|Year : 2012 | Volume
| Issue : 3 | Page : 231-236
Effectiveness of mouthrinse formulated from ethanol extract of Terminalia chebula fruit on salivary Streptococcus mutans among 12 to 15 year old school children of Belgaum city: A randomized field trial
SS Nayak1, AV Ankola1, SC Metgud2, U Bolmal3
1 Department of Public Health Dentistry, KLE VK Institute of Dental Sciences, KLE University, Belgaum, Karnataka, India
2 Department of Microbiology, JN Medical college, KLE University, Belgaum, Karnataka, India
3 Department of Pharmaceutics, KLE College of Pharmacy, Belgaum, Karnataka, India
|Date of Web Publication||21-Dec-2012|
S S Nayak
Department of Public Health Dentistry, KLE VK Institute of Dental Sciences, KLE University, Belgaum, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives : Dental caries is the most prevalent oral disease. Streptococcus mutans plays a major role in the occurrence of dental caries. Many antibacterial agents have been developed against dental caries. However, they lack the qualities of an ideal agent. Thus presently, antibacterial activity of herbal agents is being extensively studied. Materials and Methods: In the present study, ethanol extract of Terminalia chebula was prepared and mouthrinse was formulated. A total of 60 children meeting the inclusion criteria were randomly divided into study and control group and respective mouthrinses were administered. Salivary Streptococcus mutans count was assessed at 5 and 60 minutes after rinsing and compared with baseline values. Substantivity of the rinse was assessed among 10 children. Mouthrinse was given to the children and salivary Streptococcus mutans counts were assessed at baseline, 6 and 12 hours postrinsing. Mann-Whitney U test was used to assess the variance of factors like Decayed Missed Filled Teeth, plaque scores, and gingival scores. ANCOVA (Analysis of covariance) was used to determine the change in salivary Streptococcus mutans colony forming units taking baseline values as covariates. Results: It was observed that there was 44.42% reduction in salivary Streptococcus mutans colony forming units 5 minutes after rinsing as compared with baseline values and 64.14% reduction in Streptococcus mutans colony forming units at 60 minutes after rinsing as compared with baseline values. There was a reduction of 35.48% in salivary Streptococcus mutans colony forming units at 60 minutes after rinsing as compared with 5 minutes sample. Conclusion: Streptococcus mutans counts were low up to 6 hours postrinsing among 80% of the children.
Keywords: Dental caries, mouthrinse, Streptococcus mutans, Terminalia chebula
|How to cite this article:|
Nayak S S, Ankola A V, Metgud S C, Bolmal U. Effectiveness of mouthrinse formulated from ethanol extract of Terminalia chebula fruit on salivary Streptococcus mutans among 12 to 15 year old school children of Belgaum city: A randomized field trial. J Indian Soc Pedod Prev Dent 2012;30:231-6
|How to cite this URL:|
Nayak S S, Ankola A V, Metgud S C, Bolmal U. Effectiveness of mouthrinse formulated from ethanol extract of Terminalia chebula fruit on salivary Streptococcus mutans among 12 to 15 year old school children of Belgaum city: A randomized field trial. J Indian Soc Pedod Prev Dent [serial online] 2012 [cited 2020 May 25];30:231-6. Available from: http://www.jisppd.com/text.asp?2012/30/3/231/105016
| Introduction|| |
Dental caries is a major public health problem. It is the single most common chronic childhood disease - five times more common than asthma and seven times more common than hay fever.  The burden of dental caries remains for entire life; as a tooth once destroyed requires restoration and maintenance throughout life. In addition, dental caries and its sequel leads to pain, suffering, and dysfunction besides imposing an economic burden. , It has also been reported that considerable number of school hours and work hours are lost due to dental caries. 
Common oral diseases such as caries and periodontal diseases are still global problems and are documented as contributing greatly to oral health problems. This is particularly the case in under privileged sub populations in both developing and developed countries.  There is a global increase in the prevalence of dental caries. , It has also been reported that dental caries levels have increased constantly for 12-year-olds especially in developing countries, which form the major part of the world.  It has been observed that early childhood caries is more in young children, inter proximal caries is more in school-going children, and root caries is becoming a significant problem in geriatric population. Caries experience showed an increasing trend in primary dentition and in permanent dentition for poor nonHispanic Whites and Mexican Americans. 
Considering the burden exerted by dental caries, prevention of dental caries is the most cost effective approach. Dental caries can be prevented by targeting preventive measures against risk factors like micro-organisms, diet, plaque, etc. Streptococcus mutans has been known to be a etiological agent in the development of dental caries , Hence the prime target should be reduction in Streptococcus mutans in plaque and saliva. This can be effectively achieved by adoption of plaque control measures. Plaque control can be achieved using mechanical and chemical methods. It has been reported that mechanical tooth cleaning alone is not sufficient to achieve plaque control; hence, chemical methods should be used to supplement mechanical tooth cleaning. Chemical antimicrobial agents can reach difficult to clean areas such as interproximal surfaces and can also reduce the growth of biofilms on soft tissues. 
Various vehicles like toothpaste, mouthrinses, gels, varnishes, etc., are available to deliver the antimicrobial agents. Mouthrinses have been developed to deliver broadly the same functional benefits as toothpaste. The conventional mouthrinses have a number of drawbacks.  They are expensive, have adverse effects and most of them are not completely efficacious. Chlorhexidine, which is considered as a gold standard mouthrinse itself, is not free of adverse effects. It has been reported that Chlorhexidine use results in staining of the teeth and restorations, altered taste sensation, and occasionally associated with supragingival calculus build up. A case report has found that Chlorhexidine use was associated with parotitis. , Hence, there is a scope for development of alternate mouthrinses. In this aspect, the area of focus is on natural products. The reason for this is that they are relatively safe and easily available.
It has been reported that c hildren belonging to lower socioeconomic strata suffer from dental caries more than their much affluent peers. , This could be because of their inability to afford dental treatment. It may also be due to the fact that there may be loss of work hours for parents during treatment procedures, which could in turn affect income. Taking all these factors into consideration from the public health aspect, herbs provide a cost effective solution.
Various studies have demonstrated the effectiveness of herbs on Streptococcus mutans. Among the various herbs used for medicinal purpose, Terminalia chebula (also known as haritaki/harad/alalekayi) is commonly used for prevention and treatment of various diseases and is a species of Terminalia, native to southern Asia from India and Nepal east to southwestern China (Yunnan), and south to Sri Lanka, Malaysia, and Vietnam. Few studies have assessed the effect of aqueous extract of Terminalia chebula on Streptococcus mutans. ,, However, no research has been conducted to assess the effect of ethanol extract of Terminalia chebula on Streptococcus mutans. Hence the present study was conducted with an aim of determining the effect of the self formulated mouth-rinse of Terminalia chebula fruit on salivary Streptococcus mutans among 12-to 15-year-old school children of Belgaum city.
| Materials and Methods|| |
A triple blind randomized field trial was conducted among 12-to 15-year-old school children of Belgaum city. Prior to the study, ethical clearance was obtained from KLE University Research Ethics Board. Permission to conduct study among the school children was obtained from Deputy Director of Public Instructions Belgaum. The reference number is KLEU/D6576-6579. The CTRI number is CTRI/2011/04/001687. This is a part of a major study.
Preparation of mouth rinse
Dried Terminalia chebula fruits were procured. They were authenticated by a professor from Botany department. The authentication certificate number is RLSI/BOT/Consul-65. The fruits were then screened and impurities were removed. The fruits were then grinded into coarse powder form, which was then transferred to a Soxhlet apparatus. Pure ethanol was procured and then distilled water was added to make concentration of 70%. It was then transferred to Soxhlet apparatus. The fruit powder and ethanol was added in ratio of 1:6. The extract obtained after processing, was then desiccated and stored in an air tight container. Mouthrinse was formulated by adding 2.5% of the extract to distilled water. Sodium carboxymethyl cellulose was added to provide viscosity, methyl paraben preservative was added, and mannitol was used to mask the astringent taste of the extract. Control mouthrinse consisted of the above mentioned agents except ethanol extract of Terminalia chebula.
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, 30 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. From them, 60 subjects were randomly selected. Parental consent and child's assent was obtained prior to enrolling the child in the study. Children with DMFT ranging between 3 and 6, with fair plaque scores (according to Silness and Loe plaque index) and moderate gingival scores (Loe and Silness gingival index) were included in the study. Detailed medical and dental history was recorded. Oral hygiene practices and dietary practices were also recorded. Children using antibiotics within the past 1 month were excluded from the study.  Children were randomly allocated to study and control groups.
Prior to the study, the children were demonstrated the rinsing procedure. The study procedure was carried out in the school premises. The children were given 5 ml of respective rinses and they were asked to rinse for 1 minute. Rinsing was supervised so that the children actually rinsed with the mouthrinse instead of simply holding it in mouth.
Saliva collection procedure
Baseline salivary samples were collected. The subjects were informed not to eat or drink anything (except water) 2 hours prior to saliva collection. Saliva collection was done in the morning between 11 and 11.30 to match the circadian rhythm. Care was also taken to avoid saliva collection before heavy physical exercise. Salivary samples were collected again 5 and 60 minutes postrinsing. Two students at a time were made to sit comfortably on the chairs. Children were told to swallow the preexisting saliva in order to clear mouth of any residual unstimulated saliva. After this, each student was asked to collect saliva in the mouth for a minute and then it was collected using a sterile disposable syringe. The saliva was later transferred to vial containing thioglycollate transport medium.
A self-designed questionnaire was used to determine the color, odor, and taste acceptability of the extract.
| Statistics|| |
Data was entered and analyzed using SPSS version 17 (SPSS Inc., Chicago). Descriptive data was expressed in percentage. Mann-Whitney U test was used to assess whether the study group and control group varied in factors like DMFT, plaque scores, and gingival scores. ANCOVA was used to compare the effectiveness of the extract between the two groups at baseline and various intervals postrinsing. Significance was set at P < 0.05.
| Results|| |
A total of 60 subjects in the age group of 12-15 years participated in the study. Among them, 36 were male and 24 were female. The mean DMFT, plaque scores, and gingival scores of study group and control group did not differ significantly [Table 1], [Table 2], [Table 3], [Table 4].
|Table 3: Comparison of study group and control group with respect to DMFT values by t-test|
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|Table 4: Comparison of study and control group with respect to plaque scores and gingival scores using Mann– Whitney U test|
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No significant difference was observed in baseline Streptococcus mutans counts between study and control group (F value = 0.09; P = 0.7634) [Table 5]; [Figure 1].
|Figure 1: Comparison of salivary Streptococcus mutans counts of study and control group at baseline, 5 and 60 minutes postrinsing|
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|Table 5: Comparison of salivary Streptococcus mutans colony forming units among study and control group at baseline, 5 and 60 minutes postrinsing|
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Reduction of 44.42% was observed in Streptococcus mutans colony forming units when 5 minutes sample was compared with baseline sample in the study group (P < 0.05) [Table 5]; [Figure 1].
Reduction of 64.14% was observed in the Streptococcus mutans counts when 60 minutes sample was compared with baseline sample in the study group (P < 0.05) [Table 5]; [Figure 1].
Reduction of 35.48% was observed in the Streptococcus mutans counts when 5 minutes sample was compared with 60 minutes sample in the study group (P < 0.05) [Table 5]; [Figure 1].
| Discussion|| |
The present randomized controlled field trial was conducted to determine the effect of mouthrinse formulated from ethanol extract of Terminalia chebula fruit on salivary Streptococcus mutans counts among 12-to 15-year-old children of Belgaum city.
Terminalia chebula is one of the most commonly used herbs in traditional medicine. It has been popularly referred to as "King of Medicines". Various parts of Terminalia chebula plant have been used since time immemorial for prevention and cure of a number of diseases. It has been reported that Terminalia chebula exerts a range of therapeutic activities including antibacterial, antiviral, antifungal, and antioxidant activities.  It would be of interest to determine if such an herb with multiple health benefits could also have a beneficial effect on oral health. Several herbs have been studied for their effect on oral health. However, there is a lack of randomized controlled trials on the effectiveness of ethanol extract of Terminalia chebula on oral micro-organisms.
The study subjects comprised of children in the age group of 12-15 years. The prevalence of caries is relatively more in this age group. Since the intervention in the present study is a mouthrinse, it would be difficult for younger children to rinse and also the microbial flora in younger children varies during mixed dentition stage; hence, the present age group was selected.
In the present study, the extract was prepared using ethanol, as tannins are the compound of interest and most of the tannins are readily soluble in alcohol-water as compared with water alone. Ethanol was used in the present study, only as solvent, the extract that was finally used did not contain ethanol as it was recovered from the extract. Nearly 70% ethanol was used so that even those tannins, which are water soluble, are also dissolved. Previous studies conducted on Terminalia chebula have used water as solvent. ,, Moreover, in the previous studies, the sample was meager and lacked a control group. , Till date, no research has been conducted to determine the effect of ethanol extract of Terminalia chebula on Streptococcus mutans. Hence, the present study was conducted.
Prior to formulation of mouthrinse, minimum inhibitory concentration was determined using tube dilution method. It was determined thrice and average of the values was taken. The minimum inhibitory concentration was determined to be 2.5%. Previous study reported that the minimum inhibitory concentration of the aqueous extract of Terminalia chebula was 10%.  The variation in the minimum inhibitory concentration may be due to variation in the solvent used for extract preparation. Tannins dissolve more readily in ethanol, so this might be the reason for inhibition of Streptococcus mutans at lower concentration.
The efficacy of ethanol and aqueous extracts were assessed for antibacterial effect in vitro prior by the same authors of this study. It was found that ethanol extract was better than aqueous extract. Parekh also reported that ethanol extract showed maximum antibacterial activity over other extracts. However, the organisms studied were different. 
Unstimulated saliva was used to assess the parameters as it has been reported that the unstimulated saliva represents the basal salivary flow rate. To avoid contamination, the saliva was collected in sterile syringes. Thioglycollate transport media was used as a transport media so that the vitality of the organisms is maintained.
In the present study, growth of Streptococcus mutans was significantly reduced at 5 minutes postrinsing (44.42% reduction) and greater reduction was observed up to 1 hour postrinsing (64.14%) as compared with baseline values thus indicating that the antibacterial action begins immediately and increases up to 1 hour. Jagtap reported that there was inhibition of growth of Streptococcus mutans 90 minutes postrinsing. However, the study was conducted only among three subjects. Total Streptococcal counts and total bacterial counts were low up to 3 hours postrinsing.  Results similar to the present study were reported by Carounanidhy.  Moreover, in the present study, when substantivity was assessed there was reduction up to 12 hours postrinsing. The variation in the solvent used could be responsible for greater reduction in Streptococcus mutans.
Children were asked to rinse the respective mouthrinses for a period of 60 seconds. The optimal mouthrinsing time recommended is 30 seconds.  A pilot study was done 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. Hence, in the present study the children were told to rinse using the respective mouthrinses for 60 seconds.
Terminalia chebula is rich in tannins. It is estimated that it contains around 30-40% tannins. The tannins are of hydrolysable type including chebulic acid, chebulagic acid, corrilagin, and gallic acid. It also contains fructose, succinic acid, amino acid, etc. Tannins are believed to be majorly responsible for the antibacterial action.
Substantivity is an important aspect of any mouthrinse as the active ingredients of the mouthrinse have to exert effect for a considerable period after rinsing. Various studies have assessed the substantivity of Chlorhexidine. It was reported that 0.12% of Chlorhexidine showed significant reduction up to 5 hours postrinsing. , In the present study, Terminalia chebula mouthrinse showed reduction in Streptococcus mutans counts up to 6 hours postrinsing among 80% of the subjects. Binding of tannins to the oral tissues may be responsible for prolonged action of Terminalia chebula mouthrinse. These tannins might have been released into saliva as its concentration decreased over a period of time exerting prolonged action. These results indicate that the reduction in Streptococcus mutans is similar to that caused by Chlorhexidine. In addition, the advantage of Terminalia chebula would be its safety and reduced cost.
Further studies need to be conducted comparing the effect of Terminalia chebula mouthrinse with gold standard mouthrinses. In addition, long-term studies need to be conducted to determine the long-term effect of mouthrinse on oral health.
| Conclusion|| |
The results of this study indicate that Terminalia chebula ethanol extract mouth rinse exerts prolonged antimicrobial action on salivary Streptococcus mutans. Taking public health aspect into consideration, Terminalia chebula mouthrinse could be used as an adjunct for conventional tools against dental caries.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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