Laboratory of Analytical Chemistry, University of Ioannina, Ioannina, Greece.
PLoS One. 2011 Apr 4;6(4):e18127. doi: 10.1371/journal.pone.0018127.
Traditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria.
METHODOLOGY/PRINCIPAL FINDINGS: In this study we report the identification and characterization of 4',5'-O-dicaffeoylquinic acid (4',5'-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3',5'-ODCQA, 4',5'-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4',5'-ODCQA with pump inhibitory activity whereas 3',5'-ODCQA was ineffective. These initial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology.
CONCLUSIONS/SIGNIFICANCE: These techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the biological information for the inhibitory capabilities of 4',5'-ODCQA and furthermore contributed evidence that CQAs show a preferential binding to Major Facilitator Super family efflux systems, a key multidrug resistance determinant in gram-positive bacteria.
传统抗生素由于致病菌中出现的多药耐药性而日益受到影响，因此正在研究一系列新型方法来控制微生物感染，作为潜在的替代治疗方法。一种可行的抗菌替代方法可能是将传统抗菌剂/抗生素与阻止多药外排系统的小分子（称为外排泵抑制剂）结合使用。从植物来源中通过基于生物测定的纯化和化合物结构测定，已经得到了一些针对革兰氏阳性菌的泵抑制剂。
方法/主要发现：在这项研究中，我们报告了从苦艾蒿中鉴定和表征 4'，5'-O-二咖啡酰奎宁酸（4'，5'-ODCQA）作为一种泵抑制剂，它有可能针对广泛的革兰氏阳性人致病菌的外排系统。基于液质联用等色谱联用技术，对酚类化合物（绿原酸、3'，5'-ODCQA、4'，5'-ODCQA）进行分离和鉴定，如柱后固相萃取结合核磁共振波谱和质谱。微生物药敏试验和泵底物的增效作用揭示了至少两种活性化合物；绿原酸具有较弱的抗菌活性，而 4'，5'-ODCQA 具有泵抑制活性，而 3'，5'-ODCQA 则无效。这些初步发现通过棋盘试验、黄连素积累外排试验、外排相关表型和临床分离株以及分子建模方法得到了进一步验证。
结论/意义：这些技术促进了从植物提取物中直接分析活性成分，并且大大减少了分析化合物所需的时间，而无需事先分离。对接构象的计算能量学支持了 4'，5'-ODCQA 抑制能力的生物学信息，并且进一步证明 CQAs 优先与革兰氏阳性菌中的主要易化因子超家族外排系统结合，这是多药耐药性的关键决定因素。
