头花蓼内生真菌Gibberella intermedia抗多重耐药菌活性成分及其逆转细菌耐药性作用分析

Antibacterial Constituents from Endophytic Gibberella intermedia of Polygonum capitatum and Their Reversal Effect on Resistance of Multi-resistant Bacteria

目的：明确头花蓼内生真菌Gibberella intermedia抗菌活性成分，了解活性成分对临床耐药菌的逆转作用。方法：采用体外抗菌活性导向法及现代分离技术对头花蓼内生真菌G．intermedia的发酵物进行活性物质分离，并利用现代MS和NMR等波谱技术鉴定其化合物结构。采用96孔板法测试单体化合物对临床耐药菌的抗菌作用以筛选出活性成分，并在无抗菌作用的浓度下评估活性成分最低抑菌浓度（MIC）的影响，了解其逆转耐药菌的作用。结果：从头花蓼内生真菌G．intermedia的活性组分中分离得到6个化合物，分别为镰刀菌酸（1），吲哚-3-乙酸（2），对羟基苯乙酸（3），原儿茶酸（4），邻羟基苯乙酸（5）和对羟基苯甲醛（6）。化合物2-6均为从真菌G．intermedia代谢物中首次分离。化合物1对多重耐药大肠埃希菌、金黄色葡萄球菌和奇异变形杆菌的MIC分别为31．3，125，62．5mg·L^-1。且化合物1具有逆转其耐药性的作用，在其1／8M[C的浓度下，能使左氧氟沙星和环丙沙星对临床耐药大肠埃希菌的MIC分别降低4倍和2倍；在其1／4MIC的浓度下，能使左氧氟沙星和环丙沙星对耐药菌奇异变形杆菌的MIC分别降低了2倍和4倍，且对临床耐药金黄色葡萄球菌的MIC值均降低了2倍。结论：镰刀菌酸为头花蓼内生真菌G．intermedia抗耐药菌主要活性成分，并对多重耐药菌大肠埃希菌、奇异变形杆菌和金黄色葡萄球菌具有逆转耐药性作用。该研究为治疗多重耐药菌尿路感染和提高喹诺酮类抗生素在尿路感染中的疗效奠定了基础。

Objective： To clarify the antimicrobial constituents of endophytic Gibberella intermedia from the Polygonum capitatum and investigate their reversal effect on the resistance of multi-resistant bacteria. Method： The active components of G. intermedia were isolated and purified by modem separation techniques on the basis of bioassay-guided method. Their structures were identified by spectroscopic methods including modern mass spectrometry （MS） and nuclear magnetic resonance （NMR） analysis. The antibacterial activity was tested on a 96- well plate to screen the active antimicrobial constituents, while their reversal effects on the resistance of multi- resistant bacteria were determined by evaluating their effect on the minimum inhibitory concentration （MIC） of the quinolones. Result： Six compounds were isolated from the extracted metabolites of G. intermedia, and their structures were identified as fusaric acid （ 1 ）, indole-3-acetic acid （ 2 ）, p-hydroxyphenylacetic acid （3）, protocatechuic acid （4） , o-hydroxyphenylacetic acid （5） , and p-hydroxybenzaldehyde （6）. Compounds 2-6 were isolated from metabolites of G. intermedia for the first time. The MICs of compound 1 against clinically resistant Escherichia coli, Staphylococcus aureus and Proteus mirabilis were 31.3, 125, and 62.5 mg·L-1, respectively. Compound 1 showed the reversal effect on drug resistance. In the presence of 1/8 MIC of compound 1, the MICs of levofloxacin and eiprofloxacin against the resistant E. coli were reduced by g and 2 folds respectively. In the presence of 1/4 MIC of compound 1, the MICs of levofloxacin and ciprofloxacin against the resistant P. mirabilis were reduced by 2 and 4 folds respectively, and 2 folds respectively against the resistant S. aureus. Conclusion： Fusaric acid was the main antimicrobial ingredient of G. intermedia, showing reversal effects on the muhidrug- resistant E. coli, P. mirabilis and S. aureus. This study laid the foundation for treatment of multi-drug resistant urinary tract infection and improving the treatment efficiency of quinolones in urinary tract infection.