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ORIGINAL ARTICLE
Year : 2022  |  Volume : 20  |  Issue : 3  |  Page : 271-276

Antibacterial activity of garlic extract, tea tree oil, and its mouthwashes against Streptococcus mutans and Lactobacillus: An In vivo study


1 Department of Public Health Dentistry, Annoor Dental College and Hospital, Muvattupuzha, Kerala, India
2 Department of Public Health Dentistry, Indira Gandhi Institute of Dental Sciences, Kothamangalam, Kerala, India
3 Department of Community Dentistry, Faculty of Dentistry, Taif University, Taif, Saudi Arabia
4 Department of Preventive and Community Dentistry, College of Dental Sciences, Davangere, Karnataka, India
5 Department of Public Health Dentistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Date of Submission24-Oct-2021
Date of Decision12-Mar-2022
Date of Acceptance15-Jun-2022
Date of Web Publication12-Sep-2022

Correspondence Address:
Pooja Latti
Department of Public Health Dentistry, Annoor Dental College and Hospital, Muvattupuzha, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaphd.jiaphd_197_21

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  Abstract 


Background: A wide group of microorganisms is identified from carious lesions, of which Streptococcus mutans and Lactobacillus acidophilus are the main pathogenic species involved in the initiation and propagation of dental caries. Mouthrinses can deliver therapeutic ingredients and benefits to all accessible surfaces in the mouth. With the increasing use of drugs, microorganisms are attaining resistance to commonly used antibiotics, which leads to the downfall of the effectiveness of conventional medicines, and therefore, a search for new antimicrobial agents has become necessary. The objective of the study was to evaluate the effect of mouthwashes containing garlic extract (GE), tea tree oil (TTO), and chlorhexidine (CHX) on salivary S. mutans and Lactobacillus. Methods: An in vivo parallel study was conducted among 90 18–25-year-old subjects. GE (2.5%) and TTO (0.2%) mouthwashes were prepared for use in the study. Commercially available CHX mouthwash (0.12%) was used as a positive control. The study duration was 5 weeks. The participants were randomly assigned into three groups with 30 participants in each group. To assess the residual effects following discontinuation of mouthwashes, saliva samples were collected on the 18th and 24th days. One-way ANOVA was used to assess the mean colony difference. Results: No significant difference in the counts of S. mutans and Lactobacillus was observed at baseline and 6th day between all the three groups. On the 12th day, the highest reduction in S. mutans and Lactobacillus counts was observed following the use of TTO mouthwash, followed by CHX mouthwash. The reduction in the count of S. mutans was significantly higher in the TTO group on the 24th day comparable to the garlic mouthwash group, followed by the CHX mouthwash group. Conclusion: Both GE and TTO mouthwashes demonstrated a significant antibacterial activity against S. mutans and Lactobacillus in vivo, with TTO demonstrating the greatest effect.

Keywords: Chlorhexidine, garlic, Lactobacillus, Streptococcus mutans, tea tree oil


How to cite this article:
Latti P, Subramaniam R, Basha S, Prashant G M, Kumar P G, Hirekalmath S. Antibacterial activity of garlic extract, tea tree oil, and its mouthwashes against Streptococcus mutans and Lactobacillus: An In vivo study. J Indian Assoc Public Health Dent 2022;20:271-6

How to cite this URL:
Latti P, Subramaniam R, Basha S, Prashant G M, Kumar P G, Hirekalmath S. Antibacterial activity of garlic extract, tea tree oil, and its mouthwashes against Streptococcus mutans and Lactobacillus: An In vivo study. J Indian Assoc Public Health Dent [serial online] 2022 [cited 2022 Sep 28];20:271-6. Available from: https://www.jiaphd.org/text.asp?2022/20/3/271/355890




  Introduction Top


Oral diseases continue to be a major health problem worldwide.[1] Dental caries is a chronic and multifactorial disease that, although avoidable, still represents an important problem in public health, since it affects approximately 90% of the population, mainly children and adolescents, compromising their quality of life and development.[2] The prevalence and distribution of dental caries are different in developed and developing countries. India has shown an inclined trend of this disease over a relatively short period. The pooled caries prevalence was between 50.84% and 62.41% at a 5-year interval.[3] A wide group of microorganisms is identified from carious lesions, of which Streptococcus mutans, Lactobacillus acidophilus, and Actinomyces viscosus are the predominant pathogenic species associated with the initiation and propagation of dental caries.[4],[5],[6],[7],[8]

Mouthwashes (mouthrinses) are solutions or liquids used to rinse the mouth for several purposes: (a) to remove or destroy bacteria, (b) to act as an astringent, (c) to deodorize, and (d) to have a therapeutic effect by relieving infection or preventing dental caries.[9] Antimicrobial mouthrinses allow patients to deliver active agents cost-effectively when controlling dosage and timing.[10] Mouthrinses are used to deliver therapeutic ingredients and benefits to all surfaces that are accessible in the mouth including the interproximal tissues, and depending on their composition, they remain active for extended periods.[11] With the increasing use of drugs, microorganisms are attaining resistance to commonly used antibiotics, which leads to the downfall of the effectiveness of conventional medicines, resulting in the quest for new antimicrobial agents.

Garlic (Allium sativum) is used universally as a flavoring agent, traditional medicine, and functional food to enhance physical and mental health. Garlic extract (GE) inhibits the growth of oral pathogens and certain proteases, thereby possessing a therapeutic value.[12] Tea tree oil (TTO) is the essential oil steam-distilled from the Australian plant, Melaleuca Alternifolia (Myrtaceae family). TTO is effective against a high number of Gram-positive and Gram-negative bacteria, as well as fungi.[13],[14] Hence, with this background, the present study was undertaken to evaluate the effect of mouthwashes containing GE, TTO, and chlorhexidine (CHX) on salivary S. mutans and Lactobacillus in vivo, with a research hypothesis that the garlic and TTO mouthwashes have a significant effect on the salivary S. mutans and Lactobacillus counts.


  Methods Top


Study population and study design

An in vivo parallel study was conducted among 90 adult participants aged between 18 and 25 years, selected randomly from the business schools in Davanagere city, Karnataka State, India. Ethical clearance for the study was obtained from the institutional review board (ethical clearance number CODS/16/16). Written informed consent was taken from study participants.

The inclusion criteria for study participants were presence of at least 24 fully erupted teeth, absence of fixed or removable orthodontic appliances or removable prosthesis, absence of any antimicrobial agent therapy within previous 2 weeks, and participants expressing voluntary consent to cooperate and participate in the study. The exclusion criteria were smokers, participants with preexisting systemic diseases, participants under medications, participants with periodontitis, those with a known history of allergy to mouthwashes under study, and those with prosthesis or orthodontic appliances.

Preparation of mouthwashes

For the preparation of garlic mouthwash, fresh garlic was obtained from the local market, peeled, and cleaned. 50 g of cleaned fresh garlic bulbs were chopped and homogenized with 100 ml sterile distilled water. After 24 h, the mixture was filtered through a muslin cloth and then refiltered by passing through Whatman's filter paper number 1. The filtrate was concentrated by complete evaporation of the solvent on a hot water bath to yield the pure extract. This extract was considered as the 100% concentration of the extract. The TTO (100%) was obtained from the Department of Pharmaceutics, Bapuji College of Pharmacy, Davangere, India. An in vitro study was conducted to determine the minimum inhibitory concentration (MIC) for GE and TTO. MIC was determined by the serial tube dilution test.[14],[15] The GE showed a MIC of 2.5% and TTO 0.2%. Hence, these concentrations were used for the preparation of the respective test mouthwashes. The mouthwashes were prepared in sterile distilled water with sorbitol and peppermint oil as sweetener and flavoring agents, respectively.

The acceptability of the mouthwash was assessed with a pilot study, carried out among five participants. The pilot study was the basis for sample size calculation. Based on the pilot study result (mean the difference among test and controls = 4.7, ά error 5%, and power of the study 80%), the sample size (n) was calculated from the following formula:



where z = 2 (assuming the distribution is normal and confidence limit is 95%; standard normal variate)

p = expected minimum reduction in the colony counts in the treatment group = 80%

q = 100– P = 20%

L = admissible error in estimation = 20% of P = 20% of 80% =16

A sample of 20 was arrived at, which was rounded to 30 in each group keeping in mind the possible attrition of participants in follow-up visits.

A total of 148 participants were approached from S.B.C. First Grade College for Women and Bapuji Institute of Management in Davangere city, Karnataka, and the study participants fulfilling the inclusion and exclusion criteria (n = 108) were recruited. After selection of the participants, purpose of the study was explained and demographic details such as name, age, sex, address, and the number of teeth were noted in a pro forma along with consent from all the participants. The preliminary examination was done by making the patient to sit comfortably and using a plain mouth mirror and a probe in natural light. Of the 108 participants approached, 90 participants giving a voluntary informed consent were recruited and randomly allocated by lottery method into three study groups (Group I, II, and III) with 30 participants in each group. Each participant was asked to take a slip from a box containing three slips with a code for the products and they were allotted to that group. The assignment of participants to the groups was performed by a person not involved in the examination. Participants received the products according to the specified code. The three study groups were Group I (garlic mouthwash), Group II (TTO mouthwash), and Group III (CHX mouthwash).

The study was conducted over 5 weeks. The order of intervention is as follows:

  • The 1st week: Baseline, resting whole saliva samples were collected in the morning on the 3rd and 6th day from the participants directly in graduated sterilized polypropylene vials. The saliva samples were subjected to microbial analysis for the quantification of S. mutans and Lactobacillus
  • The 2nd week: All participants were submitted to a 1-min mouthwash using a control solution (sterile distilled water) and sodium fluoride dentifrice, carried out after the last tooth brushing of the day. Resting whole saliva samples were collected in the morning of the 3rd and 6th days and were subjected to microbial analysis.
  • The 3rd week: Participants were randomly allocated by lottery method into three study groups (Group A, B, and C) with 30 participants in each group. The three study groups were:


    • Group A: GE mouthwash (2.5%)
    • Group B: TTO mouthwash (0.2%)
    • Group C: CHX mouthwash (0.12%, Facmed Pharmaceuticals Private Limited, Delhi, India).


Instructions for performing oral hygiene procedure were given to all the participants, i.e., oral rinsing, in addition to their routine tooth brushing. For ensuring standardization, participants were provided with measuring cups of 10 ml and were instructed to use 10 ml of mouthwash for 1 min carried out after the last toothbrushing of the day for 7 days. Each subject was provided with a sodium fluoride dentifrice (Close-Up®, Hindustan Unilever Ltd.). All the participants were instructed to follow their routine oral hygiene practices and the assigned regimen and maintain a chart on daily product use. Compliance with their study regimen was assessed during their visit. The use of oral hygiene tools other than the attributed was strictly prohibited during the entire study period. Resting whole saliva samples were collected in the morning of the 3rd and 6th days and subjected for microbial analysis (mouthwash groups mean).

  • 4th and 5th week: The treatment was discontinued. Resting whole saliva samples were collected once a week for microbial analysis (4th and 5th week mean counts) to observe the regrowth or antimicrobial residual effects upon S. mutans and Lactobacillus.


Collection of saliva

On the day of saliva collection, participants were instructed to refrain from oral hygiene practice and breakfast until the collection of samples. To control the circadian variations, samples were collected between 7:00 am and 8:00 am. The participants were instructed to let saliva collect on the floor of the mouth without swallowing it for at least 1 min and then to expectorate into the graduated sterile vial. This procedure was continued for 5 min. The vials with the saliva samples were carried in a vaccine carrier with a freezing mixture to the laboratory, where samples' analysis was done on the same day.

Microbial analysis

The microbial analysis was conducted at the Department of Oral Pathology, College of Dental Sciences, Davangere. The saliva sample was homogenized manually using a stirrer. A 100 μl of saliva was diluted with 1 ml of sterile peptone water to obtain 1:10 dilution of saliva. 100 μl of the diluted saliva was further added to 1 ml of sterile peptone water to obtain a dilution of 1:100. This procedure was repeated to obtain a dilution of 1:1000. This dilution of saliva was used for microbial analysis. S. mutans were cultured on Mitis Salivarius Bacitracin (MSB) agar and Lactobacillus on Rogosa SL agar. Using an inoculation loop (2 mm inner diameter), 5 μl of the 1:1000 dilution sample was streaked on MSB agar under strict aseptic conditions. The MSB agar plates were incubated for 48 h at 37°C, anaerobically, in a candle jar, and Rogosa SL agar plates were incubated aerobically for 48 h at 37°C. After 48 h of incubation, S. mutans appeared on the culture plate as small, rough, raised, and adherent blue-black colonies, and Lactobacillus appeared as small white elevated round colonies. Colonies so identified based on their morphology were counted using an electronic colony counter.

Blinding

The containers with different mouthwashes were coded as I, II, and III. The participants, the person who collected the saliva sample, and the person who did the microbial and statistical analysis were blinded regarding the material used.

Statistical analysis

The data collected by experiments were computerized and analyzed using the IBM SPSS Statistics for Windows, Version 17.0.; IBM Corp. (Armonk, NY): IBM Corp. One-way analysis of variance (ANOVA) and repeated measures ANOVA were used for multiple group comparisons followed by Tukey's post hoc for group-wise comparison. For all tests, P < 0.05 was considered statistically significant.


  Results Top


The study was conducted among 90 participants with 30 in each group. There was equal distribution of male and female participants in each group.

[Table 1] compares the S. mutans count in saliva among the three mouthwash groups at various time intervals. No significant difference in the counts was observed at baseline and 6th day. On the 12th day, the highest reduction in S. mutans count was observed following the use of TTO mouthwash, followed by CHX mouthwash. The reduction in the count was significantly higher in the TTO group on the 24th day comparable to the garlic mouthwash group, followed by the CHX mouthwash group.
Table 1: Streptococcus mutans counts in saliva amongst the three mouthwash groups at different time intervals (expressed as 103 CFU/ml)

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[Table 2] compares the S. mutans counts in saliva at different time intervals in each mouthwash group. It is observed that there was a significant decrease in colony-forming unit (CFU) counts following the 1-week use of mouthwash in each group (12th day). All three types of mouthwash demonstrated a residual effect on the 24th day of sample collection.
Table 2: Mean colony counts of Streptococcus mutans in saliva at different time intervals following the use of the different mouthwashes. (expressed as 103 CFU/ml of saliva)

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[Table 3] compares the Lactobacillus counts in saliva among the three mouthwash groups at various time intervals. No significant difference in the counts was observed at baseline and 6th day. On the 12th day, the highest reduction in S. mutans count was observed following the use of TTO mouthwash, followed by CHX mouthwash. On the 18th and 24th days of saliva collection, no difference was noted in the CFU counts in any of the three mouthwash groups.
Table 3: Lactobacillus counts in saliva amongst the three mouthwash groups at different time intervals (expressed as 103 CFU/ml)

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[Table 4] compares the Lactobacillus counts in saliva at different time intervals in each mouthwash group. It is observed that there was a significant decrease in CFU counts following the 1-week use of mouthwash in each group (12th day). All three types of mouthwash demonstrated a residual effect on the 24th day of sample collection.
Table 4: Mean colony counts of Latobacillus in saliva at different time intervals following the use of the different mouthwashes. (expressed as 103 CFU/ml of saliva)

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  Discussion Top


Dental caries can be traced back to be as old as civilization with its evidence seen even in skeletal remnants of prehistoric humans. Despite improvement in the overall health status of the people in the industrialized countries, the prevalence of dental caries in school-aged children is up to 90% and the majority of adults are also affected.[1] Dental caries is one of the most common and costly diseases in the world, and although rarely life threatening, it is a major problem for health service providers.[15]

The agent that has shown the most positive antibacterial results to date is CHX, a digluconate with pronounced antiseptic properties. The literature available regarding the use of CHX for plaque control is immense and there is abundant proof of the agent's efficacy on plaque. Thus, CHX has deservedly earned its eponym as the gold standard.[16] However, the incidence of side effects of CHX mouthwash such as undesirable tooth discoloration; unpleasant taste, dryness and burning sensation in the mouth, discourage the patients to use this mouthwash[17] and this in turn has motivated the development of other natural antimicrobial agents to be used as active ingredients in the mouthwashes.

There is growing interest throughout the oral health-care profession in therapeutic agents that complement and enhance the mechanical removal of biofilms in the oral cavity.[11] Although using antimicrobial mouthrinse regularly requires a conscious participation and involvement of an individual, it does not require special dexterity or competence nor it is difficult to use. Mouthrinse formulations are generally much simpler than dentifrices, and compatibility problems are not as large an issue as they are with dentifrice products.[18] These combined properties make mouthrinses efficient for delivery of therapeutics with a particular focus on the delivery of ingredients between teeth.

An in vitro study was done before the in vivo study to assess the antimicrobial activity and determine the minimal inhibitory concentration. The antimicrobial activity of GE and TTO observed against S. mutans and L. acidophilus is in accordance with other in vitro studies.[13],[14],[19],[20],[21],[22]

Garlic antimicrobial activities have been recognized for centuries for many of their therapeutic properties, first mentioned in 1500 BC in an Egyptian recipe named Papyrus Ebers.[13] Garlic (Allium sativum) is one of the most extensively researched medicinal plants and its typical odor and antibacterial activity depends on allicin produced by enzymatic activity of alliinase on alliin after crushing or cutting garlic clove. Allicin and other thiosulfinates are believed to be responsible for the range of therapeutic effects reported for garlic.[19]

TTO is derived from the paper bark tea tree, which belongs to the family Myrtaceae; it belongs to two genera, Leptospermum and Melaleuca. It has been used for centuries by the Australian Aborigines for the treatment of bruises, insect bites, and skin infections. The main active ingredients of TTO are 1,8-cineole and terpinen-4-ol. Terpenes are volatile, aromatic hydrocarbons, and may be considered polymers of isoprene. The antimicrobial and anti-inflammatory activity of Melaleuca oil has been primarily attributed to terpinen-4-ol. The antimicrobial activity of Melaleuca may be mediated by interaction with and disruption of bacterial plasma membranes.[14],[20],[23]

In the present study, 2.5% GE mouthwash, 0.2% TTO mouthwash, and 0.12% CHX mouthwash were found to be effective in reducing the salivary S. mutans and Lactobacillus counts to a statistically significant level (P < 0.001) on the 12th day after the intervention with the mouthwash. It was also observed that there was a significant (P value < 0.001) reduction in the salivary S. mutans and Lactobacillus counts on the 18th (first follow-up) and 24th (second follow-up) day after the mouthwash was discontinued. The results of the present study are in accordance with the studies conducted by Groppo et al. in Brazil[13] with respect to the reduction in the number of salivary S. mutans counts by GE, TTO, and CHX mouthwashes, respectively, observed after the intervention and in the consecutive 2 week (4th and 5th week) follow-up period.

In a study conducted by Prabhakar et al.[24] in India, garlic and TTO mouthwashes demonstrated antimicrobial activity against S. mutans and Lactobacillus. A reduction in the number of these microorganisms was observed following the intervention with the mouthwash and in the consecutive week after the mouthwash was discontinued, the results of which are consistent with the present study. However, in the study by Prabhakar et al., the baseline samples were collected 30 min after brushing and the participants were subjected to mouth rinsing with the test solution half an hour later and saliva samples (1/2' hour sample) were collected and the age group of the participants was in between 9 and 11 years. The participants used the mouthwash twice daily for 7 days, whereas in the present study, the participants used the mouthwash once daily and still a reduction in the salivary S. mutans and Lactobacillus counts was observed. This suggests that rinsing once daily would be sufficient in reducing the salivary microbial counts.

Hence, with the observations of the this study, it can be stated that the discovery of bioactive natural products that can reduce the oral bacteria can lead in the development of new pharmaceuticals that addresses the unmet therapeutic needs in the treatment of various dental diseases in a cost-effective manner and can be used as alternatives to allopathic mouthwashes. The limitations of this study include the limited sample size and focus only on two important cariogenic microorganisms. Furthermore, the long-term effects of using the formulations need to be assessed. The study is based on the salivary microbial counts.

Medicine that is cleaner, less polluting, more renewable, safer, and more accessible to people is essential to the needs of our growing population. Thus, sustainable medicine is good for people and the environment, offering a new set of ethical values and principles for our crippled medical system[25] and hence great efforts must be put in the direction of evidence research for discovery of such medicines.


  Conclusion Top


All of the three types of mouthwashes used in the study, namely, GE (2.5%), TTO (0.2%), and CHX (0.12%), demonstrated a significant antibacterial activity against salivary S. mutans and Lactobacillus, when used in undiluted form for 1 min at a volume of 10 ml, once daily after the last tooth brushing of the day for 1 week. The antibacterial activity was evident by the reduction in the number of the respective microorganisms after the intervention when compared with the baseline counts. TTO mouthwash demonstrated the maximum antibacterial activity followed by CHX mouthwash and GE mouthwash. All the three types of mouthwashes demonstrated residual effects after the mouthwash was discontinued, over a period of 2 weeks.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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  [Table 1], [Table 2], [Table 3], [Table 4]



 

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