Antibacterial efficacy of the combination of triphala and chlorhexidine mouthwashes on salivary Streptococcus mutans of orthodontic patients: A randomized clinical trial

Amir Hooman Sadr Haghighi; Raana Sarvari; Hosein Eslami; Elaheh Fakhri; Roghayeh Nouri; Arezou Karimzadeh Sureshjani

doi:10.4103/jioh.jioh_64_22

ORIGINAL RESEARCH

Year : 2022 | Volume : 14 | Issue : 6 | Page : 636-641

Antibacterial efficacy of the combination of triphala and chlorhexidine mouthwashes on salivary Streptococcus mutans of orthodontic patients: A randomized clinical trial

Amir Hooman Sadr Haghighi¹, Raana Sarvari², Hosein Eslami³, Elaheh Fakhri⁴, Roghayeh Nouri⁵, Arezou Karimzadeh Sureshjani¹
¹ Department of Orthodontics, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz, Iran
² Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
³ Department of Oral Medicine, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz, Iran
⁴ Dental and Periodontal Research Center, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz, Iran
⁵ Department of Microbiology, Tabriz University of Medical Sciences, Tabriz, Iran

Date of Submission	19-Mar-2022
Date of Acceptance	17-Oct-2022
Date of Web Publication	30-Dec-2022

Correspondence Address:
Dr. Arezou Karimzadeh Sureshjani
Department of Orthodontics, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz
Iran

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jioh.jioh_64_22

Abstract

Aim: The aim of this study was to compare and evaluate the antibacterial efficacy of triphala mouthwash and its combination with 0.2% chlorhexidine against salivary Streptococcus mutans of orthodontic patients. Materials and Methods: A total of 54 patients under fixed orthodontic therapy were included in this randomized clinical trial and divided into three groups of 18 each. The groups were requested to use one of three mouthwashes two times a day for 10 days: chlorhexidine 0.2%, triphala 25%, and the combination of chlorhexidine and triphala (1:1). Saliva samples were collected at baseline (day 0) and after 10 days. Ultimately, the samples were diluted and cultured. After incubation, the formed colonies were counted and the CFU/mL was calculated. The results were analyzed statistically with one-way analysis of variance and t test. Results: All mouthwashes decreased the bacterial count from baseline to the end of 10 days (P < 0.05). Although triphala had the least antimicrobial effect, no significant difference was observed between the chlorhexidine and the combined mouthwash group (P = 0.251). Conclusion: It was concluded that the combination of chlorhexidine and triphala reduces the side effects of chlorhexidine while presenting an acceptable antibacterial efficacy.

Keywords: Chlorhexidine, Mouthwashes, Oral Hygiene, Plant Extracts

How to cite this article:
Sadr Haghighi AH, Sarvari R, Eslami H, Fakhri E, Nouri R, Karimzadeh Sureshjani A. Antibacterial efficacy of the combination of triphala and chlorhexidine mouthwashes on salivary Streptococcus mutans of orthodontic patients: A randomized clinical trial. J Int Oral Health 2022;14:636-41

How to cite this URL:
Sadr Haghighi AH, Sarvari R, Eslami H, Fakhri E, Nouri R, Karimzadeh Sureshjani A. Antibacterial efficacy of the combination of triphala and chlorhexidine mouthwashes on salivary Streptococcus mutans of orthodontic patients: A randomized clinical trial. J Int Oral Health [serial online] 2022 [cited 2023 Nov 30];14:636-41. Available from: https://www.jioh.org/text.asp?2022/14/6/636/366435

Introduction

Despite the various improvements in orthodontics recently, enamel decalcification, white spot lesions and tooth caries are the complications with fixed orthodontic treatment.^[1] Significant cavitations and tooth loss can cause esthetic problems and depreciate the quality of the treatment results. Orthodontic appliances increase bacterial attachment and interfere with the mechanical removal of plaque.^[2] Therefore, the lack of adequate oral hygiene predisposes patients to increased risk of tooth caries.

There is high evidence regarding the association between orthodontic appliances and alteration of the oral microbiota.^[3] Previous studies highlighted the increase of cariogenic bacteria including Streptococcus mutans and Lactobacillus spp.^[4],[5] Although changes in the oral microbial system occur during the application of all orthodontic devices, rapid modifications are detectable by using fixed appliances.^[6]

It has been reported that maintaining adequate oral hygiene by proper tooth brushing and using fluoridated dentifrice can moderate the microbiome. Nevertheless, the mentioned oral hygiene regimen is not considerably effective in preventing the development of carious lesions in orthodontic patients.^[7] Thus, orthodontists emphasize the importance of using additional chemotherapeutic agents such as antimicrobial mouth rinse. These agents affect bacterial cell viability or interfere with bacteria adhesion.^[8]

Chlorhexidine is the most common mouthwash prescribed by orthodontists.^[8] This product is widely used for decontamination of oral mucosa and tooth structures due to its magnificent antibacterial effects against oral bacteria. Hence, chlorhexidine is considered the gold standard of the chemical treatment of these bacteria.^[9] There is strong evidence regarding the anti-plaque activity of chlorhexidine in orthodontic patients.^[10],[11] Clinical studies showed a significant reduction in S. mutans level in orthodontic patients, 1 week after using chlorhexidine mouthwash.^[10] Nevertheless, the adverse effects of chlorhexidine including unpleasant taste, enamel color alteration and staining stimulated the search for alternatives.

Triphala is a well-known polyherbal compound used in Indian traditional Ayurvedic medicine. It contains equal amounts of fruits of three plants, including Emblica officinalis, Terminalia chebula, and T. bellirica, forming a fine powder.^[12] Several studies introduced triphala as an efficient antiplaque, antibacterial and anti-inflammatory mouthwash.^{[13],[14],[15]} It has been frequently reported that application of triphala in patients with gingivitis or periodontal disease reduces the plaque accumulation, gingival inflammation and bleeding equivalent to chlorhexidine.^[15] Furthermore, triphala has been used as a chemotherapeutic agent against salivary S. mutans and dental caries.^{[13],[14],[16],[17]}

Considering the various side effects of chlorhexidine, and the increasing concern for emergence of antibiotic-resistant bacterial species, application of traditional medicine for healthcare needs seems to be a potential solution. The aim of this study was to compare the antibacterial efficacy of triphala mouthwash and its combination with 0.2% chlorhexidine against salivary S. mutans of orthodontic patients. The objective was to indicate whether the combined mouthwash could be an alternative to chlorhexidine alone.

Materials and Methods

Setting and design

The design of this double-blinded randomized controlled clinical trial study was approved by the research ethics committee of Tabriz University of Medical Sciences (IR.TBZMED.REC.1400.407). An informed consent was obtained from all study participants. This study was performed at the Department of Orthodontics, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz, Iran during one year.

Sampling

Considering the power of 90% and ɑ of 0.05, the sample size was calculated based on the data from a study by Padiyar et al.^[18] In this study, 54 orthodontic patients participated. The inclusion criteria of the study were age range of 20–60 years and systematically healthy individuals. The exclusion criteria of the study were antibiotic therapy in the last 3 months, using mouthwash for the last 3 months, established periodontitis, pregnancy, and smoking habits.

Study groups

As shown in [Figure 1], patients were divided into three groups of 18 by the block randomization method.^[19] The groups with assigned mouthwash were as follows:

Figure 1: A diagrammatic representation of methodology

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A: Chlorhexidine digluconate 0.2%

B: Triphala mouthwash 25%

C: The combination of triphala 25% and chlorhexidine digluconate 0.2% (1:1)

The participants were instructed to use the mouthwash twice a day in the morning and evening after brushing for 10 days. They were asked to use 10 mL of each mouthwash for 1 min and avoid eating and drinking until 30 min.

Preparation of triphala mouthwash

Triphala was purchased in the form of a sieved powder called churna. To prepare a 25% triphala mouthwash, 250 g of pure triphala churna were dissolved in 1 L of distilled water. Then the solution was brought to a boil for 10 min, then cooled and filtered.^[20]

To prepare the combined mouthwash, chlorhexidine digluconate 0.2% and triphala 25% were mixed by a ratio of 1:1.

Microbiological evaluation

To determine the number of S. mutans colony count, 2.0 mL of non-stimulated salivary samples were taken from patients at two time points, one at the baseline and one after using the mouthwash for 10 days. The patients had refrained from eating or drinking for at least 1 h before saliva collection. Samples were collected and restored in sterilized tubes and transferred immediately to the Microbiology Laboratory of Medicine Faculty of Tabriz University of Medical Sciences.

For better dispersion, saliva samples were vortexed for 2 min. After ten-fold serial dilution, 10 µl of each sample was plated on tryptone soya-yeast agar with 20% sucrose and 0.2 U/mL bacitracin (SB₂₀ agar, Sigma, St. Louis, Missouri). Following incubation at 37°C for 24 h in anaerobic conditions, the formed colonies were counted.^[10]

Statistical analysis

Data were presented as mean values ± standard deviation. Statistical analysis was done using SPSS 17.0 software (SPSS Inc, Chicago, Illinois). The normal distribution of data was evaluated by Kolmogorov–Smirnov and Shapiro–wilk tests. The experiments were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s test for pairwise comparison. The level of statistical significance was set at 0.05.

Results

Kolmogorov–Smirnov and Shapiro–Wilk tests revealed that data are normally distributed (P > 0.05); therefore, parametric tests were used for data analysis. Application of all mouthwashes resulted in a reduced S. mutans count significantly (P < 0.05) [Table 1]. The amounts of bacterial reduction (mean ± SD) following the application of mouthwashes for 10 days are presented in [Table 2]. The one-way ANOVA test showed a statistically significant difference between all groups (P < 0.001) [Table 3]. Pairwise comparison was done by Tukey test [Table 4]. Although the combined mouthwash showed the best results in terms of reduction of S. mutans count, there was no significant difference between the combined mouthwash and chlorhexidine (P = 0.251). Chlorhexidine resulted in better bacterial reduction compared to triphala mouthwash (P < 0.05).

Table 1: Comparison of the amount of bacterial reduction from baseline to the end of 10 days

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Table 2: Bacterial reduction (mean ± SD) following the application of mouthwashes for 10 days

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Table 3: Comparison of the amount of bacterial reduction between groups by one-way ANOVA

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Table 4: Pairwise comparison of the amount of bacterial reduction between groups by Tukey test

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Discussion

A proposed novel mouthwash, with the collaboration of an herbal extract with conventional chlorhexidine mouthwash to enrich the benefits of each one and reduce the adverse effects was the climax of this study. This study showed that addition of triphala to chlorhexidine could reduce the side effects while presenting the same antibacterial efficacy against salivary S. mutans of orthodontic patients. Furthermore, subjective observation of the participants’ attitude following the application of each mouthwash showed that patients using triphala and the combination of triphala and chlorhexidine found the mouthwashes acceptable while patients using chlorhexidine complaint regarding the color alteration and unfavorable taste. It is noteworthy that all participants were in contact with researchers during the 10 days and were constantly encouraged to use the mouthwash in spite of discomfort.

Short-term evaluations on chlorhexidine have frequently showed its various outcomes including the superior efficacy against plaque accumulation.^[21] Six-month trials reported a plaque reduction of 16%–45% and a reduction in gingivitis from 27% to 80%.^[22] Furthermore, numerous studies have shown its acceptable effect against salivary S. mutans of orthodontic patients^[10],[23] and bacterial biofilm on orthodontic brackets.^[24],[25] In agreement with the literature, following 10 days of application of chlorhexidine in this study we observed a reduction of 1.732 × 10⁵ ± 1.633 × 10⁵ CFU/mL bacteria in the salivary specimen of patients, which indicates its potential antibacterial and antibiofilm efficacy. Notwithstanding, rejection by patients was a consequence of its crucial drawbacks including tooth color alteration, unfavorable taste and irritation of oral mucosa. As mentioned by Vinod et al.,^[17] these characteristics persuade researchers to investigate for an alternative mouthwash with less inconvenience for patients in long-term use.

So far, studies have evaluated triphala as a mouth rinse or topical agent in case of gingival inflammation.^[26] Previous studies have shown that daily use of triphala mouthwash reduces the gingival index, plaque index and improves gingival inflammation.^[13] Comparison of triphala and chlorhexidine in patients with gingivitis or periodontitis showed that both are equally effective.^{[13],[26],[27],[28],[29]} The anti-inflammatory efficacy of triphala is attributed to its plenty of bioactive compounds including polyphenols such as gallic acid, tannic acid, syringic acid, chebulagic acid, chebulinic acid and epicatechin along with flavonoids and ascorbic acid.^[30] Triphala is a combination of three medicinal plants, namely Emblica officinalis, T. chebula, and T. bellirica. According to Saxena et al.,^[31] the mixture of these herbal plants presents more efficient antibacterial activity against salivary S. mutans compared to each alone.

The anti-bacterial and anti-biofilm activities of triphala suggest this product as a natural source of anti-cariogenic agents. Triphala’s antimicrobial activity is also attributed to its phenolic compounds and tannins. Results of an in vitro study showed that mixed herbal powder extracts containing triphala have a greater antimicrobial effect against cariogenic microorganisms compared to the most common oral antimicrobial agent, chlorhexidine gluconate.^[32] Clinical studies investigated the potential of triphala in various concentrations in preventing plaque accumulation and its antibacterial efficacy against cariogenic bacteria including S. mutans and lactobacillus. Triphala presents an anti-plaque efficacy similar to chlorhexidine in subjects with normal gingival health and patients with chronic gingivitis or periodontitis.^{[17],[20],[31]} Long-term clinical evaluation of triphala and chlorhexidine has shown that triphala 0.6% and chlorhexidine 0.1% prevent any noticeable increase in carious status with no significant difference among them.^[33]

Pradeep et al.^[26] showed that application of 6% triphala twice a day for two months in patients with generalized chronic gingivitis, can reduce the clinical aspects of gingivitis and the microbial count of Streptococcus species including S. mitis, S. sanguis, S. intermedius, S. oralis and Actinomyces viscosus and A. naeslundii. No significant difference was found between chlorhexidine 0.2% and triphala 6% in this study. In contrast, Padiyar et al.^[18] showed that chlorhexidine has a better effect in reducing S. mutans count in 9–12 years old children compared to triphala 6%. Considering that the first study evaluated the antimicrobial efficacy of triphala for a long term, whereas the latter study was conducted for only 15 days, it can be concluded that triphala is as effective as chlorhexidine during a longer period of application. In agreement, Vinod et al.^[17] reported that herbal mouthwash containing triphala could be effective on long-term usage. Based on the present short-term evaluation due to limitations, using triphala 25% by orthodontic patients the microbial count was improved at 10 days from baseline, although chlorhexidine 0.2% presented a better antimicrobial efficacy. It should be taken into account that in this study, evaluation was on orthodontic patients, which indicate more S. mutans bacteria in their saliva in comparison to other people.^[3] A pilot in-vitro study was performed (data not shown) to evaluate the antibacterial efficacy of triphala and chlorhexidine in order to determine the optimum concentration. According to the results, triphala did not show potential antibacterial activity against S. mutans up to 25% concentration. This controversy regarding the antibacterial efficacy of triphala in various concentrations necessitates further precise investigations.

According to Srinagesh et al.,^[20] patients showed 92% compliance with triphala. Although they reported dryness of the oral cavity in the first 5 min after application, no tooth staining or unacceptable taste was mentioned. In agreement, no complaint was received regarding triphala or the combined mouthwash in this study, whereas some patients found chlorhexidine unsatisfactory.

Consequently, triphala mouthwash has been evaluated on patients with gingivitis, periodontitis and volunteer participants with normal gingival conditions. Only one study has been performed on the effects of triphala on orthodontic patients with generalized gingivitis and reported that triphala is comparable to chlorhexidine in reducing plaque accumulation.^[34] On the whole, this study is the first one evaluating the effect of the combination of chlorhexidine and triphala on salivary S. mutans count in patients under orthodontic treatments. In addition to the high acceptance by patients, the combination of triphala and chlorhexidine resulted in synergistic microbial cell growth inhibition suggesting its administration in patients with a high risk of tooth caries. For further studies, it is recommended to use a larger sample size and assess patient satisfaction by precise objective evaluation of the participants’ attitude following the application of each mouthwash.

Conclusion

Orthodontic treatments predispose patients to develop carious lesions. Due to the side effects of chlorhexidine leading to a decrease in its acceptance by patients, attempts are being made to introduce alternative mouth rinses predominantly with natural resources. This study showed that the combination of triphala 25% and chlorhexidine could be considered an alternative therapy to chlorhexidine alone in order to oral biofilm control in patients under orthodontic treatments.

Acknowledgement

Not applicable.

Financial support and sponsorship

This study was supported by Faculty of Dentistry, Tabriz University of Medical Sciences with reference number of 66048.

Conflicts of interest

There are no conflicts of interest.

Authors contributions

All authors had contribution in study design, data collection, data annotation and manuscript preparation and final editing.

Ethical policy and institutional review board statement

This study was approved by the research ethics committee of Tabriz University of Medical Sciences with reference number of IR.TBZMED.REC.1400.407. This clinical trial has been registered in Iranian Registry of Clinical Trials with URL of https://en.irct.ir/ and a registration number of IRCT20210802052055N1.

Patient declaration of consent

This clinical trial has been approved by the research ethics committee of Tabriz University of Medical Sciences at August 2, 2021, with the reference number of IR.TBZMED.REC.1400.407. All the procedures have been performed as per the ethical guidelines laid down by the Declaration of Helsinki.

Data availability statement

Not applicable.

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