的防治，无疑对提高青蒿素类衍生物的利用、增加其经济价值并改善抗生素滥用所导致的耐药现象具有重要意义。
发明内容
本发明的目的在于将青蒿素及其衍生物，特别是青蒿琥酯应用到抗菌领域，不仅拓宽青蒿素及其衍生物，特别是青蒿琥酯的用途，而且可以减轻现有抗菌药物的滥用及其导致的耐药现象。
基于上述目的，本发明使用的技术方案是将青蒿素及其衍生物中的一种与抗菌药物中的一种进行联合应用，该应用不是做为疾病的治疗方法的应用，而是在配备治疗疾病(抗菌)的药物中的应用，其中上述两种成分的比例为1∶1～4096∶1，上两种成分可以制备成组合物，也可以是两种单独的药剂。上述两种成分能够产生协同抗菌效力，能够降低抗菌药物的最低抑菌浓度(MIC)和最低杀菌浓度(MBC)。
上述菌包括革兰氏阳性菌和革兰氏阴性菌；上述衍生物包括二氢青蒿素、蒿甲醚、蒿乙醚和青蒿琥酯；上述抗菌药物包括β-内酰胺类抗生素(青霉素类、头孢菌素类、非典型β-内酰胺类抗生素)、氨基糖苷类、克林霉素类、喹诺酮类的药物，在一较佳实施例中，上述抗菌药物为青霉素G、氨苄西林、头孢呋辛、头孢匹胺、庆大霉素、克林霉素、洛美沙星、加替沙星、舒氨西林和泰能，优选为庆大霉素、舒氨西林、头孢匹胺和加替沙星。
上述应用优选为青蒿琥酯与抗菌药物的联合应用。
青蒿琥酯单独使用可抑制革兰阴性菌和革兰阳性菌的生长，与抗菌药物联合应用后可明显增强抗菌药物的抗菌效力，减少抗菌药物的使用剂量；实验表明青蒿素及青蒿琥酯与抗菌药物联合应用可明显降低细菌(包括革兰阴性、阳性菌)攻击小鼠死亡率，显著抑制小鼠血清LPS含量和细胞因子TNF-α释放。
本发现人对青蒿琥酯体内外的抗菌和抗炎作用进行了研究。
首先，本研究人员对本青蒿琥酯的体外抗菌作用进行了药理学分析，并与抗生素联合应用后发现青蒿琥酯单独使用可抑制革兰阴性菌和革兰阳性菌的生长，与抗生素联合应用后可明显增强抗生素的抗菌效力，减少抗生素的使用剂量；其次，青蒿琥酯与抗生素联合应用和可明显降低细菌(包括革兰阴性、阳性菌)攻击小鼠死亡率，显著抑制小鼠血清LPS含量和细胞因子TNF-α释放。
因此本发明拓宽了青蒿素及其衍生物，特别是青蒿琥酯的用途，提高了抗菌药物的抗菌效力，减轻了抗菌药物滥用和导致的耐药现象。
附图说明
图1表示青蒿琥酯协同庆大霉素、头孢匹胺、舒氨西林对大肠埃希菌ATCC35218的联合抑菌曲线；
图2表示青蒿琥酯协同氨苄西林、加替沙星、舒氨西林对金葡菌ATCC25923的联合抑菌曲线；
图3表示青蒿琥酯协同庆大霉素、加替沙星、舒氨西林对大肠埃希菌临床分离株的联合抑菌曲线。
具体实施方式
下面结合实施例对本发明作进一步的说明，但所述实施例仅用于说明本发明而不是限制本发明。
青蒿琥酯(AS)与抗菌药物按照表1、表2中的配比，能够产生协同抗菌效力，降低抗菌药物的最低抑菌浓度和最低杀菌浓度。
实验例1
本实验例在于研究青蒿琥酯与十种抗菌药物对不同细菌的MIC(最低抑菌浓度)和MBC(最低杀菌浓度)
采用微孔稀释法，调整细菌浓度为105CFU/ml，接种于96孔无菌培养板内，青蒿琥酯和青霉素钠、氨苄西林等10种抗菌药物分别以生理盐水稀释为5.14mg/ml。加入各种药物至含细菌培养孔内，依次倍比稀释，第1～10孔药物的最终浓度依次为256、128、64、32、16、8、4、2、1、0.5ug/ml。置37℃培养箱孵育24h和48h，读取阳性和阴性对照孔，阴性对照孔清亮，阳性对照孔混浊。药物对细菌的MIC为24h后抑制细菌肉眼可见生长的最低药物浓度，药物对细菌的MBC为48h后抑制细菌肉眼可见生长的最低药物浓度。此次实验结果表明青蒿琥酯单独使用不能完全抑制细菌生长，并观察了不同抗菌药物对该五种细菌的抑菌效力。(见表3)。
表3 10种抗菌药物和AS的单独使用时对不同细菌的MIC及MBC
实验例2
本实验例在于研究青蒿琥酯与抗菌药物联合应用后对大肠埃希菌MIC和MBC的影响。
采用棋盘式微孔稀释法，调整细菌浓度为105CFU/ml，接种于96孔无菌培养板内，将对五种细菌中等敏感的抗菌药物用低于MIC的剂量与不同浓度青蒿琥酯分别配伍，观察配伍后的药物对大肠埃希菌的MIC和MBC。此次实验结果表明青蒿琥酯单独使用虽不能完全抑制细菌生长，但与抗菌药物联合应用后可明显降低抗菌药物的MIC和MBC，说明青蒿琥酯与抗菌药物协同后对细菌有协同的抑菌效力。(见表4)。
表4 各抗菌药物与青蒿琥酯协同后的最低抑菌浓度
实验例3
本实验例在于研究青蒿琥酯与1/2 MIC浓度的不同抗菌药物协同后对大肠埃希菌生长的抑制强度。
调整细菌浓度为106CFU/ml，测定菌液OD600为0.002(1OD＝5×108CFU/ml)，参照实验例1结果，分别单独加入终浓度为256μg/ml青蒿琥酯或1/2 MIC浓度的抗菌药物，以及同时加入256μg/ml青蒿琥酯和1/2MIC浓度的抗菌药物后，至于37℃恒温摇床150rpm振摇，分别测定1、3、5、7、9、12、16、24h时菌液的OD值，计算各时间点的细菌量。研究结果表明青蒿琥酯256μg/ml可使大肠埃希菌ATCC35218，金黄色葡萄球菌ATCC25923和大肠埃希菌临床分离株生长速度明显减慢，与单独使用抗菌药物相比，联合了青蒿琥酯后明显抑制细菌生长，抑制程度比单独使用抗菌药物或青蒿琥酯均强(图1、2、3)。
实验例4
本实验例在于研究青蒿琥酯联合应用抗菌药物对革兰阴性细菌攻击小鼠的保护作用
清洁级昆明种小白鼠70只(重庆医科大学实验动物中心提供)，体重19.9±0.5g/只，雌雄各半，随机分为对照组、青蒿琥酯组、大肠埃希菌组，大肠埃希菌+庆大霉素组，青蒿琥酯(1.5、5、15mg/kg)+庆大霉素+大肠埃希菌组。每组10只动物。对照不给予任何试剂；青蒿素组肌肉注射15mg/kg的青蒿琥酯；大肠埃希菌组，给予0.8×106/kg活的大肠埃希菌ATCC35218；大肠埃希菌+庆大霉素组，给予庆大霉素0.5mg/kg肌肉注射后给予活大肠埃希菌ATCC35218；庆大霉素+青蒿琥酯+大肠埃希菌组，在给予青蒿琥酯后，立即给予庆大霉素及大肠埃希菌。给药完毕后给予正常饮食和饮水，观察7天内小鼠一般情况及死亡率(表5)。结果显示青蒿琥酯协同抗菌药物后可明显降低小鼠的死亡率，表明对青蒿琥酯与抗菌药物联合后出现协同保护作用。
表5 青蒿琥酯协同庆大霉素对大肠埃希菌ATCC35218攻击小鼠的保护作用
实验例5
本实验例在于研究青蒿琥酯联合应用抗菌药物对革兰阳性细菌攻击小鼠的保护作用
清洁级昆明种小白鼠70只(重庆医科大学实验动物中心提供)，体重19.9±0.5g/只，雌雄各半，随机分为对照组、青蒿琥酯组、金葡菌组，金葡菌+加替沙星组，青蒿琥酯(5、15、45mg/kg)+加替沙星+金葡菌组。每组10只动物。对照不给予任何试剂；青蒿素组肌肉注射45mg/kg的青蒿琥酯；金葡菌组，给予1.0×107/kg活的金葡菌ATCC25923；金葡菌+加替沙星组，给予加替沙星0.5mg/kg肌肉注射后给予活金葡菌ATCC25923；加替沙星+青蒿琥酯+金葡菌组，在给予青蒿琥酯后，立即给予加替沙星及金葡菌。给药完毕后给予正常饮食和饮水，观察7天内小鼠一般情况及死亡率(表6)。结果显示青蒿琥酯协同抗菌药物后可明显降低小鼠的死亡率，表明对青蒿琥酯与抗菌药物联合后出现协同保护作用。
表6 青蒿琥酯协同加替沙星对金葡菌ATCC25923攻击小鼠的保护作用
实验例6
本实验例在于研究青蒿琥酯对灭活大肠埃希菌攻击小鼠的保护作用。
清洁级昆明种小白鼠60只(重庆医科大学实验动物中心提供)，体重19.9±0.5g/只，雌雄各半，随机分为对照组、青蒿琥酯组、灭活大肠埃希菌组，青蒿琥酯(5、15、45mg/ml)+灭活大肠埃希菌组。每组8只动物。对照不给予任何试剂；青蒿琥酯组肌肉注射给予45mg/kg的青蒿琥酯，4h，24h，48h后重复给药一次；灭活大肠埃希菌组，给予1.25×1011/kg的灭活大肠埃希菌ATCC35218；青蒿琥酯+灭活大肠埃希菌组，在肌肉注射给予青蒿琥酯后，立即给予灭活大肠埃希菌ATCC35218，4h、24h、48h后重复肌肉注射给药青蒿琥酯1次。给药完毕后给予正常饮食和饮水，观察7天内小鼠一般情况及死亡率(表7)。结果显示青蒿琥酯可降低小鼠的死亡率，表明对致炎因子攻击的小鼠具有保护作用。
表7 青蒿琥酯对灭活大肠埃希菌攻击小鼠的保护作用
*p＜0.5；**p＜0.01 vs E.coli
实验例7
本实验例在于研究青蒿琥酯对灭活大肠埃希菌攻击小鼠血清细胞因子释放影响
清洁级昆明种小白鼠40只(重庆医科大学实验动物中心提供)，体重19.9±0.5g/只，雌雄各半，随机分为对照组、青蒿琥酯组、灭活大肠埃希菌组、青蒿琥酯(5、15、45mg/kg)+灭活大肠埃希菌组，每组动物3只。对照不给予任何试剂。青蒿琥酯组肌肉注射给予45mg/kg的青蒿琥酯；青蒿琥酯(5、15、45mg/kg)+灭活大肠埃希菌组给予青蒿琥酯后立即给予灭活大肠埃希菌ATCC35218；给药完毕4h后摘眼球取血立即离心留置上清保存于-20℃，待测定细胞因子TNF-α(表8)。结果显示青蒿琥酯可显著降低灭活大肠埃希菌攻击小鼠血清细胞因子释放。
表8 青蒿琥酯降低灭活大肠埃希菌ATCC35218攻击小鼠血清细胞因子TNF-α释放(n＝6，x±SD)
*p＜0.5；**p＜0.01 vs E.coli
本发明将青蒿琥酯与抗菌药物的联合应用，提高了抗生素的抗菌效力，将青蒿琥酯与抗生素联合应用还可以预防、治疗细菌感染。
本领域技术人员可以根据本发明所述内容，将青蒿素及其衍生物与抗菌药物的联合同样可以达到本发明所述效果。
1.青蒿琥酯与选自庆大霉素、头孢匹胺、舒氨西林、氨苄西林和加替沙星中的一种在制备抗菌药物中的应用。
2.根据权利要求1所述的应用，上述菌为革兰阳性菌或革兰阴性菌。
Combined application of artemisinin and its derivative and antibiotic medicine
The present invention discloses the new use of available antimalarial arteannuin and its derivatives dihydro arteannuin, antiannuic methyl ether, antiannuic ethyl ether and antiannuic amber. Arteannuin and its derivatives are used through combination with antibiotic medicine to inhibit bacterial growth and enhance the antibiotic effect of available antibiotic medicine. Especially, antiannuic amberis applied in preventing and treating bacterial infection diseases except being used as antimalarial.
The use in conjunction of arteannuin and derivant thereof and antibacterials
Technical field
The present invention relates to the medicinal application field, relate in particular to the antibacterial applications of arteannuin and derivant thereof.
Background technology
Infectious disease is owing in the pathogenic microorganism intrusive body, breed caused disease in vivo in large quantities, is one of major reason of clinical patient death.Cause the reason first antibacterial of death to breed the damage that the release toxin causes histoorgan in a large number; It two is that the antibacterial composition passes through immune cell activated, induce proinflammatory cytokines such as TNF-α, IL-1, IL-6, NO to discharge, cause the formed water fall effect of multiple inflammatory mediator, inflammatory reaction is enlarged even out of hand, finally causing with cell self property destruction is that the systemic inflammatory of feature is pyemic generation.Bacterial infection once was human first cause of the death, and antibiotic invention has brought human light of hope.But antibioticly generally make increasing antibacterial produce drug resistance,, wait for that human will be dark next time if do not solve the problem of antibiotic abuse.
Herba Artemisiae Annuae extract is used for treating the relevant heating of malaria the history in more than 1,000 year, and except that traditional malaria effect, Herba Artemisiae Annuae class material also has the effect of others, as relieving asthma, anticancer, schistosomicide and to immune adjusting etc.The research of relevant arteannuin and derivant thereof both at home and abroad is a lot, but research range mainly concentrates on malaria, anticancer, antischistosomal effect and the mechanism thereof.
Artesunate (Artesunate), chemistry dihydroartemisinine-1 by name, 2-α-monomester succinate, molecular formula is C19O8 H28, molecular weight 384 is the derivants with antimalarial arteannuin of sesquiterpene structure.Artesunate is novel antimalarial, and than arteannuin, artesunate can be mixed with the dosage form of any routine, comprises solid and liquid form, and administration is very convenient.Artesunate is as antimalarial, the height of not only tiring, and be difficult for producing toleration.
To the existing report of the antiinflammatory action of arteannuin and derivant thereof, discover that arteannuin can suppress the activation of the synthetic and NF-κ B of LPS/TNF-α inductivity NO synthase; Artesunate to LPS and merge that interferon stimulates Turnover of Mouse Peritoneal Macrophages NO synthetic the obvious suppression effect arranged; Artesunate also has similar protective effect to the mouse macrophage RAW264.7 that LPS stimulates; Tan Yuqing etc. find that also Herba Artemisiae Annuae extract, arteannuin can reduce endotoxin shock mice LPO, ACP, endotoxin, TNF-α, P450 concentration, the increased SOD activity, reduce mouse death rate, prolong the mean survival time of mice, Mouse Liver, lung tissue form are also had the certain protection effect; Discovery arteannuin such as Wang Jun can suppress CpG ODN, LPS, heat-inactivated EC inducing cell discharges inflammatory factor, and the sepsis model mice is had remarkable protective effect.But whether artesunate has antibacterial action, whether can improve the drug resistance of bacterial antibiotic, strengthen antibiotic antibacterial efficacy and infection that antibacterial is caused whether effective, announcement does not appear in the newspapers.China is the main grown place of arteannuin and its derivant, because this curative effect of medication height, toxicity are low, WHO has classified arteannuin as the main medicine of world's treatment malaria, China is enlarging Herba Artemisiae Annuae plantation and arteannuin production, therefore widen the indication of artemisinin derivatives, be applied to the control of bacterial infective diseases, undoubtedly to the utilization that improves artemisinin derivatives, increase its economic worth and improve the drug resistance phenomenon that the antibiotic abuse caused significant.
Summary of the invention
The objective of the invention is to arteannuin and derivant thereof, particularly artesunate is applied to antibiotic field, not only widen the purposes of arteannuin and derivant thereof, particularly artesunate, and can alleviate the abuse of existing antibacterials and the drug resistance phenomenon that causes thereof.
Based on above-mentioned purpose, the technical scheme that the present invention uses is with a kind of use in conjunction of carrying out in a kind of and antibacterials in arteannuin and the derivant thereof, this application is not the application as the treatment of diseases method, but the application in the medicine that is equipped with treatment disease (antibiotic), wherein above-mentioned two kinds of components in proportions are 1: 1～4096: 1, last two kinds of compositions can be prepared into compositions, also can be two kinds of independent medicaments.Above-mentioned two kinds of compositions can produce Synergistic antimicrobial and render a service, and can reduce the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of antibacterials.
Above-mentioned bacterium comprises gram positive bacteria and gram negative bacteria; Said derivative comprises dihydroartemisinine, Artemether, arteether and artesunate; Above-mentioned antibacterials comprise the medicine of beta-lactam antibiotic (penicillins, cephalosporins, atypia beta-lactam antibiotic), aminoglycoside, clindamycin class, quinolones, in a preferred embodiment, above-mentioned antibacterials are benzylpenicillin, ampicillin, cefuroxime, cefpiramide, gentamycin, clindamycin, lomefloxacin, Gatifloxacin, ampicillin sodium-sulbactam sodium and safe energy, are preferably gentamycin, ampicillin sodium-sulbactam sodium, cefpiramide and Gatifloxacin.
Above-mentioned application is preferably the use in conjunction of artesunate and antibacterials.
The independent use of artesunate can suppress the growth of gram-negative bacteria and gram positive bacteria, with the antibacterial efficacy that can obviously strengthen antibacterials after the antibacterials use in conjunction, reduces the using dosage of antibacterials; Experiment shows that arteannuin and artesunate and antibacterials use in conjunction can obviously reduce antibacterial (comprising Grain-negative, positive bacteria) and attack mouse death rate, significantly suppresses mice serum LPS content and cytokine TNF-α and discharges.
This finder studies the antibiotic and antiinflammatory action of artesunate inside and outside.
At first, this research worker has been carried out pharmacology's analysis to the vitro antibacterial activity of this artesunate, and with the antibiotic use in conjunction after find that the independent use of artesunate can suppress the growth of gram-negative bacteria and gram positive bacteria, with can obviously strengthen antibiotic antibacterial efficacy after the antibiotic use in conjunction, reduce antibiotic using dosage; Secondly, artesunate and antibiotic use in conjunction and can obviously reduce antibacterial (comprising Grain-negative, positive bacteria) and attack mouse death rate significantly suppress mice serum LPS content and cytokine TNF-α release.
Therefore the present invention has widened the purposes of arteannuin and derivant thereof, particularly artesunate, has improved the antibacterial efficacy of antibacterials, the drug resistance phenomenon that has alleviated the antibacterials abuse and caused.
Description of drawings
Fig. 1 represents that artesunate works in coordination with gentamycin, cefpiramide, ampicillin sodium-sulbactam sodium to the antibacterial curve of the associating of escherichia coli ATCC35218;
Fig. 2 represents that artesunate works in coordination with ampicillin, Gatifloxacin, the ampicillin sodium-sulbactam sodium antibacterial curve of associating to the golden bacterium ATCC25923 of Portugal;
Fig. 3 represents that artesunate works in coordination with gentamycin, Gatifloxacin, ampicillin sodium-sulbactam sodium to the antibacterial curve of the associating of escherichia coli clinical separation strain.
The specific embodiment
The present invention is further illustrated below in conjunction with embodiment, but described embodiment only is used to illustrate the present invention rather than restriction the present invention.
Artesunate (AS) and antibacterials can produce Synergistic antimicrobial and render a service according to the proportioning in table 1, the table 2, reduce the minimum inhibitory concentration and the minimum bactericidal concentration of antibacterials.
Experimental example 1
This experimental example is to study artesunate and ten kinds of antibacterials MIC (minimum inhibitory concentration) and the MBC (minimum bactericidal concentration) to different bacterium
Adopt the micropore dilution method, the adjustment bacterial concentration is 105CFU/ml, is inoculated in the 96 hole aseptic culture plates, and 10 kinds of antibacterials such as artesunate and penicillin sodium, ampicillin etc. are 5.14mg/ml with the normal saline dilution respectively.Add various medicines to containing in the antibacterial culturing hole, doubling dilution successively, the ultimate density of the 1st～10 hole medicine is followed successively by 256,128,64,32,16,8,4,2,1,0.5ug/ml.Put 37 ℃ of incubators and hatch 24h and 48h, read the positive and negative control hole, negative control hole is limpid, positive control hole muddiness.Medicine is the lowest concentration of drug that suppresses antibacterial naked eyes visible growth behind the 24h to the MIC of antibacterial, and medicine is the lowest concentration of drug that suppresses antibacterial naked eyes visible growth behind the 48h to the MBC of antibacterial.This time experimental result shows the independent use of artesunate bacteria growing inhibiting fully, and has observed the inhibitory effect of different antibacterials to these five kinds of antibacterials.(seeing Table 3).
During independent use of 10 kinds of antibacterials of table 3 and AS to the MIC and the MBC of different bacterium
Experimental example 2
This experimental example is to study after artesunate and the antibacterials use in conjunction influence to escherichia coli MIC and MBC.
Adopt checkerboard type micropore dilution method, the adjustment bacterial concentration is 105CFU/ml, be inoculated in the 96 hole aseptic culture plates, will be to the antibacterials of five kinds of medium sensitivities of antibacterial dosage and the variable concentrations artesunate difference compatibility that is lower than MIC, the medicine behind the observation compatibility is to the MIC and the MBC of escherichia coli.Though this time experimental result shows that artesunate uses bacteria growing inhibiting fully separately,, illustrate that there is collaborative inhibitory effect the collaborative back of artesunate and antibacterials to antibacterial with the MIC and the MBC that can obviously reduce antibacterials after the antibacterials use in conjunction.(seeing Table 4).
Minimum inhibitory concentration after each antibacterials of table 4 and artesunate are collaborative
Experimental example 3
This experimental example is to study artesunate and the inhibition strength of the collaborative back of the different antibacterials of 1/2 MIC concentration to the escherichia coli growth.
The adjustment bacterial concentration is 106CFU/ml, and measuring bacterium liquid OD600 is 0.002 (1OD=5 * 10 8CFU/ml), with reference to experimental example 1 result, adding final concentration respectively separately is the antibacterials of 256 μ g/ml artesunate or 1/2 MIC concentration, and after adding the antibacterials of 256 μ g/ml artesunate and 1/2MIC concentration simultaneously, as for 37 ℃ of constant temperature shaking table 150rpm jolting, measure 1,3,5,7,9,12,16 respectively, the OD value of bacterium liquid during 24h, calculate the amount of bacteria of each time point.Result of study shows that artesunate 256 μ g/ml can make escherichia coli ATCC35218, the staphylococcus aureus ATCC25923 and the escherichia coli clinical separation strain speed of growth obviously slow down, compare with independent use antibacterials, united behind the artesunate obviously bacteria growing inhibiting, the inhibition degree is than using antibacterials or artesunate all strong (Fig. 1,2,3) separately.
Experimental example 4
This experimental example is to study artesunate use in conjunction antibacterials are attacked mice to gram-negative bacteria protective effect
Cleaning level 70 of the kind white mice in Kunming (Medical University Of Chongqing's Experimental Animal Center provides), body weight 19.9 ± 0.5g/ only, male and female half and half, be divided into matched group, artesunate group, escherichia coli group at random, escherichia coli+gentamycin group, artesunate (1.5,5,15mg/kg)+gentamycin+escherichia coli group.Every group of 10 animals.Contrast does not give any reagent; The artesunate of arteannuin group intramuscular injection 15mg/kg; The escherichia coli group gives 0.8 * 10 6The escherichia coli ATCC35218 that/kg lives; Escherichia coli+gentamycin group gives the escherichia coli ATCC35218 that lives after the gentamycin 0.5mg/kg intramuscular injection; Gentamycin+artesunate+escherichia coli group after giving artesunate, gives gentamycin and escherichia coli immediately.Give normal diet and drinking-water after administration finishes, observe mice ordinary circumstance and mortality rate (table 5) in 7 days.The result can obviously reduce mortality of mice after showing artesunate Synergistic antimicrobial medicine, shows the coordinating protection effect occurring after artesunate and the antibacterials associating.
The collaborative gentamycin of table 5 artesunate is attacked mice to escherichia coli ATCC35218 protective effect
Experimental example 5
This experimental example is to study artesunate use in conjunction antibacterials are attacked mice to gram-positive bacteria protective effect
Cleaning level 70 of the kind white mice in Kunming (Medical University Of Chongqing's Experimental Animal Center provides), body weight 19.9 ± 0.5g/ only, male and female half and half, be divided into matched group, artesunate group, golden Portugal bacterium group at random, gold Portugal bacterium+Gatifloxacin group, artesunate (5,15,45mg/kg)+Gatifloxacin+golden Portugal bacterium group.Every group of 10 animals.Contrast does not give any reagent; The artesunate of arteannuin group intramuscular injection 45mg/kg; Gold Portugal bacterium group gives 1.0 * 10 7The golden bacterium ATCC25923 of Portugal that/kg lives; Gold Portugal bacterium+Gatifloxacin group gives the golden bacterium ATCC25923 of Portugal that lives after the Gatifloxacin 0.5mg/kg intramuscular injection; Gatifloxacin+artesunate+golden Portugal bacterium group after giving artesunate, gives Gatifloxacin and golden Portugal bacterium immediately.Give normal diet and drinking-water after administration finishes, observe mice ordinary circumstance and mortality rate (table 6) in 7 days.The result can obviously reduce mortality of mice after showing artesunate Synergistic antimicrobial medicine, shows the coordinating protection effect occurring after artesunate and the antibacterials associating.
The collaborative Gatifloxacin of table 6 artesunate is attacked mice to the golden bacterium ATCC25923 of Portugal protective effect
Experimental example 6
This experimental example is to study artesunate is attacked mice to the deactivation escherichia coli protective effect.
Cleaning level 60 of the kind white mice in Kunming (Medical University Of Chongqing's Experimental Animal Center provides), body weight 19.9 ± 0.5g/ only, male and female half and half are divided into matched group, artesunate group, deactivation escherichia coli group at random, artesunate (5,15,45mg/ml)+deactivation escherichia coli group.Every group of 8 animals.Contrast does not give any reagent; The intramuscular injection of artesunate group gives the artesunate of 45mg/kg, 4h, and 24h, repeat administration is once behind the 48h; Deactivation escherichia coli group gives 1.25 * 10 11The deactivation escherichia coli ATCC35218 of/kg; Artesunate+deactivation escherichia coli group after intramuscular injection gives artesunate, gives deactivation escherichia coli ATCC35218 immediately, repeats administered intramuscular artesunate 1 time behind 4h, 24h, the 48h.Give normal diet and drinking-water after administration finishes, observe mice ordinary circumstance and mortality rate (table 7) in 7 days.The result shows that artesunate can reduce mortality of mice, shows that the mice that pro-inflammatory cytokine is attacked has protective effect.
Table 7 artesunate is attacked the protective effect of mice to the deactivation escherichia coli
*p＜0.5；**p＜0.01?vs?E.coli
Experimental example 7
This experimental example is to study artesunate the deactivation escherichia coli is attacked the influence of mice serum release of cytokines
Cleaning level 40 of the kind white mice in Kunming (Medical University Of Chongqing's Experimental Animal Center provides), body weight 19.9 ± 0.5g/ only, male and female half and half, be divided into matched group, artesunate group, deactivation escherichia coli group, artesunate (5,15,45mg/kg)+deactivation escherichia coli group at random, 3 of every treated animals.Contrast does not give any reagent.The intramuscular injection of artesunate group gives the artesunate of 45mg/kg; Artesunate (5,15,45mg/kg)+deactivation escherichia coli group gives to give deactivation escherichia coli ATCC35218 immediately behind the artesunate; Administration finishes and plucks eyeball behind the 4h and get the centrifugal immediately indwelling supernatant of blood and be stored in-20 ℃, cytokine TNF-α to be determined (table 8).The result shows that artesunate can significantly reduce the deactivation escherichia coli and attack the mice serum release of cytokines.
Table 8 artesunate reduction deactivation escherichia coli ATCC35218 attack mice serum cytokine TNF-α release (n=6, x ± SD)
*p＜0.5；**p＜0.01?vs?E.coli
The present invention has improved antibiotic antibacterial efficacy with the use in conjunction of artesunate and antibacterials, and artesunate and antibiotic use in conjunction can also be prevented, treat bacterial infection.
Those skilled in the art can be according to content of the present invention, and the associating of arteannuin and derivant and antibacterials can be reached effect of the present invention equally.
1. Artesunate be selected from a kind of in gentamycin, cefpiramide, ampicillin sodium-sulbactam sodium, ampicillin and the Gatifloxacin in the application of preparation in the antibacterials.
2. application according to claim 1, above-mentioned bacterium are gram positive bacteria or gram-negative bacteria.
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