Open Journal of Stomatology, 2013, 3, 176-182
http://dx.doi.org/10.4236/ojst.2013.32031 Published Online May 2013 (http://www.scirp.org/journal/ojst/)

OJST

Antifungal activity of chemotype essential oils fromrosemary against Candida albicans
Yusuke Matsuzaki1, Toshiyuki Tsujisawa2, Tatsuji Nishihara3, Mari Nakamura1,
Yasuaki Kakinoki1*
1

Division of Oral Care and Rehabilitation, Department of Control of Physical Functions, Science of Physical Functions, Kyushu
Dental University, Kitakyushu, Japan2
Department of Oral Health Management, School of Oral Health Sciences, Kyushu Dental University, Kitakyushu, Japan
3
Division of Infections and Molecular Biology, Department of Health Promotion, Science of Health Improvement, Kyushu Dental
University, Kitakyushu, Japan
Email: *matsu.44.yum.44@gmail.com
Received 13 March 2013; revised 26 April 2013; accepted 8 May 2013
Copyright © 2013 Yusuke Matsuzaki et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

1. INTRODUCTION

Oral candidiasis is caused by Candida albicans. The
symptoms including pain in the oral cavity affect oureating function. While some antifungal agents arecommonly used for the treatment of candidiasis, occasional consumption of the effective dose cannot betolerated due to side effects. There is also a possibilityof induction of antifungal resistance, thus the development of replacement agents has been awaited. We
aim to explore antimicrobial activities of essential oils.
We evaluated the antifungal activities against C. albicans of essential oils from seven aromatic plantsfrom three manufacturers, and of three chemotype
essential oils from rosemary (Rosmarinus officinalis).
As a result, we found that the antifungal activity wasincreased several times by the addition of Tween 80.
All the tested essential oils showed stable antifungal
activity, however, the variation was observed among
the manufacturers of rosemary and eucalyptus. Rosemary has three chemotypes; CINEOL, CAMPHOR
and VERBENON. They derived from a same plant
species, but contain different chemical components.
The CINEOL, dose-dependently decreased the number of C. albicans in the time-kill assay. Hence weconcluded that the components of rosemary essential
oil would have an effect on its antifungal activity. A
chemotype is the first to consider in measuring antifungal activities of rosemary oil.

Candida albicans is present on the skin and mucosal surfaces as a human microbial flora [1]. C. albicans is associated with immune dysfunction in HIV infection andother diseases such as hypothyroidism, diabetes, acuteleukemia, mucosal irritation, oral cancer, xerostomia, or
the use of broad-spectrum antibiotics or steroids, and theuse of dentures with poor hygiene and trauma, in young
and elderly patients [2-4]. C. albicans causes opportunistic infections such as oral candidiasis, oesophageal candidiasis and vaginal candidiasis. It is known that oral andoesophageal candidiasis cause oral cavity pain, tongue
pain, taste disturbance, odynophagia or dysphagia etc.
[4-6]. Since symptoms of oral and oesophageal candidiasis appear in oral regions, eating function is greatly deteriorated. The reduction of food intake leads to undernutrition and impaired quality of life, and sometimes
causes life-threatening risks.
Oral candidiasis is normally treated with antifungal
agents such as nystatin, miconazole, fluconazole, itraconazole, imidazole, triazole and amphotericin B. However, occasional consumption of the effective dose cannot be tolerated due to side effects [7,8]. In addition,these agents may induce antifungal resistance in C. albicans [9-12]. As oral candidiasis is intractable and recurrent, development of substitutes for the antimicrobials is
expected. It is known that some parts and extracts of medical and aromatic plants have antimicrobial activities[13]. Essential oils are natural materials extracted fromflowers, leaves, peels, barks, roots, seeds, resins of aromatic plants, and are volatile and hydrophobic aromatic
substances containing effective compounds in high density [14]. Since each essential oil has peculiar fragrance

Keywords: Candida albicans; Essential Oil; Rosemary;Rosmarinus officinalis; Chemotype; Antifungal Activity*

Corresponding author.

OPEN ACCESSY. Matsuzaki et al. / Open Journal of Stomatology 3 (2013) 176-182

and effects, the antimicrobial activity has been examinedas the possible substitute for medicine [15,16] and foodpreservatives [17,18]. Though there have been many reports on the antimicrobial activities of essential oils, theresults have been diverse and the evaluation methods
have been inconsistent. It is known that components ofessential oils from aromatic plants of the same scientificname could be different according to the plants’ habitats,or parts and methods for extraction. That variation iscalled “chemotype” [19]. Chemotype occurs when plants
such as rosemary (Rosmarinus officinalis), thyme (Thymus vulgaris) and basil (Ocimum basilicum) etc. growunder different condition, including soils and climates[19,20]. In the previous study, Janssen et al. have described that the methods in the antimicrobial activity
studies of essential oils have been diverse and often insufficient [21]. However, the vagueness in the antimicrobial properties of essential oils is still remaining in many
reports.To eliminate ambiguities over the antifungal properties of essential oils presented in the previous studies,
we evaluated the antimicrobial activities against C. albicans of seven essential oils of the same extracting method and habitat, and of three rosemary chemotypes withor without a surfactant, using microbiologic techniques.

2. MATERIALS AND METHODS
2.1. Essential Oils and Other ReagentsSeven essential oils from aromatic plants, lemongrass(Cymbopogon citrates), eucalyptus (Eucalyptus globules),tea tree (Melaleuca alternifolia), peppermint (Mentha
piperita), sweet marjoram (Origanum majorana), geranium (Pelagonium graveolens) and rosemary (Rosmarinus officinalis) were obtained from three manufacturers(LIBRA NATUTHERAPY, Hyogo, Japan; Flavor Life,
Tokyo, Japan; TREE OF LIFE, Tokyo, Japan). The threechemotypes of rosemary were CINEOL, CAMPHOR
and VERBENON (LIBRA NATUTHERAPY, Hyogo,
Japan). The characteristic properties of seven essential
oils and three chemotypes were shown in Tables 1 and 2.
Chemical compositions of rosemary chemotypes analyzed

177

by gas chromatography mass spectrometry (GC-MS)
were kindly provided by LIBRA NATUTHERAPY (Table 3). 1,8-cineole (99% purity), α-pinene (98% purity)and camphor (96% purity) were purchased from SigmaAldrich (St. Louis, MO, USA).
Seven essential oils and three rosemary chemotypes,
α-pinene,1,8-cineole or camphor in various concentrations, were dissolved in RPMI 1640 with L-glutamineand 0.165 M MOPS without sodium bicarbonate (RPMI
1640-buffered MOPS) (Lonza, Allendale NJ, USA).
Camphor was dissolved in ethanol with density 1 µg/ml.
Polyoxethylene (20) Sorbitan Monooleate (Tween 80), a
surfactant, was purchased from Wako Pure Chemical Industries (Osaka, Japan).

2.2. Fungal Strains and Culture Conditions
C. albicans ATCC 18804 was maintained in YEASTMOLD BROTH (YM broth) (Becton, Dickinson, Franklin Lakes, NJ, USA) at 28˚C for 48 hours. The yeast colonies were adjusted before inoculation, and the final inoculum for broth microdilution assay was approximately 5- 6 × 105 CFU/ml.

2.3. Minimal Inhibitory Concentration (MIC)
Assay and Minimal Fungicidal
Concentration (MFC) Assay to C. albicans
The minimal inhibitory concentration (MIC) was measured by the two-fold serial broth dilution assay, based on
the Clinical and Laboratory Standards Institute (CLSI)
reference protocol M27-A3 with modification. The experiments were performed using sterile 96-well plates.
One hundred µl of RPMI 1640-buffered MOPS wereadded to each well, but not to the first row of wells, 200µl of 40 µl/ml seven essential oils, three rosemary chemotypes, α-pinene, 1,8-cineole or camphor with or without 20 µl/ml Tween 80 were added to the first row, andserially diluted from the first row by taking 100 µl into
the second row. The microdilution was continued to thelast row and 100 µl from the last row were discarded 100µl of C. albicans was suspended in RPMI 1640-buffered

Table 1. The characteristic properties of various essential oils from aromatic plants.
Aromatic plants

Extracted part

Extracting method

HabitatCommon nameScientific name

Lemongrass

Cymbopogon citrates

Leaf

steam distillation

Eucalyptus

Eucalyptus globules

Leafsteam distillation

Portugal

China

BrazilTea treeMelaleuca alternifolia

Leafsteam distillation

Australia

Australia

Australia

Peppermint

Mentha piperita

Leafsteam distillation

India

United States

FranceSweet marjoram

Origanum majorana

Leafsteam distillation

Egypt

Egypt

Egypt

Geranium

Pelagonium graveolens

Leaf

steam distillation

Morocco

Egypt

Egypt

Rosemary

Rosmarinus officinalis

Leafsteam distillation

Spain

Tunisia

MoroccoCopyright © 2013 SciRes.

Manufacture A

Manufacture B

Manufacture C

Nepal

India

Nepal

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178

Table 2. The characteristic properties of chemotype essential
oils of rosemary.
Chemotype

Extracted part

Extracting method

Habitat

CINEOLLeaf

steam distillation

MoroccoVERBENONE

Leaf

steam distillation

FranceCAMPHOR

Leaf

steam distillation

Spain

Table 3. Chemical composition of chemotype essential oils
from rosemary.
Components

Rosemary chemotype

MOPS and adjusted 1 - 5 × 103 CFU/ml was added toeach well. The plates were cultured at 35˚C for 48 hours
and the MICs were determined. The MIC was defined as
the lowest concentration to prevent visible growth of C.
albicans. The concentration range of essential oils, rosemary chemotypes, 1,8-cineole, α-pinene or camphor, was
20 - 0.02 µl/ml. To determine the minimal fungicidal
concentration (MFC), the fluids from all wells showing
inhibition in MIC assay were subcultured in 10 ml of
YM broth at 28˚C for 48 hours. The MFC was defined as
the lowest concentration that completely prevented thedevelopment of the inoculums.
As a control, an inoculum was added to essential oilfree culture medium, and the growth of C. albicans wasobserved. Also we confirmed that microbes did not growin the culture broth with an essential oil and Tween 80
and no inoculum. All experiments were performed in atleast triplicate.

CINEOL

VERBENONE

CAMPHOR

49.05

13.66

24.48

α-Pinene

10.08

13.05

21.03

β-Pinene

6.21

2.16

5.45

Camphen

4.09

6.94

9.15

2.4. Time-Kill Assay

d-Limonene

2.28

-

3.64

p-Cymene

1.48

2.42

1.26

β-Myrcene

0.87

-

3.18

α-Terpinene

0.35

-

-

γ-Terpinene

0.68

0.72

1.39

Terpinolene

0.33

0.97

-

Thujene

0.33

-

-

3-Carene

-

-

1.13

Tricyclene

-

0.26

-

α-Phellandrene

-

0.20

-

Camphor

12.62

15.58

19.20

α-Caryophyllene

0.30

-

-

-

10.71

1.21

To confirm the antifungal activity of CINEOL with Tween
80, time-kill procedures by Klepser with modification
were performed [22]. C. albicans was subcultured in YM
broth at 28˚C for 48 hours and prepared at 5 - 6 × 105
CFU/ml in fresh YM broth. RPMI-1640 buffered MOPS
containing 0.5%, 1%, and 2% CINEOL with Tween 80,
resuspended with the same amount of inoculation. The
concentration of CINEOL with Tween 80 used in theassay was equivalent to that in the MIC assay. At predetermined time points (0, 20, 40 minutes, 1, 2, 3, 4, 5 and6 hours) during incubation, total volume of test solution
was added to YM broth and centrifuged at 2040 × g for
10 minutes at 4˚C to remove CINEOL. The pellets were
properly diluted and spread on the surface of YM agar
plates. The plates were incubated at 28˚C for 48 hours todetermine CFU/ml. Each experiment was performed in atleast triplicate.

Borneol

3.68

3.85

2.74

3. RESULTS

Terpinene 4-ol

0.71

1.17

1.59

Linalool

0.34

1.92

-

3.1. Antifungal Activity against C. albicans of
Various Essential Oils from Aromatic Plants

α-Terpineol

0.19

1.37

-

α-Cubebene

0.22

-

-

β-Caryophyllene

3.48

1.39

2.28

δ-Cadinene

0.25

α-Cubebene

0.22
1.22

12.47

2.27

1.24

11.16

0.00

Oxides
1,8-Cineole
Monoterpenes

Ketones

Verbenone
Alcohols

Sesquiterpenes

EstersBornyl acetate
OthersEssential oils constituents as percentage (v/v) determined by GC-MS.

Copyright © 2013 SciRes.

Since essential oils contain hydrophobic components, we
examined the antifungal activity of various essential oils
with or without Tween 80. Essential oils without Tween
80 showed high MICs against C. albicansor no antifungal activity at tested concentrations. By addition of Tween
80, 2 - 16 times lower MICs were obtained, and antifungal activity was observed in all oils. The MICs against C.
albicans in the presence of Tween 80 varied in eucalyptus and rosemary oils, whereas the values were similar in
the other essential oils among the three manufacturers(Table 4).
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179

Table 4. The MICs and MFCs values of various essential oils against C. albicans.
Aromatic plants
Common nameWithout Tween 80

With Tween 80

MIC

MFC

MIC

MFC

A

B

C

A

B

C

A

B

C

A

B

C

1.25

2.5

2.5

1.25

2.5

5.0

0.16

0.16

0.31

0.16

0.31

0.31

Eucalyptus

2.5

20.0<

20.0<

2.5

20.0<

20.0<

1.25

5.0

10.0

1.25

5.0

10.0

Tea tree

20.0

20.0

20.0<

20.0

20.0

20.0<

2.5

5.0

5.0

5.0

10.0

10.0

Peppermint

10.0

20.0<

20.0

10.0

20.0<

20.0

1.25

1.25

2.5

2.5

1.25

2.5

Sweet marjoram

20.0

20.0

20.0<

20.0

20.0

20.0<

2.5

5.0

5.0

2.5

5.0

5.0

Geranium

5.0

5.0

20.0

10.0

10.0

20.0

0.63

0.63

1.25

0.63

1.25

1.25

Rosemary

10.0

20.0<

20.0<

20.0

20.0<

20.0<

1.25

10.0

10.0

2.5

10.0

10.0

Lemongrass

The numerical value was shown as μl/ml.

To examine whether the antifungal activity of essential
oils depends on bacteriostatic or germicidal action, weinvestigated the minimal fungicidal concentration (MFC).
The MFCs of each essential oil except for tea tree wereabout the same with MICs against C. albicans with orwithout Tween 80 (Table 4). The MFCs against C. albicans varied in eucalyptus and rosemary oils with Tween
80 among the three manufacturers.

3.2. The Habitats and Chemical Components ofThree Rosemary Chemotypes
The parts and method for extraction were common in thethree chemotypes, however, the habitats were different(Table 2). Three chemotypes were analyzed by GM-CS,
and 26 components in total were identified (Table 3).
The major components of three chemotypes, CINEOL,
VERBENONE, and CAMPHOR, were 1,8-cineole, α-pinene, and camphor. The contents and ratios of these components were different in the three chemotypes. CINEOL,
one of the rosemary chemotypes, includes 1,8-cineole(49.05%), α-pinene (10.08%) and camphor (12.62%).
VERBENONE includes 1,8-cineole (13.66%), α-pinene(13.05%) and camphor (15.58%). CAMPHOR includes
1,8-cineole (24.48%), α-pinene (21.03%) and camphor(19.20%).

3.3. Antifungal Activity of Three Rosemary
Chemotype
The MICs against C. albicans of CINEOL, VERBENON
and CAMPHOR without Tween 80 were 5 µl/ml, 10µl/ml, and 10 µl/ml, respectively. The MICs with Tween
80 were 1.25 µl/ml, 2.5 µl/ml, and 5.0 µl/ml, respectively.
Antifungal activity increased 2 - 4 times by adding of
Tween 80. The MFCs against C. albicans without Tween
80 were 20.0 µl/ml, however, with Tween 80, the MFC
and MIC values against C. albicans were the same (Table 5). By addition of Tween 80, 4 - 16 times lower
MFCs were obtained.
Copyright © 2013 SciRes.

3.4. Antifungal Activity of 1,8-Cineole, α-Pinene,
Camphor
The MICs of α-pinene, 1,8-cineole and camphor, major
components of rosemary chemotypes, with Tween 80
against C. albicans were 0.63 µl/ml, 5.0 µl/ml, and 5.0µl/ml, respectively. Antifungal activity increased 4 - 8times by adding of Tween 80. The MFCs of α-pinene and1,8-cineole were the same as the MICs against C. albicans, while the MFC of camphor was twice higher thanthe MIC. Alpha-pinene showed the lowest MIC and
MFC among the rosemary chemotypes (Table 6).

3.5. Time-Kill Assay
C. albicans proliferated in culture medium without
CINEOL, however by the addition of 0.5%, 1%, and 2%
CINEOL, the number of C. albicans was dose-dependently lowered. The decrease was the greatest in the 2%
CINEOL culture medium, with no viable C. albicans detected in two hours. In the 1% medium, no live C. albicans was detected in four hours, and in the 0.5% medium,C. albicans was not completely killed in six hours (Figure 1).

4. DISCUSSION
In this study, we confirmed that antifungal activities
against C. albicans of seven essential oils with a standard
method. The antifungal activities against C. albicans varied among the essential oils from the three manufacturers, though the parts and methods for extraction were thesame. Among the seven essential oils from three manufacturers, sweet marjoram and tea tree were from thesame habitats (Australia and Egypt respectively), however, the other five plants were from different habitats.
Since essential oils are natural materials from various
plants, it is suggested that the components of essential
oils could be different according to the parts and methods
for extraction and the habitats of the plants etc. [23,24].
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180

Table 5. The MICs and MFCs values of three chemotype essential oils of rosemary against C. albicans.
Rosemary(R. officinalis)

Without Tween 80

Chemotype

MIC

MFC

MIC

MFC

CINEOL

5.0

20.0

1.25

1.25

VERBENONE

10.0

20.0

2.5

2.5

CAMPHOR

10.0

20.0

5.0

5.0

With Tween 80

The numerical value was shown as μl/ml.

Table 6. The MICs and MFCs values of main aromatic components against C. albicans.
Chemotype

Without Tween 80

With Tween 80

Aromatic component

MIC

MFC

MIC

MFC

α-Pinene

5.0

5.0

0.63

0.63

1,8-Cineole

20.0<

20.0<

5.0

5.0

Camphor

non

non

5.0

10.0

The numerical value was shown as μl/ml.
107
106
0.0%

Log10 CFU/ml

105
104

0.5%

103

1.0%

102

2.0%

101
100
0

1

2

3

4
Time (h)56Figure 1. Time-kill curve plots for C. albicans following an
exposure to CINEOL. In control suspensions containing noessential oil (●), after the treatment with CINEOL at four times
the MIC (□), eight times the MIC (■) and sixteen times the
MIC (○). The MIC of CINEOL was 0.125% (v/v). The organism was suspended in RPMI 1640-buffered MOPS. Each symbol indicated the means ± SEs of at least three replicates.

We investigated the antifungal activities against C. albicans of essential oils of common extraction parts andmethods, however, we were not able to explain the differences in antifungal activity related to the plants’ habitats. Therefore, we focused on the components of thethree rosemary chemotypes.
Typical components of rosemary are 1,8-cineole, αpinene and camphor [25] and the relatively stable ratio ofthese components defines each chemotype. It is known
that the rosemary oils are widely divided into two chemotypes by the ratio of major components; one withmore than 40% of 1,8-cineole and the other with almost
the same percentage of 1,8-cineole, α-pinene and camphor[26]. That classification was consistent with that of the
Copyright © 2013 SciRes.

oils used in our study. We considered that the remarkabledifference between the components in chemotypes might
have an influence on the antifungal activity. The CINEOL
containing more than 49% of 1,8-cineole, showed thehighest antifungal activity of the three chemotypes, however, it was also found that the antifungal activity against
C. albicans was low with the 1,8-cineole alone. We foundthat VERBENONE and CAMPHOR contained 1,8cineole, α-pinene and camphor in similar ratio by a component analysis. The antifungal activity was the highestin α-pinene, but not in camphor or 1,8-cineole. The result
accorded with the report by Zuzarte et al., which claimed
that the antifungal activity against C. albicans of α-pinene was higher than those of 1,8-cineole and camphor[27]. On the other hand, the results from some studies
examining antimicrobial activity against C. albicans ofrosemary oil are conflicting [28,29]. In addition, it wasreported that α-pinene has enantiomer, (+)-α-pinene and(−)-α-pinene in the latest report [30]. Biological activities
were observed in (+)-α-pinene, but not in (−)-α-pinene.
In other studies, it was reported that the enantiomeric
distribution of the α-pinene in rosemary depends on theplant’s geographical origins [31]. Therefore we could not
explain the difference in the antifungal activities of thechemotypes, even by the major components. There arevarious kinds of components included in an essential oil,each of which is present in a small amount [32,33]. In
determining an antimicrobial mechanism, we cannot ignore the existence of these small but significant minor
components. These results explain the difficulty of elucidating active components of essential oils with antifungal activity.
Essential oils contain both water-soluble and hydrophobic components [34-36]. In this study, the addition of
Tween 80 enhanced antifungal activity of the essential
oils against C. albicans several times. It is known that thecomponents solubilized by Tween 80 increased antimicrobial activity against C. albicans. Alpha-pinene andcamphor are typical fat soluble components [34,35].
Without surfactants such as Tween 80, α-pinene and camphor do not dissolve in culture media. It is suggested thatthe presence of surfactants has a major influence on the
results of antimicrobial studies. For example, since oxygen dose not dissolve in culture media covered with insoluble ingredients, the growth of aerobes or facultative
anaerobes, such as some fungi and bacteria would beconsequently affected. Furthermore, since an essential oil
cannot be suspended when it separated from its aqueous
layer, the preparation of precise concentration is difficult
and that affects its antimicrobial activity. Thus, it was
found that the use of the surfactant, Tween 80, is necessary for antimicrobial activities of the essential oils.
In recent studies, however, there have been some reports on antimicrobial activity in the absence of surfacOPEN ACCESSY. Matsuzaki et al. / Open Journal of Stomatology 3 (2013) 176-182

181

tants. In consideration of the safety, we selected Tween
80, which is not toxic for human and is actually used as afood additive [37]. There are several reports on the effects of surfactants and solvents on antimicrobial activities of essential oils. Remmal et al. showed that the agar
dispersion is more excellent when Tween 80 and ethanol
added in investigating the antibacterial activities against
grampositive and negative bacteria of essential oils fromoregano (Origanum vulgare) and clove (Syzygium aromaticum) [36]. Inouye et al. reported that the addition of
Tween 80 inhibits the antifungal activities against Aspergillus fumigatus of perillaldehyde and citral, which are
the components of essential oils dissolve in dimethyl sulfoxide [38]. Though it seems to contradict our results, itremains unknown whether it is caused by the difference
in strains or by the influence of dimethyl sulfoxide. Further studies to explore the surfactants or solvents suitable
for the study of antimicrobial activity will be necessary.
Though we compared parts and methods for extraction,
plants’ habitats, and antifungal activities against C. albicans of seven essential oils, the variation of the components from lot to lot could not be examined. Also, whether the components of essential oils may be affected by
the status of use and preservation remains uncertain.
Janssen et al. have mentioned that the procedures of antimicrobial assays, concentrations, or presence of surfactants are not standardized in many reports on antimicrobial activities of essential oils [21]. To obtain a stable evaluation on the antimicrobial activities of essential oils,
standardization of antimicrobial activity and normalization of the essential oil components for quality maintenance would be necessary.
In this study, we evaluated antifungal activities against
C. albicans of various essential oils and found that theactivity would be enhanced by the addition of Tween 80.
Rosemary essential oil acts against C. albicans, and theactivity would be varied depending on its chemical compositions. Therefore, in examining the antifungal activityof rosemary essential oil, the chemotypes need to be considered first.

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