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Revista de Ciências
Farmacêuticas
Básica e Aplicada
Journal of Basic and Applied Pharmaceutical Sciences Rev Ciênc Farm Básica Apl., 2011;32(3):375-380
ISSN 1808-4532
Eect of the ymus vulgaris essential oil on the
growth of Streptococcus mutans
Gonçalves, G.M.S.1*; Bottaro, M.1; Nilson, A.C.1
1Faculty of Pharmaceutical Sciences, Pontical Catholic University of Campinas, Campinas, SP, Brazil
Recebido 16/05/2011 / Aceito 17/08/2011
ABSTRACT
The traditional Mediterranean herb thyme (Thymus
vulgaris) is a source of an essential oil that has been
shown to possess antimicrobial activity against many
microorganisms. A considerable part of the general
population has dental caries and Streptococcus mutans
is one of the microorganisms responsible. The aim of
this study was to assess the effect of the essential oil
extracted from thyme on the growth of S. mutans,
the main bacterium involved in the etiology of dental
caries, as well as to incorporate this oil into a toothpaste
formulation for preliminary assessment. The broth
dilution technique was used in threefold tests for
antibacterial activity. The concentrations tested were
1%, 5% and 10% essential oil diluted in ethanol or
mineral oil. The controls were triclosan at 0.25% and
0.5%, chlorhexidine digluconate at 0.06% and 0.12%,
and ethanol. The 1% solution of thyme essential oil
in ethanol proved to be the most efcient against
Streptococcus mutans and may be considered viable as
an ingredient of toothpaste, both with regard to cost
and to the sensory prole of the product. Also, analyses
of the characteristics of the formulation indicated that
the product is stable.
Keywords: Thyme/antimicrobial activity. Thymus vulgaris/
antimicrobial activity. Essential oil/pharmaceutical
applications. Streptococcus mutans. Caries/prevention.
INTRODUCTION
The history of humanity shows that the use of
medicinal plants is very old. The World Health Organization
(WHO) reports that a large portion of the population uses
plants for therapeutic purposes, owing to poverty or a bad
healthcare system (Migliato, 2007). According to Isaac et
al., (2008), it is the active plant compounds present in a
phytocosmetic product that dene the effectiveness of the
product.
The family Lamiaceae consists of approximately
150 plant genera with roughly 2800 species throughout the
world, among which Thymus vulgaris L., popularly known
as common thyme, stands out. This culinary and medicinal
herb is native to the Mediterranean region (Spain, Italy,
France, Greece, Egypt, Lebanon, Turkey) (Carreto et al.,
2007; Lorenzi & Matos, 2002). The hybridization that
occurs among Thymus species that are geographically
close and have coincident owering periods results in
great variability, which affects the homogeneity and
yield of the essential oil and its chemical composition.
Antiseptic, expectorant, carminative and antispasmodic
activities are attributed to thyme oil. Such activities are
associated with the content of thymol (2-isopropyl-5-
methylphenol) and its conformational isomer, carvacrol
(5-isopropyl-2-methylphenol), which have greater
antibacterial and antifungal activities than phenol and
are less toxic (Santurio et al., 2007; Simões et al., 2007).
Thymol, a phenolic antioxidant from plants and important
ingredient in toothpastes, has demonstrated antibacterial,
anthelminthic and antifungal activity, while carvacrol has
been investigated for its antibacterial activity (Carreto et
al., 2007; Rubin et al., 2007).
The bacteria Streptococcus mutans are oval Gram-
positive cocci of diameter 0.5 to 0.75 µm, group in pairs
or chains and require a nutritionally rich medium and an
average temperature of 37°C for optimal growth (Moreira,
2006; Neiva & Vicente, 2007). Like other streptococci,
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Thymus vulgaris and Streptococcus mutans
Rev Ciênc Farm Básica Apl., 2011;32(3):375-380
S. mutans is classied as a facultative anaerobe, that is,
an organism that can grow both in aerobic and anaerobic
conditions (Fejerskov & Kidd, 2005).
Compared to other human body sites, the oral cavity
has the highest numbers and diversity of microorganisms
(Marinho & Araújo, 2007; Fejerskov & Kidd, 2005).
Some groups of microorganisms can be found in specic
oral niches in most individuals. The normal microbiota
consists of microorganisms that are best adapted to the
environmental characteristics that are specic to each
niche (Fejerskov & Kidd, 2005). Dental caries is one of
the main oral health problems. It is localized, transmissible
and infectious, since it depends on infection by specic
cariogenic microorganisms (Fejerskov & Kidd, 2005;
Rosell et al., 2004). Dental caries is also considered a
multifactorial disease, depending on the association of
four primary factors: the host, the cariogenic microbiota,
the dietary substrates (that are metabolized by the
microorganisms) and time, since the rst three factors must
be present for a period of time for enamel demineralization
to occur. Meanwhile, secondary factors, such as reduced
salivary ow rate and tooth mineralization, inuence caries
progression and severity (Fejerskov & Kidd, 2005).
Caries begins with an imbalance in the native
microbiota, favoring the proliferation of some
opportunistic pathogenic microorganisms, since, under
normal conditions, the levels of these microorganisms are
low. Streptococcus mutans is considered the main species
involved in the development of caries. It is therefore one of
the best studied species and its only known natural habitat
is the dental surface (Fejerskov & Kidd, 2005; Marinho &
Araújo 2007).
The formation of a biolm by microorganisms
attached to a tooth surface, under continuous salivary ow,
is the initial stage in the development of dental caries (Neiva
& Vicente, 2007). Bacterial plaque is a dense, uncalcied
mass formed by microorganisms embedded in a matrix
rich in extracellular bacterial polysaccharides and salivary
glycoproteins, rmly attached to the tooth. Normally, it
develops over the salivary biolm (acquired lm) that lines
the entire oral cavity (Marinho & Araújo, 2007; Rahim &
Khan, 2006).
The cariogenic microbiota and, consequently, the
appearance of new caries, can be controlled by proper oral
hygiene allied to uoride therapy, reduced sugar intake and
intake frequency, restorative treatment of the existing caries
and use of antimicrobial substances (Marinho & Araújo,
2007; Rosell et al., 2004). According to Fejerskov & Kidd
(2005) there is little chance of suppressing the cariogenic
microbiota once it is established.
Many essential oils and their constituents have
been tested in recent years, in view of their antimicrobial
properties, resulting in their use in a range of health-related
areas, including food, cosmetics and pharmaceuticals, in
which essential oils are used as preservatives or as drugs
(Kalemba & Kunicka, 2003; Nascimento et al., 2007).
The main products and cosmetics used for oral hygiene
and to maintain the oral cavity in good working condition
are toothpaste, tooth brush, dental oss and mouthwash.
Chlorhexidine, a cationic antibacterial agent used during
dental treatments, is one of the best-studied agents and
considered to be the most effective (Marinho & Araújo,
2007; Zanatta & Rösing, 2007). Chlorhexidine inhibits
glucose incorporation by S. mutans and consequently, its
metabolism to lactic acid. Furthermore, it inhibits enzymes
that are essential for bacteria to form plaque on the dental
surfaces, such as the glycosyltransferase secreted by S.
mutans (Fejerskov & Kidd, 2005; Torres et al., 2000).
Long-term use of chlorhexidine is not recommended
because of its adverse side effects, such as tooth staining
and changes in taste sensation (Fejerskov & Kidd, 2005;
Torres et al., 2000; Zanatta & Rösing, 2007). Chlorhexidine
has been used in many formulas and innumerable studies
show that a concentration of 0.12% is enough to effectively
reduce the oral biolm (Marinho & Araújo, 2007).
Triclosan is another antimicrobial agent used in
toothpastes prophylactically to reduce plaque formation
and inhibit or slow the development of gingivitis (Fejerskov
& Kidd, 2005).
The objective of the present study was to assess
the effect of thyme (Thymus vulgaris) essential oil on the
growth of Streptococcus mutans and to prepare a toothpaste
formulation with this essential oil.
MATERIAL AND METHODS
Material
The thyme essential oil was purchased from Brasil
Portrait Cosméticos Ltda (Sorocaba, SP, Brazil) - lots 4418
and 4419. Chlorhexidine digluconate and triclosan were
purchased from Henrifarma (São Paulo, SP, Brazil). The
culture media were Brain Heart Infusion (BHI) broth and
Trypticase Soy Agar (TSA) from DifcoTM.
Samples
The test samples were: thyme essential oil in
ethanol (1%, 5% and 10%); thyme essential oil in mineral
oil at the same concentrations; triclosan in ethanol (0.25%
and 0.5%); chlorhexidine digluconate in ethanol (0.06%
and 0.12%); combinations: (A) 5% thyme essential oil in
mineral oil and 0.25% triclosan; (B) 5% thyme essential
oil in mineral oil, 0.25% triclosan and 0.06% chlorhexidine
digluconate; (C) 0.25% triclosan and 0.06% chlorhexidine
digluconate, and (D) 5% thyme essential oil in mineral oil
and 0.06% chlorhexidine digluconate.
Antimicrobial assay
The essential oil was tested against the Streptococcus
mutans strain ATCC 25175, by broth dilution assay. The
inoculum of Streptococcus mutans was prepared by
dispersing a colony in a 0.9% saline solution and adjusting
cell density to tube 0.5 on the McFarland scale (Erna Cona,
2002; Longhini et al., 2007).
To each test tube (triplicate) containing 5mL of BHI
were added 100µL of the bacterial suspension and 250µL
of a sample. The tubes were incubated at 37oC for 72
hours, under low oxygen tension in stainless steel jars. The
negative control was culture medium alone and positive
377
Thymus vulgaris and Streptococcus mutans
Rev Ciênc Farm Básica Apl., 2011;32(3):375-380
control was culture medium and microorganism. The assay
was reproduced threefold.
The presence or absence of microbial growth, which
was determined by the turbidity of the culture medium, was
recorded. If the sample was already turbid at inoculation
(5% and 10% thyme essential oil in ethanol and 0.25 and
0.5% triclosan), impairing the later reading of turbidity,
it was incubated as above and, after this period, samples
of the homogenized broth were transferred to a dish
containing TSA agar and this was incubated, under the
same conditions, to determine whether microbial growth
had occurred.
Formulation containing thyme essential oil
A toothpaste formulation was prepared for the
addition of thyme essential oil (Table 1).
Table I. Formulation of a toothpaste.
Components (INCI*) Function % w/w
Calcium carbonate Abrasive 50.0
Glycerin Humectant 20.0
Green dye Coloring agent 0.1
Methylparaben Preservative 0.10
Peppermint essence Flavoring 1.50
Sodium carboxymethyl cellulose Thickener 0.30
Sodium lauryl sulfate Surfactant 3.00
Sodium saccharin Sweetener 0.15
Thymus vulgaris essential oil Active ingredient 1.00
Water Vehicle to 100
* International Nomenclature of Cosmetic Ingredients
After a sensory test of the color, odor and appearance
of the prepared toothpaste, it was packaged in sealed plastic
tubes and stored at room temperature or in a dry Fanem®
incubator (315 SE) thermostated at 60°C for 120 days and
reassessed at 15-day intervals (Brasil, 2004; Brasil, 2007).
RESULTS
The results of all antimicrobial activity assay of
thyme essential oil are described in Table II and the results
of the formulation assessment are described in Table III.
Table II. Streptococcus mutans growth in the presence of various
concentrations of Thymus vulgaris essential oil, triclosan or
chlorhexidine digluconate, alone or combined.
Sample Reading (n=9)
1% Thyme essential oil in ethanol Absence of growth
5% Thyme essential oil in ethanol Absence of growth
10% Thyme essential oil in ethanol Absence of growth
1% Thyme essential oil in mineral oil Presence of growth
5% Thyme essential oil in mineral oil Presence of grouth
10% Thyme essential oil in mineral oil Absence of growth
Ethanol Presence of growth
0.5% triclosan Absence of growth
0.25% triclosan Absence of growth
0.06% chlorhexidine digluconate Absence of growth
0.12% chlorhexidine digluconate Absence of growth
Combination A * Absence of growth
Combination B ** Absence of growth
Combination C *** Absence of growth
Combination D **** Absence of growth
* 5% Thyme essential oil in mineral oil + 0.25% triclosan;
** 5% Thyme essential oil in mineral oil + 0.25% triclosan + 0.06% chlorhexidine digluconate;
*** 0.25% triclosan + 0.06% chlorhexidine digluconate;
**** 5% Thyme essential oil in mineral oil + 0.06 % chlorhexidine digluconate.
Table III. Color, odor and general appearance of the toothpaste
containing Thymus vulgaris essential oil at various times and two
storage temperatures.
Time (days)
Room temperature 60°C
Color Odor Appearance Color Odor Appearance
0 LG C I LG C I
15 LG C I LG C I
30 LG C I LG C I
45 LG C I LG C I
60 LG C I LGY F I
75 LG C I LGY F D
90 LG C I LGY F D
105 LG C I BY G D
120 LG C I LB G D
BY: Brownish yellow; C: Characteristic; D: Dry; F: Fading; G: gone; I: initial/unmodied ;
LB: Light brown; LG: light green; LGY: Light green yellowish
DISCUSSION
The idea for the present study came from the
population’s tendency to use plants in their search for
effective alternative medicines, which are cheaper
and possibly less aggressive to the human body. The
prophylactic use of personal hygiene products on a daily
basis is extremely important. While screening medicinal
plants that could have real antimicrobial activity, our group
came across the culinary herb, thyme (Thymus vulgaris).
Thyme is an aromatic plant whose essential oil has been an
object of considerable interest to the international scientic
community.
After preliminary tests, the concentrations of
thyme essential oil tested in the present study were 1%,
5% and 10%, since no published articles were found with
suggestions of optimal concentrations.
Thyme essential oil was diluted in ethanol and the
results showed that 0.5% triclosan (positive control) and
thyme essential oil, at all the above three concentrations,
were effective against the microorganism (Table II). When
the thyme essential oil was diluted in mineral oil, however,
the results were different and growth inhibition of S. mutans
occurred only at the highest concentration (10%).
A second positive control used in the study
was chlorhexidine digluconate, another antimicrobial
substance commonly used in oral care formulations
(Marinho & Araújo, 2007; Torres et al., 2000). Triclosan
and chlorhexidine digluconate were used at their usual
concentrations and at half strength (0.5% and 0.25%;
0.12% and 0.06%, respectively). Their concentrations were
reduced to 50% to test whether synergy occurred between
these substances and thyme essential oil.
The possibility of synergy between thyme essential
oil and the other substances was considered because the
only concentration of thyme essential oil in mineral oil that
seemed effective against the microorganism was 10%. This
concentration would be unviable in any type of formulation
because of the high cost and especially because of its
intense avor, which would render the product unpalatable.
Since 10% thyme essential oil in mineral oil is an
effective but unviable concentration for use in cosmetic
formulations, combinations of 5% thyme essential oil and
half the usual concentrations of triclosan and chlorhexidine
digluconate were tested to see if these substances would
show synergy. The concentration of 5% thyme oil was
378
Thymus vulgaris and Streptococcus mutans
Rev Ciênc Farm Básica Apl., 2011;32(3):375-380
chosen because it is considered the most viable for use in
formulations in terms of cost and interactions with the other
components of the formulation. Since this concentration
(in mineral oil) gave inadequate results when used alone,
this concentration could be used to test for synergy. Half
of the usual concentrations of triclosan and chlorhexidine
digluconate were used for the same reason: to test for
synergy, since these concentrations, alone, would not be
effective against the microorganism. Positive results were
obtained for all concentrations of these antimicrobial agents
when used alone, suggesting that the activity shown by all
the combinations was not due to synergism (Table II).
In order to test the effectiveness of thyme essential
oil dissolved in ethanol against S. mutans, it was necessary
to determine how ethanol alone affected S. mutans
growth. Ethanol did not affect microbial growth in any
of the experiments (Table II). This shows that, at all three
concentrations, the effectiveness of this solution against
bacterial growth was due to thyme essential oil and not to
ethanol.
The results obtained show that the sample that
prevented microbial growth most efciently was that
containing 1% thyme essential oil in ethanol, since even at
this low concentration, thyme essential oil showed activity
against S. mutans, inhibiting its growth. Obviously, the
same happened at concentrations of 5% and 10%.
When thyme essential oil was diluted in mineral oil,
it did not afford satisfactory action against Streptococcus
mutans. It is possible that the lipophilic nature of mineral
oil did not allow it to mix well with the BHI broth, so that
the two phases (mineral oil and BHI broth) separated.
Consequently, the thyme essential oil might not have been in
close contact with the microorganism, despite being inside
the same test tube. Ethanol promoted homogenization of
the mixture, possibly because the mixture of essential oils
has both short-chain terpenes and oxygenated compounds.
Thyme essential oil in mineral oil was investigated
in association with other commonly used antimicrobial
agents, to determine if synergism occurred, but the results
showed no improvement. The concentrations used in the
study were lower than the usual concentrations in oral
care products (mouthwash, for example), to try to avoid
microbial inhibition by these substances alone. However,
inhibition still occurred at these concentrations, so these
results were discarded, since thyme essential oil, the object
of the study, was efcient at the lowest concentration (1%)
investigated. This concentration can be considered viable
both in terms of cost and in terms of palatability.
An important factor to consider when analyzing
the results is the use of various thyme essential oil lots
because, as Nascimento et al., (2007) point out, lots can be
affected by intraspecic genetic variation and differences
in the conditions of cultivation of the plant or preparation
methods, such as climate, sowing time, soil, use of
pesticides, use of fertilizers, state of plant material (dry
or fresh) and extraction technique. These variations can
affect the chemical composition of the oil and the contents
of the active substances signicantly, thereby inuencing
their antimicrobial activity. Furthermore, adverse storage
conditions can affect the stability of the oil components and
reduce their activity. In the present study, two different lots
were mixed.
As a typical vehicle for the thyme essential oil, a
toothpaste formula was prepared and assessed, in order
to test the performance of this oil in this type of product
(Table I).
The results obtained in the present study could not
be compared with similar results in the literature because
published studies of the effect of thyme essential oil on
Streptococcus mutans were not found.
However, many investigations of the activity
of other essential oils, plant extracts and other plant
derivatives against a variety of microorganisms, including
Streptococcus mutans, were found. For example, Nogueira
et al., (2007) assessed the activity of propolis essential oils
against the cariogenic bacteria S. mutans and Lactobacillus
casei, using the agar diffusion test. The essential oils showed
signicant activity against the two microorganisms, and
some had an even larger zone of inhibition than the positive
control (1% chlorhexidine digluconate).
Vasconcelos et al., (2008) assessed the antimicrobial
activity of a dental cement containing Copaifera multijuga
Hayne oleoresin against Streptococcus mutans and
Streptococcus sanguinis by the dilution test in a liquid
medium. Inhibition of microbial growth occurred in all
tested groups of both bacterial species, showing that
copaiba oil has great potential to be used in dental cement.
Silva et al., (2008) investigated the in vitro
antimicrobial activity of a hydroalcoholic extract
of Rosmarinus ofcinalis Linn. (rosemary) against
standard Streptococcus mitis, Streptococcus sanguinis,
Streptococcus mutans, Streptococcus sobrinus and
Lactobacillus casei strains. The minimum inhibitory
concentration (MIC) was determined by the agar diffusion
test, in Petri dishes. Satisfactory results were found for
most of these microorganisms, except Streptococcus mitis,
suggesting the possibility of using rosemary extract as an
oral antimicrobial agent. However, the authors proposed to
use study models that reproduce the characteristics of the
oral cavity more accurately, for a better assessment of the
use of this extract to treat and prevent oral infections.
Studies have been published on the antimicrobial
activity of thyme essential oil against microorganisms other
than S. mutans. For example, Santurio et al., (2007) assessed
the antimicrobial activity of oregano (Origanum vulgare),
thyme (Thymus vulgaris) and cinnamon (Cinnamomum
zeylanicum) essential oils against samples of Salmonella
enterica of varied serovars, all taken from bird carcasses.
The results showed that oregano and thyme essential oils
are effective against Salmonella.
In addition to antibacterial assessments, thyme
essential oil has also been tested against viruses. Koch et
al., (2008) used a plaque reduction assay to test thyme oil
activity against the herpes simplex virus type 2 (HSV-2) in
RC-37 cells in vitro. This study showed that thyme essential
oil has satisfactory activity against the virus.
Given that no studies were found on the activity
of thyme essential oil against Streptococcus mutans, the
present study is of great relevance, since the test substance
gave very satisfactory results against this microorganism.
Thus, taking our results and the above-mentioned studies
together, thyme essential oil can be used as a broad-
spectrum prophylactic agent, protecting the oral cavity as
379
Thymus vulgaris and Streptococcus mutans
Rev Ciênc Farm Básica Apl., 2011;32(3):375-380
a whole, since the oral cavity is known to be the route of
entry for many microorganisms.
Regarding the use of this oil in a formulation,
the results obtained for the toothpaste indicate that the
formulation is stable, showing no changes when stored at
room temperature for the study period of 120 days (Table
III). However, the formulation stored at 60oC showed
considerable changes in color, odor and consistency
(darkening, fading odor and dryness) after 60 days, which
was to be expected, since the active ingredient is a highly
volatile essential oil, subject to oxidation when exposed to
high temperatures.
The incubator used in this study was a dry
incubator, which did not maintain high humidity levels
inside its chamber. Hence, any product stored in such an
environment at 60oC tends to dry up and the loss of water
raises the concentrations of all the components of the
formulation. Since the chemical decomposition kinetics
of these compounds is generally of rst order, an increase
in concentration increases the decomposition rate of the
product even more than high temperature alone, further
reducing its stability (Prista et al., 1990).
The results, in particular for the formulation stored
at room temperature, show that this formulation appears
to have good stability. Today, it is common to use active
plant ingredients in cosmetic formulations. Therefore,
the formulation prepared in this study containing thyme
essential oil, with its previously demonstrated activity, is
promising for the prophylaxis of oral diseases.
The tests carried out in this study showed that 1%
thyme essential oil diluted in ethanol performed best against
Streptococcus mutans and thus can be considered effective
at this concentration in the ght against this organism. In
the experimental conditions of this study, this concentration
proved viable, both in terms of cost and in terms of sensory
characteristics, making it suitable for use in formulations.
When the toothpaste containing thyme essential oil was
prepared and subjected to a simple stability test based on its
organoleptic characteristics, the formulation proved stable,
since, when stored at room temperature, its characteristics
remained unchanged for 120 days. When stored at 60oC,
its characteristics only started to change after 60 days,
indicating that the product was still quite stable when
stored under such a severe temperature condition.
In the experimental conditions of the present
study, Thymus vulgaris essential oil was effective against
Streptococcus mutans at the smallest concentration tested
(1%), which can be considered viable in terms of cost
and sensory characteristics. In addition, the toothpaste
formulation containing thyme essential oil was stable,
so the use of Thymus vulgaris essential oil in cosmetic
products is promising.
ACKNOWLEDGMENTS
The authors thank Pontical Catholic University of
Campinas (Brazil) for sponsoring the study.
RESUMO
Óleo essencial de Thymus vulgaris sobre o crescimento
de Streptococcus mutans
Thymus vulgaris (tomilho) é uma fonte de óleo essencial
que tem demonstrado atividade antimicrobiana. Uma
parcela considerável da população tem apresentado
problemas dentários, tais como a cárie, na qual
o Streptococcus mutans é um microrganismo de
fundamental importância. O objetivo deste trabalho
foi avaliar o efeito do óleo essencial de tomilho sobre
o crescimento do Streptococcus mutans, a principal
bactéria relacionada com a etiologia da cárie dentária,
bem como veicular este óleo essencial em uma formulação
de creme dental para estudo preliminar. O método
empregado foi diluição em caldo. As concentrações
utilizadas de óleo essencial foram 1%, 5% e 10% de óleo
essencial diluído em etanol ou óleo mineral. Os controles
foram triclosan a 0,25% e 0,5%, bem como digluconato
de clorexidina a 0,06% e 0,12%. A amostra contendo
1% de óleo essencial de tomilho diluída em etanol foi a
mais ecaz, sendo efetiva contra Streptococcus mutans
e, portanto considerada viável em relação ao custo e ao
sensorial conferido ao produto, sendo que a formulação
avaliada foi considerada estável.
Palavras-chave: Tomilho/atividade antimicrobiana.
Thymus vulgaris/atividade antimicrobiana. Óleo essencial/
aplicações farmacêuticas. Streptococcus mutans. Cárie/
prevenção.
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