The Thai herbal formulation 'Ya-Pit-Samut-Noi' containing Nigella sativa (seed), Piper retrofractum (fruit), Punica granatum (pericarp), and Quercus infectoria (nutgall) has long been traditionally used to treat diarrhea or bloody mucous diarrhea. Scientific information is needed to support its therapeutic effects and is very important for traditional drug development.This study aimed to evaluate the antibacterial activities of Ya-Pit-Samut-Noi against diarrhea-causing bacteria and determine its effects on bacterial virulence factors and in vivo acute toxicity.Ethanol and water extracts of Ya-Pit-Samut-Noi (mixed plants) and its plant components were prepared. The agar diffusion method was used for preliminary screening of antibacterial activity of the extracts against diarrhea-causing bacteria including Staphylococcus aureus, Vibrio cholerae, and Vibrio parahaemolyticus. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were investigated using the broth microdilution method. The effects on bactericidal activity, bacterial cell walls, and membranes were examined by time-kill, lysis, and leakage assays, respectively. The effects on bacterial virulence factors including quorum-sensing system, biofilm production, and swarming motility were determined. Phytochemical screening was carried out to approximately identify the group of chemical compounds present in the formulation extracts. Acute toxicity study was conducted by a single oral dose of 2000 mg/kg body weight in Wistar albino rats.The ethanol and water extracts of Ya-Pit-Samut-Noi and Quercus infectoria demonstrated antibacterial properties against all bacterial strains revealed by zones of inhibition ranging from 7.0-24.5 mm. The ethanol and water extracts of Ya-Pit-Samut-Noi and Quercus infectoria produced good bacteriostatic activity against V. parahaemolyticus (n = 11) with an MIC range of 7.81-250 μg/ml. Only the ethanol extract of Ya-Pit-Samut-Noi produced MIC and MBC values less than or equal to 1000 μg/ml against all V. parahaemolyticus. Based on time-kill study, no surviving V. parahaemolyticus (ATCC 17802 and 5268) cells were detected within 6-12 h after treatment with the ethanol extract of Ya-Pit-Samut-Noi at MBC-4MBC concentrations. The V. parahaemolyticus ATCC 17802 cells treated with the ethanol extract of Ya-Pit-Samut-Noi demonstrated no lysis and leakage through the bacterial membrane was not significantly observed. At low concentrations (0.125-0.25 μg/ml) the ethanol extract of Ya-Pit-Samut-Noi inhibited violacein production by Chromobacterium violaceum DMST 21761 without affecting bacterial growth. The ethanol (31.25-62.5 μg/ml) and water (31.25-250 μg/ml) extracts of Ya-Pit-Samut-Noi inhibited biofilm production by S. aureus. The ethanol and water extracts of Ya-Pit-Samut-Noi at 1000 μg/ml reduced the swarming motility of Escherichia coli O157: H7 by 74.98% and 52.65%, respectively. Tannins and terpenoids were detected in both the ethanol and water extracts. Flavonoids were present only in the ethanol extract. Alkaloids and antraquinones were not noticed in either extract. In the acute toxicity study, there were no significant changes in hematological and biochemical parameters nor were adverse effects on mortality, general behaviors, body weight, or organ weights detected.The scientific evidence from this study supported the therapeutic effects and safety of the traditional Thai herbal formulation 'Ya-Pit-Samut-Noi' which has been used as an alternative treatment for gastrointestinal infections.
