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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 32  |  Issue : 3  |  Page : 148-153

Comparative evaluation of antimicrobial efficacy of sesame oil irrigants on Enterococcus faecalis – An in vitro study


Department of Conservative Dentistry and Endodontics, M.A. Rangoonwala College of Dental Sciences and Research Centre, Pune, Maharashtra, India

Date of Submission15-Aug-2019
Date of Decision12-Jan-2020
Date of Acceptance13-Jun-2020
Date of Web Publication28-Oct-2020

Correspondence Address:
Dr. Hussain Mookhtiar
Department of Conservative Dentistry and Endodontics, M.A. Rangoonwala College of Dental Sciences and Research Centre, Pune - 411 001, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/endo.endo_1_19

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  Abstract 


Introduction: During chemo-mechanical preparation, irrgants play a vital role for eradication of endodontic pathogens. Due to the shortcomings of the current irrgants used, a need has come to find out whether herbal irrigants has better cytological acceptance and whether there is a decrease in biomedical waste. With the growing trends of using herbal extracts and oils as an alternate to conventional irrigants as an antimicrobial agent, the main objective of this study was to test sesame oil itself and its combination with various agents for its antimicrobial efficacy on Enterococcus faecalis (EF).
Aim: The antimicrobial efficacy of edible sesame oil and sesame oil mixture on EF using agar diffusion test.
Materials and Methods: Forty-five extracted single teeth with single canal configuration were decoronated from each tooth at the cementoenamel junction and the orifices of canals were enlarged with Gates Glidden drill size number 4. EF colonies were adjusted to 1.5 × 108 colony forming unit (CFU)/ml. Crystal violet staining and observation under stereomicroscope (BX-63 Olympus DIC) were performed to check the 100% purity of culture. The inoculated specimens were divided into three groups (n = 15). The three groups are as follows: Group A was irrigated with 5.25% sodium hypochlorite (NaOCl); Group B was irrigated with edible sesame oil; and under Group C was irrigated with prepared sesame oil mixture. After 3 weeks, following the use of F4, 2 ml experimental irrigant was left undisturbed for 60 s. A volume of 20 ml of the all three irrigants were used. Absorbent paper points of sizes similar to the last apical file size used, i.e., 40.06 (F4) were placed in the canals for 5 min and transported for microbial assessment. CFUs were checked after 24 h on blood agar plates.
Statistical Analysis: The intergroup statistical comparisons were performed using one-way analysis of variance (ANOVA) with Tukey's post hoc test for multiple group comparisons (P < 0.05). The intragroup comparisons were performed using r ANOVA in each study group (P < 0.01).
Results: The intergroup comparison of the mean CFUs of EF on blood agar plates was significantly higher in for edible sesame oil (13.30 ± 1.64) compared to NaOCl (9.40 ± 1.24) and sesame oil mixture (7.60 ± 1.43).
Conclusion: The present study confirmed the in vitro antimicrobial efficacy of prepared sesame oil mixture compared to 5.25% NaOCl and edible sesame oil.

Keywords: Colony forming units, Enterococcus faecalis, sesame oil


How to cite this article:
Jadhav SK, Mookhtiar H, Hegde V, Shanmugsundaram S. Comparative evaluation of antimicrobial efficacy of sesame oil irrigants on Enterococcus faecalis – An in vitro study. Endodontology 2020;32:148-53

How to cite this URL:
Jadhav SK, Mookhtiar H, Hegde V, Shanmugsundaram S. Comparative evaluation of antimicrobial efficacy of sesame oil irrigants on Enterococcus faecalis – An in vitro study. Endodontology [serial online] 2020 [cited 2020 Nov 28];32:148-53. Available from: https://www.endodontologyonweb.org/text.asp?2020/32/3/148/299280




  Introduction Top


Endodontic therapy aims at the elimination of these microorganisms from the infected root apex and to prevent reinfection by chemo-mechanical preparation. Microbes and microbial products are the main etiologic factors of pulpal and periapical inflammation.[1]

Enterococcus faecalis (EF) is a bacterium commonly found in endodontic infections, particularly in retreatment cases. It appears to be more resistant while it is present in an infected root canal system due to its ability to penetrate the dentinal tubules and resist every obstacle for its eradication making it the most stubborn organism. Haapasalo et al. reported have reported that EF has a depth of penetration of >10,000 um, thus making the conventional irrigants difficult to interact with them at such depths.[2],[3]

Sodium hypochlorite (NaOCl) is the gold standard endodontic irrigant due to its properties of tissue dissolution and antimicrobial activity making it the irrigating solution of choice for treatment of teeth with pulp involvement. According to literature review, these properties prove very beneficial and enable thorough disinfection till the time the NaOCl is used in the concentrations ranging from 0.5% to 5.25%.[3]

Extracts of medicinal plants such as Morinda Citrifolia, Triphala, Green Tea, Neem and various essential oils such as Eugenol have been widely used in endodontics. Studies on herbal extracts have proven that herbal plants and essential oils extracts eliminate microbes causing dental pathologies, thus proving its efficacy as an antimicrobial for oral infections.[4],[5],[6]

Sesame (Sesamum indicum L.), is a flowering plant in the genus sesamum and the family of pedaliaceae. Sesamin and sesamolin present in sesame oil are used as active ingredients in antioxidants, antiseptics, bactericides, viricides, and disinfectants. Sesame oil have been performed in dentistry such as oil pulling and its addition in various medicaments against various oral flora due to its antimicrobial and antioxidant effect.[7],[8]

Thus, the aim of this study was to compare the antimicrobial efficacy of NaOCl, edible sesame oil, and sesame oil mixture on EF which is the most common and stubborn organism in Re-Root Canal Treatment cases. The null hypothesis of this study was that edible sesame oil, prepared sesame oil irrigant, and NaOCl had no antimicrobial effect on EF.


  Materials and Methods Top


After getting Institute Ethical Clearance (IRB no. MARDC/Deptcons/DrSameer/Phd/2017), the said pilot study was conducted in the Department of Conservative Dentistry and Endodontics in association department of microbiology of the same institute.

Specimen preparation

Forty-five human extracted single-rooted teeth with single canal and well-formed root apices, extracted for orthodontic reasons were collected from the department of oral and maxillofacial surgery with informed consent of the donor. The teeth were stored in saline at room temperature to prevent dehydration of the specimens until use. The crowns were decoronated at the cemento-enamel junction using a rotating diamond disc (Carbodent; Gysi S. A, Buenos Aires, Argentina) at 20000 rpm under water-cooling and the canal lengths were standardized to 14 mm and were then thoroughly ultrasonically scaled for any deposits or remnant periodontal tissues. The root apices were left open to observe the extrusion of irrigant during the procedure. The orifices of canals were enlarged with Gates Glidden drill size number 4 (Mani Inc., Tochigi, Japan) and were randomly divided into three groups with each group comprising of 15 specimens (n = 15). The three groups are as follows: Group A was irrigated with 5.25% NaOCl (Vensons, Malleshwaram, India); Group B was irrigated with edible sesame Oil; under Group C was irrigated with prepared Sesame oil mixture. The samples were then autoclave at a temperature of 121°C and a pressure of 15lbs for 20 min to prevent contamination before inoculation of the bacterium.

Preparation of sesame oil mixture

The mixture was prepared in the Department of pharmacology of All India Shri Shivaji Memorial Society College of Pharmacy, Pune, India, under the guidance of a senior faculty member. A volume of 100 ml of cold pressed sesame oil (KNG Superman Til Oil, KNG Agro (P) Ltd., Haryana) was dispensed in a 500 ml beaker after which 38 ml of ethanol was added and mixed with a mechanical stirrer. After mixing of oil and ethanol, 75 ml of Tween 80 and 285 ml of water was added along with 50 gm of citric acid which was then mixed with a mechanical stirrer until a homogeneous solution was obtained.

Bacterial strain and microbial growth preparation

EF (ATCC 29212; Microbiologists, Medimark Europe) was incubated in Trypticase soy agar plate (HiMedia Laboratory Private Limited, Mumbai, Maharashtra, India) to improve their growth. The culture was incubated anaerobically at 37°C for 7 days. Crystal violet staining and observation under stereomicroscope (BX-63 Olympus DIC) were performed to check the 100% purity of culture. EF was then incubated in Trypticase soy broth at 36.5°C for 24 h (HiMedia Laboratory Private Limited, Mumbai, Maharashtra, India) to obtain a turbid suspension of EF and the turbidity was spectrophotometrically adjusted to 1.5 × 108 organisms/ml (equivalent to 0.5 McFarland standard).

Specimen contamination

The autoclaved roots were immersed in Eppendorf tubes containing 1 ml of 1.5 × 108 organisms/ml of Trypticase soy broth. The Eppendorf tubes were incubated under anaerobic conditions for 3 weeks at 37°C and after every 3 days, the Eppendorf tubes with roots specimens were changed with fresh bacterial broth to ensure the penetration of EF into dentinal tubules. Five specimens from each group randomly selected and were sectioned vertically for to obtain 0.8 mm thick-cross section for the verification of the growth of EF in the canals under scanning electron microscopy. The remaining specimens were used to evaluate the effectiveness of the irrigants being considered.

Specimen treatment

After 3 weeks, the root specimens were wiped with sterile gauze and were divided into three groups according to the irrigation regimen. Working length was established with No. 10 K file (Mani Inc., Tochigi, Japan) and shaping was performed with Rotary Protaper Gold (ProTaper, Dentsply Maillefer, Ballaigues, Switzerland) up to apical size #40.06 (F4) using crown-down technique. In between each filing, the canals were irrigated with 2 ml of experimental irrigant for 30 s using 30-gauge side vented needles (Prima Dental, India). Following the use of F4, 2 ml experimental irrigant was left undisturbed for 60 s. The final irrigation of the canals was performed with 5 ml saline to remove any residual debris and irrigants out of the canals. Autoclaved absorbent paper points (Diadent Group International, Korea) of sizes similar to the last apical file size used, i.e., 40.06 (F4) were placed in the canals for 5 min and were dispatched for microbial assessment in 1 ml of PBS (phosphate saline buffer). The 0.1 ml aliquot from the transport media smeared on the blood agar plates (HiMedia Laboratory Private Limited, Mumbai, Maharashtra, India) under sterile conditions were incubated anaerobically for 24 h at 37°C and colony count of EF was done from blood agar using stereomicroscope. The growth of the organisms that occurred Blood agar was verified using crystal violet staining. The 24 h colony forming units/ml (CFUs) results were statistically analyze.

Statistical analysis

The intergroup statistical comparisons were performed using one-way analysis of variance (ANOVA) with Tukey's post hoc test for multiple group comparisons. The intragroup comparisons were performed using ANOVA in each study group.


  Results Top


[Table 1] summarizes the inter- and intra-group distribution of mean CFU of EF after 24 h. Initial CFUs before the use of the irrigants was 1.5 × 108. However, all three groups showed significant reduction in the CFUs after irrigation, i.e., from 108 to 103 organisms/ml. The intergroup comparison of the mean CFUs of EF on blood agar plates which was statistically significantly higher in Group B, i.e., edible sesame oil (13.30 ± 1.64) compared to Group A, i.e., NaOCl (9.40 ± 1.24) and Group C, i.e., sesame oil mixture (7.60 ± 1.43) while Group C (7.60 ± 1.43) showed the lowest CFU (P < 0.05). [Table 2] summarizes the Significance of mean CFUs of EF on blood agar plates, which differed statistically significantly between Groups A and B, between Group A and Group C (P < 0.001) [Figure 1].
Table 1: Intergroup mean reduction of Group A, Group B, Group C of Enterococcus faecalis (standard deviation)

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Table 2: P value significance between Group A and Group B, Between Group A and Group C

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Figure 1: Intergroup distribution of mean colony forming units

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  Discussion Top


According to Haapasalo et al., an ideal irrigant should not only have low cytotoxicity and aid in debridement of root canal system but also should be antimicrobial action against endodontic pathogens during disinfection procedures. Thus, this study evaluated the antimicrobial efficacy of NaOCl, edible sesame oil, and prepared sesame oil irrigant on EF using agar diffusion test.[8],[9],[10]

Mandibular premolars with completely formed apices and straight canals were selected so that various complications, such as the loss of working length or lack of a standardized preparation, were reduced to minimal.[4],[9],[10]

EF, a facultative Gram-positive anaerobe is the most dominant bacteria implicated in root canal failures and persistent infections. EF endures prolonged period of nutritional deprivation. It binds to dentin and proficiently invades dentinal tubules.[11],[12],[13],[14],[15]

A vast majority of oral microorganisms grow and function as members of metabolically integrated communities, or biofilms.[16],[17],[18] EF is an unique microbe, which forms biofilms in conjunction with other microbes is responsible for its pathogenicity and resistance is related to proteins such as gelatinase (GelE), serine protease (SprE), and enterocin O16 which have been shown to be positively regulated by the fsr quorum-sensing system. This property of biofilms limits the efficacy of irrigating solutions.[19],[20],[21],[22]

5.25% NaOCl has been the gold standard irrigating solution in the treatment of infected root canals, because of its well-known bactericidal action. However, due to biofilm formation and ability of EF to tolerate high Ph of NaOCl and resistance factors of EF, it is difficult to eliminate from the root canals thereby increasing the possibility of re infections even after using NaOCl.[23],[24],[25],[26]

In this study, the prepared sesame oil mixture showed the best results as it has antimicrobial properties along with its anti-inflammatory properties. Possible reason for its antimicrobial efficacy is that Sesame Oil generates antioxidant action which damages the cell wall of microorganisms and kills them. Sesame oil attracts the lipid layer of bacterial cell membranes, causing it to stick or get attracted to the oil molecules allowing them to penetrate into the bacteria.[27],[28],[29],[30] Also, sesame oil also downregulates Matrix Metalloproteinase–9 expression responsible for dental caries, pulp, and periapical inflammation which play an important role in the degradation of the collagenous structure and spread of the pathology.[31],[32],[33]

The results of this study were in conjunction with a study conducted by Heidari-Soureshjani et al. who concluded that sesame oil was effective against Staphylococcus aureus.[7] Furthermore, antimicrobial studies on sesame oil has proved its effectiveness against E. coli and Candida albicans.[34],[35],[36],[37]

Edible sesame oil alone could not reduce EF to a large extent as compared to 5.25% NaOCl. This could be due to the large structure of the oil molecules which do not penetrate into the dentinal tubules and destroy EF which penetrate and adhere the dentinal tubules. Ethanol was added to sesame oil resulting in the formation of an emulsion, i.e., allowing the oil molecules to mix with water.[22],[23],[24],[25],[26] Ethanol also enhances the antibacterial process by denaturation of proteins and dissolving the lipid layer of the cell membrane. This converted the oil molecules to micelles, i.e., having a hydrophilic head and hydrophobic tail allowing to form a colloidal solution (Emulsification). Tween 80 also known as polysorbate 80 acts as a surfactant, i.e., reduces the surface tension of the oil molecules as well as stabilizing the emulsion by breaking it down allowing the oil molecules to penetrate into the dentinal tubules and increasing the contact time with the bacteria, thereby may enhance the effective cleaning of the root canals (Saponification).[31],[37]

Citric acid was added in the irrigant as a chelator in this study which aid in detaching the biofilms from the dentinal walls (Chelation). Studies have shown that ethylenediaminetetraacetic acid solubilizes in water and re-precipitates in alcohol whereas citric acid causes an ester reaction with alcohol resulting in the formation of ethyl citrate which enhances the chelation reaction.[23],[24],[25],[26] In addition, Yamaguchi et al. concluded that citric acid showed antimicrobial effects against a variety of oral microflora.[17]

Blood agar was used to evaluate the colonies of EF after 24 h. EF is a gamma-hemolytic microorganism appear as smooth, cream, or white colonies with entire edges. They lyse the RBC in the blood agar resulting in such colonies giving confirmatory results.[28],[29],[30]

In this study, NaOCl, edible sesame oil, and prepared sesame oil irrigant did show antimicrobial effects on EF, thus rejecting the null hypothesis. However, complete eradication of this organism was not achieved which is still a drawback of endodontic irrigation. Thus, prepared sesame oil irrigant can be considered as a research project in the near future.


  Conclusion Top


Within the limitations of this study, the endodontic irrigating solutions tested showed significant differences in their effectiveness against EF. The present study confirmed the in vitro antimicrobial efficacy of prepared sesame oil mixture was higher compared to 5.25% NaOCl which enables edible sesame oil and its mixture with different study designs with bigger sample size as a string for further studies in vivo studies

Acknowledgment

A special thanks to KNG Agro (P) Ltd, Haryana, RMC marketing, Pune, for providing us sesame oil to carry out this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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