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Table of Contents   
ORIGINAL ARTICLE  
Year : 2013  |  Volume : 16  |  Issue : 2  |  Page : 144-147
An in vitro evaluation of the antimicrobial efficacy of Curcuma longa, Tachyspermum ammi, chlorhexidine gluconate, and calcium hydroxide on Enterococcus faecalis


Department of Endodontics, Sudha Rustagi College of Dental Sciences, N.I.T. Faridabad, Haryana, India

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Date of Submission07-Nov-2012
Date of Decision05-Dec-2012
Date of Acceptance21-Dec-2012
Date of Web Publication7-Mar-2013
 

   Abstract 

Aim and Objectives: This in vitro study was designed to comparatively evaluate the antimicrobial efficacy of Curcuma longa (turmeric - T 1 -10%, T 2 -20%); Tachyspermum ammi (ajwain - A 1 -10%, A 2 -20%); chlorhexidine (CHX) gluconate gel (hexigel - 1%); and calcium hydroxide (10%) as intracanal medicaments against Enterococcus faecalis.
Materials and Methods: Agar plates were prepared using brain-heart infusion (BHI) agar. Cultures of E. faecalis were grown in BHI broth at 37°C. Well diffusion method was used to derive results. Plates were inoculated for 72 h at 37°C and microbial zones of inhibition were recorded. Statistical analysis was performed with repeated measures analysis of variance.
Results: C. longa (T 2 -20%) and CHX gluconate gel (hexigel - CHX-1%) showed larger zones of microbial inhibition than C. longa (T 1 -10%) that were statistically significant (P < 0.05) and were highly significant when compared to T. ammi (ajwain - A 1 and A 2 ) and calcium hydroxide.
Conclusion: C. longa can be used as intracanal medicament in endodontic failure cases.

Keywords: Chlorhexidine gluconate gel; Curcuma longa; Enterococcus faecalis; Tachyspermum ammi

How to cite this article:
Kumar H. An in vitro evaluation of the antimicrobial efficacy of Curcuma longa, Tachyspermum ammi, chlorhexidine gluconate, and calcium hydroxide on Enterococcus faecalis. J Conserv Dent 2013;16:144-7

How to cite this URL:
Kumar H. An in vitro evaluation of the antimicrobial efficacy of Curcuma longa, Tachyspermum ammi, chlorhexidine gluconate, and calcium hydroxide on Enterococcus faecalis. J Conserv Dent [serial online] 2013 [cited 2019 Dec 15];16:144-7. Available from: http://www.jcd.org.in/text.asp?2013/16/2/144/108197

   Introduction Top


Elimination of microorganisms from the root canal is the basis for successful endodontic treatment. Anaerobic bacteria especially black-pigmented Gram-negative species have been linked to the signs and symptoms of periapical disease. [1] Primary endodontic infections are polymicrobial in nature and are dominated by Gram-negative anaerobic rods while secondary endodontic infections are composed of few bacterial species. [2],[3],[4],[5] Facultative bacteria like Enterococcus faecalis is the most commonly isolated species from the root canals of the teeth with failed endodontic treatment [6] and its prevalence in such infections ranges from 24% to 77%. [7] E. faecalis is Gram-positive cocci that occur singly, in pairs or short chains, [8],[9] can survive harsh environments like extreme alkaline pH (9.6) and a temperature of 60°C for 30 min. [10] It possesses certain virulence factors such as lytic enzymes, cytolysin, pheromones, and lipotechoic acid, [9] suppresses the action of lymphocytes, potentially contributing to endodontic failure. [11] It is very small to invade and live within dentinal tubules and can endure prolonged periods of starvation. [7] When nutritional supply becomes available it can utilize serum as a nutritional source that originates from alveolar bone and periodontal ligament. [12] E. faecalis passively maintains pH homeostasis by a proton pump. It has been shown to synthesize a variety of stress proteins when exposed to adverse environmental conditions. [13] Since, its introduction in 1920 by Hermann, calcium hydroxide has been widely used as intracanal medication for the treatment of apical periodontitis. [14] Certain studies have indicated that killing of microbes in the canal can be achieved with calcium hydroxide. In a study of microbiology of root canals in retreatment cases, where calcium hydroxide-tolerant microbes were frequently isolated, it was suggested that alternative medicaments may be sometimes needed to resolve the infection to gain healing. [3] Chlorhexidine (CHX) is a broad spectrum antimicrobial agent. Its antimicrobial action is related to its cationic bisbiguanide molecular structure. At low concentration it is bacteriostatic while at higher concentration it is bactericidal as it brings about coagulation and precipitation of cytoplasm. It bears a property of substantivity and low-grade toxicity. CHX gluconate gel has been extensively used in dentistry mainly as an intracanal medicament. [15] In this modern era of dentistry, we are looking toward herbal alternatives. Extracts of plant origin have therapeutic properties since thousands of years. Use of plant parts as Folklore medicine has been trailed by traditional healers since time immemorial. WHO has reported that 80% of world's population relies mainly on traditional therapies. [16] There are many advantages of using herbs as antimicrobials: (a) They have fewer side effects; (b) less expensive; (c) better patient tolerance; and (d) renewable in nature. [17] Throughout Asia, traditional medicine has long used turmeric as an anti-inflammatory, anti-oxidant, anti-microbial agent to deal with a wide range of conditions. [18] Ajwain, the Bishop's weed containing thymol, the major phenolic compound renders it good germicidal and anti-fungal properties. [19] The purpose of this in vitro study was to evaluate the anti-microbial efficacy of Curcuma longa (turmeric - T 1 -10%, T 2 -20%); Tachyspermum ammi (ajwain - A 1 -10%, A 2 -20%); CHX gluconate gel (hexigel - CHX-1%) and calcium hydroxide (10%) as intracanal medicaments against E. faecalis.


   Materials and Methods Top


Six agar plates were prepared by using brain-heart infusion (BHI) agar (Titan Biotech Ltd. Delhi, India). Agar was mixed according to manufacturer's directions, and enough agar was poured to cover the surface of a 125 mm petridish. The BHI agar dishes were then stored at room temperature for 2 days before use to verify that they had remained sterile. BHI broth (Titan Media) was also prepared and stored in 5 ml vials for 2 days.

E. faecalis (MTCC 2729) was maintained on BHI broth and cultures of E. faecalis were grown overnight at 37°C in BHI broth for 24 h and bacterial growth was checked by presence of turbidity. The BHI broth was inoculated with E. faecalis from a freshly grown culture on an agar plate. The broth culture was incubated at 37°C for 24 h. Medicaments like C. longa (turmeric) were prepared in two concentrations of 10% (T 1 ) and 20% (T 2 ) by taking raw turmeric and grinding it into a coarse powder and then by adding sterile distilled water according to the required concentrations in two different test tubes. Similarly, T. ammi (ajwain) was prepared in two different concentrations in two different test tubes by adding sterile distilled water to ajwain crystals, thus, obtaining it in concentrations of 10% (A 1 ) and 20% (A 2 ). Other medicaments that were used were calcium hydroxide (NICE Chemicals, Kochi, India Batch 711330), which was obtained in a powdered form and then made to 10% by adding sterile distilled water in a test tube. CHX gluconate gel (hexigel - CHX-1%, ICPA Health Products Ltd. Gujarat, India, Batch 0201920712) was also used as one of the medicaments. Well diffusion method was used to derive the results. Wells of 7 mm diameter and 4 mm depth were punched in agar plates and filled with 10 μl of medicaments to be tested. Plates were inoculated for 72 h at 37°C. All manipulations of the specimens were performed under a laminar flow (Toshiba) to avoid contamination. Microbial zones of inhibition were measured in millimeters.


   Results Top


Results were recorded on the basis of diameter of zones. Statistical analysis was made using one way ANOVA and post-hoc tests. It was observed that C. longa (T 2 -20%) showed significantly (P < 0.05) more zone of microbial inhibition than C. longa (T 1 -10%), T. ammi (A 1 -10% and A 2 -20%) and calcium hydroxide (10%). Hexigel (CHX-1%) and C. longa (T 1 -10%) showed significantly larger zones of microbial inhibition than T. ammi (A 1 -10% and A 2 -20%) and calcium hydroxide (10%). The size of zone of inhibition between C. longa (T 2 -20%) and hexigel (CHX-1%) was not significant (P > 0.05). T. ammi (A 1 -10% and A 2 -20%) and control group showed no microbial inhibition [Table 1] and [Figure 1].
Figure 1: Antibacterial activity of Curcuma longa (T 1 -10%, T 2 -20%), Tachyspermum ammi (A 1 -10%, A 2 -20%), Chlorhexidine gluconate gel (CHX-1%) and Calcium hydroxide (10%)

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Table 1: Antimicrobial effect of intracanal dressing on Enterococcus faecalis

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   Discussion Top


Complete elimination of microorganisms from the root canal is not possible despite antimicrobial properties of chemomechanical preparation and intracanal medicaments. It could be because of anatomical complexities or may be due to varying vulnerabilities of involved species. [20],[21] The most commonly isolated species from root canals of teeth with failed endodontic treatment is E. faecalis.[22] It can gain entry into the root canal system during treatment, between appointments or after root canal completion. [9] Virulence factor of E. faecalis in failed endodontically treated teeth may be related to the ability of E. faecalis to invade dentinal tubules and adhere to collagen in presence of human serum. [23]

In this in vitro study, anti-microbial activity of various intracanal medicaments was compared to eliminate endodontic pathogen responsible for root canal failure. Our results verified that calcium hydroxide is relatively ineffective against E. faecalis. E. faecalis in dentinal tubules has been shown to resist intracanal dressings of calcium hydroxide for over 10 days as it is shown to withstand a high pH. [24] At pH 11.5 or greater E. faecalis does not survive, yet it can survive at a pH below 11.5. [23] In radicular dentin, alkalinity may only reach pH 10.3 after dressing the canal with calcium hydroxide. [25] It forms a biofilm that helps it resist destruction by enabling the bacteria to become more resistant to phagocytosis and antimicrobials. [26] Even though calcium hydroxide does have some antibacterial action, but under the experimental conditions it was not able to kill and eliminate sufficient cells of E. faecalis at any time. This is in accordance with findings of other reports by Haapasalo et al. [27]

In the present study, CHX in gel formulation at 1% is a powerful anti-microbial agent, showing large inhibition zones, which ranged from 19 mm to 21 mm. Although CHX showed better anti-microbial activity but its main drawback is that it does not act as a physical barrier, thus, staying in canal for a shorter period of time. This is in accordance with the study by Gomes et al., which suggested that CHX gel has a greater antibacterial activity against E. faecalis than calcium hydroxide but loses its property for longer periods. [28]

Various authors have shown the anti-microbial activity of C. longa against an array of pathogens. [18] In the present study, it was found that C. longa - turmeric (T 2 -20%) with a wide range of therapeutic action being anti-inflammatory, antibacterial, and antifungal showed highest zones of microbial inhibition ranging from 21 mm to 23 mm. The mechanism behind this has been explained by many authors on the basis of hypothesis, which states that it is due to bacterial cell wall perturbation. [29] Although T. ammi (ajwain) in both 10% (A 1 ) and 20% (A 2 ) concentrations did not show any antimicrobial effect against E. faecalis.


   Conclusion Top


With time plant extracts have been understood to encompass the attributes accounted not only for their fragrance and flavor, but also for their antimicrobial nature. In our study, C. longa (T 2 -20%) showed promising results in elimination of E. faecalis - one of the common organism responsible for root canal failure and is a good cost effective alternative to all the historical intracanal medicaments with fewer side effects and least resistance developed by the species. However, further studies should be carried out to determine the use of turmeric as an intracanal medicament in endodontics.

 
   References Top

1.Gomes BP, Drucker DB, Lilley JD. Associations of specific bacteria with some endodontic signs and symptoms. Int Endod J 1994;27:291-8.  Back to cited text no. 1
[PUBMED]    
2.Baumgartner JC, Falkler WA Jr. Bacteria in the apical 5 mm of infected root canals. J Endod 1991;17:380-3.  Back to cited text no. 2
    
3.Molander A, Reit C, Dahlén G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998;31:1-7.  Back to cited text no. 3
    
4.Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93.  Back to cited text no. 4
    
5.Hancock HH 3 rd , Sigurdsson A, Trope M, Moiseiwitsch J. Bacteria isolated after unsuccessful endodontic treatment in a North American population. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:579-86.  Back to cited text no. 5
    
6.Engstrom B. The significance of Enterococci in root canal treatment. Odontol Revy 1964;15:87-106.  Back to cited text no. 6
    
7.Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: Its role in root canal treatment failure and current concepts in retreatment. J Endod 2006;32:93-8.  Back to cited text no. 7
[PUBMED]    
8.Gilmore MS. History, Taxonomy, and Biochemical characteristics of Enterococci. The Enterococci: Pathogenesis, Molecular Biology and Antibiotic Resistance. Washington: ASM Press; 2002. p. 2.  Back to cited text no. 8
    
9.Rôças IN, Siqueira JF Jr, Santos KR. Association of Enterococcus faecalis with different forms of periradicular diseases. J Endod 2004;30:315-20.  Back to cited text no. 9
    
10.Tendolkar PM, Baghdayan AS, Shankar N. Pathogenic enterococci: New developments in the 21 st century. Cell Mol Life Sci 2003;60:2622-36.  Back to cited text no. 10
    
11.Lee W, Lim S, Son HH, Bae KS. Sonicated extract of Enterococcus faecalis induces irreversible cell cycle arrest in phytohemagglutinin-activated human lymphocytes. J Endod 2004;30:209-12.  Back to cited text no. 11
    
12.Figdor D, Davies JK, Sundqvist G. Starvation survival, growth and recovery of Enterococcus faecalis in human serum. Oral Microbiol Immunol 2003;18:234-9.  Back to cited text no. 12
    
13.Hartke A, Giard JC, Laplace JM, Auffray Y. Survival of Enterococcus faecalis in an oligotrophic microcosm: Changes in morphology, development of general stress resistance, and analysis of protein synthesis. Appl Environ Microbiol 1998;64:4238-45.  Back to cited text no. 13
    
14.Siqueira JF Jr, Lopes HP. Mechanisms of antimicrobial activity of calcium hydroxide: A critical review. Int Endod J 1999;32:361-9.  Back to cited text no. 14
    
15.Jeansonne MJ, White RR. A comparison of 2.0% chlorhexidine gluconate and 5.25% sodium hypochlorite as antimicrobial endodontic irrigants. J Endod 1994;20:276-8.  Back to cited text no. 15
    
16.World Health Organisation. Summary of WHO guidelines for the assessment of herbal medicines. Herbal Gram 1993;28:13-4.  Back to cited text no. 16
    
17.Vermani K, Garg S. Herbal medicines for sexually transmitted diseases and AIDS. J Ethnopharmacol 2002;80:49-66.  Back to cited text no. 17
    
18.Niamsa N, Sittiwet C. Antimicrobial activity of Curcuma longa aqueous extract. J Pharmacol Toxicol 2009;4:173-7.  Back to cited text no. 18
    
19.Health Benefits of Ajwain. Herbs-Treat and Taste; Available form: http://www.taste.blogspot.com. [Last accessed on 2010 Oct 15].  Back to cited text no. 19
    
20.Gomes BP, Lilley JD, Drucker DB. Associations of endodontic symptoms and signs with particular combinations of specific bacteria. Int Endod J 1996;29:69-75.  Back to cited text no. 20
    
21.Gomes BP, Pinheiro ET, Gadê-Neto CR, Sousa EL, Ferraz CC, Zaia AA, et al. Microbiological examination of infected dental root canals. Oral Microbiol Immunol 2004;19:71-6.  Back to cited text no. 21
    
22.Pinheiro ET, Gomes BP, Ferraz CC, Teixeira FB, Zaia AA, Souza Filho FJ. Evaluation of root canal microorganisms isolated from teeth with endodontic failure and their antimicrobial susceptibility. Oral Microbiol Immunol 2003;18:100-3.  Back to cited text no. 22
    
23.Bystrom A, Claesson R, Sundqvist G. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Endod Dent Traumatol 1985;1:170-5.  Back to cited text no. 23
    
24.Orstavik D, Haapasalo M. Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules. Endod Dent Traumatol 1990;6:142-9.  Back to cited text no. 24
    
25.Nerwich A, Figdor D, Messer HH. pH changes in root dentin over a 4-week period following root canal dressing with calcium hydroxide. J Endod 1993;19:302-6.  Back to cited text no. 25
    
26.Distel JW, Hatton JF, Gillespie MJ. Biofilm formation in medicated root canals. J Endod 2002;28:689-93.  Back to cited text no. 26
    
27.Haapasalo M, Orstavik D. In vitro infection and disinfection of dentinal tubules. J Dent Res 1987;66:1375-9.  Back to cited text no. 27
    
28.Gomes BP, Souza SF, Ferraz CC, Teixeira FB, Zaia AA, Valdrighi L, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J 2003;36:267-75.  Back to cited text no. 28
    
29.Lanicotti R, Gianotti A, Patrignani N, Belleti N, Guerzoni ME, Gardini F. Use of natural aroma compounds to improve shelf life of minimally processed fruits. Trends Food Sci Technol 2004;15:201-8.  Back to cited text no. 29
    

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Correspondence Address:
Hemanshi Kumar
Department of Endodontics, Sudha Rustagi College of Dental Sciences, #1B/17 A, N.I.T. Faridabad, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-0707.108197

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