Antimicrobial Activity, Biocompatibility and Anti-inflammatory Properties of Cetylpyridinium Chloride-based Mouthwash Containing Sodium Fluoride and Xylitol: An In Vitro Study.

PURPOSE: The use of a mouthwash as an adjunct to mechanical plaque removal may be useful to improve oral hygiene. In this study, cetylpyridinium chloride (CPC)-based mouthwashes containing sodium fluoride and xylitol (X-PUR Opti-Rinse 0.05% NaF and X-PUR Opti-Rinse 0.2% NaF) were evaluated for their antimicrobial activity against important oral pathogens associated with dental caries, periodontal disease, and candidiasis. Moreover, their biocompatibility and anti-inflammatory properties were assessed.
Materials and Methods: Antimicrobial activity was determined using a disk-diffusion assay, a microplate dilution assay, and the European standard protocols for antiseptics. Microbicidal properties were assessed against both planktonic and biofilm cultures. An oral epithelial cell model was used to evaluate the biocompatibility of mouthwashes and their ability to attenuate cytokine secretion.
Results: Using three different antimicrobial assays, the CPC-based mouthwashes were found to be highly active against the tested microorganisms. More specifically, the mouthwashes met the European Standard NF EN 1040 and NF EN 1275 defined as a log10 reduction ≥ 5 (≥ 99.999% killing) for bacteria and log10 reduction ≥ 4 (≥ 99.99% killing) for fungi, respectively. The CPC-based mouthwashes were also bactericidal against biofilms of S. mutans, S. sobrinus, and P. gingivalis. Using an oral epithelial cell model, the CPC-based mouthwashes were found to be less cytotoxic than a chlorhexidine-containing mouthwash used as control. Lastly, the CPC-based mouthwashes decreased the secretion of interleukin-6 and interleukin-8 by lipopolysaccharide-stimulated oral epithelial cells.
Conclusion: The CPC-based mouthwashes supplemented with sodium fluoride (0.05% or 0.2%) and xylitol (10%) were highly active against important oral pathogens. Moreover, using an oral epithelial cell model, these mouthwashes were found to be biocompatible and to exhibit anti-inflammatory activity.

Regulation of matrix metalloproteinase secretion by green tea catechins in a three-dimensional co-culture model of macrophages and gingival fibroblasts.

OBJECTIVES: Elevated levels of matrix metalloproteinases (MMPs) have been associated with the active phases of tissue and bone destruction in periodontitis, an inflammatory disease characterized by a significant breakdown of tooth support. In the present study, we used a three-dimensional (3D) co-culture model of macrophages and gingival fibroblasts to investigate the ability of a green tea extract and its major constituent epigallocatechin-3-gallate (EGCG) to regulate the secretion of MMP-3, -8, and -9. METHODS: The 3D co-culture model was composed of gingival fibroblasts embedded in a type I collagen matrix overlaid with macrophages. Two arbitrary ratios were tested. The ratio composed of 1 macrophage to 10 fibroblasts was used to mimic a slightly inflamed periodontal site while the ratio composed of 10 macrophages to 1 fibroblast was used to mimic a severely inflamed periodontal site. The 3D co-culture model was pre-treated for 2h with either the green tea extract or EGCG. It was then stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS). The model was also first stimulated with LPS for 2h and then incubated with the green tea extract or EGCG. The concentrations of secreted MMP-3, -8, and -9 were quantified by enzyme-linked immunoassays. RESULTS: When the 3D co-culture model was stimulated with A. actinomycetemcomitans LPS, the 10:1 ratio of macrophages to gingival fibroblasts was associated with a highest secretion of MMP-3 and -9 and, to a lesser extent, MMP-8, than the 1:10 ratio. Non-cytotoxic concentrations of the green tea extract or EGCG reduced the basal secretion levels of all three MMPs. A 2-h treatment with the green tea extract or EGCG prior to the stimulation with LPS resulted in a dose-dependent decrease in MMP secretion, with MMP-9 showing the most significant decrease. A decrease in MMP secretion was also observed when the green tea extract or EGCG was added following a 2-h stimulation with LPS. CONCLUSIONS: Our results suggested that green tea catechins, and more specifically EGCG, offer promising prospects for the development of a novel adjunctive treatment for periodontitis because of their ability to decrease the secretion of MMPs, which are important tissue-destructive enzymes produced by mucosal and immune cells.

Green tea catechins potentiate the effect of antibiotics and modulate adherence and gene expression in Porphyromonas gingivalis.

OBJECTIVES: A number of studies have brought evidence that green tea catechins may contribute to periodontal health. The objective of this study was to investigate the ability of a green tea extract and its principal constituent epigallocatechin-3-gallate (EGCG) to potentiate the antibacterial effects of antibiotics (metronidazole, tetracycline) against Porphyromonas gingivalis, and to modulate the adherence to oral epithelial cells and expression of genes coding for virulence factors and the high temperature requirement A (HtrA) stress protein in P. gingivalis. METHODS: A broth microdilution assay was used to determine the antibacterial activity of the green tea extract and EGCG. The synergistic effects of either compounds in association with metronidazole or tetracycline were evaluated using the checkerboard technique. A fluorescent assay was used to determine bacterial adherence to oral epithelial cells. The modulation of gene expression in P. gingivalis was evaluated by quantitative RT-PCR. The Vibrio harveyi bioassay was used for monitoring quorum sensing inhibitory activity. RESULTS: The MIC values of the green tea extract on P. gingivalis ranged from 250 to 1000 µg/ml, while those of EGCG ranged from 125 to 500 µg/ml. A marked synergistic effect on P. gingivalis growth was observed for the green tea extract or EGCG in combination with metronidazole. Both the green tea extract and EGCG caused a dose-dependent inhibition of P. gingivalis adherence to oral epithelial cells. On the one hand, green tea extract and EGCG dose-dependently inhibited the expression of several P. gingivalis genes involved in host colonization (fimA, hagA, hagB), tissue destruction (rgpA, kgp), and heme acquisition (hem). On the other hand, both compounds increased the expression of the stress protein htrA gene. The ability of the green tea extract and EGCG to inhibit quorum sensing may contribute to the modulation of gene expression. CONCLUSIONS: This study explored the preventive and therapeutic potential of green tea catechins against periodontal disease. In addition to inhibit growth and adherence of P. gingivalis, a green tea extract and its main constituent EGCG was found to decrease the expression of genes coding for the major virulence factors.

Black Tea Extract and Its Theaflavin Derivatives Inhibit the Growth of Periodontopathogens and Modulate Interleukin-8 and ß-Defensin Secretion in Oral Epithelial Cells.

Over the years, several studies have brought evidence suggesting that tea polyphenols, mostly from green tea, may have oral health benefits. Since few data are available concerning the beneficial properties of black tea and its theaflavin derivatives against periodontal disease, the objective of this study was to investigate their antibacterial activity as well as their ability to modulate interleukin-8 and human ß-defensin (hBD) secretion in oral epithelial cells. Among the periodontopathogenic bacteria tested, Porphyromonas gingivalis was found to be highly susceptible to the black tea extract and theaflavins. Moreover, our data indicated that the black tea extract, theaflavin and theaflavin-3,3'-digallate can potentiate the antibacterial effect of metronidazole and tetracycline against P. gingivalis. Using lipopolysaccharide-stimulated oral epithelial cells, the black tea extract (100 µg/ml), as well as theaflavin and theaflavin-3,3'-digallate (50 µg/ml) reduced interleukin-8 (IL-8) secretion by 85%, 79%, and 86%, respectively, thus suggesting an anti-inflammatory property. The ability of the black tea extract and its theaflavin derivatives to induce the secretion of the antimicrobial peptides hBD-1, hBD-2 and hBD-4 by oral epithelial cells was then evaluated. Our results showed that the black tea extract as well as theaflavin-3,3'-digallate were able to increase the secretion of the three hBDs. In conclusion, the ability of a black tea extract and theaflavins to exert antibacterial activity against major periodontopathogens, to attenuate the secretion of IL-8, and to induce hBD secretion in oral epithelial cells suggest that these components may have a beneficial effect against periodontal disease.

Dual Action of Myricetin on Porphyromonas gingivalis and the Inflammatory Response of Host Cells: A Promising Therapeutic Molecule for Periodontal Diseases.

Periodontitis that affects the underlying structures of the periodontium, including the alveolar bone, is a multifactorial disease, whose etiology involves interactions between specific bacterial species of the subgingival biofilm and the host immune components. In the present study, we investigated the effects of myricetin, a flavonol largely distributed in fruits and vegetables, on growth and virulence properties of Porphyromonas gingivalis as well as on the P. gingivalis-induced inflammatory response in host cells. Minimal inhibitory concentration values of myricetin against P. gingivalis were in the range of 62.5 to 125 µg/ml. The iron-chelating activity of myricetin may contribute to the antibacterial activity of this flavonol. Myricetin was found to attenuate the virulence of P. gingivalis by reducing the expression of genes coding for important virulence factors, including proteinases (rgpA, rgpB, and kgp) and adhesins (fimA, hagA, and hagB). Myricetin dose-dependently prevented NF-κB activation in a monocyte model. Moreover, it inhibited the secretion of IL-6, IL-8 and MMP-3 by P. gingivalis-stimulated gingival fibroblasts. In conclusion, our study brought clear evidence that the flavonol myricetin exhibits a dual action on the periodontopathogenic bacterium P. gingivalis and the inflammatory response of host cells. Therefore, myricetin holds promise as a therapeutic agent for the treatment/prevention of periodontitis.

Green tea extract and its major constituent epigallocatechin-3-gallate inhibit growth and halitosis-related properties of Solobacterium moorei.

BACKGROUND: Solobacterium moorei is a volatile sulfide compound (VSC)-producing Gram-positive anaerobic bacterium that has been associated with halitosis. The aim of this study was to investigate the effects of green tea extract and its major constituent epigallocatechin-3-gallate (EGCG) on growth and several halitosis-related properties of S. moorei. METHODS: A microplate dilution assay was used to determine the antibacterial activity of green tea extract and EGCG against S. moorei. Their effects on bacterial cell membrane integrity were investigated by transmission electron microscopy and a fluorescence-based permeability assay. Biofilm formation was quantified by crystal violet staining. Adhesion of FITC-labeled S. moorei to oral epithelial cells was monitored by fluorometry. The modulation of ß-galactosidase gene expression in S. moorei was evaluated by quantitative RT-PCR. RESULTS: The green tea extract as well as EGCG inhibited the growth of S. moorei, with MIC values of 500 and 250 µg/ml, respectively. Transmission electron microscopy analysis and a permeabilization assay brought evidence that the bacterial cell membrane was the target of green tea polyphenols. Regarding the effects of green tea polyphenols on the S. moorei colonization properties, it was found that biofilm formation on EGCG-treated surfaces was significantly affected, and that green tea extract and EGCG can cause the eradication of pre-formed S. moorei biofilms. Moreover, both the green tea extract and EGCG were found to reduce the adherence of S. moorei to oral epithelial cells. The ß-galactosidase activity of S. moorei, which plays a key role in VSC production, was dose-dependently inhibited by green tea polyphenols. In addition, EGCG at ½ MIC significantly decreased the ß-galactosidase gene expression. CONCLUSION: Our study brought evidence to support that green tea polyphenols possess a number of properties that may contribute to reduce S. moorei-related halitosis. Therefore, these natural compounds may be of interest to be used to supplement oral healthcare products.