In vitro evaluation of five different herbal extracts as an antimicrobial endodontic irrigant using real time quantitative polymerase chain reaction.

CONTEXT: Sodium hypochlorite is the most commonly used irrigant but it has disadvantage like high cytotoxicity. So there is a need to find an alternative to 5.25% Sodium hypochlorite against microorganism Enterococcus faecalis and Candida albicans. Literature has shown that these 5 extracts namely Terminalia chebula, Myristica frangrans, Aloe barbadensis, Curcuma longa and Azadaricta indica has good properties which can be used as a potential endodontic irrigant. AIMS: To evaluate the antimicrobial efficacy of various herbal extracts namely Curcuma longa (CL), Azadiracta indica (AI), Aloe barbadensis (AV), Myristica fragrans (MF) and Terminalia chebula (TC) as endodontic irrigant against Enterococcus faecalis and Candida albicans using real-time quantitative polymerase chain reaction (qPCR).
MATERIALS AND METHODS: Eighty-four teeth were extracted and suspended with Enterococcus faecalis and Candida albicans. A preliminary study was first performed to determine the minimum inhibitory concentration of extracts. The irrigating groups were divided into five herbal groups and 2 control groups. After irrigating the teeth the remaining microbial load was determined using qPCR. STATISTICAL ANALYSIS USED: Statistical analysis was performed using Oneway Anova/Kruskal-Wallis test with post-hoc Tukey's HSD and was statistically significant (P < 0.05).
RESULTS: It was shown that Neem was highly efficient to 5.25% NaOCl in reducing Enterococcus faecalis and Candida albicans within the root canals when compared with other extracts.
CONCLUSIONS: Neem leaf extract has a significant antimicrobial efficacy against Enterococcus faecalis and Candida albicans compared to 5.25% sodium hypochlorite.

INTRODUCTION

Pharmacological studies acknowledged the value of medicinal plants as potential source of bioactive compounds.[1] The constant increase in antibiotic resistant strains and side effects caused by synthetic drugs has prompted researchers to look for herbal alternatives. Various natural plant extracts has antimicrobial and therapeutic effects suggesting its potential to be used as an endodontic irrigant.[23]

Microorganisms and their toxic metabolic products are responsible for the development and persistence of apical periodontitis of endodontic origin.[2] Enterococcus faecalis, a facultative anaerobic gram-positive coccus and Candida albicans are the most commonly isolated species in persistent root canal infections.[45]

Sodium hypochlorite (NaOCl) has some undesirable characteristics like tissue toxicity, allergic potential and disagreeable smell and taste.[6] Recently, Murray et al.[2] evaluated Morindacitrifolia juice in conjunction with EDTA as a possible alternative to Sodium hypochlorite. Triphala has also been suggested to be used as another possible alternative.[3]

Earlier studies have used culture-based techniques to detect the presence of bacteria.[7] Low sensitivity of culturing and/or the possibility that remaining bacterial cells have been stressed and is in a viable but not cultivable state is one of its main disadvantage. To provide a solution to these difficulties, bioluminescence bacteria were inoculated into the root canals and quantified using single-tube luminometer.[8] In the past decade a major shift has occurred in oral microbiology from studies based on culturing to one that utilize molecular techniques.[9] Among the most popular molecular techniques to detect bacteria are those based on PCR amplification of the 16S or other ribosomal DNA sequences.[10] In contrast to endpoint PCR methods that essentially provide qualitative data, quantitative real-time PCR (qPCR) detects both the specific gene targets in bacteria and allows quantification of bacteria in samples.[11]

Phytochemical extracts such as Curcuma longa (CT) - Turmeric, Azadiracta indica (AI) -Neem, Myristica fragrans (MF) - Nutmeg, Terminalia chebula (TC) - Myrobolan and Aloe barbadensis (AB) - Aloe vera consists of active ingredients like curcumin,[12] nimbidin,[13] myristic acid,[14] tannins,[15] anthraquinones[16] respectively which have been reported to exert antimicrobial, anti inflammatory and antioxidant properties. However, there is lack of any documentation or data regarding the antibacterial activity of these extracts in endodontics.

Therefore the purpose of this study was to evaluate the antimicrobial efficiency of these 5 extracts against Enterococcus faecalis and Candida albicans.

MATERIALS AND METHODS

Teeth

A pre-existing archive of extracted human teeth was used for this project after ethics institutional review board approval. Eighty-four extracted human, permanent straight single-rooted, mandibular premolars with no caries, apical fractures and resorption were selected and stored in saline. This sample was based on a power analysis performed apriori. In this power analysis, the proportion efficacy to be 95% with an alpha error of 0.05 and a statistical power of 80%.[9] A rotary diamond disc was used to decoronate the teeth below the cementoenamel junction and the length was standardized to 12 mm. In order to standardize the samples, each canal was prepared upto size 30 with ProFile 0.04 taper rotary nickel-titanium instruments (Dentsply Tulsa Dental, Johnson City, TN) using crown-down technique according to manufacturer's instructions. The root apices were coated with nail varnish to seal the apical foramen. The canals were irrigated with 10 ml of 5.25% NaOCl and 10ml of 17% EDTA which was again followed by a final rinse with 10ml of NaOCl.[17] All the specimens were sterilized at 121°C for 15 minutes at 26 psi and stored aseptically in 100% humidity at 30°C until use.

Isolation of micro organisms

Pure strain of Enterococcus faecalis and Candida albicans from American Type Culture Collection (ATCC #29212 and #24433) were used. Respectively cultures were grown overnight at 37°C in brain hear infusion (BHI) broth on a rotary shaker 150 rpm and microbial growth were checked by changes in turbidity at 24 hours.[18]

Phytochemical extracts

Whole plants of TC, MF, CL, AV and AI were obtained (Yucca enterprise, Mumbai). The plant materials were washed, shade dried and powdered in a mechanical grinder. A weighed quantity (500 gm) of the air-dried powdered herbal plants were repeatedly macerated with 500 ml of 99% ethanol and filtered using Whatman filter paper. The ethanol was evaporated and the extracts were concentrated using rotary flash evaporator and stored at 4°C until used in the assay.

Antimicrobial activity test

A preliminary study was first performed in order to standardize the extracts. Cultures were grown overnight at 37°C in BHI broth on a rotary shaker 150 rpm and microbial growth were checked by changes in turbidity at 24 hours. For each extract, sterile test tubes containing 5 ml of BHI broth were inoculated with 5 × 105 E. Faecalis and C.albicans respectively. Serial dilutions were performed. E. Faecalis and C.albicans growth were determined by visual inspection of presence of turbidity. The minimum inhibitory concentration was determined to be 0.33 mg/ml for AV, MF, AI, TC and 1.25 mg/ml for CL for both E. Faecalis and C.albicans.

Contamination of the dentin specimens were carried out for 21 days at 37°C with E. Faecalis (Code A) and Candida albicans (Code B) adjusted to a degree of turbidity 1 according to McFarland scale, which corresponds to a microbial load of 3 × 108 cells/ml referent to an optical density of 550 nm. The samples were recontaminated with fresh broth containing the micro organism every second day under laminar flow.

Irrigassstion procedure

The entire experiment was conducted in duplicate separately for E. faecalis (code A) and C.albicans (code B) of 6 samples each.

Group 1: Irrigation was done with saline (negative control)

Group 2: Irrigation was done with 5.25% NaOCl (positive control)

Group 3: Irrigation was done with CL

Group 4: Irrigation was done with AI

Group 5: Irrigation was done with MF

Group 6: Irrigation was done with AV

Group 7: Irrigation was done with TC.

The contact time for all the irrigation were for 20 minutes.

25-G needle tip was placed to a depth of 1mm short of WL, followed by irrigation with 6 ml of the irrigants at a rate of approximately 3 ml/15 seconds.[8] The canals were then dried with paper points. Dentin samples were obtained at a thickness of 200 μm and 400 μm by using Gates Glidden drills no.4 and no.5. The collected dentin shavings were transferred into 1 ml of sterile Trypticase soy broth and incubated in an anaerobic environment at 37°C for 24 hours and the aliquots were analyzed by qPCR to obtain the threshold cycle (CT) value of the post-operative samples.

Real-Time quantitative polymerase chain reaction

The PCR reaction was performed in a final volume of 20 μl and loaded in an optical 96-well plate, which was then covered with an optical adhesive sheet. The primers used amplified enterococcal DNA sequences in the tuf gene. The PCR conditions were as follows: The initial denaturation was at 94°C for 15 seconds, annealing at a temperature of 55°C and extension at 72°C for 45 seconds. The final extension was at 72 for 5 minutes and then cooled to 4°C until removed. All PCR experiments had positive and negative controls. The qPCR assay was carried out in a thermal cycler (7900 HT Real-time PCR system). The reaction mix contained 16Sr DNA primers, sterile water, template and SYBR Green master mix.

RESULTS

Polymerase chain reaction determines the results in threshold cycle (CT). This is inversely proportional to the amount of target DNA and hence, the number of micro organism in the sample. The pre-operative microbial load inoculated in each sample was 3 × 108 cells/ml which gave a mean CT value of 18.14. CT values and its percentage of remaining bacteria for all the groups against E. Faecalis and C.albicans are listed in [Tables 1 and 2] respectively. Statistical analysis was performed using One way Anova/Kruskal-Wallis test with post-hoc Tukey's HSD and was statistically significant (P < 0.005).

Table 1

Influence of herbal extracts on the bacterial load within the root canals-code A (E. Faecalis)

Table 2

Influence of herbal extracts on the fungal load within the root canals-code B (C.albicans)

In our study of Group A, 0.033% AI was equally efficient to 5.25% NaOCl in reducing E. Faecalis within the root canals when compared with other extracts. The efficiency of the extracts in descending order are as follows: AI, CL, MF, TC and AV. In our study of Group B, 0.033% AI was highly efficient to 5.25% NaOCl in reducing C.albicans within the root canals when compared with other extracts. The efficiency of the extracts in descending order are as follows: AI, CL, MF, TC and AV.

DISCUSSION

The available scientific evidence suggests that irrigating solution must be effective against this organism to become successful in clinical endodontic practice.[56] In qPCR the release of the fluorescent dye during each amplification round allows the products to be detected and measured in real-time when the amplification is first detected.[11]

In this study for root dentin sampling, the model proposed by Haapasalo and Orstavik was modified. This model has been proved to be quite sensitive and is suitable for in vitro testing of intracanal medicaments.[19] The root canal specimens were incubated for 3 weeks which has been shown to produce a dense infection reaching 200 μm to 400 μm into the dentinal tubules.[20]

These 5 extracts used in this study are proven to be safe, containing active constituents that have beneficial property such as antimicrobial, antioxidant and anti-inflammatory activity. It would appear prudent to replace the traditional root canal irrigant with these potential extracts.

Curcumin, a yellow bioactive pigment, is the major constituent of turmeric which has a wide spectrum of biological actions such as anti-inflammatory, antioxidant, antifungal and antibacterial activities.[12] The constituent responsible for MF for its antibacterial activity is myristic acid.[14] The chief constituent; tannin is responsible for the antibacterial action of TC. AV consists of the chemical constituent an thraquinones which is responsible for its antibacterial, antiviral and analgesic effects.[16] The reason for its significant increase in microbial load thereby showing reduced efficacy against both E. faecalis and C.albicans is that though AV possess antibacterial effect, the concentration of substances are affected however, by growth, harvesting, and processing of the aloe leaves therefore it does not have sufficient efficacy due to its dissolution nature. It loses its antibacterial property once it is exposed to the environment.[21]

In our study, 0.033% AI was highly efficient to 5.25% NaOCl in reducing both E. faecalis and C.albicans within the root canals when compared with other extracts. Interest on AI is based onits properties like antibacterial, antifungal, antiviral, antioxidant, anti inflammatory, antipyretic and analgesic effects.[1322] The AI extracts has undergone extensive pharmacological screening and found to have several pharmacological activities due to the presence of several active constituents like nimbidin, nimbin, nimbolide, gedunin, azadirachtin, mahmoodin, margolone and cyclictrisulphide responsible for its antibacterial action.[22] Its anti-adherence activity by altering bacterial adhesion and the ability of organism to colonize has resulted in AI having the maximum reduction in adherence of E. faecalis to dentin.[23]

Use of AI as an endodontic irrigant might be advantageous because it is a biocompatible antioxidant and thus not likely to cause the severe injuries to patients that might occur via NaOCl accidents. Bitter taste associated with this plant can be altered by different formulations due to addition of sweeteners and flavors to increase the patient's compliance and acceptability.

Having used Real-time PCR assay in a root dentin model which is thwe most acceptable methodology, it can be concluded thatneem leaf extract has a significant antimicrobial efficacy against both E. faecalis and C.albicans. However, preclinical and clinical trials are needed to evaluate biocompatibility and safety before AI can conclusively be recommended as an intracanal irrigating solution, but in vitro observation of AI effectiveness appears promising.