| Abstract|| |
Context: Sodium hypochlorite is the most widely used irrigant in endodontic practice, but it has various disadvantages. Literature has shown that herbal products such as Propolis, Azadirachta indica (AI), Triphala, Curcuma longa, and Morinda citrifolia (MC) possess good antimicrobial properties and thus can be used as potential endodontic irrigants.
Aim: To evaluate and compare the antimicrobial activity of five herbal extracts, i.e., Propolis, AI, Triphala, C. longa, and MC with that of 2.5% sodium hypochlorite against Enterococcus faecalis.
Materials and Methods: E. faecalis American Type Culture Collection 21292 was inoculated onto brain heart infusion agar plate. Discs impregnated with herbal medicaments were placed on the inoculated plates and incubated at 37°C aerobically for 24 h and growth inhibition zones were measured.
Statistical Analysis: Mean zone of inhibition in descending order was found as sodium hypochlorite > Propolis > AI > Triphala > C. longa = MC > ethanol. Statistical analysis was performed using one-way analysis of variance which showed a significant difference in the zone of inhibition of sodium hypochlorite and Propolis (P < 0.001).
Results: Propolis showed highest zone of inhibition among all the herbal extracts next to sodium hypochlorite.
Conclusion: Propolis and AI have significant antimicrobial activity against E. faecalis.
Keywords: Antimicrobial activity, Enterococcus faecalis, herbal extracts
|How to cite this article:|
Saxena D, Saha SG, Saha MK, Dubey S, Khatri M. An in vitro evaluation of antimicrobial activity of five herbal extracts and comparison of their activity with 2.5% sodium hypochlorite against Enterococcus faecalis. Indian J Dent Res 2015;26:524-7
Elimination of microorganisms from infected root canals always remains a challenging task. The chances of successful root canal treatment are significantly superior if microbes are eradicated effectively before the root canal system is obturated. However, tenacious existence of micro-organisms in the canal leads to treatment failure. 
|How to cite this URL:|
Saxena D, Saha SG, Saha MK, Dubey S, Khatri M. An in vitro evaluation of antimicrobial activity of five herbal extracts and comparison of their activity with 2.5% sodium hypochlorite against Enterococcus faecalis. Indian J Dent Res [serial online] 2015 [cited 2019 Nov 14];26:524-7. Available from: http://www.ijdr.in/text.asp?2015/26/5/524/172080
Enterococcus faecalis is most frequently isolated from persistent apical periodontitis  and has many virulence factors. It remains viable and is more resistant to endodontic treatment with the capability to invade the dentinal tubules and adhere to collagen in the presence of human serum, thus acting as a pathogen in failed endodontic treatment. 
Various measures to reduce the number of microorganisms in the root canal system include the use of various instrumentation techniques, irrigation regimens, and intracanal medicaments. Irrigation is complementary to instrumentation in facilitating the removal of pulp tissue and/or microorganisms. Sodium hypochlorite (NaOCl) has been universally used as a root canal irrigant. However, this popular irrigant has various side effects such as unpleasant taste and odor, tissue toxicity, inability to remove smear layer, inability to fully eradicate microbes from the infected canals, allergic potential, risk of emphysema on overfilling, and discoloration of clothes. 
Due to potential side effects, safety concerns, and ineffectiveness of conventional endodontic irrigants, we are now looking for herbal alternatives. Extracts of plant origin have been found to have therapeutic properties since thousands of years. As per WHO report, 80% of world's population relies mainly on traditional therapies.  There are many advantages of using herbs as antimicrobials: (a) They have minimal side effects; (b) are cost effective; (c) have better patient tolerance; and (d) are renewable in nature. Phytochemical extracts such as Curcuma longa (CT) -Turmeric, Azadirachta indica (AI) - Neem, Morinda citrifolia (MC) - Noni, Propolis (P), and Triphala (T) have been reported to exert antimicrobial properties suggesting their potential to be used as endodontic irrigants. However, there is a lack of sufficient supporting documentation regarding the antibacterial activity of these extracts in endodontics.
Therefore, the purpose of this study was to evaluate and compare the antimicrobial activity of these extracts with the standard irrigant (2.5% sodium hypochlorite) against E. faecalis so as to assess their utility as root canal irrigants.
| Materials and methods|| |
Preparation of herbal medicaments
CT (Vindhya Herbals, India), AI (Vindhya Herbals, India), Triphala (Vindhya Herbals, India), and MC (Vestige, India) extracts available in powder form were used for the study. 20 g of each powder were mixed with 25 ml of ethanol and kept in airtight containers for 48 h. 20 g of Propolis, which is available in crude cake form was mixed with 25 ml of ethanol (Changshu Yangyuan Chemical, China) and kept for 48 h in an airtight container.
After 48 h, extracts were filtered using Whatman filter papers and filtrates were collected in Petri dishes. From each Petri dish, 20 mg of extract was taken and mixed with 2 ml ethanol and kept in test tubes. From each test tube, 10 μL solution was loaded on sterile discs (Hi-Media Labs Mumbai, India) till the filtrate got completely absorbed by the discs. Discs were then allowed to dry. Discs of sodium hypochlorite (Vishal Laboratories, India) and ethanol were also similarly prepared, which served as control.
Isolation of microorganism
Pure strain of E. faecalis from American Type Culture Collection number 29212 was used. Culture was grown overnight at 37°C in brain heart infusion (BHI) broth (Hi-Media Labs, Mumbai, India) and microbial growth was checked by changes in turbidity at 24 h.
Disc diffusion method/Kirby-Bauer method
The BHI broth containing E. faecalis was inoculated on BHI agar plate (Hi-Media Lab, Mumbai, India) using lawn culture method with the help of a sterile glass spreader. Five test herbal discs (CT, AI, MI, P, and T); and two control discs (ethanol and sodium hypochlorite) were placed on the culture plate. The plate was incubated aerobically for 24 h at 37°C. After 24 h, microbial zone of inhibition was measured in millimeters.
| Results|| |
Results were recorded on the basis of the diameter of zones. Mean zone of inhibition in descending order for each drug was found to be: NaOCl (22.0 ± 1.53) > Propolis (12.2 ± 1.53) > AI (08.8 ± 1.60) > Triphala (05.1 ± 0.94) > CT (02.2 ± 0.40) = MC (02.2 ± 0.40) > Ethanol (00.0 ± 0.00) as shown in [Figure 1]. Statistical analysis was made using one-way analysis of variance, which showed the significant difference in the zone of inhibition of NaOCl and Propolis (P < 0.001) with no significant difference was observed among Triphala, CT, and MC.
|Figure 1: Zone of inhibition (in millimeter) of herbal extracts, sodium hypochlorite, and ethanol against Enterococcus faecalis|
Click here to view
| Discussion|| |
Long-term predictable success of root canal treatment depends on complete removal of causative microorganisms from complex root canal anatomical areas. An attempt was made to select representative bacteria that have been classically isolated from necrotic canals. E. faecalis was chosen as a test organism because it is a facultative Gram-positive organism that rapidly colonizes dentinal tubules. It has been extensively used in endodontic research because it has been found to be evident in 63% of teeth with endodontic failures. 
The agar diffusion test has been used in this study as it is one of the most commonly used methods for assessment of the anti-microbial activity of endodontic irrigants. It allows direct comparisons of the test materials against the test microorganisms, suggesting which material has the ability to eliminate bacteria in the local microenvironment of the root canal system. ,,, The five herbal extracts used in the present study have been proven to be safe, having active components that possess beneficial antimicrobial properties. Thus, they have shown to have potential to replace the conventional root canal irrigant, i.e., NaOCl. ,,,,
In this study, sodium-hypochlorite showed the largest zone of inhibition, which may be because of its volatile nature and high diffusibility on agar plate. However, the size of the inhibition zones does not indicate the absolute antimicrobial activity of the material. Various factors also play an important role, such as solubility and diffusibility in the agar, loading dose on agar plate, method of extraction of herb, and volatile nature of drugs. 
In the present study, Propolis showed the highest zone of inhibition among all the herbal extracts, which has already shown good antimicrobial activity in previous studies.  The Propolis contains important pharmacologically active constituents such as flavonoids, phenolics, and aromatics. It is believed that flavonoids account for maximum biological activity in Propolis.  Some of the important biological activities of Propolis are antimicrobial, anti-inflammatory, antioxidant, anesthetic action, and cytotoxicity.  Ethanol extract of Propolis presents good antimicrobial properties for endodontic use. It enhances bone regeneration and induces hard tissue formation in pulpotomies or pulp capping. Propolis, being a good antimicrobial and anti-inflammatory agent, shows promise as a effective intracanal irrigant and intracanal medicament. A comparative evaluation of the microbial efficacy of Propolis, NaOCl, and saline when used as intracanal irrigants indicated that Propolis has antimicrobial activity comparable to that of NaOCl.  AI has various active constituents such as nimbidin, nimbin, nimbolide, gedunin, azadirachtin, mahmoodin, margolone, and cyclictrisulphide, which are responsible for its antibacterial action. It has been proved to be effective against E. faecalis. Its antioxidant and antimicrobial properties make it a potential agent for root canal irrigation as a substitute to sodium hypochlorite.  It showed second highest zone of inhibition which may be because of its active constituents possessing antimicrobial effects. Triphala is an Indian Ayurvedic herbal combination of dried and powdered fruits of three medicinal plants, Terminalia bellerica, Terminalia chebula, and Emblica officinalis. It is rich in citric acid, which may aid in removal of smear layer thereby acting as a chelating agent, which can be used as an adjunct to sodium hypochlorite for root canal irrigation.  Curcumin, a yellow bioactive pigment, which is the major constituent of CT, has a wide spectrum of biological actions such as anti-inflammatory, antioxidant, antifungal, and antibacterial activities.  Antibacterial properties of MC is attributed to the presence of L-asperuloside and alizarin.  Murray et al. evaluated MC juice in conjunction with EDTA as a possible alternative to NaOCl.  However, in the present study, Triphala, Turmeric, and Morinda showed minimum antimicrobial activity when compared to other studies,  which may be because of (1) minimum loading dose, i.e., 10 μL was used to prepare discs (2) unlike previous studies, in which aqueous extracts were used,  in this study, alcoholic extracts were used.
| Conclusion|| |
Recent researches on the herbal products have proven that herbs are not only known for their flavor and fragrance, but also for their antimicrobial activity and medicinal value. Within the limitations of the present study, Propolis and AI showed promising results in the elimination of E. faecalis. However, their activity was found to be inferior to NaOCl. As these drugs have several advantages over NaOCl such as easy availability, increased shelf life, minimal side effects, and no reported microbial resistance, these extracts appear to be promising in the field of endodontics. Further, in vitro and clinical studies are required to evaluate their efficacy, biocompatibility, and safety factors before they can conclusively be recommended as root canal irrigants.
I wish to acknowledge Dr. Savita Vyas, Associate professor, Department of Pharmacology, M.G.M. Medical College, Indore, for her guidance.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Sjögren U, Figdor D, Persson S, Sundqvist G. Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int Endod J 1997;30:297-306.
Adib V, Spratt D, Ng YL, Gulabivala K. Cultivable microbial flora associated with persistent periapical disease and coronal leakage after root canal treatment: A preliminary study. Int Endod J 2004;37:542-51.
Elsner HA, Sobottka I, Mack D, Claussen M, Laufs R, Wirth R. Virulence factors of Enterococcus faecalis
and Enterococcus faecium
blood culture isolates. Eur J Clin Microbiol Infect Dis 2000;19:39-42.
Murray PE, Farber RM, Namerow KN, Kuttler S, Garcia-Godoy F. Evaluation of Morinda citrifolia
as an endodontic irrigant. J Endod 2008;34:66-70.
World Health Organization. Summary of WHO guidelines for the assessment of herbal medicines. Herb Gram 1993;28:13-4.
Love RM. Enterococcus faecalis
- A mechanism for its role in endodontic failure. Int Endod J 2001;34:399-405.
Grossman L. Antimicrobial effect of root canal cements. J Endod 1980;6:594-7.
Gomes BP, Pedroso JA, Jacinto RC, Vianna ME, Ferraz CC, Zaia AA, et al. In vitro
evaluation of the antimicrobial activity of five root canal sealers. Braz Dent J 2004;15:30-5.
Gomes BP, Vianna ME, Sena NT, Zaia AA, Ferraz CC, de Souza Filho FJ. In vitro
evaluation of the antimicrobial activity of calcium hydroxide combined with chlorhexidine gel used as intracanal medicament. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:544-50.
Abdulkader A, Duguid R, Saunders EM. The antimicrobial activity of endodontic sealers to anaerobic bacteria. Int Endod J 1996;29:280-3.
Oncag O, Cogulu D, Uzel A, Sorkun K. Efficacy of propolis as an intracanal medicament against Enterococcus faecalis
. Gen Dent 2006;54:319-22.
Prabhakar J, Senthilkumar M, Priya MS, Mahalakshmi K, Sehgal PK, Sukumaran VG. Evaluation of antimicrobial efficacy of herbal alternatives (Triphala and green tea polyphenols), MTAD, and 5% sodium hypochlorite against Enterococcus faecalis
biofilm formed on tooth substrate: An in vitro
study. J Endod 2010;36:83-6.
Gul N, Mujahid TY, Jehan N, Samia A. Studies on the antibacterial effect of different fractions of Curcuma longa
against urinary tract infection isolates. Pak J Biol Sci 2004;7:2055-60.
Dubey S, Chaodary M, Gupta P. Comparative study of the antimicrobial efficiency of neem leaf extract, sodium hypochlorite and Biopure MTAD - An in vitro
study. Indian J Dent Adv 2012;4:740-3.
Kumar H. An in vitro
evaluation of the antimicrobial efficacy of Curcuma longa
, Tachyspermum ammi
, chlorhexidine gluconate, and calcium hydroxide on Enterococcus faecalis
. J Conserv Dent 2013;16:144-7.
Dumani A, Yoldas O, Yilmaz S, Akcimen B, Seydaoglu G, Kipalev A, et al. In vitro
susceptibility of E. faecalis
and C. albicans
isolates from apical periodontitis to common antimicrobial agents, antibiotics and antifungal medicaments. J Clin Exp Dent 2012;4:e1-7.
Ferreira FB, Torres SA, Rosa OP, Ferreira CM, Garcia RB, Marcucci MC, et al.
Antimicrobial effect of propolis and other substances against selected endodontic pathogens. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:709-16.
Koo H, Rosalen PL, Cury JA, Park YK, Bowen WH. Effects of compounds found in propolis on Streptococcus mutans
growth and on glucosyltransferase activity. Antimicrob Agents Chemother 2002;46:1302-9.
Polaquini SR, Svidzinski TI, Kemmelmeier C, Gasparetto A. Effect of aqueous extract from neem (Azadirachta indica
A. Juss) on hydrophobicity, biofilm formation and adhesion in composite resin by Candida albicans
. Arch Oral Biol 2006;51:482-90.
Leach AJ, Leach DN, Leach GJ. Antibacterial activity of some medicinal plants of Papua New Guinea. Sci N G 1988;14:1-7.
Department of Conservative Dentistry and Endodontics, College of Dental Science and Hospital, Indore, Madhya Pradesh
Source of Support: None, Conflict of Interest: None