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Padjadjaran Journal of Dentistry 2013;25(1):21-25.
Corresponding author: Ahmad Alif Wazir bin Jumali, Department of Oral Biology Faculty of Dentistry Universitas Padjadjaran
Jl. Sekeloa Selatan No.1 Bandung, West Java-Indonesia, Ph./Fax.: +6222-2504985/2532805
Antibacterial eect of clove (Eugenia aromaticum) oil
extracted from clove cigarettes towards Streptococcus
mutans
Ahmad Alif Wazir bin Jumali*, Mieke Hermiawati Satari*, Warta Dewi*
*Department of Oral Biology Faculty Of Dentistry Universitas Padjadjaran, Indonesia
ABSTRACT
Streptococcus mutans is a commensal bacteria of the oral cavity, particularly found in dental
plaque attached to the tooth surface, and can also found in the saliva, buccal mucosa, tongue, and the
gingival sulcus. Clove cigarette contains the clove oil used worldwide as a herbal remedy for a variety of
health disorders due to its antibacterial, antifungal, and antiviral properties. The purpose of this study
was to determined the antibacterial eect of the clove oil contained in the clove cigarette towards
Streptococcus mutans as a cariogenic bacteria. The research was an experimental laboratory, which
tested the sensitivity of the Streptococcus mutans bacteria taken from the saliva of 10 clove cigarette
smokers towards the clove oil extract of the clove cigarette in-vitro by using the Kirby-Bauer method. The
study showed that the clove oil extract of the clove cigarette which contained eugenol has antibacterial
towards the growth of Streptococcus mutans, and there was a dierence of the antibacterial activity
between clove oil extracted from minced and combusted clove cigarette. The conclusion of this study
was the clove oil extracted from minced clove cigarette had a better antibacterial eect than the
combusted clove cigarette.
Keywords: Antibacterial eect, clove oil, Streptococcus mutans, clove cigarette
INTRODUCTION
Nowadays, smoking has become the
worldwide health concerning problem. The
smoking habit has a high tendency for the
respiratory and gastrointestinal epithelium cancer
development. There is a possibility that cigarette
smoke may directly aect the commensal ora
of the oral cavity. Smokers also have tendencies
to develop gingival infections. These infections
occurred as a result of complex interaction of
the host, bacteria, and the short- and long-term
eects of cigarette smoke.1
There are two types of cigarettes, a
ltered-cigarette, and a non-ltered cigarette.
The only dierence between a ltered and non-
ltered cigarettes is that non-ltered cigarettes
allow slightly higher levels of nicotine into the
smoker’s system.2
Furthermore, in Indonesia, there are several
types of cigarettes available in the market. One of
them is the clove cigarettes. Clove cigarette which
is also known as kretek, produced in Indonesia
and distributed worldwide, and often mistakenly
thought as a safe smoking alternative because the
clove oil contained in the cigarettes is well known
22
Antibacterial eect of clove (Eugenia aromaticum) oil extracted from clove cigarettes (Ahmad Alif Wazir bin Jumali et al.)
as a herbal remedy for various health disorders
including toothaches, indigestion, cough, asthma,
headache, stress, and blood impurities.3
In the research conducted by Keene and
Johnson,4 the amount of nicotine in cigarettes may
aected the bacteria growth because of the high
level of nicotine volume in the saliva of smokeless
tobacco users. Thus, the smokeless tobacco use
could stimulate the growth of Streptococcus
mutans and increased risk for dental caries.
The health benets of clove oil attributed
to its antibacterial, antifungal, and antiviral
properties.3 Clove oil has the ability to inhibit
the growth of Streptococcus mutans by disrupting
bacterial cell membrane, thus increasing the
permeability of the membrane and causing
bacteria to lysis.5
Clove cigarettes are composed of a mixture
of tobacco (60–80%), ground clove buds (20–40%),
clove oil, and other additives.6 Clove cigarettes
are available with or without lters, and usually
machine rolled in the white, brown, or black paper,
with pungently sweet odor and taste (Malson et
al., 2002). Clove cigarettes deliver higher tar
content than most cigarettes, ranging from 34-
65 mg; nicotine ranging from 1.9-2.6 mg; and
carbon monoxide from 18-28 mg per cigarette.3,7
The high deliveries of tar probably caused by
the combination of four factors: the nature of
Indonesian tobacco as the cigarette compound;
the high weight of the cigarettes; the number of
pus required to smokes the cigarette; and the
tar residues left by the clove bud.3 Much of the
smoke derived from a clove cigarette contains a
simple distillation of clove oil, without oxidation
of the elements. Distilled clove oil composition
obtained from the combustion of 100% clove
cigarette, consists primarily of eugenol and
related compounds which also identied in the
natural clove oil.3
According to the research conducted
by Walsh and Tsang,8 smoking will create the
low oxygen environment and cause salivary
dysfunction, because the eects of nicotine on
salivary gland ow that will cause a reduction in
the pH value of the oral cavity. These conditions
are a favourable parameter for Streptococcus
mutans growth.
Streptococcus mutans is a gram-positive
bacteria live in the oral cavity. It can thrive in the
temperature ranging from 18-40ºC. It metabolizes
dierent kinds of carbohydrates, creating an
acidic environment in the oral cavity. Thus, these
acidic environments in the mouth that will cause
the tooth decay. This is the leading cause of
dental caries worldwide. Streptococcus mutans is
considered to be the most cariogenic of all of the
oral Streptococci.9 Streptococcus mutans would
cause dental caries and eventually lead to the
tooth decay and the tooth loss. The decayed tooth
will lose its function and would cause multiple
infections such as periapical abscess thus needs to
be extracted. Tooth decay will cause mastication
problem and psychological problems such as low
self-esteem and poor socialization. Tooth decay
is one of the most common infectious diseases in
humans.10
METHODS
The research method was an experimental
laboratory to determine the antibacterial eect
of the clove oil of the clove cigarette towards the
growth of Streptococcus mutans. The population of
this research was clove cigarettes from all brand.
The sample of this research was taken by a random
sampling method of the population. Materials
used In this research were clove cigarettes, clove
oil (as the control group), Streptococcus mutans
bacteria isolated from saliva, Trypticase Yeast
Cysteine Sucrose Bacitracin (TYCSB) agar, and
blood agar with sugar addition.
RESULTS
The saliva sample taken from smokers
were cultured on TYCSB agar plate and incubated
in candle jar to create a facultative anaerobes
environment at 37ºC for 72 hours. After 72 hours,
the TYCSB agar plate showed a tiny white, crystal-
like cauliower shape colonies attached rmly
to the medium. The result of such reactions
was a characteristic indicated that the colony
was Streptococcus mutans. This reaction was a
result of the Bacitracin antibiotic which added to
the medium that suppresses the growth of most
species besides Streptococcus mutans to grow
(Figure 1).11
Figure 2 showed the result of microscopic
examination of the colonies with gram staining
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Padjadjaran Journal of Dentistry 2013;25(1):21-25.
Sample Repetition Inhibitory zone
(mm)
a b c
A 1 2.95 1.20 11.36
2 3.13 1.20 10.75
B 1 2.56 1.29 10.27
2 2.85 1.25 8.05
C 1 3.97 1.53 12.73
2 3.65 1.40 9.31
D 1 2.49 1.25 12.69
2 2.64 2.57 9.90
E 1 3.22 1.50 9.70
2 3.26 1.75 10.26
Average 3.23 1.89 10.39
Note: a = clove cigarette extract; b = combusted clove ciga-
rette; c = pure clove oil
Figure 1. Streptococcus mutans colony on TYCSB agar plate
Figure 2. Microscopic views of Streptococcus mutans
Figure 3. Antibacterial inhibitory zone of clove-
cigarette-extracted clove oil and pure clove oil towards
Streptococcus mutans
Table 1 The result of the inhibition zone measurement of
clove oil extract toward Streptococcus mutans
Source of Variation SS df MS F Value F Table
Between Groups 451.961 2 225.982 241.840 3.34
Within Groups 25.230 27 0.934
Total 477.194
Table 2. Varians analysis
Variables Mean
Dierence Signicant
a B 1.57700 0.001
C -7.33120 0.000
b A -1.57700 0.001
C -8.90820 0.000
c A 7.33120 0.000
B 8.90820 0.000
Table 3. Comparison test between variables
characterized the cocci-shaped gram-positive
bacteria with the chain formation.
The assessment of the antibacterial activity
of clove oil towards Streptococcus mutans was
performed with the Kirby-Bauer agar diusion
method. The bacteria inhibitory zone around the
hole lled with a mixture of clove cigarette and
pure clove oil after incubated in the facultative
anaerobic environment at 37°Celcius for 24 hours
were presented in Figure 3. The measurement
results of the clove oil antibacterial inhibitory
zone towards Streptococcus mutans in 5 samples
tested were shown in Table 1.
Based on the results shown in Table 1, there
were dierences in the inhibitory zone of each sam-
ple. The average inhibitory area of the clove cig-
arette-extracted clove oil was 3.23mm, while the
combusted clove cigarette-clove oil showed the av-
erage value was 1.89mm. The control group (pure
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Antibacterial eect of clove (Eugenia aromaticum) oil extracted from clove cigarettes (Ahmad Alif Wazir bin Jumali et al.)
clove oil) showed the average value of 10.39mm.
According to Sudjana,12 the results of the
statistical analysis with 95% condence level (α
= 0.05) showed signicant results because the
countable F value was greater than the F table as
shown in Table 2. Since the test statistic (F value)
was higher than the critical value (F table), there
were signicant dierences in the antibacterial
eect of clove oil extract extracted from clove
cigarette and pure clove oil towards Streptococcus
mutans.
If the result of a comparison test between
variables showed the signicant value lower than
0.05 (Sig<0.05), suggested signicant dierences.
Table 3 showed that signicant dierences
between variable a-b, variable a-c, and variable
b-c. Based on data in Table 1, Table 2. and Table
3, were concluded that the clove oil extracted
from both clove cigarette and combusted clove
cigarette have antibacterial activity towards the
growth of Streptococcus mutans.
DISCUSSION
According to Table 1, Streptococcus
mutans were less found in the mixture of clove
oil extracted from clove cigarette and combusted
clove cigarette compare to the pure clove oil. This
nding was caused by the mixture of nicotine and
other compound and less concentration of clove
oil in the clove cigarette. Furthermore, in the
combusted clove cigarette, the eect of eugenol
was less due to the evaporation eugenol compound
during combustion.
In Table 2 and 3 presented a dierence
between clove oil extract, combusted clove
cigarette, and pure clove oil. This nding was
caused by the amount of the clove oil contained in
the extract. The higher concentration of clove oil,
the higher antibacterial eect towards the growth
of Streptococcus mutans. The low antibacterial
eect shown from clove cigarette extract means
that only less concentration of clove oil was found
in the clove cigarette.
Clove oil consists of eugenol which is
the phenol group. This component is a highly
active antibacterial agent.13 The mechanism
of antibacterial activity of eugenol towards
bacteria causes by the interaction of eugenol
on the bacterial cell membrane by increasing
the permeability of the membrane.5 Eugenol
damages the membrane of bacteria by causing
protein and lipid breakage. This action is the
result of its hydrophobicity characteristic which
enables eugenol to breaks the lipids of bacteria
cell membrane, disturbing the cell structure, and
rendering them to becomes more permeable.14
According to the results obtained in Table
1, there were variations in the inhibitory zone
diameters of each sample. This nding was
probably due to the mechanism by bacteria to
resist the bactericidal and bacteriostatic action of
the antibacterial agent. One of the mechanisms
is antibiotic eux pumps. Microorganisms have
adapted antibiotic eux pumps from their
original purpose which is to expel waste products
or toxins to a very ecient means of antibiotic
resistance. More than 50 such eux systems
(multidrug eux pumps, cytoplasmic membrane
eux proteins) have been described operating
in many microorganisms such as Escherichia
coli, Pseudomonas aeruginosa, Enterococci,
Staphylococci, and Streptococci.15
Smoking creates anaerobiosis within the
oral cavity, and the low oxygen environment
favours the growth of Streptococcus mutans.
There are two additional components involved
in the impact of smoking on dental caries. The
rst is salivary dysfunction which will cause a
decreasing salivary ow. This is the result of the
pharmacological eects of nicotine on salivary
gland ow. Reduced ow linked with reduced pH
value, which is also a favourable parameter for
Streptococcus mutans growth. The second factor
is the direct eect of nicotine in the salivary
milieu on Streptococcus mutans. There was
some evidence that nicotine itself can directly
aect the growth of Streptococcus mutans, with
concentrations of nicotine of 0.1-1.0 mmol/L
was able to stimulate growth, although higher
concentrations will aected adversely.8
Smokes derived from a clove cigarette was
a simple distillation of clove oil, without oxidation
of the compounds. The composition of distilled
clove oil, as obtained by the combustion of a 100%
clove cigarette, consists primarily of eugenol and
related compounds which are also identied in
natural clove oil.3
There are still controversial issues of the
use of clove cigarette. In this research, the in-
25
Padjadjaran Journal of Dentistry 2013;25(1):21-25.
vitro test showed that the clove oil extracted
from the clove cigarette inhibited the growth of
Streptococcus mutans, but the cigarette smoke
and nicotine in the clove cigarette can eventually
increase the growth of Streptococcus mutans.
CONCLUSION
The conclusion of this study was the clove
oil extracted from minced clove cigarette had a
better antibacterial eect than the combusted
clove cigarette.
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