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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 11  |  Issue : 3  |  Page : 119-123

Comparative evaluation of Calendula officinalis and 2% chlorhexidine against Enterococcus faecalis and Candida albicans


1 Professor and Head of the Department, SRM Dental College, SRM Institute of Science and Technology, Chennai, India
2 Department of Conservative Dentistry and Endodontics, SRM Dental College, Chennai, India
3 Reader, Rajas Dental College and Hospital, Tirunelveli, India
4 Clinical Head, Fathima Clinic, Coimbatore, Tamil Nadu, India

Date of Submission19-Jul-2021
Date of Decision29-Nov-2021
Date of Acceptance29-Nov-2021
Date of Web Publication22-Dec-2021

Correspondence Address:
Dr. Selvanathan M. J. Vinola
SRM Dental College, SRM Institute of Science and Technology, Ramapuram Campus, Bharathi Salai, Ramapuram, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jid.jid_28_21

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   Abstract 

Background: Endodontic infections require effective removal of microorganisms from the root canal system for long-term prognosis. Chlorhexidine (CHX) is one of the most effective irrigants, but it's few drawbacks warrant search for newer alternatives. Aim: The aim of this study is to evaluate the antimicrobial efficacy of Calendula officinalis (CO) and 2% CHX against Enterococcus faecalis (E. faecalis) and Candida albicans (C. albicans). Materials and Methods: The antimicrobial activity of CO extract and 2% CHX against E. faecalis and C. albicans was checked by the agar diffusion method and the zone of inhibition was statistically analyzed. Results: CHX has significantly more antimicrobial activity than CO against E. faecalis and C. albicans. However, CO also possesses reasonable antimicrobial activity against E. faecalis and antifungal activity against C. albicans. Conclusion: Under the limitations of this study, it can be concluded that CO has antimicrobial and antifungal activity against E. faecalis and C. albicans; however, CHX is more potent.

Keywords: Antimicrobial activity, Calendula Officinalis, Candida albicans, chlorhexidine, Enterococcus faecalis


How to cite this article:
Vinola SM, Sekar M, Renganathan SK, Dhiraviam S. Comparative evaluation of Calendula officinalis and 2% chlorhexidine against Enterococcus faecalis and Candida albicans. J Interdiscip Dentistry 2021;11:119-23

How to cite this URL:
Vinola SM, Sekar M, Renganathan SK, Dhiraviam S. Comparative evaluation of Calendula officinalis and 2% chlorhexidine against Enterococcus faecalis and Candida albicans. J Interdiscip Dentistry [serial online] 2021 [cited 2022 Jan 25];11:119-23. Available from: https://www.jidonline.com/text.asp?2021/11/3/119/333337




   Clinical Relevance to Interdisciplinary Dentistry Top


Calendula officinalis can be used as an effective irrigant along with other conventional irrigants.


   Introduction Top


The main reason for endodontic failure is the persistence of microorganisms.[1],[2] Enterococcus faecalis (E. faecalis) and Candida albicans (C. albicans) are the most resistant pathogens found in endodontic failure cases.[3] The presence of E. faecalis and C. albicans was found in primary and refractory endodontic infections.[4] E. faecalis is persistent in cases of apical periodontitis occasionally, even as the only single isolated bacteria.[5],[6] The endodontic biofilms protect the bacteria against the irrigants making it difficult for removal.[7],[8] C. albicans, a yeast has been isolated from the root canal in cases of persistent apical periodontitis both in pure culture and together with bacteria.[9] According to the literature, clinically, C. albicans species grow in vitro over a pH range of 3.0–8.0.[10],[11]

Although shaping and cleaning of the root canal system is done to achieve the biological objective of the complete elimination of bacteria and their endotoxins, there is some residual pulpal tissue, bacteria, fungi, and dentinal debris within the deep-seated irregularities and isthmuses.[3] The complex anatomy of the root canal system demands the use of antimicrobial irrigants along with mechanical preparation of root canal space for effective disinfection.[12] Various irrigants have been used in root canal preparation to remove debris and necrotic pulp tissue and to help eliminate microorganisms that cannot be reached by mechanical instrumentation.[13],[14],[15] One of the most commonly used irrigants is chlorhexidine (CHX).[16],[17],[18]

CHX gluconate, a less malodorous and toxic agent, has been suggested as a potent irrigant based on its antibacterial effects, substantivity, and lower cytotoxicity than NaOCl, whilst demonstrating efficient clinical performance.[19],[20],[21] It has been an irrigant of choice in certain cases of apical periodontitis and endodontic reinfection. CHX is a broad-spectrum antimicrobial agent which possesses the property of substantivity and low-grade toxicity. It is a cationic substance that kills the bacteria by acting at the microbial cell wall or outer membrane and has a wide range of activity against both Gram-positive/Gram-negative bacteria. It has been recommended as a root canal irrigant and medicament in certain cases of open apex, root resorption, foramen enlargement, and root perforation due to its biocompatibility, or in cases of allergy related to bleaching solutions. Due to its drawbacks such as inability to dissolve pulp tissue or remove debris, its use as a routine irrigant is limited. Herbal irrigants have been tried as an alternative to the chemical irrigants with varying results, such as Propolis, Azadirachta Indica (Neem), Aloe vera (Aloe barbadensis miller), Morinda citrifolia (Noni), Triphala and Green tea polyphenols, Salvadora persica solution (Miswak-Siwak), and Allium sativum (Garlic). However, no one irrigant fulfills all the requirements of an ideal irrigant.[22],[23],[24] Due to the constant increase in antibiotic-resistant strains and the side effects caused by synthetic drugs, there is the need for an alternative disinfecting measure. To overcome the disadvantages of currently known irrigants, the use of herbal alternatives is suggested. The rationale of this study was to find an alternative to the currently available CHX irrigant.

Since ancient times, herbal products have been used for their antibacterial, antifungal, antioxidant, and anti-inflammatory action.[4] In dentistry, homeopathic remedies have been proposed for oral ulcers, sialorrhea, neuralgia, temporomandibular joint disorders, xerostomia, and lichen planus.[25],[26] Calendula officinalis (CO) is the extract of the flowers of pot marigold (Asteraceae Family), usually prepared in alcoholic tincture.[27],[28],[29] It has been used for its anti-inflammatory, antipyretic, antitumor, and cicatrizing effects.[30],[31],[32] They contain flavonoids, coumarins, essential oils, carotenoids, glycosides, sterols, and fatty acids.[33] It possesses potent antimicrobial property.[34],[35],[36] Efstratiou et al. evaluated the efficacy of CO against certain clinical microbes and proved to be effective against E. faecalis and C. albicans, which are most predominantly seen in the root canal system.[37] However, its efficacy against current root canal irrigants is not yet done. Thus, the aim of this study is to compare the antimicrobial activity of CO with 2% CHX against E. faecalis and C. albicans by the agar diffusion method.


   Materials and Methods Top


Preparation of test materials or solutions or liquids

The CO extract (SBL CO Mother Tincture Q, SBL Pvt Ltd, India) and 2% CHX (Prevest DenPro, Jammu, India) were used in the study. CO solution was mixed with sterile saline to obtain different concentrations (10, 20, 30, and 50 μL/ml).

Agar well-diffusion test

Agar well diffusion method was used in this study to determine the antibacterial activity of test solutions against E. faecalis and antifungal activity against C. albicans. Pure culture of E. faecalis (American Type Culture Collection [ATCC] 29212) (King Institute, Chennai, India) and C. albicans (ATCC 10231) (King Institute, Chennai, India) were used for the study. Fifty microliters of respective microbial inoculum were taken using a micropipette, and were loaded evenly onto the Mueller-Hinton agar plates. The agar plates were inoculated with the respective microorganisms by three times evenly streaking the swab over the entire surface of the plate rotating the Petri plates at 60° approximately after each application. The periphery of the agar surface was also finally swabbed. Six wells of 7 mm diameter and 4 mm depth were made at an equal distance in each Petri plate, and 50 μL of test solution was added to the wells with the help of micropipettes [Figure 1]. The plates were then incubated at 37°C for 24 h under aseptic conditions. The Petri plates were observed for the zone of inhibition, which were measured using a scale in millimeters under illuminated reflected light. The tests were repeated three times to minimize errors. This was done in accordance with the criteria laid down by the National Committee for Clinical Laboratory Standards. The results were statistically analyzed.
Figure 1: (a) IZ (mm) of Enterococcus faecalis with Chlorhexidine, (b) IZ (mm) of Enterococcus faecalis with Calendula officinalis, (c) IZ (mm) of Candida albicans with Chlorhexidine, (d) IZ (mm) of Candida albicans with Calendula officinalis

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Statistical analysis

The normality tests Kolmogorov Smirnov and Shapiro Wilk test results reveal that the inhibition zone (IZ) does not follow normal distribution. Therefore, to analyze the data nonparametric method was applied. To compare IZ values between groups, the Mann-Whitney U-test was applied. To analyze the data SPSS (IBM SPSS Statistics for Windows, Version 26.0, Armonk, NY, USA: IBM Corp. Released 2019) was used. Significance level was fixed as 5% (α = 0.05)


   Results Top


As shown in the bar diagram [Figure 2], MannWhitney U-test of the data revealed that CHX was found to possess more antibacterial activity which was statistically significant (P < 0.05) compared to CO extract against E. faecalis at 10 μL/well, whereas at concentrations of 20 μL/well, 30 μL/well, and 50 μL/well the difference was not statistically significant. The antifungal activity of CHX was found to be significantly higher than CO extract at all concentrations against C. albicans (P < 0.05).
Figure 2: Bar diagram – Zone of Inhibition (mm)

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   Discussion Top


The removal of the residual pulp, cleaning of the root canal, and complete sanitization of the root canal are the main objectives of chemomechanical preparation.[38] The cleaning process cannot be accomplished without the use of an antimicrobial irrigant due to the persistence of microbes postmechanical root canal preparation.[39],[40] Despite the high success rate of endodontic treatments, clinical data have shown that the presence of biofilms after the chemomechanical process in complex anatomies is not uncommon. Multidrug-resistant microbes such as enterococci are important nosocomial pathogens and a growing clinical challenge. These organisms have developed resistance to virtually all antimicrobials currently used in clinical practice using a diverse number of genetic strategies. The presence of exotoxins and endotoxins in the biofilm leads to the resistance of the microorganisms to the irrigants, thereby leading to the failure of root canal therapy. In this context, the use of irrigants that eliminate smear layer and present antimicrobial activity appears highly desirable.

C. albicans and E. faecalis are the most commonly isolated organisms in failed root canal treatments.[41] They possess an inherent antimicrobial resistance providing the ability to adapt to harsh environmental changes and to grow in root canal walls as biofilm.[6] The biofilms are highly organized structures that consist of bacterial cells enclosed in a self-produced exopolymeric matrix which hinder the penetration of agents into the biofilm, thereby limiting their effectiveness to the superficial layer.[7],[8] They possess the capacity to neutralize immunoglobulins, lyse cells, and tissues.[42] C. albicans is versatile with properties such as adapting to a range of pH, changing gene expression in response to environmental conditions, adhering to a variety of surfaces, producing degradative enzymes, and changing morphologic forms to evade the immune system.[4] Thus, both the microorganisms act as potent barriers for the success of root canal treatment and there is a need for irrigation solution to be augmented with mechanical preparation of the root canal. CHX is one of the most commonly used irrigants for root canal treatments; hence, it was used in this study.[40]

CO is the extract of the flowers of pot marigold (Asteraceae Family). A number of phytochemical studies have demonstrated the presence of several chemical compounds, the main ones being terpenoids, flavonoids, coumarins, quinones, volatile oil, carotenoids, and amino acids.[33] The extract is found to have potent effect as an antimicrobial agent. In this study, the methanolic form of the extract was used due to its efficient antibacterial and antifungal activities as evidenced by Kemper.[32]

The MannWhitney U-test used in the present study revealed that there was no significant difference between CHX and CO at 20 and 30 μL/well, while CHX showed significantly higher antibacterial activity against CO at 10 and 50 μL/well volumes, indicating a volume-dependent antibacterial activity of CO. The antifungal activity of CHX was found to be significantly higher than CO extract at all concentrations against C. albicans (P < 0.05).

The results reveal that extracts of CO leaves were significantly effective against both Gram-positive organisms and fungi. The results were in agreement with the study done by Chakraborthy.[34] The activity can be attributed to the presence of triterpenoids, the three most active compounds of which were the esters of faradiol-3-myristic acid, faradiol-3-palmitic acid, and 4-taraxasterol.[43],[44] Kavitha et al. stated that the action was due to the flavonoid fraction in the extract.[45] When the essential oil of the flowers was tested (using disc diffusion technique) against various fungal strains of C. albicans, CO showed potent antifungal activity (15 μL/disc).[46] Some other reports in the literature have shown varying degree of antibacterial and antifungal activities of CO extracts.[46],[47] Our results are in agreement with the finding of Chakraborthy, stating the presence of profound antimicrobial activity of different forms of CO (petroleum ether, chloroform, ethanol, and water extracts) against Bacillus subtilis, Staphylococcus aureus,  Escherichia More Details coli, Klebsiella pneumoniae, C. albicans, and Aspergillus niger.[34] On the contrary, the author has also stated the decreased antifungal activity of CO against fungal strains. Goyal et al. observed good inhibition effect of C. officinalis extracts against E. coli, Pseudomonas aeruginosa, Enterococcus sp., coagulase-positive Staphylococcus sp., as well as coagulase-negative Staphylococcus sp., C. albicans and Candida parapsilosis. Varying degrees of antimicrobial activity might be due to the variation of sources of microorganisms. Schmidgall et al., 1999 has demonstrated in an ex vivo laboratory model the presence of substantivity in CO.[48] Thus, even though CHX is an eminent irrigant against CO, the presence of substantivity and potent antimicrobial activity of CO makes it an adjuvant for use as a root canal irrigant. However, further properties such as cytotoxicity, penetration depth, and diffusibility of the substance into root dentin have to be analyzed. Further in vitro and in vivo studies should be analyzed before its use in clinical practice as an endodontic irrigant.


   Conclusion Top


Under the limitations of this study, it can be concluded that CO does possess antimicrobial activity but however, CHX still continues to be significantly better. CO can be used as a safe and effective alternative irrigant. However, further in vitro and in vivo studies are required before their usage in addition to endodontic sealer or as an endodontic irrigant.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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