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Table of Contents   
ORIGINAL RESEARCH  
Year : 2014  |  Volume : 25  |  Issue : 4  |  Page : 475-479
Evaluation of antimicrobial efficacy of Triphala (an Indian Ayurvedic herbal formulation) and 0.2% chlorhexidine against Streptococcus mutans biofilm formed on tooth substrate: An in vitro study


1 Department of Conservative Dentistry, Tagore Dental College and Hospital, Chennai, Tamil Nadu, India
2 Department of Periodontics, Tagore Dental College and Hospital, Chennai, Tamil Nadu, India
3 Undergraduate Student, Tagore Dental College and Hospital, Chennai, Tamil Nadu, India
4 Department of Bioproducts, Central Leather Research Institute, Chennai, Tamil Nadu, India

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Date of Submission13-Feb-2014
Date of Decision23-Mar-2014
Date of Acceptance05-Jun-2014
Date of Web Publication10-Oct-2014
 

   Abstract 

Background: Streptococcus mutans is one of the most important cariogenic species of the human oral microbial flora. Biofilm style of microbial growth thought to resist the actions of antimicrobials.
Aim: The purpose of this study was to evaluate the antimicrobial efficacy of Triphala, and 0.2% chlorhexidine against S. mutans biofilm formed on tooth substrate.
Settings and Design: Randomized control trial.
Methods: Extracted human mandibular premolars sectioned below the cemento-enamel junction were placed in the tissue culture wells exposing the crown surface to S. mutans to form a biofilm. At the end of 3 rd and 7 th day, all groups were treated for 10 min with the test solutions and control and were analyzed qualitatively and quantitatively.
Statistical Analysis Used: One-way ANOVA.
Results: Qualitative assay with 3 days biofilm showed complete inhibition of bacterial growth with Triphala, but 0.2% chlorhexidine and saline showed the presence of bacterial growth. In quantitative analysis, 0.2% chlorhexidine and Saline treated tooth samples have shown 1052 Χ 10 4 ± 15.1 Χ 10 4 CFU/ml, 141.3 Χ 10 9 ± 2.1Χ10 9 CFU/ml, respectively. Qualitative assay with 7 days biofilm on crown portion showed dense growth when treated with 0.2% chlorhexidine and saline, whereas Triphala has shown minimal growth. In Quantitative analysis, Triphala showed statistically significant result when compared with 0.2% chlorhexidine and saline.
Conclusion: Triphala showed statistically significant antibacterial activity against S. mutans biofilm formed on tooth substrate. The incorporation of Triphala in mouth rinse could prove to be effective in reducing S. mutans count in the oral cavity.

Keywords: Biofilm on tooth substrate, chlorhexidine, herbal mouth rinse, mouth rinse, Streptococcus mutans, Triphala

How to cite this article:
Prabhakar J, Balagopal S, Priya M S, Selvi S, Senthilkumar M. Evaluation of antimicrobial efficacy of Triphala (an Indian Ayurvedic herbal formulation) and 0.2% chlorhexidine against Streptococcus mutans biofilm formed on tooth substrate: An in vitro study. Indian J Dent Res 2014;25:475-9

How to cite this URL:
Prabhakar J, Balagopal S, Priya M S, Selvi S, Senthilkumar M. Evaluation of antimicrobial efficacy of Triphala (an Indian Ayurvedic herbal formulation) and 0.2% chlorhexidine against Streptococcus mutans biofilm formed on tooth substrate: An in vitro study. Indian J Dent Res [serial online] 2014 [cited 2019 Nov 22];25:475-9. Available from: http://www.ijdr.in/text.asp?2014/25/4/475/142539
Dental caries is a chronic infectious disease caused by the formation of biofilm on tooth surfaces. Among the oral bacteria, mutans streptococci are considered to be causative agents of dental caries. [1] Streptococcus mutans is considered one of the most important cariogenic species of the human oral microbial flora. [2] There is ample evidence for the association between S. mutans and dental caries. [3]

It is known that bacteria with efficient adaptive mechanisms adjust to new environmental and biological circumstances. Biofilm style of microbial growth is such an adaptation where a community of microbes gets adsorbed to a solid surface and is embedded in a common matrix. [4] The altered microbial genetic and metabolic processes, along with the extracellular matrix, are thought to resist the actions of antimicrobials. [5] Thus, the concept of bacterial biofilm is gaining more attention in recent times and is associated with a wide range of persistent infections. [4]

In 1986, Loesche described caries and periodontal disease as "perhaps the most expensive infections that most individuals have to contend with during a lifetime". The primary goal of a caries prevention program should be to reduce the number of cariogenic bacteria. Thus, the addition of an antimicrobial mouthrinse to daily oral hygiene regimens would help to reduce the total oral bacterial burden and would be a meaningful, cost-effective addition to mechanical oral hygiene methods. [6]

Studies have demonstrated the effectiveness of rinsing with chlorhexidine antimicrobial mouthrinse in significantly reducing salivary levels of S. mutans. [7] However, the adverse side-effects of chlorhexidine have prompted to look for herbal alternatives.

Plants are known to produce a variety of compounds to protect themselves against a variety of pathogens. It is expected that plant extracts showing target sites other than those used by antibiotics will be active against drug resistant pathogens .[8] Herbal products have been proven to be safe, containing active constituents that have beneficial physiological effect apart from its curative property. Triphala is an Indian ayurvedic herbal formulation consisting of dried and powdered fruits of three medicinal plants Terminalia bellerica, Terminalia chebula and Emblica officinalis .[9]

The purpose of this in vitro study was to evaluate the antimicrobial efficacy of Triphala, 0.2% chlorhexidine against S. mutans biofilm formed on tooth substrate of extracted human teeth.


   Methods Top


A pure culture of S. mutans (MTCC 497) (IMTECH, MTCC, Chandigarh, India) was grown on Brain Heart Infusion Agar (BHIA, Himedia, India), inoculated into Brain Heart Infusion Agar Broth (BHIAB, Himedia, India), incubated at 37°C overnight and adjusted to an optical density (OD 600 ) of 1 with sterile BHIAB.

Triphala (IMPCOPS Ltd., Chennai, India) powder was made into a solution by dissolving in 10% Dimethyl Sulfoxide (DMSO) (S.D. Fine Chem Pvt. Ltd., India). 0.2% chlorhexidine was prepared by diluting calypso 20% chlorhexidine mouthwash (Septodent, Cedex, France) with sterile distilled water. The antibacterial activity of Triphala and 0.2% chlorhexidine were initially tested on planktonic cells before evaluating them against S. mutans biofilm formed on tooth substrate.

The antibacterial sensitivity test was performed by disc diffusion method (National Committee for Clinical Laboratory Standard, 2000). Sterile blank discs (6 mm diameter, Himedia, India) were impregnated with 10 μl of test solutions (Triphala and 0.2% chlorhexidine). The broth culture of S. mutans was swabbed on sterile Brain Heart Infusion Agar plates using sterile swabs. With the help of sterile forceps the test solutions incorporated discs were placed on the medium and the plates were incubated at 37°C overnight. A standard vancomycin disc (30 mcg) was included for comparison. A disc with 10% DMSO was also included to see if it shows any significant zone of inhibition.

The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the test solutions were determined by the tube dilution method. Double dilution was made from a higher dilution 100 mg/ml to a lower dilution in a series of test tubes. Each tube was inoculated with bacterial suspensions and incubated at 37°C overnight. The MIC was regarded as the lowest concentration in the series of dilutions, which did not permit the growth of the susceptible bacteria. The subcultures were made from the tubes, which did not yield any visible turbidity (growth) in the MIC assay on freshly prepared BHIA plates. After 24 h incubation at 37°C, the MBC was regarded as the lowest concentration of the test solution that allowed <0.1% of the original inoculum to grow on the surface of the medium. In each experiment test solutions were tested in triplicate.

The time kill study was done, which determined the time required for killing S. mutans by exposing the bacteria with the bactericidal concentration of test solutions for 30 min. At regular intervals (2 min), a loop full of the sample was inoculated on BHIA plate, incubated at 37°C for 24 h and observed for growth.

Biofilm formation on tooth substrate

Single rooted human mandibular premolars with fully formed apices were used in this study. The teeth were cleaned of superficial debris, calculus, tissue tags and stored in normal saline to prevent dehydration before use. The tooth specimens were sectioned below the cemento-enamel junction with a diamond disc to obtain a standardized crown length of 4 mm for uniform specimen.

The tooth surface was minimally grounded to achieve a flat surface to enable placement in the tissue culture wells exposing the crown surface to S. mutans to form a biofilm. The sectioned samples were then divided into three experimental groups. Each group consisted of 30 samples each and assigned as Group 1 (Triphala), Group 2 (0.2% chlorhexidine) and Group 3 (saline). Then the samples were placed in the wells of tissue culture plates (Zellkuthur - test plate 24, Europe/Switzerland) and sterilized by Gamma irradiation (BI 2000, BARC, Cancer Institute, Chennai, India).

The bacterium was cultured as described above and the wells containing tooth samples were inoculated with 2 ml of bacterial solution and incubated at 37°C. The culture medium (Brain Heart Infusion Agar Broth) was replaced every alternate day to avoid nutrient depletion and accumulation of toxic end products. The samples were taken from each well with a sterile paper point and inoculated onto Brain Heart Infusion Agar plates and incubated at 37°C for 24 h to check for cell viability and purity of culture.

At the end of 3rd day all groups were treated for 10 min as follows; Group 1: Immersed in 3 ml of Triphala (80 mg/ml in 10% DMSO); Group 2: Immersed in 3 ml of 0.2% chlorhexidine; Group 3: Immersed in 3 ml sterile saline. Then, the biofilm on the crown portion was scraped and inoculated on Brain Heart Infusion Agar plates and incubated for 24 h at 37°C for qualitative analysis where n = 5 for each group. The quantitative analysis was done by vortexing the tooth samples with sterile saline for few minutes followed by serial dilution method for all the groups where n = 10 for each group. The same procedure was repeated for all groups once again at the end of the 7 th day to analyze qualitatively and quantitatively.

Statistics

Statistical analysis was performed using the one-way ANOVA using IBM SPSS (Statistical Package for the Social Sciences)  (student version 7.01). The criterion for statistical significance was defined as P < 0.05.


   Results Top


[Table 1] shows the zone of inhibition, MIC and MBC of test solutions for S. mutans. All test solutions have shown significant zone of inhibition in the disc diffusion assay when compared with vancomycin. No zone of inhibition was shown by 10% DMSO. 0.2% chlorhexidine achieved 100% killing of S. mutans at 2 min, whereas Triphala took 6 min.
Table 1: Susceptibility of Streptococcus mutans MTCC 497 against the test solutions


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Qualitative assay with 3 days biofilm on crown portion showed complete inhibition of bacterial growth when treated with Triphala. However, the samples treated with 0.2% chlorhexidine and saline has shown the presence of bacterial growth. In quantitative analysis, 0.2% chlorhexidine and saline treated tooth samples have shown 1052 × 10 5 ± 15.1 × 10 5 CFU/ml, 141.3 × 10 9 ± 2.1 × 10 9 CFU/ml (mean ± standard deviation) respectively [Table 2].
Table 2: Quantitative analysis of 3 days Streptococcus mutans biofilm formed on tooth substrate for different groups


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Qualitative assay with 7 days biofilm on crown portion showed dense growth when treated with 0.2% chlorhexidine and saline, whereas Triphala has showed minimal growth. [Table 3] shows the bacterial population in quantitative assay with 7 days biofilm for Triphala, 0.2% chlorhexidine and saline treated tooth samples. Group 1 (Triphala) have shown significant reduction of bacterial population compared to control group, which showed 138.9 × 10 9 ± 3.6 × 10 9 CFU/ml [Table 3].
Table 3: Quantitative analysis of 7 days Streptococcus mutans biofilm formed on tooth substrate for different groups


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   Discussion Top


Dental caries is a common oral bacterial pathology caused by a biofilm consisting of microorganisms present on the tooth surface. [10] S. mutans is the most common microorganism associated with dental caries. [2] When S. mutans grows as a biofilm, the altered genetic and metabolic processes of bacteria along with its complex matrix prevent the entry and action of several antimicrobial agents. [5] The antibiotic resistance has been found to increase up to 1500 times when compared to planktonic cells. [11],[12] Therefore, testing the effect of an antibacterial agent on planktonic cells will not fulfill its effectiveness in in vivo conditions.

It is established that the biofilm forming capacity and its structural organization are influenced by the chemical nature of the substrate. Biofilm experiments conducted on polycarbonate or glass substrate will not provide a true indication of the bacteria-substrate interaction. [13] Hence, S. mutans biofilm was formed on a tooth substrate in this study. All the groups were tested in direct contact with the biofilm formed on tooth substrate at different durations (3 days and 7 days).

The antibacterial activity was directly proportional to the concentration of the test solutions. In the initial antibacterial sensitivity test on planktonic cells, chlorhexidine maintains its efficacy against S. mutans at 0.2 μg whereas the Triphala required more concentration (5 mg/ml). Chlorhexidine killed the bacterial cells more rapidly than the herbal formulation. The concentration of the herbal solutions was increased, as the fact that sessile bacteria on surfaces or present within biofilm are much less readily inactivated than planktonic cells. A Biocide gradient is produced throughout the biofilm, so that in thick biofilm there will be an "in-use" concentration as the biocide penetrates into the community. [13] The concentration of 80 mg/ml used in this study was found to be effective as an antibacterial against S. mutans and further reduction in concentration, when used in vivo is still feasible as the bacterial count is expected to be much less than what we have used.

Dimethyl sulfoxide was used as a solvent for Triphala though it was readily soluble in water. DMSO is a clean, highly polar, aprotic solvent, which helps in bringing out the pure properties of all the components of the herb being dissolved. [14],[15] Antibacterial inertness of 10% DMSO was confirmed with the disc diffusion test.

About 0.2% chlorhexidine showed good antibacterial sensitivity against S. mutans planktonic cells and showed 4-log reduction against 3 days old biofilm, but failed to show significant antibacterial activity against 7 days old biofilm. Side-effects such as loss of taste, burning sensation of the oral mucosa, subjective dryness of the oral cavity and discoloration of teeth and tongue were reported with 0.2% chlorhexidine. [16],[17] Resistance of pathogens to synthetic drugs is posing an ever increasing therapeutic problem. [18] and increased MICs of chlorhexidine for S. mutans have been reported. [19] The resistance of Staphylococcus aureus and Streptococcus sanguis to chlorhexidine has been documented. [20],[21]

Triphala showed 100% efficiency against 3 days biofilm and eight log reduction against 7 days old biofilm. Though Triphala and 0.2% chlorhexidine exhibited similar antibacterial sensitivity on S. mutans planktonic cells, Triphala showed more potency on S. mutans biofilm. This may be attributed to its formulation, which contains three different medicinal plants in equal proportions. In such formulations, different compounds maybe of help in enhancing the potency of the active compounds resulting in an additive or synergistic positive effect. Recently, Prabhakar et al. have reported excellent antibacterial efficacy of Triphala against 3 weeks and 6 weeks old Enterococcus faecalis biofilm formed on tooth substrate. [22]

Healing potential in plants is an ancient idea, but in recent times it has gained renewed interest and importance. Triphala is proven to be safe, containing active constituents that have beneficial physiological effect apart from its curative property such as antioxidant activity, antiinflammatory, and radical scavenging activity [23],[24],[25] and may have an added advantage over the synthetic drugs. The major advantages of using herbal alternatives are easy availability, cost-effectiveness, increased shelf life, low toxicity and lack of microbial resistance reported so far. [26]

Within the limitations of this study, Triphala showed maximum antibacterial activity against 3 and 7 days S. mutans biofilm formed on tooth substrate. 0.2% chlorhexidine was found to be ineffective against 7 days biofilm. The use of Triphala as a mouthwash could prove to be effective in reducing the S. mutans count in caries risk patients. It is very important to evaluate the antibacterial efficacy of Triphala dissolved in water to conclusively recommend Triphala as a mouth rinse.

 
   References Top

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14.Jacob SW, Herschler R. Biological actions of dimethyl sulfoxide. Ann N Y Acad Sci 1975;243:1-508.  Back to cited text no. 14
    
15.Biological actions and medical applications of dimethyl sulfoxide. Ann N Y Acad Sci 1983;411:1-404.  Back to cited text no. 15
    
16.Hepsø HU, Bjørnland T, Skoglund LA. Side-effects and patient acceptance of 0.2% versus 0.1% chlorhexidine used as post-operative prophylactic mouthwash. Int J Oral Maxillofac Surg 1988;17:17-20.  Back to cited text no. 16
    
17.Gürgan CA, Zaim E, Bakirsoy I, Soykan E. Short-term side effects of 0.2% alcohol-free chlorhexidine mouthrinse used as an adjunct to non-surgical periodontal treatment: A double-blind clinical study. J Periodontol 2006;77:370-84.  Back to cited text no. 17
    
18.Guillemot D. Antibiotic use in humans and bacterial resistance. Curr Opin Microbiol 1999;2:494-8.  Back to cited text no. 18
    
19.Dever JG, Beck DJ, Tagg JR. Oral changes associated with six months' exposure to chlorhexidine. J Dent Res 1982;61:529.  Back to cited text no. 19
    
20.Yamamoto T, Tamura Y, Yokota T. Antiseptic and antibiotic resistance plasmid in Staphylococcus aureus that possesses ability to confer chlorhexidine and acrinol resistance. Antimicrob Agents Chemother 1988;32:932-5.  Back to cited text no. 20
    
21.Westergren G, Emilson CG. In vitro development of chlorhexidine resistance in Streptococcus sanguis and its transmissibility by genetic transformation. Scand J Dent Res 1980;88:236-43.  Back to cited text no. 21
    
22.Prabhakar J, Senthilkumar M, Priya MS, Mahalakshmi K, Sehgal PK, Sukumaran VG. Evaluation of antimicrobial efficacy of herbal alternatives (Triphala and green tea polyphenols), MTAD, and 5% sodium hypochlorite against Enterococcus faecalis biofilm formed on tooth substrate: An in vitro study. J Endod 2010;36:83-6.  Back to cited text no. 22
    
23.Vani T, Rajani M, Sarkar S, Shishoo CJ. Antioxidant properties of the ayurvedic formulation triphala and its constituents. Int J Pharmacogn 1997;35:313-7.  Back to cited text no. 23
    
24.Rasool M, Sabina EP. Antiinflammatory effect of the Indian ayurvedic herbal formulation Triphala on adjuvant-induced arthritis in mice. Phytother Res 2007;21:889-94.  Back to cited text no. 24
    
25.Jagetia GC, Malagi KJ, Baliga MS, Venkatesh P, Veruva RR. Triphala, an ayurvedic rasayana drug, protects mice against radiation-induced lethality by free-radical scavenging. J Altern Complement Med 2004;10:971-8.  Back to cited text no. 25
    
26.Abascal K, Yarnell E. Herbs and drug resistance Part 2 - Clinical implications of research on microbial resistance to antibiotics. Altern Complement Ther 2002;8:284-90.  Back to cited text no. 26
    

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Correspondence Address:
S Balagopal
Department of Conservative Dentistry, Tagore Dental College and Hospital, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.142539

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    Tables

  [Table 1], [Table 2], [Table 3]

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