|Year : 2015 | Volume
| Issue : 4 | Page : 274-278
Comparative evaluation of antimicrobial activity of hydroalcoholic extract of Aloe vera, garlic, and 5% sodium hypochlorite as root canal irrigants against Enterococcus faecalis: An in vitro study
Swati Ramesh Karkare1, Nivedita Pramod Ahire2, Smita Uday Khedkar3
1 Department of Pedodontics, MGV's KBH Dental College and Hospital, Nashik, Maharashtra, India
2 Department of Preventive Dentistry, MGV's KBH Dental College and Hospital, Nashik, Maharashtra, India
3 Department of Microbiology, MGV's KBH Dental College and Hospital, Nashik, Maharashtra, India
|Date of Web Publication||18-Sep-2015|
Dr. Nivedita Pramod Ahire
B-37/38, Garden View, Devidayal Road, Opposite Mulund Bus Depot, Mulund-West, Mumbai - 400 080, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Enterococcus faecalis are the most resistant and predominant microorganisms recovered from root canals of teeth where previous treatment has failed. Over the past decade, interest in drugs derived from medicinal plants has markedly increased. In dentistry, phytomedicines has been used as an anti-inflammatory, antibiotic, analgesic, sedative, and also as an endodontic irrigant. In endodontics, because of the cytotoxic reactions of most of the commercial intracanal medicaments and their inability to eliminate bacteria completely from dentinal tubules, the trend is shifting toward use of biologic medication extracted from natural plants. Aim: To compare the antimicrobial efficacy of newer irrigating agents which would probably be as effective or more and at the same time less irritating to the tissues than sodium hypochlorite (NaOCl). The objective of this study was to compare the antimicrobial activity of saturated and diluted (1:1) hydroalcoholic extract of Aloe vera, garlic, and 5% NaOCl against E. faecalis using the commonly used agar diffusion method. Results: Saturated hydroalcoholic extract of A. vera showed the highest zone of inhibition against E. faecalis. NaOCl, which is considered as gold standard, also showed higher zones of inhibition.
Keywords: Agar diffusion, Aloe vera, Enterococcus faecalis, garlic, herbal, root canal irrigants
|How to cite this article:|
Karkare SR, Ahire NP, Khedkar SU. Comparative evaluation of antimicrobial activity of hydroalcoholic extract of Aloe vera, garlic, and 5% sodium hypochlorite as root canal irrigants against Enterococcus faecalis: An in vitro study. J Indian Soc Pedod Prev Dent 2015;33:274-8
|How to cite this URL:|
Karkare SR, Ahire NP, Khedkar SU. Comparative evaluation of antimicrobial activity of hydroalcoholic extract of Aloe vera, garlic, and 5% sodium hypochlorite as root canal irrigants against Enterococcus faecalis: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2015 [cited 2019 Jul 22];33:274-8. Available from: http://www.jisppd.com/text.asp?2015/33/4/274/165658
| Introduction|| |
The effectiveness of endodontic files, rotary instrumentation, irrigating solutions, and chelating agents to clean, shape, and disinfect root canals underpins the success, longevity, and reliability of modern endodontic treatments. The role of microorganisms in the development and perpetuation of pulp and periapical diseases has clearly been demonstrated in animal models and human studies. ,,, Elimination of microorganisms from infected root canals is a complicated task. The chances of a favorable outcome with root canal treatment are significantly higher if the infection is eradicated effectively before the root canal system is obturated. However, if microorganisms persist at the time of obturation or if they penetrate into the canal after obturation, there is a high risk of treatment failure. , Numerous measures have been described to reduce the number of microorganisms in the root canal system, including the use of various instrumentation techniques, irrigation regimens, and intracanal medicaments. The use of chemical agents during instrumentation to completely clean all aspects of the root canal system is central to successful endodontic treatment.  The main aim of an endodontic treatment is to remove the diseased tissue, eliminate bacteria from the root canal system, and prevent its recontamination.  Irrigation is carried out to reduce the number of bacteria in the root canal system and to control the periapical disease.  A wide variety of synthetic antimicrobial agents have been used over the years as endodontic irrigants. Because of the increased antibiotic resistance to these antimicrobial agents, toxic and harmful side effects of few common antibacterial agents, there is a need for alternative agents which are affordable, nontoxic, and effective. It has been found that natural plant extracts could be used as effective endodontic irrigants. 
Sodium hypochlorite (NaOCl) is one of the most widely used endodontic irrigant because of its ability to destroy a broad spectrum of microbes but it has some undesirable characteristics such as tissue toxicity, allergic potential, disagreeable taste, and inability to remove the smear layer. ,, Primary root canal infections are polymicrobial, typically dominated by obligatory anaerobic bacteria.  The most frequently isolated microorganisms before root canal treatment include Gram-negative anaerobic rods, Gram-positive anaerobic cocci, Gram-positive anaerobic and facultative rods, Lactobacillus species, and Gram-positive Streptococcus species. The obligate anaerobes are rather easily eradicated during root canal treatment. On the other hand, facultative bacteria such as Staphylococcus, Enterococci, once established, are more likely to survive chemomechanical instrumentation and root canal.  In particular, Enterococcus faecalis has gained attention in the endodontic literature, as it is likely to be isolated from root canals in cases of failed root canal treatments. Herbal products have been used since ancient times in folk medicine, involving both Eastern and Western medicinal traditions. Many plants with biological and antimicrobiological properties have been studied since there has been a relevant increase in the incidence of antibiotic overuse and misuse. In dentistry, phytomedicines has been used as anti-inflammatory, antibiotic, analgesic, and sedative agents.  In this in vitro study, the herbal agents explored are Aloe vera and garlic. The microorganism used in the study is E. faecalis.
| Materials and Methods|| |
The following materials were used in the study:
5% of NaOCl, hydroalcoholic extract of A. vera and hydroalcoholic extract of garlic (Tulsi Amrit, Indore), E. faecalis (ATCC 11420) (National Chemical Laboratories, Pune), Mueller Hinton Agar (High Medium Laboratories, Mumbai). Sterile beakers, sterile test-tubes, sterile cork borer, and sterile droppers [Figure 1].
Preparation of saturated hydroalcoholic extract of Aloe vera and garlic
Five grams of the available hydroalcoholic extract was dissolved in 10 ml of distilled water in sterile containers. Containers were shaken well, precipitate was left beneath, and only the supernatant was taken for the study.
Preparation of diluted hydroalcoholic extract of Aloe vera and garlic
Preparation was same as that of saturated hydroalcoholic extract of A. vera and garlic. Later, 1:1 dilution was done by adding 1 part of saturated solution of hydroalcoholic extract to 1 part of sterile distilled water.
Preparation of microbial inocula
The density of selected organisms was adjusted equal to that of the 0.5 McFarland standards (1.5 × 108 CFU/ml) by adding them to nutrient broth for E. faecalis. A 24 h old culture was used for the preparation of bacterial suspension. McFarland standards were used as a reference to adjust the turbidity of microbial suspension so that the number of microorganisms would be within a given range.
Antimicrobial susceptibility test
Agar well diffusion method was used to conduct the antimicrobial susceptibility test.  E. faecalis was cultured on Mueller Hinton agar plate. Four different concentrations (saturated and diluted form) of each of the prepared hydroalcoholic extracts were made using distilled water. The agar media plates were inoculated with the organisms by even streaking of the swab over the entire surface of the plate 3 times, rotating the plate approximately 60° after each application to ensure an even distribution of the inoculums. 3 wells of 8 mm size were made with sterile borer into each agar plate containing the bacterial inoculums. 100 μl volume of each of the hydroalcoholic extract prepared in saturated concentrations was dispensed into the wells of inoculated plates. 5% NaOCl was the positive control since it is the commonly used irrigant and has excellent antimicrobial activity against the above-mentioned organisms. The plates, thus prepared were incubated for 24 h at 37°C. The zone of inhibition was measured and expressed in millimeters [Figure 2]. Antibacterial activity was recorded if the zone of inhibition was >8 mm. Experiment was performed 3 times and mean of the zone of inhibition was recorded in mm. The antibacterial activity results were expressed in terms of the diameter of zone of inhibition and <9 mm zone was considered as inactive; 9-12 mm as partially active; whereas 13-18 mm as active; and >18 mm as very active.  The mean and standard deviation of the diameter of inhibition zones were calculated.
| Discussion|| |
To understand whether there is any significant difference among saturated A. vera, diluted A. vera, and NaOCl when compared for inhibition zone, ANOVA test was applied at 95% significance level and 2 and 6 degrees of freedom. The calculated value of F is 9.3749 which is found significant with P = 0.000. Knowing that there is significant difference among the three samples saturated; A. vera, diluted A. vera, and NaOCl, further unpaired t-test is applied at 95% confidence level and 4 degrees of freedom, the results obtained are shown in the [Table 1].
|Table 1: Comparison of mean inhibition zone between saturated A. vera, diluted A. vera and NaOCl|
Click here to view
Observing [Table 1], it can be said that there is significant difference between mean inhibition zone for saturated A. vera and diluted A. vera as well as diluted A. vera, and NaOCl, while there is no significant difference between the mean inhibition zone obtained by saturated A. vera and diluted A. vera. Looking at the mean values of all the three, it can be said that the best solution is NaOCl followed by saturated A. vera and lastly diluted A. vera.
Similarly, another group of saturated garlic and NaOCl is tested for mean inhibition region. ANOVA test is applied at 95% confidence level and 2 and 6 degrees of freedom where the calculated value of 'F' statistic is 4.75 which is found insignificant with P = 0. 582. The statistics hence shows that there is no significant difference between the readings of mean inhibition obtained using three different samples.
Further, unpaired t-test is applied to understand if there is any significant difference between groups at 95% confidence level and 4 degrees of freedom. The results obtained are shown in [Table 2].
|Table 2: Comparison of mean inhibition zone between saturated garlic, diluted garlic, and NaOCl|
Click here to view
Observing [Table 2], it can be seen that there is no significant difference between the mean readings of inhibition zone obtained using saturated garlic, diluted garlic, and NaOCl . All the three are equal.
Finally, saturated A. vera and saturated garlic solution and diluted A. vera and diluted garlic solutions were compared for the mean readings of inhibition region. The results obtained are shown in [Table 3], after applying unpaired t-test with 95% confidence level and 4 degrees of freedom.
|Table 3: Mean of zone of inhibition in saturated and diluted (1:1) form of hydroalcoholic extract of A. vera and garlic|
Click here to view
From [Table 3], it can be seen that there is no significant difference found between the saturated solutions of A. vera and garlic when mean inhibition region reading is compared as well as there was no significant difference found for dilute solutions of A. vera and garlic both are at equal level.
An ideal irrigating solution should possess maximal antimicrobial and tissue solving properties and minimal toxic effects.  NaOCl has been used as long as a root canal irrigator because of its enormous antibacterial activity.  Several studies have revealed that the antibacterial effect of NaOCl is much greater than normal saline and other root canal irrigating solutions.  A. vera is a kind of plant, where its leaf is composed of two major parts. The outer green rind contains vascular bundles and the inner colorless part, named as aloe gel, includes the parenchymal cells which contain viscous clear liquid.  The aloe gel consists of 99.5% of water. The remaining solid material includes mineral, vitamins, enzymes, polysaccharides, phenolic compounds, and organic acids.  It has been proved to have multiple therapeutic and antimicrobial properties, especially on E. faecalis. ,,,, Furthermore, it has been successfully used for decontamination of Gutta-percha.  In the present in vitro study, E. faecalis, which is a Gram-positive bacterial strain, was used to evaluate the antibacterial effect of irrigating solutions due to its proved resistance to antimicrobial agents.  Several studies have shown that A. vera extract, especially acts against Gram-positive bacteria.  The antibacterial activity of A. vera is related to anthraquinones which are phenolic derivatives.  According to Sureshchandra and Kumar, A. vera extract is effective against E. faecalis in agar medium. 
According to earlier reports, garlic has traditional dietary and medicinal applications as an anti-infective agent.  In vitro evidence of the antimicrobial activity of fresh and freeze dried garlic extracts against many bacteria,  fungi and viruses,  supports these applications. Allicin, the active ingredient of garlic, acts by partially inhibiting DNA and protein synthesis and also totally inhibiting RNA synthesis as a primary target.  Organosulfur compounds and phenolic compounds have been reported to be involved in the garlic antimicrobial activity. ,,,,,
The antimicrobial potency of plants is believed to be due to tannins, saponins, phenolic compounds, essential oils, and flavonoids.  It is interesting to note that crude extracts of these plants showed good activity against multidrug-resistant strains where modern antibiotic therapy has limited effect. The effect of these spices on these organisms in vivo cannot be predicted from this study. Moreover, though paper disk assays are practical approach to study potential antibacterial compounds, using the size of inhibition zone to indicate relative antibacterial activity is not adequate. The zone must be affected by the solubility and rate of diffusion in agar medium or its volatilization; and thus the results could be affected. Thus, there is a need for detailed scientific study of traditional medical practices to ensure that valuable therapeutic knowledge of some plants is preserved and also to provide scientific evidence for their efficacies.
To conclude, there is wide body of scientific evidence to show that garlic has great potential in the treatment of many microbial diseases. Since the garlic extract used in the present study was dried hydroalcoholic, the exact concentration of the active ingredient, that is, allicin cannot be assessed, so it did not show good antibacterial activity. Freshly prepared extract of garlic is always better than commercially available products, as no preservatives are added in the former one which may give biased results. We prophesize that these plants have an extraordinary potential to yield biologically active materials which could be valuable in the treatment of many microbial diseases and this should be fully explored in proper approach. However, it is necessary to isolate the active constituents, and determine their toxicity, side effects, and pharmaco-kinetic properties.
Special thanks to my guide Dr. Swati Karkare for her encouragement and support throughout my study. I am also thankful to Dr. Smita Khedkar and Mr. Kiran Erende for their enormous help and guidance.
Also, I would like to thank Dr. Kartik R. Lote, Dr. Yogesh Khandelwal, Dr. Khushboo Patel and Dr. Priyanka Harshal Gajeshwar for their kind assistance throughout the study.
Financial support and sponsorship
National Chemical Laboratories, Pune, Tulsi Amrit Manufacturing Limited, Indore.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965;20:340-9.
Möller AJ, Fabricius L, Dahlén G, Ohman AE, Heyden G. Influence on periapical tissues of indigenous oral bacteria and necrotic pulp tissue in monkeys. Scand J Dent Res 1981;89:475-84.
Sundqvist G. Ecology of the root canal flora. J Endod 1992;18:427-30.
Mohammadi Z. An update on the antibiotic-based root canal irrigation solutions. Iran Endod J 2008;3:1-7.
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.
El Karim I, Kennedy J, Hussey D. The antimicrobial effects of root canal irrigation and medication. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:560-9.
Shen Y, Stojicic S, Qian W, Olsen I, Haapasalo M. The synergistic antimicrobial effect by mechanical agitation and two chlorhexidine preparations on biofilm bacteria. J Endod 2010;36:100-4.
Calt S, Serper A. Time-dependent effects of EDTA on dentin structures. J Endod 2002;28:17-9.
Hülsmann M, Heckendorff M, Lennon A. Chelating agents in root canal treatment: Mode of action and indications for their use. Int Endod J 2003;36:810-30.
Kamat S, Rajeev K, Saraf P. Role of herbs in endodontics. An update. Endodontology 2011;23:98-102.
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.
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.
Mohammadi Z, Yasd I. Sodium hypochlorite in endodontics: An update review. IDJ 2008;58:329-41.
Zehnder M. Root canal irrigants. J Endod 2006;32:389-98.
Groppo FC, Bergamaschi Cde C, Cogo K, Franz-Montan M, Motta RH, de Andrade ED. Use of phytotherapy in dentistry. Phytother Res 2008;22:993-8.
Leonardo MR, da Silva LA, Tanomaru Filho M, Bonifácio KC, Ito IY. In vitro
evaluation of antimicrobial activity of sealers and pastes used in endodontics. J Endod 2000;26:391-4.
Junior A, Zanil C. Biological screening of Brazilian medicinal plants. Braz J Sci 2000;95:367-73.
Onçag O, Hosgör M, Hilmioglu S, Zekioglu O, Eronat C, Burhanoglu D. Comparison of antibacterial and toxic effects of various root canal irrigants. Int Endod J 2003;36:423-32.
Siqueira JF Jr, Machado AG, Silveira RM, Lopes HP, de Uzeda M. Evaluation of the effectiveness of sodium hypochlorite used with three irrigation methods in the elimination of Enterococcus faecalis
from the root canal, in vitro
. Int Endod J 1997;30:279-82.
Hamman JH. Composition and applications of Aloe vera
leaf gel. Molecules 2008;13:1599-616.
Agarry OO, Olaleye MT, Bello-Michael CO. Comparative antimicrobial activities of Aloe vera
gel and leaf. Afr J Biotechnol 2005;4:1413-4.
Sureshchandra B, Kumar AJ. Antibacterial efficacy of Aloe vera
extract on resistant antimicrobial strains in endodontics. Endodontology 2011;23:56-60.
Thirupathi S, Ramasubramanian V, Sivakumar T, Thirumalaiarasu VJ. Anti-bacterial and antifungal activity of Aloe vera
gel extract. J Biol Sci Res 2010;1:251-8.
Athiban PP, Borthakur BJ, Ganesan S, Swathika B. Evaluation of antimicrobial efficacy of Aloe vera
and its effectiveness in decontaminating Gutta percha cones. J Conserv Dent 2012;15:246-8.
Love RM. Enterococcus faecalis:
A mechanism for its role in endodontic failure. Int Endod J 2001;34:399-405.
Lawrence R, Tripathi P, Jeyakumar E. Isolation, purification and evaluation of antibacterial agents from Aloe vera
. Braz J Microbiol 2009;40:906-15.
Ross ZM, O′Gara EA, Hill DJ, Sleightholme HV, Maslin DJ. Antimicrobial properties of garlic oil against human enteric bacteria: Evaluation of methodologies and comparisons with garlic oil sulfides and garlic powder. Appl Environ Microbiol 2001;67:475-80.
Rees LP, Minney SF, Plummer NT, Slater JH, Skyrme DA. A quantitative assessment of the antimicrobial activity of garlic (Allium sativum
). World J Microbiol Biotechnol 1993;9:303-7.
Weber ND, Andersen DO, North JA, Murray BK, Lawson LD, Hughes BG. In vitro
virucidal effects of Allium sativum
(garlic) extract and compounds. Planta Med 1992;58:417-23.
Eja ME, Asikong BE, Abriba C, Arikpo GE, Anwan EE, Enyi-Idoh KH. A comparative assessment of the antimicrobial effects of garlic (Allium sativum
) and antibiotics on diarrheagenic organisms. Southeast Asian J Trop Med Public Health 2007;38:343-8.
Griffiths G, Trueman L, Crowther T, Thomas B, Smith B. Onions - A global benefit to health. Phytother Res 2002;16:603-15.
Oyedemi SO, Afolayan AJ. Antibacterial and antioxidant activities of hydroalcoholic stem bark extract of Schotia latifolia
Jacq. Asian Pac J Trop Med 2011;4:952-8.
Raja RD, Jeeva S, Prakash JW, Antonisamy JM, Irudayaraj V. Antibacterial activity of selected ethnomedicinal plants from South India. Asian Pac J Trop Med 2011;4:375-8.
Johnson M, Wesely EG, Kavitha MS, Uma V. Antibacterial activity of leaves and inter-nodal callus extracts of Mentha arvensis
L. Asian Pac J Trop Med 2011;4:196-200.
Nweze EI, Ezute S, Emeka NC, Ogbonna CC, Eze C. Bacteria etiological agents causing respiratory tract infections in children and their resistance patterns to a panel of ten antibiotics. Asian Pac J Trop Dis 2011;2:18-23.
Jombo GT, Emanghe UE, Amefule UE, Damen JG. Antimicrobial susceptibility profiles at a university hospital in Sub-Saharan Africa. Asian Pac J Trop Dis 2011;2:7-11.
Aboaba O, Efuwape BM. Antibacterial properties of some Nigerian species. Biol Res Commun 2001;13: 183-8.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]