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ORIGINAL ARTICLE
Year : 2016  |  Volume : 34  |  Issue : 3  |  Page : 210-216
 

Evaluation of antimicrobial effect of azadirachtin plant extract (Soluneem ) on commonly found root canal pathogenic microorganisms (viz. Enterococcus faecalis) in primary teeth: A microbiological study


1 Department of Pedodontics and Preventive Dentistry, College of Dental Sciences and Research Centre, Ahmedabad, Gujarat, India
2 Department of Dentistry, Faculty of Dentistry, SEGi University, Kota Damansara PJU 5, 47810 Petaling Jaya, Selangor, Malaysia

Date of Web Publication25-Jul-2016

Correspondence Address:
Karthik Venkataraghavan
Department of Pedodontics and Preventive Dentistry, College of Dental Sciences and Research Centre, Ahmedabad, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-4388.186741

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   Abstract 

Aim: The aim of this study is to evaluate the antimicrobial activity of Soluneem when used as an irrigating solution along with other commonly used irrigating solution sodium hypochlorite (NaOCl) against Enterococcus faecalis. Materials and Methods: Microorganism used in this study was E. faecalis (Microbial Type Culture Collection 439). Test substance used was Soluneem , which was obtained from Vittal Mallya Scientific Research Foundation (VMSRF), Bengaluru. This study was conducted in a microbiology laboratory (Biocare Research India Pvt., Ltd. Laboratory, Ahmedabad, Gujarat) to evaluate the antimicrobial effect of Soluneem (Azadirachtin) on E. faecalis. Antimicrobial activity testing was performed using the macrobroth dilution method according to the Clinical Laboratory Standards Institute guidelines. All determinations were performed thrice. Results: Minimum bactericidal concentration (MBC) was seen as 2.6% for Soluneem while the same was seen at 0.1% for NaOCl. Independent sample t-test was carried out to compare the MBC of Soluneem and NaOCl, which showed that there was no statistically significant difference between them, i.e., 2.6% Soluneem was as effective as 0.1% NaOCl. Conclusion: Soluneem showed antimicrobial activity against E. faecalis at various concentrations. It was also found that the efficacy of Soluneem at 2.6% concentration and above was relatively similar to that of gold standard irrigating solution (NaOCl) on inhibition of E. faecalis.


Keywords: Antimicrobial efficacy, Enterococcus faecalis, macrobroth dilution, sodium hypochlorite, Soluneem


How to cite this article:
Shah S, Venkataraghavan K, Choudhary P, Mohammad S, Trivedi K, Shah SG. Evaluation of antimicrobial effect of azadirachtin plant extract (Soluneem ) on commonly found root canal pathogenic microorganisms (viz. Enterococcus faecalis) in primary teeth: A microbiological study. J Indian Soc Pedod Prev Dent 2016;34:210-6

How to cite this URL:
Shah S, Venkataraghavan K, Choudhary P, Mohammad S, Trivedi K, Shah SG. Evaluation of antimicrobial effect of azadirachtin plant extract (Soluneem ) on commonly found root canal pathogenic microorganisms (viz. Enterococcus faecalis) in primary teeth: A microbiological study. J Indian Soc Pedod Prev Dent [serial online] 2016 [cited 2019 Nov 22];34:210-6. Available from: http://www.jisppd.com/text.asp?2016/34/3/210/186741



   Introduction Top


Complete debridement and effective disinfection of the root canal space is an important prerequisite for achieving long-term success of nonsurgical endodontics.[1] Microorganisms and their by-products are considered to be the primary etiologic agents in endodontic diseases. Failure, during and after endodontic treatment, is linked to the presence of bacteria in the root canal.[2]

Enterococcus faecalis and Candida albicans are known to be important resistant species in infected root canals, and they may cause treatment failures.[2]

E. faecalis, a Gram-positive cocci, is associated with different forms of periradicular diseases, including primary endodontic infections and persistent infections.[3]

Numerous irrigants have been recommended for use in the treatment of root canal infections. Sodium hypochlorite (NaOCl) has been widely used as an irrigant since its introduction in endodontics by Walker in 1936.[2] NaOCl is extremely toxic to periapical tissues if injected beyond apex.[3]

Hence, there is a need to find a substance which can be used to reduce or eradicate Streptococcus mutans and E. faecalis with least amount of side effects.

A predominant trend in modern dentistry has been to search for biocompatible agents, especially those to be used in direct contact with tissues. In this context, phytotherapy has evolved as a science, and there has been growing interest in evaluating plant extracts with a potential therapeutic application in dentistry.[4]

The antibacterial activity of neem has been evaluated and known from ancient times. It has been considered to have various activities such as astringent, antiseptic, insecticidal, anti ulcer and for cleaning the teeth in pyorrhea and other dental diseases. Apart from this the leaf extract of neem showed superior antiviral and antihyperglycemic activity in vitro and in vivo on animals. It had showed good in vitro broad range antibacterial activity.[5]

Azadiractin is a secondary metabolite present in neem tree seeds.

Soluneem is a unique, world's first water-soluble antimicrobial agent consisting of various limonoids, primarily azadirachtin — A, derived from neem seed kernel. It is produced by a novel patented technology that enhances thermostability and bioavailability, and has a greater shelf life. It is highly stable even at 110°C (thermostable) whereas technical Neem Seed Kernel Extracts are thermolabile. Soluneem is available as off-white, amorphous powder, which could be stored for more than 2 years without loss in bioactivity. It instantly dissolves in water to give a clear solution that is ready for use.[6]

Aim

The aim of this study was to evaluate the antimicrobial activity of Soluneem when used as an irrigating solution along with other commonly used irrigating solution (NaOCl) against E. faecalis.


   Materials and Methods Top


Microorganism used in this study was E. faecalis. The strain was purchased from Microbial Type Culture Collection (MTCC 439), Institute of Microbial Technology (IM-TECH), Chandigarh, India. Soluneem was obtained from Vittal Mallya Research Institute, Bengaluru. The study was conducted in a microbiology laboratory (Biocare Research India Pvt., Ltd. Laboratory, Ahmedabad, Gujarat) to evaluate the antimicrobial effect of Soluneem (Azadirachtin) on E. faecalis, which is responsible for root canal failure.

The substances used in the study were divided into following groups:

  • Group 1: Soluneem
  • Group 2: NaOCl
  • Group 3: Normal saline (negative control).


Antimicrobial activity testing was performed using the macrobroth dilution method according to the Clinical Laboratory Standards Institute guidelines. By adjusting density standards (Densimate Nephelometer, Densimat Biomerieux France), the microorganism suspensions were prepared to equal the density of a 0.5 McFarland standard (1.5 × 108 CFU/ml for bacteria). Cultures of E. faecalis were maintained on brain heart infusion (BHI) broth and agar at 37°C.

Stock solutions were used as follows:

  • Group 1: Soluneem (20 mg/100 ml of distilled water, i.e., 20%)
  • Group 2: NaOCl (3%)
  • Group 3: Normal saline.


Microorganisms were then inoculated as 1.5 × 105 CFU/ml for E. faecalis, in the final concentrations. E. faecalis was incubated at 37°C for 24 h. All determinations were performed thrice. This was done to find out the inhibitory effect of various test substances against E. faecalis.

Minimum inhibitory concentration

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 tubes.

Minimum bactericidal concentration

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 BHI agar plates for E. faecalis, and incubating it at 37°C for 24 h. The MBC was determined to be the lowest concentration that precluded bacterial growth on the BHI agar plate [Figure 1].
Figure 1: Subculturing for minimum bactericidal concentration

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   Results Top


The results obtained were statistically analyzed using one-way ANOVA followed by Tukey honest significant difference (HSD) test.

Normal saline was included as the negative control in the study and it did not have any inhibitory effect against E. faecalis.

About 0.1-10% concentration of Soluneem was tested for their inhibitory effect on E. faecalis. Concentration below 2% showed no inhibitory effect against E. faecalis, whereas at 2% first inhibitory effect was observed [Table 1] and [Graph 1 [Additional file 1]].
Table 1: Correlation between various concentrations of Soluneem solution and microbial load encountered in respective samples (using one-way ANOVA test)

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One-way ANOVA done for Soluneem showed no statistically significant difference between samples of different concentrations but showed no statistically significant difference between samples of same concentrations [Table 2].
Table 2: Statistical analysis through one-way ANOVA test of the data collected for seeing the inhibitory effect of Soluneem. solution on Enterococcus faecalis

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Multiple comparisons using Tukey HSD test showed statistically significant difference between 2% concentration and concentrations 3% and above (P< 0.05, [Table 3]). No statistically significant difference was observed between concentration 3% and above (i.e., P > 0.05, [Table 3]).
Table 3: Multiple comparison between various concentrations of Soluneem by Tukey honest significant difference (HSD) test

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Hence to find out at which particular concentration complete inhibition was obtained, further test was carried out using concentration between 2.5% and 3% [Table 4]. Data were then subjected to one-way ANOVA followed by Tukey HSD test and it was found that maximum inhibition of E. faecalis was at 2.6% concentration and all the concentrations above that were similar [Table 3] and [Graph 2 [Additional file 2]]. The 0.1-3% concentration of NaOCl was tested for their inhibitory effect on E. faecalis. Concentration of 0.1% and above showed an inhibitory effect against E. faecalis [Table 5], [Graph 3 [Additional file 3]] and [Graph 4 [Additional file 4]].
Table 4: Correlation between 2.6% and 3% concentrations of Soluneem. solution and microbial load encountered in respective samples (using one-way ANOVA test)

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Table 5: Correlation between various concentrations of sodium hypochlorite solution and microbial load encountered in respective samples (using one-way ANOVA test)

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One-way ANOVA done for NaOCl showed no statistically significant difference between samples of same concentrations and also there was no statistically significant difference seen between various concentrations [Table 6]. This was followed by Tukey HSD test which showed that there was no statistically significant difference between concentrations 0.1% and above (i.e., P > 0.05, [Table 7]), which means that 0.1% concentration was as effective as concentrations higher than that.
Table 6: Statistical analysis through one-way ANOVA test of the data collected for seeing the inhibitory effect of sodium hypochlorite solution on Enterococcus faecalis

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Table 7: Multiple comparisons between various concentrations of sodium hypochlorite by Tukey highly significant difference test

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MBC was seen as 2.6% for Soluneem while the same was seen at 0.1% for NaOCl.

Independent sample t-test was carried out to compare the MBC of Soluneem and NaOCl, which showed that there is no statistically significant difference between them. Hence, 2.6% Soluneem is as effective as 0.1% NaOCl [Table 8].
Table 8: Independent sample t-test

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   Discussion Top


Root canal irrigation plays a key role in the success of endodontic treatment, as it helps in progressive removal of the smear layer and neutralizes the root canal microbial flora.[7]

Total cleaning of root canal system using mechanical instrumentation is ineffective due to extremely complex root canal anatomy. Irrigants serve several purposes including tissue solvent, disinfection, flushing of gross debris, and lubrication. Proper irrigation of root canal system during endodontic treatment is essential for successful treatment.[8]

Various irrigating solutions have been used in root canal treatment. Among them the most commonly used are NaOCl and chlorhexidine, and this can be attributed to their antimicrobial action.[4]

This in vitro study was designed to assess the antibacterial effects of most commonly used root canal irrigant (i.e., NaOCl) along with a newer herbal extract of neem (Soluneem ) against E. faecalis.

E. faecalis is a facultative anaerobic Gram-positive coccus and is implicated in persistent root canal infections [9],[10],[11],[12],[13] and has been used in several previous studies on the efficacy of endodontic irrigants, especially for its high level of resistance to a wide range of antimicrobial agents.[7]

In this study, MTCC strain was used because it was also utilized in previous in vitro studies investigating the antibacterial effects of endodontic irrigants. BHI medium was used in this study.[1],[14],[15],[16]

NaOCl is a commonly used intracanal irrigating solution, and its antimicrobial properties are attributed to HOCl acid.[17] It has been used for a long time in endodontics as a root canal irrigating solution at different concentrations ranging from 0.5% to 5%. In this study, 0.05-3% NaOCl was used. Several other studies have demonstrated the antimicrobial action of NaOCl on E. faecalis with concentration as low as 0.1% which is in accordance with this study.[7]

Even though antibacterial effects of NaOCl are recognized, the exact mechanism of microbial killing is not well elucidated. When NaOCl is added to water, HOCl acid is formed, which contains active chlorine, a strong oxidizing agent.[18]

However, on the other hand, NaOCl presents negative properties such as corrosion of endodontic instruments, ineffectiveness for some microorganisms when used at low concentrations and not able to differentiate between necrotic and vital tissue when in contact with apical and periapical tissue,[7] it has various other disadvantages such as unpleasant taste and potential weakening of tooth structure by decreasing hardness and structural integrity of dentin within the root canal.[8]

There is a growing interest throughout the oral health-care profession in incorporating herbal products in oral health-care products for eradicating caries, decreasing plaque, and root canal irrigation.[8]

The major advantages of using herbal alternatives are easy availability, cost effectiveness, increased shelf life, low toxicity, and lack of microbial resistance reported so far.[18]

Neem is of particular interest in the field of dentistry for it has a long history treating teeth and gum problems.[3] It has been investigated due to its antimicrobial potential against oral microorganisms.

The presence of active constituents such as nimbidin, nimbin, nimbolide, gedunin, azadirachtin, mahmoodin, margolone, and cyclic trisulfide contributes to the antibacterial activity of neem. These active constituents uncouple mitochondrial oxidative phosphorylation, thus inhibiting the respiratory chain. This resulted in its anti-adherence activity by altering the bacterial adhesion and the ability of the microorganism to colonize thereby causing maximum reduction in adherence of E. faecalis to dentin.[18]

Besides the antimicrobial action, this group of compounds also demonstrates anti-inflammatory function (ability to prevent the production of prostaglandins, especially prostaglandin E1 and 5-hydroxytryptamine) which is a desirable characteristic of the irrigant. An immunomodulatory function has also been suggested for the neem drug that enables antigen presentation to immunocompetent cells.[19]

Use of neem as an endodontic irrigant might be advantageous because it is a biocompatible antioxidant thus not likely to cause severe injuries to patients that might occur via NaOCl accidents.[20]

This study also demonstrated the antimicrobial action of Soluneem on E. faecalis with MIC of 2% and MBC of 2.6%. This is supported by other studies done on neem extracts.[15], 16, [18],[19],[20],[21]

Furthermore, in this study, NaOCl showed bactericidal effect at concentration as low as 0.1%. Soluneem at 2.6% concentration was equally effective as 0.1% NaOCl.


   Conclusion Top


In this study which was done to check the antimicrobial action of Soluneem (Azadirachtin) on most commonly found root canal pathogen, i.e., E. faecalis, it was concluded that Soluneem showed antimicrobial activity against E. faecalis at various concentrations. It was also found that the efficacy of Soluneem at 2.6% concentration and above is relatively similar to that of Gold Standard irrigating solution (NaOCl) on inhibition of E. faecalis.

Limitations

  1. 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 Soluneem as a root canal irrigating solution.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Dubey S, Chaodary M, Gupta P. Comparative study of the antimicrobial efficiency of neem leaf extract, sodium hypochlorite and biopure MTAD: An in vitro study. Indian J Dent Adv 2012;4:740-3.  Back to cited text no. 1
    
2.
Bhardwaj A, Ballal S, Velmurugan N. Comparative evaluation of the antimicrobial activity of natural extracts of Morinda citrifolia, papain and Aloe vera (all in gel formulation), 2% chlorhexidine gel and calcium hydroxide, against Enterococcus faecalis: An in vitro study. J Conser Dent 2012;15:293-7.  Back to cited text no. 2
    
3.
Kumar N, Sidhu P. The antimicrobial activity of Azardirachta indica, Glycyrrhiza glabra, Cinnamum zeylanicum, Syzygium aromaticum, Acacia nilotica on Streptococcus mutans and Enterococcus faecalis — An in vitro study. Endodontology 2011;23:18-25.  Back to cited text no. 3
    
4.
Costa EM, Evangelista AP, Medeiros AC, Dametto FR, Carvalho RA. In vitro evaluation of the root canal cleaning ability of plant extracts and their antimicrobial action. Braz Oral Res 2012;26:215-21.  Back to cited text no. 4
    
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Pai MR, Acharya LD, Udupa N. Evaluation of antiplaque activity of Azadirachta indica leaf extract gel — A 6-week clinical study. J Ethnopharmacol 2004;90:99-103.  Back to cited text no. 5
    
6.
Soluneem TM. An Eco-friendly Water Soluble Bioinsecticide from Neem Seeds. Available from: . [Last cited on 2013 Mar 01].  Back to cited text no. 6
    
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Kumar GV, Uppin V, Shenoy A. Comparison of antibacterial effects of various root canal irrigants on Enterococcus faecalis. World J Dent 2011;2:211-5.  Back to cited text no. 7
    
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Laxmi T. Antibacterial activity of two herbal extracts and 2% sodium hypochlorite against Enterococcus faecalis: An in vitro comparative study. J Chem Pharm Res 2013;5:782-6.  Back to cited text no. 8
    
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Pinheiro ET, Gomes BP, Ferraz CC, Sousa EL, Teixeira FB, Souza-Filho FJ. Microorganisms from canals of root-filled teeth with periapical lesions. Int Endod J 2003;36:1-11.  Back to cited text no. 9
    
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Kuzekanani M, Moaddab S. Isolation of Enterococcus faecalis in previously root-filled canals in Kerman population. Dent News 2006;13:32-6.  Back to cited text no. 10
    
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Pirani C, Bertacci A, Cavrini F, Foschi F, Acquaviva GL, Prati C, et al. Recovery of Enterococcus faecalis in root canal lumen of patients with primary and secondary endodontic lesions. New Microbiol 2008;31:235-40.  Back to cited text no. 11
    
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Gajan EB, Aghazadeh M, Abashov R, Salem Milani A, Moosavi Z. Microbial flora of root canals of pulpally-infected teeth: Enterococcus faecalis a prevalent species. J Dent Res Dent Clin Dent Prospects 2009;3:24-7.  Back to cited text no. 12
    
13.
Singamaneni V, Guna Shekhar M, Himagiri S. In vitro effectiveness of different endodontic irrigants on the reduction of Enterococcus faecalis in root canals. J Clin Exp Dent 2010; 2:169-72. Available from: http://www.medicinaoral.com/odo/volumenes/v2i4/jcedv2i4p169.pdf. [Last cited on 2014 Dec 13].  Back to cited text no. 13
    
14.
Nayak A, Ranganathan N, Sowmya GB, Kishore B, Kudalkar M. Evaluation of antibacterial and anticandidial efficacy of aqueous and alcoholic effect of neem (Azadirachta Indica): An Invitro study. Int J Res Ayurveda Pharm 2011;2:230-5.  Back to cited text no. 14
    
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Bohora A, Hegde V, Kokate S. Comparison of the antibacterial efficiency of neem leaf extract and 2% sodium hypochlorite against E. faecalis, C. albicans and mixed culture-An in vitro study. Endodontology 2010;22: 8-12.  Back to cited text no. 15
    
16.
Ghonmode WN, Balsaraf OD, Tambe VH, Saujanya KP, Patil AK, Kakde DD. Comparison of the antibacterial efficiency of neem leaf extracts, grape seed extracts and 3% sodium hypochlorite against E. feacalis — An in vitro study. J Int Oral Health 2013;5:61-6.  Back to cited text no. 16
    
17.
Ahangari Z, Samiee M, Yolmeh MA, Eslami G. Antimicrobial activity of three root canal irrigants on Enterococcus faecalis: An in vitro study. Iran Endod J 2008;3:33-7.  Back to cited text no. 17
    
18.
Rosaline H, Kandaswamy D, Gogulnath D, Rubin M. Influence of various herbal irrigants as a final rinse on the adherence of Enterococcus faecalis by fluorescence confocal laser scanning microscope. J Conserv Dent 2013;16:352-5.  Back to cited text no. 18
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19.
Dutta A, Kundabala M. Comparative anti-microbial efficacy of Azadirachta indica irrigant with standard endodontic irrigants: A preliminary study. J Conserv Dent 2014;17:133-7.  Back to cited text no. 19
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20.
Hegde V, Kesaria D. Comparative evaluation of antimicrobial activity of neem, propolis, turmeric, liquorice and sodium hypochlorite as root canal irrigants against E. faecalis and C. albicans — An in vitro study. Endodontology 2013;25:38-45.  Back to cited text no. 20
    
21.
Vinothkumar TS, Rubin MI, Balaji L, Kandaswamy D. In vitro evaluation of five different herbal extracts as an antimicrobial endodontic irrigant using real time quantitative polymerase chain reaction. J Conserv Dent 2013;16:167-70.  Back to cited text no. 21
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]



 

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