# Inhibitory Effects ofChrysanthemum boreale Essential Oil on Biofilm Formation and Virulence Factor Expression of Streptococcus mutans

Authors: Beom-Su Kim; Sun-Ju Park; Myung-Kon Kim; Young-Hoi Kim; Sang-Bong Lee; Kwang-Hee Lee; Na-Young Choi; Young-Rae Lee; Young-Eun Lee; Yong-Ouk You
Journal: Evidence-Based Complementary and Alternative Medicine
 (2015)

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## Abstract

The aim of the study was to evaluate the antibacterial activity of essential oil extracted fromChrysanthemum boreale (C. boreale) on Streptococcus mutans (S. mutans). To investigate anticariogenic properties, and bacterial growth, acid production, biofilm formation, bacterial adherence of S. mutans were evaluated. Then gene expression of several virulence factors was also evaluated. C. boreale essential oil exhibited significant inhibition of bacterial growth, adherence capacity, and acid production of S. mutans at concentrations 0.1–0.5 mg/mL and 0.25–0.5 mg/mL, respectively. The safranin staining and scanning electron microscopy results showed that the biofilm formation was also inhibited. The result of live/dead staining showed the bactericidal effect. Furthermore, real-time PCR analysis showed that the gene expression of some virulence factors such as gtfB, gtfC, gtfD, gbpB, spaP, brpA, relA, and vicR of S. mutans was significantly decreased in a dose dependent manner. In GC and GC-MS analysis, seventy-two compounds were identified in the oil, representing 85.42% of the total oil. The major components were camphor (20.89%), β-caryophyllene (5.71%), α-thujone (5.46%), piperitone (5.27%), epi-sesquiphellandrene (5.16%), α-pinene (4.97%), 1,8-cineole (4.52%), β-pinene (4.45%), and camphene (4.19%). These results suggest that C. boreale essential oil may inhibit growth, adhesion, acid tolerance, and biofilm formation of S. mutans through the partial inhibition of several of these virulence factors.


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## Body

## 1. Introduction

Dental caries, known as tooth decay or a cavity, is a plaque-related disease of teeth and slowly progressive infectious disease in the dental area [1, 2]. The dental caries disease is caused by specific types of acid-producing bacteria that cause demineralization and destruction of the teeth [3].S. mutans are generally regarded as one of the primary pathogenic bacteria in dental caries [4]. TheS. mutans adhere to the colonizer and accumulate on the tooth enamel surface by generation of extracellular polysaccharide from fermentable carbohydrates such as sucrose, by action of glucosyltransferases (GTFase) [1, 5]. The carbohydrate metabolism promotes bacteria aggregation to the tooth surface and acid production [1]. The produced acids initiate dissolution of the enamel surface of teeth subsequently leading to localized decalcification [6].Therefore, inhibition of the growth and biofilm formation of theS. mutans is one of the strategies for prevention of dental caries. Although several antiplaque agents have been used, the attempt to search for an effective agent still continued [7, 8]. For example, some studies reported that several natural products derived herb, such asMentha longifolia L.,Aralia continentalis, andCurcuma longa L, showed the inhibitory effect of dental plaque [9–11].C. boreale is a perennial herb with yellow flowers and belongs to the Asteraceae family. It is widely distributed in wild fields and mountains of East Asia. It is also usually have been used as tea or wine in Korea. TheChrysanthemum species herb has been reported as having potential medicinal properties including anti-inflammatory, antiviral, and antibacterial [12–14]. In previous study [15], the essential oil was extracted fromC. boreale and eighty-seven constituents were identified. Furthermore, the essential oil showed antibacterial activity against several bacteria includingS. mutans. However there is poorly scientific evidence about effect of essential oil fromC. boreale onS. mutans causing dental plaque formation. Therefore, in this study, we examined influence of essential oil of extracted fromC. boreale on the growth, acid-production, bacterial attachment, and biofilm formation ofS. mutans. Furthermore, several virulence factors ofS. mutans, associated with dental plaque and caries formation, were assessed, and the detailed chemical constituents ofC. boreale essentialoil were also analyzed by GC and GC-MS.


## 2. Materials and Methods

### 2.1. Plant Material and Essential Oil

C. boreale was collected in October, 2013, at the full flowering stage from plants grown wild in Iksan district in Korea and the aerial parts were used to isolate essential oil. The identity was confirmed by Young-Hoi Kim at the College of Environmental & Bioresource Sciences, Chonbuk National University. Voucher specimen (number: 10-24-13) has been deposited at the Herbarium of College of Environmental & Bioresource Sciences, Chonbuk National University. The aerial parts (leaves, stems, and flowers) ofC. boreale (1 kg) were finely chopped. The chopped plant materials ofC. boreale were placed in 5 L round-bottom flask and distilled water was added (3 L). Hydrodistillation was carried out in a Clevenger-type apparatus for 3 hours. The yield of the essential oil ofC. boreale was 0.84%, based on fresh weight of the plant. The essential oil was stored in a deep freezer (−70°C) to minimize the escape of volatile compounds.


### 2.2. Inhibition of Bacterial Growth

S. mutans (ATCC 25175) was purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA) and cultured in brain heart infusion (BHI; Difco, Detroit, MI) broth under aerobic condition at 37°C. To determine inhibitory effect ofC. boreale on bacterial growth,S. mutans was cultured at 37°C in 0.95 mL of BHI broth containing 1% glucose and various concentrations of the essential oil ofC. boreale. These tubes were inoculated with 0.05 mL of an overnight culture grown in BHI broth (final: 5 × 105 colony-forming units (CFU)/mL), and incubated for 24 h. Also, 0.1% of sodium fluoride (NaF) was used as a positive control. The optical density (OD) of cells was measured at 550 nm using a spectrophotometer. Three replicates were made for each concentration of the test extracts.


### 2.3. Acid Production

Acid production byS. mutans was examined to evaluate the effect of the essential oil ofC. boreale, as described by a previous study [16]. Briefly, theC. boreale essential oil was filtered to sterilization using membrane filter with 0.2 μm pore size and added to 0.95 mL of the phenol red broth containing 1% glucose, which was then inoculated with 0.05 mL of the seed culture ofS. mutans. After 24 h of cultivation, the pH was directly determined in the bacterial growth media using a pH meter (Corning Inc, Corning, NY, USA). The initial pH of BHI with various concentrations ofC. boreale essential oil was also determined before inoculation ofS. mutans. Each concentration of the extract was tested in triplicate.


### 2.4. Bacterial Adherence

The effect ofC. boreale essential oil on bacterial adherence was determined using hydroxyapatite beads (diameter of 80 μm; Bio-Rad, Hercules, CA, USA) in a previously described method [17]. Briefly, hydroxyapatite beads were coated with clarified human saliva and the saliva-coated hydroxyapatite beads (S-HAs) were immersed in bacterial suspension (1 × 107 CFU/mL) with various concentrations ofC. boreale essential oil. To allow bacteria to be adherent, the mixture was gently agitated for 90 min at 37°C. Following this, S-HAs was rinsed to remove nonadherent bacteria and was transferred to a new tube that contained potassium phosphate buffer. The adherentS. mutans onto the S-HAs were dispersed using a sonicator (Fisher Scientific, Springfield, NJ, USA) at 50 W for 30 sec and the supernatants were spread on bacitracin (3.2 mg/mL) contained MSA plate. After 48 h of cultivation, the numbers of colonies were counted.


### 2.5. Biofilm Formation Assay

Biofilm formation was measured by staining with safranin [18] and observation was done by scanning electron microscopy (SEM). Briefly, various concentrations ofC. boreale essential oil were added to 0.1% sucrose containing BHI broth in 35 mm polystyrene dish or 24-well plate that contained resin teeth (Endura, Shofu Inc., Kyoto, Japan). Then, the culture was created in the allotted broths by inoculating them with seed cultures ofS. mutans (5 × 105 CFU/mL) and incubated for 24 h. After incubation, the supernatants were removed and the culture dish or resin teeth were rinsed with distilled water. Biofilm formation was stained with 0.1% safranin and photographed. In addition, to observe the biofilm formation using a SEM, the biofilms formed polystyrene dishes were rinsed with distilled water, fixed with 2.5% glutaraldehyde solution, and dehydrated in ethanol gradient series. Then, the samples were sputter-coated with gold and observed by SEM (JOM-6360, JEOL, Tokyo, Japan).


### 2.6. Confocal Laser Scanning Microscopy

To determine the bactericidal effect ofC. boreale essential oil onS. mutans live and dead staining were performed. Briefly, approximately 1 × 107 CFU/mL ofS. mutans was treated with various concentrations ofC. boreale essential oil for 24 h at 37°C under aerobic conditions. Then the bacteria were washed with PBS and stained with LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Eugene, OR, USA) according to the manufacture’s protocol. After 15 min of staining, the bacteria were observed using a confocal laser scanning microscopy (LSM 510, Zeiss, Germany).


### 2.7. Real-Time Polymerase Chain Reaction (PCR) Analysis

A real-time PCR was performed to evaluate the effect ofC. boreale essential oil on gene expression ofS. mutans. The subminimal inhibitory concentration (0.5–0.25 mg/mL) of the essential oil was treated. After 24 h of culture, total RNA was isolated fromS. mutans using a Trizol reagent (Bibco-BRL) and cDNA was synthesized. The amplification was performed using a StepOnePlus Real-Time PCR system with QPCR SYBR Green Mixes (Applied Bio system, Foster City, CA, USA). 16S rRNA was used as an internal control. The primer pairs were described by previous report [19] and are listed in Table 1.Table 1
Oligonucleotide primers that were used in this study.
Gene*
Gene description
Primer sequences (5′-3′)
gtfB
Glucosyltransferase-I
Forward:Reverse:
AGCAATGCAGCCAATCTACAAATACGAACTTTGCCGTTATTGTCA
gtfC
Glucosyltransferase-SI
Forward:Reverse:
GGTTTAACGTCAAAATTAGCTGTATTAGCCTCAACCAACCGCCACTGTT
gtfD
Glucosyltransferase-S
Forward:Reverse:
ACAGCAGACAGCAGCCAAGAACTGGGTTTGCTGCGTTTG
brpA
Biofilm regulatory protein A
Forward:Reverse:
GGAGGAGCTGCATCAGGATTCAACTCCAGCACATCCAGCAAG
spaP
Cell surface antigen SpaP
Forward:Reverse:
GACTTTGGTAATGGTTATGCATCAATTTGTATCAGCCGGATCAAGTG
gbpB
Secreted antigen GbpB/SagA
Forward:Reverse:
ATGGCGGTTATGGACACGTTTTTGGCCACCTTGAACACCT
relA
GTP pyrophosphokinase
Forward:Reverse:
ACAAAAAGGGTATCGTCCGTACATAATCACGCTTGGTATTGCTAATTG
vicR
Response regulator
Forward:Reverse:
TGACACGATTACAGCCTTTGATGCGTCTAGTTCTGGTAACATTAAGTCCAATA
16S rRNA
16S rRNA
Forward:Reverse:
CCTACGGGAGGCAGCAGTAGCAACAGAGCTTTACGATCCGAAA
*Based on the NCBI S. mutans genome database.


### 2.8. GC and GC-MS Analysis

GC analysis was performed on Hewlett-Packard (HP) model 6890 series gas chromatograph, with a flame ionization detector (FID), a split ratio of 30 : 1 using two different fused silica capillary columns, Supelcowax 10 (30 m × 0.32 mm, i.d., 0.25μm film thickness) and SPB-1 (30 m × 0.32 mm, i.d., 0.25 μm film thickness). The temperature of the column was programmed from 50°C to 230°C at 2°C/min and then kept constant at 230°C for 30 min for Supelcowax 10 column and SPB-1 column was programmed from 40°C to 230°C at 2°C/min and then kept constant at 230°C for 20 min. The injector and detector temperatures for both analyses were 250°C, respectively. The gas carrier was nitrogen at a flow rate of 1.50 mL/min for Supelcowax 10 column and nitrogen at a flow rate of 1.20 mL/min for SPB-1 column. Peak areas were measured by electronic integration. The relative amounts of the individual components are based on the peak areas. The GC-MS was carried out on Agilent 7890A GC and Agilent 5975C mass selective detector (MSD) operating in EI mode at 70 eV, fitted a DB-Wax column (30 m × 0.25 mm, i.d., 0.25 μm film thickness) and SPB-1 column (30 m × 0.25 mm, i.d., 0.25 μm film thickness). The temperature of the column were programmed from 40°C to 230°C at 2°C/min and then kept constant at 230°C for 30 min for both analyses. The injector and interface temperatures were 250°C, respectively. The gas carrier was helium at a flow rate of 1.50 mL/min for both analyses. The identification of the chemical constituents was based on comparison of their mass spectral spectra with those of Wiley7n/NIST05 mass spectra libraries, and then the compounds of MS matching similarity ≥ 90% were selected as results. Linear retention indices were calculated for each component with the retention time ofn-alkane series (C6–C26) [20] under same GC operating conditions with the sample. They were compared with their retention indices available in the literatures [21, 22] or NIST gas chromatographic retention data webbook (http://webbook.nist.gov/chemistry/) database.


### 2.9. Statistical Analysis

All experiments were performed in triplicate. Data were analyzed using the statistical package for social sciences (SPSS, Chicago, IL, USA). The data were expressed as the mean ± standard deviation (SD) values. The statistical analysis was evaluated by one-way ANOVA. Values ofP<0.05 were considered as statistically significant.


## 2.1. Plant Material and Essential Oil

C. boreale was collected in October, 2013, at the full flowering stage from plants grown wild in Iksan district in Korea and the aerial parts were used to isolate essential oil. The identity was confirmed by Young-Hoi Kim at the College of Environmental & Bioresource Sciences, Chonbuk National University. Voucher specimen (number: 10-24-13) has been deposited at the Herbarium of College of Environmental & Bioresource Sciences, Chonbuk National University. The aerial parts (leaves, stems, and flowers) ofC. boreale (1 kg) were finely chopped. The chopped plant materials ofC. boreale were placed in 5 L round-bottom flask and distilled water was added (3 L). Hydrodistillation was carried out in a Clevenger-type apparatus for 3 hours. The yield of the essential oil ofC. boreale was 0.84%, based on fresh weight of the plant. The essential oil was stored in a deep freezer (−70°C) to minimize the escape of volatile compounds.


## 2.2. Inhibition of Bacterial Growth

S. mutans (ATCC 25175) was purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA) and cultured in brain heart infusion (BHI; Difco, Detroit, MI) broth under aerobic condition at 37°C. To determine inhibitory effect ofC. boreale on bacterial growth,S. mutans was cultured at 37°C in 0.95 mL of BHI broth containing 1% glucose and various concentrations of the essential oil ofC. boreale. These tubes were inoculated with 0.05 mL of an overnight culture grown in BHI broth (final: 5 × 105 colony-forming units (CFU)/mL), and incubated for 24 h. Also, 0.1% of sodium fluoride (NaF) was used as a positive control. The optical density (OD) of cells was measured at 550 nm using a spectrophotometer. Three replicates were made for each concentration of the test extracts.


## 2.3. Acid Production

Acid production byS. mutans was examined to evaluate the effect of the essential oil ofC. boreale, as described by a previous study [16]. Briefly, theC. boreale essential oil was filtered to sterilization using membrane filter with 0.2 μm pore size and added to 0.95 mL of the phenol red broth containing 1% glucose, which was then inoculated with 0.05 mL of the seed culture ofS. mutans. After 24 h of cultivation, the pH was directly determined in the bacterial growth media using a pH meter (Corning Inc, Corning, NY, USA). The initial pH of BHI with various concentrations ofC. boreale essential oil was also determined before inoculation ofS. mutans. Each concentration of the extract was tested in triplicate.


## 2.4. Bacterial Adherence

The effect ofC. boreale essential oil on bacterial adherence was determined using hydroxyapatite beads (diameter of 80 μm; Bio-Rad, Hercules, CA, USA) in a previously described method [17]. Briefly, hydroxyapatite beads were coated with clarified human saliva and the saliva-coated hydroxyapatite beads (S-HAs) were immersed in bacterial suspension (1 × 107 CFU/mL) with various concentrations ofC. boreale essential oil. To allow bacteria to be adherent, the mixture was gently agitated for 90 min at 37°C. Following this, S-HAs was rinsed to remove nonadherent bacteria and was transferred to a new tube that contained potassium phosphate buffer. The adherentS. mutans onto the S-HAs were dispersed using a sonicator (Fisher Scientific, Springfield, NJ, USA) at 50 W for 30 sec and the supernatants were spread on bacitracin (3.2 mg/mL) contained MSA plate. After 48 h of cultivation, the numbers of colonies were counted.


## 2.5. Biofilm Formation Assay

Biofilm formation was measured by staining with safranin [18] and observation was done by scanning electron microscopy (SEM). Briefly, various concentrations ofC. boreale essential oil were added to 0.1% sucrose containing BHI broth in 35 mm polystyrene dish or 24-well plate that contained resin teeth (Endura, Shofu Inc., Kyoto, Japan). Then, the culture was created in the allotted broths by inoculating them with seed cultures ofS. mutans (5 × 105 CFU/mL) and incubated for 24 h. After incubation, the supernatants were removed and the culture dish or resin teeth were rinsed with distilled water. Biofilm formation was stained with 0.1% safranin and photographed. In addition, to observe the biofilm formation using a SEM, the biofilms formed polystyrene dishes were rinsed with distilled water, fixed with 2.5% glutaraldehyde solution, and dehydrated in ethanol gradient series. Then, the samples were sputter-coated with gold and observed by SEM (JOM-6360, JEOL, Tokyo, Japan).


## 2.6. Confocal Laser Scanning Microscopy

To determine the bactericidal effect ofC. boreale essential oil onS. mutans live and dead staining were performed. Briefly, approximately 1 × 107 CFU/mL ofS. mutans was treated with various concentrations ofC. boreale essential oil for 24 h at 37°C under aerobic conditions. Then the bacteria were washed with PBS and stained with LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Eugene, OR, USA) according to the manufacture’s protocol. After 15 min of staining, the bacteria were observed using a confocal laser scanning microscopy (LSM 510, Zeiss, Germany).


## 2.7. Real-Time Polymerase Chain Reaction (PCR) Analysis

A real-time PCR was performed to evaluate the effect ofC. boreale essential oil on gene expression ofS. mutans. The subminimal inhibitory concentration (0.5–0.25 mg/mL) of the essential oil was treated. After 24 h of culture, total RNA was isolated fromS. mutans using a Trizol reagent (Bibco-BRL) and cDNA was synthesized. The amplification was performed using a StepOnePlus Real-Time PCR system with QPCR SYBR Green Mixes (Applied Bio system, Foster City, CA, USA). 16S rRNA was used as an internal control. The primer pairs were described by previous report [19] and are listed in Table 1.Table 1
Oligonucleotide primers that were used in this study.
Gene*
Gene description
Primer sequences (5′-3′)
gtfB
Glucosyltransferase-I
Forward:Reverse:
AGCAATGCAGCCAATCTACAAATACGAACTTTGCCGTTATTGTCA
gtfC
Glucosyltransferase-SI
Forward:Reverse:
GGTTTAACGTCAAAATTAGCTGTATTAGCCTCAACCAACCGCCACTGTT
gtfD
Glucosyltransferase-S
Forward:Reverse:
ACAGCAGACAGCAGCCAAGAACTGGGTTTGCTGCGTTTG
brpA
Biofilm regulatory protein A
Forward:Reverse:
GGAGGAGCTGCATCAGGATTCAACTCCAGCACATCCAGCAAG
spaP
Cell surface antigen SpaP
Forward:Reverse:
GACTTTGGTAATGGTTATGCATCAATTTGTATCAGCCGGATCAAGTG
gbpB
Secreted antigen GbpB/SagA
Forward:Reverse:
ATGGCGGTTATGGACACGTTTTTGGCCACCTTGAACACCT
relA
GTP pyrophosphokinase
Forward:Reverse:
ACAAAAAGGGTATCGTCCGTACATAATCACGCTTGGTATTGCTAATTG
vicR
Response regulator
Forward:Reverse:
TGACACGATTACAGCCTTTGATGCGTCTAGTTCTGGTAACATTAAGTCCAATA
16S rRNA
16S rRNA
Forward:Reverse:
CCTACGGGAGGCAGCAGTAGCAACAGAGCTTTACGATCCGAAA
*Based on the NCBI S. mutans genome database.


## 2.8. GC and GC-MS Analysis

GC analysis was performed on Hewlett-Packard (HP) model 6890 series gas chromatograph, with a flame ionization detector (FID), a split ratio of 30 : 1 using two different fused silica capillary columns, Supelcowax 10 (30 m × 0.32 mm, i.d., 0.25μm film thickness) and SPB-1 (30 m × 0.32 mm, i.d., 0.25 μm film thickness). The temperature of the column was programmed from 50°C to 230°C at 2°C/min and then kept constant at 230°C for 30 min for Supelcowax 10 column and SPB-1 column was programmed from 40°C to 230°C at 2°C/min and then kept constant at 230°C for 20 min. The injector and detector temperatures for both analyses were 250°C, respectively. The gas carrier was nitrogen at a flow rate of 1.50 mL/min for Supelcowax 10 column and nitrogen at a flow rate of 1.20 mL/min for SPB-1 column. Peak areas were measured by electronic integration. The relative amounts of the individual components are based on the peak areas. The GC-MS was carried out on Agilent 7890A GC and Agilent 5975C mass selective detector (MSD) operating in EI mode at 70 eV, fitted a DB-Wax column (30 m × 0.25 mm, i.d., 0.25 μm film thickness) and SPB-1 column (30 m × 0.25 mm, i.d., 0.25 μm film thickness). The temperature of the column were programmed from 40°C to 230°C at 2°C/min and then kept constant at 230°C for 30 min for both analyses. The injector and interface temperatures were 250°C, respectively. The gas carrier was helium at a flow rate of 1.50 mL/min for both analyses. The identification of the chemical constituents was based on comparison of their mass spectral spectra with those of Wiley7n/NIST05 mass spectra libraries, and then the compounds of MS matching similarity ≥ 90% were selected as results. Linear retention indices were calculated for each component with the retention time ofn-alkane series (C6–C26) [20] under same GC operating conditions with the sample. They were compared with their retention indices available in the literatures [21, 22] or NIST gas chromatographic retention data webbook (http://webbook.nist.gov/chemistry/) database.


## 2.9. Statistical Analysis

All experiments were performed in triplicate. Data were analyzed using the statistical package for social sciences (SPSS, Chicago, IL, USA). The data were expressed as the mean ± standard deviation (SD) values. The statistical analysis was evaluated by one-way ANOVA. Values ofP<0.05 were considered as statistically significant.


## 3. Results

### 3.1. Bacterial Growth Inhibition byC. boreale

In the study, we firstly investigated the antibacterial activity of the essential oil ofC. boreale againstS. mutans. The bacteria were treated with 0.05, 0.1, 0.25, and 0.5 mg/mL ofC. boreale essential oil. When treated with 0.1% NaF, as a positive control, the manifested significant inhibition was shown. When treated with 0.1 mg/mL of the essential oil, the bacterial growth was significantly inhibited. In addition, significant inhibition was shown at concentrations 0.25 mg/mL and 0.5 mg/mL of essential oil in comparison to the control group (Figure 1) (P<0.05).Figure 1
Effect ofChrysanthemum boreale(C. boreale) essential oil on growth ofStreptococcus mutans(S. mutans). S. mutans was inoculated into BHI broth with various concentrations ofC. boreale essential oil and incubated for 24 h. Antibacterial activity againstS. mutans was shown in presence ofC. boreale essential oil at concentration ranging from 0.1 mg/mL to 0.5 mg/mL. Each value is expressed as a mean ± standard deviation (SD). 0.1% of sodium fluoride (NaF) was used as a positive control. Significance was determined at *P<0.05 when compared with the control.Furthermore, the manifested significant inhibition was shown at concentrations higher than 0.25 mg/mL and 0.5 mg/mL in comparison to the control group.


### 3.2. Inhibition of Acid Production

To determine whether theC. boreale essential oil inhibits the acid production inS. mutans, the bacteria were cultured in the presence of various concentrations (0.05–0.5 mg/mL) of the essential oil and the pH change was measured. As shown in Table 2, the pH was significantly decreased at control group (pH 5.47±0.05). However, the pH decrease was significantly inhibited at positive group (0.1% NaF, pH 7.37±0.05). Although the pH decrease was not inhibited at 0.05–0.1 mg/mL ofC. boreale essential oil, when treated with 0.25 mg/mL and 0.5 mg/mL ofC. boreale essential oil, the pH decrease was also significantly inhibited and the inhibition levels was similar to the positive group. These results indicate that theC. boreale essential oil may inhibit the organic acid production byS. mutans.Table 2
Effect of essential oil ofC. boreale on acid production of S. mutans.
Conc. (mg/mL)
pH (before incubation)
pH (after incubation)
Control
7.00 ± 0.00
5.47 ± 0.05
0.05
7.00 ± 0.00
5.43 ± 0.05
0.1
7.00 ± 0.00
5.33 ± 0.05
0.25
7.00 ± 0.00
7.33 ± 0.05*
0.5
7.00 ± 0.00
7.33 ± 0.05*
0.1% NaF
7.00 ± 0.00
7.37 ± 0.05*
Data (pH) are represented as mean ± standard deviation.*P < 0.05 when compared with the control group after incubation.


### 3.3. Inhibitory Effect ofC. boreale Essential Oil onS. mutans Adherence

We tested the inhibitory effect ofC. boreale essential oil on the ability ofS. mutans to adhere to S-HAs. When treated withC. boreale essential oil, theS. mutans was significantly inhibited in a dose dependent manner. The adherence onto S-HAs particularly was significantly inhibited at concentration of 0.1–0.5 mg/mL ofC. boreale essential oil (Figure 2).Figure 2
Effect ofChrysanthemum boreale(C. boreale) essential oil on colony-forming units (CFU) ofStreptococcus mutans(S. mutans). S. mutans was inoculated into BHI broth with various concentrations ofC. boreale essential oil and incubated for 24 h. The CFU ofS. mutans that adhered to saliva-coated hydroxyapatite beads that were treated with various concentration ofC. boreale essential oil are shown. When treated with 0.1–0.5 mg/mL ofC. boreale essential oil, adherence was significantly repressed. Each value is expressed as a mean ± standard deviation (SD). Significance was determined at *P<0.05 when compared with the control. 0.1% of sodium fluoride (NaF) was used as a positive control.


### 3.4. Bactericidal Effect ofC. boreale Essential Oil onS. mutans

To evaluate bactericidal effect ofC. boreale essential oil,S. mutans were cultured in presence of various concentrations (0.05–0.5 mg/mL) of the essential oil and stained with LIVE/DEAD BacLight Bacterial Viability Kit and they were observed using confocal laser scanning microscopy. Treatment withC. boreale essential oil decreases living bacteria (green fluorescence labeled cell stained by SYTO 9) and increases dead bacteria (red fluorescence labeled cell stained by PI) in a dose dependent manner (Figure 3). This result suggests thatC. boreale essential oil has bactericidal effect onS. mutans.Figure 3
Bactericidal effect ofChrysanthemum boreale(C. boreale) essential oil. CulturedStreptococcus mutans(S. mutans) were treated withC. boreale essential oil and stained with LIVE/DEAD BacLight Bacterial Viability Kit. Treatment withC. boreale essential oil showed bactericidal effect onS. mutans in a dose dependent manner. Living bacteria was stained by SYTO 9 as green color and dead bacteria was stained by PI as a red color. Scale Bar = 100 μm.


### 3.5. Inhibitory Effect ofC. boreale Essential Oil on Biofilm Formation

To determine whetherC. boreale essential oil inhibits biofilm formation byS. mutans, the bacteria had been cultured in the presence of various concentrations ofC. boreale essential oil in polystyrene dishes. As a result of safranin staining, the biofilm formation byS. mutans was significantly inhibited by treatment withC. boreale essential oil in a dose dependent manner. When treated with 0.1% NaF (positive control), complete inhibition was shown. In addition, the biofilm formation was also significantly inhibited at 0.1 mg/mL and 0.5 mg/mL of the essential oil (Figure 4). Also, we observed biofilm formation on the surface of resin teeth by safranin staining and SEM observation. Treatment with 0.05 mg/mL ofC. boreale essential oil slightly inhibited biofilm formation byS. mutans and significantly inhibited at concentration 0.1–0.5 mg/mL ofC. boreale essential oil (Figure 5(a)). Also the SEM image showed consistent result with safranin staining of resin teeth (Figure 5(b)).Figure 4
Effect ofChrysanthemum boreale(C. boreale) essential oil on biofilm formation on polystyrene dishes byStreptococcus mutans(S. mutans). S. mutans was inoculated into BHI broth with various concentrations ofC. boreale essential oil and cultured for 48 h. The biofilm that formed on the polystyrene dish surface was measured by staining with 0.1% safranin. Biofilm formation was also significantly inhibited at 0.1 mg/mL and 0.5 mg/mL of theC. boreale essential oil. 0.1% of sodium fluoride (NaF) was used as a positive control.Effect ofChrysanthemum boreale(C. boreale) essential oil on biofilm formation on resin teeth surface.Streptococcus mutans(S. mutans) biofilm on resin tooth surface were incubated in various concentration ofC. boreale essential oil (a). Biofilm formation was significantly inhibited at 0.05–0.25 mg/mL ofC. boreale essential oil. Also biofilm formation was completely inhibited at 0.5 mg/mL ofC. boreale essential oil. Scanning electron microscopy image ofS. mutans biofilm formation on resin tooth surfaces (b). 0.1% of sodium fluoride (NaF) was used as a positive control. Scale bar represents 25 μm.
(a)
(b)


### 3.6. Inhibitory Effect ofC. boreale Essential Oil on Expression of Virulence Factor

To assess the effect ofC. boreale essential oil on the gene expression of virulence factors,S. mutans was cultured in presence of 0.05–0.25 mg/mL ofC. boreale essential oil and the gene expressions of virulence factors were evaluated by real-time PCR (Figure 6). After treatment withC. boreale essential oil, firstly genetic expression ofgtfB,gtfC, andgtfD, which encode GTFase B, C, and D proteins, respectively, was evaluated. The expression ofgtfB was significantly decreased whenS. mutans was treated with 0.1 mg/mL and 0.25 mg ofC. boreale essential oil andgtfC was significantly decreased at 0.25 mg/mL ofC. boreale essential oil. However, the expression ofgtfD was significantly decreased byC. boreale essential oil at concentration of 0.05–0.25 mg/mL. The expression ofSpaP andgbpB, which contribute to bacterial adherence, was also decreased at 0.25 mg/mL and 0.05–0.25 mg/mL ofC. boreale essential oil, respectively. The expression ofbrpA andrelA, which are related with acid tolerance andvicR, which is associated with regulating the expression ofgbpB,gtfB,gtfC, andgtfD, was also decreased byC. boreale essential oil treatment at the concentration of 0.05–0.25 mg/mL.Figure 6
Real-time PCR analysis of mRNA expressions of several virulence factor genes.Streptococcus mutans(S. mutans) was cultured and treated with various concentrations ofChrysanthemum boreale(C. boreale) essential oil and real-time PCR analysis was performed as described in the Materials and Methods.gtfB,gtfC,and gtfD were significantly inhibited at 0.1–0.25 mg/mL, 0.25 mg/mL, and 0.05–0.25 mg/mL ofC. boreale essential oil, respectively. In addition,gbpB,brpA,relA, andvicR expressions were significantly inhibited at 0.05–0.25 mg/mL. The expression ofspaP was significantly inhibited at 0.25 mg/mL ofC. boreale essential oil. Each value is expressed as a mean ± standard deviation. Significance was determined at *P<0.05 when compared with the control.


### 3.7. GC and GC-MS Analysis

The chemical composition ofC. boreale essential oil identified by GC and GC-MS analysis was shown in Table 3 together with their major constituent. Seventy-two compounds were identified in the oil, representing 85.42% of the total oil. All unidentified compounds were minor components. The major components were camphor (20.89%), β-caryophyllene (5.71%), α-thujone (5.46%), piperitone (5.27%),epi-sesquiphellandrene (5.16%), α-pinene (4.97%), 1,8-cineole (4.52%), β-pinene (4.45%), and camphene (4.19%) (Table 3).Table 3
GC and GC-MS analysis of the essential oil isolated fromC. boreale.
Peakno.a
Components
Retention index
Peak area(%)d
Polarb
Apolarc
Monoterpene hydrocarbons
(19.66)
1
Tricyclene
1009
920
0.18
2
α-Pinene
1027
933
4.97
3
α-Thujene
1030
925
0.23
4
Camphene
1070
945
4.19
5
β-Pinene
1110
970
4.45
6
Sabinene
1123
966
0.61
10
Myrcene
1167
984
0.92
11
α-Terpinene
1181
1007
0.48
13
Limonene
1199
1020
0.65
16
cis-β-Ocimene
1239
1029
0.87
17
γ-Terpinene
1249
1050
0.47
18
p-Cymene
1276
1022
1.34
19
Terpinolene
1286
1078
0.30
Oxygenated monoterpenes
(47.23)
12
2,3-Dehydro-1,8-cineole
1191
976
0.05
14
1,8-Cineole
1212
1020
4.52
22
α-Thujone
1427
1090
5.46
23
β-Thujone
1441
1096
1.04
28
Camphor
1518
1124
20.89
30
Linalool
1537
1101
0.10
31
trans-Sabinene hydrate
1562
1051
0.40
32
trans-Chrysanthenyl acetate
1569
1186
0.46
33
Bornyl acetate
1576
1268
0.69
35
Terpinen-4-ol
1599
1165
0.72
36
Lavandulyl acetate
1608
—
0.08
37
Myrtenal
1621
1167
0.15
38
Umbellulone
1639
1149
0.28
39
Pinocarveol
1651
1124
0.29
40
p-Mentha-1,5-dien-8-ol
1662
1169
0.60
43
α-Terpineol
1697
1174
0.38
44
Borneol
1701
1146
1.94
48
Piperitone
1725
1224
5.27
49
Carvone
1731
1212
1.14
50
cis-Chrysanthenol
1761
1157
2.07
53
Myrtenol
1795
1179
0.32
54
trans-Carveol
1833
1181
0.09
55
Geraniol
1852
—
0.07
56
Geranyl acetone
1854
—
0.08
57
cis-Carveol
1860
1196
0.14
Sesquiterpene hydrocarbons
12.51
25
α-Guaiene
1470
—
0.09
27
α-Copaene
1493
1370
0.32
29
Berkheyaradulen
1527
1377
0.19
34
β-Caryophyllene
1590
1412
5.71
41
cis-β-Farnesene
1671
—
0.38
42
β-Selinene
1676
1488
0.15
45
epi-Sesquiphellandrene
1707
—
5.16
46
Widdrene
1710
—
0.09
47
Zingiberene
1714
1496
0.42
51
ar-Curcumene
1780
1484
0.28
Oxygenated Sesquiterpenes
(4.44)
58
Caryophyllene oxide
1975
1561
2.08
59
Viridiflorol
2042
1569
0.10
60
Nerolidol
2049
1555
0.38
61
Elemol
2072
—
0.40
62
Spathulenol
2118
1563
0.42
64
Torreyol
2167
1606
0.15
67
epi-Globulol
2217
1589
0.44
68
Farnesol
2244
1688
0.03
70
α-Costol
2301
—
0.29
71
Caryophyllenol II
2349
—
0.07
72
Farnesol (isomer)
2353
1704
0.08
Others
(1.58)
5
n-Hexanal
1087
835
0.07
8
Butyl benzene
1126
938
0.11
9
2-Methylpropylbenzene
1133
1050
0.14
15
2-Pentyl furan
1236
981
0.04
20
6-Methyl-5-hepten-2-one
1341
—
0.06
21
n-Hexanol
1356
882
0.10
24
1-Octen-3-ol
1454
966
0.21
26
2,2,4-Trimethyl-2-cyclohexene carbaldehyde
1474
1040
0.04
52
Methyl salicylate
1789
1169
0.07
63
Eugenol
2164
1327
0.29
65
Thymol
2185
1275
0.34
66
Carvacrol
2212
1275
0.05
69
Decanoic acid
2263
—
0.06
Total identified
(85.42)
aNumbering refers to the elution order on Supelcowax 10 column.
bRetention index on polar Supelcowax 10 column.
cRetention index on apolar SPB-1 column.
dPeak area percentage is based on polar Supelcowax 10 column, and values represent averages of three determinations.


## 3.1. Bacterial Growth Inhibition byC. boreale

In the study, we firstly investigated the antibacterial activity of the essential oil ofC. boreale againstS. mutans. The bacteria were treated with 0.05, 0.1, 0.25, and 0.5 mg/mL ofC. boreale essential oil. When treated with 0.1% NaF, as a positive control, the manifested significant inhibition was shown. When treated with 0.1 mg/mL of the essential oil, the bacterial growth was significantly inhibited. In addition, significant inhibition was shown at concentrations 0.25 mg/mL and 0.5 mg/mL of essential oil in comparison to the control group (Figure 1) (P<0.05).Figure 1
Effect ofChrysanthemum boreale(C. boreale) essential oil on growth ofStreptococcus mutans(S. mutans). S. mutans was inoculated into BHI broth with various concentrations ofC. boreale essential oil and incubated for 24 h. Antibacterial activity againstS. mutans was shown in presence ofC. boreale essential oil at concentration ranging from 0.1 mg/mL to 0.5 mg/mL. Each value is expressed as a mean ± standard deviation (SD). 0.1% of sodium fluoride (NaF) was used as a positive control. Significance was determined at *P<0.05 when compared with the control.Furthermore, the manifested significant inhibition was shown at concentrations higher than 0.25 mg/mL and 0.5 mg/mL in comparison to the control group.


## 3.2. Inhibition of Acid Production

To determine whether theC. boreale essential oil inhibits the acid production inS. mutans, the bacteria were cultured in the presence of various concentrations (0.05–0.5 mg/mL) of the essential oil and the pH change was measured. As shown in Table 2, the pH was significantly decreased at control group (pH 5.47±0.05). However, the pH decrease was significantly inhibited at positive group (0.1% NaF, pH 7.37±0.05). Although the pH decrease was not inhibited at 0.05–0.1 mg/mL ofC. boreale essential oil, when treated with 0.25 mg/mL and 0.5 mg/mL ofC. boreale essential oil, the pH decrease was also significantly inhibited and the inhibition levels was similar to the positive group. These results indicate that theC. boreale essential oil may inhibit the organic acid production byS. mutans.Table 2
Effect of essential oil ofC. boreale on acid production of S. mutans.
Conc. (mg/mL)
pH (before incubation)
pH (after incubation)
Control
7.00 ± 0.00
5.47 ± 0.05
0.05
7.00 ± 0.00
5.43 ± 0.05
0.1
7.00 ± 0.00
5.33 ± 0.05
0.25
7.00 ± 0.00
7.33 ± 0.05*
0.5
7.00 ± 0.00
7.33 ± 0.05*
0.1% NaF
7.00 ± 0.00
7.37 ± 0.05*
Data (pH) are represented as mean ± standard deviation.*P < 0.05 when compared with the control group after incubation.


## 3.3. Inhibitory Effect ofC. boreale Essential Oil onS. mutans Adherence

We tested the inhibitory effect ofC. boreale essential oil on the ability ofS. mutans to adhere to S-HAs. When treated withC. boreale essential oil, theS. mutans was significantly inhibited in a dose dependent manner. The adherence onto S-HAs particularly was significantly inhibited at concentration of 0.1–0.5 mg/mL ofC. boreale essential oil (Figure 2).Figure 2
Effect ofChrysanthemum boreale(C. boreale) essential oil on colony-forming units (CFU) ofStreptococcus mutans(S. mutans). S. mutans was inoculated into BHI broth with various concentrations ofC. boreale essential oil and incubated for 24 h. The CFU ofS. mutans that adhered to saliva-coated hydroxyapatite beads that were treated with various concentration ofC. boreale essential oil are shown. When treated with 0.1–0.5 mg/mL ofC. boreale essential oil, adherence was significantly repressed. Each value is expressed as a mean ± standard deviation (SD). Significance was determined at *P<0.05 when compared with the control. 0.1% of sodium fluoride (NaF) was used as a positive control.


## 3.4. Bactericidal Effect ofC. boreale Essential Oil onS. mutans

To evaluate bactericidal effect ofC. boreale essential oil,S. mutans were cultured in presence of various concentrations (0.05–0.5 mg/mL) of the essential oil and stained with LIVE/DEAD BacLight Bacterial Viability Kit and they were observed using confocal laser scanning microscopy. Treatment withC. boreale essential oil decreases living bacteria (green fluorescence labeled cell stained by SYTO 9) and increases dead bacteria (red fluorescence labeled cell stained by PI) in a dose dependent manner (Figure 3). This result suggests thatC. boreale essential oil has bactericidal effect onS. mutans.Figure 3
Bactericidal effect ofChrysanthemum boreale(C. boreale) essential oil. CulturedStreptococcus mutans(S. mutans) were treated withC. boreale essential oil and stained with LIVE/DEAD BacLight Bacterial Viability Kit. Treatment withC. boreale essential oil showed bactericidal effect onS. mutans in a dose dependent manner. Living bacteria was stained by SYTO 9 as green color and dead bacteria was stained by PI as a red color. Scale Bar = 100 μm.


## 3.5. Inhibitory Effect ofC. boreale Essential Oil on Biofilm Formation

To determine whetherC. boreale essential oil inhibits biofilm formation byS. mutans, the bacteria had been cultured in the presence of various concentrations ofC. boreale essential oil in polystyrene dishes. As a result of safranin staining, the biofilm formation byS. mutans was significantly inhibited by treatment withC. boreale essential oil in a dose dependent manner. When treated with 0.1% NaF (positive control), complete inhibition was shown. In addition, the biofilm formation was also significantly inhibited at 0.1 mg/mL and 0.5 mg/mL of the essential oil (Figure 4). Also, we observed biofilm formation on the surface of resin teeth by safranin staining and SEM observation. Treatment with 0.05 mg/mL ofC. boreale essential oil slightly inhibited biofilm formation byS. mutans and significantly inhibited at concentration 0.1–0.5 mg/mL ofC. boreale essential oil (Figure 5(a)). Also the SEM image showed consistent result with safranin staining of resin teeth (Figure 5(b)).Figure 4
Effect ofChrysanthemum boreale(C. boreale) essential oil on biofilm formation on polystyrene dishes byStreptococcus mutans(S. mutans). S. mutans was inoculated into BHI broth with various concentrations ofC. boreale essential oil and cultured for 48 h. The biofilm that formed on the polystyrene dish surface was measured by staining with 0.1% safranin. Biofilm formation was also significantly inhibited at 0.1 mg/mL and 0.5 mg/mL of theC. boreale essential oil. 0.1% of sodium fluoride (NaF) was used as a positive control.Effect ofChrysanthemum boreale(C. boreale) essential oil on biofilm formation on resin teeth surface.Streptococcus mutans(S. mutans) biofilm on resin tooth surface were incubated in various concentration ofC. boreale essential oil (a). Biofilm formation was significantly inhibited at 0.05–0.25 mg/mL ofC. boreale essential oil. Also biofilm formation was completely inhibited at 0.5 mg/mL ofC. boreale essential oil. Scanning electron microscopy image ofS. mutans biofilm formation on resin tooth surfaces (b). 0.1% of sodium fluoride (NaF) was used as a positive control. Scale bar represents 25 μm.
(a)
(b)


## 3.6. Inhibitory Effect ofC. boreale Essential Oil on Expression of Virulence Factor

To assess the effect ofC. boreale essential oil on the gene expression of virulence factors,S. mutans was cultured in presence of 0.05–0.25 mg/mL ofC. boreale essential oil and the gene expressions of virulence factors were evaluated by real-time PCR (Figure 6). After treatment withC. boreale essential oil, firstly genetic expression ofgtfB,gtfC, andgtfD, which encode GTFase B, C, and D proteins, respectively, was evaluated. The expression ofgtfB was significantly decreased whenS. mutans was treated with 0.1 mg/mL and 0.25 mg ofC. boreale essential oil andgtfC was significantly decreased at 0.25 mg/mL ofC. boreale essential oil. However, the expression ofgtfD was significantly decreased byC. boreale essential oil at concentration of 0.05–0.25 mg/mL. The expression ofSpaP andgbpB, which contribute to bacterial adherence, was also decreased at 0.25 mg/mL and 0.05–0.25 mg/mL ofC. boreale essential oil, respectively. The expression ofbrpA andrelA, which are related with acid tolerance andvicR, which is associated with regulating the expression ofgbpB,gtfB,gtfC, andgtfD, was also decreased byC. boreale essential oil treatment at the concentration of 0.05–0.25 mg/mL.Figure 6
Real-time PCR analysis of mRNA expressions of several virulence factor genes.Streptococcus mutans(S. mutans) was cultured and treated with various concentrations ofChrysanthemum boreale(C. boreale) essential oil and real-time PCR analysis was performed as described in the Materials and Methods.gtfB,gtfC,and gtfD were significantly inhibited at 0.1–0.25 mg/mL, 0.25 mg/mL, and 0.05–0.25 mg/mL ofC. boreale essential oil, respectively. In addition,gbpB,brpA,relA, andvicR expressions were significantly inhibited at 0.05–0.25 mg/mL. The expression ofspaP was significantly inhibited at 0.25 mg/mL ofC. boreale essential oil. Each value is expressed as a mean ± standard deviation. Significance was determined at *P<0.05 when compared with the control.


## 3.7. GC and GC-MS Analysis

The chemical composition ofC. boreale essential oil identified by GC and GC-MS analysis was shown in Table 3 together with their major constituent. Seventy-two compounds were identified in the oil, representing 85.42% of the total oil. All unidentified compounds were minor components. The major components were camphor (20.89%), β-caryophyllene (5.71%), α-thujone (5.46%), piperitone (5.27%),epi-sesquiphellandrene (5.16%), α-pinene (4.97%), 1,8-cineole (4.52%), β-pinene (4.45%), and camphene (4.19%) (Table 3).Table 3
GC and GC-MS analysis of the essential oil isolated fromC. boreale.
Peakno.a
Components
Retention index
Peak area(%)d
Polarb
Apolarc
Monoterpene hydrocarbons
(19.66)
1
Tricyclene
1009
920
0.18
2
α-Pinene
1027
933
4.97
3
α-Thujene
1030
925
0.23
4
Camphene
1070
945
4.19
5
β-Pinene
1110
970
4.45
6
Sabinene
1123
966
0.61
10
Myrcene
1167
984
0.92
11
α-Terpinene
1181
1007
0.48
13
Limonene
1199
1020
0.65
16
cis-β-Ocimene
1239
1029
0.87
17
γ-Terpinene
1249
1050
0.47
18
p-Cymene
1276
1022
1.34
19
Terpinolene
1286
1078
0.30
Oxygenated monoterpenes
(47.23)
12
2,3-Dehydro-1,8-cineole
1191
976
0.05
14
1,8-Cineole
1212
1020
4.52
22
α-Thujone
1427
1090
5.46
23
β-Thujone
1441
1096
1.04
28
Camphor
1518
1124
20.89
30
Linalool
1537
1101
0.10
31
trans-Sabinene hydrate
1562
1051
0.40
32
trans-Chrysanthenyl acetate
1569
1186
0.46
33
Bornyl acetate
1576
1268
0.69
35
Terpinen-4-ol
1599
1165
0.72
36
Lavandulyl acetate
1608
—
0.08
37
Myrtenal
1621
1167
0.15
38
Umbellulone
1639
1149
0.28
39
Pinocarveol
1651
1124
0.29
40
p-Mentha-1,5-dien-8-ol
1662
1169
0.60
43
α-Terpineol
1697
1174
0.38
44
Borneol
1701
1146
1.94
48
Piperitone
1725
1224
5.27
49
Carvone
1731
1212
1.14
50
cis-Chrysanthenol
1761
1157
2.07
53
Myrtenol
1795
1179
0.32
54
trans-Carveol
1833
1181
0.09
55
Geraniol
1852
—
0.07
56
Geranyl acetone
1854
—
0.08
57
cis-Carveol
1860
1196
0.14
Sesquiterpene hydrocarbons
12.51
25
α-Guaiene
1470
—
0.09
27
α-Copaene
1493
1370
0.32
29
Berkheyaradulen
1527
1377
0.19
34
β-Caryophyllene
1590
1412
5.71
41
cis-β-Farnesene
1671
—
0.38
42
β-Selinene
1676
1488
0.15
45
epi-Sesquiphellandrene
1707
—
5.16
46
Widdrene
1710
—
0.09
47
Zingiberene
1714
1496
0.42
51
ar-Curcumene
1780
1484
0.28
Oxygenated Sesquiterpenes
(4.44)
58
Caryophyllene oxide
1975
1561
2.08
59
Viridiflorol
2042
1569
0.10
60
Nerolidol
2049
1555
0.38
61
Elemol
2072
—
0.40
62
Spathulenol
2118
1563
0.42
64
Torreyol
2167
1606
0.15
67
epi-Globulol
2217
1589
0.44
68
Farnesol
2244
1688
0.03
70
α-Costol
2301
—
0.29
71
Caryophyllenol II
2349
—
0.07
72
Farnesol (isomer)
2353
1704
0.08
Others
(1.58)
5
n-Hexanal
1087
835
0.07
8
Butyl benzene
1126
938
0.11
9
2-Methylpropylbenzene
1133
1050
0.14
15
2-Pentyl furan
1236
981
0.04
20
6-Methyl-5-hepten-2-one
1341
—
0.06
21
n-Hexanol
1356
882
0.10
24
1-Octen-3-ol
1454
966
0.21
26
2,2,4-Trimethyl-2-cyclohexene carbaldehyde
1474
1040
0.04
52
Methyl salicylate
1789
1169
0.07
63
Eugenol
2164
1327
0.29
65
Thymol
2185
1275
0.34
66
Carvacrol
2212
1275
0.05
69
Decanoic acid
2263
—
0.06
Total identified
(85.42)
aNumbering refers to the elution order on Supelcowax 10 column.
bRetention index on polar Supelcowax 10 column.
cRetention index on apolar SPB-1 column.
dPeak area percentage is based on polar Supelcowax 10 column, and values represent averages of three determinations.


## 4. Discussion

C. boreale are frequently used as a tea or wine in oriental medicine and their medicinal effects such as anti-inflammatory, ant-viral, and antibacterial have been reported [12–14]. Previously, we reported that theC. boreale essential oils were extracted and it was identified that the essential oils were composed of eighty-seven constituents where major components were camphor, α-thujone, cis-chrysanthenol, 1,8-cineole, α-pinnen, and β-caryophyllene. Furthermore, the essential oil exhibited the inhibitory effect on growth of several bacteria includingS. mutans [15]. However, there is no report on its potential effect on the cariogenic properties such as bacterial growth, adherence, biofilm formation, and acid production.To evaluate anticariogenic properties ofC. boreale essential oil,S. mutans was used because the bacteria is considered as a major bacterium for the formation of dental caries [5, 23]. Our results showed that growth ofS. mutans was suppressed by treatment withC. boreale essential oil. Furthermore, the live/dead staining results also showed thatC. boreale essential oil has an antibactericidal effect againstS. mutans. These results suggested thatC. boreale essential oil has a potential for anticariogenic effect because the inhibition of the growth ofS. mutansis one of the strategies for prevention of dental caries.In dental plaque formation, pH is one of the major causes because low pH leads to demineralized tooth enamel and favors the occurrence of the dental caries.S. mutans can metabolize dietary sugars and produce organic acid and the produced acids and it is induced by acidic environment in the mouth [24]. Therefore, the alternation of pH is used as an indicator to determine the effect of anticariogenic agents. In this study,C. boreale essential oil inhibited the decrease of pH induced byS. mutans and the result suggests thatC. boreale essential oil may be inhibiting dental caries through inhibition of acid production byS. mutans.Furthermore, in the creation of dental plaque process, synthesized extracellular glucan byS. mutans is generally regarded as being a major factor [25]. Glucans induce bacterial adherence and result in the formation of dental biofilm [26]. Herein, we examined whetherC. boreale essential oil can inhibit the ability ofS. mutans to adhere to S-HAs. Our result showed thatC. boreale essential oil significantly inhibited bacterial adhesion. In addition, biofilm formation byS. mutans was also inhibited by treatment withC. boreale essential oil cultured both on polystyrene dishes and on surface of resin teeth. These results suggested thatC. boreale essential oil directly inhibits the attachment and biofilm formation byS. mutans.Several virulence factors ofS. mutans are associated with cariogenicity such as bacterial adhesion [27], biofilm formation [28], and acid tolerance [29]. In this study, to evaluate correlation between inhibitory effect byC. boreale essential oil and virulence factors expression, we determined the mRNA expression level of several virulence factors such asgtfB,gtfC,gtfD,gbpB,spaP,brpA,relA, andvicR, using a real-time PCR analysis. Firstly we evaluated the gene expression level ofgtfB,gtfC, andgtfD, which encode the glucosyltransferases (GTFase) B, C, and D. GTFase are recognized as essential virulence factor because these enzymes synthesize glucan from sucrose; the synthesized glucans provide binding site for bacterial adhesion [27]. Besides GTFase virulence factors,gbpB, which encodes surface-associated glucan binding protein (GBP), are also a required factor for bacterial adhesion because the protein mediates interaction between cell surface and glucan [30]. Furthermore,S. mutans expressedspaP gene which encodesSpaP protein and the protein contributes adhesion ofS. mutans [31, 32]. In this study,C. boreale essential oil significantly inhibited the transcription level ofgtfB,gtfC,gtfD,gbpB, andspaP. In biofilm formation process byS. mutans,brpA andrelA gene play critical roles.brpA is associated with biofilm regulation [28] andrelA gene plays a major role in several processes including biofilm formation, glucose uptake, and acid tolerance [33, 34]. AlsovicR gene is reportedas a regulatory gene of other virulence factors such asgbpB,gtfB,gtfC, andgtfD [19]. Based on our results of real-time PCR, the expression ofbrpA,relA, andvicR was also repressed when treated withC. boreale essential oil. The chemical constituents ofC. boreale were analyzed with GC and GC-MS. Seventy-two compounds were identified in the oil, representing 85.42% of the total oil. All unidentified compounds were minor components. The major components were camphor (20.89%), β-caryophyllene (5.71%), α-thujone (5.46%), piperitone (5.27%),epi-sesquiphellandrene (5.16%), α-pinene (4.97%), 1,8-cineole (4.52%), β-pinene (4.45%), and camphene (4.19%). Some previous results reported that the essential oils fromC. coronarium andC. indicum contain monoterpene hydrocarbons and oxygenated monoterpenes such as α-pinene, β-pinene, camphene, 1,8-cineole, α-thujone, camphor, and sesquiterpene hydrocarbon β-caryophyllene as major components and these components contribute to antimicrobial and antifungal properties of the oil [35, 36].


## 5. Conclusion

This study has proved thatC. boreale essential oil exhibited significant inhibition of bacterial growth, adherence capacity, and acid production ofS. mutans. Furthermore,C. boreale essential oil also inhibited the transcription level of several virulence factors such asgtfB,gtfC,gtfD,gbpB,spaP,brpA,relA, andvicR ofS. mutans. In GC and GC-MS analysis, the major components were camphor, β-caryophyllene, α-thujone, piperitone,epi-sesquiphellandrene, α-pinene, 1,8-cineole, β-pinene, and camphene. Therefore,C. boreale essential oil appears to be a promising new agent that may prevent dental caries.



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*Source: 616309-2015-02-11.xml*