An in vitro evaluation of the antimicrobial efficacy of Curcuma longa, Tachyspermum ammi, chlorhexidine gluconate, and calcium hydroxide on Enterococcus faecalis.

AIM AND
OBJECTIVES: This in vitro study was designed to comparatively evaluate the antimicrobial efficacy of Curcuma longa (turmeric - T1-10%, T2-20%); Tachyspermum ammi (ajwain - A1-10%, A2-20%); 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 (T2-20%) and CHX gluconate gel (hexigel - CHX-1%) showed larger zones of microbial inhibition than C. longa (T1-10%) that were statistically significant (P < 0.05) and were highly significant when compared to T. ammi (ajwain - A1 and A2) and calcium hydroxide.
CONCLUSION: C. longa can be used as intracanal medicament in endodontic failure cases.

INTRODUCTION

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.[2-5] 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,[89] 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 – T1-10%, T2-20%); Tachyspermum ammi (ajwain – A1-10%, A2-20%); CHX gluconate gel (hexigel – CHX-1%) and calcium hydroxide (10%) as intracanal medicaments against E. faecalis.

MATERIALS AND METHODS

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% (T1) and 20% (T2) 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% (A1) and 20% (A2). 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

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 (T2-20%) showed significantly (P < 0.05) more zone of microbial inhibition than C. longa (T1-10%), T. ammi (A1-10% and A2-20%) and calcium hydroxide (10%). Hexigel (CHX-1%) and C. longa (T1-10%) showed significantly larger zones of microbial inhibition than T. ammi (A1-10% and A2-20%) and calcium hydroxide (10%). The size of zone of inhibition between C. longa (T2-20%) and hexigel (CHX-1%) was not significant (P > 0.05). T. ammi (A1-10% and A2-20%) and control group showed no microbial inhibition [Table 1 and Figure 1].

Table 1

Antimicrobial effect of intracanal dressing on Enterococcus faecalis

Figure 1

Antibacterial activity of Curcuma longa (T1-10%, T2-20%), Tachyspermum ammi (A1-10%, A2-20%), Chlorhexidine gluconate gel (CHX-1%) and Calcium hydroxide (10%)

DISCUSSION

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.[2021] 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 (T2-20%) 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% (A1) and 20% (A2) concentrations did not show any antimicrobial effect against E. faecalis.

CONCLUSION

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 (T2-20%) 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.