Figure 4.

Carvacrol induces cytoplasmic content leakage. (A) Carvacrol causes the leakage of cytoplasmic nucleic materials from Streptococcus pyogenes in a concentration-dependent manner. Agarose gel (1%, w/v) electrophoresis and gel red staining of leaked nucleic acid from cell suspensions (1 × 10⁶, OD = 0.02) of ATCC 19615 and clinical isolate strains (a) exposed to MIC (125 μg/mL), 1/2 MIC, and 1/4 MIC of carvacrol or vehicle control (0.25% DMSO) over 24 h. 1 kb ladder as reference. (b) Agarose gel (0.8%, w/v) electrophoresis and ethidium bromide staining of genomic DNA recovery of the DNA by ethanol precipitation from bacteria suspension (OD = 0.6) of followed by 2 h carvacrol treatment. Carvacrol concentration is adjusted to the high bacterial density as MIC = 3750 μg/mL and 1 kb ladder were used as a reference. (B) Carvacrol causes leakage of lactate dehydrogenase (LDH) from ATCC 19615 and a clinical isolate of Streptococcus pyogenes. Overnight incubated cells were treated with different carvacrol or vehicle (0.25% DMSO) for 4 h. A standard lysis buffer (9% Triton X-100) was included as a positive control and was defined as 100% LDH release. The carvacrol-induced LDH release into culture media was measured using the Promega LDH Cytotoxicity Assay Kit. Data expressed as % LDH release and represented mean ± SE (n = 3), ***P < 0.001, compared among means (ANOVA, Tukey’s test). (C) Carvacrol does not cause the release of LDH from cultured human tonsil epithelial cells. The seeded human tonsil epithelium cells (TonEpiCs) for 24 h were treated with different concentrations of carvacrol or DMSO vehicle for 4 h. A standard lysis buffer was included as a positive control and defined as 100% LDH release. The carvacrol-induced LDH release into cell supernatant was measured using the Promega LDH Cytotoxicity Assay Kit. Data expressed as % LDH release and represented mean ± SEM (n = 3), ***P < 0.001, *P < 0.05, compared among means (ANOVA, Tukey’s test).