| Abstract|| |
Background: Gutta-percha cones used directly from the manufacturer's sealed packs harbour micro-organisms. During clinical use, the chances of contamination of gutta-percha increases. Hence, disinfection of gutta-percha cones before use is essential to prevent canal recontamination. Aim: To evaluate the antimicrobial efficacy of babul against chemical disinfectants, and its effectiveness in disinfecting gutta-percha cones. Materials and Methods: The extract of babul was used to assess its antimicrobial activity against E-faecalis, S. aureus and C. albicans using the agar diffusion method. A total of 120 cones acquired from freshly opened manufacturers' pack were cut into three equal parts and divided into group I which was directly placed into a nutrient broth, group II was clinically contaminated with gloves for 30 s and placed into a nutrient broth, and the third part was clinically contaminated with gloves and decontaminated for 5 min using the experimental disinfectants to check for the presence of turbidity. Statistical Analysis: The data were analysed by an Unpaired t-test and Chi-square test at a significance level of P < 0.05 using software SPSS 20. Results: Two percent chlorhexidine showed maximum antibacterial action against E. faecalis and S. aureus followed by babul extract. It was also found more efficacious in decontaminating gutta-percha cones followed by babul and 3% sodium hypochlorite in 5 min. Conclusion: Babul herbal extract is indeed effective in decontaminating gutta-percha cones and is a better alternative to chemical disinfectants.
Keywords: Antimicrobial efficacy, babul, C. albicans, chlorhexidine, E. fecalis, gutta-percha, S. aureus, sodium hypochlorit
|How to cite this article:|
Jagyasi DR, Chandwani ND, Gunwal MK, Ranka AS. Antimicrobial efficacy of acacia Nilotica (Babul) extract and its effectiveness in disinfecting gutta percha cones - An in vitro study. Indian J Dent Res 2021;32:221-5
|How to cite this URL:|
Jagyasi DR, Chandwani ND, Gunwal MK, Ranka AS. Antimicrobial efficacy of acacia Nilotica (Babul) extract and its effectiveness in disinfecting gutta percha cones - An in vitro study. Indian J Dent Res [serial online] 2021 [cited 2021 Dec 8];32:221-5. Available from: https://www.ijdr.in/text.asp?2021/32/2/221/330849
| Introduction|| |
For optimum infection control, every instrument and material used or placed in the root canals should be sterile. This holds true for obturating materials also. Gutta-percha (GP) is the most commonly used obturating material due to its biocompatibility, dimensional stability, radiopacity, thermoplasticity. GP cones have antimicrobial properties owing to the zinc component. Despite GP cones being manufactured and packaged under sterile conditions, they can get contaminated by aerosols, inadvertent storage, and physical handling. Higgins et al., reported that GP cones can be directly used from their packages with no concern for their sterility. GP cones cannot be sterilized by moist or dry heat owing to their thermoplastic properties.
As GP is heat-labile, chemical disinfectants such as ethyl alcohol, paraformaldehyde, formocresol, chlorhexidine, and sodium hypochlorite are routinely used, which usually take 3–25 min for disinfection. Sodium hypochlorite (NaOCl) being an oxidizing agent, inactivates the bacterial enzymes making it an effective antibacterial and sporicidal agent. Even though most studies affirm the potency of 5.25% NaOCl for rapid disinfection the main concern seems to be the variation seen in the structural and physical properties which were not enviable. Chlorhexidine (CHX) gluconate is a broad-spectrum antimicrobial agent with substantivity and relatively low toxicity. Although sensitivity to CHX is unlikely, contact dermatitis is a common adverse reaction to CHX. Also, topographic changes are seen on the surfaces of GP cones after disinfection with CHX which may impair the radicular seal. Acacia nilotica (Leguminosae) commonly known as babul is an evergreen tree found in different parts of India in abundance. This plant finds an important place in the indigenous system of medicine and is used in the treatment of various systemic diseases including dental problems. It has exhibited antibacterial, antifungal, antihypertensive, antispasmodic, hypoglycemic activity, antioxidant, antiviral and gastroprotective activity. The bark extract is acrid, astringent, and used as a gargle for sore throat, cough, and toothache.
Gutta-percha cones taken directly from the manufacturer's sealed package harbour micro-organisms. During clinical use, the contamination of gutta-percha increases. Therefore, disinfection of gutta-percha cone before use is essential to prevent canal recontamination. Indiscriminate use of antimicrobial agents has led to the development of many resistant microbial strains which forced scientists to search for new antimicrobial substances from herbs. Herbal agents have been used in dental and medical specialties and are prevalent due to their high antimicrobial activity, biocompatibility, anti-inflammatory, and antioxidant properties. Owing to the above-mentioned beneficial properties of 'babul' the current study was devised and executed in three stages. In its first stage, comparison of antimicrobial properties of NaOCl, CHX and babul herbal extract against E-faecalis, Staphylococcus aureus, and Candida Albicans was done. The second stage of the study was to verify whether the GP cones are indeed contaminated during storage and glove handling. In the third stage of the study, a comparison of the disinfection efficacy of 3% NaOCl, 2% CHX and babul for GP cones within the stipulated time was made.
| Materials and Method|| |
The current study was approved by the institutional Ethical Committee of Dental College and Hospital (CDCRI/DEAN/ETHICS/COMMITTEE/CONS-02/2017). The study was designed (i) to evaluate the antimicrobial property of 3% NaOCl (Prime dental products Pvt. Ltd, Mumbai, India), 2% CHX (Exawash 500, Xenon, Biomed India R. N Avenue, Kolkata, India), saline (positive control) and babul herbal extract against Enterococcus faecalis (ATCC 29212)), Staphylococcus aureus (ATCC-25923) and Candida albicans (ATCC-90028); (ii) to verify the contamination of GP cones (size-60, taper-2%; Sure-endo, Suretech city, Korea) during storage and handling with gloves; and (iii) to evaluate the efficacy of NaOCl, CHX and babul herbal extract for disinfection of GP cones.
A total of 120 gutta-percha cones (size-60, taper-2%; Sure-endo, Suretech city, Korea) were obtained from freshly opened manufacturers' packs. Each gutta-percha cone was cut into three equal parts (06 mm) with a sterile stainless scissor (Forgesym Golden sports and surgical industries, Jalandhar, India) and placed in three different sterile containers with sterile tweezer (Forgesym Golden sports and surgical industries, Jalandhar, India); Group 1: The first part was directly placed into nutrient broth (n = 120) Group 2: second part was clinically contaminated with gloves for 30 s and then transferred into the nutrient broth (n = 120); and Group 3: third part was clinically contaminated with gloves for 30 s and divided into four groups of 30 each to be placed in four different disinfectant agents for 5 min. They were then placed into nutrient broth; Group 3a: placed in 3% NaOCl (n = 30); Group 3b placed in 2% CHX (n = 30); Group 3c: placed in babul herbal extract (n = 30); and Group 3d: placed in 0.9% sodium chloride (saline – positive control) (n = 30).
Preparation of babul herbal extract
The herbal extract of babul bark was obtained from Mahaveer College of Ayurvedic Science, Sundra, Rajnandgaon. The babul bark was dried in a hot air oven at 30°C and made in powder form in a pulverizer. A total of 25 gm of formed powder was acquired and mixed with 100 mL of distilled water. This solution was kept for 24 h in a sterile container. The solution was brought to one-fourth of its original volume by boiling up to 300°C in a heating melter. The concentrated extract was filtered with filter paper.
Evaluation of antimicrobial efficacy of disinfecting agents
The inoculums of Staphylococcus aureus and E. faecalis were incubated overnight in nutrient broth (Laxminarayan Institue of Technology, Nagpur, India) to collect a sufficient number of microbial colonies. Each species was then lawn cultured on 12 Petri-dishes (Laxminarayan Institue of Technology). The strain of C. albicans species was cultured on Sabouraud dextrose agar medium (Laxminarayan Institue of Technology). Each petri-dish was punched with a sterile template on its surface to make four wells of 5 × 5 mm. Each well was marked according to the disinfectant dispensed as Group A: 3% sodium hypochlorite (Prime dental products Pvt. ltd. dental division, Mumbai, India) Group B: 2% chlorhexidine (Indoco remedies ltd. dental division, City, India), Group C: babul, Group D: saline (positive-control). About 0.5 mL of the disinfectant to be tested was dispensed into the wells using a micropipette. All strains were tested using the agar well diffusion technique (Kirby-Bauer method) incubated at 37°C for 24 h, and observed for the development of clear zones around the samples used. The antibacterial activity was assessed by measuring the diameter of the clear zone of inhibition in centimeters (cm) against light [Figure 1].
|Figure 1: The zones of inhibition in the Petri dish More Detailses after testing of antibacterial and antifungal activity of 3% NaOCl, 2% CHX, babul extract, and normal saline against E. Faecalis, S. Aureus, and C. Albicans|
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Verification of contamination of GP cones
A total of 120 GP cones were selected from freshly opened manufacturers' packs. Each cone was cut into three parts with a sterile scissor. The first parts of the cones (n = 120) were placed in individual test tubes containing nutrient broth. To achieve contamination, students performing endodontic treatment were asked to handle the second part of gutta-percha (n = 120) for 30 s with their glove hand. These cones were then collected in a sterile container and carried to the Department of Biotechnology (Laxminarayan Institue of Technology, Nagpur) to verify the contamination of gutta-percha cone by placing into the nutrient broth. The third part of the gutta-percha cone was first clinically contaminated with gloves for 30 s and then placed into four different disinfectants for 5 min (n = 30) [Figure 2]. The antimicrobial activity of disinfectants was evaluated by observing turbidity.
|Figure 2: (a) Cutting of the GP cones into 3 sections, (b) Placing the noncontaminated GP section in the nutrient broth, and (c) Placing the glove contaminated GP section in the nutrient broth|
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Verification of antimicrobial activity of disinfecting agents to disinfect the glove handled gutta-percha cones
The third part of gutta-percha cones was first clinically contaminated with gloves for 30 s and divided into four groups containing 30 gutta-percha cones each group. They were divided according to the disinfectant used as Group IIIa: 3% sodium hypochlorite (n = 30, 5 min), Group IIIb: 2% chlorhexidine (n = 30, 5 min), Group IIIc: babul (n = 30, 5 min), Group IIId: saline (n = 30, 5 min). The treated cones were placed on absorbent paper to remove excess disinfecting agents [Figure 3]. They were then immersed in nutrient broth incubated for 24 h and observed for the presence or absence of turbidity. The presence of turbidity at 24 h was considered positive.
|Figure 3: Clinically contaminated GP cone placed in (a) sodium hypochlorite (b) CHX 2% (c) Babul|
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| Results|| |
For the current three tailed evaluation, an unpaired t-test was used to test the difference between an antimicrobial zone of inhibition of 3% NaOCl, 2% CHX, babul extract, and 0.9% sodium chloride. Chi-square test was performed for pairwise comparisons between 3% NaOCl, 2% CHX, babul extract, and 0.9% sodium chloride against E. faecalis, Staphylococcus aureus and Candida albicans. Chi-square test was also used to evaluate the effectiveness of 3% NaOCl, 2% CHX, babul extract, and 0.9% sodium chloride to decontaminate the glove-handled contaminated gutta-percha cones. The significance level was set at P < 0.05. The software version used was SPSS 20.
Evaluation of the antimicrobial activity of disinfecting agents by measuring the zone of inhibition (Stage I)
In this stage, the antimicrobial and antifungal activity of the solution was tested. Here, 2% CHX showed maximum antimicrobial efficacy against E. faecalis and S. aureus followed by babul, whereas 3% NaOCl showed the least antimicrobial efficacy (P < 0.01). Also, 2% CHX exhibited better antifungal activity against Candida albicans (P < 0.01) [Table 1].
|Table 1: Evaluation of antimicrobial activity of disinfecting agents by measuring the zone of inhibition|
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Verification of contamination of gutta-percha cone (Stage II)
In this stage, the GP cones were tested for contamination by the percentage of turbidity created. The contaminated and non-contaminated gutta-percha cones did not show a significant difference in the percentage of turbidity (P = 0.10) [Table 2].
|Table 2: Pairwise comparison between the first part and second part of gutta-percha|
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Evaluating the effectiveness of 3% sodium hypochlorite, 2% chlorhexidine, babuland saline to decontaminate gutta-percha cones (Stage III)
In this stage, the decontamination of GP cones was evaluated using the different solutions tested. Here, 2% CHX showed maximum efficacy in decontamination of gutta-percha cones in 5 min followed by babul and then sodium hypochlorite [Table 3].
|Table 3: Pairwise comparison between 3% sodium hypochlorite, 2% chlorhexidine, babul and saline in decontamination of gutta-percha cone|
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| Discussion|| |
Gutta-percha cones which are used as root canal filling material for over 100 years may be contaminated right from the manufacturing process, packaging, and to its use. In an observation done by Pang et al., where gutta-percha cones were stored in clinical conditions for more than 3 months, they reported that a 19.4% contamination rate in hospital-based endodontic clinics. On the contrary to Pang et al., Montgomery and Gomes et al., reported in their studies that even freshly opened boxes were contaminated with microorganisms from 8% to 25%. Hence, through constant surveillance, it is confirmed that even though GP cones are produced under aseptic conditions, they cannot entirely guarantee complete sterility and can still be contaminated by handling, aerosols and physical sources during the storage process. Studies have shown that freshly opened packages of gutta-percha cones could also be domiciling microorganisms with further contamination on clinical usage.
In the stage I of the present study, the antimicrobial activity of 3% sodium hypochlorite, 2% chlorhexidine, babul herbal extract and saline was assessed against E. faecalis, Staphylococcus aureus and Candida albicans. This was assessed by measuring the zone of inhibition by the agar diffusion method. Amongst all the experimental groups, 3% sodium hypochlorite showed the lowest zone of inhibition, whereas 2% chlorhexidine showed the highest zone of inhibition against E. faecalis and Staphylococcus aureus which implies it having more antibacterial activity when compared to other experimental groups. The highest antimicrobial activity shown by 2% chlorhexidine can be attributed to the fact that it kills bacteria by disruption of the cell membranes and by inducing precipitation of the cytoplasm.,
While evaluating the antimicrobial efficacy against Candida albicans, it was found that 2% chlorhexidine showed a zone of inhibition, whereas 3% sodium hypochlorite, babul extract and saline did not show any zone of inhibition against Candida albicans.
In the stage II of the present study, the contamination of gutta-percha cones when they are directly retrieved from sealed gutta-percha boxes and their further contamination on clinical glove handling. The results showed that 58.33% gutta-percha cones showed turbidity which confirms that almost half of the gutta-percha cones were not sterile. This result is in accordance with the result obtained by Montgomery S et al. and Namazikhah et al. On the other hand, it is expected that contamination would increase upon opening of the package and its usage in the clinical environment. Hence, the second part of gutta-percha cone was intentionally contaminated with gloves for 30 s and then placed into nutrient broth incubated at 37°C for 24 h to evaluate turbidity. The result of the present study showed that 68.33% of gutta-percha cones showed turbidity during clinical glove handling. The finding of this study is in agreement with the previous reported literature., Hence, it justifies the need for disinfection of gutta-percha cone before its use.
In the stage III of the present study, the effectiveness of 3% sodium hypochlorite, 2% chlorhexidine, babul and saline to disinfect glove-handled gutta-percha cone was evaluated. The third part of gutta-percha cone was first intentionally contaminated with gloves for 30 s followed by decontamination with 3% sodium hypochlorite, 2% chlorhexidine, babul and saline for 5 minutes. After decontamination, gutta-percha cones were placed in nutrient broth incubated at 37°C for 24 h. Two percent chlorhexidine showed maximum efficacy in decontamination of gutta-percha cones followed by babul and sodium hypochlorite. Babul showed better efficacy in the decontamination of gutta-percha cones as it contains phenolics, condensed tannin and phlobatannin, gallic acid, protocatechuic acid which are responsible for its antimicrobial activity. Babul extract showed antimicrobial activity against E. faecalis, Staphylococcus aureus, and it also had the capacity to disinfect glove handled–contaminated gutta-percha cones.
| Conclusion|| |
Based on these outcomes, it seems that babul extract can potentially be used during endodontic practice for disinfection of gutta-percha cones. However, within the limitations of the study, further studies are needed to evaluate the antimicrobial action of babul extract against other microorganisms that may contaminate gutta-percha cone surfaces.
| Acknowledgments|| |
The authors wish to thank Mrs. Aarti Shanware and Miss Shreya Jajoo, Department of Biotechnology, Laxminarayan Institute of Technology, Nagpur for their valuable support during the research.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Neelam D Chandwani
Flat no 301, Kanha Vihar, Gokul Housing Society, Gorewada Road, Borgaon, Nagpur - 440 013, Maharashtra
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]