|Year : 2015 | Volume
| Issue : 2 | Page : 104-107
Antimicrobial activity of Triphala on Lactobacilli and Candida albicans: An in vitro study
Swati Chainani, Sunitha Siddana, Chhava Venkatkonda Reddy, Manjunathappa Thippeswamy, Manjunath Maurya, Sushma Rudraswamy
Department of Public Health Dentistry, JSS Dental College and Hospital, Mysore, Karnataka, India
|Date of Web Publication||17-Nov-2015|
201, Veronica, Regency Estate, Kalyan Shil Road, Dombivali (East), Mumbai, Maharashtra
Source of Support: None, Conflict of Interest: None
Aim: To determine whether Triphala (Terminalia bellerica, Terminalia chebula, Emblica officinalis) extract has an antimicrobial activity against Lactobacilli and Candida albicans. Materials and Methods: Ethanolic extract of Triphala was prepared by using the cold extraction method. The extract was diluted with an inert solvent, dimethylformamide, to obtain 15 different concentrations of the extract. 0.2% chlorhexidine was used as a positive control and dimethylformamide was used as a negative control. The extract, along with the controls, was subjected to microbiological investigation to determine which concentration among the 15 different concentrations of the extract gave a wider inhibition zone against Lactobacilli and C. albicans. The zones of inhibition were measured in millimeters using a Vernier caliper. Results and Conclusions: Triphala extract demonstrated antimicrobial property against Lactobacilli and C. albicans with maximum zone of inhibition of 22 mm at 6% and 20 mm at 9%.
Keywords: Antimicrobial activity, Candida albicans, Lactobacilli, Triphala
|How to cite this article:|
Chainani S, Siddana S, Reddy CV, Thippeswamy M, Maurya M, Rudraswamy S. Antimicrobial activity of Triphala on Lactobacilli and Candida albicans: An in vitro study. J Orofac Sci 2015;7:104-7
|How to cite this URL:|
Chainani S, Siddana S, Reddy CV, Thippeswamy M, Maurya M, Rudraswamy S. Antimicrobial activity of Triphala on Lactobacilli and Candida albicans: An in vitro study. J Orofac Sci [serial online] 2015 [cited 2017 Mar 27];7:104-7. Available from: http://www.jofs.in/text.asp?2015/7/2/104/169757
| Introduction|| |
Dental caries is a chronic and multifactorial disease that, although avoidable, still represents an important problem in public health.  The prevalence of dental caries in India is 80% with five decayed teeth per child on an average at the age of 16 years. 
It results from the interplay of three main factors over time : d0 ietary carbohydrates, cariogenic bacteria in plaque, and susceptible hard tooth surfaces. Among the three factors stated above, bacteria have been suggested to have a major effect on the prevalence or incidence of dental caries. 
Streptococcus mutans has been implicated as a causative organism of dental caries. Lactobacillus acidophilus has been isolated from carious teeth and its number in plaque has been shown to increase only after caries has developed.  A certain body of clinical and microbiological evidence has suggested a correlation between high prevalence of yeast Candida species in dental plaque and saliva and the development of active carious lesions. On the basis of these facts, it has been hypothesized that Candida albicans and Lactobacilli are relevant secondary pathogen involved in the progression of caries. 
A variety of therapeutic agents have been examined for their ability to control oral microorganisms.  The main classes which have been tested are bisbiguanides, cationic detergents, phenolic compounds, plant extracts, essential oils, enzymes, antibiotics, halogens, etc.  The emergence of antimicrobial resistance as a global public health problem and side effects of the conventional therapies in controlling the infectious disease of the oral cavity have led to search of newer alternatives.
There has been a change in thinking globally, with a growing tendency to "go green." Medicinal plants have been a part and parcel of Indian civilization for combating a variety of diseases. Research has been focused recently on herbal medicines due to their wide range of biological and medicinal activities, ease of availability, higher safety margins, and lower cost. 
One such herbal cocktail is Triphala, an equiproportional mixture of Terminalia chebula, Terminalia belerica, and Emblica officinalis. It has been used extensively in Indian system of medicine as a potent anti-inflammatory,  antioxidant,  and antimicrobial agent against a wide spectrum of microbes.
In the context of oral microbes, Tandon et al. compared the effect of Triphala mouthwash with chlorhexidine on caries status among 1,501 students aged 8-12 years. It was concluded that there was no significant difference between Triphala and chlorhexidine mouthwash in the prevention of dental caries. 
Srinagesh et al conducted an in vivo study to evaluate the antibacterial efficacy of 6% Triphala mouthwash against oral streptococci as against 0.2% chlorhexidine. The result showed 44% and 45% reduction in CFUs/mL at the end of 7 days. It was concluded that antibacterial efficacy of Triphala closely parallels chlorhexidine. 
Though many studies are conducted on the effect of Triphala on streptococcus mutans, the literature on its action on Lactobacilli and Candida is unavailable. Hence, an attempt is made.
| Materials and Methods|| |
Preparation of Triphala extract
Fruits of T. bellerica (Vibhitaki), T. chebula (Haritaki), and E. officinalis (Amalaki) were obtained from the local market and were authenticated at JSS College of Ayurveda, Mysore. Seeds from the individual fruits were removed, and dried fruit pulp was crushed to semicoarse powder using a grinder. Triphala was prepared from these powders by mixing them in equal proportions (1:1:1) based on ayurvedic formulary of India. 
Alcoholic extract of Triphala was prepared using cold maceration technique. The coarse powder of Triphala was soaked in 97% ethanol for 72 h. The extract was then filtered using Whatman's filter paper. The filtrate was concentrated by complete evaporation of solvent on a hot water bath to yield the pure extract. This extract was considered as the 100% concentration of the extract.
Preparation of 15 different concentrations of Triphala extract
One gram of extract was dissolved in 10 mL of dimethylformamide to obtain a 10% concentration of extract. One milliliter of the extract was transferred to a sterilized test tube and labeled as 10%. The remaining 9 mL of the extract was then diluted further with dimethylformamide to obtain 14 different concentrations (0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, and 9%) of Triphala extract. A total of 15 concentrations ranging from 0.5 to10% were prepared. 0.2% chlorhexidine was used as a positive control and dimethylformamide was used as a negative control.
Pure strains of Lactobacilli (MTCC 10307) and C. albicans (MTCC 4748) were obtained from Microbial Type Culture Collection, Chandigarh. Brain heart infusion media and Saboraud dextrose agar (SDA) were used to grow and dilute the microbial suspensions. Bacterial strains were grown at exponential phase at 37°C for 18 h and adjusted to a final density of 0.5 McFarland units.
The antimicrobial activity of different concentrations of Triphala was determined by modified agar well diffusion method. Rogosa agar for Lactobacilli and Sabroud's dextrose agar was used to culture C. albicans. Agar plates were seeded with 0.5 mL of 24 h broth cultures of each isolate. The plates were allowed to dry for 1 h. A sterile 8 mm cork borer was used to cut three wells at equidistance in each of the plates. A total of six such plates were prepared for each organism under investigation. 0.2 mL of extract dilutions were introduced into each of these wells. Dimethylformamide was used as a negative control and 0.2% chlorhexidine as a positive control. The plates were incubated at 37°C for 48 h. The antimicrobial activity was evaluated by measuring the diameter of zones of inhibition (in mm). All the plates were made in duplicates and the experiments repeated twice.
| Results|| |
This study was undertaken to evaluate the antimicrobial activity of Triphala extract against Lactobacilli and C. albicans using agar well diffusion method. Triphala showed a maximum zone of inhibition measuring 22 mm at a concentration of 6% against Lactobacilli. Maximum zone of inhibition for Candida species was 20 mm at 9% concentration. Increasing the concentration further above these percentages did not produce any increase in the zone of inhibition. Both species were found to be resistant to the action of Triphala at lower concentrations. For 0.2% chlorhexidine (positive control), a much wider zone of inhibition of 32 mm was achieved for Lactobacilli and 30 mm for Candida. Both species were resistant to the action of dimethylformamide (negative control) [Table 1].
|Table 1: Zone of inhibitions of Triphala extracts on Lactobacilli and Candida|
Click here to view
| Discussion|| |
Current advancements in drug development technology and search for novel chemical diversity have intensified the efforts for exploring leads from Ayurveda, the traditional system of medicine in India. Triphala is one such herbal mix used extensively in Indian system of medicine. Biradar et al. reported that tannic acid, chebulic acid, and flavonoids - the active constituents present in Triphala - perhaps are largely responsible for the therapeutic potential of Triphala.  In this study, we found that Triphala at 6% concentration was effective against Lactobacilli, and similar results were obtained by Bajaj who studied the efficacy of Triphala on Streptococcus and Lactobacilli. It was reported that Triphala at 0.6% concentration showed a significant reduction in CFU/mL of Lactobacilli from the salivary isolates.  Hegde and Vasavi found a 71.7% reduction in oral lactobacilli in an in vitro study studying antibacterial effect of Triphala against lactobacilli.  We also found Triphala in 9% concentration to be highly effective against yeast candida. This is in accordance to the study conducted by Shetty who tested anticandidal activity of Triphala in the tablet formulation and found it to be highly effective. 
The antimicrobial activity of Triphala can be attributed to these constituents. The phenolic ring of these phytochemicals is shown to be toxic to microorganisms. The site(s) and number of hydroxyl and the phenol group are thought to be related to their relative toxicity to microorganisms on the evidence that increased hydroxylation results in increased toxicity. In addition, some authors have found that most of the highly oxidized phenols are inhibitory. The mechanisms thought to be responsible for phenolic toxicity to microorganisms include enzyme inhibition by the oxidized compounds, possibly through reaction with sulfhydryl groups or through more nonspecific interactions with the proteins. 
In this study, we used ethanol as a solvent because the polarity of antibacterial compounds present in Triphala makes them more readily extracted by organic solvents, and it does not negatively affect the bioactivity against bacterial species.  Dimethylformamide, an inert solvent, was used to dilute the extract to neutralize the effect of alcohol, which itself is an antiseptic, attributing the result solely to Triphala. In this study, chlorhexidine was found to be more effective when compared to Triphala extract. However, the well-known side effect of Chlorhexidine, that is, staining of teeth and restoration, alteration of taste sensation, and development of resistant microorganisms, may limit the long-term use of chlorhexidine.  In comparison with herbal medicines, Triphala is traditionally accepted, culturally amiable, economically feasible, and possess minimal side effects, hence it can be recommended for long-term use in various formulations for oral care.
Studies regarding the mode of action for these compounds in the bacterial cell should be done. There is also a need to pursue the characterization of active principles, to optimize the observed activity.
| Conclusion|| |
Triphala extract demonstrated antimicrobial property against Lactobacilli and C. albicans at 6% and 9%, respectively.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Martins AC, Maluf ML, Svidzinski TI. Prevalence of yeast species in the oral cavity and its relationship to dental caries. Acta Sci Health Sci 2011;33:107-12.
Jain P, Pundir RM. Strategies to prevent and treat dental caries and periodontal disease. J Pharm Res 2009;2:1223-8.
Shivakumar KM, Vidya SK, Chandu GN. Dental caries vaccine. Indian J Dent Res 2009;20:99-106.
Akortha EE, Ibadin OK. Microbial flora in orodontal infections and their susceptibility to some commonly used antibiotics. Bull Sci Assoc Niger 2007;28:58-63.
Signoretto C, Burlacchini G, Faccioni F, Zanderigo M, Bozzola N, Canepari P. Support for the role of Candida
spp. in extensive caries lesions of children. New Microbiol 2009;32:101-7.
Aneja KR, Joshi R, Sharma C. The antimicrobial potential of ten often used mouthwashes against four dental caries pathogens. Jundishapur J Microbiol 2010;3:15-27.
Groppo FC, Ramacciato JC, Simões RP, Flório FM, Sartoratto A. Antimicrobial activity of garlic, tea tree oil, and chlorhexidine against oral microorganisms. Int Dent J 2002;52:433-7.
McCoy LC, Wehler CJ, Rich SE, Garcia RI, Miller DR, Jones JA. Adverse events associated with chlorhexidine use: Results from the department of veterans affairs dental diabetes study. J Am Dent Assoc 2008;139:178-83.
Jagadish L, Anandkumar VK, Kaviyarasan V. Role of Triphala
on dental biofilms. Indian J Sci Technol 2009;2:30-3.
Asmawi MZ, Kankaanranta H, Moilanen E, Vapaatalo H. Anti-inflammatory activities of Emblica officinalis
Gaertn leaf extracts. J Pharm Pharmacol 1993;45:581-4.
Tandon S, Gupta K, Rao S, Malagi KJ. Effect of Triphala
mouthwash on the caries status. Int J Ayurveda Res 2010;1:93-9.
Srinagesh J, Krishnappa P, Somanna SN. Antibacterial efficacy of Triphala
against oral streptococci: An in vivo
study. Indian J Dent Res 2012;23:696.
Ayurvedic Formulary of India, Part-I, 6;14:90-5.
Biradar YS, Jagatap S, Khandelwal KR, Singhania SS. Exploring of antimicrobial activity of Triphala
mashi-an ayurvedic formulation. Evid Based Complement Alternat Med 2008;5:107-13.
Bajaj N, Tandon S. The effect of Triphala
and chlorhexidine mouthwash on dental plaque, gingival inflammation, and microbial growth. Int J Ayurveda Res 2011;2:29-36.
Hegde V, Vasavi V. A study to assess the antibacterial effect of Triphala
churna against Streptococcus mutans
- An in vitro
study. J Postgrad Dent 2011;1:13-4.
Shetty PJ, Hegde V, Gomes L. Anticandidal efficacy of denture cleansing tablet, Triphala
, Aloe vera
, and Cashew leaf on complete dentures of institutionalized elderly. J Ayurveda Integr Med 2014;5:11-4.
Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev 1999;12:564-82.
Lalla JK, Hamrapurkar PD, Mamania HM. Mineral content and microbial impurity of Triphala
and its raw materials. Indian J Tradit knowl 2004;3:86-91.