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

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.

RCT Entities:   Population Interventions Outcomes

INTRODUCTION

Dental caries is a worldwide oral disease, especially 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 process (2).

Due to the difficulties 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). Considering all the disadvantages of using different chemical 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 officinalis) extract on reduction of SM in dental plaque in a group of school-aged children.

MATERIALS AND METHODS

Enrollment.

A double blind randomized clinical 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 officinalis 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; Schwabach, Germany) at 90 rpm for 48 hours. Thereafter, the solution was passed through a strainer and transferred to a rotar y 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 containing 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 Significance, and the Efficacy 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 first 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.

Prior to the study, the children 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 par ticipants were instr ucted 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 samples 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 specific 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 significance was set at 0.05.

RESULTS

The mean colony count scores of the study and control group, before and after mouth wash application are presented in Tables 1 and 2.

Table 1.

Means of colony counts of 11–14 year-old children, before and after using test mouth rinse

Index	Colony-pre	Colony-post
N	35	35
Mean	3900	600
SD	1465.7	665.1

Table 2.

Means of colony counts of 11–14 year-old children, before and after using placebo mouth rinse

Index	Colony-pre	Colony-post
N	35	35
Mean	4071.4	3174.3
SD	1630.7	1628.3

A significant difference was observed in post treatment SM counts between the study and control group (P = 0.00) and also between baseline and post treatment samples in the study group (P = 0.001). Although, no significant difference was observed in baseline SM counts between study and control group (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).

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	35	1465.7	3900	Colony-pre
			35	665.1	600	Colony-post
control	0.11	1.63	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	35	1630.7	4071.43	control
			35	1465.7	3900	study
Colony-post	0.00	10.48	35	1628.3	3714.29	control
			35	665.1	600	study

DISCUSSION

The present randomized controlled clinical trial was conducted to determine the effect of mouth rinse formulated from Sage extract on dental plaque SM counts among 11 –14 year-old children in Hamadan, Iran.

Salvia officinalis 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 including antibacterial, antiviral, antifungal, and antioxidant effects (13–15). It would be of interest to determine if such an herb could also have a beneficial effect on oral health.

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 variability, depending on the sensitivity of microorganisms and the efficiency of the tested compounds. Salvia species rich in essential oils (such as S. officinalis) with volatile monoterpenoid as their major constituents are reported to be effective antibacterial (18).

Generally, Gram-positive bacteria are more sensitive 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 predisposes the enamel of the tooth to caries (20). The sensitivity 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 membrane, 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 flora 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 dental 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.