| Summary: | Monterey cypress (<i>Cupressus macrocarpa</i>) is a decorative plant; however, it possesses various pharmacological activities. Therefore, we explored the phytochemical profile of <i>C. macrocarpa</i> root methanol extract (CRME) for the first time. Moreover, we investigated its antidiarrheal (in vivo), antibacterial, and antibiofilm (in vitro) activities against <i>Salmonella enterica</i> clinical isolates. The LC-ESI-MS/MS analysis of CRME detected the presence of 39 compounds, besides isolation of 2,3,2″,3″-tetrahydro-4′-<i>O</i>-methyl amentoflavone, amentoflavone, and dihydrokaempferol-3-<i>O</i>-α-<span style="font-variant: small-caps;">l</span>-rhamnoside for the first time. Dihydrokaempferol-3-<i>O</i>-α-<span style="font-variant: small-caps;">l</span>-rhamnoside presented the highest antimicrobial activity and the range of values of MICs against <i>S. enterica</i> isolates was from 64 to 256 µg/mL. The antidiarrheal activity of CRME was investigated by induction of diarrhea using castor oil, and exhibited a significant reduction in diarrhea and defecation frequency at all doses, enteropooling (at 400 mg/kg), and gastrointestinal motility (at 200, 400 mg/kg) in mice. The antidiarrheal index of CRME increased in a dose-dependent manner. The effect of CRME on various membrane characters of <i>S. enterica</i> was studied after typing the isolates by ERIC-PCR. Its impact on efflux and its antibiofilm activity were inspected. The biofilm morphology was observed using light and scanning electron microscopes. The effect on efflux activity and biofilm formation was further elucidated using qRT-PCR. A significant increase in inner and outer membrane permeability and a significant decrease in integrity and depolarization (using flow cytometry) were detected with variable percentages. Furthermore, a significant reduction in efflux and biofilm formation was observed. Therefore, CRME could be a promising source for treatment of gastrointestinal tract diseases.
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