# Chamaecyparis obtusa Suppresses Virulence Genes inStreptococcus mutans

Authors: Eun-Hee Kim; Sun-Young Kang; Bog-Im Park; Young-Hoi Kim; Young-Rae Lee; Jin-Hee Hoe; Na-Young Choi; Ji-Young Ra; So-Youn An; Yong-Ouk You
Journal: Evidence-Based Complementary and Alternative Medicine
 (2016)

---

## Abstract

Chamaecyparis obtusa (C. obtusa) is known to have antimicrobial effects and has been used as a medicinal plant and in forest bathing. This study aimed to evaluate the anticariogenic activity of essential oil ofC. obtusa onStreptococcus mutans, which is one of the most important bacterial causes of dental caries and dental biofilm formation. Essential oil fromC. obtusa was extracted, and its effect on bacterial growth, acid production, and biofilm formation was evaluated.C. obtusa essential oil exhibited concentration-dependent inhibition of bacterial growth over 0.025 mg/mL, with 99% inhibition at a concentration of 0.2 mg/mL. The bacterial biofilm formation and acid production were also significantly inhibited at the concentration greater than 0.025 mg/mL. The result of LIVE/DEAD® BacLight™ Bacterial Viability Kit showed a concentration-dependent bactericidal effect onS. mutans and almost all bacteria were dead over 0.8 mg/mL. Real-time PCR analysis showed that gene expression of some virulence factors such asbrpA, gbpB, gtfC, andgtfD was also inhibited. In GC and GC-MS analysis, the major components were found to be α-terpinene (40.60%), bornyl acetate (12.45%), α-pinene (11.38%), β-pinene (7.22%), β-phellandrene (3.45%), and α-terpinolene (3.40%). These results show thatC. obtusa essential oil has anticariogenic effect onS. mutans.


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## Body

## 1. Introduction

Dental caries is the most common infectious oral disease that has afflicted humans including children and adolescents [1]. It is a multifactorial disease, which is caused by detrimental changes in bacterial ecology due to formation of a biofilm that adheres to the tooth surface [2]. During the past few decades, many reports worldwide showed an overall decreasing trend of dental caries. However, recent studies have reported an alarming increase in caries prevalence, especially among the underprivileged groups [3].S. mutans can colonize the oral cavity and form bacterial biofilm. It has the ability to survive in an acidic environment and interact with other microorganisms colonizing this ecosystem [2].Caries results from an imbalance between demineralization and remineralization of tooth structure. Acidogenic bacteria ferment dietary carbohydrates, thereby producing organic acids, which initiate dissolution of tooth enamel and breakdown of dental tissue [4]. The extent of the pH fall is influenced by numerous factors, including the composition of the microflora, as well as the type and frequency of sugar intake [5].S. mutans produce glucosyltransferase (GTF) enzyme which is recognized as virulence factors in the etiology of dental caries. GTF enzymes synthesize extracellular glucans and contribute significantly to the dental plaque matrix’s polysaccharide formation [6]. The sucrose-dependent mechanism of plaque formation is based on GTF produced byS. mutans in combination with glucan-binding proteins (GBPs). The synthesized glucans provide the possibility of both bacterial adhesion to the tooth enamel and adhesion of the microorganisms to each other [2].Demineralization can be reversed by calcium and phosphate, together with fluoride, diffusing into the tooth and depositing a new veneer on the crystal remnants in the noncavitated lesion, and is known as remineralization [4]. Fluoride has been used as the “first choice” for the prevention of dental caries [7], and other anticariogenic natural products or compounds like xylitol have also been introduced [8].C. obtusa is a tropical tree species found in Japan and the southern region of South Korea, and essential oil is extracted from leaves and twigs of theC. obtusa tree. The essential oil has several types of terpenes and has been commercially used in soaps, toothpaste, and cosmetics as a functional additive [9]. The essential oil ofC. obtusa is a concentrated hydrophobic liquid containing volatile compound with natural antibiotic properties that protect against harmful insects, animals, and microorganisms. Inhalation of this essential oil is known asC. obtusa aromatherapy orC. obtusa forest bathing [10] and has been shown to exert antibacterial and antifungal effects [11].This study was performed to analyze anticariogenic effect ofC. obtusa onS. mutans and to determine its chemical composition using a gas chromatography (GC)/gas chromatography-mass spectrometry (GC-MS) analysis.


## 2. Materials and Methods

### 2.1. Plant Material and Essential Oil

C. obtusa was collected in October 2013 from the Jeollanam-do province, South Korea. Fresh leaves and twigs ofC. obtusa (1 kg) were ground mechanically and hydrodistilled for 3 hours using a Clevenger-type apparatus. The yield of theC. obtusa essential oil was 1.08% of yellow pale oil, based on the fresh weight of the plant. TheC. obtusa essential oil was stored in a deep freezer (−70°C) to minimize the loss 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, USA) broth under aerobic condition at 37°C. The growth ofS. mutans was examined at 37°C in 0.95 mL of brain heart infusion broth containing various concentrations of theC. obtusa. These tubes were inoculated with 0.05 mL of an overnight culture grown in the BHI broth (final: 5 × 105 colony-forming units [CFU]/mL) and incubated at 37°C for 24 h. The optical density (OD) of cells was measured at 550 nm using a spectrophotometer. Each concentration of the extract was tested in triplicate.


### 2.3. Acid Production

Acid production byS. mutans was examined to evaluate the effect ofC. obtusa essential oil using a modification of previously described method [12]. TheC. obtusa essential oil was filter-sterilized 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 of the cultures was determined using a pH meter (Corning, Inc., Corning, NY, USA). Three replicates were measured for each concentration of the test extract.


### 2.4. Biofilm Formation Assay

The biofilm assay was based on a previously described method [13, 14]. Biofilm formation was measured by staining with safranin and observed by scanning electron microscopy (SEM).C. obtusa essential oil was added to BHI broth containing 0.1% sucrose in 35 mm polystyrene dishes. The cultures were then inoculated with a seed culture ofS. mutans (final: 5 × 105 CFU/mL) and incubated for 24 h at 37°C. After incubation, the supernatants were removed, and the dishes were rinsed with distilled water. Biofilm formation were stained with 0.1% safranin and photographed. The bound safranin was released from the stained cells with 30% acetic acid and the absorbance of the solution was measured at 530 nm. In addition, biofilm on 35 mm polystyrene dishes was observed by SEM [15]. The biofilm formed on the dishes was rinsed with distilled water, fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at 4°C for 24 h, and dehydrated with ethanol gradient series (60%, 70%, 80%, 90%, 95%, and 100%). Then, the samples were freeze-dried, sputter-coated with gold (108A sputter coater; Cressington Scientific Instruments, Inc., Watford, England, United Kingdom), and observed by SEM (JSM-6360, JEOL, Tokyo, Japan).


### 2.5. Confocal Laser Scanning Microscopy

To determine the bactericidal effect ofC. obtusa essential oil onS. mutans, staining with LIVE/DEAD®BacLight™ Bacterial Viability Kit was performed and examined by confocal laser scanning microscopy. The culturedS. mutans in BHI was diluted using BHI media to 1 × 107 CFU/mL and treated with the essential oil at 37°C under aerobic conditions. After 30 min of incubation, the bacteria were washed with PBS and stained with the LIVE/DEAD®BacLight™ Bacterial Viability Kit (Molecular Probes, Eugene, OR, USA), prepared according to the manufacturer’s protocol. After 15 min of staining, bacteria were observed using confocal laser scanning microscopy (LSM 510, Zeiss, Oberkochen, Germany).


### 2.6. Real-Time Polymerase Chain Reaction Analysis

A real-time PCR was performed to examine the effect ofC. obtusa essential oil on virulence factor gene expression ofS. mutans. The subminimal inhibitory concentration (0.025–0.1 mg/mL) of the essential oil was used. After 24 h of culture, total RNA was isolated fromS. mutans using a TRIzol® Reagent (Life Technologies, Carlsbad, CA, USA) and cDNA was synthesized. The amplification was performed using a StepOnePlus Real-Time PCR system with SYBR® Green Master Mix (Applied Biosystems, Carlsbad, CA, USA). 16S rRNA was used as an internal control.


### 2.7. GC and GC-MS Analysis

GC analysis was performed on a Hewlett-Packard model 6890 series gas chromatograph with a flame ionization detector (FID) and a split ratio of 30 : 1 using DB-5HT fused silica capillary columns (30 m × 0.32 mm, i.d., 0.10μm film thickness). The temperature of 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 were 230°C and 250°C, respectively. The gas carrier used was nitrogen, at a flow rate of 0.80 mL/min. Peak areas were measured by electronic integration and the relative amounts of the individual components were determined based on the peak areas. The GC-MS analysis was carried out on an Agilent Technologies 7890A GC and 5975C mass selective detector (MSD) operating in EI mode at 70 eV, fitted with a DB-5MS fused silica capillary column (30 m × 0.25 mm, i.d., 0.25 μm film thickness). The column temperature 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 ion source temperatures were 250°C, respectively. The gas carrier was helium at a flow rate of 1.0 mL/min. The identification of individual components was based on comparisons with Wiley 7n/NIST 05 mass spectra libraries and retention indices with reference to literature data. Linear retention indices were calculated against those of n-paraffin (C6–C26) series [16].


### 2.8. Statistical Analysis

All experiments were performed in triplicate. Data were analyzed using SPSS software 12.0 (Chicago, IL, USA). The data are expressed as the mean ± standard deviation values. The statistical analysis was done using Student’st-test. Values of p
<
0.01 were considered statistically significant.


## 2.1. Plant Material and Essential Oil

C. obtusa was collected in October 2013 from the Jeollanam-do province, South Korea. Fresh leaves and twigs ofC. obtusa (1 kg) were ground mechanically and hydrodistilled for 3 hours using a Clevenger-type apparatus. The yield of theC. obtusa essential oil was 1.08% of yellow pale oil, based on the fresh weight of the plant. TheC. obtusa essential oil was stored in a deep freezer (−70°C) to minimize the loss 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, USA) broth under aerobic condition at 37°C. The growth ofS. mutans was examined at 37°C in 0.95 mL of brain heart infusion broth containing various concentrations of theC. obtusa. These tubes were inoculated with 0.05 mL of an overnight culture grown in the BHI broth (final: 5 × 105 colony-forming units [CFU]/mL) and incubated at 37°C for 24 h. The optical density (OD) of cells was measured at 550 nm using a spectrophotometer. Each concentration of the extract was tested in triplicate.


## 2.3. Acid Production

Acid production byS. mutans was examined to evaluate the effect ofC. obtusa essential oil using a modification of previously described method [12]. TheC. obtusa essential oil was filter-sterilized 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 of the cultures was determined using a pH meter (Corning, Inc., Corning, NY, USA). Three replicates were measured for each concentration of the test extract.


## 2.4. Biofilm Formation Assay

The biofilm assay was based on a previously described method [13, 14]. Biofilm formation was measured by staining with safranin and observed by scanning electron microscopy (SEM).C. obtusa essential oil was added to BHI broth containing 0.1% sucrose in 35 mm polystyrene dishes. The cultures were then inoculated with a seed culture ofS. mutans (final: 5 × 105 CFU/mL) and incubated for 24 h at 37°C. After incubation, the supernatants were removed, and the dishes were rinsed with distilled water. Biofilm formation were stained with 0.1% safranin and photographed. The bound safranin was released from the stained cells with 30% acetic acid and the absorbance of the solution was measured at 530 nm. In addition, biofilm on 35 mm polystyrene dishes was observed by SEM [15]. The biofilm formed on the dishes was rinsed with distilled water, fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at 4°C for 24 h, and dehydrated with ethanol gradient series (60%, 70%, 80%, 90%, 95%, and 100%). Then, the samples were freeze-dried, sputter-coated with gold (108A sputter coater; Cressington Scientific Instruments, Inc., Watford, England, United Kingdom), and observed by SEM (JSM-6360, JEOL, Tokyo, Japan).


## 2.5. Confocal Laser Scanning Microscopy

To determine the bactericidal effect ofC. obtusa essential oil onS. mutans, staining with LIVE/DEAD®BacLight™ Bacterial Viability Kit was performed and examined by confocal laser scanning microscopy. The culturedS. mutans in BHI was diluted using BHI media to 1 × 107 CFU/mL and treated with the essential oil at 37°C under aerobic conditions. After 30 min of incubation, the bacteria were washed with PBS and stained with the LIVE/DEAD®BacLight™ Bacterial Viability Kit (Molecular Probes, Eugene, OR, USA), prepared according to the manufacturer’s protocol. After 15 min of staining, bacteria were observed using confocal laser scanning microscopy (LSM 510, Zeiss, Oberkochen, Germany).


## 2.6. Real-Time Polymerase Chain Reaction Analysis

A real-time PCR was performed to examine the effect ofC. obtusa essential oil on virulence factor gene expression ofS. mutans. The subminimal inhibitory concentration (0.025–0.1 mg/mL) of the essential oil was used. After 24 h of culture, total RNA was isolated fromS. mutans using a TRIzol® Reagent (Life Technologies, Carlsbad, CA, USA) and cDNA was synthesized. The amplification was performed using a StepOnePlus Real-Time PCR system with SYBR® Green Master Mix (Applied Biosystems, Carlsbad, CA, USA). 16S rRNA was used as an internal control.


## 2.7. GC and GC-MS Analysis

GC analysis was performed on a Hewlett-Packard model 6890 series gas chromatograph with a flame ionization detector (FID) and a split ratio of 30 : 1 using DB-5HT fused silica capillary columns (30 m × 0.32 mm, i.d., 0.10μm film thickness). The temperature of 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 were 230°C and 250°C, respectively. The gas carrier used was nitrogen, at a flow rate of 0.80 mL/min. Peak areas were measured by electronic integration and the relative amounts of the individual components were determined based on the peak areas. The GC-MS analysis was carried out on an Agilent Technologies 7890A GC and 5975C mass selective detector (MSD) operating in EI mode at 70 eV, fitted with a DB-5MS fused silica capillary column (30 m × 0.25 mm, i.d., 0.25 μm film thickness). The column temperature 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 ion source temperatures were 250°C, respectively. The gas carrier was helium at a flow rate of 1.0 mL/min. The identification of individual components was based on comparisons with Wiley 7n/NIST 05 mass spectra libraries and retention indices with reference to literature data. Linear retention indices were calculated against those of n-paraffin (C6–C26) series [16].


## 2.8. Statistical Analysis

All experiments were performed in triplicate. Data were analyzed using SPSS software 12.0 (Chicago, IL, USA). The data are expressed as the mean ± standard deviation values. The statistical analysis was done using Student’st-test. Values of p
<
0.01 were considered statistically significant.


## 3. Results

### 3.1. Inhibition of Bacterial Growth

After extraction ofC. obtusa essential oil by hydrodistillation, the antibacterial activity of the oil was tested againstS. mutans.C. obtusa essential oil significantly inhibited the growth ofS. mutans in a concentration-dependent manner. The bacteria were treated with 0.025, 0.05, 0.1, and 0.2 mg/mL ofC. obtusa essential oil. When treated with 0.025 mg/mL of the essential oil, the bacterial growth was significantly inhibited in comparison to the control group (p
<
0.05). The positive control (0.1% NaF) also showed antibacterial activity (Figure 1).Figure 1
Bacterial growth inhibition ofStreptococcus mutans (S. mutans) byChamaecyparis obtusa (C. obtusa) essential oil. The optical density (A550) was read using a spectrophotometer. p
∗
<
0.01 compared to the control group.


### 3.2. Inhibition of Acid Production

To investigate whetherC. obtusa essential oil can inhibitS. mutans organic acid production, the bacteria were cultured in the presence of various concentrations (0.025–0.2 mg/mL) of the essential oil and the change in pH was measured. The pH of the control declined to 5.30 after bacterial culture, while the initial pH of the media before bacterial culture was 7.40. However, the addition of 0.025, 0.05, 0.1, and 0.2 mg/mL ofC. obtusa essential oil resulted in pH levels of 5.38, 5.73, 7.12, and 7.40, respectively. These results indicate thatC. obtusa essential oil may inhibit the organic acid production byS. mutans. NaF (0.1%) used for the positive control also inhibited acid production, resulting in a pH of 7.10 (Table 1).Table 1
Inhibitory effect ofC. obtusa essential oil on acid production by S. mutans.
Con. (mg/mL)
pH (before incubation)
pH (after incubation)
Control
7.40 ± 0.00
5.30 ± 0.00
0.025
7.40 ± 0.00
5.38 ± 0.01∗
0.05
7.39 ± 0.00
5.73 ± 0.00∗
0.1
7.40 ± 0.00
7.12 ± 0.00∗
0.2
7.40 ± 0.00
7.40 ± 0.00∗
0.1% NaF
7.40 ± 0.00
7.10 ± 0.00∗
Data (pH) are represented as mean ± standard deviation.
⁢p
∗
<
0.01 when compared with the control group after incubation.


### 3.3. Inhibition of Biofilm Formation

To determine whetherC. obtusa essential oil inhibits biofilm formation byS. mutans, the bacteria were cultured in the presence of various concentration ofC. obtusa essential oil in polystyrene dishes. Biofilm formation was studied using safranin staining, and absorbance was measured at 530 nm. The biofilm formation byS. mutans was significantly inhibited by treatment withC. obtusa essential oil in a dose-dependent manner over 0.025 mg/mL ofC. obtusa essential oil. When treated with 0.1% NaF (positive control), complete inhibition was shown (Figure 2). SEM results were consistent with those of safranin staining (Figure 3).Figure 2
Inhibitory effect ofC. obtusa essential oil on biofilm formation byS. mutans, as observed by safranin staining. (a) Control; (b) 0.025 mg/mL; (c) 0.05 mg/mL; (d) 0.1 mg/mL; (e) 0.2 mg/mL essential oil ofC. obtusa; and (f) positive control (0.1% NaF). S
i
g
n
i
f
i
c
a
n
c
e
∗ was determined at p
∗
<
0.01 compared to the control group.Figure 3
Inhibitory effect ofC. obtusa essential oil on biofilm formation byS. mutans, as observed by scanning electron microscopy. (a) Control; (b) 0.025 mg/mL; (c) 0.05 mg/L; (D) 0.1 mg/mL; (e) 0.2 mg/mL; and (f) positive control (0.1% NaF). Scale bar = 10 μm.
(a)
(b)
(c)
(d)
(e)
(f)


### 3.4. Bactericidal Effect

To evaluate bactericidal effect ofC. obtusa essential oil,S. mutans were cultured in presence of high concentrations (0.2–1.6 mg/mL) of the essential oil, stained with LIVE/DEAD®BacLight™ Bacterial Viability Kit and observed using confocal laser scanning microscopy. Treatment withC. obtusa essential oil decreases living bacteria (green fluorescence labeled cell stained by SYTO® 9) and increases dead bacteria (red fluorescence labeled cell stained by PI). The bactericidal effect ofC. obtusa essential oil was also observed in a dose-dependent manner (Figure 4). However, low concentration (lesser than 0.2 mg/mL) of the essential oil did not show bactericidal effect (data not shown).Figure 4
Bactericidal effect ofC. obtusa essential oil. CulturedS. mutans was treated withC. obtusa extract (0.2–1.6 mg/mL) and stained with LIVE/DEAD®BacLight™ Bacterial Viability Kit. The stained bacteria were observed by confocal laser scanning microscopy. Treatment withC. obtusa decreased green-labeled living bacteria (SYTO® 9 stain) and increased red-labeled dead bacteria (PI stain) in a dose-dependent manner. (a) Control; (b) 0.2 mg/mL; (c) 0.4 mg/mL; (d) 0.8 mg/mL; (e) 1.6 mg/mL; and (f) positive control (0.1% NaF). Bar = 100 μm.
(a)
(b)
(c)
(d)
(e)
(f)


### 3.5. Inhibition of Virulence Factor Gene Expression

To evaluate the influence ofC. obtusa on the gene expression of virulence factors inS. mutans, the bacteria were treated with subminimal inhibitory concentration (0.025–0.1 mg/mL) ofC. obtusa, and the gene expressions of various virulence factors were assessed by real-time PCR. We evaluated the genetic expressions ofbrpA,gbpB,gtfC, andgtfD. The expressions ofbrpA, which contribute to the control of the phosphotransferase system (PTS) and are related with acid tolerance, andgbpB which contribute to bacterial adherence, were significantly decreased at 0.025 mg/mL ofC. obtusa. The expression ofgtfC andgtfD which encode GTFase C and D proteins was significantly decreased whenS. mutans was treated with 0.025 mg/mL ofC. obtusa (Figure 5).Figure 5
Inhibitory effect ofC. obtusa essential oil on virulence factor gene expression.S. mutans was cultured and treated with subminimal inhibitory concentration (0.025–0.1 mg/mL) ofC. obtusa extract, and real-time polymerase chain reaction (PCR) analysis was performed. ThebrpA, gbpB, gtfC, andgtfD expressions were significantly inhibited at 0.025 mg/mL ofC. obtusa. Each value is expressed as mean ± standard deviation. S
i
g
n
i
f
i
c
a
n
c
e
∗ was determined at p
∗
<
0.01 when compared with the control group.


### 3.6. The Composition ofC. obtusa Essential Oil by GC and GC-MS Analysis

Fifty-nine constituents were identified in theC. obtusa essential oil, accounting for up to 97.38% of the total material. Thus, all unidentified compounds were only minor components. The major components included α-terpinene (40.60%), bornyl acetate (12.45%), α-pinene (11.38%), β-pinene (7.22%), β-phellandrene (3.45%), and α-terpinolene (3.40%) (Table 2).Table 2
Gas chromatography and gas chromatography-mass spectrometry (GC/GC-MS) analysis of the essential oil isolated fromC. obtusa.
Retention time (min)
Retention indexa
Compound
Peak area (%)
8.849
801
n-Hexanal
0.18
11.982
852
trans-2-Hexenal
0.28
13.110
870
n-Hexanol
0.06
15.125
902
Bornylene
0.12
16.419
919
Tricyclene
0.77
16.763
925
α-Thujene
0.12
17.742
936
α-Pinene
11.38
18.650
952
Camphene
2.87
20.228
975
Sabinene
0.46
21.345
980
δ-3-Carene
0.37
22.047
983
β-Pinene
7.22
24.483
1023
α-Terpinene
40.60
24.927
1029
Limonene
0.57
25.474
1036
β-Phellandrene
3.45
27.317
1061
γ-Terpinene
0.21
28.393
1072
cis-Sabinene hydrate
0.35
29.742
1089
α-Terpinolene
3.40
29.983
1092
Dehydro-p-cymene
0.53
30.874
1102
trans-Sabinene hydrate
0.09
31.769
1115
1-Octen-3-yl acetate
0.06
33.199
1133
trans-p-2-Menthen-1-ol
0.05
36.312
1173
Cryptone
0.11
36.461
1175
p-Cymen-8-ol
0.10
36.965
1181
Terpinen-4-ol
0.23
37.889
1193
α-Terpineol
0.11
38.960
1207
Verbenone
0.09
39.699
1217
Fenchyl acetate
0.07
42.535
1256
Linalyl acetate
0.08
45.552
1296
Bornyl acetate
12.45
49.502
1352
α-Terpinyl acetate
0.62
51.422
1380
α-Copaene
0.28
53.780
1414
α-Cedrene
0.52
54.015
1417
β-Caryophyllene
0.16
54.331
1422
β-Cedrene
0.25
55.009
1431
cis-Thujopsene
0.07
55.419
1434
β-Gurjunene
0.06
56.378
1453
α-Humulene
0.37
56.976
1462
cis-Muurola-4(14),5-diene
0.24
57.656
1472
β-Cadinene
0.06
57.927
1476
Germacrene D
0.13
58.150
1490
β-Himachalene
0.10
59.003
1492
α-Muurolene
0.16
59.519
1497
α-Chamigrene
0.05
60.024
1508
γ-Cadinene
0.38
61.079
1525
δ-Cadinene
0.15
61.522
1530
Cadina-1,4-diene
0.09
61.937
1539
Selina-3,7(11)-diene
0.07
63.011
1551
Elemol
0.07
63.406
1562
trans-Nerolidol
0.25
64.651
1578
Spathulenol
0.05
64.916
1586
Caryophyllene oxide
0.10
66.435
1603
Cedrol
0.97
67.079
1622
1-epi-Cubenol
0.08
67.835
1635
γ-Eudesmol
0.58
68.123
1239
τ-Cadinol
0.57
68.964
1648
α-Cadinol
0.09
69.973
1670
Bulnesol
0.07
70.829
1685
α-Bisabolol
0.13
82.676
1898
Rimuene
0.27
84.836
1926
Hibaene
2.33
85.085
1945
Pimaradiene
0.10
86.747
1977
13-Isopimaradiene
1.42
88.068
1998
Dolabradiene
0.09
90.446
2051
Dehydroabietane
0.07
Total
97.38
aRetention index on DB-5HT column.


## 3.1. Inhibition of Bacterial Growth

After extraction ofC. obtusa essential oil by hydrodistillation, the antibacterial activity of the oil was tested againstS. mutans.C. obtusa essential oil significantly inhibited the growth ofS. mutans in a concentration-dependent manner. The bacteria were treated with 0.025, 0.05, 0.1, and 0.2 mg/mL ofC. obtusa essential oil. When treated with 0.025 mg/mL of the essential oil, the bacterial growth was significantly inhibited in comparison to the control group (p
<
0.05). The positive control (0.1% NaF) also showed antibacterial activity (Figure 1).Figure 1
Bacterial growth inhibition ofStreptococcus mutans (S. mutans) byChamaecyparis obtusa (C. obtusa) essential oil. The optical density (A550) was read using a spectrophotometer. p
∗
<
0.01 compared to the control group.


## 3.2. Inhibition of Acid Production

To investigate whetherC. obtusa essential oil can inhibitS. mutans organic acid production, the bacteria were cultured in the presence of various concentrations (0.025–0.2 mg/mL) of the essential oil and the change in pH was measured. The pH of the control declined to 5.30 after bacterial culture, while the initial pH of the media before bacterial culture was 7.40. However, the addition of 0.025, 0.05, 0.1, and 0.2 mg/mL ofC. obtusa essential oil resulted in pH levels of 5.38, 5.73, 7.12, and 7.40, respectively. These results indicate thatC. obtusa essential oil may inhibit the organic acid production byS. mutans. NaF (0.1%) used for the positive control also inhibited acid production, resulting in a pH of 7.10 (Table 1).Table 1
Inhibitory effect ofC. obtusa essential oil on acid production by S. mutans.
Con. (mg/mL)
pH (before incubation)
pH (after incubation)
Control
7.40 ± 0.00
5.30 ± 0.00
0.025
7.40 ± 0.00
5.38 ± 0.01∗
0.05
7.39 ± 0.00
5.73 ± 0.00∗
0.1
7.40 ± 0.00
7.12 ± 0.00∗
0.2
7.40 ± 0.00
7.40 ± 0.00∗
0.1% NaF
7.40 ± 0.00
7.10 ± 0.00∗
Data (pH) are represented as mean ± standard deviation.
⁢p
∗
<
0.01 when compared with the control group after incubation.


## 3.3. Inhibition of Biofilm Formation

To determine whetherC. obtusa essential oil inhibits biofilm formation byS. mutans, the bacteria were cultured in the presence of various concentration ofC. obtusa essential oil in polystyrene dishes. Biofilm formation was studied using safranin staining, and absorbance was measured at 530 nm. The biofilm formation byS. mutans was significantly inhibited by treatment withC. obtusa essential oil in a dose-dependent manner over 0.025 mg/mL ofC. obtusa essential oil. When treated with 0.1% NaF (positive control), complete inhibition was shown (Figure 2). SEM results were consistent with those of safranin staining (Figure 3).Figure 2
Inhibitory effect ofC. obtusa essential oil on biofilm formation byS. mutans, as observed by safranin staining. (a) Control; (b) 0.025 mg/mL; (c) 0.05 mg/mL; (d) 0.1 mg/mL; (e) 0.2 mg/mL essential oil ofC. obtusa; and (f) positive control (0.1% NaF). S
i
g
n
i
f
i
c
a
n
c
e
∗ was determined at p
∗
<
0.01 compared to the control group.Figure 3
Inhibitory effect ofC. obtusa essential oil on biofilm formation byS. mutans, as observed by scanning electron microscopy. (a) Control; (b) 0.025 mg/mL; (c) 0.05 mg/L; (D) 0.1 mg/mL; (e) 0.2 mg/mL; and (f) positive control (0.1% NaF). Scale bar = 10 μm.
(a)
(b)
(c)
(d)
(e)
(f)


## 3.4. Bactericidal Effect

To evaluate bactericidal effect ofC. obtusa essential oil,S. mutans were cultured in presence of high concentrations (0.2–1.6 mg/mL) of the essential oil, stained with LIVE/DEAD®BacLight™ Bacterial Viability Kit and observed using confocal laser scanning microscopy. Treatment withC. obtusa essential oil decreases living bacteria (green fluorescence labeled cell stained by SYTO® 9) and increases dead bacteria (red fluorescence labeled cell stained by PI). The bactericidal effect ofC. obtusa essential oil was also observed in a dose-dependent manner (Figure 4). However, low concentration (lesser than 0.2 mg/mL) of the essential oil did not show bactericidal effect (data not shown).Figure 4
Bactericidal effect ofC. obtusa essential oil. CulturedS. mutans was treated withC. obtusa extract (0.2–1.6 mg/mL) and stained with LIVE/DEAD®BacLight™ Bacterial Viability Kit. The stained bacteria were observed by confocal laser scanning microscopy. Treatment withC. obtusa decreased green-labeled living bacteria (SYTO® 9 stain) and increased red-labeled dead bacteria (PI stain) in a dose-dependent manner. (a) Control; (b) 0.2 mg/mL; (c) 0.4 mg/mL; (d) 0.8 mg/mL; (e) 1.6 mg/mL; and (f) positive control (0.1% NaF). Bar = 100 μm.
(a)
(b)
(c)
(d)
(e)
(f)


## 3.5. Inhibition of Virulence Factor Gene Expression

To evaluate the influence ofC. obtusa on the gene expression of virulence factors inS. mutans, the bacteria were treated with subminimal inhibitory concentration (0.025–0.1 mg/mL) ofC. obtusa, and the gene expressions of various virulence factors were assessed by real-time PCR. We evaluated the genetic expressions ofbrpA,gbpB,gtfC, andgtfD. The expressions ofbrpA, which contribute to the control of the phosphotransferase system (PTS) and are related with acid tolerance, andgbpB which contribute to bacterial adherence, were significantly decreased at 0.025 mg/mL ofC. obtusa. The expression ofgtfC andgtfD which encode GTFase C and D proteins was significantly decreased whenS. mutans was treated with 0.025 mg/mL ofC. obtusa (Figure 5).Figure 5
Inhibitory effect ofC. obtusa essential oil on virulence factor gene expression.S. mutans was cultured and treated with subminimal inhibitory concentration (0.025–0.1 mg/mL) ofC. obtusa extract, and real-time polymerase chain reaction (PCR) analysis was performed. ThebrpA, gbpB, gtfC, andgtfD expressions were significantly inhibited at 0.025 mg/mL ofC. obtusa. Each value is expressed as mean ± standard deviation. S
i
g
n
i
f
i
c
a
n
c
e
∗ was determined at p
∗
<
0.01 when compared with the control group.


## 3.6. The Composition ofC. obtusa Essential Oil by GC and GC-MS Analysis

Fifty-nine constituents were identified in theC. obtusa essential oil, accounting for up to 97.38% of the total material. Thus, all unidentified compounds were only minor components. The major components included α-terpinene (40.60%), bornyl acetate (12.45%), α-pinene (11.38%), β-pinene (7.22%), β-phellandrene (3.45%), and α-terpinolene (3.40%) (Table 2).Table 2
Gas chromatography and gas chromatography-mass spectrometry (GC/GC-MS) analysis of the essential oil isolated fromC. obtusa.
Retention time (min)
Retention indexa
Compound
Peak area (%)
8.849
801
n-Hexanal
0.18
11.982
852
trans-2-Hexenal
0.28
13.110
870
n-Hexanol
0.06
15.125
902
Bornylene
0.12
16.419
919
Tricyclene
0.77
16.763
925
α-Thujene
0.12
17.742
936
α-Pinene
11.38
18.650
952
Camphene
2.87
20.228
975
Sabinene
0.46
21.345
980
δ-3-Carene
0.37
22.047
983
β-Pinene
7.22
24.483
1023
α-Terpinene
40.60
24.927
1029
Limonene
0.57
25.474
1036
β-Phellandrene
3.45
27.317
1061
γ-Terpinene
0.21
28.393
1072
cis-Sabinene hydrate
0.35
29.742
1089
α-Terpinolene
3.40
29.983
1092
Dehydro-p-cymene
0.53
30.874
1102
trans-Sabinene hydrate
0.09
31.769
1115
1-Octen-3-yl acetate
0.06
33.199
1133
trans-p-2-Menthen-1-ol
0.05
36.312
1173
Cryptone
0.11
36.461
1175
p-Cymen-8-ol
0.10
36.965
1181
Terpinen-4-ol
0.23
37.889
1193
α-Terpineol
0.11
38.960
1207
Verbenone
0.09
39.699
1217
Fenchyl acetate
0.07
42.535
1256
Linalyl acetate
0.08
45.552
1296
Bornyl acetate
12.45
49.502
1352
α-Terpinyl acetate
0.62
51.422
1380
α-Copaene
0.28
53.780
1414
α-Cedrene
0.52
54.015
1417
β-Caryophyllene
0.16
54.331
1422
β-Cedrene
0.25
55.009
1431
cis-Thujopsene
0.07
55.419
1434
β-Gurjunene
0.06
56.378
1453
α-Humulene
0.37
56.976
1462
cis-Muurola-4(14),5-diene
0.24
57.656
1472
β-Cadinene
0.06
57.927
1476
Germacrene D
0.13
58.150
1490
β-Himachalene
0.10
59.003
1492
α-Muurolene
0.16
59.519
1497
α-Chamigrene
0.05
60.024
1508
γ-Cadinene
0.38
61.079
1525
δ-Cadinene
0.15
61.522
1530
Cadina-1,4-diene
0.09
61.937
1539
Selina-3,7(11)-diene
0.07
63.011
1551
Elemol
0.07
63.406
1562
trans-Nerolidol
0.25
64.651
1578
Spathulenol
0.05
64.916
1586
Caryophyllene oxide
0.10
66.435
1603
Cedrol
0.97
67.079
1622
1-epi-Cubenol
0.08
67.835
1635
γ-Eudesmol
0.58
68.123
1239
τ-Cadinol
0.57
68.964
1648
α-Cadinol
0.09
69.973
1670
Bulnesol
0.07
70.829
1685
α-Bisabolol
0.13
82.676
1898
Rimuene
0.27
84.836
1926
Hibaene
2.33
85.085
1945
Pimaradiene
0.10
86.747
1977
13-Isopimaradiene
1.42
88.068
1998
Dolabradiene
0.09
90.446
2051
Dehydroabietane
0.07
Total
97.38
aRetention index on DB-5HT column.


## 4. Discussion

Fluoride plays an important role in the prevention and control of dental caries. However, an unfortunate side effect of fluoride is fluorosis. Ingestion of fluoride before 2 to 3 years of age is considered critical for possible fluorosis in the permanent dentition [17]. Therefore, natural products are currently receiving special attention as a good alternative to synthetic chemical substances for the prevention of dental caries [18].Essential oils are volatile components mainly obtained by distillation of plant and consist of a mixture of various terpenoids. Terpenes are the therapeutic chemical substances present in medicinal plants [19]. Essential oils extracted from various plants are known to have antimicrobial activity [20]. Some natural derivatives like extracts fromMyristica fragrans,Lippia sidoides,Hyptis pectinata,Curcuma longa, andBaccharis dracunculifolia have been proved to be effective againstS. mutans [21–25]. This study was performed to evaluate anticariogenic activity ofC. obtusa essential oil onS. mutans.To evaluate anticariogenic properties ofC. obtusa essential oil,S. mutans was used because these bacteria is considered as a major cause for the formation of dental caries [2]. Our results showed that growth ofS. mutans was suppressed by treatment withC. obtusa essential oil. Furthermore, the results of LIVE/DEAD®BacLight™ Bacterial Viability Kit also showed thatC. obtusa essential oil has a bactericidal effect againstS. mutans. These results suggested thatC. obtusa essential oil has a potential for anticariogenic effect, which is interesting since the inhibition of the growth ofS. mutans is one of the strategies for prevention of dental caries.Growth ofS. mutans was suppressed by treatment withC. obtusa essential oil in a concentration-dependent manner above the concentration of 0.025 mg/mL. In dental plaque formation, pH is one of the major factors, since low pH leads to demineralized hydroxyapatite and favors the cariogenicity [26].S. mutans can metabolize dietary sugars and produce organic acid. Low-pH environment in the biofilm matrix results in dissolution of enamel, thus initiating the pathogenesis of dental caries. Therefore, the alteration of pH is used as an indicator to determine the effect of anticariogenic agents [27]. In this study,C. obtusa essential oil inhibited the decrease of pH induced byS. mutans and the result suggests thatC. obtusa essential oil may inhibit dental caries through inhibition of acid production byS. mutans.Biofilms are communities of microorganisms that adhere to biological or abiotic substrata and produce an extracellular matrix typically comprising of polysaccharides and proteins. Dental plaque is a kind of biofilm found on a tooth surface, embedded in a matrix of host and bacterial polymers [28]. In biofilm, known as plaque in the oral cavity, the interaction of specific bacteria with constituents of the diet results in caries [27]. Biofilm formation byS. mutans was also inhibited by treatment withC. obtusa essential oil cultured on polystyrene dishes. These results suggested thatC. obtusa essential oil directly inhibits the biofilm formation byS. mutans.Furthermore, several virulence gene factors ofS. mutans are associated with various aspects of cariogenicity such as acid tolerance, bacterial adhesion, and biofilm formation [2]. ThebrpA has been shown to contribute to biofilm formation and plays a major role in cell envelope biogenesis/homeostasis and regulation of stress response as well as in acid tolerance [29, 30]. The gbpB, which encodes surface-associated glucan-binding protein (GBP), mediates binding of bacteria to glucans and enables development of biofilm. The gtfC and gtfD encode glucosyltransferases (GTFase) which are essential virulence factor in plaque development and are responsible for glucans formation from sucrose [2]. In this study, to evaluate correlation between inhibitory effect byC. obtusa essential oil and virulence factors expression, we determined the mRNA expression level of several virulence factors using a real-time PCR analysis. We evaluated the gene expression level ofbrpA,gbpB,gtfC, andgtfD. C. obtusa essential oil significantly inhibited the transcription level ofbrpA,gbpB,gtfC, andgtfD.Based on our results of GC/GC-MS analysis, the major components includedα-terpinene (40.60%), bornyl acetate (12.45%), α-pinene (11.38%), β-pinene (7.22%), β-phellandrene (3.45%), and α-terpinolene (3.40%) (Table 2). Although the biological activities ofC. obtusa essential oil are not yet fully understood, some previous study reported that several types of terpenes ofC. obtusa have been shown to exert antibacterial and antifungal effect [11]. Recent studies also reported other beneficial properties ofC. obtusa, such as pharmacological activities for the treatment of atopic dermatitis [31], improvement on cognitive function of the central nervous system on rat experimental model [10], and promotion of hair growth [32]. In addition,C. obtusa has antinociceptive and anti-inflammatory properties, which increases its applicability in oral care [9, 33].Based on our results, we conclude thatC. obtusa may have a possible practical use against the cariogenic bacteria within the mouth and recommend further investigation ofC. obtusa on periodontopathic and cariogenic bacteria.


## 5. Conclusions

This study has proved thatC. obtusa essential oil exhibited significant inhibition of bacterial growth, acid production, and biofilm formation byS. mutans. AlsoC. obtusa essential oil showed bactericidal effect. Furthermore,C. obtusa essential oil also inhibited the transcription level of several virulence factors such asbrpA,gbpB,gtfC, andgtfD ofS. mutans. In GC and GC-MS analysis, the major components were α-terpinene (40.60%), bornyl acetate (12.45%), α-pinene (11.38%), β-pinene (7.22%), β-phellandrene (3.45%), and α-terpinolene (3.40%). Therefore, the results of this study indicate thatC. obtusa essential oil showed good anticariogenic effect onS. mutans and appear to be a promising new agent that may prevent dental caries. Further studies are needed to develop the new medicine for clinical use.



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*Source: 2396404-2016-05-11.xml*