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Table of Contents   
ORIGINAL RESEARCH  
Year : 2020  |  Volume : 31  |  Issue : 1  |  Page : 124-128
Comparison of antibacterial efficacy of cinnamon extract, neem extract as irrigant and sodium hypochlorite against Enterococcus fecalis: An in vitro study


1 Department of Pediatric and Preventive Dentistry, Saveetha Dental College, Saveetha University, Saveetha Institute of Technical and Medical Sciences, Chennai, Tamil Nadu, India
2 Department of Microbiology, Saveetha Dental College, Saveetha University, Saveetha Institute of Technical and Medical Sciences, Chennai, Tamil Nadu, India

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Date of Submission04-Mar-2018
Date of Acceptance03-Oct-2018
Date of Web Publication02-Apr-2020
 

   Abstract 


Aim: The aim of the study is to compare the antibacterial effectiveness between cinnamon extract as irrigant with neem extract irrigant and sodium hypochlorite against E. fecalis. Materials and Methods: The present study is a randomized controlled in vitro study conducted on 60 extracted permanent single rooted teeth. The teeth were divided into three groups- Group 1 was irrigated with cinnamon extract, Group 2 was irrigated with neem extract irrigant and Group 3 with 3% sodium hypochlorite. The colonies in the reduction of E. feacalis was noted as CFU values per ml. Wilcoxon signed ranked test was used to compare in between the three groups. Mann- Whitney's test was used for inter group comparison between the three groups. Results: Cinnamon extract had the maximum reduction in the CFU colonies followed by sodium hypochlorite. Neem extract irrigant had the least reduction in the colonies of E. feacalis. (P < 0.05). Conclusion: Cinnamon extract irrigant show better reduction in E. fecalis as compared to 3% sodium hypochlorite and neem extract irrigant.

Keywords: Cinnamon extract, endodontic irrigant, Enterococcus feacalis, neem extract, sodium hypochlorite

How to cite this article:
Panchal V, Gurunathan D, Muralidharan N P. Comparison of antibacterial efficacy of cinnamon extract, neem extract as irrigant and sodium hypochlorite against Enterococcus fecalis: An in vitro study. Indian J Dent Res 2020;31:124-8

How to cite this URL:
Panchal V, Gurunathan D, Muralidharan N P. Comparison of antibacterial efficacy of cinnamon extract, neem extract as irrigant and sodium hypochlorite against Enterococcus fecalis: An in vitro study. Indian J Dent Res [serial online] 2020 [cited 2021 May 11];31:124-8. Available from: https://www.ijdr.in/text.asp?2020/31/1/124/281798



   Introduction Top


Pulpal and periapical pathology is the major cause of pain among patients.[1] The primary cause of pulpal and periapical infection is the colonization of the micro-organisms.[1] The foci of infection consists of gram positive, gram negative and anaerobic micro-organisms.[2] Among all the colonies of micro-organisms, Enterococcus fecalis is the most predominant species leading to infection and reinfection in the root canal systems.[3] The resistance of E. fecalis was due to its increased penetration in the dentinal tubules, thus making the accessibility difficult with mechanical cleaning alone.[4] Thus, the objective of root canal treatment involves elimination of microbes from the root canal system.[5]

Biomechanical preparation involves removal of infected dentin and debridement of the root canals.[6] Instrumentation of the root canals shapes and prepares the canal to aid in better debridement.[6] An addition of antimicrobial irrigant to the biomechanical preparation will aid in flushing the debris out of the canals with extended antibacterial action.[7] The ideal requirements of irrigants include its ability to dissolve pulp along with removal of smear layer and an extended antimicrobial action.[8]

Sodium hypochlorite fulfils most of the requirements of the ideal endodontic irrigant and is widely used in dentistry.[9],[10] It has been the choice of irrigant due to its pulp dissolving capacity, antimicrobial action and ability to remove smear layer. However, cases have been reported of allergic reaction due to the use of sodium hypochlorite.[11],[12] There have been cases of tissue toxicity and accidental mishaps associated with sodium hypochlorite.[13],[14] A bad smell and taste is also associated with irrigation using sodium hypochlorite. To overcome this disadvantage, other herbal alternatives have been considered over time.

Neem also known as Azadirachta indica is widely used in clinical dentistry due to it antibacterial and antiviral properties.[15] Studies have been done to evaluate the antibacterial effectiveness of neem against wide variety of micro-organism.[15],[16] The advantage of using neem as an endodontic irrigant is due to its antibacterial and antioxidant properties. It shows a wide spectrum of action against gram positive and gram negative oraganism.[16]

Cinnamon also known as C. zeylanicum has gained popularity in recent time due its antibacterial activity.[17] The antibacterial action of cinnamon is due to the presence of vanillic, caffeic, gallic, protocatechuic, p-coumaric, and ferulic acids in the extracts.[17] It has shown effectiveness against S. mutans and E. fecalis organisms.[18] It is also known to show a wide spectrum of action on gram positive and gram negative organism.[18]

Due to a various choice of herbal irrigants available and its advantage over the traditional chemical agents, a wide number of studies have evaluated the effectiveness between herbal agents and chemical agents.[15],[16],[18],[19] However there needs to be a comparison between the effectiveness of two herbal irrigants against specific micro-organism.

In this regard, the present study aims to compare neem extract, cinnamon extract and sodium hypochlorite as irrigant. The comparison is based on the effectiveness of these irrigants against E. fecalis in extracted permanent teeth.


   Materials and Methods Top


The present study followed an in vitro study design. The study design was approved by the institutional review board of the dental institution. The present study was a single blinded study. Two different personnel was assigned for the study. The shaping of the canals was done by a single operator. The same operator irrigated the study groups of irrigants into the canals. The antibacterial efficacy was evaluated using colony forming units. The colonies were counted by a separate operator who was unaware of the three groups.

The present study uses ethanolic extract of neem and cinnamon to prepare the irrigation solution.[20] The ethanolic extract of neem was obtained from neem leaf ground into coarse and medium coarse particles. These particles were mixed with 50% ethanol and this suspension was placed on the magnetic stirrer for 4 hours. Whitman's filter paper was used to obtain the filtrate which was then dried to obtain the extract. The extract was dried to evaporate the solvent to prevent the influence of extracting agent (ethanol) on the final extract. Cinnamon extract was obtained using cinnamon bark which was pulverized into medium coarse granules. The extracting agent and the procedure to obtain the final extract was similar to that for neem extract. These extracts were stored in dark colored bottles at 4 degree Celsius until use.

The irrigating solution was prepared my mixing the crude extract in DMSO (dimethyl sulfoxide) solvent. Previous studies have noted MIC for neem extract[21] to be 25% and for cinnamon extract to be 20%.[22] Neem extract was prepared by dissolving 25 grams of crude extract in 100 ml DMSO solution. Similarly, cinnamon extract was prepared by dissolving 20 grams of crude extract in 100 ml of DMSO. The final solution were refrigerated at 4 degree Celsius.

The present study includes a total of 60 extracted teeth. The teeth included were single rooted permanent teeth extracted for orthodontic purpose or due to any periodontal problems. The included teeth were inspected for intact root surface. Any tissue tags, calculus or debris present on the teeth were removed to obtain a clean external surface. These teeth were stored in normal saline until use to prevent desiccation of the tooth surface.

The extracted teeth were mounted on individual block of modelling wax. Access opening was done with No. 4 round carbide bur (DENTSPLY Maillefer, OK, USA). The dentinal overhangs of the roof of the pulp chamber was removed used a safe ended tungsten carbide bur (Endo-Z, FG, DENTSPLY Maillefer, OK, USA). 20 no stainless steel K- file (DENTSPLY Maillefer, OK, USA) was used to check the patency of the canals up to the apex. The working length was determined using radiograph and was kept 0.5 mm short of the radiographic apex. After initial instrumentation up to no 30 stainless steel hand files, rotary instrumentation was done using ProTaper (DENTSPLY Maillefer, OK, USA) up to F3. The sequential order for ProTaper was followed. Saline was used to remove the debris in between the instrumentation sequence. After complete instrumentation the tooth were scheduled for the inoculation of E. fecalis

Before the inoculation of E. fecalis in the tooth, all the teeth were autoclaved twice at 121 degree Celsius for the elimination of all the microbes from the tooth and prevent contamination of the study with other microbes. A pure culture of test strain of E. fecalis ATCC 29212 (Department of Microbiology, PGIMS) was prepared in sterile nutrient broth. The presence of E. fecalis was confirmed in the nutrient broth by pipetting 10 micro liter of the broth and observing its presence under microscope. Nutrient broth inoculated with E. fecalis was transferred in a sterile container. The level of the broth was marked. The extracted teeth placed in the nutrient broth containing E. fecalis and incubated for 21 days at 37 degree Celsius. The level of the nutrient broth was checked daily, and new broth inoculated with E. fecalis was added to maintain all the extracted teeth completely submerged in the inoculated broth for 21 days.

The inoculated teeth were divided into three groups for the irrigation protocol. 20 teeth were randomly assigned to each group. Each tooth was assigned a serial number. The allocation of each sample into individual groups was done using block randomization using lots. Group 1 was irrigated using 3% sodium hypochlorite, group 2 was irrigated using neem extract irrigant and group 3 was irrigated using cinnamon extract irrigant. The solution was irrigated using flexible tip attached to a 5 ml irrigating syringe. Each tooth was irrigated with 5 ml of irrigating solution. The irrigating solution was left in contact with the tooth surface for 20 minutes.

After 20 minutes, the sample of dentin was taken to evaluate the reduced colony forming units. No 2 Gates-Glidden drill was used to remove the dentin sample from each tooth. A fresh sterilized Gates-Glidden drill was used for every tooth to prevent cross-contamination. The dentin debris from the gates Glidden drill was transferred to 1 ml of saline. All the micro-test tubes were kept in water bath for 30 minutes at 62 degree Celsius to prevent cross- contamination. 25 microliters of the solution from the micro test tubes were pipetted and inoculated on the nutrient agar. This agar was incubated at 37 degree Celsius and the colonies of E. fecalis was calculated under magnifying glass. The presence of E. fecalis was verified using an eosinophilic and gentian violet staining to verify gram positive organism.

The data obtained were entered in the spreadsheet and and statistical analysis was done using SPSS software version 22. (IBM Corp, Armont, NY, USA). Kruskal-Wallis test was used to compare the CFU values per ml between the three groups. Mann-Whitney's test was used was used in multiple pairwise comparison between the groups.


   Results Top


A total of 60 permanent single rooted teeth are included in the study. Out of the total teeth included 52 were single rooted lower premolars. 5 were upper central incisors and 3 were lateral incisors. Out of the 20 teeth irrigated with neem extract irrigant, 16 were premolars, 3 central incisor and 1 lateral incisor. Out of the 20 teeth irrigated with cinnamon extract irrigant, 18 were premolars, 1 central incisor and 1 lateral incisor. Out of the 20 teeth irrigated with sodium hypochlorite, 18 were premolars, 1 lateral incisor and 1 central incisor. The outcome variable for all the groups is the CFU values per ml.

The Normality tests Kolmogorov-Smirnov and Shapiro-Wilks tests results reveal that the variable follows a non-normal distribution of data. Therefore to analyze the data non-parametric methods are applied.

The mean CFU values obtained using neem as irrigant was 0.72 × 103 having a standard deviation of 0.34. The mean CFU values obtained using cinnamon extract as irrigant was 0.25 × 103 having a standard deviation of 0.08. The mean CFU values obtained using sodium hypochlorite as irrigant is 0.40 × 103 having a standard deviation of 0.30 [Table 1].
Table 1: Comparision of colony forming units values between cinnamon extract irrigant, neem extract irrigant and sodium hypochlorite

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Kurskal-Wallis test was used to compare CFU values between the three groups. Kruskal Wallis test showed a significant difference between the three groups with cinnamon extract irrigant having the least CFU values (P = 0.007) [Table 1] and [Figure 1].
Figure 1: Comparison of CFU values between cinnamon extract irrigant, neem extract irrigant and sodium hypochlorite

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Mann-Whitney's test was used for multiple pairwise comparison between the three groups. Cinnamon extract irrigant showed significantly less CFU values as compared to neem extract irrigant (P = 0.004). However, cinnamon extract irrigant as compared to sodium hypochlorite showed lower CFU values but the results was not statistically significant (P = 0.185) [Table 2]. On the other hand sodium hypochlorite irrigant showed statistically significant lower CFU values as compared to neem extract irrigant (P = 0.031). Thus, cinnamon extract showed statistically significant difference to reduce the E. fecalis colonies as compared to other two groups [Table 3] and [Table 4].
Table 2: Intergroup comparison of colony forming units values between hypochlorite irrigant and neem extract irrigant

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Table 3: Intergroup comparison of colony forming units values between cinnamon irrigant and hypochlorite irrigant

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Table 4: Intergroup comparison of colony forming units values between cinnamon irrigant and hypochlorite irrigant

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   Discussion Top


The root canal of infected teeth is the harbor of various colonies of micro-organisms.[3] Out of all the inhabitant species, E. fecalis is the major cause of infection and reinfection of the root canal.[23] Elimination of E. fecalis from the root canal has always been a challenge for complete disinfection of the root canal due to its deeper penetration in the dentinal tubules.[4]

The primary objective of root canal treatment is complete disinfection of root canals and elimination of microbes.[24] Biomechanical preparation is the primary step for the elimination of microbes. The biomechanical preparation involves instrumentation which aids in the removal of infected dentin and use of irrigants to aid in complete removal of debris and disinfection.[7]

Chemical irrigants have been in practice over a long time in the disinfection of infected root canal.[10] Due to its antibacterial action and pulp dissolving ability, sodium hypochlorite is being used as an ideal irrigant for root canal treatment. Other irrigants with increased increased antibacterial effectiveness such as chlorhexidine has also been advocated in necrotic teeth.[25] However, there has been an increased allergic reaction and adverse effects associated with the use of chemical irrigants.[14] To overcome this, the use of herbal alternatives having comparable antibacterial effectiveness should be considered.

Neem extract is the most common herbal extract used as an irrigant due to its antioxidant properties which aid in antibacterial action against E. fecalis. Dutta and Kundabala in 2014 showed similar antibacterial efficacy of neem extract as irrigant with sodium hypochlorite and chlorhexidine irrigant against E. fecalis.[26] Babaji et al. have found comparable effectiveness of neem irrigant with sodium hypochlorite irrigation.[27]

Cinnamon extract has recently gained a limelight as an irrigant in endodontics due its antibacterial properties. Gupta-Wadhwa et al. have demonstrated comparable antibacterial effectiveness of cinnamon than other herbal irrigants.[28] Gupta et al. also shows higher antibacterial effectiveness of cinnamon extract as compared to other herbal agents.[18]

The literature reveals no existing study which compares the antimicrobial effectiveness between neem and cinnamon as irrigant. Thus this study aims in comparing the recently used trends of cinnamon extract as irrigant to the traditionally used herbal irrigants. Sodium hypochlorite being the ideal chemical agent is used as a comparative agent, to evaluate the comparative antibacterial effectiveness between the three.

The present study is a randomized controlled trial with the in vitro study design. Randomization was followed in the present study to avoid selection bias. Only the observer was blinded in the present study. Due to the color and odor of individual irrigants, blinding of the operator was not possible.

The present study shows cinnamon extract irrigant to have better antibacterial effectiveness followed by sodium hypochlorite. The neem showed to have least antibacterial effectiveness. These results are contradictory to previous study by Gupta-Wadhwa et al., where sodium hypochlorite showed higher effectiveness as compared to cinnamon extract.[28] This can be attributed to a lower concentration of sodium hypochlorite used in the present study as compared to other study. The present study showed similar results in relation to comparison of neem and sodium hypochlorite. According to Sinha et al., neem extract showed less antibacterial effectiveness as compared to sodium hypochlorite irrigant. The present study shows cinnamon extract to have higher antibacterial effectiveness against E. fecalis than neem irrigant. This study however does not show the pharmacological characteristics of the agent to determine the exact mechanism of action the herbal agents. The present study is thus a preliminary study comparing the antibacterial effectiveness of two herbal irrigants.

The present study compares the antibacterial effectiveness of only two herbal agents. Further improvement in the study can be done by including more herbal agents in the comparison. The present study evaluates the colony forming units per ml to determine the antibacterial efficacy. However, further improvement in the study can done by evaluation of the antibacterial efficacy by more standardized methods like PCR. This can aid in achieving more standardized results.

This study thus acts as a preliminary study to compare cinnamon extract, neem extract irrigant and sodium hypochlorite irrigant. This study recommends further studies at a molecular level to understand the exact mechanism of the action of pharmacological agents.


   Conclusion Top


The present study shows cinnamon extract to have better effectiveness followed by sodium hypochlorite and neem extract irrigant. This study thus acts as a preliminary study to compare the antibacterial effectiveness of cinnamon extract irrigant and neem extract irrigant with sodium hypochlorite.

Acknowledgement

The authors acknowledge the department of microbiology Saveetha dental college for their support for the laboratory work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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27.
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Veerale Panchal
Clinic 28, Saveetha Dental College, Poonamallee High Road, Velappanchavadi, Chennai - 600 077, Tamil Nadu
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_177_18

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