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Table of Contents   
ORIGINAL RESEARCH  
Year : 2015  |  Volume : 26  |  Issue : 5  |  Page : 528-532
Antimicrobial and antifungal efficacy of Spilanthes acmella as an intracanal medicament in comparison to calcium hydroxide: An in vitro study


1 Department of Pedodontics, KVG Dental College and Hospital, Dakshina Kannada, Karnataka, India
2 Practitioner, Specialist Pedodontist, International Hospital of Bahrain, Manama, Bahrain

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Date of Submission02-Dec-2014
Date of Decision17-Dec-2014
Date of Acceptance06-Aug-2015
Date of Web Publication17-Dec-2015
 

   Abstract 

Background: Repeated endodontic failures are due to pathogens like Enterococcus faecalis and Candida albicans which are resistant to the common intracanal medicaments like calcium hydroxide (Ca[OH] 2 ).
Aims: To determine the role of commonly used folk medicine, Spilanthes acmella (SPA) against root canal pathogens like E. faecalis, C. albicans, Staphylococcus aureus, Streptococcus sp, and to compare its efficacy with Ca(OH) 2 , a popularly used intracanal medicament.
Materials and Methods: Bacterial strains of S. aureus, Streptococcus sp., E. faecalis, and fungal strains of C.albicans were tested against different concentrations of SPA and Ca(OH) 2 . Seven days old cultures of test organisms were seeded onto agar plates and uniformly spread with a spreader. Five to six wells (8 mm) were made on agar plate to which different concentrations of the test solutions were added. The inoculated plates were kept in an incubator at 37°C for 48 h and inhibition zones were measured.
Statistical Analysis: Results of SPA were compared with results of Ca(OH) 2 statistically using Mann-Whitney U test.
Results: A significant zone of inhibition was obtained with SPA and was found to increase as the concentration increased for C. albicans. SPA showed a significant zone of inhibition at 2-5% and 10% whereas Ca(OH) 2 showed a zone of inhibition only at 10% for E. faecalis. SPA showed a zone of inhibition only at 10%, whereas, Ca(OH) 2 showed a significant zone of inhibition at 5% and 10% for S. aureus and Streptococcus sp., respectively.
Conclusion: SPA possesses remarkable antibacterial and antifungal activity against common root canal pathogens which are responsible for repeated endodontic failures such as E. faecalis and C. albicans when compared with medicaments like Ca(OH) 2 .

Keywords: Calcium hydroxide, intracanal medicament, Spilanthes acmella, zone of inhibition

How to cite this article:
Sathyaprasad S, Jose BK, Chandra H S. Antimicrobial and antifungal efficacy of Spilanthes acmella as an intracanal medicament in comparison to calcium hydroxide: An in vitro study. Indian J Dent Res 2015;26:528-32

How to cite this URL:
Sathyaprasad S, Jose BK, Chandra H S. Antimicrobial and antifungal efficacy of Spilanthes acmella as an intracanal medicament in comparison to calcium hydroxide: An in vitro study. Indian J Dent Res [serial online] 2015 [cited 2019 Dec 6];26:528-32. Available from: http://www.ijdr.in/text.asp?2015/26/5/528/172081
Endodontic therapy is based on nonspecific elimination of intraradicular microorganisms. [1] However, bacteria located in ramifications, isthmi, and other irregularities are likely to escape the effects of chemomechanical procedures and may remain viable, multiplying in period between treatments leading to endodontic failures. [2] Repeated endodontic failures are due to pathogens like Enterococcus faecalis and Candida albicans which are resistant to the common intracanal medicaments like calcium hydroxide (Ca[OH] 2 ). [3] Studies with flower head of Spilanthes acmella (SPA), have shown strong antimicrobial activity against common pathogens as E. faecalis, Streptococcus mutans, Staphylococcus aureus, C. albicans, Escherichia Coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus albus. [4] However, no study has been done so far to test the efficacy of the SPA against the commonly occurring endodontic pathogens. This study aims at testing the antimicrobial and antifungal effect of SPA against common intracanal pathogens and comparing its efficacy with Ca(OH) 2 .


   Materials and methods Top


Bacterial and fungal strains obtained from the Department of Microbiology, were used for the testing antibacterial and antifungal activity of SPA and Ca(OH) 2 .

Investigation design

Plant material and chemical extraction

Flower heads were air dried, and the extract was obtained with petroleum ether (40-60°C) using a Soxhlet apparatus. [5]

A Soxhlet extractor is a laboratory apparatus invented in 1879 by Franz von Soxhlet, [6] originally designed for the extraction of a lipid from a solid material [Figure 1]. However, the extraction was not limited to only lipids. Typically, a Soxhlet extraction is only required where the desired compound has a limited solubility in a solvent, and the impurity is insoluble in that solvent.
Figure 1: Schematic representation of Soxhlet extractor[6]

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Air dried flower heads of SPA were placed inside a thimble made from thick filter paper, which was loaded into the main chamber of the Soxhlet extractor. The Soxhlet extractor was placed onto a flask containing the extraction solvent, petroleum ether (40-60°C). The Soxhlet was then equipped with a condenser.

The solvent was heated to reflux, and the vapour travelled up a distillation arm and floods into the chamber containing flower head. Some of the desired compound from flower heads then dissolved into the warm solvent. When the Soxhlet chamber was almost full, the chamber was automatically emptied by a siphon side arm, with the solvent running back down to the distillation flask. This cycle was allowed to repeat many times, over hours or days and during each of these cycles, a portion of the nonvolatile compounds dissolved into the solvent. After many cycles, the desired compound was concentrated in the distillation flask.

After extraction the solvent was removed, typically by means of a rotary evaporator, yielding the extracted compound. The nonsoluble portion of the extracted flower head remains in the thimble and was usually discarded. [7]

Different concentrations of plant extract (0.1%, 0.4%, 1%, 1.6%, 2-5%, and 10%) were made by dissolving in dimethyl sulfoxide.

Test organisms

Four test organisms, bacterial strains of S. aureus, Streptococcus sp., E. faecalis, and fungal strain of C. albicans were obtained from Department of Microbiology.

Bioassay

Agar cup bioassay was employed for testing antibacterial and antifungal activity of plant extract. The readymade Mueller-Hinton agar and Sabouraud's dextrose agar medium was suspended in distilled water and autoclaved at 121°C, pressure of 15 lb/inc 2 for 20 min. Seven days old cultures of test organisms inoculated on to peptone water and incubated for 4 h, were seeded onto a plate and uniformly spread with a spreader. Five to six wells (8 mm) were made on Mueller-Hinton agar and Sabouraud's dextrose agar plate with sterile cork borer. To each well, different concentrations of test solutions SPA extract and Ca(OH) 2 paste (0.1-l0%) were added. The inoculated plates were kept in an incubator at 37°C for 48 h and inhibition zones were measured. As a positive control, 4 plates contained just the organisms to ensure bacterial life cycle does not become inactive before 24-48 h. SPA extract and Ca(OH) 2 paste placed on two plates that had not been inoculated with bacteria and fungi were used as negative control.

Statistical analysis

All the results were expressed as mean ± standard deviation. Statistical significance was determined by Mann-Whitney U test. P < 0.05 was considered significant.


   Results Top


The outcome of the study was that SPA extract at different concentrations has antimicrobial efficacy against E. faecalis and antifungal efficacy on C. albicans which are responsible for repeated endodontic failures when compared to Ca(OH) 2 .

It was determined by measuring the extent of the zone of inhibition on the culture medium in millimeters.

For Candida albicans

The highly significant statistical difference (P < 0.05) for the zone of inhibition was observed between Ca(OH) 2 group and SPA group at all the concentrations tested. Zone of inhibition for SPA increased as the concentration increased while for Ca(OH) 2 zone of inhibition was present only at 10%. The result shows that SPA is effective against C. albicans in all concentration compared to Ca(OH) 2 [Table 1].
Table 1: Group comparison of values for zone of inhibition of Candida albicans Ca(OH)2 and SPA showing mean and SD (P<0.05)


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For Enterococcus faecalis

Statistically significant difference (P < 0.05) was observed for the zone of inhibition at concentrations of 2-5% and 10% for Ca(OH) 2 group compared to SPA group. No difference was seen at concentrations of 0.1%, 0.4%, 1%, and 1.6% between the two groups. The results show that SPA is more effective than Ca(OH) 2 at concentration 2-5% and 10% [Table 2].
Table 2: Group comparison of values for zone of inhibition of Enterococcus faecalis for Ca(OH)2 and SPA showing mean and SD (P<0.05)


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For Streptococcus sp.

Statistically significant difference (P < 0.05) was observed for the zone of inhibition at concentration of 5% and 10% for Ca(OH) 2 group compared to SPA group. No significant difference was seen at concentrations of 0.1%, 0.4%, 1%, 1.6%, 2%, and 3% between Ca(OH) 2 group and SPA group. The result shows that Ca(OH) 2 is more effective than SPA at concentrations of 10% [Table 3].
Table 3: Group comparison of values for zone of inhibition of Streptococcus sp. for Ca(OH)2 and SPA showing mean and SD (P<0.05)


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For Staphylococcus aureus

Statistically significant difference (P < 0.05) was observed for the zone of inhibition at concentrations of 5% and 10% for Ca(OH) 2 group compared to SPA group. No significant difference was seen at concentrations of 0.1%, 0.4%, 1%, 1.6%, 2%, and 3% between Ca(OH) 2 group and SPA group. The result shows that Ca(OH) 2 is more effective than SPA at concentrations of 5% and 10% [Table 4].
Table 4: Group comparison of values for zone of inhibition of Staphylococcus aureus for Ca(OH)2 and SPA showing mean and SD (P<0.05)


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   Discussion Top


Repeated endodontic failures are said to be due to inefficiency of the root canal medicaments against few pathogens such as E. faecalis and C. albicans which are resistant to the common intracanal medicaments like Ca(OH) 2 . [3],[8]

Ca(OH) 2 , a popular intracanal medicament is known from many decades for its efficiency in eliminating both aerobic and anaerobic bacteria by its ionic dissociation into Ca + and OH ions and for its cytotoxic effect by consequent change in the organic component and in nutrient transport. [3] However, repeated endodontic failures are reported due to the ineffectiveness of this medicament against E. faecalis and C. albicans. [3],[5],[9] According to Lana et al. even after treatment with Ca(OH) 2 , polymicrobial infections with Candida and Streptococcus persisted within the root canals. [10] Estrela et al. showed that Ca(OH) 2 had no antimicrobial effect on E. faecalis and S. aureus. [11] Behnen et al. also suggest that thin preparations of Ca(OH) 2 is not effective in the elimination of E. faecalis from root cana1. [12] Hence, in the quest to find an alternative medicament, extract of the flower head of SPA was sought to check its potential for microbial eradication when used as an intracanal medicament. Hence, the present study was done to evaluate the antibacterial and antifungal efficacy of SPA against common root canal pathogens S. aureus, Streptococcus sp., E faecalis, and C.albicans using agar well diffusion method and compare it with antibacterial and antifungal efficacy of Ca(OH) 2 .

Results of the present study showed that SPA had a significant antibacterial action at different concentrations of 2-5% and 10% for E. faecalis (P < 0.05) whereas Ca(OH) 2 showed antibacterial action only at the higher concentration of 10%. This was in accordance with the studies done by Gomes et al., Haapasalo and Orstavik, and Safavi et al. who showed that E. faecalis was resistant to Ca(OH) 2 . [2],[13],[14] Siqueira and Uzeda demonstrated that Ca(OH) 2 was ineffective in eliminating E. faecalis cells inside dentinal tubules, even after 1-week of contact. [15] The limited effectiveness of Ca(OH) 2 is due to factors such as low solubility and diffusibility, varying alkaline potential of different formulations, dense biofilms of bacteria and the ability of E. faecalis to evade the hydroxyl ions due to their presence in ramifications, isthmuses, and irregularities. [3] Evans et al. showed that Ca(OH) 2 eliminated E. faecalis only when present in low numbers in infected teeth without previous root fillings and not in teeth with previous root fillings where E. faecalis was found in higher numbers. The probable reason that E. faecalis is able to survive intracanal treatment of Ca(OH) 2 could be (a) E. faecalis passively maintain pH homeostasis by cytoplasm's buffering capacity and by the proton pump. (b) As a result of the buffering capacity of dentin, pH of Ca(OH) 2 cannot be maintained constantly at 11.5. [16] However, studies done by Almyroudi et al. compared the susceptibility of intracanal medicaments containing Ca(OH) 2 , found that Ca(OH) 2 was effective against E. faecalis. [l7] Onçag et al. in his study to evaluate the efficacy of different intracanal medicaments also showed that Ca(OH) 2 was effective against E. faecalis. [18] These studies were contradictory to this present study which showed that Ca(OH) 2 was not effective against E. faecalis.

Microbiological investigations have revealed that yeasts can be isolated from root canal together with bacteria [19] and Waltimo et al. showed this in his in vitro study that the most common species isolated from the root-canal were C. albicans. [8] Studies done by Valera et al. and Ferguson et al. have shown that C. albicans are resistant to Ca(OH) 2 . [20],[21] Ashford et al. and Rojas et al. in their study have proved the antifungal property of SPA. [22],[23] Similarly, the effect of SPA on C. albicans was studied at various concentrations and compared with the antifungal property of Ca(OH) 2 .The results of this study showed that SPA was effective against C. albicans for all the concentrations tested (0.1%, 0.4%, 1%, 1.6%, 2-5%, and 10%) and the zone of inhibition was found to increase as the concentration increased. However, for Ca(OH) 2 the antifungal property was present only at higher concentration of 10%. The result showed statistically significant difference in the zone of inhibition between the two medicaments (P < 0.05). Thus, it can be concluded that SPA is effective against C. albicans in all concentration compared to Ca(OH) 2 which is effective only at higher concentrations.

Our results are in accordance with the studies done by Waltimo et al. who concluded that C. albicans was resistant to aqueous Ca(OH) 2 . [8] Study was done by Miglani et al. also showed that Ca(OH) 2 based sealers has less antifungal effect when compared with other sealers. [24] The probable reason could be due to the resistance shown by Candida species in a wide range of pH values. [25] In contrast, Barbosa et al. found that a saturated solution of Ca(OH) 2 was effective against C. albicans. [26]

Coagulase-negative Staphylococci, Streptococci, and P. aeruginosa were most frequently isolated microflora in endodontically treated teeth associated with asymptomatic periapical lesions. [27] Studies were done by Podbielski et al. and Adre et al. have shown the effectiveness of Ca(OH) 2 against these microorganisms. [28],[29]

The antibacterial property of SPA and Ca(OH) 2 against S. aureus and Streptococcus sp. were evaluated in the present study. The results showed SPA antibacterial activity only at 10%, whereas Ca(OH) 2 showed a significant zone of inhibition at 5% and 10%, respectively, (P < 0.05). Hence, Ca(OH) 2 was more effective than SPA on these species of S. aureus and Streptococcus sp. even in lower concentrations. This is in accordance with the study done by Podbielski et al. and Adre et al. who showed that Ca(OH) 2 is effective against Coagulase negative Staphylococci, Streptococci, and P. aeruginosa. [28],[29]

The present study has shown that the flower head extract of SPA possesses remarkable antibacterial and antifungal activity against root canal pathogens like E. faecalis and C. albicans responsible for repeated failures when compared with Ca(OH) 2 . Nakatani and Nagashiwa showed that the antibacterial and antifungal activity of different concentration of SPA extract might be due to the presence of amide spilanthol and alkamides. [30] Krishnaswami et al. and Mukharya et al. also reported the presence of nonvolatile sesquiterpenoids and saponins which may be responsible for antibacterial and antifungal activity of SPA. [31],[32]

A study done by Rani and Murty showed antifungal activity in lower concentrations of 0.1-2 mg. [5] However, our study with a concentration of 0.1-2.0 mg showed less antibacterial and antifungal efficacy. Hence a higher concentrations of 3-10 mg was used for this study. The low activity of extract at lower concentrations may be due to the crude nature of the test solution. [5]

The present study has shown that the flower head extract of SPA possesses remarkable antibacterial and antifungal activity and has the potential for development of an intracanal medicament against the common root canal pathogens that cause repeated failures in both primary and permanent dentition. However, this study is in a preliminary stage, further investigations are necessary to identify the bioactive principles and their mechanism of action on inhibition of these bacterial and fungal agents.


   Conclusion Top


The following were the significant finding in our study group:

  • Flower head extract of SPA possesses antibacterial and remarkable antifungal activity
  • SPA possesses remarkable antibacterial and antifungal activity against common root canal pathogens which are responsible for repeated endodontic failures like E. faecalis and C. albicans when compared with medicaments like Ca(OH) 2
  • Ca(OH) 2 was effective against S. aureus, Streptococcus sp. but ineffective against E. faecalis and C. albicans at low concentrations.
  • However, further research and studies are needed to highlight the antibacterial and antifungal efficiency of SPA before its use as an ideal intracanal medicament.
Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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[PUBMED]    
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Correspondence Address:
Savitha Sathyaprasad
Department of Pedodontics, KVG Dental College and Hospital, Dakshina Kannada, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.172081

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]

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