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Year : 2018  |  Volume : 22  |  Issue : 6  |  Page : 498-502  

Determining the antibacterial substantivity of Triphala mouthwash and comparing it with 0.2% chlorhexidine gluconate after a single oral rinse: A crossover clinical trial

1 Department of Periodontics, Goa Dental College and Hospital, Bambolim, Goa, India
2 Department of Microbiology, Tatyasaheb Kore Dental College and Research Centre, Kolhapur, Maharashtra, India
3 Department of Periodontics, Tatyasaheb Kore Dental College and Research Centre, Kolhapur, Maharashtra, India
4 Department of Preventive and Social Medicine, Krishna Institute of Dental Sciences, Karad, Maharashtra, India

Date of Submission17-Apr-2018
Date of Acceptance22-Jun-2018
Date of Web Publication1-Nov-2018

Correspondence Address:
Dr. Ritam Sanvala Naiktari
Department of Periodontics, Goa Dental College and Hospital, Bambolim, Goa
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jisp.jisp_265_18

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Context: Triphala has been extensively used in dentistry as a mouthwash because of its antiplaque and antigingivitis properties.
Aim: The present study is designed to determine the duration of its antibacterial substantivity after a single oral rinse and to compare it with the substantivity of 0.2% chlorhexidine gluconate (CHX). Materials and Methods: In this clinical crossover trial, unstimulated saliva from 30 individuals was collected 2 h after routine oral hygiene procedures but not rinsing (pre-sample) with randomly selected mouthwash, (10% Triphala mouthwash, 0.2% CHX, and normal saline) and 5 min after rinsing (postsample). A washout period of 1 week was kept between two rinses. The sampling was repeated after every 2 h for 12 h (post 1, post 2, post 3, post 4, post 5, and post 6) and was checked for microbial count. Statistical Analysis Used: Friedman test, Kruskal–Wallis test, and post hoc analysis were used to assess the effect of different mouthrinses on colony forming units at different times. Results: Ten percent Triphala showed statistically significant results when the antibacterial effect at post, post 1 and post 2 were compared to pre-assessment count (P < 0.05). After which the effect was at par with normal saline (P > 0.05). The results for CHX were statistically significant at all times when compared to pre-assessment count (P < 0.05) and it showed the maximum substantivity of 7 h. Conclusion: After a single rinse with no eating and drinking restrictions over the day, 10% Triphala mouthwash had an antibacterial effect for 3–4 h. It can be used three times daily for its maximum antibacterial effect.

Keywords: Antibacterial substantivity, chlorhexidine, triphala

How to cite this article:
Naiktari RS, Dharmadhikari C, Gurav AN, Kakade S. Determining the antibacterial substantivity of Triphala mouthwash and comparing it with 0.2% chlorhexidine gluconate after a single oral rinse: A crossover clinical trial. J Indian Soc Periodontol 2018;22:498-502

How to cite this URL:
Naiktari RS, Dharmadhikari C, Gurav AN, Kakade S. Determining the antibacterial substantivity of Triphala mouthwash and comparing it with 0.2% chlorhexidine gluconate after a single oral rinse: A crossover clinical trial. J Indian Soc Periodontol [serial online] 2018 [cited 2020 May 29];22:498-502. Available from:

   Introduction Top

Triphala is a polyherbal preparation, which consists of equal amounts of fruits of three plants, namely amalaki Phyllanthus emblica (Emblica officinalis) Phyllanthaceae family, Haritaki (Terminalia chebula) Combretaceae family, and Bahera (Terminalia bellirica) Combretaceae family, in a fine powder form. It contains tannins, quinones, flavones, flavonoids, flavonols phenols, glycosides, Gallic acid, and Vitamin C which are responsible for its strong antioxidant activity apart from its antimicrobial, antistress, purgative, anthelmintic, hypolipidemic, antifungal, antioxidant, antiulcer, immunomodulatory, antitumor, anti-inflammatory antihistaminic, antibacterial, antispasmodic, antiasthmatic, analgesic, and antimutagenic properties.[1],[2] It has been used in dentistry as a mouthwash because of its antiplaque and antigingivitis properties.[3],[4],[5] Several studies have compared its efficacy with the gold standard, chlorhexidine (CHX) and are proven to be equivalent to CHX with additional advantages over CHX, such as cost-effectiveness, easy availability, and with no side effects.[6],[7] Abraham et al.[8] indicated the strong inhibitory activity of Triphala against PMN-type collagenases, especially matrix metalloproteinase-9 and corroborated the use of Triphala in periodontal diseases. Since antibacterial substantivity of Triphala has not been investigated so far, we could not determine the frequency of its use for maximum efficacy. Thus, the present study was designed to determine the duration of its antibacterial substantivity and to compare it with the substantivity of 0.2% CHX, a positive control, and normal saline, a negative control.

   Materials and Methods Top

In this single-blind, randomized, crossover clinical trial the participants were dental students staying in the hostel of the college. The study population was selected based on the inclusion criteria like healthy controls in the age group of 18–25 years with <4 teeth missing and the controls having good oral hygiene. The individuals with high caries index, any systemic illness, using antibiotics, or any other mouthwashes for 3 months, using orthodontic/prosthetic appliances and with xerostomia were excluded from the study. An ethical approval was incurred from the institutional review board, and an informed written consent was obtained from the students after explaining the methodology of the clinical trial. Oral prophylaxis was performed before carrying out the study. All participants were trained and instructed regarding the usage of mouthrinses before commencing the study. Students were instructed to rinse

  • 10 ml of 0.2% CHX for 1 min
  • 50 ml 10% Triphala for 4 min
  • 15 ml of normal saline for 1 min.

Unstimulated saliva sample (baseline, pre-sample) was collected in sterile penicillin bulbs in the morning 2 h after their routine oral hygiene procedures. After that, the individuals were given a randomly selected mouthrinse using a chit pull system. Five minutes after rinsing with an allotted mouthrinse, a second sample (postsample) was collected, and both the samples were sent to the Microbiology unit for culture and microbial count. Individuals were asked to continue with their routines without any limitations over their eating and drinking habits. The sampling was repeated in a similar manner after every 2 h for 12 h (post 1, post 2, post 3, post 4, post 5, and post 6 samples) and was checked for microbial count. This was a crossover study with each participant using all mouthwashes. A washout period of 1 week[9],[10] was kept between two mouthwashes. The same procedure was repeated for the analysis of microbial count after 0.2% CHX and normal saline rinse [Figure 1].
Figure 1: Study design flow chart. n – sample size

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Preparation of Triphala mouthwash

For the preparation of 50 ml solution (decoction), 10 gm of Triphala powder containing equal quantity of Terminalia bellirica, Terminalia chebula, and Emblica officinalis was added to 100 ml of water.

Evaluation of microbial load count

Bacterial count (colony forming units [CFUs]) in each sample was determined by culture and microscopy at the Department of Medical Microbiology and Pathology, Gandhi Medical. The collected saliva sample was inoculated on nutrient agar plate. For inoculation of saliva, standard quantity 0.01 ml of saliva was taken using standard diameter nichrome wire loop. Inoculated nutrient agar plates were incubated at 37°C for 24 h. The grown colonies on nutrient agar were counted against standard inoculum used.

Preparation of nutrient agar plate

For this research work, commercially available dehydrated nutrient agar from Hi-media was used. Twenty-eight grams of dehydrated media was mixed into 1000 ml of distilled water. Media was sterilized by autoclave at 121°C for 15 min. Thereafter, 20 ml of sterilized media was dispensed in each of the sterile Petri plates (90 mm × 15 mm in dimension). These plates were stored in a refrigerator.

Counting the colony forming units

For the counting, semi-quantitative method was used. The microbiologist was kept blind to avoid the bias.

Statistical analysis

All data were entered into a computer by giving coding system, proofed for entry errors and analyzed with statistical package, statistical package for the social sciences (SPSS Inc, IBM Corporation, New York, USA). Chi-square test was used to assess the age-wise and gender-wise distribution of study individuals in all three groups. Friedman test was used to assess the effect of different mouthrinses on CFU at different times. Kruskal–Wallis test was to assess CFU at different times for different mouthrinses. If Friedman test and Kruskal–Wallis test was found significant post hoc test, Dunn's multiple comparison test was carried out to find out the time of CFU significantly different at various times as compared to CFU at pre-assessment. P < 0.05 was considered statistically significant.

The following coding system was used for CFU:

  • Nil: 0 1–10: 1 11–20: 2
  • 21–30: 3 31–40: 4 41–50: 5
  • 51–60: 6 61–70: 7 71–80: 8
  • 81–90: 9 91–100: 10>100: 11.

   Results Top

The present randomized clinical crossover trial recruited 30 students (7 males/23 females) in the age group of 19–23 years [Figure 1]. All the participants were instructed to use 0.2% CHX, 10% Triphala and normal saline mouthrinse with a washout period of 1 week. 0.2% CHX demonstrated statistically significant results in terms of CFU count at different times, as compared to baseline [Table 1]. 0.2% CHX also exhibited a statistically significant difference (P < 0.005), when compared with 10% Triphala and normal saline [Table 2]. Ten percent Triphala presented with a statistically significant result (P < 0.005) in the initial 2–3 h only. However, post this period, the results with 10% Triphala were noted to be statistically insignificant, corresponding to normal saline. Normal saline showed statistically insignificant results at all time.
Table 1: Comparison of colony forming unit at different times for various mouthrinses

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Table 2: Effect of chlorhexidine, Triphala, and saline on colony forming unit at different times as compared to preassessment

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   Discussion Top

Studies have demonstrated that, in situ, 0.2% CHX has a greater immediate antibacterial effect and substantivity than other antiseptics used in the oral cavity.[11],[12],[13],[14] Our study also demonstrated the same. Its antibacterial mode of action is explained by the fact that the positively charged bis-biguanide molecule gets rapidly attracted by the negatively charged bacterial tooth surfaces and oral mucosal cell surfaces, increasing substantivity through controlled release of the agent.[15],[16] The persistence of CHX on the oral surfaces and its ability to suppress salivary bacterial counts was demonstrated to last for than 12 h.[17] Thus, CHX in a mouthrinse (0.12% or 0.2% solution) is administered at 12-h intervals and retains its ability to retard/prevent plaque formation.[18] In this clinical crossover trial, 0.2% CHX substantivity was noticed till the 7th h. At the 8th h, the count of CFU was found to be increased. These results were in accordance with Tsuchiya et al.,[15] König et al.,[16] Boulos et al.,[18] Tomás et al.,[19] Addy M et al.,[20] and Tomás et al.[19] found that the practice of eating, chewing, and drinking significantly decreased the substantivity of 0.2% CHX, with complete recovery of the salivary flora at 3–7 h after the mouthrinse. This study demonstrated that 0.2% CHX was the most effective agent both in terms of magnitude of effects and duration of action. Similar results were found by Elworthy et al.,[13] Herrera et al.[21] There were no colonies found soon after rinsing with 0.2% CHX in our clinical trial. This suggested its bactericidal effect which remained for at least first 2 h. For next 6–7 h, the number of colonies remained static, thus proving its bacteriostatic effect. 0.2% CHX also demonstrated statistically significant results in terms of CFU count at different times, as compared to baseline (P < 0.05).

In the present scenario, Triphala is considered as an alternative for CHX in the prevention and treatment of periodontal diseases. Triphala has been used as a mouthrinse in many trials including healthy controls with gingivitis and periodontitis.[3],[6],[22] Triphala presented an antiplaque efficacy similar to that of CHX and was more effective at inhibiting plaque formation with lesser or no side effects.[4],[7] However, unlike CHX, antibacterial substantivity of Triphala was not evaluated so far. Hence, the present study was required to determine its substantivity thereby promoting its frequency of usage for the maximum efficiency. In this study, Triphala showed statistically significant results when the antibacterial effect at post, post 1 and post 2 were compared to preassessment count (P < 0.05). This suggested that Triphala has anti-bacterial activity for first 3–4 h only. After which the effect was at par with normal saline (P > 0.05). Jagadish et al.[4] had conducted a study to determine the effect of Triphala on dental biofilm. The authors concluded that Triphala has potent antioxidant and antimicrobial activity. It was also inferred that Triphala inhibits the growth of Streptococcus mutans, Gram-positive cocci, involved in plaque formation when it gets adsorbed to the tooth surface. Triphala effectively controlled the plaque formation by inhibiting 83.72% growth of S. mutans in 5% solution and 86.34% in 10%. The authors reported that this effect of Triphala is because of the tannic acid, which gets adsorbed well to the hydroxyapatite of the tooth or the salivary mucins, alternately it binds to anionic groups on the surface of the bacterial cells. This results in protein denaturation and ultimately to the bacterial cell death.[4] In one more bacteriological study with the Triphala decoction, the authors demonstrated that Triphala has a good antibacterial property and it is sensitive to 72.7% bacteria that were investigated.[23] Gupta et al.[24] reported that all the concentrations of ethanolic and aqueous extracts of Triphala inhibited the growth of S. mutans, Streptococcus sanguis, and Streptococcus salivarius and there was no statistically significant difference in the mean diameter of inhibition zone between the ethanolic and aqueous extracts of Triphala against S. mutans. The authors also highlighted that the efficacy increased with increasing concentration with maximum inhibition at 50% concentration. According to Kirubanandan et al.[25] Triphala could be potentially used as a therapeutic agent for methicillin-resistant S. aureus (MRSA)-infected dermal wounds because of its inhibitory effect on different MRSA strains and their enzymes such as serine protease and metalloprotease. They found that the topical application of Triphala ointment on an infected wound not only effectively heals the wound but also promotes collagen synthesis at the wound site. With regards to antimicrobial activity, the activity of Polyphenols present in the Triphala might be exerted by direct binding to peptide structure of bacterial components, and enzymes.

However, with this clinical trial, it may be emphasized that this antimicrobial-antibacterial effect remains only for 3–4 h after a single use. In almost all studies till date, Triphala had been used twice daily as a mouthrinse like CHX. In the authors' opinion, it may be used thrice a day to accentuate results.

Normal saline which was used as a negative control did not show any antibacterial effect at any point of time. This was confirmed by the fact that the results were statistically insignificant (P > 0.05) when CFU counts at different times were compared with the pre-assessment counts.

However, in all three groups of this crossover trial, there was a common observation that last three samples which were taken at 5 pm, 7 pm, and 9 pm had comparatively less bacterial count than other samples, and it showed statistically significant results when compared to preassessment count. This may be related to the decreased salivary flow due to the stress during college hours, normally observed in students.[26],[27],[28]

When Triphala and normal saline were compared to CHX at different times, there was a statistically significant difference (P < 0.001). When Triphala and normal saline were compared to each other, there was statistically significant difference only in the initial few hours. i.e., 3–4 h. Thus, we could say that Triphala has antibacterial effect for 3–4 h in an oral cavity. Thus, it may be suggested through this clinical trial that Triphala can be rinsed for at least three times a day for its maximum antibacterial effect. In future, similar studies are required with more sample size for determining the effect of Triphala mouthrinse on the different strains of microorganisms present in saliva. Further research is warranted for evaluating the efficiency of Triphala using a rinse thrice daily.

   Summary and Conclusion Top

Triphala mouthrinse has an antibacterial effect for 3–4 h after a single rinse. It can be used for at least 3 times daily for its maximum antibacterial effect. CHX remains the gold standard providing maximum antibacterial substantivity of 7–8 h.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

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  [Table 1], [Table 2]


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