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Mahidol University Journal of Pharmaceutical Sciences 2007; 34(1-4): 60-64.
Original Article
Antimicrobial Activity of Essential Oils Against
Five Strains of Propionibacterium acnes
S. Luangnarumitchai,1 S. Lamlertthon1,2* and W. Tiyaboonchai1
1Faculty of Pharmaceutical Sciences, 2Faculty of Medical Sciences, Naresuan University,
Phitsanulok, Thailand.
Abstract The present study was conducted to evaluate the in vitro antibacterial activities of 22 essential
oils from Thai medicinal plants against 5 strains of Propionibacterium acnes (P. acnes). Antibacterial
activity of essential oils was investigated using disc diffusion and agar dilution methods. The results
showed that 19 oils could inhibit the growth of P. acnes. According to the inhibition zone, kaffir lime leaf
(Citrus hystrix DC.), lemongrass oil (Cymbopogon citratus (DC.) Stapf), clove oil (Syzygium aromaticum (L.)
Merr. & Perry) and michelia oil (Michelia alba DC.) had the strongest antibacterial activity. The results
form the agar dilution method showed the same trend in which lemongrass oil and citronella oil showed the
lowest minimum inhibitory concentration against 5 strains bacteria at 0.125% v/v. Therefore, these two
essential oils are an interesting source for further study and possibly as an alternative acne treatment. ©All
right reserved.
Keywords: acne vulgaris, antibacterial activity, essential oils, Propionibacterium acnes
INTRODUCTION
Acne vulgaris is an inflammatory skin disease
of pilosebaceous units. Propionibacterium
acnes (P. acnes) plays an important role in
the pathogenesis of acne inflammation by
producing polymorphonuclear leukocyte and
monocyte or macrophage to produce pro-
inflammatory mediators.1 Moreover, P. acnes
can also induce follicular keratinocytes to
release interleukin-1, which causes keratino-
cytes to proliferate and contributes to the
formation of the preclinical micromedo.2
Therefore, the compounds for targeting acne
vulgaris should be able to inhibit P. acnes.
For many years, antibiotics have been used to
treat acne vulgaris; however, the presence of
antibiotic resistance strains has been
increasing as shown in many reports.3-6
Hence, there is a challenge for the discovery
of new substances from natural sources to
overcome this problem.
Essential oils are complex natural mixtures
that are isolated by steam distillation. The
majority of them are used as flavoring agents
in perfume, food and beverage. Additionally,
they have been traditionally used as natural
preservatives in medicine, food and
cosmetics. Moreover, several reports have
indicated antibacterial activities of essential
oils.7-15 Therefore, researchers are interested
in searching for essential oils from medicinal
plants which have been traditionally used as
antibacterial agents by examining against the
microorganism predominately involved in
acne inflammation, P. acnes.
MATERIALS AND METHODS
Materials
Samples of pure essential oils with known
compositions which were obtained via steam
distillation were purchased from Thai China
Flavors & Fragrances Industry Co. (Thailand).
*Corresponding author: Department of Microbiology and Parasitology, Faculty of Medical Sciences, Naresuan
University, Phitsanulok 65000, Thailand. Email: paolamlertthon@hotmail.co
m
Antimicrobial Activity of Essential Oils Against Five Strains of Propionibacterium acnes
61
The test organisms used in this study were P.
acnes (DMST No. 14916, 14917, 14918, 21823,
21824). These bacteria were obtained from
Department of Medical Sciences, Ministry of
Public Health, Bangkok, Thailand.
Disc Diffusion Method
This experiment was performed by following
the method of Hayes and Markovic (2002)1
with some modifications. Briefly, P. acnes
was incubated in Brain heart infusion broth
supplemented with 1% glucose and adjusted
to yield approximately 1.0 x 108 CFU/ml.
The inoculum suspension was swabbed on
the entire surface of brain heart infusion agar
containing 1% glucose and 0.5% polysorbate
80. Sterile filter paper discs (Macherey-
Nagel, MN®) were aseptically placed on
inoculated plate and impregnated with 15 μl
of each essential oil. The plates were left at
ambient temperature for 30 minutes to allow
exceed prediffusion prior to incubation at
37oC for 72 hours under anaerobic conditions
using the gas generation kit (Oxoid, UK).
Standard discs of tetracycline (30 μg/disc),
erythromycin (15 μg/disc), and clindamycin
(2 μg/disc) (Oxoid, English) were used
individually as positive controls. The diameters
of the inhibition were measured (mm) and
recorded as the mean ± S.D. (standard
deviation). All experiment was performed in
duplicate and repeated three times.
Agar Dilution Method
The minimal inhibitory concentration (MIC)
values were determined by agar dilution
method. Test materials were added
aseptically to 20 ml aliquots of appropriate
sterile molten agar containing 0.5%
polysorbate 80 at the appropriate volumes to
produce the required concentration range of
test material (0.015-4.0% v/v). The resulting
agar solutions were vortexed at high speed
for 15 seconds or until completely dispersed,
immediately poured into sterile petri dishes
then allowed to set for 30 minutes. The plates
were then spotted inoculated with multipoint
inoculator to transfer the desired test
organisms (1 μl) from the prepared inoculum
onto the plates. Inoculated plates were left
until the inoculum had set and then incubated
at 37oC for 72 hours under anaerobic condition
as previous described. Following the
incubation period, the plates were observed
and recorded for presence or absence of
growth. From the results, the MIC was
recorded as the lowest concentration of test
materials where absence of growth was observed.
RESULTS AND DISCUSSION
The present results indicated that 19 essential
oils could effectively inhibit the growth of P.
acnes. Among these essential oils, kaffir lime
leaf (Citrus hystrix DC.), lemongrass oil
(Cymbopogon citratus (DC.) Stapf), clove oil
(Syzygium aromaticum (L.) Merr. & Perry)
and michellia oil (Michelia alba DC.) showed
the strongest antibacterial activity with
inhibition zones of more than 20 mm (Table
1). Subsequent experiments were conducted
to determine inhibitory concentrations of all
essential oils. Lemongrass and citronella oil
(Cymbopogon nardus L.) revealed the highest
antibacterial effect as they possessed the MIC
value of 0.125% v/v for all of 5 strains
bacterial tested (Table 2). Even though kaffir
lime leaf (Citrus hystrix DC.) showed the
largest inhibition zone, its MIC value was
greater than that of lemongrass and citronella
oil. This may be due to their abilities to
diffuse from paper disc and solubilize in agar;
however, the disc diffusion method was used
for screening the antibacterial activity of the
essential oils.16,17
Lertsatithanakorn et al.18 reported that the
MICs against P. acnes of lemongrass, kaffir
lime, holy basil and plai, determined by a
broth microdilution method, were 0.6 µl/ml,
5.0 µl/ml, 5.0 µl/ml and 25.0 µl/ml,
respectively. The lower MICs from that study
may be due to the different method used.
Generally, essential oils comprise of a large
number of active components. The difference
in antibacterial activity of the essential oils
may be due to the difference in chemical
compositions. The main components of
essential oils exhibited high activity in this
study were previously reported for their
S. Luangnarumitchai et al.
62
marked antibacterial activity against various
types of bacteria. Citral, a main component in
lemongrass oil, showed activity against
several microorganisms such as Staphylo-
coccus aureus, Escherichia coli, Bacillus subtilis
and Candida albicans.12,19-21 Similarly, geraniol,
trans-citral, cis-citral, geranyl acetate,
citronellal and citronellol in citronella oil22
Table 1. Antibacterial activity of essential oils against 5 strains of Propionibacterium acnes
a Values for inhibition zone are presented as mean ± S.D. of three independent experiments.
- Inhibition zone was not more than 6 mm (diameter of disc).
Inhibition zone (mm)a ± S.D.
Test samples
P. acnes
DMST
14916
P. acnes
DMST
14917
P. acnes
DMST
14918
P. acnes
DMST
21823
P. acnes
DMST
21824
Black pepper oil (Piper nigrum L.) - - - - -
Canaga abs oil (Cananga odorata
Hook.f. & Thomson var. fruticosa
(Craib) Corner)
- - - - -
Citronella oil (Cymbopogon nardus L.) 18.1± 0.6 18.1± 0.5 17.9 ± 0.4 18.4 ± 0.8 19.5 ± 0.5
Clove oil (Syzygium aromaticum (L.)
Merr. & Perry) 25.3 ± 2.5 23.6 ± 4.3 21.8 ± 2.1 25.3 ± 1.3 23.3 ± 0.3
Coriander oil (Coriandrum sativum L.) 13.7 ± 2.2 12.8 ± 2.2 11.6 ± 1.4 9.9 ± 1.5 12.3 ± 2.2
Eucalyptus oil (Eucalyptus globulus
Labill.) - - - - -
Galanga oil (Alpia galangal (L.)
Swatz) 8.6 ± 0.8 9.4 ± 1.4 9.6 ± 1.2 9.8 ± 2.0 8.9 ± 0.7
Ginger oil (Zingiber offinale Roscoe) 9.6 ± 0.7 9.6 ± 1.5 8.9 ± 0.1 9.0 ± 0.4 9.0 ± 0.7
Guava leaf oil (Psidium guajava L.) 13.1 ± 0.5 12.5 ± 1.5 11.6 ± 1.1 14.7 ± 1.8 12.4 ± 1.9
Holy basil oil (Ocimum tenuiflorum L.) 16.5 ± 3.3 17.3 ± 1.4 17.3 ± 3.8 16.3 ± 3.6 17.2 ± 3.7
Jasmine oil (Jasminum sambac Ait. ) 12.8 ± 0.6 12.0 ± 1.2 11.3 ± 0.8 12.9 ± 1.4 12.8 ± 1.0
Kaffir lime oil (Citrus hystrix DC.) 14.1 ± 3.0 18.0 ± 1.4 16.7 ± 1.5 15.1 ± 2.6 17.3 ± 0.4
Kaffir lime leaf oil (Citrus hystrix DC.) > 90 > 90 > 90 > 90 > 90
Lavender oil (Lavandula angustifolia) 17.8 ± 1.1 17.7 ± 2.2 17.8 ± 2.0 20.2 ± 2.3 15.5 ± 2.8
Lemongrass oil (Cymbopogon citratus
(DC.) Staphf) 35.8 ± 3.9 34.4 ± 2.9 35.6 ± 4.1 31.9 ± 3.9 36.9 ± 3.8
Lesser galangal (Alpinia officinarum
Hance) 14.8 ± 0.2 14.6 ± 0.8 15.3 ± 0.3 13.6 ± 2.7 15.0 ± 2.7
Michellia oil (Michelia alba DC.) 23.8 ± 1.6 21.5 ± 2.9 22.7 ± 2.3 28.3 ± 4.6 22.0 ± 3.0
Plai oil (Zingiber montanum (Koenig)
Link ex Dietr.) 13.5 ± 2.0 13.9 ± 0.9 14.7 ± 1.7 13.3 ± 0.7 15.7 ± 2.2
Sweet basil oil (Ocimum basilicum L.) 11.7 ± 0.7 10.4 ± 1.3 10.4 ± 2.0 10.9 ± 1.8 10.5 ± 0.6
Tea tree oil (Melaleuca alternifolia) 16.2 ± 1.2 15.2 ± 1.7 15.6 ± 2.6 17.4 ± 1.7 15.3 ± 1.9
Tumeric oil (Curcuma longa L.) 10.3 ± 0.4 11.0 ± 0.1 9.6 ± 0.7 10.1 ± 1.5 9.3 ± 0.9
Ylang ylang oil (Cananga odorata
(Lam.) Hook. f. & T. Thomson var.
odorata)
8.8 ± 0.7 9.4 ± 0.1 9.5 ± 0.7 9.4 ± 0.1 9.2 ± 0.1
Clindamycin (2 μg/disc) 31.0 ± 1.3 31.2 ± 1.5 30.4 ± 1.6 29.4 ± 0.8 29.1 ± 1.7
Erythromycin (15 μg/disc) 30.9 ± 1.4 31.3 ± 2.8 29.1 ± 1.3 29.2 ± 1.1 28.6 ± 1.5
Tetracycline (30 μg/disc) 35.5 ± 4.6 35.3 ± 3.1 35.5 ± 3.8 35.3 ± 3.0 35.8 ±2.8
Antimicrobial Activity of Essential Oils Against Five Strains of Propionibacterium acnes
63
Table 2. The minimum inhibitory concentration values of essential oils against 5 strains of Propioni-
bacterium acnes
and eugenol, β-caryophyllene and acetyl-eugenol
in clove oil23 also showed activities against
various microorganism such as S. aureus,
Klebsiella pneumoniae, Pseudomonas
aeruginosa, Clostridium perfringens and E.
coli.13-15 Therefore, antibacterial activity
against P. acnes of these essential oils may be
due to the activity of those key constituents.
CONCLUSION
As previously mentioned, compounds
exhibiting activity against P. acnes can be
used to treat acne vulgaris. Lemongrass oil
and citronella grass oil had the highest
inhibitory activity against P. acnes by which
their MIC values were the same, i.e. 0.125%
v/v. Therefore, these two essential oils may
have a potential for further study as an
alternative acne treatment.
Minimum inhibotory concentration (% v/v)
Test samples
P. acnes
DMST
14916
P. acnes
DMST
14917
P. acnes
DMST
14918
P. acnes
DMST
21823
P. acnes
DMST
21824
Black pepper oil (Piper nigrum L.) > 4 > 4 > 4 > 4 > 4
Canaga abs oil (Cananga odorata
Hook.f. & Thomson var. fruticosa
(Craib) Corner)
> 4 > 4 > 4 > 4 > 4
Citronella oil (Cymbopogon nardus L.) 0.125 0.125 0.125 0.125 0.125
Clove oil (Syzygium aromaticum (L.)
Merr. & Perry) 0.25 0.25 0.25 0.25 0.25
Coriander oil (Coriandrum sativum L.) 1 1 1 1 1
Eucalyptus oil (Eucalyptus globulus
Labill.) 4 4 4 4 4
Galanga oil (Alpia galangal (L.) Swatz) > 4 > 4 > 4 > 4 > 4
Ginger oil (Zingiber offinale Roscoe) > 4 > 4 > 4 > 4 > 4
Guava leaf oil (Psidium guajava L.) > 4 > 4 > 4 > 4 > 4
Holy basil oil (Ocimum tenuiflorum L.) 2 2 2 2 2
Jasmine oil (Jasminum sambac Ait. ) 2 2 2 2 2
Kaffir lime oil (Citrus hystrix DC.) 2 2 2 2 2
Kaffir lime leaf oil (Citrus hystrix DC.) 0.25 0.25 0.25 0.25 0.25
Lavender oil (Lavandula angustifolia) 2 2 2 2 2
Lemongrass oil (Cymbopogon citratus
(DC.) Staphf) 0.125 0.125 0.125 0.125 0.125
Lesser galanga (Alpinia officinarum
Hance) 1 1 1 1 1
Michellia oil (Michelia alba DC.) 1 1 1 1 1
Plai oil (Zingiber montanum (Koenig)
Link ex Dietr.) 4 4 4 4 4
Sweet basil oil (Ocimum basilicum L.) > 4 > 4 > 4 > 4 > 4
Tea tree oil (Melaleuca alternifolia) 1 1 1 1 1
Tumeric oil (Curcuma longa L.) > 4 > 4 > 4 > 4 > 4
Ylang ylang oil (Cananga odorata
(Lam.) Hook. f. & T. Thomson var.
odorata)
> 4 > 4 > 4 > 4 > 4
S. Luangnarumitchai et al.
64
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