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Background: Curcumin is a multi-functional polyphenol with anti-bacterial and anti-inflammatory effects and may have potential for treatment of periodontal diseases. The present study was conducted to examine the molecular basis of the anti-bacterial effect of curcumin against Porphyromonas gingivalis using metabolome analysis. Materials and Methods: P. gingivalis were incubated with 10 µg/mL curcumin, and then metabolites were analyzed with CE-TOF/MS. Expression levels of sigma factors were also evaluated using RT-PCR assays. The activities of dipeptidyl peptidases (DPPs) were assessed by examining the degradation reactions of MCA-labeled peptides. Results: The relative amounts of various glycogenic amino acids were significantly decreased when P. gingivalis was incubated with curcumin. Furthermore, the metabolites on the amino acid degradation pathway, including high-energy compounds such as ATP, various intermediate metabolites of RNA/DNA synthesis, nucleoside sugars and amino sugars were also decreased. Additionally, the expression levels of sigma-54 and sigma-70 were significantly decreased, and the same results as noted following nutrient starvation. Curcumin also significantly suppressed the activities of some DPPs, while the human DPP-4 inhibitors markedly inhibited the growth of P. gingivalis and activities of the DPPs. Conclusions: Curcumin suppresses the growth of P. gingivalis by inhibiting DPPs and also interferes with nucleic acid synthesis and central metabolic pathways, beginning with amino acid metabolism.

RESUMEN

New bis-quaternary ammonium compounds (bis-QACs) 3-(3-hydroxy-2-(hydroxymethyl)-2-{[(1-dodecylpyridinium-3-yl)oxy]methyl}propoxy)-1-dodecylpyridinium dibromide (3HHDMP-12) and 3,3'-[1,4-phenylenebis(oxy)]bis(1-dodecylpyridinium) dibromide (3PHBO-12) were compared with commonly-used antiseptics such as benzalkonium chloride (BAC), octenidine dihydrochloride (OCT), chlorhexidine digluconate (CHG) and polyhexamethylene biguanide (PHMB), to evaluate their potential to cause skin irritation. The cytotoxicity of these compounds in various cultured cells, as well as their effect on the expression of inflammatory cytokine genes such as IL-1α were evaluated. The cytotoxic effect of these bis-QACs on several types of human-derived cells was lower than that of common quaternary ammonium compounds (BAC and OCT), although the bis-QACs showed higher cytotoxicity than the biguanide-based compounds (CHG and PHMB). In addition, IL-1α mRNA expression was more strongly induced by BAC and OCT than by the new bis-QACs, at concentrations below the IC50 obtained in normal human epidermal keratinocytes. Furthermore, even at the actual therapeutic concentration, the new bis-QACs did not alter inflammatory cytokine mRNA expression or IL-1α secretion as demonstrated using the reconstructed human epidermis model LabCyte EPI-MODEL. The results suggested that the potential of 3PHBO-12 and 3HHDMP-12 to induce skin irritation is comparable to or less than that of existing antiseptics, and these bis-QACs may be useful antiseptics with few side effects.

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RESUMEN

　With the object of developing new biocides milder for human use than the current antiseptics, we synthesized a series of bis-quaternary ammonium compounds (bis-QACs). The antimicrobial activity of the newly synthesized bis-QACs and common biocides used as antiseptics was compared by examining minimum inhibitory concentrations and minimum bactericidal concentrations (MBCs). Moreover, the cytotoxicity of these compounds against human cells was determined to calculate the biocompatibility index (BI) of these compounds. BI was the ratio of the concentration of a biocide giving a 50% lethal effect on normal human epidermal keratinocytes to its MBC against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The commonly used antiseptics tested were benzalkonium chloride (BAC), octenidine dihydrochloride (OCT), chlorhexidine digluconate (CHG) and polyhexamethylene biguanide (PHMB). In comparison with these antiseptics, it was shown that some of new bis-QACs exhibited a wider and more potent antimicrobial spectrum than OCT. The cytotoxicity of these bis-QACs was equal or lower compared to that of the quaternary ammonium compounds (BAC and OCT), although these bis-QACs showed higher toxicity than the biguanide-based compounds (CHG and PHMB). Finally, the comparison of BIs revealed that new bis-QACs such as N-dodecyl ｛4,4'-(2,4,8,10-tetraoxaspiro［5.5］undecan-3,9-diyl) ｝dipyridinium dibromide (4TOSU-12), 3,3'-［1,4-Phenylenebis (oxy)］bis (1-dodecylpyridinium) dibromide (3PHBO-12) and 3-(3-Hydroxy-2-(hydroxymethyl)-2-｛［(1-dodecylpyridinium-3-yl) oxy］methyl｝propoxy)-1-dodecylpyridinium dibromide (3HHDMP-12) had equal or greater biocompatibility than the commonly used biocides tested. Thus, these results strongly suggested that 4TOSU-12, 3PHBO-12 and 3HHDMP-12 could be useful as antiseptics for topical application to the skin.

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Most critical instruments are not designed for heat sterilization and autoclaving. These items are usually treated with chemical agents such as peracetic acid(PAA), glutaraldehyde (GA) and ortho-phthalaldehyde (OPA). MTT assay is often used to evaluate the in vitro cytotoxicity of these chemical agents. In this study, disinfectants were allowed to come in direct contact with cells. Their cytotoxicity was evaluated based on cell viability and adhesive properties. The results obtained from the direct contact method were compared with those obtained from the conventional MTT assay wherein the disinfectants were added into a nutrient medium. It was found that the two methods yielded very different results, especially when aldehyde- and halogen-containing disinfectants were tested, and that toxicity may be underestimated in the MTT assay. Hence, it can be assumed that the direct contact assay is more accurate when evaluating the cytotoxicity of residual chemicals. It was also observed that the cytotoxicity of PAA was lower than that of GA and OPA.

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