Table of Contents Author Guidelines Submit a Manuscript
Evidence-Based Complementary and Alternative Medicine
Volume 2017 (2017), Article ID 2850947, 10 pages
https://doi.org/10.1155/2017/2850947
Research Article

Antibiofilm and Anti-Inflammatory Activities of Houttuynia cordata Decoction for Oral Care

1Department of Pharmacognosy, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima 770-8505, Japan
2Department of Oral Microbiology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima 770-8504, Japan
3Department of Conservative Dentistry, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima 770-8504, Japan

Correspondence should be addressed to Keiji Murakami; pj.ca.u-amihsukot@imakarumk

Received 29 March 2017; Revised 23 July 2017; Accepted 10 September 2017; Published 16 October 2017

Academic Editor: Subash C. Gupta

Copyright © 2017 Yasuko Sekita et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Dental biofilms that form in the oral cavity play a critical role in the pathogenesis of several infectious oral diseases, including dental caries, periodontal disease, and oral candidiasis. Houttuynia cordata (HC, Saururaceae) is a widely used traditional medicine, for both internal and external application. A decoction of dried HC leaves (dHC) has long been consumed as a health-promoting herbal tea in Japan. We have recently reported that a water solution of HC poultice ethanol extract (wHCP) exerts antimicrobial and antibiofilm effects against several important oral pathogens. It also exhibits anti-inflammatory effects on human keratinocytes. In our current study, we examined the effects of dHC on infectious oral pathogens and inflammation. Our results demonstrated that dHC exerts moderate antimicrobial effects against methicillin-resistant Staphylococcus aureus (MRSA) and other oral microorganisms. dHC also exhibited antibiofilm effects against MRSA, Fusobacterium nucleatum (involved in dental plaque formation), and Candida albicans and inhibitory effects on interleukin-8, CCL20, IP-10, and GROα productions by human oral keratinocytes stimulated by Porphyromonas gingivalis lipopolysaccharide (a cause of periodontal disease), without cytotoxic effects. This suggests that dHC exhibits multiple activities in microorganisms and host cells. dHC can be easily prepared and may be effective in preventing infectious oral diseases.

1. Introduction

Dental biofilms that form in the oral cavity play a critical role in the pathogenesis of numerous infectious oral diseases, including periodontal disease. This can be due to the absorption of antimicrobial and antiseptic drugs and development of resistance to host immune cells [13]. Predominant fungi such as Candida albicans in the oral cavity can also contribute to the development of infectious oral diseases ranging from denture stomatitis [4, 5] to life-threatening invasive infections, including aspiration pneumonia. This is particularly apparent in immunocompromised and elderly patients [68]. We have previously reported a higher prevalence of Candida spp., Pseudomonas aeruginosa, and Staphylococcus spp. in the oropharyngeal microflora of patients with cerebrovascular infarction and dysphagia [6]. Reducing adherence and biofilm formation by oral microorganisms can contribute to the prevention of chronic oral infections that may lead to potentially severe, systemic opportunistic diseases, particularly in the elderly [9, 10].

Oral keratinocytes play an important role as the first physical barrier to bacterial invasion by organizing the local innate immune system against colonizing microorganisms. They also secrete several proinflammatory mediators (i.e., chemokines and cytokines) in response to various stimuli, including microbial infections and chemical or thermal irritations [1113]. These mediators ultimately cause periodontal inflammation. Lipopolysaccharide (LPS) from Gram-negative bacteria, such as the important periodontal pathogen Porphyromonas gingivalis, upregulates the production of various proinflammatory mediators via signal cascades in the gingival epithelium. These include interleukin-8 (IL-8), CCL20, IFN-γ-inducible protein 10 (IP-10), and growth related oncogene-α (GROα) [1422]. Therefore, the development of oral care products that reduce biofilm formation and subsequent proinflammatory responses is essential for improving health and preventing disease.

Previous studies have demonstrated that extracts from medicinal plants exhibit various pharmacological activities including antimicrobial effects [2326], antiadherence effects against oral microorganisms [2729], and anti-inflammatory effects [3033]. Houttuynia cordata Thunb. (HC, Saururaceae) is widely used as a traditional medicine, both internally and externally [34]. However, evidence to indicate that HC extract exerts pharmacological effects against oral microorganisms is limited. We have focused on identifying and characterizing any activity of HC against infectious diseases caused by oral microorganisms. We have recently reported that a HC poultice ethanol extract (eHCP) exerted antibacterial effects against cutaneous infection-related bacteria and anti-inflammatory effects on human keratinocytes [34]. However, caution is required when suggesting that eHCP could be applied to oral care because ethanol found in mouthwashes has been suggested to increase the risk of oral cancer [35].

Fortunately, we have also shown that a water solution of eHCP (wHCP) exhibits antimicrobial and antibiofilm effects on oral microorganisms and anti-inflammatory effects on oral keratinocytes [36]. A decoction of dried HC leaves (dHC), commonly consumed as a health-promoting herbal tea in Japan, is simpler to prepare than wHCP. We therefore examined the antimicrobial and antibiofilm effects of dHC on several important infectious oral pathogens and investigated the anti-inflammatory effects of dHC on P. gingivalis LPS-stimulated human oral keratinocytes.

2. Material and Methods

2.1. Plant Materials and Sample Preparation

HC used in this study was collected in Kochi City and identified by Dr. K. Fujikawa (the Kochi Prefectural Makino Botanical Garden, Kochi, Japan). Voucher specimens (FOS-007536, FOS-007537, and FOS-010389) were also deposited here.

2.2. Preparation of dHC

dHC was prepared as follows: 3 g of dried HC leaves was decocted with 130 mL of sterile purified water at 90–95°C for 30 min (EK-SA 10, ZOJIRUSHI, Osaka, Japan). The decoction was then centrifuged for 15 min at 1,500 ×g. After centrifugation, the clear supernatant layer was filtered through a 0.45-μm filter and stored at 4°C until being assayed.

2.3. Flavonoid Glycosides

Quercitrin and rutin were purchased from Sigma-Aldrich (St. Louis, MO) and Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), respectively. Isoquercitrin and hyperin were isolated from the aerial parts of Hypericum sikokumountanum [37]. Flavonoid glycosides were dissolved in dimethyl sulfoxide (DMSO, nacalai tesque, Kyoto, Japan).

2.4. Bacterial Strains and Growth Conditions

The bacterial strains used in this study are shown in Table 1. P. aeruginosa was grown in Muller-Hinton broth (Becton Dickinson, Sparks, MD, USA) that was supplemented with 50 μg/mL CaCl2 and 25 μg/mL MgCl2. Methicillin-resistant Staphylococcus aureus (MRSA) strains were grown in Muller-Hinton broth supplemented with 25 μg/mL CaCl2, 12.5 μg/mL MgCl2, and 2% NaCl [38]. Streptococcus spp. were grown anaerobically in brain heart infusion (Becton Dickinson). Fusobacterium nucleatum and P. gingivalis were grown anaerobically in brain heart infusion supplemented with 5 μg/mL hemin and 0.5 μg/mL menadione. Aggregatibacter actinomycetemcomitans was grown anaerobically in Todd Hewitt Broth (OXOID Ltd., Hampshire, UK). C. albicans was grown in Sabouraud dextrose medium composed of 10 g/L peptone and 40 g/L glucose. For biofilm formation assays, trypticase soy broth (Becton Dickinson) supplemented with 5 μg/mL hemin and 0.5 μg/mL menadione, trypticase soy broth supplemented with 0.3% glucose, and yeast nitrogen base medium at pH 7 containing 2.5 mmol/L N-acetylglucosamine [39] were used for F. nucleatum, MRSA-T31, and C. albicans, respectively.

Table 1: Bacterial strains.
2.5. Susceptibility Assay

The minimum inhibitory concentration (MIC) of dHC was assessed using a microbial broth dilution method. Approximately 106 colony-forming units (CFU)/mL of each bacterial culture were inoculated into 100 μL of medium containing a twofold serial dilution of dHC in 96-well plates (TPP, Trasadingen, Switzerland) and incubated either anaerobically (for Streptococcus spp., A. actinomycetemcomitans, F. nucleatum, and P. gingivalis) or aerobically (for MRSA T31, MRSA COL, P. aeruginosa, and C. albicans) at 37°C for 20 or 48 h. The MIC was defined as the lowest concentration that showed no bacterial growth.

2.6. Biofilm Formation Assay

A crystal violet biofilm assay was performed to quantify the biofilm mass as previously described [40]. A 2-μL (107 CFU/mL) sample of MRSA T31 or C. albicans CAD1 in the stationary phase or a 5-μL (107 CFU/mL) sample of F. nucleatum JCM8532 in the stationary phase was transferred into a 96-well plate (Cellstar, Greiner Bio-One, Frickenhausen, Germany) from the primary 150 L suspensions of broth or media. dHC was then added to a final concentration of 10%. Quercitrin, isoquercitrin, hyperin, and rutin were added to a final concentration of 200 μg/mL. Bacterial suspensions were incubated either anaerobically (for F. nucleatum) at 37°C for 24 h or aerobically (for MRSA T31 and C. albicans) at 37°C for 6 and 24 h. For the positive control, 2 or 1 μg/mL of cetylpyridinium chloride (CPC) (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was used [41]. For the negative control, 10% distilled H2O was used. After incubation, any biofilms that formed were washed with purified water twice without disturbing the adherent biofilm. They were then stained with 150 μL of 0.1% crystal violet at 25°C for 10 min. Excess staining was removed by gentle washing with purified water. After drying, stained biofilms were extracted from each well by adding 150 μL of ethanol, and the absorbance of the extract from the stained biofilm was measured at 595 nm using a microplate reader (model 680; Bio-Rad Laboratories, Hercules, CA, USA).

2.7. Cell Culture

RT-7 cells, an immortalized human keratinocyte cell line kindly provided by Dr. Kamata (Hiroshima University, Hiroshima, Japan) [42], were cultured in Keratinocyte-SFM (Gibco BRL, Gaithersburg, MD, USA) supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco BRL), at 37°C in a water-saturated atmosphere of 95% air and 5% CO2. Confluent monolayers were cultured with 1 μg/mL P. gingivalis LPS (InvivoGen, San Diego, CA, USA), with and without the addition of 1% dHC or 50 μg/mL of quercitrin, isoquercitrin, hyperin, or rutin.

2.8. Lactate Dehydrogenase (LDH) Cytotoxicity Assay

The effects of dHC on cell cytotoxicity were assessed using a LDH assay. Confluent RT-7 cell monolayers in a 24-well plate were cultured in Keratinocyte-SFM medium supplemented with 10% and 1% dHC at 37°C for 24 h in a water-saturated atmosphere of 95% air and 5% CO2. As a positive control, RT-7 cells were treated with 0.1% Triton X-100 at 25°C for 10 min. In the cytotoxicity assay, the levels of LDH released into the recovered cell culture supernatants were measured using an LDH cytotoxicity assay kit (Cayman Chemical, Ann Arbor, MI, USA) following the manufacturer’s instructions. Absorbance was measured at 490 nm using a microplate reader (Bio-Rad Laboratories).

2.9. Enzyme-Linked Immunosorbent Assay

Enzyme-linked immunosorbent assay (ELISA) kits were used to quantify the levels of IL-8, CCL20, IP-10, and GROα (R&D Systems, Minneapolis, MN, USA) in cell culture supernatants.

2.10. Statistical Analysis

All statistical analyses were performed using an unpaired Student’s -test. Differences were considered significant when the probability value was less than 5%  .

3. Results

3.1. Moderate Antimicrobial Effects of dHC

We examined the antimicrobial effects of dHC against several oral microorganisms. As shown in Table 2, dHC exerted a moderate antimicrobial effect against MRSA T31, MRSA COL, S. intermedius, S. mitis, F. nucleatum, and P. gingivalis (MIC; 375–1500 μg/mL).

Table 2: MIC of dHC.
3.2. Antibiofilm Effects of dHC

We next investigated whether dHC had any antibiofilm effects. In this experiment, we used a culture of the MRSA T31 clinical isolate. This strain was selected because MRSA T31 exhibits increased biofilm formation compared to MRSA COL (data not shown). We also examined F. nucleatum and C. albicans. Both of these species form biofilms in the human oral cavity, including on denture surfaces [43, 44]. A biofilm formation assay at 6 and 24 h revealed that 10% dHC significantly inhibited biofilm formation by MRSA T31 (Figure 1(a)) and C. albicans (Figure 1(b)). We also showed that 10% dHC significantly inhibited 24-h biofilm formation by F. nucleatum (Figure 1(c)). In each experiment, 10% dHC did not affect the growth of these microorganisms (data not shown). These results revealed that dHC exhibits an antibiofilm effect against MRSA T31, F. nucleatum, and C. albicans. However, dHC did not exert any antibiofilm effects against S. mutans MT8148, a cause of dental caries (data not shown). The MICs of dHC against MRSA T31, F. nucleatum, and C. albicans were 5% (750 μg/mL), 2.5% (375 μg/mL), and >10% (>1500 μg/mL), respectively. In the biofilm formation assay, bacterial abundance was approximately 100-fold higher than that in the MIC assay. We also observed that bacteria grew despite high concentrations of dHC.

Figure 1: Antibiofilm effects of dHC on MRSA T31, C. albicans, and F. nucleatum. Antibiofilm effects of a water decoction of Houttuynia cordata (dHC) on biofilm formation by MRSA T31 (a) and CAD1 (b) at 6 or 24 h. Antibiofilm effects of dHC on biofilm formation by F. nucleatum JCM8532 (c) at 24 h. As a positive control, 2 μg/mL (for MRSA T31, F. nucleatum) or 1 μg/mL (for CAD1) of cetylpyridinium chloride (CPC) was used. A negative control of 10% distilled H2O was used. differences between the indicated groups at . differences between the indicated groups at using a Student’s -test ().
3.3. No Cytotoxic Effects of dHC on Oral Keratinocytes

To investigate the cytotoxicity of dHC on oral keratinocytes, we measured the levels of LDH released from RT-7 cells. As shown in Figure 2, dHC did not exert any cytotoxic effects, up to a concentration of 10%. These results suggest that dHC could be applied to oral care.

Figure 2: No cytotoxic effects of dHC on oral keratinocytes. The cytotoxic effects of dHC on RT-7 cells were assessed by a lactate dehydrogenase (LDH) cytotoxicity assay. 0.1% Triton X-100 treatment and gentle agitation at 25°C for 10 min were used as a positive control. differences between the indicated groups at using a Student’s -test ().
3.4. Inhibitory Effects of dHC on Chemokine Production by Oral Keratinocytes

We have previously demonstrated that CCL20 produced by inflamed gingival epithelial cells appears to be closely connected to the proinflammatory response of the gingiva. This is due to an important regulatory role in specific lymphocyte migration into diseased periodontal tissue [15]. In addition, TLR2, a pattern recognition receptor for LPS from Gram-negative bacteria such as P. gingivalis, is strongly expressed in the pocket epithelium of periodontal tissues with chronic periodontitis. This participates in a signaling cascade that upregulates the production of IL-8, IP-10, and GROα [12, 14, 1622, 45]. We therefore examined whether dHC inhibits the production of IL-8, CCL20, IP-10, and GROα in RT-7 cells stimulated by P. gingivalis LPS. We found that 1% dHC significantly inhibited IL-8, CCL20, IP-10, and GROα productions by RT-7 cells stimulated with P. gingivalis LPS after 24 h (Figures 3(a), 3(b), 3(c), and 3(d)). This suggests that dHC may be clinically useful as an oral care product to prevent the infectious oral inflammation that is observed during periodontal disease.

Figure 3: Inhibitory effects of dHC on chemokine production by P. gingivalis LPS-stimulated oral keratinocytes. Inhibitory effects of a water decoction of Houttuynia cordata (dHC) on IL-8 (a), CCL20 (b), IP-10 (c), and GROα (d) production by oral keratinocytes stimulated with P. gingivalis LPS for 24 h. differences between the indicated groups at . differences between the indicated groups at using a Student’s -test ().
3.5. Antibiofilm and Anti-Inflammatory Effects of Flavonoid Glycosides

Previous reports have shown that the leaves of HC contain flavonoid glycosides such as quercitrin, isoquercitrin, hyperin, and rutin [46, 47]. In this study, antibacterial, antibiofilm, and anti-inflammatory assays of four flavonoid glycosides (quercitrin, isoquercitrin, hyperin, and rutin) were performed. As shown in Table 3, these flavonoid glycosides had little antibacterial activities. However, 200 μg/mL of quercitrin, isoquercitrin, and hyperin significantly inhibited 24-h biofilm formation by MRSA T31 and F. nucleatum but rutin showed inhibitory effect of biofilm formation only by MRSA T31 (Figures 4(a) and 4(c)). In biofilm formation by C. albicans, we could not observe antibiofilm effects by these flavonoid glycosides (Figure 4(b)). Moreover, 50 μg/mL of quercitrin, isoquercitrin, hyperin, and rutin significantly inhibited CCL20, IP-10, and GROα productions by RT-7 cells stimulated with P. gingivalis LPS (Figures 5(b), 5(c), and 5(d)). In IL-8 production, suppressive effect was observed only in rutin (Figure 5(a)).

Table 3: MIC of flavonoid glycosides.
Figure 4: Antibiofilm effects of flavonoid glycosides on MRSA T31, C. albicans, and F. nucleatum. Antibiofilm effects of flavonoid glycosides on biofilm formation by MRSA T31 (a), CAD1 (b), and F. nucleatum (c) at 24-h. A negative control of 2% DMSO was used. differences between the indicated groups at . differences between the indicated groups at . differences between the indicated groups at using a Student’s -test ().
Figure 5: Anti-inflammatory effects of flavonoid glycosides on chemokine production by P. gingivalis LPS-stimulated oral keratinocytes. Inhibitory effects of flavonoid glycosides on IL-8 (a), CCL20 (b), IP-10 (c), and GROα (d) productions by oral keratinocytes stimulated with P. gingivalis LPS for 24 h. differences between the indicated groups at . differences between the indicated groups at . differences between the indicated groups at using a Student’s -test ().

4. Discussion

Our study has successfully demonstrated that dHC exerts a moderate antimicrobial effect against microorganisms that normally colonize the oral cavity (Table 2). We have also shown that dHC exhibits antibiofilm effects against MRSA, F. nucleatum, and C. albicans (Figures 1(a), 1(b), and 1(c)). Finally, we have shown that dHC can inhibit IL-8 and CCL20 production by P. gingivalis LPS-stimulated human oral keratinocytes, with no apparent cytotoxic effects (Figures 2, 3(a), 3(b), 3(c), and 3(d)).

Previous studies have reported that medicinal plant extracts can exert moderate antimicrobial effects against oral microorganisms. These include a crude aqueous extract of ripe Morinda citrifolia fruit (Indian noni), a methanol extract of Polygonum cuspidatum, and a methanol extract of Syzygium aromaticum (clove) [2326]. However, it has been suggested that the use of organic extracts that include ethanol for oral care increases the risks of adverse reactions [35]. Therefore, a water solution of HC poultice ethanol extract (wHCP) used in our study may be safer than the ethanol based extract (eHCP) [36]. Our study has examined the properties of dHC, which is simpler to prepare than wHCP and has been used as health-promoting herbal tea without any reported adverse reactions from our interview survey [34, 36]. Previous studies have demonstrated that medicinal plants also exert antiadherence effects against oral microorganisms [2729]. In addition to a moderate antimicrobial effect, our results have shown that dHC significantly inhibits adherence after 6 h (Figures 1(a) and 1(b)) and biofilm formation by MRSA and C. albicans at 24 h (Figures 1(a) and 1(b)). It also inhibited biofilm formation by F. nucleatum (Figure 1(c)).

Finally, HC has also been shown to have an effect on host responses, with previous studies reporting that a 70% ethanol extract of HC dried aerial parts inhibits the production of several inflammatory biomarkers by lung epithelial cells, including IL-6 and nitric oxide (NO), and HC also inhibited lung inflammatory responses in a mouse model of LPS-induced acute lung injury [32]. Furthermore, a HC ethanol extract reduced the production of proinflammatory cytokines through the NF-κB signaling pathway in human mast cells [31]. A water extract of HC has also been shown to exert strong anti-inflammatory effects against S. aureus lipoteichoic acid-induced inflammatory responses that are partly attributed to the inhibition of tumor necrosis factor (TNF) expression in dermal fibroblasts [30]. Finally, a powdered extract of HC was recently found to modulate innate oral immune mediators in oral epithelial cells [33], and the mRNA abundance of IL-8 and CCL20 (used as inflammatory mediators) was upregulated in a dose-dependent manner. However, our results demonstrated that dHC inhibited IL-8, CCL20, IP-10, and GROα production by P. gingivalis LPS-stimulated human oral keratinocytes. This indicates that dHC can exhibit multiple different activities against microorganisms and host cells and may be useful as an oral care product to prevent infectious oral diseases.

Previous reports have shown that HC contains aldehydes, such as capric aldehyde (decanal), lauryl aldehyde (dodecanal), and decanoyl acetaldehyde (3-oxo-dodecanal, houttuynin), and flavonoid glycosides, such as quercitrin, isoquercitrin, hyperin, and rutin [4649]. Aldehydes have antibacterial activity; however, the dried leaves of HC could not contain aldehydes because of their instability and volatility. This will be why the antimicrobial effects of dHC were weaker than those of wHCP. Conversely, the antibiofilm and anti-inflammatory effects demonstrated by dHC were similar to those of wHCP. This suggests that the antibiofilm and anti-inflammatory constituents of dHC and wHCP would be flavonoid glycosides. These effects of each flavonoid glycoside are different with bacterial species and cytokines. The use of dHC containing flavonoid glycosides represents a simpler preparation method than wHCP for self-medication.

In Japan, aspiration pneumonia is a serious medical issue in immunocompromised patients, particularly the elderly. We have previously reported a higher prevalence of Candida spp., P. aeruginosa, and Staphylococcus spp. in the oropharyngeal microflora of patients with cerebrovascular infarction and dysphagia [6]. Reducing adherence and biofilm formation by oral microorganisms can contribute to the prevention of chronic oral infections and potentially severe, systematic opportunistic diseases, particularly in the elderly [9, 10]. Therefore, the use of dHC as herbal tea may strongly contribute to the prevention of aspiration pneumonia.

Povidone iodine, chlorhexidine, and benzethonium chloride are typically used as antiseptic mouthwashes or rinses to prevent oral infections including dental caries and periodontal diseases [5055]. However, these antiseptics exhibit mucosal cytotoxicity, have a bad flavor, and can cause anaphylactic reactions [54, 5658]. Therefore, dHC prepared as herbal tea is likely a safer mouthwash than these antiseptics. It also has a milder taste, without odor or cytotoxicity.

The results of our study contribute to the evaluation of dHC as an effective herbal tea that may help prevent infectious oral diseases. Further studies are needed to fully characterize the constituents of dHC and identify the specific factors that exhibit the antibiofilm and anti-inflammatory activities.

5. Conclusion

This study demonstrated that dHC exerts a moderate antibacterial effect against MRSA and other microorganisms. It also exhibited antibiofilm effects against MRSA, F. nucleatum, and C. albicans. We have shown that dHC exerts inhibitory effects on IL-8 and CCL20 production by P. gingivalis LPS-stimulated human oral keratinocytes, without cytotoxicity. Our results suggest that dHC has multiple activities in microorganisms and host cells and a herbal tea preparation may be effective in preventing infectious oral diseases.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

References

  1. M. Wilson, “Susceptibility of oral bacterial biofilms to antimicrobial agents,” Journal of Medical Microbiology, vol. 44, no. 2, pp. 79–87, 1996. View at Publisher · View at Google Scholar · View at Scopus
  2. J. W. Costerton, P. S. Stewart, and E. P. Greenberg, “Bacterial biofilms: a common cause of persistent infections,” Science, vol. 284, no. 5418, pp. 1318–1322, 1999. View at Publisher · View at Google Scholar · View at Scopus
  3. P. E. Kolenbrander, “Oral microbial communities: biofilms, interactions, and genetic systems,” Annual Review of Microbiology, vol. 54, pp. 413–437, 2000. View at Publisher · View at Google Scholar · View at Scopus
  4. D. R. Radford, S. J. Challacombe, and J. D. Walter, “Denture plaque and adherence of Candida albicans to denture-base materials in vivo and in vitro,” Critical Reviews in Oral Biology and Medicine, vol. 10, no. 1, pp. 99–116, 1999. View at Publisher · View at Google Scholar · View at Scopus
  5. G. Ramage, K. Tomsett, B. L. Wickes, J. L. López-Ribot, and S. W. Redding, “Denture stomatitis: a role for Candida biofilms,” Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontology, vol. 98, no. 1, pp. 53–59, 2004. View at Publisher · View at Google Scholar · View at Scopus
  6. K. Hirota, T. Yoneyama, M. Sakamoto et al., “High prevalence of Pseudomonas aeruginosa from oropharyngeal biofilm in patients with cerebrovascular infarction and dysphagia,” Chest, vol. 138, no. 1, pp. 237-238, 2010. View at Publisher · View at Google Scholar · View at Scopus
  7. E. Martori, R. Ayuso-Montero, J. Martinez-Gomis, M. Viñas, and M. Peraire, “Risk factors for denture-related oral mucosal lesions in a geriatric population,” Journal of Prosthetic Dentistry, vol. 111, no. 4, pp. 273–279, 2014. View at Publisher · View at Google Scholar · View at Scopus
  8. A. Rekhi, C. M. Marya, S. S. Oberoi, R. Nagpal, C. Dhingra, and S. Kataria, “Periodontal status and oral health-related quality of life in elderly residents of aged care homes,” in Proceedings of the in Delhi , Geriatrics Gerontology International, 2015.
  9. M. P. Cullinan and G. J. Seymour, “Periodontal disease and systemic illness: will the evidence ever be enough?” Periodontology 2000, vol. 62, no. 1, pp. 271–286, 2013. View at Publisher · View at Google Scholar · View at Scopus
  10. K. R. Atanasova and Ö. Yilmaz, “Prelude to oral microbes and chronic diseases: past, present and future,” Microbes and Infection, vol. 17, no. 7, pp. 473–483, 2015. View at Publisher · View at Google Scholar · View at Scopus
  11. M. Wilson, K. Reddi, and B. Henderson, “Cytokine-inducing components of periodontopathogenic bacteria,” Journal of Periodontal Research, vol. 31, no. 6, pp. 393–407, 1996. View at Publisher · View at Google Scholar · View at Scopus
  12. Y. Kusumoto, H. Hirano, K. Saitoh et al., “Human gingival epithelial cells produce chemotactic factors interleukin-8 and monocyte chemoattractant protein-1 after stimulation with Porphyromonas gingivalis via Toll-like receptor 2,” Journal of Periodontology, vol. 75, no. 3, pp. 370–379, 2004. View at Publisher · View at Google Scholar · View at Scopus
  13. Y. Taguchi and H. Imai, “Expression of β-defensin-2 in human gingival epithelial cells in response to challenge with Porphyromonas gingivalis in vitro,” Journal of Periodontal Research, vol. 41, no. 4, pp. 334–339, 2006. View at Publisher · View at Google Scholar · View at Scopus
  14. R. Schwandner, R. Dziarski, H. Wesche, M. Rothe, and C. J. Kirschning, “Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2,” Journal of Biological Chemistry, vol. 274, no. 25, pp. 17406–17409, 1999. View at Publisher · View at Google Scholar · View at Scopus
  15. Y. Hosokawa, T. Nakanishi, D. Yamaguchi et al., “Macrophage inflammatory protein 3α-CC chemokine receptor 6 interactions play an important role in CD4+ T-cell accumulation in periodontal diseased tissue,” Clinical and Experimental Immunology, vol. 128, no. 3, pp. 548–554, 2002. View at Publisher · View at Google Scholar · View at Scopus
  16. L. Ren, W. K. Leung, R. P. Darveau, and L. Jin, “The expression profile of lipopolysaccharide-binding protein, membrane-bound CD14, and toll-like receptors 2 and 4 in chronic periodontitis,” Journal of Periodontology, vol. 76, no. 11, pp. 1950–1959, 2005. View at Publisher · View at Google Scholar · View at Scopus
  17. Y. Takahashi, M. Davey, H. Yumoto, F. C. Gibson III, and C. A. Genco, “Fimbria-dependent activation of pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial cells,” Cellular Microbiology, vol. 8, no. 5, pp. 738–757, 2006. View at Publisher · View at Google Scholar · View at Scopus
  18. K. Ohta, H. Shigeishi, M. Taki et al., “Regulation of CXCL9/11 in oral keratinocytes and fibroblasts,” Journal of Dental Research, vol. 87, no. 12, pp. 1160–1165, 2008. View at Publisher · View at Google Scholar · View at Scopus
  19. R. Peyyala, S. S. Kirakodu, K. F. Novak, and J. L. Ebersole, “Oral microbial biofilm stimulation of epithelial cell responses,” Cytokine, vol. 58, no. 1, pp. 65–72, 2012. View at Publisher · View at Google Scholar · View at Scopus
  20. A. Fukui, K. Ohta, H. Nishi et al., “Interleukin-8 and CXCL10 expression in oral keratinocytes and fibroblasts via toll-like receptors,” Microbiology and Immunology, vol. 57, no. 3, pp. 198–206, 2013. View at Publisher · View at Google Scholar · View at Scopus
  21. S. W. Hong, J. E. Baik, S.-S. Kang, C.-H. Yun, D.-G. Seo, and S. H. Han, “Lipoteichoic acid of Streptococcus mutans interacts with Toll-like receptor 2 through the lipid moiety for induction of inflammatory mediators in murine macrophages,” Molecular Immunology, vol. 57, no. 2, pp. 284–291, 2014. View at Publisher · View at Google Scholar · View at Scopus
  22. G. Ramage, D. F. Lappin, E. Millhouse et al., “The epithelial cell response to health and disease associated oral biofilm models,” Journal of Periodontal Research, vol. 52, no. 3, pp. 325–333, 2017. View at Publisher · View at Google Scholar · View at Scopus
  23. L. Cai and C. D. Wu, “Compounds from Syzygium aromaticum possessing growth inhibitory activity against oral pathogens,” Journal of Natural Products, vol. 59, no. 10, pp. 987–990, 1996. View at Publisher · View at Google Scholar · View at Scopus
  24. J.-H. Song, S.-K. Kim, K.-W. Chang, S.-K. Han, H.-K. Yi, and J.-G. Jeon, “In vitro inhibitory effects of Polygonum cuspidatum on bacterial viability and virulence factors of Streptococcus mutans and Streptococcus sobrinus,” Archives of Oral Biology, vol. 51, no. 12, pp. 1131–1140, 2006. View at Publisher · View at Google Scholar · View at Scopus
  25. S.-H. Ban, Y.-R. Kwon, S. Pandit, Y.-S. Lee, H.-K. Yi, and J.-G. Jeon, “Effects of a bio-assay guided fraction from Polygonum cuspidatum root on the viability, acid production and glucosyltranferase of mutans streptococci,” Fitoterapia, vol. 81, no. 1, pp. 30–34, 2010. View at Publisher · View at Google Scholar · View at Scopus
  26. B. Kumarasamy, S. Manipal, P. Duraisamy, A. Ahmed, S. P. Mohanaganesh, and C. Jeevika, “Role of aqueous extract of Morinda Citrifolia(Indian Noni) ripe fruits in inhibiting dental caries-causing Streptococcus Mutans and Streptococcus Mitis,” Journal of Dentistry, vol. 11, no. 6, pp. 703–710, 2014. View at Google Scholar
  27. J. Limsong, E. Benjavongkulchai, and J. Kuvatanasuchati, “Inhibitory effect of some herbal extracts on adherence of Streptococcus mutans,” Journal of Ethnopharmacology, vol. 92, no. 2-3, pp. 281–289, 2004. View at Publisher · View at Google Scholar · View at Scopus
  28. Z. H. A. Rahim and N. Thurairajah, “Scanning electron microscopic study of Piper betle L. leaves extract effect against Streptococcus mutans ATCC 25175,” Journal of Applied Oral Science, vol. 19, no. 2, pp. 137–146, 2011. View at Publisher · View at Google Scholar · View at Scopus
  29. M.-A. Nordin, W. H. A. W. Harun, and F. A. Razak, “An in vitro study on the anti-adherence effect of Brucea javanica and Piper betle extracts towards oral Candida,” Archives of Oral Biology, vol. 58, no. 10, pp. 1335–1342, 2013. View at Publisher · View at Google Scholar · View at Scopus
  30. J. Y. Choi, J. A. Lee, J. B. Lee, S. J. Yun, and S. C. Lee, “Anti-Inflammatory Activity of Houttuynia cordata against lipoteichoic acid-induced inflammation in human dermal fibroblasts,” Chonnam Medical Journal, vol. 46, no. 3, p. 140, 2010. View at Publisher · View at Google Scholar
  31. H. J. Lee, H.-S. Seo, G.-J. Kim et al., “Houttuynia cordata Thunb inhibits the production of pro-inflammatory cytokines through inhibition of the NFκB signaling pathway in HMC-1 human mast cells,” Molecular Medicine Reports, vol. 8, no. 3, pp. 731–736, 2013. View at Publisher · View at Google Scholar · View at Scopus
  32. J. H. Lee, J. Ahn, J. W. Kim, S. G. Lee, and H. P. Kim, “Flavonoids from the aerial parts of Houttuynia cordata attenuate lung inflammation in mice,” Archives of Pharmacal Research, vol. 38, no. 7, pp. 1304–1311, 2015. View at Publisher · View at Google Scholar · View at Scopus
  33. S. Satthakarn, W. Chung, A. Promsong, and W. Nittayananta, “Houttuynia cordata modulates oral innate immune mediators: Potential role of herbal plant on oral health,” Oral Diseases, vol. 21, no. 4, pp. 512–518, 2015. View at Publisher · View at Google Scholar · View at Scopus
  34. Y. Sekita, K. Murakami, H. Yumoto et al., “Anti-bacterial and anti-inflammatory effects of ethanol extract from Houttuynia cordata poultice,” Bioscience, Biotechnology, and Biochemistry, vol. 80, no. 6, pp. 1205–1213, 2016. View at Publisher · View at Google Scholar
  35. D. W. Lachenmeier, “Safety evaluation of topical applications of ethanol on the skin and inside the oral cavity,” Journal of Occupational Medicine and Toxicology, vol. 3, no. 1, article 26, 2008. View at Publisher · View at Google Scholar · View at Scopus
  36. Y. Sekita, K. Murakami, H. Yumoto et al., “Preventive effects of Houttuynia cordata extract for oral infectious diseases,” BioMed Research International, vol. 2016, Article ID 2581876, 2016. View at Publisher · View at Google Scholar · View at Scopus
  37. N. Tanaka, Y. Kashiwada, T. Nakano et al., “Chromone and chromanone glucosides from Hypericum sikokumontanum and their anti-Helicobacter pylori activities,” Phytochemistry, vol. 70, no. 1, pp. 141–146, 2009. View at Publisher · View at Google Scholar · View at Scopus
  38. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, s Institute, 8th edition, 2009.
  39. D. A. Hogan, Å. Vik, and R. Kolter, “A Pseudomonas aeruginosa quorum-sensing molecule influences Candida albicans morphology,” Molecular Microbiology, vol. 54, no. 5, pp. 1212–1223, 2004. View at Publisher · View at Google Scholar · View at Scopus
  40. A. Nur, K. Hirota, H. Yumoto et al., “Effects of extracellular DNA and DNA-binding protein on the development of a Streptococcus intermedius biofilm,” Journal of Applied Microbiology, vol. 115, no. 1, pp. 260–270, 2013. View at Publisher · View at Google Scholar · View at Scopus
  41. J. Latimer, J. L. Munday, K. M. Buzza, S. Forbes, P. K. Sreenivasan, and A. J. McBain, “Antibacterial and anti-biofilm activity of mouthrinses containing cetylpyridinium chloride and sodium fluoride,” BMC Microbiology, vol. 15, no. 1, article no. 169, 2015. View at Publisher · View at Google Scholar · View at Scopus
  42. R. Fujimoto, N. Kamata, K. Yokoyama et al., “Establishment of immortalized human oral keratinocytes by gene transfer of a telomerase component,” Journal of Japanese Society for Oral Mucous Membrane, vol. 8, no. 1, pp. 1–8, 2002. View at Publisher · View at Google Scholar
  43. Y. Yoshijima, K. Murakami, S. Kayama et al., “Effect of substrate surface hydrophobicity on the adherence of yeast and hyphal Candida: original article,” Mycoses, vol. 53, no. 3, pp. 221–226, 2010. View at Publisher · View at Google Scholar · View at Scopus
  44. A. Yoshida, M. Niki, Y. Yamamoto, A. Yasunaga, and T. Ansai, “Proteome analysis identifies the Dpr protein of Streptococcus mutans as an important factor in the presence of early streptococcal colonizers of tooth surfaces,” PLoS ONE, vol. 10, no. 3, Article ID e0121176, 2015. View at Publisher · View at Google Scholar · View at Scopus
  45. O. Takeuchi, K. Hoshino, T. Kawai et al., “Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components,” Immunity, vol. 11, no. 4, pp. 443–451, 1999. View at Publisher · View at Google Scholar · View at Scopus
  46. T. Kawamura, Y. Hisata, K. Okuda et al., “Pharmacognostical studies of Houttuyniae Herba (1). Flavonoid glycosides contents of Houttuynia cordata Thunb.,” Natural Medicines, vol. 48, no. 3, pp. 208–212, 1994. View at Google Scholar · View at Scopus
  47. J. Fuse, H. Kanamori, I. Sakamoto, and S. Yahara, “Studies on flavonol glycosides in Houttuynia cordata,” Natural Medicines, vol. 48, no. 4, pp. 307–311, 1994. View at Google Scholar · View at Scopus
  48. T. Kosuge, “Structure of an Antimicrobial Substance isolated from Houttuynia cordata Thunb,” Yakugaku Zasshi, vol. 72, no. 10, pp. 1227–1231, 1952. View at Publisher · View at Google Scholar
  49. H. Lu, X. Wu, Y. Liang, and J. Zhang, “Variation in chemical composition and antibacterial activities of essential oils from two species of Houttuynia THUNB,” Chemical and Pharmaceutical Bulletin, vol. 54, no. 7, pp. 936–940, 2006. View at Publisher · View at Google Scholar · View at Scopus
  50. T. Oyanagi, J. Tagami, and K. Matin, “Potentials of mouthwashes in disinfecting cariogenic bacteria and biofilms leading to inhibition of caries,” Open Dentistry Journal, vol. 6, no. 1, pp. 23–30, 2012. View at Publisher · View at Google Scholar · View at Scopus
  51. H. Mahmoud Hashemi, F. Mohammadi, M. Hasheminasab, A. Mahmoud Hashemi, S. Zahraei, and T. Mahmoud Hashemi, “Effect of Low-Concentration Povidone Iodine on Postoperative Complications After Third Molar Surgery: A Pilot Split-Mouth Study,” Journal of Oral and Maxillofacial Surgery, vol. 73, no. 1, pp. 18–21, 2015. View at Publisher · View at Google Scholar
  52. J. Kanagalingam, R. Feliciano, J. H. Hah, H. Labib, T. A. Le, and J.-C. Lin, “Practical use of povidone-iodine antiseptic in the maintenance of oral health and in the prevention and treatment of common oropharyngeal infections,” International Journal of Clinical Practice, vol. 69, no. 11, pp. 1247–1256, 2015. View at Publisher · View at Google Scholar · View at Scopus
  53. C. Mor-Reinoso, A. Pascual, J. Nart, and M. Quirynen, “Inhibition of de novo plaque growth by a new 0.03 % chlorhexidine mouth rinse formulation applying a non-brushing model: a randomized, double blind clinical trial,” Clinical Oral Investigations, vol. 20, no. 7, pp. 1459–1467, 2016. View at Publisher · View at Google Scholar · View at Scopus
  54. F. Sadat Sajadi, M. Moradi, A. Pardakhty, R. Yazdizadeh, and F. Madani, “Effect of Fluoride, Chlorhexidine and Fluoride-chlorhexidine Mouthwashes on Salivary Streptococcus mutans Count and the Prevalence of Oral Side Effects,” Journal of Dental Research, Dental Clinics, Dental Prospects, vol. 9, no. 1, pp. 49–52, 2015. View at Publisher · View at Google Scholar
  55. Y. Iwamura, J.-I. Hayashi, T. Sato et al., “Assessment of oral malodor and tonsillar microbiota after gargling with benzethonium chloride,” Journal of Oral Science, vol. 58, no. 1, pp. 83–91, 2016. View at Publisher · View at Google Scholar · View at Scopus
  56. H. Nagamune, T. Maeda, K. Ohkura, K. Yamamoto, M. Nakajima, and H. Kourai, “Evaluation of the cytotoxic effects of bis-quaternary ammonium antimicrobial reagents on human cells,” Toxicology in Vitro, vol. 14, no. 2, pp. 139–147, 2000. View at Publisher · View at Google Scholar · View at Scopus
  57. P. Chen, W. Huda, and N. Levy, “Chlorhexidine anaphylaxis: Implications for post-resuscitation management,” Anaesthesia, vol. 71, no. 2, pp. 242-243, 2016. View at Publisher · View at Google Scholar · View at Scopus
  58. G. Sharp, S. Green, and M. Rose, “Chlorhexidine-induced anaphylaxis in surgical patients: A review of the literature,” ANZ Journal of Surgery, vol. 86, no. 4, pp. 237–243, 2016. View at Publisher · View at Google Scholar · View at Scopus