title: The anthraquinone rhein exhibits synergistic antibacterial activity in association with metronidazole or natural compounds and attenuates virulence gene expression in Porphyromonas gingivalis.
abstract: OBJECTIVE
  Rhein is a major anthraquinone found in rhubarb root. As a continuation of our ongoing studies aimed to identify beneficial properties of this anthraquinone for periodontal disease, in this study, we investigated the ability of rhein to (i) exhibit antibacterial synergy towards the periodontopathogen Porphyromonas gingivalis when used in combination with metronidazole or polyphenols belonging to different families, and (ii) attenuate virulence factor gene expression in P. gingivalis.
  
  
  METHODS
  The minimal inhibitory concentrations (MIC) of compounds under investigation were determined by a broth microdilution assay. The synergistic effects of rhein in association with either metronidazole or polyphenols of various families were evaluated using the chequerboard technique to determine the fractional inhibitory concentration index (FICI). The effect of rhein on virulence factor gene expression in P. gingivalis was determined by quantitative RT-PCR.
  
  
  RESULTS
  Rhein showed a MIC of 2.5 μg/mL, which was similar to that of metronidazole. Except for the association with epigallocatechin-3-gallate that gave an additive effect, all the other combinations (licochalcone A, glabridin, myricetin, and metronidazole) resulted in synergistic effects. The strongest synergy was observed when rhein was used in association with myricetin (FICI=0.12) and licochalcone A (FICI=0.19). At a sub-MIC of rhein (0.5 μg/mL), a significant decrease in the expression of fimA, hagA, and hagB genes, which are involved in host colonization, was observed. Moreover, the expression of rgpA and kgp, two protease genes related to inactivation of host defense mechanisms, tissue destruction, and nutrient acquisition, was also down-regulated.
  
  
  CONCLUSIONS
  The data presented in our study indicate that rhein possessed antibacterial activity, which can be potentiated in combination with metronidazole or other polyphenols. In addition, rhein can impair the pathogenicity of P. gingivalis by reducing transcription of genes coding for important virulence factors.
authors.affiliation: Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine Dentaire, Université Laval, Quebec City, Quebec, Canada.
authors.forename: Jabrane
authors.initials: J
authors.lastname: Azelmat
authors.affiliation: Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine Dentaire, Université Laval, Quebec City, Quebec, Canada.
authors.forename: Jade Fournier
authors.initials: JF
authors.lastname: Larente
authors.affiliation: Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine Dentaire, Université Laval, Quebec City, Quebec, Canada. Electronic address: Daniel.Grenier@greb.ulaval.ca.
authors.forename: Daniel
authors.initials: D
authors.lastname: Grenier
mesh_terms: D000880:Anthraquinones / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004357:Drug Synergism; D015870:Gene Expression; D008795:Metronidazole / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D059808:Polyphenols / Q000494:pharmacology; D016966:Porphyromonas gingivalis / Q000187:drug effects* / Q000235:genetics* / Q000472:pathogenicity; D014774:Virulence; D037521:Virulence Factors
chemical_list: D000880:Anthraquinones; D000900:Anti-Bacterial Agents; D059808:Polyphenols; D037521:Virulence Factors; D008795:Metronidazole; C020491:rhein
keywords: Anthraquinone; Periodontitis; Porphyromonas gingivalis; Rhein; Synergy