838 Regular Article Biol. Pharm. Bull. 36(5) 838–844 (2013) Vol. 36, No.

Suppression of Inflammatory Reactions by Terpinen-4-ol, a Main

Constituent of Tea Tree Oil, in a Murine Model of Oral Candidiasis and
Its Suppressive Activity to Cytokine Production of Macrophages in Vitro
Kentaro Ninomiya,*,a Kazumi Hayama,a Sanae A. Ishijima,a Naho Maruyama,a,b Hiroshi Irie,c
Junichi Kurihara,d and Shigeru Abea,c
a
 Teikyo University Institute of Medical Mycology; c Faculty of Medical Technology, Teikyo University; 359 Otuka,
Hachioji, Tokyo 192–0395, Japan: b Faculty of Health and Medical Science, Teikyo Heisei University; 2–51–4
Higashi-ikebukuro, Toshima-ku, Tokyo 170–8445, Japan: and d Faculty of Pharma Sciences, Teikyo University;
2–11–1 Kaga, Itabashi-ku, Tokyo 173–8605, Japan.
Received January 10, 2013; accepted February 19, 2013

The onset of oral candidiasis is accompanied by inflammatory symptoms such as pain in the tongue,
edema or tissue damage and lowers the quality of life (QOL) of the patient. In a murine oral candidiasis
model, the effects were studied of terpinen-4-ol (T-4-ol), one of the main constituents of tea tree oil, Mela-
leuca alternifolia, on inflammatory reactions. When immunosuppressed mice were orally infected with Can-
dida albicans, their tongues showed inflammatory symptoms within 24 h after the infection, which was moni-
tored by an increase of myeloperoxidase activity and macrophage inflammatory protein-2 in their tongue
homogenates. Oral treatment with 50 µL of 40 mg/mL terpinen-4-ol 3h after the Candida infection clearly
suppressed the increase of these inflammatory parameters. In vitro analysis of the effects of terpinen-4-ol on
cytokine secretion of macrophages indicated that 800 µg/mL of this substance significantly inhibited the cyto-
kine production of the macrophages cultured in the presence of heat-killed C. albicans cells. Based on these
findings, the role of the anti-inflammatory action of T-4-ol in its therapeutic activity against oral candidiasis
was discussed.
Key words terpinen-4-ol; anti-inflammation; Candida albicans; myeloperoxidase; macrophage inflammatory
protein-2

Candida albicans is known as an opportunistic infectious
microorganism that causes oral candidiasis.1) The major afflic-
tions induced by onset of oral candidiasis are inflammatory
symptoms accompanied by discomfort, pain of the tongue,
dysgeusia and bad breath all of which greatly lower the qual-
ity of life of the patient. In most cases, oral candidiasis is
effectively treated with anti-fungal drugs,2–4) but recurrence
after termination of chemotherapy which arises in favor of C.
albicans, the commensal yeast, and emergence of an anti-fun-
gal drug resistant species as a result of repeated application of
that drug have become serious issues.5,6) Therefore, develop-
ment of a new type of treatment is highly sought after.
As reported previously, some of the essential oils,7,8) fatty
acids9) or other substances10,11) are therapeutically efficacious
to murine oral candidiasis. In preceding papers, we have
reported that oral treatment with tea tree oil (TTO) and its
main component, terpinen-4-ol (T-4-ol), improved Candida
infected lesions on the tongue’s surface and lowered the num-
ber of Candida cells in the murine oral cavity.7,12) Interest-
ingly, although TTO and T-4-ol showed an efficacy in oral
candidiasis, the recovery of lesion score and the decrease in
viable Candida cell number were not correlated7); that led us
to assume that there might be several factors affecting the
therapeutic results other than the direct anti-Candida efficacy.
In our previous histopathological research, oral treat-
ment with T-4-ol was suggested to inhibit accumulation of
inflammatory cells at local sites in Candida infected tongue
mucosa.7) Experimental findings in vitro indicated that TTO
and/or T-4-ol suppress hyphal growth of C. albicans at low
concentrations12,13) or possess anti-inflammatory activity.14,15)
The authors declare no conflict of interest.
 To whom correspondence should be addressed.  e-mail: kninomiya@daiwa-pharm.com
*  © 2013 The Pharmaceutical Society of Japan
In this study, we investigated quantitatively: 1. inflammatory
responses in murine oral candidiasis, and 2. suppressive ef-
ficacy of T-4-ol against the inflammation. We believe that our
findings about the anti-inflammatory activity of T-4-ol on mu-
rine oral candidiasis as given here explain part of the mecha-
nism of the therapeutic efficacy of this terpenoid.
Materials and Methods
Organisms ​The Candida albicans strain TIMM 1768 was
clinically isolated from patients and maintained at Teikyo Uni-
versity Institute of Medical Mycology. This strain was stored
at −80°C in Sabouraud dextrose broth (Becton Dickinson,
MD, U.S.A.) containing 0.5% yeast extract (Becton Dickin-
son) and 10% glycerol until the experiment was performed.
Agents ​Terpinen-4-ol was purchased from Tokyo Chemi-
cal Industry Co. (Tokyo, Japan). Lipopolysaccharide (LPS),
hexadecyltrimethylammonium bromide (HTAB), human my-
eloperoxidase (MPO) and tetramethylbenzidine (TMB) were
purchased from Sigma-Aldrich Japan (Tokyo, Japan).
Animals ​Animal experiments were performed according
to the guidelines for the care and use of animals approved by
Teikyo University and guidelines for animal experiments con-
ducted at research institutions by the Ministry of Education,
Culture, Sports, Science and Technology of Japan. Six week-
old female ICR mice (Charles River Japan, Kanagawa, Japan)
were used for all animal experiments. The photoperiods were
adjusted to 12 h of light and 12 h darkness daily, and the en-
vironmental temperature was constantly maintained at 21°C.
The mice were kept in cages housing 3 animals and given ac-
cess to food and water ad libitum.
Murine Oral Candidiasis ​Experimental procedure of
May 2013839
the murine oral candidiasis model was described previously16)
and partly modified. Briefly, immunosuppressed mice were
induced by subcutaneous treatment with a 100 mg/kg dose of
prednisolone (Kyoritsu Seiyaku Corporation, Tokyo, Japan)
1 d prior to oral infection. Tetracycline hydrochloride (Sumika
Enviro-Science, Tokyo, Japan) in drinking water at a dose of
4.2 mg/mL was given to the animals from 1 d before infection
until the experiment ended. On the day of Candida inocula-
tion, mice were anesthetized by intramuscular injection with
14.4 mg/kg of chlorpromazine chloride (Wako Pure Chemical
Industries, Ltd., Osaka, Japan) in the left femur; subsequently,
oral infection was induced with about 2×108 cells/mL vi-
able cells of C. albicans (TIMM1768) in RPMI1640 medium
containing fetal bovine serum (FBS), the complete medium.
The oral infection was performed by means of a cotton swab
(baby cotton buds; Johnson & Johnson, Tokyo, Japan) rolled
over the entire tongue in the mouth. The cell number of C.
albicans inoculated in the oral cavity was calculated to be ap-
proximately 1×106 cells/mouse by the difference in viable cell
number adhering to the cotton swabs before and just after oral
inoculation.16)
Oral Treatment ​T-4-ol was suspended in 1% Tween 80
(Wako Pure Chemical Industries, Ltd.) and applied to the oral
cavity with feeding needles 3 h after C. albicans inoculation
as previously reported.7,17) One percent Tween 80 was applied
to the control group in the same manner.
Quantitation of Viable C. albicans from Oral Cavity and
Homogenization of Infected Tongues ​Twenty-four hours
after Candida inoculation, groups of mice were sacrificed and
the Candida cells in the oral cavity were collected with a cot-
ton swab. After swabbing this cavity, the end of the swab was
cut off and placed in a tube containing 5 mL sterile saline.
The Candida cells were re-suspended by mixing with a vortex
mixer and diluted with a series of 20 and 100-fold dilution
of sterile saline. Fifty microliters of each dilution was incu-
bated on a Candida GS agar plate for 20 h at 37°C followed by
counting of the CFU (colony forming units).
To measure MPO activity as well as the quantity of cyto-
kines in the Candida infected tongues, the supernatants of
the homogenized tongues were frozen until the experiments.
The resected tongues were placed in 2 mL of 0.5% HTAB for
MPO assay or the complete medium for enzyme-linked im-
munosorbent assay (ELISA) and were homogenized followed
by centrifugation at 3400 rpm for 10 min. The retrieved super-
natant was stored at −80°C until measured.
MPO Assay ​ Measurement of MPO activity was based
on the method of Maruyama et al.18) and partly modified. The
samples from −80°C were thawed and poured into 96 well
micro plates (30 µL/well). Subsequently, 200 µL of a mixture
containing 100  µL of phosphate buffered saline, 85  µL of
0.22 m sodium phosphate buffer and 15 µL of 0.017% hydrogen
peroxide was added to the wells. The reaction was started by
the addition of 20 µL of 18.4 m m TMB in 8% aqueous dimeth-
ylformamide. Plates were incubated at 37°C for 3 min and then
placed on ice where the reaction was stopped by addition to
each well of 30 µL of 1.46 m sodium acetate. The absorbance
of samples was measured with 620 nm (optical density (OD)
value) and converted into MPO values per 0.1 g of murine
tongues.
ELISA ​ An ELISA assay kit was purchased from BD
Biosciences (CA, U.S.A.) for tumor necrosis factor-alpha
(TNF-α) and R&D Systems (Minnesota, U.S.A.) for macro-
phage inflammatory protein-2 (MIP-2). The supernatants of
tongues were adjusted as described earlier and ELISA was
conducted according to the manufacturer’s instructions.
Preparation of Heat-Killed Candida albicans for in Vitro
Assay ​Heat-killed Candida albicans (HKCA) prepared using
the following protocol was applied for in vitro experiments.
C. albicans was grown on Sabouraud dextrose for 16h and
harvested in RPMI1640 medium containing 2.5% FBS. Half
of the culture medium containing Candida cells was im-
mersed in warmed 95°C water for 20 min and stored at −80°C
until experiments (HKCA yeast). The other half was adjusted
to 1×107 cells/mL and poured into petri dishes followed by
incubation at 37°C, 5% CO2 for 3 h. This incubation allowed
the Candida to form germ tubes. The germinated cells were
collected, heated and stored in the same manner until further
experiments (HKCA hyphae).
Preparation of Murine Macrophage Monolayer ​Murine
macrophages were prepared from peritoneal exudate by the
method described previously19) and partly modified. Briefly,
ICR mice were intraperitoneally injected with 2 mL of 3%
Fluid thioglycollate medium (Becton Dickinson) in distilled
water 3 d before the collection of peritoneal exudate cells
(PEC). The collected PEC were centrifuged at 1500 rpm, 4°C
for 5 min and adjusted to 1×106 cells/mL with a hemocytome-
ter followed by applying 200 µL/well into 96-well microplates.
The cells were incubated for 2.5 h at 37°C in 5% CO2; non-
adherent cells were gently washed out with 37°C PBS result-
ing in a monolayer which consisted of macrophages (>95%).19)
Measurement of Anti-inflammatory Activity of T-4-ol
in Vitro ​ HKCA, LPS and T-4-ol were prepared with the
complete medium and added to the macrophage-monolayer
followed by 37°C in 5% CO2 incubation. The tested concentra-
tion of T-4-ol was adjusted to 200, 400 and 800 µg/mL, since
a previous report20) showed that epithelial cells or fibroblasts
were remained viable at least after short time exposure of less
than 1000 µg/mL of T-4-ol. After the incubation, collected
culture supernatants were centrifuged for 2000 rpm at 4°C
for 5 min and the supernatants were stored at −80°C until the
measurement.
Histopathological Observation ​The murine tongues were
fixed in 4% paraformaldehyde solution and embedded in par-
affin for histopathological study. Five-micron sections were
obtained from the paraffin block and stained with periodic
acid-Schiff (PAS) stain.
Statistical Analysis ​All results of statistical analysis were
analyzed using unpaired t-tests. In all cases, p<0.05 was con-
sidered to be significant.
Results
Kinetics of MPO Activity in Murine Oral Candidiasis
and MPO Suppression of T-4-ol ​First, the neutrophil accu-
mulation was evaluated by measuring MPO activity, the mark-
er of neutrophils, as an inflammatory response in Candida-in-
fected tongues; these tongues showed a clear increase of MPO
activity on average although it varied among individual mice.
The mean value of MPO activity was 101.21±​59.39 munits/
mouse tongue (n=6) in a Candida-infected group compared
with 7.11±​3.64 munits/mouse tongue (n=6) in an uninfected
group 24 h after Candida inoculation, which means that MPO
840 Vol. 36, No. 5

Fig.  1. Effects of T-4-ol Treatment on MPO Activity and Viable Candida Cell Number in Murine Oral Candidiasis
T-4-ol was orally applied 3 h after Candida inoculation. The infected tongues were homogenized 24 h after infection followed by determination of their MPO activity. C.
albicans cells in an oral cavity were collected by swabbing before tongue resection (n=6). (a) MPO activity. (b) Viable Candida cell number. * p<0.05.

Fig.  2.  Microscopic Observation of Typical Lesion of PAS-Stained Tongues Treated with or without T-4-ol at 24 h after Candida Inoculation
Accumulation of inflammatory cells is arrowed. (a) uninfected; (b) infected tongue (control); (c) treated with 10 mg/mL of T-4-ol; (d) treated with 40 mg/mL of T-4-ol.

activity of Candida infected tongues increase 14 folds to the
uninfected group.
Oral treatments with 50 µL of T-4-ol solution at the concen-
tration of 10 mg/mL significantly decreased MPO activity of
infected tongues (Fig. 1a). T-4-o1 was applied once 3h after
the Candida inoculation. In this experiment, we observed
that viable Candida cell numbers in the oral cavity were also
decreased significantly at 40 mg/mL, but not at 10 mg/mL, al-
though a decreasing tendency was appeared (Fig. 1b).
Histopathological Observation of Candida Infected
Tongues Treated by T-4-ol ​ PAS stained sections of
tongues, prepared 24 h after Candida infection, were observed
as in Fig. 2. Hyphal extension of C. albicans and accumulation
of inflammatory cells were observed in the control group (Fig.
2b), but not in the uninfected group (Fig. 2a). The amount of
hyphae was diminished dose dependently by oral application
of T-4-ol although some were still present on the mucosal
surface (Figs. 2c, d). No accumulation of inflammatory cells
was observed in the local infected site, which seemed clearly
different from the control group.
T-4-ol Suppresses Production of MIP-2 in Murine Oral
Candidiasis ​ Since it is known that MIP-2 functions as a
major chemotactic factor for neutrophils to inflammatory sites
in a murine body, the kinetics of the amount of MIP-2 pro-
duced in Candida-infected tongues was evaluated. In a control
experiment, the tongues of Candida-uninfected mice with or
without T-4-ol (40 mg/mL) were homogenized to determine
their MIP-2 amount followed by the ELISA assay. The average
quantity of MIP-2 was 0±​1.01 or 6.95±​20.89 pg/tongue (0.1 g)
(n=6), which indicated that T-4-ol treatment to uninfected
tongues did not significantly affect the level of MIP-2. The
amount produced in Candida-infected groups treated with 10
or 40 mg/mL of T-4-ol was suppressed significantly compar-
ing to the control group as shown in Fig. 3.
May 2013841
Measurement of TNF-α Production by Murine Mac-
rophages Stimulated with Heat-Killed Candida Cells in
Vitro ​As described above, oral application of T-4-ol seemed
to suppress local inflammation in Candida infected tongues.
From these results, we performed in vitro studies to examine
whether T-4-ol possesses direct suppressive activity to pro-
duction of inflammatory cytokines from Candida-stimulated
macrophages. It is reported that tissue invasion of Candida
hyphae causes production of inflammatory cytokines.21) As
a major inflammatory cytokine, TNF-α which is reportedly
secreted in a short time (2 h) after exposure to C. albicans,22)
was evaluated by supernatants from murine peritoneal macro-
phages cultured with HKCA.
To determine whether there is a difference of production
amount of the cytokine between HKCA yeast and hyphae,
Fig.  3. Suppression of MIP-2 Production in Murine Tongues by T-4-ol
Treatment
T-4-ol was orally applied 3 h and tongues were resected 24 h after Candida in-
oculation. The amount of MIP-2 in supernatants was determined by ELISA (n=6).
** p<0.01.
Fig. 4.  TNF-α Production by Murine Macrophages Stimulated with HKCA in Vitro
Macrophage monolayer was prepared as in Materials and Methods. HKCA yeast and hyphae were applied followed by 2 h of 37°C incubation and the amounts of TNF-α
in the culture supernatants were determined by ELISA (n=6). * p<0.05, ** p<0.01.
each of stimulatory activities was tested to murine macro-
phages. Adjusted concentrations of HKCA yeast or hyphae
were added to a macrophage monolayer followed by 2  h
incubation at 37°C, and TNF-α amount in the culture super-
natants was then measured with ELISA. The results showed
that TNF-α was produced more effectively after 2 h culture
of macrophages stimulated with HKCA hyphae, than with
HKCA yeast (Fig. 4) .
The effects of T-4-ol on TNF-α production by murine
macrophages were examined. Macrophage monolayer was cul-
tured in the presence of HKCA hyphae with or without T-4-ol
for 2, 6, or 20 h cultivation and then the culture supernatants
were obtained for measurement of TNF-α secretion (Fig. 5a).
T-4-ol suppressed the production of TNF-α dose dependently
and its 50% inhibitory concentration (IC50) at 2 or 6 h culture
was estimated to be under 800 µg/mL. The level of TNF-α in
20 h of culture supernatant seemed to be lowered. Figure 5b
shows that stimulation with LPS elicited a greater production
of TNF-α than HKCA hyphae and that TNF-α production in
the case of LPS-stimulation was not similarly suppressed by
800 µg/mL of T-4-ol. Lowered suppression efficacy of T-4-ol
(800 µg/mL) at with 6 or 20 h incubation was also confirmed.
T-4-ol Suppresses MIP-2 Production in Vitro ​The
effects of T-4-ol on MIP-2 production by macrophages was
also evaluated in vitro. In the presence of T-4-ol, macrophages
were cultured with 1×106 cells/mL of HKCA hyphae for 6h
and the production amount of MIP-2 in the supernatants was
evaluated with ELISA. Figure 6 clearly shows that a non-
stimulated macrophage monolayer produced little MIP-2 in
contrast to the groups that cultured with HKCA hyphae. T-
4-ol suppressed these MIP-2 productions to less than 50% at
the concentration of 800 µg/mL.
Discussion
Candida albicans, especially Candida cells with hyphal
growth, causes oral candidiasis by invading oral mucosal
tissues; the Candida-infected lesions are often accompanied
by pain in the tongue, redness and swelling.23) In experimen-
842
Fig. 5. Effects of T-4-ol on TNF-α Production by Macrophages Stimulated with HKCA or Lipopolysaccharide Were Determined Time and Dose
Dependently in Vitro
HKCA hyphae and/or T-4-ol were applied to the macrophage monolayers. Amounts of TNF-α in their culture supernatants were measured with ELISA (n=6). (a) Stimu-
lated with HKCA hyphae. (b) Stimulated with LPS. Concentration of T-4-ol was as follows: □ 0, 200, 400, 800 μg/mL. * p<0.05, ** p<0.01.
Fig. 6. Suppression of MIP-2 Production from Murine Macrophages
Stimulated by HKCA Hyphae of T-4-ol in Vitro
Macrophage monolayer was prepared with microplates as in Materials and Meth-
ods. HKCA hyphae were adjusted to 1×106 cells/well and were cultured with T-4-ol
for 6 h. Amounts of MIP-2 in the culture supernatants were determined by ELISA
(n=4). ** p<0.01.
tal murine oral candidiasis, it was reported previously that
inflammatory cells, mainly consisting of neutrophils, accu-
mulated in Candida-infected lesions.7,24) Since inflammatory
responses with these cells would cause major pathological
changes in oral candidiasis, we believe that the inflammatory
severity of infected tongues should be evaluated quantitatively
in order to understand the pathological conditions of this dis-
ease. In this study, the following two findings were obtained:
1. a marker of neutrophils, the MPO activity, was increased in
the tongue homogenates after 24 h of oral Candida infection;
2. oral treatment with T-4-ol suppressed this increase of MPO
activity, which might be caused by inhibition of the produc-
Vol. 36, No. 5
tion of MIP-2, a mouse homologue of human IL-8 which
could stimulate the migration of neutrophils. The decrease of
MPO activity in tongue tissues by T-4-ol application indicates
that this monoterpene alcohol possesses not only anti-Candida
activity but also anti-inflammatory efficacy in its therapy
against oral candidiasis.
Since cellular distribution of MPO is limited mainly to
neutrophils,25) it can be used as a marker of these cells. Our
experiments showed that MPO activity in Candida-infected
tongues at 24 h increased to more than 14 times that of a non-
infected group. This means that neutrophils, the inflammatory
cells, were accumulated in the local sites of Candida infec-
tion. As far as we know, this is the first report enabling quan-
titation of the degree of inflammatory symptoms of tongues in
murine oral candidiasis.
Treatment of T-4-ol suppressed the increase of MPO activ-
ity in tongues 24 h after Candida infection (Fig. 1a), which
might correspond to the histological observation suggesting
inhibition of the local accumulation of neutrophils. We specu-
lated that this suppression might be caused by the inhibited
production of MIP-2 in the tongue tissue, which may be pro-
duced by macrophages, epithelial cells and other inflamma-
tory cells in the event of Candida infection. Our experimental
results showed that T-4-ol treatment suppressed production of
MIP-2 in tongues with murine oral candidiasis (Fig. 3), which
is apparently related to diminution of MPO activity. These
findings indicate that T-4-ol exerts anti-inflammatory activity
in oral candidiasis therapy.
Cytokine production by macrophages, which is considered
to play an essential role in the early stage of inflammation,26)
and the effects of T-4-ol on the production were examined in
vitro. Comparison of TNF-α production from murine macro-
phages stimulated by HKCA yeast and hyphae revealed that
the hyphae form more strongly induced the cytokine produc-
tion than did the yeast form (Fig. 4). The secretion of TNF-α
and MIP-2 from macrophages stimulated with HKCA hyphae
was suppressed by T-4-ol in vitro. This suppression was dem-
onstrated in the case of 2 h culture for TNF-α and 6 h culture
for MIP-2 (Figs. 5a, 6). It was previously reported that Can-
May 2013843
dida hyphae started to invade the mucosal tissues of tongues
3 h after Candida-oral inoculation.27) This evidence appears to
explain the mechanism of prompt suppression of inflammatory
responses in vivo, that is, T-4-ol treatment 3 h after Candida
inoculation suppresses the increase of MIP-2 and MPO ac-
tivity very rapidly for up to 24 h after the infection. In this
context, we wish to note that the low molecular weight of this
terpenoid must enable its quick infiltration into the mucosal
tissues of the tongues, as true of cinnamaldehyde.8)
We have assumed that the therapeutic activities of ter-
penoids including T-4-ol on candidiasis depend on its anti-
Candida-cell actions. In fact, we have reported that minimum
inhibitory concentration (MIC) of T-4-ol for yeast-form
growth of TIMM 1768 is about 40 mg/mL and even 800 µg/
mL of T-4-ol inhibits mycelial growth of the Candida cells in
vitro.12) Therapeutical activity of T-4-ol, especially decrease
of viable candida cell numbers collected from oral cavity,
should attribute to inhibition of mycelial growth of this fun-
gus.7) In this report, we showed that T-4-ol application at a
dose of 50 µL of 40 mg/mL, significantly decreased the viable
Candida cell number in the oral cavity (Fig. 1b). Therefore,
we can speculate that decrease of the Candida burden on the
tongue surface by T-4-ol might negatively affect the inflam-
matory responses including MIP-2 production. Although this
speculation is possible, we postulate the importance of the
anti-inflammatory activity of T-4-ol in our therapeutic model,
because the increase of MPO activity in the infected tongues
was inhibited even with a lower dose (10 mg/mL) of T-4-ol.
Moreover, histopathological findings indicate that the exis-
tence of mycelia of Candida cells in tongue tissue treated with
T-4-ol did not induce an accumulation of inflammatory cells
as shown in Figs. 2c and d. Since suppression of accumulation
of inflammatory cells by T-4-ol was demonstrated at lower
concentrations, we may consider that the impact of anti-in-
flammatory activity of T-4-ol must be equal to or more potent
than direct anti-Candida action in improving the symptoms of
oral candidiasis. To support this assumption, we found there
was no correlation between MPO activity and viable Candida
cell number in the oral cavity of individual mice treated with
T-4-ol (data not shown).
Since T-4-ol possesses anti-inflammatory activity in vitro
and in vivo, we must discuss the possibility that this terpene
alcohol may suppress the self-defense mechanisms against
microbial infection. As shown in Fig. 5b, the presence of T-
4-ol (800 µg/mL) did not suppress TNF-α production from
the macrophage monolayer stimulated by LPS. This result
indicates that anti-inflammatory activity of T-4-ol might be
varied by different stimulants such as HKCA hyphae or LPS,
a water-soluble material. We also wish to note that in the pro-
duction amount of TNF-α in LPS, the control group did not
differ from the T-4-ol treated groups cultured for 6 or 20 h.
This means that T-4-ol did not exert cytotoxicity non-specif-
ically to macrophages, at least under our experimental condi-
tions. Therefore, we can speculate that T-4-ol does not non-
specifically affect defense mechanisms to various infections;
this opinion can also be supported by excellent traditional
estimates of the therapeutic efficacy of TTO on various cu-
taneous infectious diseases.28–30) However, in this context we
should note that further precise study on influences of T-4-ol
to killing activity of macrophages and neutrophils is needed.
Our study showed that the anti-inflammatory activity of T-
4-ol is one of the key roles of its therapeutic efficacy against
oral candidiasis. TTO, the essential oil mainly composed of
T-4-ol, has been employed empirically against various dis-
eases that include inflammation-related disorders.28,31) The
anti-inflammatory activity of T-4-ol could explain the reason
why this terpene alcohol has been used for those treatments.
Although further study is needed, we hope that T-4-ol can
serve not only in oral candidiasis therapy but against various
inflammatory diseases; the significance of this agent would
be increased in terms of its prevention or therapy of disorders
and amelioration of the weaker QOL caused by inflammation.
References
  1) Odds FC. Candida and Candidosis, a review and bibliography. 2nd
ed., Bailliere Tindale, London, pp. 115–152 (1988).
  2) Garber GE. Fluconazole: a new option in the treatment of Candida
mucositis and esophageal candidiasis. J. Otolaryngol., 21, 92–94
(1992).
  3) Lewis MA, Samaranyake LP, Lamey PJ. Diagnosis and treatment of
oral candidosis. J. Oral Maxillofac. Surg., 49, 996–1002 (1991).
 4) Ghannoum MA, Kuhn DM. Voriconazole—better chances for pa-
tients with invasive mycoses. Eur. J. Med. Res., 31, 242–256 (2002).
 5) Masci JR. Complete response of severe, refractory oral candi-
diasis to mouthwash containing lactoferrin and lysozyme. AIDS, 14,
2403–2404 (2000).
 6) Rautemaa R, Richardson M, Pfaller M, Koukila-Kähkölä P, Per-
heentupa J, Saxén H. Decreased susceptibility of Candida albicans
to azole antifungals: a complication of long-term treatment in
autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
(APECED) patients. J. Antimicrob. Chemother., 60, 889–892 (2007).
  7) Ninomiya K, Maruyama N, Inoue S, Ishibashi H, Takizawa T, Os-
hima H, Abe S. The essential oil of Melaleuca alternifolia (tea tree
oil) and its main component, terpinen-4-ol protect mice from ex-
perimental oral candidiasis. Biol. Pharm. Bull., 35, 861–865 (2012).
 8) Taguchi Y, Takizawa T, Ishibashi H, Sagawa T, Arai R, Inoue S,
Yamaguchi H, Abe S. Therapeutic effects on murine oral candidia-
sis by oral administration of cassia (Cinnamomum cassia) prepara-
tion. Nippon Ishinkin Gakkai Zasshi, 51, 13–21 (2010).
  9) Takahashi M, Inoue S, Hayama K, Ninomiya K, Abe S. Inhibition
of Candida mycelia growth by a medium chain fatty acids, capric
acid in vitro and its therapeutic efficacy in murine oral candidiasis.
Med. Mycol. J., 53, 255–261 (2012), in Japanese.
10) Kamagata-Kiyoura Y, Abe S, Yamaguchi H, Nitta T. Protective
effects of human saliva on experimental murine oral candidiasis. J.
Infect. Chemother., 10, 253–255 (2004).
11) Ishijima SA, Hayama K, Burton JP, Reid G, Okada M, Matsushita
Y, Abe S. Effect of Streptococcus salivarius K12 on the in vitro
growth of Candida albicans and its protective effect in an oral can-
didiasis model. Appl. Environ. Microbiol., 78, 2190–2199 (2012).
12) Ninomiya K, Hayama K, Ishijima SA, Takahashi M, Kurihara J,
Abe S. Effects of inhibitory activity on mycelial growth of Candida
albicans and therapy for murine oral candidiasis by the combined
use of terpinen-4-ol and a middle-chain fatty acid, capric acid.
Yakugaku Zasshi, 133, 133–140 (2013).
13) Hammer KA, Carson CF, Riley TV. Melaleuca alternifolia (tea tree)
oil inhibits germ tube formation by Candida albicans. Med. Mycol.,
38, 355–362 (2000).
14) Brand C, Ferrante A, Prager RH, Riley TV, Carson CF, Finlay-
Jones JJ, Hart PH. The water-soluble components of the essential
oil of Melaleuca alternifolia (tea tree oil) suppress the production of
superoxide by human monocytes, but not neutrophils, activated in
vitro. Inflamm. Res., 50, 213–219 (2001).
15) Hart PH, Brand C, Carson CF, Riley TV, Prager RH, Finlay-Jones
JJ. Terpinen-4-ol, the main component of the essential oil of
844
Melaleuca alternifolia (tea tree oil), suppresses inflammatory me-
diator production by activated human monocytes. Inflamm. Res., 49,
619–626 (2000).
16) Takakura N, Sato Y, Ishibashi H, Oshima H, Uchida K, Yamagu-
chi H, Abe S. A novel murine model of oral candidiasis with local
symptoms characteristic of oral thrush. Microbiol. Immunol., 47,
321–326 (2003).
17) Hisajima T, Maruyama N, Tanabe Y, Ishibashi H, Yamada T, Ma-
kimura K, Nishiyama Y, Funakoshi K, Oshima H, Abe S. Protective
effects of farnesol against oral candidiasis in mice. Microbiol. Im-
munol., 52, 327–333 (2008).
18) Maruyama N, Sekimoto Y, Ishibashi H, Inouye S, Oshima H,
Yamaguchi H, Abe S. Suppression of neutrophil accumulation in
mice by cutaneous application of geranium essential oil. J. Inflamm.
(Lond.), 2, 1–11 (2005).
19) Tokuda Y, Tsuji M, Yamazaki M, Kimura S, Abe S, Yamaguchi
H. Augmentation of murine tumor necrosis factor production by
amphotericin B in vitro and in vivo. Antimicrob. Agents Chemother.,
37, 2228–2230 (1993).
20) Ramage G, Milligan S, Lappin DF, Sherry L, Sweeney P, Williams
C, Bagg J, Culshaw S. Antifungal, cytotoxic, and immunomodula-
tory properties of tea tree oil and its derivative components: poten-
tial role in management of oral candidosis in cancer patients. Front
Microbiol., 3, 1–8 (2012).
21) Blander JM, Sander LE. Beyond pattern recognition: five immune
checkpoints for scaling the microbial threat. Nat. Rev. Immunol., 12,
215–225 (2012).
22) Kim HS, Choi EH, Khan J, Roilides E, Francesconi A, Kasai M,
Vol. 36, No. 5
Sein T, Schaufele RL, Sakurai K, Son CG, Greer BT, Chanock S,
Lyman CA, Walsh TJ. Expression of genes encoding innate host
defense molecules in normal human monocytes in response to Can-
dida albicans. Infect. Immun., 73, 3714–3724 (2005).
23) Yamaguchi H. Byougenshinkin to Shinkinsho. 4th ed., Nanzando,
Tokyo, pp. 236–241 (2007).
24) Okada M, Hisajima T, Ishibashi H, Miyasaka T, Abe S, Satoh T.
Pathological analysis of the Candida albicans-infected tongue tis-
sues of a murine oral candidiasis model in the early infection stage.
Arch. Oral Biol., 58, 444–450 (2013).
25) Klebanoff SJ. Myeloperoxidase: friend and foe. J. Leukoc. Biol., 77,
598–625 (2005).
26) Romani L. Immunity to fungal infections. Nat. Rev. Immunol., 4,
1–23 (2004).
27) Hisajima T, Ishibashi H, Yamada T, Nishiyama Y, Yamaguchi
H, Funakoshi K, Abe S. Invasion process of Candida albicans to
tongue surface in early stages of experimental murine oral candi-
diasis. Med. Mycol., 46, 697–704 (2008).
28) Inoue S, Abe S. An invitation to anti-infectious aromatherapy. 1st
ed., Fragrance Journal, Tokyo, pp. 141–224 (2011).
29) Markum E, Baillie J. Combination of essential oil of Melaleuca al-
ternifolia and iodine in the treatment of molluscum contagiosum in
children. J. Drugs Dermatol., 11, 349–354 (2012).
30) Soukoulis S, Hirsch R. The effects of a tea tree oil-containing gel
on plaque and chronic gingivitis. Aust. Dent. J., 49, 78–83 (2004).
31) Schnaubelt K. Advanced Aromatherapy: the science of essential oil
therapy. 1st ed., Healing Arts Press, Vermont, pp. 31–124 (1998).