The antibacterial effect of sage extract (Salvia officinalis) mouthwash against Streptococcus mutans in dental plaque: A randomized clinical trial

Abstract

Background and objective:
The aim of the study was to evaluate the clinical effects of a mouthwash containing Sage (Salvia officinalis) extracts on Streptococcus mutans (SM) causing dental plaque in school-aged children.

Material and methods:
A double blind clinical trial study was conducted in a dormitory on 70 girls aged 11-14 years having the same socioeconomic and oral hygiene conditions. These students were randomly divided into 2 groups; the first group (N=35) using Sage mouthwash, and the second group (N=35) using placebo mouthwash without active any ingredients. At the baseline, plaque samples obtained from the buccal surfaces of teeth were sent to laboratory to achieve SM colony count. These tests were reevaluated after 21 days of using the mouthwashes. Statistical data analysis was performed using t-student tests with p<0.05 as the level of significance.

Results:
Sage mouthwash significantly reduced the colony count (P=0.001). Average number of colonies in test group was 3900 per plaque sample at the baseline, and 300 after mouthwash application. In the control group, pre-test colony count was 4400 that was reduced to 4000; although this reduction wasn't significant.

Conclusion:
The Sage mouthwash effectively reduced the number of Streptococcus mutans in dental plaque.

Full text

173
*Corresponding author: Dr. Mohadese Azarsina
Address: Department of Operative Dentistry, Faculty of
Dentistry, International Branch of Shahid Beheshti Universi-
ty of Medical Sciences, South Jamalzade Street, Tehran, Iran.
Tel: +98-21-66917171
Fax: +986134433715
E-mail: azarsina2012@yahoo.com, beheshtirouym@ya-
hoo.com
The antibacterial effect of sage extract (Salvia ofcinalis)
mouthwash against Streptococcus mutans in dental plaque: a randomized
clinical trial
Maryam Beheshti-Rouy1, Mohadese Azarsina2*, Loghman Rezaie-Sou1, Mohammad Yousef Alikhani3,
Ghodratollah Roshanaie4, Samira Komaki1
1Department of Operative Dentistry, Faculty of Dentistry, Hamadan University of Medical Sciences,
Hamadan, Iran.
2Department of Operative Dentistry, Faculty of Dentistry, International Branch of Shahid Beheshti University
of Medical Sciences, Tehran, Iran.
3Mohammad-Yousef Alikhani: Department of Microbiology, Faculty of Medicine, Hamadan University of
Medical Sciences, Hamadan, Iran.
4Ghodratollah Roshanaie: Department of Public Health, Faculty of Medicine, Hamadan University of Medical
Sciences, Hamadan, Iran.
Received: June 2013, Accepted: October 2014
ABSTRACT
Background and Objective: The aim of the study was to evaluate the clinical effects of a mouthwash containing Sage
(Salvia ofcinalis) extracts on Streptococcus mutans (SM) causing dental plaque in school-aged children.
Material and Methods: A double blind clinical trial study was conducted in a dormitory on 70 girls aged 11-14 years having
the same socioeconomic and oral hygiene conditions. These students were randomly divided into 2 groups; the rst group
(N=35) using Sage mouthwash, and the second group (N=35) using placebo mouthwash without active any ingredients. At
the baseline, plaque samples obtained from the buccal surfaces of teeth were sent to laboratory to achieve SM colony count.
These tests were reevaluated after 21 days of using the mouthwashes. Statistical data analysis was performed using t-student
tests with p<0.05 as the level of signicance.
Results: Sage mouthwash signicantly reduced the colony count (P=0.001). Average number of colonies in test group was
3900 per plaque sample at the baseline, and 300 after mouthwash application. In the control group, pre-test colony count was
4400 that was reduced to 4000; although this reduction wasn’t signicant.
Conclusion: The Sage mouthwash effectively reduced the number of Streptococcus mutans in dental plaque.
Keywords: anti-bacterial agents; dental plaque; Salvia ofcinalis; Streptococcus mutans;
INTRODUCTION
Dental caries is a worldwide oral disease, espe-
cially in developing countries, which form the major
part of the world. Bacterial plaque is considered as
an etiologic factor for caries, and oral self-care for
plaque control is an essential step in the prevention
from caries (1). Streptococcus mutans (SM) is the
main bacteria in dental plaque, responsible for caries
Volume 7 Number 3 (June 2015) 173-177
ORIGINAL ARTICLE

http://ijm.tums.ac.ir
process (2).
Due to the difculties for teens in achieving
complete plaque control, the administration of
some antiplaque agents such as chemical or herbal
antimicrobial dental products was suggested as an
auxiliary protocol to tooth brushing (3). Consider-
ing all the disadvantages of using different chemi-
cal agents, many studies are being conducted on the
effectiveness of herbal materials (4).
Recently, antimicrobial effect of sage extract has
been shown experimentally (5, 6). Dry sage leaves
were used in folk medicine for a variety of disorders
(7). Today, sage is also used as a traditional remedy
for many diseases (8, 9).
According to the results of previous studies (10,
11), and considering lack of randomized controlled
trials on the effectiveness of sage extract on oral
microorganisms, the aim of the present study was to
evaluate the clinical effectiveness of a mouthwash
containing Sage (1% Salvia ofcinalis) extract on
reduction of SM in dental plaque in a group of school-
aged children.
MATERIALS AND METHODS
Enrollment. A double blind randomized clin-
ical trial was conducted among female 11-14 year-
old school children of Hamadan, Iran during the
year 2012. Prior to the study, ethical clearance was
obtained from Hamadan University Research
Ethics Board (Protocol No: 1010/9/35/16). Permission
to conduct the study among the school children was
obtained from their guardians. The IRCT number is
2012070710204N. Each subject was provided with a
written informed consent and all of the researchers
undertook Helsinki treaty.
Mouth rinse preparation. Sage mouthwash was
extracted from the plant Salvia ofcinalis in the
laboratory of a pharmaceutical company (Jahanghir,
Tehran) by an expert pharmacologist. Leaves of the
plant were chopped, fragmented, and broken into
small pieces, and each 50 g of leaves were soaked in
1500 ml of solvent (50% water/ 50%ethanol [96%])
in a shaker apparatus (Heidolph Unimax; Schwa-
bach, Germany) at 90 rpm for 48 hours. Thereaf-
ter, the solution was passed through a strainer and
transferred to a rotary evaporator apparatus
(Heidolph WD2000; Schwabach, Germany) to
separate the solvent from the extract. The 5% Sage
mouthwash was prepared (0.5 g of extract in 100 ml
distilled water) and poured into bottles each contain-
ing 240 ml of the solution. Normal saline mouthwash
was prepared in the bottles with the same shape and
color, to be used as control.
Selection and allocation of subjects. Sample
size was determined using Biometrika table for
proportions, which is based on three factors: Power
of the study, Level of Signicance, and the Efcacy
values in the previous studies. Based on this
estimation, 35 subjects were included in each group.
Two stage random sampling was done to select the
subjects. In the rst stage, all the subjects were screened
for inclusion criteria (11-14 year-old girls under the
supervision of a welfare organization with same
socioeconomic and nutritional conditions). Children
with systemic physical or mental problems or using
antibiotics within the past 1 month were excluded
from the study. Children were randomly allocated to
study and control groups.
Rinsing procedure. Pr ior to the study, the ch ild ren
were demonstrated the rinsing procedure. The study
procedure was carried out in the school premises.
The mouth rinse bottles given to the participants were
unlabeled. The participants were instructed to
continue their usual oral hygiene measures and not
to use any other mouth rinse for the duration of
the study. The subjects were demonstrated to use
the mouth wash for 60 seconds, twice daily (once
taken at night just before the bed time) over the
3-week study period. The participants' compliance
was evaluated by measuring the remaining volume
of the mouth wash that they brought back during
their recalls. They were also asked to report any
adverse reactions experienced during the use of their
mouth wash.
Plaque sample collection. Baseline plaque sam-
ples were collected. The subjects were informed not
to brush 24 hours prior to plaque collection. Plaque
collection was done in the morning. Plaque samples
were collected using sterile disposable sticks from
the buccal surface of anterior teeth. The plaque was
placed in a vial containing a transport medium and
transported to 1ml Brain-Heart Infusion (BHI)
[BHI; Difco, Sparks, MD, USA] culture medium.
Afterwards, the samples were cultured in MSB
174 IRAN. J. MICROBIOL. Volume 7 Number 3 (June 2015) 173-177
MARYAM BEHESHTI-ROUY ET AL .

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specic medium (A.L. Norway) containing 0.2 units per
milliliter Bacitracin. The numbers of the SM colonies
grown in Bacitracin culture medium were counted
visually. Data were statistically analyzed by t-student
test, using SPSS software (Version 16, SPSS Inc.,
Chicago, USA). Level of signicance was set at 0.05.
RES ULTS
The mean colony count scores of the study
and control group, before and after mouth wash
application are presented in Tables 1 and 2.
A signicant difference was observed in post treat-
ment SM counts between the study and control group
(P = 0.00) and also between baseline and post treat-
ment samples in the study group (P = 0.001). Al-
though, no signicant difference was observed in
baseline SM counts between study and control group
ANTIBACTERIAL EFFECT OF SAGE EXTRACT
IRAN. J. MICROBIOL. Volume 7 Number 3 (June 2015) 173-177 175 http://ijm.tums.ac.ir
Table 1. Means of colony counts of 11-14 year-old children,
before and after using test mouth rinse
Index
N
Mean
SD
Colony-pre
35
3900
1465.7
Colony-post
35
600
665.1
Table 2. Means of colony counts of 11-14 year-old children,
before and after using placebo mouth rinse
Index
N
Mean
SD
Colony-pre
35
4071.4
1630.7
Colony-post
35
3174.3
1628.3
(P = 0.65), and between baseline and post treatment
in control group (P = 0.11) despite the drastic colony
count reduction (Tables 3 and 4).
DISCUSSION
The present randomized controlled clinical trial was
conducted to determine the effect of mouth rinse for-
mulated from Sage extract on dental plaque SM counts
among 11 -14 year-old children in Hamadan, Iran.
Salvia ofcinalis is one of the most commonly used
herbs in traditional medicine (9, 11, 12). It has been
popularly referred to as "Sage". It has been reported
that sage exerts a range of therapeutic activities in-
cluding antibacterial, antiviral, antifungal, and antiox-
idant effects (13-15). It would be of interest to deter-
mine if such an herb could also have a benecial effect
on oral health.
Table 3. Multiple comparisons of the Colony count test among the study and control group.
Group p.value t N SD Mean index
study 0.001 13.4
control 0.11 1.63
35 1465.7 3900 Colony-pre
35 665.1 600 Colony-post
35 1630.7 4071.4 Colony-pre
35 1628.3 3714.3 Colony-post
Table 4. Multiple comparisons of the Colony count test before and after using test or control mouth rinse.
Group p.value T N SD Mean Index
Colony-pre 0.65 0.46
Colony-post 0.00 10.48
35 1630.7 4071.43 control
35 1465.7 3900 study
35 1628.3 3714.29 control
35 665.1 600 study

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Several herbs have been studied for their effect
on oral health (16, 17). Studies on the antimicrobial
potential of the Salvia genus reveal a broad variabil-
ity, depending on the sensitivity of microorganisms
and the efciency of the tested compounds. Salvia
species rich in essential oils (such as S. ofcinalis) with
volatile monoterpenoid as their major constituents are
reported to be effective antibacterial (18).
Generally, Gram-positive bacteria are more sensi-
tive to sage essential oil compared to other kinds of
bacteria (19). SM is an anaerobic, Gram-positive
bacterium with the ability to metabolize sucrose and
release lactic acid. This acidic environment predispos-
es the enamel of the tooth to caries (20). The sensitiv-
ity of bacteria is related to the morphological structure
and chemical composition of their membrane (21).
Essential oils can inhibit microorganisms by various
mechanisms, in part due to their hydrophobicity. They
get partitioned into the lipid bi layer of the cell mem-
brane, making it more permeable, causing leakage of
vital cell contents (22). The loss of the differential
permeability character of the cytoplasmic membrane
is the cause of cell death (22, 23).
The subjects of the present study were children in
the age group of 11-14 years old. The prevalence of
caries is relatively more in this age group (24). The
microbial ora in younger children varies during
mixed dentition stage (25). A pilot study was
performed to determine the maximum time up to
which children could rinse without any discomfort.
It was observed that children could rinse up to 60
seconds.
Sage mouth rinse can be used as an adjunct for
conventional methods of plaque control against den-
tal caries. Although chlorhexidine has a proven role
in reducing plaque accumulation, tooth staining is the
major limiting factor for its daily use (26). Further
studies need to be conducted comparing the effect of
sage mouth rinse to gold standard mouth rinses.
Considering the limitations of the present study, it
was concluded that sage extract mouth rinse exerted
antibacterial action against Streptococcus mutans in
dental plaque.
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