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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 8  |  Issue : 4  |  Page : 375-382

Comparison of the efficacy of homemade herbal disinfectants with chlorhexidine for decontamination of toothbrush: a randomized controlled trial


1 Department of Public Health Dentistry, Babu Banarasi Das College of Dental Sciences, Lucknow 227015, Uttar Pradesh, India
2 Microbiology, Babu Banarasi Das College of Dental Sciences, Lucknow 227015, Uttar Pradesh, India

Date of Submission16-Jul-2021
Date of Acceptance25-Oct-2021
Date of Web Publication22-Dec-2021

Correspondence Address:
Dr. Ankita Singh
Department of Public Health Dentistry, Babu Banarasi Das College of Dental Sciences, Lucknow 227015, Uttar Pradesh.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mgmj.mgmj_52_21

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  Abstract 

Introduction: A toothbrush is the most common oral hygiene aid used, but maintaining and storing the toothbrush hygienically is commonly neglected. In India, people are not aware of the contamination of toothbrushes. Contaminated toothbrushes can act as a vector for the transmission or reinfection of various bacteria, viruses, and fungi. The herbs, which have pharmaceutical properties, can be used as an alternative. The study aimed to compare the antimicrobial efficacy of 3% neem and 3% garlic on Streptococcus mutans and to compare with 0.2% chlorhexidine mouthwash as toothbrush decontaminants. Materials and Methods: A triple-blinded randomized controlled trial was done on 120 subjects. The subjects were divided into four groups: Group I (distilled water), i.e., control, three study groups having 3% neem (Group II), 3% garlic extract (Group III), and 0.2% chlorhexidine gluconate (Group IV). Subjects were provided toothbrushes and toothpaste for both baseline and intervention phases. The toothbrushes were collected after 14 and 28 days for microbial analysis in both phases. The data were analyzed and compared using appropriate t-test and analysis of variance. The level of significance was set at P < 0.05. Results: Garlic (Group III) was most effective at 98%, followed by chlorhexidine (Group IV) 96% and neem (Group II) 88% in reducing the level of S. mutans. Distilled water (Group I) showed only a 3% reduction. Conclusion: Neem and garlic proved to be as effective as chlorhexidine, and these herbal products can be used as an alternative to chlorhexidine as disinfectants for toothbrushes. These are the common ingredients that can be easily found in Indian households. It is herbal, cost-effective, and has no side effects so it can be easily used by every individual as a toothbrush decontaminant.

Keywords: Blood agar, chlorhexidine, garlic, neem, Streptococcus mutans, toothbrush


How to cite this article:
Singh A, Palshikar A, Chatterji T. Comparison of the efficacy of homemade herbal disinfectants with chlorhexidine for decontamination of toothbrush: a randomized controlled trial. MGM J Med Sci 2021;8:375-82

How to cite this URL:
Singh A, Palshikar A, Chatterji T. Comparison of the efficacy of homemade herbal disinfectants with chlorhexidine for decontamination of toothbrush: a randomized controlled trial. MGM J Med Sci [serial online] 2021 [cited 2022 Jan 18];8:375-82. Available from: http://www.mgmjms.com/text.asp?2021/8/4/375/333320



The article has been transcribed out of a dissertation submitted to Babu Banrasi Das College of Dental Sciences, Lucknow, U.P., India for the award of MDS (Master of Dental Surgery) Degree. It is certified that neither the dissertation has been published in any format anywhere nor it will be published in the future. Permission has been obtained from the management of the college for the publication of the manuscript in the proposed journal.


  Introduction Top


Oral health is an integral part of general health. It, directly and indirectly, reflects the overall well-being of an individual. Poor oral health can affect the course and pathogenesis of several systemic diseases such as cardiovascular disease, bacterial pneumonia, diabetes mellitus, and low birth weight. Thus, maintaining good oral hygiene becomes very important.[1]

The article has been transcribed out of a dissertation submitted to Babu Banrasi Das College of Dental Sciences, Lucknow, Uttar Pradesh, India for the award of MDS (Master of Dental Surgery) Degree. It is certified that neither the dissertation has been published in any format anywhere nor it will be published in the future. Permission has been obtained from the management of the college for the publication of the manuscript in the proposed journal.

Even though there are different oral hygiene aids available in the market, none of them gives complete protection. The most commonly used aid for maintaining oral hygiene is a toothbrush, but maintaining and storing the toothbrushes hygienically is commonly neglected.[2] In India, people are not aware of the contamination of toothbrushes.

A newly manufactured toothbrush is free of microorganisms. But after a single use, they may become contaminated by a wide array of microorganisms present both in the oral cavity and in the external environment.[3] Microorganisms can remain viable on toothbrush bristles for periods ranging from 24 h to 7 days.[4] Contaminated toothbrushes can act as a vector for the transmission or reinfection of various bacteria, viruses, and fungi.[5]

In the majority of Indian households, the toothbrushes of family members are stored in the same container. The routine use of contaminated toothbrushes might lead to disseminate microorganisms within the oral cavity of the same person or between different individuals. A method to reduce contamination of toothbrushes might be a helpful means to avoid infection and cross-infection risks among individuals. So there is a need for disinfection methods, which are rapidly effective, cost-effective, non-toxic, and can be easily implemented.[6]

With time, there is an increase in the incidence of drug resistance in the prevalent pathogens and an associated risk with chemotherapeutic agents, thus it is essential to find an alternative to existing drugs.[7] The herbs, which have known pharmaceutical properties, can be the best source of these alternative drugs.[7] In recent years, the antimicrobial properties of medicinal plants are being increasingly reported from different parts of the world.[8] Garlic (Allium sativum) is an essential part of cuisine and is used in many parts of the country. It is native to central Asia, and it has been used in native medicine from ancient times for the treatment of various diseases.[9]

Neem is employed in traditional medicine as a source of therapeutic agents in the Indian culture and grows well in tropical countries.[10] Every part of the tree has been used as traditional medicine for a household remedy against various diseases in India. This tree is also regarded as a “village dispensary” in India.[11]

Chlorhexidine gluconate (CHX) is the biguanide compound having topical antibacterial activity. It is considered as a “gold standard,” but it is associated with a few side effects such as discoloration of teeth, irritation of mucosa, altered taste sensation, swelling of the parotid, and increased supragingival calculus formation.[12]

There is limited information available comparing the potency of garlic, neem, and chlorhexidine gluconate as toothbrush disinfectants. Hence, the present investigation was designed to study the antimicrobial efficacy of 3% neem and 3% garlic on Streptococcus mutans and compare them with 0.2% chlorhexidine as toothbrush disinfectants. Hence, the null hypothesis of the study was that there is no difference in the antibacterial effect of neem, garlic, and chlorhexidine as toothbrush disinfectants.


  Materials and methods Top


This study was a single-center, multiple arm, triple-blinded, parallel in-vitro comparative experimental trial. The study population consisted of 18–25-year-old students of Babu Banarasi Das University. The subjects aged between 18-25 years, both male and female, had at least three single-rooted and two multi-rooted functional teeth per quadrant (excluding third molar), and decayed, missing, and filled teeth (DMFT) score of <3 was included in the study.

Those subjects using antibiotics or antiseptic mouthwashes for at least 3 months before the study, undergoing any dental treatment, with orthodontic or with an extensive intraoral prosthesis, medically compromised patients (e.g., in diabetic patients), subjects using neem, garlic, and chlorhexidine in any form as an oral hygiene aid were excluded from the study.

Ethical clearance was obtained from the Institutional Ethical Committee of Babu Banarasi Das College of Dental Sciences, Babu Banarasi Das University, Lucknow, Uttar Pradesh (IEC 16) BBDCODS/10/2020. The purpose of the study was explained, and informed written consent was obtained from all the study participants.

The sample size was calculated with the help of G-Power analysis software, keeping the power of the study at 80%, alpha error at 5%, effect size at 0.25,[6] beta error 20%, and confidence interval at 95%. A sample size of 28 was obtained for each group. The final sample size was decided to be 30 in each group. There are four groups in the study, so a total of 120 healthy volunteers were involved in the study.

The pilot was conducted on the toothbrush of 20 participants to check for the feasibility of the study, and they were not included in the final study. Before the study, the investigator (1) was trained and calibrated in the department of Public Health Dentistry. Type III clinical examination was done. A study-specific proforma was used to record demographic details and medical and dental history of each participant.

The caries experience was calculated using the DMFT index by Klein, Palmer, and Knutson (WHO modified 1997), and participants who had a DMFT score of <3 were included in the study. Out of the 325 students screened, 152 students fulfilled the inclusion criteria. From the eligible 152 study participants, the final required 120 subjects were randomly selected.

Trial design

The allocation ratio is 1:1:1:1.

Allocation sequence: Participants were allocated to the group by the envelope method.

Type of randomization is simple random method.

Unit of randomization: This includes DMFT scores <3.

Blinding

It was a triple-blinded clinical trial. The study subjects remained blind to further procedures after the collection of toothbrushes. The subjects, microbiologists, and statisticians remained blind regarding the disinfectant solutions the toothbrush was kept in.

Implementation

Participants were assigned to the group and all the records were maintained by the organizing clerk.

Preparation of solutions

3% Neem

About 100 g of neem leaves was blended in a mixing jar, and the extract was filtered first through a muslin cloth and then was filtered into a jar using a Whatman’s No. 1 filter paper. To make it 3%, 3 mL of neem leaves extract was mixed with 100 mL of distilled water. The solution was prepared freshly on the day of the experiment.

3% Garlic

For preparing 3% garlic extract, 100 g of fresh garlic obtained from the local market was peeled and blended in a mixer and the extract was first filtered by using a muslin cloth and then into another jar using a Whatman’s No. 1 filter paper. To make it 3%, 3 mL of garlic extract was mixed with 100 mL of distilled water. Garlic solution was prepared freshly on the day of the experiment.

0.2% Chlorhexidine

Chlorhexidine 0.2% (Hexidine, ICPA Health Products Ltd, India) was used, which was readily available in the market.

Five new toothbrushes (Oral B Shine) were freshly opened from the packets and were subjected to microbial analysis to check for S. mutans colonies on bristles. This was done to ensure that the new toothbrushes were free from contamination before their use by study participants.

The selected 120 subjects were divided into four groups (30 × 4):

Group I—distilled water (control);

Group II—3% neem extract;

Group III—3% of garlic extract;

Group IV—0.2% chlorhexidine gluconate.

All the subjects were provided with pre-color-coded toothbrushes (Oral B Shine-soft) and toothpaste (Colgate) during the entire study procedure. All of them were instructed to brush twice daily using a horizontal scrub method for 2–3 min and to rinse the toothbrushes under running tap water for 20 s after brushing. They were instructed to keep their toothbrushes in such a manner that the head of the brush with the bristles should face outside and be left open for drying. Subjects were periodically reminded to follow the instructions via mobile phone. The study was conducted in two phases.

First phase

In the first phase, all participants were provided with a new set of pre-color-coded toothbrushes and toothpaste. The toothbrushes were collected after 14 days. These toothbrushes were then stored in separate disposable sterile sealed plastic pouches and then transferred to the laboratory for microbial analysis. This was done to obtain the baseline analysis.

Second phase

In the second stage, all 120 subjects were again provided with a new set of toothbrushes and toothpaste. The toothbrushes were collected after 28 days. The collected toothbrushes were again transferred to the laboratory for microbial analysis.

Intervention

None. All the participants reverted after 14 and 28 days.

Microbial analysis

The bristles from one tuft from each toothbrush of Group I participants have separated aseptically and were aseptically introduced directly into test tubes containing 5 mL of distilled water (control). Similarly, the bristles from the tufts of toothbrushes were collected from subjects in Group II, Group III, and Group IV and were immersed in the test tubes containing 5 mL of 3% neem, 3% of garlic, and 0.2% of chlorhexidine mouthwash for 12 h.

The samples were then vortexed for 15 s to dislodge the bacteria from the bristles to the media using a cyclomixer, and the solution was inoculated using a platinum inoculation loop. An aliquot of 10 μL of the vortexed sample was transferred to a blood agar medium by the spread plate method. The plate was incubated for 24–48 h at 37°C in an incubator. After 24 h, the colonies were counted through the manual method and it was expressed as colony-forming units (CFU)/mL. After inoculation and incubation, total colony count was calculated. The total colony count was calculated using the formulae: colony-forming unit (CFU)/mL=total colony count × 1000. Further, the colony count was subjected to statistical analyses to observe the efficacy best obtained among the three decontaminants. The laboratory procedure was similar for both stages.

Data analysis

Data were entered in Microsoft Excel, and the analysis was performed using SPSS software version 20 (IBM Corporation). The t-test was used for comparing the baseline and test values and analysis of variance (ANOVA) was used for multiple group comparisons. In all the above tests, P < 0.05 was considered statistically significant.


  Results Top


There were a total of 120 subjects involved in this study. It consists of 78 males (65%) and 42 females (35%). All the subjects were present from the beginning to the end of the study, and there was no loss to follow-up. The mean level of contamination of toothbrush by S. mutans (baseline level) in each group is as follows: Group I had 14.56 ± 0.76, Group II had 14.82 ±0.54, Group III had 15.06 ± 0.46, and Group IV had 14.68 ± 0.36, as shown in [Table 1]. After decontamination, the mean level of contamination of toothbrush by S. mutans in each group is as follows: Group I had 14.12 ± 1.58, Group II had 1.82 ±2.4, Group III had 0.34 ± 2.2, and Group IV had 0.58 ± 2.5, as shown in [Table 2]. The mean difference in the level of S. mutans from baseline and intervention groups is shown in [Table 3].
Table 1: Mean CFU of S. mutans in different groups (baseline)

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Table 2: Mean CFU of S. mutans in different groups after decontamination

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Table 3: Mean difference in the level of S. mutans from baseline and intervention groups

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On comparing the level of S. mutans in Group I (distilled water) before and after decontamination of toothbrush bristles, it was found to be non-significant (P > 0.05). On comparing the level of S. mutans in Group II (neem), Group III (garlic), and Group IV (chlorhexidine) before and after decontamination of toothbrush bristles, it was found to be statistically significant (P < 0.05).

The percentage of reduction done by different solutions in the level of S. mutans is shown in [Figure 1]. Group I (distilled water) showed only 3% reduction, Group II (neem) showed a reduction of 88%, Group III (garlic) showed a maximum reduction of 98%, and Group IV (chlorhexidine) showed a reduction of 96% in the level of S. mutans. The highest reduction in S. mutans was seen in the garlic group, and distilled water group showed the least reduction. When comparing the mean percentage reduction in S. mutans, CFUs between the control group (I) and each experimental group (II, III, and IV), the differences noted were all statistically significant (P < 0.05), as shown in [Table 4].
Figure 1: Mean percentage reduction of S. mutans CFUs in four study groups from baseline

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Table 4: Comparison of the mean percentage reductions in S. mutans count between different groups after decontamination

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When the mean percentage reduction produced by experimental Groups II and III were compared with experimental Group IV, the differences noted were not statistically significant (P > 0.05), as shown in [Table 4].

When the mean percentage reduction produced by experimental Group II was compared with experimental Group III, the differences noted were not statistically significant (P > 0.05), as shown in [Table 4].


  Discussion Top


The main etiological agent for the initiation of dental caries is S. mutans. Cobb[13] in 1920 stated that toothbrush is a major cause of repeated infection in the mouth. Toothbrush and toothpaste were used in this study as an oral hygiene aid as it is the most commonly used aid and it is the most effective and safest device for maintaining oral hygiene. In Indian household, toothbrushes of family members are kept in the same containers, so there is a further risk of cross-infection; microorganisms can get transmitted from one toothbrush to others if they are kept nearby or even on sharing toothbrushes.[14]

In routine households, for preventing contamination of toothbrush, rinsing and drying is usually adopted. Glass[15] in his study states that the air-drying method for decontamination of toothbrushes is incomplete. So we need an alternative method that should be effective, economical, and does not have any side effects. American Dental Association (ADA) in 1996 recommended that for every 3 months everyone should discard their old toothbrush.[16] Glass and Jensen[17] and Denny[18] had advised that patients undergoing chemotherapy should change their toothbrushes after every 3 days, and those patients who had to undergo major surgeries should change their toothbrush every day, and those who are sick should change their toothbrushes at the beginning of their illness, when they first feel better and when they are completely well.

Many people change their toothbrushes on the flaring of bristles as there is no awareness regarding decontamination of toothbrushes. Despite millions of toothbrushes sold worldwide, there is very little public awareness that the bristles get contaminated with use.

Each subject was provided with an individual toothbrush and toothpaste to maintain uniformity in the study. For maintaining the sterility control, five new toothbrushes that were freshly removed from the packets were subjected to microbial analysis, and none of the unused toothbrushes showed any colonies of S. mutans. This suggests that the origin of these microorganisms is from the oral cavity. All the study subjects involved in this study had a DMFT score of less than 3 as WHO classifies less than 3 DMFT to be ideal for 18-year-old individuals.[19]

In our study, the toothbrushes were collected from study participants after 14 days. Many studies have chosen different study intervals such as 48 h,[20] 5 days,[21],[22] 7 days,[23],[24] and 3 months.[25] In our study, we have taken 14 days because a study conducted by Sogi et al.[20],[22] states that maximum microbial contamination was found in toothbrushes after 14 and 28 days. The toothbrushes were immersed in disinfecting solutions for 12 h, which was similar to other studies.[24],[25] Twice-a-day tooth brushing was used in this study. Hence, a soaking period of 12 h of toothbrushes in disinfectant solutions was employed. However, other studies have used different soaking times of 20 h,[21] 24 h,[26] or 20 min,[20] and all had employed once-a-day brushing.

Neem (Azadirachta indica) possesses various therapeutic properties and is widely available in most rural and urban areas of our country. In our study, 3% neem was found to be an effective antimicrobial solution against S. mutans on toothbrush bristles and showed an 88% reduction. This was similar to the studies conducted by Anand et al.,[6] Balappanavar et al.,[25] and Bhat et al.,[2] where it showed 88%, 86%, and 86% reductions, respectively. This reduction could be due to the presence of polyphenolic tannins which are present in the extract. It gets bound to the surface-associated bacterial proteins and results in bacterial aggregation and also loss of glucosyltransferase activity. This bacterial aggregate reduces the count of S. mutans. Aarati et al.[27] conducted a study and stated that both aqueous and alcoholic extracts of neem have significant antibacterial activity against S. mutans.

Garlic showed a maximum reduction of 98% in the S. mutans count in our study, and it was similar to the study conducted by Anand et al.[6] and Chandrdas et al.,[28] which shows 96% and 100% reductions, respectively. This antibacterial activity of garlic is due to the presence of allicin. Allicin possesses strong anti-S. mutans activity which was reported by Nikolić and co-workers.[29]

On doing the literature search on 3% garlic, not many studies were found. Fani et al. conducted a study on mouthwash containing garlic and stated that garlic mouthwashes can be used for the prevention of dental caries.[30] Garlic extracts of 2.5%[31],[32] and 10%[33] were used as a mouthwash in studies, which also showed a significant reduction in salivary S. mutans counts. Despite having antimicrobial properties, garlic has side effects like unpleasant taste, halitosis, and nausea.[34]

Chlorhexidine is a cationic agent and it exhibits broad-spectrum antimicrobial effects. It has both bacteriostatic (inhibits bacterial growth) and bactericidal (kills bacteria) mechanisms of action, depending on its concentration.[3] It is considered as a benchmark control in various studies. In our study, chlorhexidine showed a 96% reduction in the S. mutans count. It kills bacteria by disrupting the cell membrane.[35]

In studies conducted by Bhat et al.[2],[26] and Nanjunda-Swamy et al.,[36] chlorhexidine produced 100% reduction of the S. mutans count, and a study conducted by Anand et al.[6] showed 92% reduction. The study conducted by Sogi et al.[20] found that chlorhexidine produces 88% of bacterial reduction in toothbrushes after 14 days. But the result of the present study was not in concurrence with the study reported by Balappanavar et al.,[25] Bhat et al.,[2] and Chandrdas et al.,[28] in which the percentages of reduction in S. mutans count were only 64%, 65%, and 78.3%, respectively, which were less than the present study.

As per the results of the present study, neem and garlic have been proved to be as effective disinfectants as chlorhexidine for toothbrushes. Kudva et al.[37] reported that garlic and chlorhexidine are effective against S. mutans. Fani et al.[30] showed that the inhibitory activity of garlic on S. mutans was comparable with chlorhexidine. In the present investigation, garlic showed the maximum reduction in the S. mutans count. It produced 98% reduction with garlic followed by chlorhexidine 96% and neem 88%. Distilled water showed only a 3% reduction. When comparing the mean difference and also the mean percentage reduction obtained in S. mutans colony count between the groups, only the comparison with distilled water showed a statistically significant difference. But when comparing the mean difference or mean percentage reduction in CFUs of S. mutans in garlic and neem, with chlorhexidine, the differences were not statistically significant from each other. So the present investigation showed that immersion of toothbrush for 12 h in 3% neem and 3% garlic was as efficacious as 0.2% chlorhexidine for disinfection. So neem and garlic can be used as toothbrush disinfectants as chlorhexidine.


  Limitations Top


The present study considered only S. mutans for evaluation of the efficacy of disinfecting agents and did not take into account all other microorganisms present on toothbrushes. It also did not assess the acceptability of toothbrushes dipped in these solutions by the study subjects as antimicrobial agents are known to have unpleasant smells, bitter and altered taste, especially with garlic. It also did not test whether the properties of toothbrush bristles can be affected by the herbal solutions they are dipped in.


  Conclusion Top


Based on the results of the study, it can be concluded that neem and garlic are equally efficacious as chlorhexidine and these herbal products can be used as potent alternatives to chlorhexidine as disinfectants for toothbrushes. It is seen that toothbrushes can act as a carrier of infection in the oral cavity. Thus, every individual must disinfect his/her brush at regular intervals to maintain good oral hygiene.

Neem and garlic are the common ingredients that can be easily found in Indian society and it is herbal, cost-effective, and having no side-effect so it can be easily used by every individual as toothbrush decontaminants.

Further clinical research is required to broaden our understanding of various antimicrobial agents, especially the natural/herbal agents for the prevention of dental caries. Comparison with green tea extract and vinegar can also be taken into consideration for the future. Also, further research is required to analyze various antimicrobial agents with different concentrations and different soaking times.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Ethical consideration

Ethical clearance was obtained from the Institutional Ethics Committee of Babu Banarasi Das College of Dental Sciences, Lucknow, Uttar Pradesh, India vide their letter no. (IEC 16) BBDCODS/10/2020. It is submitted that the purpose of the study was explained and informed written consent was obtained from all the study participants.



 
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