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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 28  |  Issue : 2  |  Page : 137-142

Efficacy of calcium hydroxide, mushroom, and Aloe vera as an intracanal medicament against Enterococcus faecalis: An in vitro study


1 Department of Conservative Dentistry and Endodontics, Dayananda Sagar College of Dental Sciences, Bengaluru, Karnataka, India
2 Department of Botany, Bangalore University, Bengaluru, Karnataka, India

Date of Web Publication9-Dec-2016

Correspondence Address:
D V Swapna
Department of Conservative Dentistry and Endodontics, Dayananda Sagar College of Dental Sciences, Bengaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-7212.195427

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  Abstract 


Introduction: To evaluate and compare antimicrobial efficacy of calcium hydroxide, extracts of mushroom and Aloe vera leaves against Enterococcus faecalis, and to assess the minimal inhibitory concentration (MIC) of extracts of mushroom and A. vera against E. faecalis.
Materials and Methods: Ninety freshly extracted single-rooted teeth were decoronated and canals enlarged up to F3 (ProTaper). The samples were sterilized and infected with E. faecalis and incubated for 21 days. Teeth samples were then divided into three groups. Group 1: calcium hydroxide, Group 2: mushroom extract, and Group 3: A. vera extract. At the end of 1, 3, and 5 days, microbiological sampling and culturing were done from the root canal dentinal shavings obtained and colony forming units (CFUs) were counted. The MIC was determined for two plant extracts against E. faecalis strains using ELISA microdilution method. Statistical analysis was done using analysis of variance technique and multiple comparisons were done using (post hoc test) Bonferroni test.
Results: The number of CFUs was statistically significant in all the groups. Percentage reduction of CFUs was highest in mushroom followed by A. vera and calcium hydroxide. MIC for calcium hydroxide was 40 mg/ml, A. vera extract 60 mg/ml, and for mushroom extract 40 mg/ml.
Conclusions: Antibacterial activity of the mushroom extract was highest followed by A. vera extract and then calcium hydroxide, at all time periods tested in this study. Antibacterial activity of all the groups increased with time.

Keywords: Aloe vera; calcium hydroxide; Enterococcus faecalis; minimal inhibitory concentration; mushroom.


How to cite this article:
Kurian B, Swapna D V, Nadig RR, Ranjini M A, Rashmi K, Bolar SR. Efficacy of calcium hydroxide, mushroom, and Aloe vera as an intracanal medicament against Enterococcus faecalis: An in vitro study. Endodontology 2016;28:137-42

How to cite this URL:
Kurian B, Swapna D V, Nadig RR, Ranjini M A, Rashmi K, Bolar SR. Efficacy of calcium hydroxide, mushroom, and Aloe vera as an intracanal medicament against Enterococcus faecalis: An in vitro study. Endodontology [serial online] 2016 [cited 2019 May 25];28:137-42. Available from: http://www.endodontologyonweb.org/text.asp?2016/28/2/137/195427




  Introduction Top


Favorable outcome of endodontic treatment in teeth with apical periodontitis is significantly higher when infection is eradicated effectively before the root canal system is obturated. Even with rigorous procedures, root canal infection may still persist because of the complexity of the root canal system, which is difficult to instrument.[1],[2] Therefore, the use of intracanal medicament in such cases eliminates bacteria that persist even after cleaning and shaping thereby provides an environment conducive for periapical tissue repair.[3]

Various chemicals have been tried as intracanal medicaments. The majority of these preparations except calcium hydroxide is not used routinely in contemporary endodontic practice due to reported toxicity, allergic reactions, and resistance.[4]

Calcium hydroxide has been advocated as an intracanal medicament because of its bactericidal properties. Its high pH (12.5) has a destructive effect on bacterial cell membranes and intracellular protein structure. However, calcium hydroxide is not effective in eliminating Enterococcus faecalis from dentinal tubules.[5],[6]

E. faecalis is associated with different forms of periradicular diseases, both primary (4–44%) and persistence endodontic infections (24–74%). It is a Gram-positive cocci and facultative anaerobe, which can survive extreme pH (9.6), salt concentration and survive a temperature of 60°C for 30 min.[7],[8] Starvation also increases the resistance of the E. faecalis from 1000-fold to 10,000-fold.[9]

Studies have shown that E. faecalis is highly resistant to commonly used intracanal medicament, such as calcium hydroxide (pH 12.5). E. faecalis passively maintains the pH homeostasis. Dentine has also been found to have a buffering effect on high pH, further compromising the antimicrobial effect of calcium hydroxide in the elimination of E. faecalis.[7],[10]

Antibiotics can be considered as an adjunct to endodontic treatment in retreatment and resistant cases. However, the potential for bacterial resistance, the risk of hypersensitivity, allergic reaction, and the potential to mask certain etiological factor limit their use.[4]

In the search of a novel antimicrobial compound, traditional plants have been proved to be a better source. High antimicrobial, anti-inflammatory, antioxidant (due to the presence of wide variety of active phytochemicals, including flavonoids, terpenoids, lignans, sulfides, polyphenolics, carotenoids, coumarins, saponins, plant sterols, curcumins, and phthalides), and biocompatible properties make their use in dentistry more extensive. Judicious use of these herbal formulations has been found to mitigate the E. faecalis count, which could in turn reduce its posttreatment infections. Hence, it can be considered as one of the possible alternatives or a replacement for the synthetic chemical formulations.[11]

Aloe barbadensis Miller (Aloe vera) belongs to the liliaceae family. It contains active components such as curcumin, nimbidin, myristic acid, tannin, and anthraquinone that have anti-inflammatory, antibacterial, antifungal, and antiviral properties.[12] Compared to other natural extracts, A. vera has broad antibacterial activity against various oral pathogens.[13]

Mushrooms contain low molecular weight (LMW) and high molecular weight (HMW) active compounds. Data available from the literature review indicate that LMW and HMW possess medicinal properties such as immune modulatory, anti-inflammatory, antiviral, antioxidant, and antimicrobial properties with higher antimicrobial activity against Gram-positive bacteria.[14],[15]

Hence, the objective of this study was to evaluate antimicrobial efficacy of extracts of mushroom and A. vera leaves against E. faecalis and compare it with calcium hydroxide and to evaluate the minimal inhibitory concentrations (MICs) of these herbal extracts.


  Subjects and Methods Top


Pure strains of (ATCC 29212) E. faecalis were obtained from microbiology laboratory. Herbal extracts were authenticated by the Department of Botany. Fifty grams of powdered herbs was boiled in 500 ml of deionized distilled water and reduced to a volume of 10–20 ml to obtain the aqueous extract. This was stored in an airtight bottle in a refrigerator till it was used for the antimicrobial study.

Preparation of tooth specimens

One hundred freshly extracted single-rooted teeth were cleaned of debris using an ultrasonic scaler (NSK, Varios 750). Teeth were then decoronated to a standard of 10 mm length with a safe-sided diamond disc (SHOFU). Patency of apical foramen was established by inserting a size 15 K file (Mani, Japan) till it will be visible at the apical foramen and the working length was set 0.5 mm short of the apex. Canals were prepared using rotary ProTaper Files (Dentsply India) to an apical size of F3. Canals were irrigated with 3% sodium hypochlorite (VIP, Vensons India) solution and RC Help (Prime Dental products) was used as a lubricant. The canals were irrigated with 17% ethylenediaminetetraacetic acid (Fisher Scientific, Fair Lawn, NJ, USA) to remove the smear layer. The outer surfaces of teeth were covered with two coats of nail varnish to avoid environmental contamination. Apical foramen was sealed with composite resin (Brilliant Coltene).

Contamination of the blocks

Prior to inoculation, the samples were sterilized in an autoclave for two cycles. The first cycle was at 121°C and the second was with the samples immersed in 1 ml of nutrient broth in an individual microcentrifuge. To provide an enriched environment for the bacterial growth in the dentinal tubules, samples were agitated in an ultrasonic bath (for better penetration of the nutrient medium into dentinal tubules) for 15 min. Two control teeth were cultured in nutrient broth to confirm the sterility of the samples. The cultured E. faecalis was then suspended in 5 ml of TS broth and incubated for 4 h at 37° C, and its turbidity was adjusted to 0.5 McFarland standard. All the teeth samples were placed in culture media containing E. faecalis in round bottom flask, agitated for 10 min, and then incubated at 37°C for 21 days.

Antimicrobial assessment

After 21 days, the infected samples were irrigated with 5 ml of sterile saline (Infutec Health Care Limited, Hoshiarpur, Punjab, India) to remove the incubation broth. Four teeth were similarly treated and infected with E. faecalis as test samples and incubated at 37°C for 1 week without any medication to check the growth of E. faecalis. Teeth were then assigned into three experimental groups (n = 30 teeth) and a control group (n = 10 teeth). Group 1: Calcium hydroxide, Group 2: Mushroom extract, and Group 3: A. vera extract. Calcium hydroxide (Sigma-Aldrich, Mumbai, India) was mixed with sterile saline in a ratio of 1.5:1 (weight/volume) to obtain a paste-like consistency for Group 1. Thoroughly washed mushroom (Agaricus bisporus) and A. vera (aloe barbadensis miller) were shade dried and then powdered with the help of blender. Fifty grams of powder was boiled in 500 ml of deionized distilled water and allowed to boil to a final volume of 10–20 ml. The concentrated mixture was filtered and the clear extract was stored in an airtight bottle in the refrigerator for antimicrobial studies. Hydroxyethyl cellulose (Encore Natural Polymers Pvt. Ltd., Gujarat, India) was used as a thickening agent in the ratio of 2:1 (volume/weight) for Group 2 (mushroom) and Group 3 (A. vera) to obtain paste-like consistency. The respective intracanal medicaments were injected into the canal with a syringe. The control group did not receive any intracanal medicament. The canal orifice was sealed with paraffin wax. Teeth were placed in eppendorf tube and incubated at 37°C for 1 week.

Culturing of samples

At the end of 3, 5, and 7 days, the antimicrobial assessment was carried out with ten specimens at each time interval. The root canal orifice was re-opened and rinsed with sterile saline. Harvesting of dentin was carried out with 30 size H file (Mani, Japan). The H file was placed to the working length, and with filling motion, the dentinal shavings were scraped from apical third to a coronal third of the canal. The dentin shavings were then collected into test tubes containing nutrient broth. Serial 10-fold dilution was made with nutrient broth as diluent. From serial dilution, 1 ml was transferred and placed on Mueller–Hinton agar and incubated at 37°C for 24 h. Colony forming units (CFUs) were then counted.

The MIC was determined for the two plant extracts against E. faecalis using ELISA microdilution method.

Statistical analysis

Statistical analysis was done using analysis of variance technique. To evaluate the significant difference between the groups, multiple comparisons were done using (post hoc test) Bonferroni test.


  Results Top


The difference in mean CFU among the groups was found to be statistically significant (P< 0.001).

Mushroom demonstrated a better antimicrobial efficacy followed by A. vera and calcium hydroxide.

Mean (colony forming unit/ml) * (10^4) value recorded for the 3rd day results among the groups [Graph 1*]



[Graph 1*] Higher mean (CFU/ml) * (10^4) value was recorded in the control group followed by A. vera extract group, calcium hydroxide group, and mushroom extract group, respectively. The difference in mean (CFU/ml) * (10^4) value among the groups was found to be statistically significant (P< 0.001).

The difference in mean (CFU/ml) * (10^4) value was found to be statistically significant between control and calcium hydroxide (P< 0.001), control and mushroom extract (P< 0.001), control and A. vera extract (P< 0.001), calcium hydroxide and mushroom extract (P< 0.001), and also between mushroom extract and A. vera extract (P< 0.001).

Mean (colony forming unit/ml) * (10^4) value recorded for the 5th day results among the groups [Graph 2**]



Graph 2**] Higher mean (CFU/ml) * (10^4) value was recorded in the control group followed by calcium hydroxide group, A. vera extract group, and mushroom extract group, respectively. The difference in mean (CFU/ml) * (10^4) value among the groups was found to be statistically significant (P< 0.001).

The difference in mean (CFU/ml) * (10^4) value was found to be statistically significant between control and calcium hydroxide (P< 0.001), control and mushroom extract (P< 0.001), control and A. vera extract (P< 0.001), calcium hydroxide and mushroom extract (P< 0.001), calcium hydroxide and A. vera extract (P< 0.05), and also between mushroom extract and A. vera extract (P< 0.001).

Mean (colony forming unit/ml) * (10^4) value recorded for the 7th day results among the groups [Graph 3***]



[Graph 3***] Higher mean (CFU/ml) * (10^4) value was recorded in the control group followed by calcium hydroxide group, A. vera extract group, and mushroom extract group, respectively. The difference in mean (CFU/ml) * (10^4) value among the groups was found to be statistically significant (P< 0.001).

The difference in mean (CFU/ml) * (10^4) value was found to be statistically significant between control and calcium hydroxide (P< 0.001), control and mushroom extract (P< 0.001), control and A. vera extract (P< 0.001), calcium hydroxide and mushroom extract (P< 0.001), as well as between calcium hydroxide and A. vera extract (P< 0.01).

MIC for calcium hydroxide was found to be 40 mg/ml, A. vera extract 60 mg/ml, and for mushroom extract 40 mg/ml.


  Discussion Top


Persistent endodontic infection might be attributed to the retention of microorganism in dentinal tubules. The etiological spectrum and treatment options of persistent apical periodontitis are broader than those of teeth that have not undergone previous root canal treatment.[1]

E. faecalis was chosen as the test organism in this study because it is commonly found in persistent root canal infection (63% of teeth with posttreatment disease) and is resistant to commonly used medicament, calcium hydroxide.[1] Heling et al. have found that calcium hydroxide did not show any antibacterial activity against E. faecalis present inside dentinal tubule and failed to disinfect the dentine or prevent secondary infection.[16] Endodontic literature shows that the low antibacterial effectiveness of calcium hydroxide against E. faecalis is due to the buffering action of dentin and deep penetration of E. faecalis into the dentinal tubules.[17] Kowalski et al. found that the E. faecalis penetrates the dentinal tubules to the depth of 1483.33 μm in nutrient rich aerobic condition, 1166.66 μm in nutrient rich anaerobic condition, and 620 μm in nutrient deprived anaerobic condition.[18]

Intracanal medicaments help in reducing the remaining bacteria from the root canal and prevent re-infection. In this study, calcium hydroxide group was compared with newer intracanal medicaments because of its prevalent clinical application as an intracanal medicament.[5]

Herbal extracts of A. vera and mushroom have been tested forin vitro andin vivo antimicrobial activity.[14] Abidin et al. described their mechanism of action was predominantly on the cell membrane by disrupting its structure, blocking membrane synthesis, and inhibition of cellular respiration thereby causing cell leakage and cell death.[19] Hence, the use of an intracanal medicament made from natural extracts is of great significance.

Human permanent teeth were used as the samples to simulate the clinical scenario. The method used in thisin vitro study simulates the same clinical condition as persistent apical periodontitis.[1] In this study, for root dentin sampling, modified model of Haapasalo and Orstavik was followed. This model has been proved to be sensitive and is suitable forin vitro testing of intracanal medicaments.[20]

The samples were placed in the culture media and agitated for better penetration of the E. faecalis into the root canal dentinal tubules. This resulted in dense infection reaching into the dentinal tubules.[21]

While most of the intracanal medicaments lose their efficacy after 24 h and others (calcium hydroxide) efficacy in turn increases with time. Therefore, this study was conducted to check the efficacy of the medicaments after 3rd, 5th, and 7th day.[17],[22]

The results revealed that all the medicaments tested reduced E. faecalis from the root canal after intracanal medication at 3rd, 5th, and 7th days. Mean CFUs of each group decreased over a period of time from 3rd to 7th day thus confirming the traditional claim.[23]

In the present study, the percentage of reduction of E. faecalis from 3rd to 7th day for calcium hydroxide (MIC for calcium hydroxide was 40 mg/ml) was less compared to other groups. Siqueira and Lopes demonstrated that calcium hydroxide was ineffective in eliminating E. faecalis from inside dentinal tubules even after 1 week.[5] The reason could be the limited action of calcium hydroxide against facultative anaerobes, buffering action of dentin, and the arrangement of bacterial cells colonizing the root canal walls (cells located at the periphery of colonies can protect those located more deeply inside the tubules).[6]

A. vera (MIC 60 mg/ml) showed less antibacterial activity than calcium hydroxide on the 3rd day. The gel form of A. vera would have taken time to release the active ingredients into the canal. A. vera showed less antibacterial activity than mushroom at all points of time tested. The reason could be the molecular weight (1000 kDa) of the A. vera, which is more than the mushroom (12 kDa), so lesser penetration of A. vera into the dentinal tubules and hence lesser action. A. vera showed better antibacterial activity than calcium hydroxide on the 5th and 7th day (mean CFUs 82.1%, 60.8%, 36.3% on 3rd, 5th, and 7th day) and a 20% increase in a bacterial reduction from 3rd to 7th day. The reason may be that A. vera gel contains active ingredients such as anthraquinones, tannins, myristic acid, curcumin, and nimbidin that show high antibacterial action and antiadherence activity that reduces bacterial colonization and adhesion to dentin.[12] In anin vitro study, similar to our study by Bhardwaj et al., compared the antimicrobial activity of natural extracts such as A. vera, morida citrifolia, papain along with calcium hydroxide, and chlorhexidine against E. faecalis. Results showed that A. vera gel had 78.9% of antibacterial activity as compared to 64.3% for calcium hydroxide and 100% chlorhexidine.[23]

Mushroom extract (MIC 40 mg/ml) showed the highest antibacterial activity against E. faecalis at all the time period tested (3rd, 5th, and 7th day). The reason could be that mushroom extracts have some of the active compounds, including low molecular weight and high molecular weight compounds. The low molecular weight compounds (plectasin, confuentin, grifolin, and neogrfolin) show antibacterial action.[15] A study done by Signoretto et al. showed that low molecular weight mass fraction of an aqueous mushroom extract had antimicrobial activity against potential oral pathogens.[14] Low molecular weight fraction of mushroom extract (molecular weight around 12 kDa) may have the capacity to penetrate deep into dentinal tubules and hence better antibacterial property (Lipinski's rule of active drug).[24],[25] The literature shows that mushroom has the highest activity against Gram-positive bacteria, and the gel form of extract increases the contact time, which enhances its performance.[15]

Based on the results of the present study, aqueous extracts of A. vera and mushroom can be considered as intracanal medicaments for resistant cases. As endodontic infection is polymicrobial, studies with regards to its action on other microbial flora need further evaluation. Since mushroom showed greater effectiveness at all time period tested, it can be considered in situ ations where there is a limitation of time exist; this may be added advantage as well. Furtherin vitro andin vivo studies regarding cytotoxicity, penetrability, easy formulation and delivery, structural alteration, interference with the bonding of obturating material, and discoloration need to be evaluated.


  Conclusions Top


Within the limitation of the present study, mushroom showed the highest antibacterial activity at all the time periods tested, whereas A. vera showed the least activity at the 3rd day, but by 5th and 7th day, anti-bacterial activity was higher than that of calcium hydroxide. Antibacterial activity of all the three groups increased with time. The percentage decrease in bacterial count from the 3rd to 7th day was highest with mushroom followed by A. vera and calcium hydroxide. MIC for calcium hydroxide was 40 mg/ml, A. vera extract 60 mg/ml, and for mushroom extract 40 mg/ml. Hence, mushroom and A. vera extracts can be considered as an alternative intracanal medicament for resistant and retreatment cases.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Athanassiadis B, Abbott PV, Walsh LJ. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. Aust Dent J 2007;52 1 Suppl: S64-82.  Back to cited text no. 4
    
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Siqueira JF Jr., de Uzeda M. Disinfection by calcium hydroxide pastes of dentinal tubules infected with two obligate and one facultative anaerobic bacteria. J Endod 1996;22:674-6.  Back to cited text no. 6
    
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