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Year : 2019  |  Volume : 9  |  Issue : 3  |  Page : 181-185

An in vitro evaluation of antimicrobial efficacy of 5% sodium hypochlorite, 2% chlorhexidine, and herbal extracts of neem and Aloe vera in disinfection of gutta-percha cones

Department of Conservative Dentistry and Endodontics, Sinhgad Dental College and Hospital, Pune, Maharashtra, India

Date of Web Publication16-Aug-2019

Correspondence Address:
Dr. Meera Uday Kulkarni
Department of Conservative Dentistry and Endodontics, Sinhgad Dental College and Hospital, S. No. 44/1, Vadgaon Budruk, Off Sinhgad Road, Pune - 411 041, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/sej.sej_110_18

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Aim: An in vitro evaluation of the antimicrobial efficacy of 5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and herbal extracts of neem and Aloe vera in disinfection of gutta-percha cones for 3 min.
Materials and Methods: Ninety gutta-percha cones were used for the study. They were divided into four groups (Group A, Group B, Group C, and Group D) based on the decontaminant used. Each group had 20 cones which were further divided into two subgroups. The microbial species used were Enterococcus faecalis and Staphylococcus aureus. Ten cones in each subgroup were contaminated with E. faecalis and S. aureus, respectively. From the remaining 10 cones, five were contaminated with E. faecalis and five with S. aureus and were disinfected with normal saline which was used as a control. These concentrated extracts were obtained using Soxhlet extraction procedure and were tested for their antimicrobial efficacy by counting the colony-forming units (CFU). The count was taken on day 0 (immediately after disinfection) and day 7 (after incubation for 7 days) for each organism.
Results: The number of CFU on day 0 was not more than five for both E. faecalis and S. aureus for all the disinfectants used. However, on day 7 after incubation, only the neem group showed full growth of colonies for the E. faecalis.
Conclusion: Long-term effects of A. vera were found to be superior and comparable to 5% NaOCl and 2% CHX. However, the long-term effect of neem as a disinfectant for gutta-percha needs further investigation.

Keywords: Colony-forming units, gutta-percha cones, herbal disinfectants, soxhlation procedure

How to cite this article:
Kulkarni MU, Desai N. An in vitro evaluation of antimicrobial efficacy of 5% sodium hypochlorite, 2% chlorhexidine, and herbal extracts of neem and Aloe vera in disinfection of gutta-percha cones. Saudi Endod J 2019;9:181-5

How to cite this URL:
Kulkarni MU, Desai N. An in vitro evaluation of antimicrobial efficacy of 5% sodium hypochlorite, 2% chlorhexidine, and herbal extracts of neem and Aloe vera in disinfection of gutta-percha cones. Saudi Endod J [serial online] 2019 [cited 2020 Jul 2];9:181-5. Available from: http://www.saudiendodj.com/text.asp?2019/9/3/181/264639

  Introduction Top

The success of endodontic therapy is dependent on the maintenance of aseptic environment.[1] It is of utmost importance that the practitioner must be concerned with the exogenous as well as endogenous oral microbial flora.[1] Every material and instrument that is placed in the root canals should be sterile including the obturating material used for optimum infection control. The most commonly used core material for the obturation of the root canal system is gutta-percha.[2] Although these are manufactured under sterile conditions, they may get easily contaminated by improper storage, aerosols, and physical handling.[2],[3] Several studies have found the Staphylococcus genus to be the most common microorganism contaminating gutta-percha cones during their handling with gloves.[4],[5] The incidence was found to be around 15.7% according to Guimaraes et al. and Mukka et al.[6],[7] Studies have also found Enterococcus faecalis to be the most resistant intracanal pathogen in root canal cases that have failed and hence serves as a gold standard organism in endodontic research.

E. faecalis has a superior virulence property, and hence, it was selected for the study to represent the other possible microorganisms that may contaminate gutta-percha cones.[8] Gutta-percha cones are heat labile, thus moist and dry heat sterilization cannot be used as it causes alteration of gutta-percha structure.[9],[10] Hence, it is necessary to focus on cold disinfection techniques for rapid chair-side sterilization of gutta-percha. Furthermore, herbal solutions have been commonly used to treat a variety of conditions, and their popularity has increased as they are cheaper, more accessible, and provide a natural form of alternatives without causing any side effects.[11]

Sodium hypochlorite (NaOCl) is the most commonly used disinfectant and has been considered as a gold standard, as it does not alter the properties of gutta-percha. However, if left within the canal, it can alter the properties of dentin such as the flexural strength and elastic modulus.[12]

Natural products such as Azadirachta indica (neem), Aloe vera, baicalein extracted from the root of Scutellaria baicalensis plant, and lime and lemon oils have been used in endodontics in cleaning and disinfection of root canals either as intracanal medicaments or as irrigants, storage media for avulsed teeth, sealer cements to lubricate and assist in bonding of gutta-percha material, removal of obturation material through softening and dissolving it, removal of smear layer and pulp, and dentin repair.[13]

Therefore, the purpose of the present investigation was to compare the efficacy of A. vera and neem leaf extract with chlorhexidine (CHX) and NaOCl in disinfecting gutta-percha cones.

  Materials and Methods Top

Ninety gutta-percha cones (size 80 and 2% taper, Dentsply, Maillefer) from a freshly opened box under sterile conditions were used for the study. Damaged cones were discarded. They were divided into four groups based on the decontaminant used. Each group had 20 cones which were further divided into two subgroups based on the microorganism used. E. faecalis and Staphylococcus aureus in nutrient broth (HiMedia Laboratories) were the test organisms used.

The decontaminant used for each group was as follows:

  • Group A 1: 5% NaOCl used as a disinfectant against E. faecalis
  • Group A 2: 5% NaOCl used as a disinfectant against S. aureus
  • Group B 1: 2% CHX used as a disinfectant against E. faecalis
  • Group B 2: 2% CHX used as a disinfectant against S. aureus
  • Group C 1: Neem extract used as a disinfectant for E. faecalis
  • Group C 2: Neem extract used as a disinfectant for S. aureus
  • Group D 1: A. vera extract used as a disinfectant against E. faecalis
  • Group D 2: A. vera extract used as a disinfectant against S. aureus.

All the test procedures were carried out in a laminar airflow chamber (Micro-Filt, India). The inoculum containing the microorganisms was serially diluted 10-fold. Aliquots of the dilutions were plated on nutrient agar medium and incubated to note the initial colony count in the suspension.

Preparation of herbal disinfectants

For the preparation of the herbal extracts, fresh neem leaves were dried to evaporate the water and then ground into fine powder [Figure 1]a,[Figure 1]b,[Figure 1]c,[Figure 1]d,[Figure 1]e. Dried A. vera stones were used [Figure 1]f,[Figure 1]g,[Figure 1]h,[Figure 1]i. The dried neem powder and A. vera stones were subjected to Soxhlet extraction procedure to obtain the extracts. These concentrated extracts were thick with a gel-like consistency. The extracts were then dissolved in dimethyl sulfoxide (DMSO) of molecular grade as they are immiscible in saline. DMSO dissolves both polar and nonpolar compounds in the extract and also does not alter their properties. The solution was then filtered through a nylon membrane filter (Medical Millex-GV Syringe Filter Unit) having a pore size of 0.22 microns. The final concentration of neem and A. vera was decided based on the minimum inhibitory concentration levels for each organism based on previous studies.[14],[15] Minimum inhibitory concentration is the lowest concentration of neem that would restrict the bacterial growth of the organisms in the culture media. 5 ml of each disinfectant was divided into four Eppendorf vials [Figure 1]i. 5 ml of physiologic saline solution was filled in the fifth vial and was used as a control.
Figure 1: (a and b) Fresh neem leaves dried and powdered, (c) soxhlet apparatus, (d) evaporation of alcohol residues from extract, (e) pure neem extract, (f and g) dried Aloe vera stones, (h) soxhlet apparatus, and (i) extracts mixed with dimethyl sulfoxide

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Artificial contamination and disinfection of gutta-percha cones

Ten cones in each subgroup were contaminated using fresh isolates of E. faecali s and S. aureus, respectively. The cones in each subgroup were contaminated by immersion in the broth containing each of the microorganisms for 20 min. Then, they were kept in contact with each of the disinfectant for 3 min, rinsed with normal saline, and each cone was individually transferred to sterile Eppendorf tubes containing 1.5 ml of nutrient broth and incubated at 37°C for 7 days. Control procedures were performed in parallel with the test procedures. The purpose of placing the cones individually in the tubes was to obtain standardization. Before incubation, 100 μl of the nutrient broth from each tube was transferred to individual  Petri dish More Detailses containing nutrient agar to note if there were any viable organisms present immediately after disinfection. The plates were incubated and the colony count on day 0 was noted after 24 h. The procedure was repeated on the 7th day, where 100 μl was pipetted from each of the vials that were incubated onto nutrient agar plates. A sterile cotton tip was used to spread the media in a thin layer over the agar. The plates were then incubated for 48 h aerobically at 37°C, and the colony-forming units (CFU) were counted on day 7 [Figure 2] and [Figure 3].
Figure 2: Colonies of Enterococcus faecalis on day 0 and day 7

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Figure 3: Colonies of Staphylococcus aureus on day 0 and day 7

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  Results Top

The antimicrobial efficacy was assessed by the number of CFU for E. faecalis and S. aureus, respectively. From the data presented in [Table 1], we can infer that the CFU for S. aureus on day 0 (immediately after disinfection) and day 7 (after incubation) did not show any significant difference for all the four disinfectants within the stipulated period of 3 min. On the other hand, CFU for the E. faecalis subgroup for all four disinfectants failed to show growth on day 0 but showed growth of colonies on day 7 for the neem group only. The control group showed complete colonization on both day 0 and day 7 for both the organisms.
Table 1: Microbial counts on day 0 and day 7 for Enterococcus faecalis and Staphylococcus aureus after application of the four disinfectants

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  Discussion Top

Endodontic treatment procedures, such as cleaning, shaping, and obturation, require the maintenance of an aseptic protocol.[16] However, since it is difficult to completely eradicate microorganisms from root canal system, it is important to avoid possible sources of exogenous contamination by the use of antimicrobial agents to minimize the chances of reinfection and to enhance peri-radicular healing.[17] Since gutta-percha cones are the most commonly used core material for the obturation of the root canal system,[18] the assurance of its sterility is of critical concern as they are placed in the final step of root canal procedures.[17],[19]

NaOCl (5.25%) is a known disinfectant for effective disinfection of gutta-percha cones.[2] Investigators have also advocated the use of various solutions such as Milton's solution (1%) and Dakin liquid (0.5%) with time varying from 3 to 25 min for disinfection.[1],[2] Investigations on the antimicrobial activity of 0.5%, 1%, 2.5%, 5% NaOCl, and 2% CHX in endodontics have been found in literature.[2] However, there are fewer of studies on the use of natural substances, such as neem and A. vera, which do not permit more objective conclusions about their use.[1],[19] Neem and A. vera being herbal reagents possess several properties such as anti-inflammatory, antifungal, analgesic, antipyretic, and antibacterial. The compounds which impart antiseptic and antibacterial properties to A. vera include lupeol, salicylic acid, urea nitrogen, cinnamic acid, phenols, and sulfur while for neem volatile sulfides provide the antibacterial action.[20],[21] There are several ways to carry out the extraction process from various natural substances such as Soxhlet extraction, hydrodistillation, and ultrasonic extraction. Soxhlet extraction is one of the methods used for conventional extraction of solid samples introduced by Franz Ritter von Soxhlet.[22]

Soxhlet extraction can be carried out using an aqueous or an alcoholic solvent. In the current study, the herbal extracts were prepared using an alcoholic solvent, pure ethanol (99.99%). The advantage of using an alcoholic solvent is that maximum isolation of antioxidant, antibacterial properties, and phenolic compounds from the plant can be obtained since these are generally soluble in polar solvents such as methanol.[23] Till date, the use of Soxhlet extracts of neem and A. vera for disinfection of gutta-percha cones has not been studied fully. Most researches have used commercially available herbal products or oils for testing their antimicrobial efficacy for disinfection of gutta-percha cones.[6],[10]

In this study, fresh neem leaves and dried A. vera stones were used, thus both the extracts obtained had no additional compounds that would alter the antimicrobial properties of the herbal extracts. Moreover, majority of the studies have evaluated the zones of microbial inhibition of various organisms for checking the efficacy of herbal extracts.[1],[19] On the contrary, in the present study, the CFU was counted which gives an accurate measure of the number of organisms that have been destroyed.

The microbial colony count was evaluated on day 0 and day 7 which gave a count of the organisms that were immediately killed and also showed if there were any viable organisms that had multiplied by day 7. Most studies have allowed an incubation period of 48 h rather than 7 days.[1],[19] However, this may not be enough to complete the growth cycle of the organisms.

In this investigation, the comparison of the antimicrobial efficacy of neem leaves and A. vera extracts demonstrated that neem extracts caused a substantial reduction in the microbial count for E. faecalis on day 0; whereas after incubation, the viable bacteria multiplied and showed growth of colonies on day 7. [Table 1] substantiates the fact that there was a significant difference in the values when compared on day 0 and day 7 for the neem group tested for E. faecalis, owing to its superior virulence properties.

For the S. aureus group, the individual microbial colonies for each sample on day 7 were less compared to day 0; however, when the average values of the colonies in each group were taken, they were 0 for both day 0 and day 7 [Table 1]. Thus, we can infer that the neem extract was effective against S. aureus but was not able to show a long-term antimicrobial activity against E. faecalis. For the A. vera, NaOCl and CHX groups, there was no difference in the values when compared on day 0 and day 7 for both E. faecalis and S. aureus. Hence, it showed that the antimicrobial efficacy was not affected even after incubation for 7 days. This indicates that even if there were any viable microorganism they were either killed or their number remained the same; however, there was no increase in the number of colonies.

Shenoi et al. conducted a similar study to compare the efficacy of neem bark extract with 5.25% NaOCl against E. faecalis and S. aureus within a minute.[19] However, in their work, the long-term antimicrobial action of neem bark was not assessed. Mukka et al., in their study, concluded that Pancha Tulsi possesses superior antibacterial activity when compared with A. vera juice and amla juice.[6] Athiban et al., in their study, proved that A. vera is effective against E. coli, E. faecalis, and S. aureus even after 48 h of incubation.[1] The results of their study were in accordance with the results of our study, where A. vera showed antimicrobial effect even after 7 days of incubation. Chandrappa et al. found Mixture of Doxycycline, citric acid and a detergent to be as effective as NaOCl and CHX for disinfection of gutta-percha cones.[2] Makade et al. found lemongrass oil to have the highest antimicrobial activity as compared to commercially available basil oil and obicure tea extract.[10]

Gutta-percha cones can easily get contaminated if handled incorrectly. Neem and A. vera may serve as potent antimicrobial agents for chair-side rapid disinfection of gutta-percha cones and may serve as an alternative to NaOCl.

  Conclusion Top

The study found disinfection with A. vera for 3 min to be the most effective herbal disinfectant against the two most commonly found microorganisms, E. faecalis and S. aureus. Antimicrobial activity of neem for a longer duration against a resistant pathogen such as E. faecalis would require further investigation. Herbal extracts may serve as a promising tool for rapid chair-side disinfection of gutta-percha cones before obturation as they are eco-friendly and cost-effective.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1]


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