Introduction: Recently, researches and uses of medicinal plants are ...

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Feb 20, 2013 (4 years and 3 months ago)

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1


Introduction: Recently, researches and uses of medicinal plants are increasing.

The most important
bacteria in dental plaque are Streptococcus mutans, Streptococcus salivarius and Streptococcus
sanguis
. Thus the present study was performed to determine the invitro antibacterial effect of
Satureja hortensis extract and it’s essential oil against these bacteria.

Method: In this experimental study, after the collection of the aerial parts of Satureja horte
nsis,
different concentrations of it’s extract and essential oil were prepared by Double Dilution method.
The antibacterial activity of Satureja hortensis extract and it’s essential oil against the test bacteria
was determined by Disk
-
Diffusions method. Th
e inhibition zones for all concentrations were
measured in diameter (mm) after the incubation at 37°c for 18 hours. According to these
measurements the MIC (Minimal Inhibitory Concentration) for each bacteria was reported.
Tetracycline 30μg and Erythromyci
n 15μg were used as positive control. Data were analyzed by
ANOVA test.

Results: Aqueous extract and methanol extract did not show significant antibacterial activity against
the test bacteria but the essential oil significantly inhibited the growth of the
test bacteria. The MIC
values of the essential oil from Satureja hortensis were measured: Streptococcus mutans %3.125,
Streptococcus salivarius %1.5625 and Streptococcus sanguis %1.5625, respectively. For
Streptococcus mutans, the inhibition effect of Tetr
acycline 30μg was similar with 50% and %25
dosages of the essential oil (p>0.05). For Streptococcus salivarius the effect of Tetracycline 30μg
was very close to 50% dosages of the essential oil (p>0.05). For Streptococcus sanguis the effect of
Erythromyc
in 15μg was higher than 50% and 25% dosages of the essential oil (p<0.001).

Conclusion: Due to strong antibacterial effect of Satureja hortensis essential oil against the growth
of oral bacteria, it may serve as a herbal mouthrinse. In vivo clinical testin
g is necessary to confirm
this antibacterial effect.

Keywords: Antibacterial effect, Satureja hortensis, Streptococcus mutans, Streptococcus salivarius,
Streptococcus sanguis.





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Introduction

Dental caries and Periodontal diseases are the most common infec
tious diseases affecting
humankind(Lamster 2006). The majority of the population may not perform mechanical plaque
removal sufficiently. Thus, antimicrobial mouthrinses that augment daily home care may provide an
effective means of removing or controlling
bacterial plaque to limit gingivitis and periodontitis
(Barnett 2003). Clinicians frequently recommend mouthrinses to their patients as useful in helping
reduce dental plaque and control gingivitis but some patients avoid chemical mouthrinses because of
th
e presence of alcohol,artificial preservation,

or artificial color in mouthrinses(Haffajee 2008).
Recently, researches and uses of medicinal plants are increasing.

The genus Satureja L. (savory, saturei) includes more than 30 species belonging to the famil
y
lamiaceae, subfamily Nepetoideae, tribe Mentheae

Satureja hortensis,
one of these species, is an
annual plant (10

35 cm tall) and aromatic herb with lilac, purplish or white flowers and linear to
linear
-
oblanceolate leaves. It grows wild on rocky or erod
ed slopes, screes, gravelly places and
coastal dunes, fallow fields and roadsides (Davis 1982). In earlier investigations, Satureja species
have been studied with respect to essential oil composition and show to be rich in components such
as carvacrol,γ
-
t
erpinene, thymol, and p
-
cymene(Dardioti et al. 1997; Slavkovska et al. 1997; Tument
et al. 1998; Akgul et al. 1999).

It is well known that chemical composition and yield of essential oils
are affected by exogenous factors such as geophysical position, alti
tude, climate and soil
composition(Baser
et al. 2004)
.

Recently,many studies have demonstrated that the genusSatureja
has antimicrobial activity against human, food, and plant pathogens ( Deans & Svoboda 1990; Ciani
et al. 2000; Ozcan & Erkmen 2001; Azaz
et al. 2002). These studies have focused on antibacterial
and antifungal activity of the essential oil or extracts of Satureja species.

Thus, we undertook a
study to test the in vitro efficacy of Satureja hortensis extract and essential oil against three

oral
bacteria , as determined by the minimum inhibitory
(MIC).


Materials and Methods

Plant Material: The plant (Satureja hortensis L.) used in this work was collected from Hamidieh
,
Ahvaz, Iran in February in 2011. The species were identified by the staffs of the herbarium section,
Ahvaz.
Then the collected plant materials were dried in shadow, and the plant leaves were separated
from the stems and ground, Then were powdered in a gr
inder.

Preparation of the aqueous and methanol extracts: The dried and powdered plant leaves (100 g) were
extracted successively with 500cc of methanol and 500cc of water using Soxhlet extractor for 48 h
at a temperature not exceeding the boiling point of
the solvent. The aqueous and methanol extracts
were filtered through Whatman filter paper and then concentrated in vacuo at 40°C by means of a
Rotary Evaporator.The residues obtained were stored in a freezer until future tests( Lin et al. 1999).


3


Isolatio
n of the essential oil
: The fresh aerial parts of the plants collected were submitted to water
-
distillation for 5 h usig a Clevenger
-
type apparatus. Then, The essential oil was stored until future
tests.

Microbial strains: The extracts and the essential oi
l were tested against three oral bacteria
(Streptococcus mutans PTCC1683, Streptococcus salivarius PTCC1448, Streptococcus sanguis
PTCC1449). These bacteria were provided by the Iranian Type Culture Collection then the
lyophilized strains were inoculated o
n blood agar and then incubated for 18 h at 37°C. The pre
-
cultures of bacteria were prepared for the susceptibility tests.For this purpose,the bacteria strains
were taken by sterile inoculating loop,touching to 4
-
5 colonies raised from pure microorganism
c
ulture and these strains were inoculated at the concentration of 1×


cfu/ml (in order to achieve
the Mc. Farland No:o.5 density) and then incubated at 37°C.

Disc
-
diffusion assay:
Extracts and essential oil were diluted in DMSO (Dimethyl sulfoxid) to the

different test concentrations (for extract test consentrations were 5, 2.5, 1.25, 0.625, 0.3125, 0.15625
mg/ml and for essential oil test concentration were: 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5625%).
Antimicrobial tests were carried out by the disc
-
diff
usion method ( Murray
et al.1995)
. The discs
(6mm diameter) were impregnated with 30 μl of the Extracts and oil dilution and placed on the
inoculated blood agar. Negative controls were prepared using the same solvents to dissolve the
extracts and essent
ial oil (DMSO). Tetracycline (30 μg) and Erithromycin (15 μg ) were used as
positive reference standards to determine the sensitivity of a strain of each tested microbial species.
The inoculated plates were incubated at
37°C


for 18 hours. Antimicrobial
activity was evaluated
by measuring the zone of inhibition against the test microorganisms.
The least concentration of each
extracts and essential oil showing a clear zone of inhibition were taken as the MIC. The assays were
performed three times for each
bacteria.

Statistical Analysis:

Analysis of variance (ANOVA) was used to determine the significance
(p≤0.05)

of the data obtained in all experiments.


Results

According to the results of this study, aqueous extract and methanol extract did not show significant
antibacterial activity against the test bacteria but the essential oil significantly inhibited the growth
of the test bacteria. Average of the inhibition
zones of the bacteria by essential oil, negative control(
DMSO) and positive control (Tetracycline 30μg, Erythromycine 15μg) are summarized in Table 1.






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Table 1
-

Average of inhibition zone of bacteria


Microorganism

Dosage of essential oil (%)

Negative
control
(
-
)

Positive
control
(+)

1.5625

3.125

6.25

12.5

25

50

Average
of
inhibition
zone
(mm)

Streptococcus
mutans

--

--

13.3

15.3

18

20.3

--

20

Streptococcus
salivarius

--

11.5

12.16

16.3

19.16

23.3

--

26

Streptococcus
sanguis

--

11.3

17

18.6

22

28.83

--

17


Negative control: Dimethyl sulfoxide

Positive control: Tetracycline 30 µg/disc for mutans and salivarius, Erythromycine 15 µg/disc for
sanguis.


According to the table 1, the high concentrations of essential oil processed greater antimicrobial
effects against 3 oral bacteria than other low concentrations. Generally, the inhibition effect of
essential oil increased by the increase of the dosages for

all bacteria. For Streptococcus mutans, the
inhibition effect of Tetracycline 30μg was similar with 50% and %25 dosages of the essential oil
(p>0.05). For Streptococcus salivarius the effect of Tetracycline 30μg was very close to 50%
dosages of the esse
ntial oil (p>0.05). For Streptococcus sanguis the effect of Erythromycin 15μg
was higher than 50% and 25% dosages of the essential oil (p<0.001).For all bacteria, Negative
control DMSO didn't show any inhibition zone. The MIC values of essential oil on ta
rget bacteria
were as follow: Streptococcus mutans 3.125%, Streptococcus salivarius 1.5625%, Sterptococcus
sanguis 1.5625%.(Table 2).

Table 2
-

MIC values of Satureja hortensis essential oil against oral bacteria

Microorganism

PTCC

MIC (%)

Streptococcus
mutans

1683

3.125

Streptococcus salivarius

1448

1.5625

Streptococcus sanguis

1449

1.5625






5


Discussion

The primary cause of gingival inflammation and dental caries is bacterial plaque. Some of people
avoid chemical mouthrinse

because of the presence of alcohol,artificial preservation,

, or artificial
color in mouthrinses(Haffajee 2008). so, recently, researches and uses of medicinal plants are
increasing. In vitro evaluation of the antimicrobial activity of test agents or mixt
ures of test agents is
a common first step in determining potential therapeutic efficacy .
Several studies have been
performed concerning the antimicrobial activity of essential oils or extracts of other Satureja
species. Many of the previous studies demo
nstrated that the members of the genus Satureja show a
high antimicrobial activity due to the presence of thymol, carvacrol,and their precursors (Azaz et al.
2002; Gulluce et al. 2003; Sahin et al. 2003).

The data of this study clearly indicated that aque
ous extract and methanol extracts of Satureja
hortensis

did not show significant antibacterial activity against the test bacteria but the essential oil
significantly inhibited the growth of the test

oral bacteria. Furthermore,

it may serve as a natural
an
timicrobial mouthrinse alternative for patients who wish to avoid alcohol, artificial preservatives,
and artificial flavors and colors in chemical mouthrinses. But

In vivo clinical testing is necessary to
confirm this antibacterial effect. Also,several asp
ects need to be studied before adding plant
essential oil into mouthrinses,such as their toxicity , their allergenicity and the concentration of oil
required in mouthrinses.


Acknowledgments:

This study is financially has supported by research affairs, Ahv
az Jundishapur University of

Medical sciences.


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