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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 28  |  Issue : 2  |  Page : 166-170

Antimicrobial activity of different biological extracts as intracanal medicament against Enterococcus faecalis: An in vitro study


1 Department of Endodontic and Conservative Dentistry, People's Dental Academy, People's University, Bhopal, Madhya Pradesh, India
2 Department of Paedodontics and Preventive Dentistry, People's College of Dental Sciences, People's University, Bhopal, Madhya Pradesh, India

Date of Web Publication9-Dec-2016

Correspondence Address:
Muktishree Mahendra
Department of Endodontic and Conservative Dentistry, People's Dental Academy, People's University, Bhopal - 462 037, Madhya Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-7212.195433

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  Abstract 


Introduction: A successful endodontic treatment depends upon complete debridement of microflora from the root canal system. However, due to complex root canal configuration, complete debridement through mechanical instrumentation alone cannot remove entire bacterial load. So the aim is in vitro evaluation of antimicrobial efficacy of biological extracts against Enterococcus faecalis MTCC-439 strain when used as intracanal medicaments. The medicaments used were Nissin, an antibiotic peptide; neem known for its antiseptic properties; platelet rich plasma (PRP) known for its regenerative properties and propolis, a resin extract derived from bees.
Materials and Methods: Sixty single rooted lower premolar teeth which were extracted for orthodontic purpose were collected. Tooth specimens were sectioned at cement-enamel junction with a diamond saw to obtain a standard root length. The root canals of the specimen were instrumented with K3 rotary files followed by inoculation of E. faecalis strains and sealed with dental wax. The specimens were then kept in incubator for 21 days at 37°C and after that randomly divided into six treatment groups: Group I, 5 μL Normal saline; Group II, 5 μL Nisin (Vasta Biotech, Chennai); Group III, 5 μL propolis; Group IV, 5 μL neem; Group V, 5 μL PRP; Group VI, 5 μL Calcium hydroxide. Roots were then incubated for 7 days at 37°C. On 8th day, to evaluate the degree of infection, dentin chips from root canal of specimens were extracted with a sterile 6% K3 rotary file. Kruskal–Wallis test and Mann–Whitney U-test were applied for difference in colony forming units (CFUs) count for different medicaments.
Results: In present in vitro study, Nisin showed no CFU while neem and propolis showed significantly less growth as compared to PRP and calcium hydroxide against E. faecalis.
Conclusions: Nisin outreach propolis and neem in eliminating the E. faecalis when used as intracanal medicaments.

Keywords: Calcium hydroxide; Enterococcus faecalis; herbs; intracanal medicaments; Nisin.


How to cite this article:
Mahendra M, Agrawal N, Munaga S, Tyagi S. Antimicrobial activity of different biological extracts as intracanal medicament against Enterococcus faecalis: An in vitro study. Endodontology 2016;28:166-70

How to cite this URL:
Mahendra M, Agrawal N, Munaga S, Tyagi S. Antimicrobial activity of different biological extracts as intracanal medicament against Enterococcus faecalis: An in vitro study. Endodontology [serial online] 2016 [cited 2019 Mar 26];28:166-70. Available from: http://www.endodontologyonweb.org/text.asp?2016/28/2/166/195433




  Introduction Top


A successful endodontic treatment depends upon complete debridement of microflora from the root canal system.[1] However, due to complex root canal configuration, complete debridement through mechanical instrumentation alone cannot remove entire bacterial load.

Contemporarily use of chemical intracanal irrigators and medicaments are requisite to debride infected tissues and eradicate microorganisms from the root canal system.[2] Moreover, low oxygen tension, less nutrient availability, and enormous bacterial interactions lead to predominant colonization of facultative anaerobic species prevailing in the root canals.[3] In persistent periradicular infections, Enterococcus faecalis had been isolated in about 24%–77% cases that perpetually resulted in failure of root canal therapy. This could be because of the ability of E. faecalis to survive at high alkaline environment and deeper tubular invasion. It grows through adhering on biofilm and colonizes on to the surface.[4]

Calcium hydroxide is a gold standard traditionally when used as intracanal medicaments. Although its antibacterial activity is on wide range of microflora of the root canal, but was found less effective against E. faecalis.[5],[6],[7],[8] Moreover, increasing rates of cytotoxic reactions and inability to eliminate the microorganism from dentinal tubules by commercially available medicaments had laid a need of introduction of novel molecules used as intracanal medicaments. In last few decades, the use of alternative therapeutic agents had considerably increased and are derived from plants, insects, microorganisms, etc.[1],[3],[9]

A natural occurring peptide isolated from strains of Lactococcus lactis termed as Nisin had been recently introduced. This antibiotic peptide is a Class I bacteriocin. It is effective against Gram-positive bacteria and spores,[7],[3] including strains of E. faecalis.[10] Nisin intensively used as a food preservative in over 40 countries and is safe to humans. Recent documents are suggestive that nisin is effective in eradication of E. faecalis from root canals.[11]

Resin extract “Propolis” is collected by bees from poplars and conifers trees and clusia flowers. It contains flavonoids, phenolics, and aromatics compounds that are pharmacologically active and aids in its antimicrobial, anti-inflammatory, antioxidant, and anesthetic activities.[12] In a recent study, when compared with NaOCl, propolis showed almost equal antimicrobial efficacy.[13] Its better antimicrobial and anti-inflammatory properties aid in effective eradication of microbes and can serve as a better intracanal medicament.[14],[15]

Azadirachta indica is a commonly known “Indian neem/margosa tree” or “Indian lilac” in India. This holy tree is of great medicinal value. It contains “isoprenoid” group out of 135 compounds which have anti-inflammatory, antibacterial, antifungal, and immunomodulatory properties.[16]

A biologically active substance platelet rich plasma (PRP) contains high concentration of platelets and growth factors. It also releases thrombocidins, platelet microbicidal proteins, platelet factor-4 chiefly, which are immunomodulatory agents with antimicrobial activity. Further studies had shown the potential inhibitory effect of these agent of PRP on bacterial load.[17]

Thus, the aim of present study is to compare antimicrobial properties of various biological extract Nisin, neem, propolis, and PRP as intracanal medicament when compared with calcium hydroxide.


  Subjects and Methods Top


The medicaments used in the study were calcium hydroxide powder, neem leaves, propolis tablets (forever living products), Nisin (2.5%, Sigma Aldrich, USA), PRP. Materials used were absolute ethanol, brain heart infusion broth, tripticase soy blood agar plates, and E. faecalis (MTCC-439) strain.

Preparation of teeth

Sixty single-rooted permanent mandibular premolars previously extracted for orthodontic purpose, free from caries and developmental defects were included in the study. The crowns were sectioned from the root at cementoenamel junction with a diamond disc under saline irrigation. The specimens were then randomly divided into six groups (n = 10). The working length measurement was done by measuring file length 1 mm less until tip of file was visible at the apical foramen. To standardize, the root were cut to make working length as 10 mm.

The biomechanical preparation of root canals was done using 4% K3 rotary files (No. 25) engine-driven nickel titanium (SybronEndo, USA) in crown down technique for apical size standardization. After that copious intracanal irrigation was done with 2.5% sodium hypochlorite and ethylenediamine tetraacetic acid (17% w/v) using 5 mL luer lock syringe followed by irrigating with 0.9% normal saline solution (Marck Biosciences, India) to remove smear layer.

The specimens were then cleaned in distil water for 30 min in an ultrasonic bath. After rinsing, teeth were stored in sterile water. Then the roots were dried, coated externally with clear nail varnish, and sterilized in an autoclave for 30 min at a temperature of 121°C and at a pressure of 15 psi.

Root canal infection

Each root canal was inoculated with 24 h old cultured broths of bacterial solution up till the canal orifice using a sterile endodontic needle in a microbiological safety cabinet. After inoculation, the samples were kept in cotton plugged test tube and incubated at 37°C for 21 days. Every 3rd day, the canals were reinoculated with fresh bacterial samples.

Root canal medication

The canal contents were aspirated after 21 days of incubation, then rinsed with 5 mL saline and patted dry with sterile paper points. The specimens were then randomly divided into six groups (n = 10 each) for intracanal medicaments:

  • Group I: (control): Normal saline
  • Group II: Calcium hydroxide mixed with normal saline
  • Group III: Nisin mixed with distilled water
  • Group IV: Neem extract
  • Group V: Propolis
  • Group VI: PRP.


Preparation of calcium hydroxide mix-calcium hydroxide intracanal medicament is prepared by mixing powder with normal saline in the ratio of 1:1 to obtain the paste.

Preparation of neem leaf extract-neem leaves, ripped were taken from the medicinal garden of People's naturopathy. To remove dust and debris, leaves were washed in distilled water and shade dried in laboratory. After weighing, neem leaves of 25 g were added to 50 mL of absolute ethanol for soxhlet extraction. Extract obtained was subjected to evaporation of solvent to get extract in crystalline/slurry form which were suitably diluted and used for studies of their antimicrobial activity.

Preparation of aqueous solution of propolis-one tablet of propolis (forever living products) was dissolved in 60 mL of warm normal saline to prepare 1:60 aqueous solution of propolis.

Preparation of aqueous Nisin - 200 mg/mL concentration of Nisin was prepared by dissolving it in distilled water.

Preparation of platelet plasma-prior informed consent was taken to draw 30 cc of blood from a patient with a sterile syringe (DispoVan, Mantri Pharma, India) and then centrifuged (spun) to obtain components into three layers. The bottom layer composed of red blood cells, the middle layer consists of platelets and white blood cells, and the topmost layer consist of plasma.

In all the samples, the prepared medicaments of 5 µL were injected in the root canals and completely filled. The canals were then sealed with sticky wax (Pyrax, India) and incubated at 37°C for 7 days. After 7 days of incubation, the wax was removed from each of the canals entrance. The bacterial samples from each canal were retrieved with sterile paper points and inoculated on brain-heart infusion broth (HiMedia Laboratories, India) and incubated at 37°C for 24 h. After that, each canal was irrigated with 5 mL of saline and sterile paper points were used to dry the canal.

To evaluate the degree of infection of the canal wall and its radicular dentine, specimens of dentine chips were retrieved from entire root canal length of all the specimens using a sterile #25 size 0.06 taper K3XF (SybronEndo, USA) instrument. The dentin chips were transferred by placing files into 0.5 mL of brain-heart infusion broth through sterile Eppendorf tubes and incubated at 37°C for 24 h. After 24 h, 5 µL of solution were inoculated on TSY-blood agar plates from each tube and incubated at 37°C for 24 h to obtain bacterial colony forming unit (CFU) count. Statistical analysis was done using Kruskal–Wallis test in SPSS version 21.0 (IBM Corporation, Armonk, New York, USA) windows with P value considered significant at ≤0.05.


  Results Top


The means CFUs count of the present study were showed in [Table 1] and [Graph 1]. Nisin showed no CFUs of E. faecalis. The negative control is saline group showed 120 CFU (105/mL) of E. faecalis. While neem and propolis showed significantly less CFU count when compared with PRP and calcium hydroxide which is seen in [Figure 1]. Statistically significant difference was present in CFU count when different medicaments used against E. faecalis by Kruskal–Wallis test. Mann–Whitney U-test showed that CFU was higher in saline group followed by PRP, Ca(OH)2, propolis, neem extract, and Nisin. Nisin showed significantly least CFU count among all the medicaments.
Table 1: Mean, standard deviation, minimum and maximum values of Enterococcus faecalis colony forming units in different medicaments in permanent teeth

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Figure 1: Colony forming units seen in treatment groups

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  Discussion Top


Formerly since a decade or more intracanal medicament had been used as an interim appointment dressing. The commonly used commercial synthetic medicaments are calcium hydroxide, phenolic compounds (eugenol and camphorated monochlorophenol), aldehydes (formocresol), halides (iodine potassium iodide), antibiotics, etc.[2] However, majority had been reported for its toxic effect, development of resistant strains and depletion of immune response. This had led the shift in paradigm from synthetic to naturally derived medicaments.

E. faecalis is a Gram-positive cocci, been a facultative anaerobic, it can penetrate deeper inside the dentinal tubules which could possibly results in root canal reinfection.[18] It resist chemico-mechanical instrumentation and can survive as a monoculture inside the root canals without any mutualism with other bacterias. E. faecalis can even withstand high alkaline pH during calcium hydroxide dressing.[19] Thus, rendering a need of an alternative method for eradicating this species and will be beneficial for the prognosis of endodontic treatment.

The present study showed Nisin as the most effective medicament against E. faecalis as there were none of the CFUs with Nisin. Nisin is an antimicrobial peptide, naturally occurring produced by Streptococcus lactis. It is commonly used as a food preservative in meat and dairy industry. Previously, studies had reported the antimicrobial activity of Nisin against E. faecalis both in vitro and in vivo.[12] Nisin exerts its bactericidal activity through pores formation by interacting with a specific molecule “Lipid II” and inhibit cell wall synthesis. The principal component of Gram-positive bacterial cell membrane is Lipid II. At a nanomolecular level, Nisin target this Lipid II as a “docking molecule” to form pores on the cell membrane surface and effectively kills the bacteria.[20],[21] Turner et al. showed significantly lower infected dentinal shaving of E. faecalis with Nisin when compared to calcium hydroxide.[11] According to Somanath et al., when Nisin was compared with chlorhexidine is found to be effective after 1 week interval which was confirmed by Hemadri et al.[22],[23]

Propolis pharmacological active components like flavonoids, phenolics and aromatics are known for its antibacterial properties. In the present study, less amount of E. faecalis CFUs was present with propolis. Previously, many in vitro studies had demonstrated its antibacterial activity against E. faecalis. This suggests that propolis can be used as an alternative intracanal medicament in root canal treatment.[23] The specificity against E. faecalis was accounted on the presence of flavanoids in propolis.[24] Ramani et al. showed propolis was more effective against E. faecalis when compared with chlorhexidine when used as an intracanal medicament.[25] Moreover, when compared with calcium hydroxide, significantly less CFUs were present as documented by Jahromi et al.[26]

In the present study, lesser CFUs of E. faecalis was found with neem extract when compared with PRP and calcium hydroxide. This suggests that neem has better antimicrobial potential against oral microorganisms. Moreover, neem alter bacterial adhesion and colonization as an anti-adherence activity. Its biocompatibility to human periodontal ligament fibroblasts is a key factor favoring its clinical application.[27] According to Hegde et al., neem was more effective than propolis were compared against E. faecalis which was akin to present study.[28]

Calcium hydroxide was ineffective against E. faecalis in the present study. The results were in accordance with the studies done by Harrison et al. and Hemadri et al.[23],[29]E. faecalis is resistant to highly alkaline environment due to the presence of proton pump as a primary resistance mechanism.[30] At pH of 11.5 E. faecalis cannot survive, however calcium hydroxide medicament result in alkalinity of pH 10.3 within radicular dentine as being reported in vitro.[31] This is due to buffering capacity of dentine which provides a decreasing pH from inner to peripheral root dentine.[30]

The regenerative potential of plasma components has been documented considerably during the last two decades. In this study, the CFUs with PRP are very high when compared with other treatment groups. On the contrary, in the available literature, few reports can be found about their antimicrobial effects.


  Conclusions Top


Under the limits of the present study, nisin, propolis, and neem are effective in eliminating the E. faecalis when used as intracanal medicaments while calcium hydroxide, PRP, and normal saline are not effective in eliminating the E. faecalis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Koo H, Rosalen PL, Cury JA, Park YK, Bowen WH. Effects of compounds found in propolis on Streptococcus mutans growth and on glucosyltransferase activity. Antimicrob Agents Chemother 2002;46:1302-9.  Back to cited text no. 12
    
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Ferreira FB, Torres SA, Rosa OP, Ferreira CM, Garcia RB, Marcucci MC, et al. Antimicrobial effect of propolis and other substances against selected endodontic pathogens. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:709-16.  Back to cited text no. 14
    
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24.
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Hegde V, Kesaria DP. 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;25:39-45.  Back to cited text no. 28
    
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30.
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