# Antibiofilm and Anti-Inflammatory Activities ofHouttuynia cordata Decoction for Oral Care

Authors: Yasuko Sekita; Keiji Murakami; Hiromichi Yumoto; Kouji Hirao; Takashi Amoh; Natsumi Fujiwara; Katsuhiko Hirota; Hideki Fujii; Takashi Matsuo; Yoichiro Miyake; Yoshiki Kashiwada
Journal: Evidence-Based Complementary and Alternative Medicine
 (2017)

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## 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-resistantStaphylococcus aureus (MRSA) and other oral microorganisms. dHC also exhibited antibiofilm effects against MRSA,Fusobacterium nucleatum (involved in dental plaque formation), andCandida albicans and inhibitory effects on interleukin-8, CCL20, IP-10, and GROα productions by human oral keratinocytes stimulated byPorphyromonas 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.


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## Body

## 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 [1–3]. Predominant fungi such asCandida 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 [6–8]. We have previously reported a higher prevalence ofCandida spp.,Pseudomonas aeruginosa, andStaphylococcus 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 [11–13]. These mediators ultimately cause periodontal inflammation. Lipopolysaccharide (LPS) from Gram-negative bacteria, such as the important periodontal pathogenPorphyromonas 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) [14–22]. 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 [23–26], antiadherence effects against oral microorganisms [27–29], and anti-inflammatory effects [30–33].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 onP.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 Table1.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-resistantStaphylococcus 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 andP. 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 forF.nucleatum, MRSA-T31, andC.albicans, respectively.Table 1
Bacterial strains.
Strain
Source
Methicillin-resistantStaphylococcus aureus T31
Clinical isolate
Methicillin-resistantStaphylococcus aureus COL
Wild type
Streptococcus mutans MT8148
Clinical isolate
Streptococcus mutans UA159
Clinical isolate
Streptococcus sobrinus 1310
Clinical isolate
Streptococcus gordonii ATCC10558
Type strain
Streptococcus oralis ATCC10557
Type strain
Streptococcus constellatus 4528
Clinical isolate
Streptococcus intermedius 40138
Clinical isolate
Streptococcus mitisJCM 12971
Wild type
Aggregatibacter actinomycetemcomitans Y4
Wild type
Fusobacterium nucleatumJCM8532
Wild type
Porphyromonas gingivalis ATCC33277
Type strain
Pseudomonas aeruginosa PAO1
Wild type
Candida albicans CAD1
Clinical isolate


### 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 (forStreptococcus spp.,A. actinomycetemcomitans,F. nucleatum, andP. gingivalis) or aerobically (for MRSA T31, MRSA COL,P. aeruginosa, andC. 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 orC. albicans CAD1 in the stationary phase or a 5-μL (107 CFU/mL) sample ofF. nucleatum JCM8532 in the stationary phase was transferred into a 96-well plate (Cellstar, Greiner Bio-One, Frickenhausen, Germany) from the primary 150 μL 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 (forF. nucleatum) at 37°C for 24 h or aerobically (for MRSA T31 andC. 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/mLP. 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’st-test. Differences were considered significant when the probability value was less than 5%  (P∗<0.05).


## 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 Table1.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-resistantStaphylococcus 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 andP. 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 forF.nucleatum, MRSA-T31, andC.albicans, respectively.Table 1
Bacterial strains.
Strain
Source
Methicillin-resistantStaphylococcus aureus T31
Clinical isolate
Methicillin-resistantStaphylococcus aureus COL
Wild type
Streptococcus mutans MT8148
Clinical isolate
Streptococcus mutans UA159
Clinical isolate
Streptococcus sobrinus 1310
Clinical isolate
Streptococcus gordonii ATCC10558
Type strain
Streptococcus oralis ATCC10557
Type strain
Streptococcus constellatus 4528
Clinical isolate
Streptococcus intermedius 40138
Clinical isolate
Streptococcus mitisJCM 12971
Wild type
Aggregatibacter actinomycetemcomitans Y4
Wild type
Fusobacterium nucleatumJCM8532
Wild type
Porphyromonas gingivalis ATCC33277
Type strain
Pseudomonas aeruginosa PAO1
Wild type
Candida albicans CAD1
Clinical isolate


## 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 (forStreptococcus spp.,A. actinomycetemcomitans,F. nucleatum, andP. gingivalis) or aerobically (for MRSA T31, MRSA COL,P. aeruginosa, andC. 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 orC. albicans CAD1 in the stationary phase or a 5-μL (107 CFU/mL) sample ofF. nucleatum JCM8532 in the stationary phase was transferred into a 96-well plate (Cellstar, Greiner Bio-One, Frickenhausen, Germany) from the primary 150 μL 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 (forF. nucleatum) at 37°C for 24 h or aerobically (for MRSA T31 andC. 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/mLP. 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’st-test. Differences were considered significant when the probability value was less than 5%  (P∗<0.05).


## 3. Results

### 3.1. Moderate Antimicrobial Effects of dHC

We examined the antimicrobial effects of dHC against several oral microorganisms. As shown in Table2, dHC exerted a moderate antimicrobial effect against MRSA T31, MRSA COL,S. intermedius,S. mitis,F. nucleatum, andP. gingivalis (MIC; 375–1500 μg/mL).Table 2
MIC of dHC.
Bacterial strain
MIC
%
∗
(μg/mL)
Methicillin-resistantStaphylococcus aureus T31
5
750
Methicillin-resistantStaphylococcus aureus COL
10
1500
Streptococcus mutans MT8148
>10
>1500
Streptococcus mutans UA159
>10
>1500
Streptococcus sobrinus 1310
>10
>1500
Streptococcus gordonii ATCC10558
>10
>1500
Streptococcus oralis ATCC10557
>10
>1500
Streptococcus constellatus 4528
>10
>1500
Streptococcus intermedius 40138
10
1500
Streptococcus mitis JCM 12921
10
1500
Aggregatibacter actinomycetemcomitans Y4
>10
>1500
Fusobacterium nucleatumJCM8532
2.5
375
Porphyromonas gingivalis ATCC33277
10
1500
Pseudomonas aeruginosa PAO1
>10
>1500
Candida albicans CAD1
>10
>1500
C
o
n
c
e
n
t
r
a
t
i
o
n
∗ of dHC in medium v/v.


### 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 examinedF. nucleatum andC.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)) andC. albicans (Figure 1(b)). We also showed that 10% dHC significantly inhibited 24-h biofilm formation byF. 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, andC. albicans. However, dHC did not exert any antibiofilm effects againstS. mutans MT8148, a cause of dental caries (data not shown). The MICs of dHC against MRSA T31,F. nucleatum, andC.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 ofHouttuynia cordata (dHC) on biofilm formation by MRSA T31 (a) and CAD1 (b) at 6 or 24 h. Antibiofilm effects of dHC on biofilm formation byF.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. Significant∗∗∗ differences between the indicated groups at p<0.001. Significant∗∗ differences between the indicated groups at p<0.01 using a Student’s t-test (n=8).
(a)
(b)
(c)


### 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 Figure2, 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. Significant∗∗∗ differences between the indicated groups at p<0.001 using a Student’s t-test (n=4).


### 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 asP. 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, 16–22, 45]. We therefore examined whether dHC inhibits the production of IL-8, CCL20, IP-10, and GROα in RT-7 cells stimulated byP. gingivalis LPS. We found that 1% dHC significantly inhibited IL-8, CCL20, IP-10, and GROα productions by RT-7 cells stimulated withP. 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 ofHouttuynia cordata (dHC) on IL-8 (a), CCL20 (b), IP-10 (c), and GROα (d) production by oral keratinocytes stimulated withP. gingivalis LPS for 24 h. Significant∗∗ differences between the indicated groups at p<0.01. Significant∗ differences between the indicated groups at p<0.05 using a Student’s t-test (n=6).
(a)
(b)
(c)
(d)


### 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 andF. nucleatum but rutin showed inhibitory effect of biofilm formation only by MRSA T31 (Figures 4(a) and 4(c)). In biofilm formation byC. 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 withP. 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.
Bacterial strain
MIC (μg/mL)
Quercitrin
Isoquercitrin
Hyperin
Rutin
Methicillin-resistantStaphylococcus aureus T31
>512
>512
>512
>512
Candida albicans CAD1
>512
>512
>512
>512
Fusobacterium nucleatumJCM8532
256
512
512
256Figure 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), andF.nucleatum (c) at 24-h. A negative control of 2% DMSO was used. Significant∗∗∗ differences between the indicated groups at p<0.001. Significant∗∗ differences between the indicated groups at p<0.01. Significant∗ differences between the indicated groups at p<0.05 using a Student’s t-test (n=4).
(a)
(b)
(c)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 withP. gingivalis LPS for 24 h. Significant∗∗∗ differences between the indicated groups at p<0.001. Significant∗∗ differences between the indicated groups at p<0.01. Significant∗ differences between the indicated groups at p<0.05 using a Student’s t-test (n=3).
(a)
(b)
(c)
(d)


## 3.1. Moderate Antimicrobial Effects of dHC

We examined the antimicrobial effects of dHC against several oral microorganisms. As shown in Table2, dHC exerted a moderate antimicrobial effect against MRSA T31, MRSA COL,S. intermedius,S. mitis,F. nucleatum, andP. gingivalis (MIC; 375–1500 μg/mL).Table 2
MIC of dHC.
Bacterial strain
MIC
%
∗
(μg/mL)
Methicillin-resistantStaphylococcus aureus T31
5
750
Methicillin-resistantStaphylococcus aureus COL
10
1500
Streptococcus mutans MT8148
>10
>1500
Streptococcus mutans UA159
>10
>1500
Streptococcus sobrinus 1310
>10
>1500
Streptococcus gordonii ATCC10558
>10
>1500
Streptococcus oralis ATCC10557
>10
>1500
Streptococcus constellatus 4528
>10
>1500
Streptococcus intermedius 40138
10
1500
Streptococcus mitis JCM 12921
10
1500
Aggregatibacter actinomycetemcomitans Y4
>10
>1500
Fusobacterium nucleatumJCM8532
2.5
375
Porphyromonas gingivalis ATCC33277
10
1500
Pseudomonas aeruginosa PAO1
>10
>1500
Candida albicans CAD1
>10
>1500
C
o
n
c
e
n
t
r
a
t
i
o
n
∗ of dHC in medium v/v.


## 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 examinedF. nucleatum andC.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)) andC. albicans (Figure 1(b)). We also showed that 10% dHC significantly inhibited 24-h biofilm formation byF. 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, andC. albicans. However, dHC did not exert any antibiofilm effects againstS. mutans MT8148, a cause of dental caries (data not shown). The MICs of dHC against MRSA T31,F. nucleatum, andC.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 ofHouttuynia cordata (dHC) on biofilm formation by MRSA T31 (a) and CAD1 (b) at 6 or 24 h. Antibiofilm effects of dHC on biofilm formation byF.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. Significant∗∗∗ differences between the indicated groups at p<0.001. Significant∗∗ differences between the indicated groups at p<0.01 using a Student’s t-test (n=8).
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(b)
(c)


## 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 Figure2, 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. Significant∗∗∗ differences between the indicated groups at p<0.001 using a Student’s t-test (n=4).


## 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 asP. 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, 16–22, 45]. We therefore examined whether dHC inhibits the production of IL-8, CCL20, IP-10, and GROα in RT-7 cells stimulated byP. gingivalis LPS. We found that 1% dHC significantly inhibited IL-8, CCL20, IP-10, and GROα productions by RT-7 cells stimulated withP. 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 ofHouttuynia cordata (dHC) on IL-8 (a), CCL20 (b), IP-10 (c), and GROα (d) production by oral keratinocytes stimulated withP. gingivalis LPS for 24 h. Significant∗∗ differences between the indicated groups at p<0.01. Significant∗ differences between the indicated groups at p<0.05 using a Student’s t-test (n=6).
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## 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 andF. nucleatum but rutin showed inhibitory effect of biofilm formation only by MRSA T31 (Figures 4(a) and 4(c)). In biofilm formation byC. 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 withP. 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.
Bacterial strain
MIC (μg/mL)
Quercitrin
Isoquercitrin
Hyperin
Rutin
Methicillin-resistantStaphylococcus aureus T31
>512
>512
>512
>512
Candida albicans CAD1
>512
>512
>512
>512
Fusobacterium nucleatumJCM8532
256
512
512
256Figure 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), andF.nucleatum (c) at 24-h. A negative control of 2% DMSO was used. Significant∗∗∗ differences between the indicated groups at p<0.001. Significant∗∗ differences between the indicated groups at p<0.01. Significant∗ differences between the indicated groups at p<0.05 using a Student’s t-test (n=4).
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(c)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 withP. gingivalis LPS for 24 h. Significant∗∗∗ differences between the indicated groups at p<0.001. Significant∗∗ differences between the indicated groups at p<0.01. Significant∗ differences between the indicated groups at p<0.05 using a Student’s t-test (n=3).
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## 4. Discussion

Our study has successfully demonstrated that dHC exerts a moderate antimicrobial effect against microorganisms that normally colonize the oral cavity (Table2). We have also shown that dHC exhibits antibiofilm effects against MRSA,F. nucleatum, andC. albicans (Figures 1(a), 1(b), and 1(c)). Finally, we have shown that dHC can inhibit IL-8 and CCL20 production byP. 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 ripeMorinda citrifolia fruit (Indian noni), a methanol extract ofPolygonum cuspidatum, and a methanol extract ofSyzygium aromaticum (clove) [23–26]. 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 [27–29]. 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 andC. albicans at 24 h (Figures 1(a) and 1(b)). It also inhibited biofilm formation byF. 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 againstS. 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 byP. 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 [46–49]. 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 ofCandida spp.,P. aeruginosa, andStaphylococcus 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 [50–55]. However, these antiseptics exhibit mucosal cytotoxicity, have a bad flavor, and can cause anaphylactic reactions [54, 56–58]. 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, andC. albicans. We have shown that dHC exerts inhibitory effects on IL-8 and CCL20 production byP. 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.



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*Source: 2850947-2017-10-16.xml*