Original Article Comparison of antimicrobial efficacy of Irrigants -Sodium hypochlorite

Original Article
Comparison of antimicrobial efficacy of Irrigants -Sodium hypochlorite, Curcuma Longa (Turmeric), Momordica charantia (Bitter Gourd) in the dentinal tubules infected with Enterococcus faecalis-an in-vitro study
Department of Conservative Dentistry and Endodontics, ITS Dental College and Hospital, Greater Noida, Uttar Pradesh, India
Aims: To Compare the antimicrobial efficacy of Irrigants-Sodium Hypochlorite, Curcuma longa (Turmeric), Momordica charantia (Bitter Gourd) against Enterococcus faecalis.
Materials and Methods: Herbal Extracts of Turmeric and Bitter Gourd were prepared and strains of E. faecalis (ATCC 29212) were grown in Tryptic Soya Broth. Minimum Inhibitory Concentration (MIC) of extracts was determined using broth dilution method. Forty single rooted teeth were prepared. The dentin specimens were contaminated for 21 days with E.faecalis and divided into 4 Groups:- Group-I was irrigated with Bitter Gourd irrigant, Group-II with Turmeric irrigant, Group-III with Sodium Hypochlorite and Group IV-(Control group) with Distilled water. Dentin shavings were collected and transferred into 1 ml of sterile tryptic soya broth and incubated at 37º for 24 hours. Minimal bactericidal count (MBC) was determined. Study was done in triplet. Colonies on plates were counted. The data was tabulated & subjected to statistical analysis using one way Anova and Post-hoc test.
Results: The Curcuma longa (Turmeric) extract had a significant antimicrobial efficacy in reducing E.faecalis as compared to Momordica charantia (Bitter Gourd) extracts.

Conclusions: Curcuma longa (Turmeric) extract was equally efficient to 5% NaOCl in reducing E. Faecalis within the root canals when compared with Momordica charantia (Bitter Gourd) extracts.

Keywords: Endodontic irrigant; E. faecalis; Herbal extracts, Colony Forming Units (CFU)

Introduction :

The main purpose of root canal treatment is to remove microorganisms and their products from the complex root canal system as they cause reinfection and result in endodontic treatment failure. Biomechanical cleaning and shaping of the canal with adequate irrigation is necessary to reduce the number of bacteria in the root canal system and to control the periapical disease.

Various irrigants like sodium hypochlorite, chlorhexidine, citric acid, phosphoric acid etc. have been used for canal disinfections. Irrigant solutions help in disinfecting and lubricating the root canal, flushing out debris from the canal system, and dissolving organic and inorganic tissues.1,2

Haapasalo., et al3. in their study discussed the roles of different irrigants in endodontic treatment. They explained that these have a washing effect and help in reducing friction while using instruments, improving cutting efficiency and cooling the working area.

E. faecalis is a Gram?positive cocci. It resists chemico?mechanical instrumentation and can grow at 100ºC – 450ºC at pH 9.6. It plays a major role in the etiology of persistent periradicular lesions after root canal treatment It has the capacity to produce biofilms where they aggregate and co-aggregate with other micro-organisms and are embedded in extracellular matrix.4, 5. It is non-fastidious and easy to culture. It has been previously shown to infect dentinal tubules rapidly and persist within it as a mono infection for up to 10 days without any nutrition.6,7, 8, 9, 10

Several chemicals and therapeutic agents are used to disinfect the root canal system but complete sterilization of pulp space is not always achieved due to complex anatomy. Sodium hypochlorite (NaOCl) has been widely used as an irrigant to eliminate E.faecalis from root canal space and dentinal tubules since its introduction in endodontics by Alfred Walker in 1936.11
Sodium hypochlorite has excellent properties of tissue dissolution and antimicrobial activity. Grossman and Meiman12 were one of the first to mention the use of sodium hypochlorite (NaOCl) as an endodontic irrigant in their investigations in dissolving pulp tissue and this role of hypochlorite is one of the most important aspects.
Dunavant et al.13 have showed that concentrations of 1% and 6% sodium hypochlorite were the most effective irrigant against E faecalis biofilm.
The most widely used endodontic irrigant is 0.5% to 6% sodium hypochlorite (NaOCl), because of its bactericidal activity and ability to dissolve vital and necrotic organic tissue.14,15,16
Radcliffeet et al.17 and Gomes et al.18 have evaluated the effectiveness of five concentrations of NaOCl (0.5%, 1%, 2.5%, 4% and 5.25%) and found that all irrigants were effective in killing E. faecalis, but at different times.

Sodium hypochlorite is extremely toxic to periapical tissues if injected beyond the apex of the teeth. It has some disadvantages like – Unpleasant smell & taste, corrosive to instruments, Allergic potential, Inability to remove the smear layer, reduction in elastic modulus and flexural strength of dentin. 19, 20 It alters the properties of dentin due to the dissolving action of NaOCl to the organic material such as dentin collagen. 21 It is very toxic to vital tissues due to the release of chlorine and causes pain, swelling and haemorrhage.22, 23

To overcome the problems associated with currently used irrigants, the use of natural plant extracts as endodontic irrigants have become popular due to easy availability, low toxicity, less expensive and better tolerated by patients. In dentistry, phytomedicines have been used as anti-inflammatory, antibiotic, analgesic, and sedative agents and suggesting their potential to be used as an endodontic irriganat. Hence, it seems beneficial to evaluate the antibacterial efficiency of Turmeric and Bitter Gourd extracts when used as root canal irrigants on E. faecalis.

Curcuma longa known as Turmeric belongs to family Zingiberaceae. The main component of turmeric is Curcuminoids which contains curcumin (diferuloylmethane), demethoxycurcumin, and bisdemethoxycurcumin. The curcumin is responsible for biological properties such as anti-inflammatory, antioxidant, antifungal, anti-carcinogenic, antimutagenic, anticoagulant, antidiabetic, antifertility and antibacterial activities.24, 25, 26 Turmeric is useful for bronchial asthma & chronic cough. It makes the gums & Teeth strong, provides relief from toothache, Gingivitis & periodontitis.

Momordica charantia (Bitter Gourd) belongs to the family of Cucurbitaceae. It contains insulin-like peptides, alkaloids and mixture of steroid saponins, known as charantin. Bitter Gourd is an antipyretic, anti-viral, anti-bacterial, anthelmintic and appetizer. It treats diabetes, lowers cholesterol, strengthens the immune system, cures skin & blood diseases, ulcers etc
Hence, the aim of the study was to evaluate the antimicrobial efficacy of herbal irrigating extracts of Turmeric and Bitter Gourd which would be more efficacious and less irritating to the periapical tissues compared to Sodium hypochlorite.
Null Hypothesis: There is no difference in the antimicrobial efficacy among Sodium hypochlorite, Curcuma Longa (Turmeric) and Momordica charantia (Bitter Gourd).

Material & Methods –
Preparation of Herbal Extracts : Whole rhizome of Turmeric and Bitter Gourd were washed and dried in shade for one week and powdered in a mechanical grinder. A 25 gms. of Herbal powder were macerated with 50 ml of ethanol. Extracts were filtered by using Whatman filter paper and concentrated by evaporating ethonal till the volume about 25 ml. and stored in conical flasks under 4oC.
The E. faecalis strain was grown aerobically in Tryptic Soya Broth. The density of the organism was adjusted equal to that of 0.5 McFarland standard (1.5 x 108 CFU/ml). McFarland was used as a reference to adjust the turbidity of microbial suspension so that the number of microorganisms will be within the given range.
Antimicrobial activity test : For each extract, sterile test tubes containing 5 ml of sterile broth were inoculated with E. faecalis. 10 fold serial dilutions for each extract were performed and E. faecalis growth were determined by visual inspection of presence of turbidity. The minimum inhibitory concentration (MIC) was determined. The minimum inhibitory concentration (MIC) was defined as the lowest concentration that did not result in any visible growth of the microorganisms compared with the growth in the control tube.
Teeth preparation : Forty single rooted permanent teeth were extracted and radiographed to confirm the presence of patent canal. Teeth were decoronated with diamond disc and instrumented till 40k – file. The root apices were coated with nail varnish to seal the apical foramen. 10 ml of 5% NaOCL and 10 ml of 17% EDTA were used for cleaning and shaping. All teeth were sterilized at 121°C for 15 minutes and stored aseptically in 100% humidity at 30°C until use.
Teeth apices were sealed with composite resin and the root surface were covered with a double layer of nail polish to prevent leakage of the microbial suspension and also to maintain the extracts within the root canal.

Contamination of Dentin Specimen : Contamination of the dentin specimens were carried out for 21 days at 37°C with E. faecalis. The entire experiment was divided into 4 Groups and each group was irrigated with respective irrigants as per MIC concentration. The contact time for all the irrigation were 20 minutes.
Group I and Group II were irrigated with herbal irrigants – Momordica charantia (Bitter Gourd) and Curcuma longa (Turmeric) respectively, Group III was irrigated with conventional irrigants (Sodium Hypochlorite) and Group IV (Control group) with Distilled water.
All root canals were dried with paper points. Dentin shavings were collected by using Gates Glidden drills and transferred into 1 ml of sterile Tryptic soya broth and incubated at 37ºC for 24 hours. The minimum bactericidal concentration (MBC) was determined by spreading samples from each tube with a concentration equal, or higher than the MIC (i.e. tubes that showed no visible growth) onto the surface of blood agar plates for E. faecalis, and incubating it at 37°C for 24 hrs to 48 hrs. Study was done in triplet. Colonies on plates were counted. The data was tabulated & subjected to statistical analysis using one way Anova and Post-hoc test.
Result :
Irrigant Study 1 Study 2 Study 3 Total Mean ± SD P value
Momordica charantia (Bitter Gourd) 72000 78000 70000 220000 73330 ± 4163.33 0.002
Curcuma longa (Turmeric) 68000 60000 57000 185000 61660 ± 5684.24
Sodium Hypochlorite (NaOCl) 62000 59000 50000 171000 57000 ± 6244.99
Distilled Water 80000 75000 80000 235000 78330 ±2886.75

The above table showed that there is a significant difference in mean organisms growth among the four groups (P=0.002) using ANOVA test.

Graph showing Comparison of mean organism growth among the four groups
Group I – Teeth irrigated with Momordica charantia (Bitter Gourd) showed mean colony counts of organisms of 73330 ± 4163.33 which is more than Curcuma longa (Turmeric) (Group II) and Sodium Hypochlorite (NaOCl) (Group III) and less than Distilled Water (Group IV)

Group II – Teeth irrigated with Curcuma longa (Turmeric) showed mean colony counts of organisms of 61660 ± 5684.24 which is more than sodium hypochlorite (NaOCl) (Group III) and less than Momordica charantia (Bitter Gourd) (Group 1) and Distilled water (Group IV).

Group III: Teeth irrigated with Sodium Hypochlorite (NaOCl) showed mean colony count of organisms of 57000 ± 6244.99 which is significantly less when compared to Momordica charantia (Bitter Gourd) (Group I), Curcuma longa (Turmeric) (Group II) and distilled water irrigated group (Group IV).

Group IV: Teeth irrigated with distilled water showed mean colony count of organisms of 78330 ±2886.75 which is significantly more when compared to Momordica charantia (Bitter Gourd) (Group I), Curcuma longa (Turmeric) (Group II) and Sodium Hypochlorite (NaOCl) (Group III).

Further Post-Hoc Bonferroni analysis showed that there is significant mean difference of organisms growth which is found to be more in Bitter Gourd and Distilled water as compared to NaOCl and Turmeric.

Comparison of mean differences between various irrigants Mean Difference Std. Error P value
Sodium Hypochlorite (NaOCl) Curcuma longa (Turmeric) -4666.667 4020.779 1.000
Momordicacharantia (Bitter Gourd) – 6333.333 4020.779 0.022
Distilled Water -21333.333 4020.779 0.004
Curcuma longa (Turmeric) Momordicacharantia (Bitter Gourd) -11666.667 4020.779 0.119
Distilled Water -16666.667 4020.779 0.019
Momordica charantia (Bitter Gourd) Distilled Water -5000.000 4020.779 1.000

Sodium Hypochlorite (NaOCl) showed significantly less colony forming units (CFU/ml count), when compared with all tested plant extracts. This was followed by a Curcuma longa (Turmeric) which showed significantly less CFU/ml count when compared with Momordica charantia (Bitter Gourd) and Distilled water. Distilled water (Control) showed the significantly highest CFU/ml count when compared to all the tested groups.

The Curcuma longa (Turmeric) extract has a significant antimicrobial efficacy and equally efficient to Sodium Hypochlorite in reducing Enterococcus faecalis.

Discussion :

In the endodontic treatment root canal irrigants helps in removing smear layer and neutralizing the root canal microbial flora. In the present study, we have used extracts of Momordica charantia (Bitter Gourd) and Curcuma longa (Turmeric) as herbal irrigants. These plants have been tested for their medicinal properties for the treatment of various human ailments. However, the studies proving their effectiveness as an endodontic irrigants are very rare. Hence, the purpose of this study was to compare these plant extracts for their antimicrobial effectiveness with NaOCl against E.faecalis.

In this study, the single rooted teeth with single canal were selected to exclude the anatomical variations and complexity factors. E.faecalis was selected as the test organism because it is easy to grow and rapidly colonizes the tubules.27, 28, 10 It is recognized as the associated pathogen and resistant to NaOCl. It has been used extensively in endodontic research because it has been detected in 63% of post treatment diseases.29

In this study the teeth were inoculated with the organism and incubated for 21 days because E.faecalis can penetrate dentinal tubules to a depth of 300-400 ?m within 3 weeks. Prolonged incubation period increased the number of infected dentinal tubules but depth of penetration of bacteria increases slowly with time.6 (Hapsalo)

The Microbial Culture technique was used to detect E. faecalis inside the root canal because it is widely available method and it allows quantification of all major viable cultivable microorganisms in a sam¬ple.30

The results of the study showed that all experimental groups showed a difference between them and reject the Null Hypothesis.

Unfortunately, none of the reviewed studies tested the antimicrobial efficacy of the Bitter Gourd, but this study has proven that the Bitter Gourd showed less antimicrobial efficacy than Turmeric against E. faecalis.

This study has proven that the Turmeric inhibited the growth of E. faecalis. Such antimicrobial activity may be due to the presence of yellow bioactive component (Curcumin). This study is
in agreement with the findings of Neelakantan et al31 He found in his study that curcumin had significant antibacterial activity against E.faecalis and concluded that the antibacterial activity of curcumin was similar to sodium hypochlorite and thus herbal medicine can be used as an alternative to Sodium Hypochlorite in endodontics for root canal failure.

A study by Gul et al32 on the antibacterial efficacy of turmeric found that ionic, resin and ethanolic fractions of turmeric are 100% effective against all tested gram positive organism, which are resistant to most of the broad spectrum antibiotics used.

Conclusions :
In this present study, Sodium hypochlorite (5.25%) showed the highest antimicrobial effect among all of the tested groups. Curcuma longa (Turmeric) extract is equally efficient to NaOCl in reducing E. faecalis within the root canals when compared with Momordica charantia (Bitter Gourd) extracts. The efficiency of the extracts in descending order is as follows:
NaOCl > Curcuma longa > Momordica charantia > Distilled Water

So, within the limitation of our study, we can conclude that turmeric has a great potential to be used as an intracanal medicament.
Limitation of the study:
1. The present study was an in-vitro study with a limited sample size. Further in-vitro studies with a larger sample size as well as incorporating other microorganisms responsible for root canal infection need to be carried out.
2. In addition to this, long-term in vivo studies also need to be done to establish the role of Curcuma longa (Turmeric) as a root canal irrigating solution.