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Note
Antimicrobial effect of linalool and
a
-terpineol against periodontopathic and
cariogenic bacteria
Soon-Nang Park
a
, Yun Kyong Lim
a
, Marcelo Oliveira Freire
b
, Eugene Cho
a
, Dongchun Jin
c
,
Joong-Ki Kook
a
,
*
a
Department of Oral Biochemistry, School of Dentistry, Chosun University, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Republic of Korea
b
Herman Ostrow School of Dentistry, University of Southern California, 925 West 34th Street, Los Angeles, CA 90089, USA
c
Department of Veterinary Medicine, College of Agriculture, Yanbian University, Jilin Province 133002, China
article info
Article history:
Received 19 December 2011
Received in revised form
4 April 2012
Accepted 6 April 2012
Available online 17 April 2012
Keywords:
Antimicrobial effect
Linalool
a
-Terpineol
Periodontopathogens
Mutans streptococci
abstract
Linalool and
a
-terpineol exhibited strong antimicrobial activity against periodontopathic and cariogenic
bacteria. However, their concentration should be kept below 0.4 mg/ml if they are to be used as
components of toothpaste or gargling solution. Moreover, other compounds with antimicrobial activity
against periodontopathic and cariogenic bacteria should be used in combination.
Crown Copyright Ó2012 Published by Elsevier Ltd. All rights reserved.
Dental caries and periodontal diseases are major oral infectious
diseases caused by bacteria colonizing the tooth surface. The major
causative bacteria of dental caries are mutans group streptococci
(MS), such as Streptococcus mutans and Streptococcus sobrinus,in
the case of humans [1]. The major causative agents of periodontal
diseases are gram negative anaerobic bacteria, such as Porphylo-
monas gingivalis,Prevotella intermedia,Aggregatibacter actimony-
cetemcomitans,Fusobacterium nucleatum [2,3]. Dental plaque
control is an essential strategy for preventing dental caries and
periodontal diseases. Tooth-brushing and gargling are the most
accepted and effective methods for controlling plaque. Recently,
many studies have attempted to identify effective chemical
compounds extracted from plants that can be used as antimicrobial
agents to prevent dental caries and periodontal diseases [4e6].
Essential oils are odorous, volatile products of plant secondary
metabolism found in many leaves and stems [7]. It has been
reported that essential oils possess antibacterial, antifungal, anti-
leishmanial, and antiviral activities [8e16]. Linalool (3,7-
dimethylocta-1,6-dien-3-ol) and
a
-terpineol [2-(4-Methyl- 1-
cyclohex- 3-enyl) propan-2-ol] are terpene alcohols that have
been shown to display broad spectrum antimicrobial activity
[7,9,17e19]. Nevertheless, there is only limited information on the
antimicrobial effect of linalool and
a
-terpineol against cariogenic
and periodontopathic bacteria [7,9]. In this study, the antibacterial
effects of linalool and
a
-terpineol against cariogenic and perio-
dontopathic bacteria and the cytoxicity of both essential oils on
human oral tissue cells were evaluated to test whether these
compounds can be used as candidates for pre-clinical and clinical
trials in future oral hygiene products.
The following bacterial strains were used: Porphyromonas
gingivalis ATCC 33277
T
,P. gingivalis ATCC 49417, P. gingivalis ATCC
53978, P. intermedia ATCC 25611
T
,P. intermedia ATCC 49046,
Prevotella nigrescens ATCC 33563
T
,P. nigrescens ATCC 25261,
F. nucleatum subsp. nucleatum ATCC 25586
T
,F. nucleatum
subsp. polymorphum ATCC 10953
T
,F. nucleatum subsp. vincentii
ATCC 49046
T
,F. nucleatum subsp. fusiforme ATCC 51190
T
,
F. nucleatum subsp. animalis ATCC 51191
T
,Aggregatibacter actino-
mycetemcomitans ATCC 33384
T
,A. actinomycetemcomitans ATCC
43717, A. actinomycetemcomitans ATCC 43718, S. mutans ATCC
25175
T
and S. sobrinus ATCC 33478
T
. All reference strains were
purchased from the American Type Culture Collection (ATCC, USA).
The clinical strains of S.mutans (KCOM 1054, KCOM 1111, KCOM
*Corresponding author. Tel.: þ82 62 230 6877; fax: þ82 62 224 3706.
E-mail address: jkkook@chosun.ac.kr (J.-K. Kook).
Contents lists available at SciVerse ScienceDirect
Anaerobe
journal homepage: www.elsevier.com/locate/anaerobe
1075-9964/$ esee front matter Crown Copyright Ó2012 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.anaerobe.2012.04.001
Anaerobe 18 (2012) 369e372
Author's personal copy
1113, KCOM 1116, KCOM 1126, KCOM 1128, KCOM 1136, KCOM 1197,
KCOM 1202, KCOM 1207, KCOM 1217) and S.sobrinus (KCOM 1157,
KCOM 1196, KCOM 1221) were obtainedfrom the Korean Collection
for Oral Microbiology (KCOM, Gwangju, Korea).
With the exception of Streptococcus spp., the above strains were
cultured in Tryptic Soy broth supplemented with 0.5% yeast extract,
0.05% cysteine HCleH
2
O, 0.5 mg/ml of hemin and 2
m
g/ml of
vitamin K
1
at 37o
C in an anaerobic chamber (Bactron I, Sheldon
Manufacturing Inc., Cornelius, OR, USA) under an atmosphere
containing 10% H
2
,5%CO
2
and 85% N
2
. All Streptococcus spp. strains
were cultured in Todd Hewitt (TH) broth or on agar plates (Difco,
Lab., Detroit, MI, USA) in a 37o
C incubator that had an atmosphere
composition of 90% air and 10% CO
2
.
The MIC and MBC were determined using a microdilution assay
according to the NCCLS standard [20]. The bacterial strains were
cultured in the described above media at 37o
C in an incubator for
24 h, and added to a 96-well plate to a final concentration of
110
6
CFU/ml. Solutions of linalool (Sigma, St. Louis, MO, USA) or
a
-terpineol (Sigma) dissolved in dimethyl sulfoxide (DMSO; Sigma)
were added to each well to a final concentration of 0.1, 0.2, 0.4, 0.8,
1.6, 3.2 and 6.4 mg/ml. The final concentration of DMSO in each
well was 1%. 1% DMSO in the medium was used as the negative
control and ampicillin (concentration of 100
m
g/ml) was used as
a positive control. After 24 h of incubation under the appropriate
conditions, the lowest concentration of essential oil that inhibited
visible growth was considered the MIC. To determine the MBC
values, 10
m
l of the bacterial culture solution from each well at the
MIC value determined above was diluted 100- or 10,000-fold and
plated onto an appropriate agar plate for each bacterial strain. The
agar plate was incubated in a 37o
C incubator for 24 h and the
number of bacterial colonies was then counted. The concentration
that killed 99.9% of the bacteria was considered the MBC value. All
experiments were carried out in triplicate.
The KB cell line, which is an oral epithelial carcinoma cell line,
was obtained from ATCC. The KB cells were grown in MEM medium
(Gibco, Grand Island, NY, USA) supplemented with 10% heat-
inactivated fetal bovine serum (PAA Laboratories, Etobicoke,
Ontario, Canada), 100 U/ml penicillin and 100
m
g/ml streptomycin
(Gibco) at 37o
C in a humidified atmosphere containing 5% CO
2
.
A MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide, a tetrazole) assay was used to assess cell toxicity of
linalool and
a
-terpineol on the KB cells as described previously [21]
at different linalool and
a
-terpineol concentrations (0.2, 0.4, 0.8,1.6,
3.2 and 6.4 mg/ml).
Linalool or
a
-terpineol showed remarkable antibacterial effects
against the periodontopathic and cariogenic bacteria. The MIC and
MBC values of linalool or
a
-terpineol against 5 species of perio-
dontopathogens ranged from 0.1 to 1.6 mg/ml or 0.1e0.8 mg/ml,
respectively (Table 1). Significant toxicity was observed when
the KB cells were treated with 0.8 mg/ml of linalool or 0.8 mg/ml
of
a
-terpineol, and a mild decrease in cytotoxicity was observed
when 0.4 mg/ml of linalool (76.4%) or 0.2 mg/ml of
a
-terpineol
(73.7%) were used (Fig. 1(A) and (B)). Considering the cell cyto-
toxicity of linalool, all periodontopathogen strains were sensitive
to <0.4 mg/ml of linalool except for the P. gingivalis ATCC 33277
T
,
P. intermedia ATCC 49046, and two P. nigrescens strains. The
sensitivity of the P. gingivalis and P. intermedia strains to linalool
varied according to the strain, whereas the effects on the
P. nigrescens,F. nucleatum, and A. actinomycetemcomitans species
were similar. In addition,
a
-terpineol had a similar antimicrobial
effect on the periodontopathic strains (Table 1).
In this study, the bacterial model system [22] was used to
examine antimicrobial activity and determine the optimal linalool
and
a
-terpineol concentration. The MIC and MBC values of linalool
or
a
-terpineol against mutans streptococci of the bacterial model
system ranged from 0.1 to 3.2 mg/ml and 0.1e1.6 mg/ml,
respectively (Table 2). Of the strains included in the bacterial
model system, S. mutans KCOM 1113, S. mutans KCOM 1136, and
S. sobrinus KCOM 1221 strains were much more sensitive than the
other type strains. The remaining clinical isolates had the same or
lower MIC and MBC values compared to the type strains (Table 2).
In a previous study, the MBC value of the three natural extracts,
Phellodendron amurense,Coptidis rhizome and Galla rhois, against
the clinical strains of S. mutans and S. sobrinus was higher than that
against the type strains, which ranged from 7.5% to 90% and from
60% to 93.3%, respectively [22]. This suggests that the antimicrobial
efficacy of the chemical compounds is dependent on the mutans
streptococci strain.
Essential oils were previously shown to exert their antimicrobial
activity by disrupting bacterial cell walls, inhibiting bacterial
enzyme activity, and suppressing translation of certain regulatory
gene products [23e25]. These findings suggest that the cytotoxic
effect of linalool and
a
-terpineol on human tissue cells occurs
through a similar mechanism. Considering the cytotoxicity to KB
cells, linalool or
a
-terpineol should not be used alone as compo-
nents of oral hygiene products at high concentration (Table 2) and
(Fig. 1). Various essential oils have been shown to display syner-
gistic activities when combined with chlorhexidine digluconate or
Table 1
Antimicrobial effects of essential oils against periodontopathogens.
Species and strains Linalool
a
-Terpineol
MIC (mg/ml) MBC (mg/ml) MIC (mg/ml) MBC (mg/ml)
Porphylomonas gingivalis ATCC 33277
T
0.8 1.6 0.4 0.4
P. gingivalis ATCC 49417 0.1 0.2 0.1 0.2
P. gingivalis ATCC 53978 0.1 0.1 0.4 0.4
Prevotella intermedia ATCC 25611
T
0.2 0.4 0.4 0.4
P. intermedia ATCC 49046 1.6 1.6 0.8 0.8
Prevotella nigrescens ATCC 33563
T
0.8 0.8 0.4 0.4
P. nigrescens ATCC 25261 0.8 0.8 0.4 0.4
Fusobacterium nucleatum subsp. nucleatum ATCC 25586
T
0.2 0.4 0.4 0.4
F. nucleatum subsp. polymorphum ATCC 10953
T
0.2 0.4 0.4 0.4
F. nucleatum subsp. vincentii ATCC 49046
T
0.1 0.1 0.1 0.1
F. nucleatum subsp. fusiforme ATCC 51190
T
0.2 0.4 0.2 0.4
F. nucleatum subsp. animalis ATCC 51191
T
0.2 0.2 0.4 0.4
Aggregatibacter actinomycetemcomitans ATCC 33384
T
0.1 0.2 0.2 0.2
A. actinomycetemcomitans ATCC 43717 0.1 0.1 0.2 0.2
A. actinomycetemcomitans ATCC 43718 0.1 0.1 0.2 0.4
ATCC, America Type Culture Collection.
S.-N. Park et al. / Anaerobe 18 (2012) 369e372370
Author's personal copy
antibiotics [26,27]. A combinatorial approach might be used in
future studies, in which low concentrations of the compounds will
allow for the safe use of the material and enhance the antimicrobial
activity (Table 2) and (Fig. 1).
Based on the in vitro studies, linalool and
a
-terpineol not only
displayed strong antimicrobial activity but were also cytotoxic to
mammalian cells. In general, the effects of chemicals or natural
extracts on human cell viability are often examined in vitro.
However, human oral tissue cells might be more resistant to these
chemicals in vivo than in vitro because oral tissue cells in vivo are
continuously supplied with nutrients through the blood, which can
better support repair mechanisms than those in vitro [4].In
particular, the epithelium of the human oral mucosa is composed of
three to four layers. Although, the superficial layer of the epithe-
lium was detached due to the cytotoxicity of essential oils, in vitro
systems are limited and in vivo outer layers are recovered by the
growth of other layers, particularly epithelial cells of the germinal
layer. More studies will be needed to demonstrate the efficacy and
safety of these compounds.
Acknowledgments
This study was supported by a grant from the Korea Healthcare
technology R&D Project, Ministry for Health, Welfare & Family
Affairs, Republic of Korea (A100960).
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Fig. 1. Effects of (A) Linalool and (B)
a
-Terpineol on the cell viability of KB cells.
Table 2
Antimicrobial effects of essential oils against mutans streptococci.
Species and strains Linalool
a
-Terpineol
MIC
(mg/ml)
MBC
(mg/ml)
MIC
(mg/ml)
MBC
(mg/ml)
Streptococcus mutans ATCC 25175
T
1.6 3.2 0.8 1.6
S. mutans KCOM 1054 1.6 3.2 0.8 0.8
S. mutans KCOM 1111 0.8 1.6 0.8 1.6
S. mutans KCOM 1113 0.4 0.4 0.4 0.4
S. mutans KCOM 1116 0.4 0.8 0.8 0.8
S. mutans KCOM 1126 0.8 0.8 0.4 0.4
S. mutans KCOM 1128 3.2 3.2 0.8 0.8
S. mutans KCOM 1136 0.1 0.1 0.1 0.1
S. mutans KCOM 1197 0.4 0.8 0.8 1.6
S. mutans KCOM 1202 1.6 1.6 0.8 0.8
S. mutans KCOM 1207 0.8 1.6 0.8 0.8
S. mutans KCOM 1217 1.6 1.6 0.8 0.8
Streptococcus sobrinus ATCC 33478
T
1.6 1.6 0.8 0.8
S. sobrinus KCOM 1157 1.6 1.6 0.4 0.8
S. sobrinus KCOM 1196 1.6 1.6 1.6 1.6
S. sobrinus KCOM 1221 0.4 0.8 0.8 0.8
ATCC, America Type Culture Collection; KCOM, Korean Collection for Oral
Microbiolgoy.
S.-N. Park et al. / Anaerobe 18 (2012) 369e372 371
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