Preclinical evaluation of 6-Gingerol in modulating gut microbiota and SCFAs to mitigate Clostridium difficile-associated diarrhea in mice

<i>Clostridium difficile</i> associated diarrhea (CDAD) is a growing healthcare concern with limited effective treatments. 6-Gingerol, a major bioactive compound in ginger, exhibits notable antibacterial and anti-inflammatory properties, making it a potential alternative therapy. This study combines <i>in vitro</i> and <i>in vivo</i> approaches to evaluate its efficacy against CDAD. <i>In vitro</i> assays determined the half-maximal inhibitory concentration (IC₅₀) and minimum inhibitory concentration (MIC) of 6-Gingerol against <i>C. difficile</i>, which were 61.99 μM and 173.3 μM, respectively, indicating direct antibacterial activity. <i>In vivo</i>, a mouse model of CDAD was used to assess the therapeutic effects of 6-Gingerol. Outcomes included clinical symptoms, <i>C. difficile</i> load, inflammation, intestinal barrier integrity, gut microbiota composition, and short-chain fatty acids (SCFAs) levels. The results showed that in the CDAD mouse model, high-dose 6-Gingerol significantly alleviated CDAD symptoms, reduced <i>C. difficile</i> load (<i>P</i> < 0.001), improved gut barrier function, and suppressed intestinal inflammation. Although it did not notably increase microbial diversity, 6-Gingerol modulated gut microbiota structure-markedly increasing beneficial bacteria such as <i>Lactobacillus acidophilus</i> (<i>P</i> < 0.01) and <i>Bacteroides thetaiotaomicron</i>, while reducing harmful bacteria including <i>Klebsiella pneumoniae</i> and <i>Proteus mirabilis</i>. Targeted quantification revealed restored levels of key SCFAs, particularly acetate (<i>P</i> < 0.001), butyrate (<i>P</i> < 0.01), and valerate (<i>P</i> < 0.001), which are closely linked to gut health and recovery from CDAD. In summary, 6-Gingerol exerts therapeutic effects against CDAD through direct inhibition of <i>C. difficile</i>, regulation of gut microbiota, restoration of SCFA levels, and protection of the intestinal barrier, highlighting its potential as a novel natural treatment for CDAD.