The present study was an ex-vivo study conducted in the Department of Pedodontics, Government Dental College, Kozhikode, Kerala, India.

Collection of sample: The infected root canal specimen from 60 primary molar teeth indicated for pulpectomy were collected from children belonging to the age group of 5 to 7 years. A barbed broach with the infected pulp was transferred into a sterile vial with a screw cap containing 1ml saline and taken to microbiological laboratory within 2 hours.

Preparation of 100% neem leaf extract and 40% honey: 100% Neem leaf extract and 40% honey was prepared in the College of Pharmaceutical Sciences, Government Dental College, Kozhikode, India. Fresh neem leaves were collected and washed in sterile distilled water. Leaves were measured up to 25gms in sterile disposable cup to which 50ml of absolute ethanol was added. This mixture was macerated for 1-2minutes and the extract obtained was filtered using filter paper. The coarse residue obtained was again subjected to the same process using 25ml of ethanol. The extracts obtained in the above two steps were mixed together and filtered. The alcohol content in the extracts was removed by allowing the extract to boil on water bath till the volume was about 25ml. The prepared extract was stored in an air tight plastic container.

Pure honey was filled in the measuring jar up to 50ml. Aqueous solution of 40% honey was prepared by adding 20ml of distilled water to it and stirred well. The prepared 40% honey was stored in a glass container.

Microbiological procedure: The microbiological procedure was done in the Department of Microbiology, Government Dental College, Kozhikode, India. The infected sample was then streak cultured on Mitis Salivarius (MS) agar and blood agar which were incubated aerobically at 37⁰C. After incubation for 24 hours, the agar plates were examined for microbial growth [Table/Fig-1].

[Table/Fig-1]:

Microbial colonies in culture media.

1a: Microbial colonies in blood agar.

1b: Microbial colonies in Mitis salivarius agar.

Microbiological analysis: The different microbial colonies were identified by gram staining [Table/Fig-2]. Pure culture of each microbial colony was obtained and was inoculated in glucose broth in test tube separately for 2 hours. To check the antimicrobial efficacy of 40% honey, 100% neem leaf extract and 5.25% NaOCl, agar diffusion method was performed. After 24 hours the largest diameter of the zones of inhibition of microbial growth around each well was measured using a transparent scale [Table/Fig-3].

[Table/Fig-2]:

Identification of microbial colonies by gram staining.

2a: Streptococci, 2b: Gram negative bacilli, 2c: Staphylococci, 2d: Micrococci

[Table/Fig-3]:

Zones of inhibition produced by (→) Neem leaf extract, (→) Honey and (→) Sodium hypochlorite.

3a: Zone of inhibition against Gram negative bacilli, 3b: Zone of inhibition against Streptococci, 3c: Zone of inhibition against stapylococci, 3d: Zone of inhibition against micrococci.

Observations from the microbiological procedure were made and results were statistically analysed and tabulated. Statistical analysis was done by Repeated Analysis of Variance (ANOVA) and Bonferroni method.

Mean and standard deviation of the zones of inhibition produced by each irrigant against each microorganism were calculated and mean was compared by Repeated Analysis of Variance (ANOVA). Bonferroni test was done to find out which among the three had significant difference.

[Table/Fig-4] shows the means of the zones of inhibition produced by the three root canal irrigants used in this study. There was a significant difference among the groups as p-value was <0.001. Bonferroni test showed that the significance was greater between sodium hypochlorite and the other two agents as p-value was <0.001, whereas there was no significant difference between honey and neem leaf extract as p-value was 1.000. This indicates that sodium hypochlorite showed the maximum inhibition to growth of the microorganisms, neem leaf extract and honey showed only minimal inhibition to the growth of microorganisms.

[Table/Fig-5] shows zones of inhibition produced by 40% honey, 100% neem leaf extract and 5.25% sodium hypochlorite against each microorganism isolated. It indicates that sodium hypochlorite showed the maximum inhibition to growth of α hemolytic streptococci, gram negative bacilli, Candida and Staphylococci, neem leaf extract and honey showed only minimal inhibition to the growth of these microorganisms. The p-value was insignificant > 0.05 with the zones of inhibition produced by sodium hypochlorite, honey and neem leaf extract against lactobacilli, spore bearing gram positive bacilli, Micrococci, Enterococci. Thus, sodium hypochlorite, honey and neem were ineffective against lactobacilli, spore bearing gram positive bacilli, Micrococci, Enterococci according to this study.

The infective microorganisms are considered as the main etiologic factor in the development of pulp and periapical lesions. The root canal irrigants aid in the removal of pulpal tissue, dentinal debris and infective microorganisms especially in root canals with complex internal anatomy [5].

Sodium hypochlorite, the most commonly used root canal irrigant has broad spectrum of antimicrobial activity and excellent pulpal tissue dissolving potential. But its cytotoxicity due to its pH of 11-12 causes oxidation of proteins resulting in hemolysis and necrosis. Several NaOCl accidents such as damage to patient’s clothing, patient’s or operator’s eye, air emphysema when injected into root canals and allergic reaction to the irrigant [6,7]. These disadvantages led to the need to identify a biocompatible and effective root canal irrigant.

Honey has a broad-spectrum antibacterial activity suggested by several mechanisms. The hydroxyl ions present in honey as similar to hydrogen peroxide is considered to be responsible for its antibacterial activity. In addition, the osmotic pressure exerted by honey affects the survival of the microbes. The non peroxide components such as complex phenols and organic acids are also responsible for the antibacterial activity [3,8,9]

Neem tree [Azadirachta indica] is a tree in the mahogany family Meliaceae is well known for its antimicrobial and therapeutic effects. The neem bark extract has immunomodulatory activity. The extract from neem bark, leaves, fruits and flowers contain flavonoids, flavonoglycosides, dihydrochalocones and tannins [10–13].

The present study evaluated the effectiveness of 40% honey, 100% neem leaf extract and 5.25% sodium hypochlorite as intracanal irrigant. The neem leaf extract was prepared as suggested by Ghonmode et al., [14] in their study to evaluate the antimicrobial property of neem leaf extract, grape seed extract and sodium hypochlorite against E.fecalis.

The pure cultures of various microorganisms were obtained from the inoculation of the infected root canal specimens. Agar diffusion test is one of the most commonly used tests to study the antimicrobial activity of endodontic irrigants. In our study three wells of 6mm diameter were made in the blood agar. The different root canal irrigants to be evaluated were filled in the different wells. These zones of inhibition signify the presence of antimicrobial activity of the given chemical agent against the selected microorganisms. In the present study the largest diameter of the zone of inhibition was measured using a transparent ruler. Sodium hypochlorite showed the maximum inhibition to growth of the microorganisms followed by neem and honey which showed only moderate inhibition to the growth of microorganisms. The findings of the present study were compared with other experimental studies and tabulated in [Table/Fig-6]. The results of the present study show that NaOCl has maximum antimicrobial activity when compared with neem and honey which is in contrast with the results obtained by the authors shown in [Table/Fig-6]. Neem and honey though not as effective as NaOCl in their antimicrobial activity can still be considered as root canal irrigant due to their biocompatibility with the soft tissues and honey has an added advantage of sweet taste which would increase the patient acceptance.

The limitations of the present study are that chemical agent’s formulation (liquid or gel), its molecular size, diffusion property, viscosity of the agar medium, storage conditions and the incubation time might affect the reliability and reproducibility of the agar diffusion method. This indirectly affects the antimicrobial activity assessed against the test specimens.

This is an era of evidence based research, so in the near future clinical trials comparing the effectiveness of ethnopharmacological products with sodium hypochlorite might bring into limelight the antibacterial effectiveness of these products. The results obtained in this ex-vivo study indicated that sodium hypochlorite is an effective root canal irrigant whereas neem leaf extract and honey have only moderate antibacterial activity against the root canal pathogens. The disadvantage of sodium hypochlorite is that it is cytotoxic, causes tissue irritation, unpleasant taste and damages clothing. Neem leaf extract and honey have an added advantage in this aspect as they are biocompatible to the tissues. The sweet taste of honey further favors the patient acceptance and this plays an important role in case of pediatric patients. The success of endodontic treatment depends on the elimination of root canal pathogens. Sodium hypochlorite has a wide antimicrobial spectrum and it can be recommended as an effective root canal irrigant, but 100% neem leaf extract and 40% honey were not as effective as 5.25% sodium hypochlorite as root canal irrigants.