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Table of Contents
Year : 2018  |  Volume : 6  |  Issue : 4  |  Page : 81-84

Antibacterial efficacy of herbal alternatives and 2% chlorhexidine on biofilm: An in vitro study

1 Department of Conservative Dentistry and Endodontics, Chhattisgarh Dental College and Research Institute, Sundara, India
2 Department of Conservative Dentistry and Endodontics, Maitri College of Dentistry and Research Centre, Anjora, India
3 Department of Oral Medicine and Radiology, Chhattisgarh Dental College and Research Institute, Sundara, Chhattisgarh, India

Date of Web Publication14-May-2019

Correspondence Address:
Dr. Pooja P Bopche
Department of Conservative Dentistry and Endodontics, Bopche Chawl, Balaghat Road, Gondia 441614, Maharashtra.
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/INJO.INJO_1_19

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Aim: To evaluate the efficacy of natural agents for disinfection of root canal system, such as the effect of Azadirachta indica (neem), thyme oil, and 2% chlorhexidine gluconate (CHX) on multispecies biofilm of most commonly found pathogens such as Streptococcus mutans and Enterococcus faecalis in the field of endodontics. Materials and Methods: To visualize the antibacterial effectualness of the methanolic extracts of the medicated plants and CHX, an in vitro dentin model was used. A polymicrobic biofilm was grown on human extracted teeth for 21 days. Microbial load in the form of colony-forming units per milliliter (CFU/mL) after the antimicrobial treatment was calculated. Data were statistically analyzed using analysis of variance and Bonferroni post hoc tests. Results: Plant extracts showed remarkable antibacterial efficacy along with 2% CHX compared with that of control. A significant statistical difference was found with 2% CHX. Conclusion: The methanolic extract of A. indica, thyme oil, and CHX has shown considerable antimicrobial activity against polymicrobic dentinal biofilm of S. mutans and E. faecalis.

Keywords: Azadirachta indica, chlorhexidine gluconate, Enterococcus faecalis, Streptococcus mutans, thyme oil

How to cite this article:
Chandwani N, Bopche PP, Maurya N, Ranka A, Pal A, Fuladi T. Antibacterial efficacy of herbal alternatives and 2% chlorhexidine on biofilm: An in vitro study. Int J Oral Care Res 2018;6:81-4

How to cite this URL:
Chandwani N, Bopche PP, Maurya N, Ranka A, Pal A, Fuladi T. Antibacterial efficacy of herbal alternatives and 2% chlorhexidine on biofilm: An in vitro study. Int J Oral Care Res [serial online] 2018 [cited 2020 Feb 19];6:81-4. Available from: http://www.ijocr.org/text.asp?2018/6/4/81/254620

  Introduction Top

In necrotic root canal systems, bacterial biofilms, which are embedded in an extracellular matrix material,[1],[2] are the main cause of root canal failure (22%–77%). Biofilms are disrupted and the microbial load is reduced by mechanical instrumentation, irrigation with tissue-lytic and microbicidal solutions, and medicaments in the root canal.[3]

The use of antimicrobial irrigating solutions, other disinfecting agents, and medicaments plays a main role in the removal of microorganisms.[3] Chemo-mechanical instrumentation can eradicate a significant number of bacterial colonies from the root canal system.[4] According to current literature, sodium hypochlorite and 2% chlorhexidine seems to be the most commonly used and popular irrigating solutions in endodontics.[5] Chlorhexidine gluconate (CHX) has a wide range of antimicrobial spectrum. At 2% concentration, it may cause deleterious effects if it is expressed beyond the root canal system and it can impair with the healing of periradicular tissue.[6]

A vital interest has been noticed in the past few years in the use of different plant extracts known for their antimicrobial properties against microorganisms causing tooth pathology and root canal failure. Azadirachta indica (neem) is the most popular and traditional medicinal plant.[7] The tree is still regarded as sacred as well as most useful in India. Its fruits, seeds, and even every part give a different medicinal components having antiseptic, antiviral, antipyretic, anti-inflammatory, anti-ulcer, and antifungal properties.[7]

Thyme (Thymus vulgaris) is known for its use in household and industrial purposes. Thyme possesses antiseptic, bronchiolitis, antispasmodic, and antimicrobial properties that make it popular as a medicinal herb and as a preservative for foods.[8],[9] Flavonoids, thymol, eugenol, and aliphatic phenols, namely saponins, luteolin, and tetramethoxylated flavones are the therapeutic potential contents of thyme.[8],[9]

This study aims at the evaluation of antimicrobial efficacy of methanolic extracts of selected medicinal plants such as A. indica (neem) and thyme oil along with 2% CHX against polymicrobic biofilm formed on root canal walls of extracted human teeth.

  Materials and Methods Top

Sample collection and storage

The leaves of neem were collected, then washed with distilled water, and dried under shade for 10–15 days. They were ground in an electric grinder to produce a powder. Thyme oil is easily available in markets, which is sold for commercial purposes.

Preparation of extracts

The powdered material was again dried in sunlight with special considerations for 7h and used for extraction. Veraciously weighed 50g of the powdered leaf sample was mixed with 500mL of methanol and its extract was collected. This process was repeated until only the organic residue was left and then the residue was separated and evaporated in rotary evaporator [Figure 1]. The extracts were kept under most suitable conditions with zero moisture for dryness, leaving behind the thick semisolid residue. This extract was dissolved in dimethyl sulfoxide (DMSO) to get six different concentrations [Figure 2].
Figure 1: Rotary evaporator

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Figure 2: Neem extract

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Biofilm formation

A total of 31 single-rooted single canal, intact, noncarious human anterior teeth with fully formed apices were selected. The pulp chamber was opened with a round bur. The root canals were instrumented using the step back technique with rotary instruments, and canals were prepared to an apical size 30 using ProTaper F3 (Dentsply Maillefer, Ballaigues, Switzerland). During the preparation, 2mL of 3% NaOCl was used as an irrigant. The teeth were then sectioned at the cementoenamel junction as well as vertically along the midsagittal plane into two halves to allow formation of a biofilm. The opposite concave tooth surfaces were then minimally ground to achieve a flat surface.

Microbial culture procedure

The strains used were Enterococcus faecalis (MTCC 439), Streptococcus mutans (MTCC 497). A nutrient agar plate was divided into six wells (6mm in diameter) and inoculums having 1×105 CFU/mL of bacteria were laid on the plate with the bacterial suspension. With the help of a micropipette, 100 µL of the solution of various medicated plant extracts with different concentrations of the medicine was inserted in the wells. The plates were then incubated at 37°C for 24h in an aerobic environment. After overnight incubation, the plates were observed for the zone of inhibition and its diameter (in millimeters) was calculated using a scale. The mean values were recorded after testing each extract for 3–4 times. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration were evaluated by dilution method, namely micro-broth dilution method.

Test procedure

The sterilized tooth sections were kept in 2-mL Eppendorf tubes. The vials containing tooth sections were inoculated with 2mL of a bacterial suspension and incubated at 37°C for 21 days to allow biofilm formation. The samples were taken from each well with a sterile paper point, inoculated onto Mueller-Hinton agar plates, and incubated at 37°C for 24h to check for cell viability and purity of culture.

Sixty sections were then divided into four groups according to the medicaments to be tested. Normal saline was served as negative control.

The four groups (15 each) were divided as follows:

  • Group 1: 2% CHX
  • Group 2: A. indica extract (75mg/mL in 10% DMSO)
  • Group 3: Thyme extract (75mg/mL in 10% DMSO)
  • Group 4: Normal saline

All the samples were immersed in the 2mL of test solutions according to their groups for 10min. Then, the biofilm on the root canal portion was scraped off with the help of a scalpel to gather all the well-attached, deeply seated as well as loosely attached superficial bacteria from the formed biofilm. The scrapings were inoculated on Mueller-Hinton agar plates and incubated for 24h at 37°C for qualitative analysis, where n = 2 for each group.

The quantitative analysis was performed by vortexing the scraped biofilm of the treated tooth samples with sterile saline for a few minutes, followed by serial dilution method and colony-forming unit (CFU) counts, where n = 13 for each group. The mean values and standard deviations (SDs) were calculated.

  Statistical Analysis Top

The collected data were tabulated and analyzed using the Statistical Package for the Social Sciences (SPSS) software, version 17 (SPSS, Chicago, Illinois), for analysis of variance. Multiple comparisons were carried out using Bonferroni post hoc test (difference between the groups).

  Results Top

Both the plant extracts showed considerable antimicrobial activity against S. mutans and E. faecalis in agar diffusion test, whereas 2% CHX was the most consistent of all the tested agents, showing antimicrobial effects against the microorganisms. However, the difference between the CHX and plant extracts was not statistically significant [Table 1] and [Table 2].
Table 1: Comparison between MIC

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Table 2: Comparison between CFU

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  Discussion Top

Chemo-mechanical preparation is of very importance in successful endodontic treatment. Completely eliminating intracanal bacterial populations or reducing them to the levels that can impair periradicular tissue healing is the main microbiologic goal of the chemo-mechanical preparation of infected root canals.[10] The most frequently isolated microorganisms before root canal treatment include gram-negative anaerobic rods, gram-positive anaerobic cocci, gram-positive anaerobic and facultative rods, Lactobacillus species, and gram-positive facultative Streptococcus species.[11] The involvement of microorganisms in our study was basically based on their remarkable presence in endodontic infections as well as their key mark in the failure of the endodontic treatment.

Maximum studies performed to check the antimicrobial efficacy of various medicaments have been performed either on single or multiple culture biofilm of E. faecalis. E. faecalis has been noted to exist with other several groups in infected tooth.[12],[13]S. mutans is markedly noted with preoperative symptoms and inflammatory changes.[14] It may have a major impact on both the initiation of pulpal lesion and subsequent pulpal pathology.[15]

In this study, polymicrobic biofilm model constituting these microorganisms is used to evaluate the antimicrobial activity of methanolic extracts of A. indica and thyme oil along with 2% CHX. CHX is known to be the best with significant difference in terms of elimination of microorganisms against various groups. It is widely preferred in endodontics as both an irrigant and an intracanal medicament because of its antimicrobial efficacy against a wide range of microorganisms. Its efficacy is based on the interaction mechanism between cell wall and negatively charged molecule, which allows the CHX molecule to perforate into the bacteria with its harmful effects.[16]

Plant extracts showed remarkable antibacterial efficacy along with 2% CHX compared with that of control. Methanolic extracts exhibited increase in activity because more phytoconstituents were leached from them in comparison to other extracts. A. indica and thyme oil showed considerable efficacy against biofilm bacteria as compared to control; however, the difference between them was not significant. A. indica has been noted to be of high importance in treating periodontal diseases. The use of neem being an endodontic irrigant may be recommended as a biocompatible antioxidant.[17]

Thyme is an effective antimicrobial agent. Thyme consists of substances such as thymol, terpenes, eugenol, flavones, glycosides of phenolic monoterpenoids, and aliphatic alcohols among other elements. These substances acting alone or in combination may result in a broad spectrum of antimicrobial activity, which was shown for both bacteria and fungi. Thyme possesses antimicrobial activity against Streptococcus at different dilutions of the extracts. These findings also agree with the work of other researchers.[18] Microorganisms present in plant extract develop certain genetic and phenotypic variations in biofilm growth as compared to their planktonic counterpart, which result in significantly less effect of extract as compared with that of 2% CHX.[19],[20],[21] These changes result in increased resistance of the microorganisms to topical antimicrobials.[5]

  Conclusion Top

Within the limitations of this study, 2% CHX showed maximum antimicrobial activity against 3-week-old polymicrobic biofilm formed on tooth substrate. Methanolic extracts of thyme oil and A. indica showed considerable efficacy as an antimicrobial agent. However, further research is required to recommend natural plant extracts as a root canal irrigant.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Nair PNR. Light and electron microscopic studies of root canal flora and periapical lesions. J Endod 1987;13:29-39.  Back to cited text no. 1
Costerton W, Veeh R, Shirtliff M, Pasmore M, Post C, Ehrlich G. The application of biofilm science to the study and control of chronic bacterial infections. J Clin Invest 2003;112:1466-77.  Back to cited text no. 2
Xie Q, Johnson BR, Wenckus CS, Fayad MI, Wu CD. Efficacy of berberine, an antimicrobial plant alkaloid, as an endodontic irrigant against a mixed-culture biofilm in an in vitro tooth model. J Endod 2012;38:1114-7.  Back to cited text no. 3
Ercan E, Ozekinci T, Atakul F, Gül K. Antibacterial activity of 2% chlorhexidine gluconate and 5.25% sodium hypochlorite in infected root canal: In vivo study. J Endod 2004;30:84-7.  Back to cited text no. 4
Mistry KS, Sanghvi Z, Parmar G, Shah S, Pushpalatha K. Antibacterial efficacy of Azadirachta indica, Mimusops elengi and 2% CHX on multispecies dentinal biofilm. J Conserv Dent 2015;18:461-6.  Back to cited text no. 5
[PUBMED]  [Full text]  
Faria G, Celes MR, De Rossi A, Silva LA, Silva JS, Rossi MA. Evaluation of chlorhexidine toxicity injected in the paw of mice and added to cultured l929 fibroblasts. J Endod 2007;33:715-22.  Back to cited text no. 6
Arora DS, Kaur J. Antimicrobial activity of spices. Int J Antimicrob Agents 1999;12:257-62.  Back to cited text no. 7
Briozzo J, Núñez L, Chirife J, Herszage L, D’Aquino M. Antimicrobial activity of clove oil dispersed in a concentrated sugar solution. J Appl Bacteriol 1989;66:69-75.  Back to cited text no. 8
Cosentino S, Tuberoso CI, Pisano B, Satta M, Mascia V, Arzedi E, et al. In vitro antimicrobial activity and chemical composition of Sardinian thymus essential oils. Lett Appl Microbiol 1999;29:130-5.  Back to cited text no. 9
Mustafa M. Antibacterial efficacy of neem (Azadirachta indica) extract against Enterococcus faecalis: an in vitro study. J Contemp Dent Pract 2016;17:791-4.  Back to cited text no. 10
Sundqvist G. Taxonomy, ecology, and pathogenicity of the root canal flora. Oral Surg Oral Med Oral Pathol 1994;78:522-30.  Back to cited text no. 11
Rôças IN, Siqueira JF Jr, Santos KR. Association of Enterococcus faecalis with different forms of periradicular diseases. J Endod 2004;30:315-20.  Back to cited text no. 12
Munson MA, Pitt-Ford T, Chong B, Weightman A, Wade WG. Molecular and cultural analysis of the microflora associated with endodontic infections. J Dent Res 2002;81:761-6.  Back to cited text no. 13
Fouad AF, Barry J, Caimano M, Clawson M, Zhu Q, Carver R, et al. PCR-based identification of bacteria associated with endodontic infections. J Clin Microbiol 2002;40:3223-31.  Back to cited text no. 14
Hahn CL, Best AM, Tew JG. Cytokine induction by Streptococcus mutans and pulpal pathogenesis. Infect Immun 2000;68: 6785-9.  Back to cited text no. 15
Gomes BP, Pinheiro ET, Gadê-Neto CR, Sousa EL, Ferraz CC, Zaia AA, et al. Microbiological examination of infected dental root canals. Oral Microbiol Immunol 2004;19:71-6.  Back to cited text no. 16
Botelho M, Santos AD, Martin J, Carvalho C, Paz M, Azenha C, et al. Efficacy of a mouthrinse based on leaves of neem in the treatment of patients with chronic gingivitis. J Med Plant Res 2008;2:341-6.  Back to cited text no. 17
Gislene GF, Paulo C, Giuliana L. Antibacterial activity of plant extracts and phytochemicals on antibiotic resistant bacteria. Braz J Microbiol 2000;31:314-25.  Back to cited text no. 18
Narayanan LL, Vaishnavi C. Endodontic microbiology. J Conserv Dent 2010;13:233-9.  Back to cited text no. 19
[PUBMED]  [Full text]  
Deans SG, Ritchie GA. Antimicrobial properties of plant essential oils. Int J Food Microbiol 1987;5:165-80.  Back to cited text no. 20
Dorman HGD, Deans SG. Antimicrobial agents from plants: Antibacterial activity of plant volatile oils. J Appl Microbiol 2000;88:308-16.  Back to cited text no. 21


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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