title: Synergism of coumarins from the Chinese drug Zanthoxylum nitidum with antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA).
abstract: BACKGROUND
  Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious therapeutic challenge in current clinic and new drug development. Natural coumarins have diverse bioactivities and the potential of resistance modifying effects.
  
  
  OBJECTIVE
  This study is to present in-depth evaluations of in vitro antimicrobial activities of four natural coumarins 5-geranyloxy-7-methoxycoumarin (Gm, 1), (5,7-dimethoxy-8-prenyloxycoumarin (artanin, Ar, 2)), isopimpinellin (Is, 3) and phellopterin (Ph, 4) from Zanthoxylum nitidum (Roxb.) DC. (Rutaceae) extracts, focusing on their potential restoration the activity of conventional antibacterial agents against clinical MRSA strains.
  
  
  METHODS
  Bioactivity-guided fractionation and spectral analyses were used to isolate the coumarins and identify the structures, respectively. The double broth microdilution method was used to assay the coumarins' alone activity. The classic checkerboard microdilution and dynamic time-killing methods were used to evaluate combinatory effects.
  
  
  RESULTS
  The four plant coumarins Gm (1), Ar (2), Is (3) and Ph (4) were isolated and identified from Z. nitidum extracts. Coumarins 1-4 displayed promising inhibition against both MSSA and MRSA with minimal inhibitory concentrations (MICs) of 8-64µg/ml, but very weak against Gram-negative pathogen and yeast with MICs of 256 to ≥1024µg/ml. The geranyloxy and prenyloxy substitutions showed to be more active than the methoxy substitution on the coumarin skeletons. 1-4 also showing different extent of synergism with a total of eight conventional antibacterial agents, i.e. chloramphenicol (CL), gentamicin (CN), fosfomycin (FF), levofloxacin (LE), minocycline (MI), piperacillin/tazobactam (P/T), teicoplanin (TE) and vancomycin (VA) against ten clinical MRSA strains. Four to ten of the tested MRSA strains showed bacteriostatic synergy in the eleven combinations. The anti-MRSA modifying effects were related to different arrangement in the combinations with fractional inhibitory concentration indices (FICIs) from 0.187 to 1.125 and the three combinations CN (Is), CL (Ph) and MI (Gm) were the best ones. The enhancement of activity was also shown by 2-64 of dose reduction indices (DRIs) of the combined MICs, with VA (Ph) combination resulted the biggest DRI. The resistance of MRSA to antibacterial agents could be reversed in the combinations of CL (Gm or Ph), LE (Ph) and MI (Is) following the Clinical and Laboratory Standards Institute (CLSI) criteria. Six combinations P/T (Gm), TE (Ar), CN (Is), VA (Ph) and CL (Gm or Ph) also showed bactericidal synergy with Δlog10CFU/ml >2 at 24h incubation.
  
  
  CONCLUSIONS
  The coumarins showed high potentiating effects of the antibacterial agents against multi-drug resistant SA. The resistance reversal effect of CL, LE and MI warrants further pharmacological investigation on combinatory therapy for the sake of fighting against MRSA infections.
authors.affiliation: Research Center for Natural Medicines, Kunming General Hospital of Chengdu Military Command, Kunming 650032, China. Electronic address: zuoguoying@263.net.
authors.forename: Guo-Ying
authors.initials: GY
authors.lastname: Zuo
authors.affiliation: Research Center for Natural Medicines, Kunming General Hospital of Chengdu Military Command, Kunming 650032, China; School of Pharmacy, Kunming Medical University, Kunming 550004, China.
authors.forename: Chun-Juan
authors.initials: CJ
authors.lastname: Wang
authors.affiliation: School of Basic Medical Sciences, Yunnan Traditional Chinese Medical College, Kunming 650500, China. Electronic address: hanzjn@126.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Han
authors.affiliation: School of Basic Medical Sciences, Yunnan Traditional Chinese Medical College, Kunming 650500, China.
authors.forename: Yu-Qing
authors.initials: YQ
authors.lastname: Li
authors.affiliation: Research Center for Natural Medicines, Kunming General Hospital of Chengdu Military Command, Kunming 650032, China.
authors.forename: Gen-Chun
authors.initials: GC
authors.lastname: Wang
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D003374:Coumarins / Q000494:pharmacology*; D004357:Drug Synergism; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D008712:Methicillin / Q000494:pharmacology; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects*; D008730:Methoxsalen / Q000031:analogs & derivatives / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D013211:Staphylococcus aureus / Q000187:drug effects; D032106:Zanthoxylum / Q000737:chemistry*
chemical_list: C442483:5-geranyloxy-7-methoxycoumarin; C436726:5-hydroxy-6-methoxy-7-(3-methyl-but-2-enyloxy)-2H-1-benzopyran-2-one; D000900:Anti-Bacterial Agents; D003374:Coumarins; D004365:Drugs, Chinese Herbal; D007093:Imidazoles; C517283:coumarin 7; C104601:phellopterin; C039409:pimpinellin; D008712:Methicillin; D008730:Methoxsalen
keywords: Antibacterial agents; Antimicrobial activity; Coumarin; MRSA; Synergy