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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 31  |  Issue : 1  |  Page : 9-12

In silico evaluation of the efficacies of two different medicaments against Enterococcus faecalis


Department of Conservative Dentistry and Endodontics, Rajarajeswari Dental College and Hospital, Bengaluru, Karnataka, India

Date of Web Publication19-Jun-2019

Correspondence Address:
A S Anjum Husna
Department of Conservative Dentistry and Endodontics, Rajarajeswari Dental College and Hospital, No. 14, Ramohalli Cross, Kumbalgodu, Mysore Road, Bengaluru - 560 074, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/endo.endo_88_18

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  Abstract 


Aim: The aim of the study is to evaluate the efficacies of two different medicaments, such as ledermix and licorice pastes, against Enterococcus faecalis using in silico method.
Methodology: Procurement of medicaments is from pharmaceutical shop. Culturing of E. faecalis is done under sterile conditions using brain–heart infusion broth. Bacterial growth check is done at 600 nm for further processing. Medicaments are mixed in double distilled for the analyses. After autoclaving at 121°C, the butylated hydroxyanisole is poured into Petri dishes to solidify. After solidification, wells are punched using sterile punchers. The punched plates are ultraviolet treated for 10 min before inoculating with the organism. About 100 mL of culture sample is inoculated and made a fine spread inside the Petri dish. The prepared medicaments are poured and incubated at 37°C for analysis. After incubation, the clear zone is measured mechanically for antimicrobial effect at the duration of 24 h and 3, 5, and 7 days for virulence factor. The growth of the bacteria is subjected to microscopic analysis. Molecular docking studies are to be carried out using in silico method to find out the action of medicaments against E. faecalis at the molecular level. Groups are Group I: ledermix paste and Group II: licorice paste + Ca(OH)2 at 1:1 ratio.
Results: The zone of inhibition of licorice paste + Ca(OH)2 was higher at 24 and 48 h, whereas ledermix showed less significant zone of inhibition against E. faecalis.
Conclusion: Combination of licorice + Ca(OH)2 completely inhibited the growth of E. faecalis. Licorice + Ca(OH)2 compounds show best results both in Microbial inhibition concentration (MIC) (6 ± 1 cm) and in docking mechanism (−7 K cal/mol). Comparatively, ledermix showed only (4 ± 1 cm) in Microbial inhibition concentration and only (−6.8 K cal/mol) in docking mechanism.

Keywords: Enterococcus faecalis, intracanal medicaments, ledermix, licorice + Ca(OH)2, molecular docking


How to cite this article:
Geeta I B, Anjum Husna A S, Sha S S, Muhammed AM, Moses J. In silico evaluation of the efficacies of two different medicaments against Enterococcus faecalis. Endodontology 2019;31:9-12

How to cite this URL:
Geeta I B, Anjum Husna A S, Sha S S, Muhammed AM, Moses J. In silico evaluation of the efficacies of two different medicaments against Enterococcus faecalis. Endodontology [serial online] 2019 [cited 2019 Oct 17];31:9-12. Available from: http://www.endodontologyonweb.org/text.asp?2019/31/1/9/260679




  Introduction Top


The fundamental goal of root canal treatment is to eliminate bacteria from the root canal and prevent reinfection. A wide range of microorganism presents in the root canal of which Enterococcus faecalis is highly resistant. The ability of E. faecalis to cause periapical disease and chronic failure of an endodontically treated tooth is due to its ability to bind to the collagen of the dentinal tubule and remains viable within the tubule.

Intracanal medicaments such as calcium hydroxide and antibiotic pastes are used to inhibit the microbial growth of which Ca(OH)2 has no potential effect against E. faecalis. Few herbal products such as licorice are gaining popularity due to their biocompatibility, antimicrobial, and anti-inflammatory properties and effective against E. faecalis as intracanal medicament.

This study emphasizes the use of licorice in combination with Ca(OH)2 and compares the antimicrobial effect with ledermix against E. faecalis using in silico method.

Aim

The aim of the study is to evaluate the efficacies of two different medicaments, such as ledermix and licorice pastes, against E. faecalis andunderstand the mechanism of action of herbal medicaments using in silico method of molecular docking technique.


  Methodology Top


  • Procurement and preparation of medicaments as per the manufacture norms
  • Subculturing E. faecalis strain using sterile brain–heart infusion broth
  • Incubation for 24 h at 37°C
  • Bacterial growth checked using ultraviolet–visible spectrophotometer at 600 nm
  • For testing microbial inhibition concentration, the culture is plated on a sterile brain–heart infusion agar medium with different concentrations of the medicaments
  • The plates were incubated 37°C before analysis
  • The microbial inhibition concentration is measured under dark background for the zone of clearance. The mechanism of bactericidal is by in silico method using molecular docking technique.


The study is divided into two groups: Group I: ledermix paste and Group II: licorice paste + Ca(OH)2 at 1:1 ratio.


  Results Top


The diameter of zone of inhibition formed around samples in each group was given in [Table 1]. Combination of licorice + Ca(OH)2 completely inhibited the growth of E. faecalis. Microbial inhibition concentration in each group was given in [Table 2]. Licorice + Ca(OH)2 compounds show best results both in microbial inhibition concentration (6 ± 1 cm) and in docking mechanism (−7 K cal/mol). Comparatively, ledermix showed only (4 ± 1 cm) in Microbial inhibition concentration and only (−6.8 K cal/mol) in docking mechanism.
Table 1: The zone of inhibition of ledermix paste and licorice paste + calcium hydroxide

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Table 2: Binding affinity of ledermix and licorice paste+calcium hydroxide against Enterococcus faecalis

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  Discussion Top


Microbial invasion of the root canal system is time related and bacterial species dependent. Hence, early endodontic treatment of a tooth should minimize the number of microorganisms lodged in the dentinal tubules. E. faecalis are enteric facultative anaerobic Gram-positive cocci that have frequently been associated with persistent endodontic infections. Microorganisms continue to be the most common causative agents in etiology of pulpal diseases. Hence, it is of paramount importance to achieve a root canal space free of pathogenic microbes during endodontic procedures, in order to achieve a predictable treatment outcome. Cleaning and shaping techniques seldom achieve complete cleaning of canal space due to the failure of endodontic instruments in reaching the complex canal intricacies, especially in the apical region. Therefore, canal disinfection protocols assume a prime role during root canal treatment.[1]

Ledermix

Ledermix paste (Lederle Pharmaceuticals) is a combination of 3.2% demeclocycline HCl and a corticosteroid (1% triamcinolone acetonide) in polyethylene glycol base. The function of corticosteroid was to reduce inflammation, and antibiotic agent was added to counteract the reduced immune response and limit the possibility of infection.[2] The concentration of antibiotic is sufficient to limit the growth of bacteria in immediate vicinity of the applied medicament inside the canal space. Although the components of paste are able to diffuse through dentinal tubules and cementum and reach the periodontal ligament space and periradicular tissues, still the levels of demeclocycline are inadequate to suppress the bacterial growth. A modification of ledermix paste is available in cement form consisting of 0.7% triamcinolone, 3% demeclocycline, and calcium salts. It has been used for pulp capping and pulpotomy and as a liner. Ledermix has also shown the favorable result in management of cracked teeth with irreversible pulpitis. When used as an intracanal medicament, it also appears to reduce the incidence of postoperative pain in patients with preoperative pain. Research has shown the absence of any systemic adverse effects or any detrimental changes in periodontal tissues after application of ledermix paste.[3]

Intracanal medicaments such as calcium hydroxide and antibiotic pastes are used to inhibit the microbial growth of which Ca(OH)2 has no potential effect against E. faecalis alone.[4] Few herbal products such as licorice are gaining popularity due to their biocompatibility, antimicrobial, and anti-inflammatory properties and effective against E. faecalis as intracanal medicament.[5] Calcium hydroxide (Ca[OH]2) has been used extensively as an interappointment endodontic medicament. The antibacterial action of Ca(OH)2 is based on its ability to maintain an elevated pH in the root canal space and in the dentinal tubules through the slow release of hydroxyl ions. The ability to maintain high levels of hydroxyl ions in the dentinal tubules is influenced by both the amount of Ca(OH)2 present and by the type of vehicle in which it is delivered.

Recently, computational strategies for structure-based drug discovery offer a valuable alternative to the costly and time-consuming process of random screening. Dihydrofolate reductase (DHFR) plays a vital role in the DNA synthesis by reducing the dihydrofolic acid to tetrahydrofolic acid which is an essential component [Figure 1]. In silico program directed toward the design of DHFR inhibitors opens up novel medicinal approach involving bioinformatics study in predicting applications of the herbal medicaments. DHFR is an enzyme crucial for cell proliferation and cell growth. It uses nicotinamide adenine dinucleotide phosphate as electron donor to reduce dihydrofolate to tetrahydrofolate. In the present docking study, it is seen that herbal compounds bind to DHFR with higher affinity [Figure 2] and [Figure 3] which can be taken further as antimicrobial drug targeting toward DHFR.[6]
Figure 1: The active component of ledermix is triamcinolone, and the active component of licorice is licopyranocoumarin. The target protein for both the components was dihydrofolate reductase

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Figure 2: Docking site of triamcinolone from ledermix

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Figure 3: Docking site of licopyranocoumarin from licorice

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Thus, in this in vitro study, licorice fortified with calcium hydroxide exhibited high antibacterial activity against E. faecalis specious. Furthermore, molecular docking demonstrated high-binding efficacy for licorice with calcium hydroxide. Licorice a principle extract of has been shown to be the key reason for its antibacterial activity. Glycyrrhizin is an active compound in licorice which is also responsible for the anti-inflammatory action of licorice.[6] Licorice extract promotes regulatory T-cells, suggesting that licorice can protect against autoimmune and inflammatory diseases.[7]


  Conclusion Top


Antibiotic agents have been tried with varying success in the management of pulpal and periapical lesions. Since systemic administration of antibiotics has little value due to compromised pulpal blood flow during inflammatory condition, local or topical placement of these drugs inside the root canal appears to be more judicious. One of the most successfully employed drugs is tetracycline class of drugs that have several unique properties other than bacteriostatic action, such as inhibition of clastic cells and matrix metalloproteinases. Thus, the combination of licorice + Ca(OH)2 completely inhibited the growth of E. faecalis. Licorice + Ca(OH)2 compounds show best results both in Microbial inhibition concentration (6 ± 1 cm) and in docking mechanism (−7 K cal/mol). Comparatively, ledermix showed only (4 ± 1 cm) in Microbial inhibition concentration and only (−6.8 K cal/mol) in docking mechanism.

Acknowledgment

The author would like to thank Mr. Aravind Ganessin from Dextrose Technology Pvt. Ltd., for assisting in molecular docking studies and result analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Jyoti A, Amit M, Abhimanyu S, Chirag B. Tetracycline: A useful class of antibiotics for successful endodontics. Int J Ayurveda Pharma Res 2015;3:13-8.  Back to cited text no. 1
    
2.
Kundabala M, Jagadish S, Ramya S. Efficacy of Ledermix as a root canal medicament in symptomatic teeth: A clinical study. J Interdiscip Dent 2014;4:85.  Back to cited text no. 2
    
3.
Pierce A, Heithersay G, Lindskog S. Evidence for direct inhibition of dentinoclasts by a corticosteroid/antibiotic endodontic paste. Endod Dent Traumatol 1988;4:44-5.  Back to cited text no. 3
    
4.
Athanassiadis B, Abbott PV, Walsh LJ. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. Aust Dent J 2007;52:S64-82.  Back to cited text no. 4
    
5.
Ehrmann EH, Messer HH, Adams GG. The relationship of intracanal medicaments to postoperative pain in endodontics. Int Endod J 2003;36:868-75.  Back to cited text no. 5
    
6.
Yamini L, Vijjulatha M. Inhibitors of human dihydrofolate reductase: A computational design and docking studies using glide. J Chem 2008;5:263-70.  Back to cited text no. 6
    
7.
Sedighinia F, Afshar AS, Soleimanpour S, Zarif R, Asili J, Ghazvini K, et al. Antibacterial activity of Glycyrrhiza glabra against oral pathogens: An in vitro study. Avicenna J Phytomed 2012;2:118-24.  Back to cited text no. 7
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]



 

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