|Year : 2015 | Volume
| Issue : 1 | Page : 28-32
Antimicrobial activity of herbal extracts against recalcitrant endodontic pathogens: An original in vitro study
Taruna Arora1, Raghubir Singh Kang1, Jagvinder Singh Mann1, Navjot Singh Khurana1, Rishi Aggarwal2, Geeta Walia3
1 Department of Conservative Dentistry and Endodontics, Government Dental College, Patiala, Punjab, India
2 Department of Conservative Dentistry and Endodontics, Guru Nanak Dev Dental College, Sunam, Punjab, India
3 Department of Microbiology, Government Medical College, Patiala, Punjab, India
|Date of Web Publication||30-Jul-2015|
Dr. Taruna Arora
Department of Conservative Dentistry and Endodontics, Government Dental College, Patiala - 147 001, Punjab
Source of Support: None, Conflict of Interest: None
Introduction: Plants have been used for health disorders and to prevent diseases including epidemics since times immemorial. The knowledge of their healing properties has been transmitted over the centuries within and among human communities.
Aims and Objectives: To evaluate and compare the antimicrobial potential of herbal extracts, namely neem (Azadirachta indica), tulsi (Ocimum sanctum), bitter gourd (Momordia charantia), and arka (Calotropis procera) as endodontic irrigants against Enterococcus faecalis and Candida albicans in vitro.
Materials and Methods: Agar well diffusion test was performed. The inoculums of E. faecalis and C. albicans were streaked on the blood agar plate, and wells were made using cork borers. The prepared herbal extracts of the test samples were loaded onto agar plate. The plates were incubated at 37°C for 24 h. The inhibition zones indicating the antimicrobial potential were measured using a millimeter scale and results were analyzed statistically.
Results: Bitter gourd showed the maximum zones of inhibition followed by neem, tulsi, and calotropis for both E. faecalis and C. albicans.
Conclusion: The tested irrigants showed significant antimicrobial efficacy against E. faecalis and C. albicans. Thus, the use of herbal alternatives as root canal irrigants might prove advantageous considering the undesirable characteristics of presently used irrigants.
Keywords: Antimicrobial potential, Candida albicans, Enterococcus faecalis, herbal extracts, root canal irrigant
|How to cite this article:|
Arora T, Kang RS, Mann JS, Khurana NS, Aggarwal R, Walia G. Antimicrobial activity of herbal extracts against recalcitrant endodontic pathogens: An original in vitro study. Saint Int Dent J 2015;1:28-32
|How to cite this URL:|
Arora T, Kang RS, Mann JS, Khurana NS, Aggarwal R, Walia G. Antimicrobial activity of herbal extracts against recalcitrant endodontic pathogens: An original in vitro study. Saint Int Dent J [serial online] 2015 [cited 2018 Dec 11];1:28-32. Available from: http://www.sidj.org/text.asp?2015/1/1/28/161798
Microorganisms and their by-products are considered to be the primary etiologic agents in endodontic diseases.  Failures during and after endodontic treatment are linked to the presence of bacteria in the root canal.  Thus, for an optimal outcome of the endodontic treatment to be achieved, bacterial population within the root canal should ideally be eliminated or at least significantly reduced to levels that are compatible with peri-radicular tissue healing.
Enterococcus faecalis (E. faecalis) is a facultative anaerobic Gram-positive cocus and is the most common species cultured from nonhealing endodontic cases.  E. faecalis can even tolerate the antibacterial action of calcium hydroxide.  Candida albicans (C. albicans) is the most common opportunistic oral fungal infection and has dentinophilic properties with an affinity for smear layer.  E. faecalis and C. albicans are known to be important resistant species in infected root canals and may cause treatment failures. ,
The most effective way to achieve complete sterilization of root canals is by means of thorough instrumentation followed by irrigation using effective endodontic irrigants.  The intricacies and fine aspects of root canal anatomy tend to compromise the physical removal of bacterial load through instrumentation, shedding responsibilities of disinfection on irrigants.  Hence, the role of irrigants holds a major place in successful endodontic treatment.
Several chemicals and therapeutic agents are used to disinfect the root canal system. , Sodium hypochlorite, due to its excellent properties of tissue dissolution and antimicrobial activity, has been the most widely used irrigant since its introduction in endodontics by Walker in 1936. However, unpleasant taste and odor, toxicity, resorption, inability to remove smear layer and fully eradicate microbes from the infected canals are the main disadvantages of this popular irrigant.  Chlorhexidine has a reasonably wide range of activity against aerobic and anaerobic organisms as well as the Candida species. But the presence of inflammatory exudates and killed microorganisms can inhibit the action of chlorhexidine in root canals.  A new irrigant Biopure MTAD, (Biopure MTAD, Dentsply Tulsa Dental Specialties, USA) a mixture of tetracycline isomer, citric acid, and Tween 80 has been found to remove smear layer effectively.  Recently, Murray et al.  evaluated Morinda citrifolia juice in conjunction with ethylene-diamine-tetra-acetate (EDTA) as a possible alternative to NaOCl. There is an ongoing search for suitable pure herbal alternatives owing to the constant increase in antibiotic resistant strains and side effects caused by synthetic drugs.
Medicinal plants have been identified as the backbone of treatment since the dawn of civilization.  The natural products derived from herbal heaven have abundant healing capabilities with a wider safety margin.  Such plants are potential sources of bioactive compounds with a proven history of therapeutic properties and if explored cautiously stand to form the basis for the development of new medicinal chemicals. 
Phytodentistry is an emerging branch in dentistry. It implies the use of medicinal plants and their products for treating disease directly or indirectly.  In dentistry, the potential herbal extracts have been used as anti-inflammatory, antibiotic, analgesic, and sedative agents. , Further, phytodentistry is recognized as an effective way to discover future medicines.
Studies have demonstrated the anti-microbial, antiviral, analgesic, anti-inflammatory, wound healing properties of herbal drugs. ,, Neem (Azadirachta indica) has a long history of treating teeth and gum problems.  Tulsi (Ocimum sanctum) is gifted with enormous antimicrobial substances and is used to treat a variety of illnesses.  Arka (Calotropis procera) has been used in the treatment of toothaches, vertigo, rheumatoid/joint swelling and paralysis.  Bitter gourd (Momordia charantia) has antibacterial, anti-inflammatory, anti-oxidant, triglyceride lowering properties.  These properties suggest the use of these herbal plants as alternative, inexpensive, simple, and effective anti-microbial agents. Furthermore, literature has shown that herbal medicaments have antimicrobial and therapeutic effects suggesting their potential to be used as endodontic irrigants, but there is lack of any documentation or data regarding research in dentistry, particularly in endodontics.
Considering the advantages of herbal medicines and multiple drug resistance against synthetic antibiotics, use of biologically active herbal extracts derived from medicinal plants seems to be the way forward. Therefore, the present study aimed to evaluate and compare the antimicrobial activity of neem (A. indica), tulsi (O. sanctum), bitter gourd (M. charantia), and arka (C. procera) extracts as potential irrigants against E. faecalis and C. albicans for use in endodontics.
| Materials and Methods|| |
The present study was conducted in the Department of Endodontics, Govt. Dental College, Patiala with appreciable cooperation from the Department of Microbiology, Government Medical College, Patiala (Punjab) India. The standard procedures for evaluating the antimicrobial potential of tested herbal extracts against E. faecalis and C. albicans were followed.
The pure cultures of the reference strains of E. faecalis (American Type Culture Collection [ATCC] 29212) and C. albicans (ATCC 10231) were procured from the Department of Microbiology on documented request. Cultures were grown overnight in nutrient broth and adjusted to 0.5 turbidity on McFarland's scale.
The powdered extracts of neem (A. indica), tulsi (O. sanctum), bitter gourd (M. charantia), and ark (C. procera) were taken from Ayush Herbs Pvt. Ltd, (Nagrota, Himachal Pradesh, India). The powdered extracts were made into solution form by dissolving them in 10% (v/v) dimethyl sulfoxide (DMSO) (S.D. Fine Chem. Pvt. Ltd, Mumbai, India). The solutions were sterilized by filter sterilization through membrane filter, labeled and stored in sterile airtight containers for further evaluations.
In vitro antimicrobial activities of the tested herbal extracts against resistant endodontic species (E. faecalis and C. albicans) were determined using the agar well diffusion method. The recommendations given by National Committee for Clinical Laboratory Standard (2000) were followed. The broth culture of reference microorganisms was swabbed on sterile blood agar plates using sterile swabs. Wells of 6 mm diameter and 4 mm depth were made in the agar using sterile cork borers. A premeasured volume (50 μl) of tested organic herbal extracts (neem, tulsi, bitter gourd, and arka) was seeded into their respective wells. The 10% solution of solvent (DMSO) was used as control. Culture plates were marked and incubated at 37°C (98.6 °F) for 24 h under aerobic conditions.
The antibacterial activity for each extract was expressed in terms of the average diameter of zone of inhibition after incubation. The zone of inhibition refers to the size of the clear circular zone without bacterial or fungal growth around the wells. The size of cleared zones was measured in millimeters using a ruler with lid off. Final readings regarding the zone of inhibition were given by measuring it in reflected light from back of the plate. The experiment was repeated three times under strict aseptic conditions, and the readings were averaged for statistical purpose.
Statistical analysis was performed using one-way analysis of variance and compared by the Post-hoc Tukey test. SPSS software version 17 (SPSS Inc., Chicago, III., USA) was used. The level of significance was set to 5% (P < 0.05).
| Results|| |
The organic extract of bitter gourd showed the maximum zone of inhibition, followed by neem, tulsi, and calotropis against E. faecalis and C. albicans, respectively. No zone of inhibition was shown by 10% DMSO. The results are shown in [Table 1]. Graphic representation is shown in [Figure 1].
When compared statistically, all the tested natural plant extracts showed a statistically very highly significant antimicrobial activity against E. faecalis and C. albicans (P < 0.001).
|Figure 1: Graphical comparison of zones of inhibition (millimeter) reflecting antimicrobial activity of extracts of tested plants|
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|Table 1: Mean zones of inhibition (in mm) of Enterococcus faecalis and Candida albicans by the tested herbal extracts |
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On intra-group comparison for antimicrobial activity against E. faecalis, bitter gourd extract showed a very highly significant (P < 0.001) and significant antimicrobial activity (P = 0.005) when compared with calotropis and tulsi extracts, respectively. However, neem and bitter gourd extracts have comparable statistically nonsignificant (P < 0.668) antimicrobial activity.
On intra-group comparison for antimicrobial activity against C. albicans, bitter gourd extract showed a very highly significant (P = 0.001) antimicrobial activity when compared with tulsi and calotropis whereas it showed significant antimicrobial activity (P = 0.023) when compared with neem extract.
| Discussion|| |
Plants provide a natural blueprint for the development of new drugs. Screening the active compounds from plants has led to the discovery of new medicinal drugs. Currently, the use of natural extracts in dentistry has gained importance as the shift is toward natural health remedies.  It is estimated that plant materials have provided models for more than 50% Western drugs today. According to the World Health Organization, approximately 25% of modern drugs used in the United States have been derived from plants. 
Enterococcus faecalis is the most frequently isolated species in root canals with persistent infection and is often resistant to traditional antibiotics.  Adaptive process of microbial growth as biofilm enables the microorganisms to survive the harsh growth conditions.  C. albicans is an opportunistic pathogen in immunodeficient individuals and those receiving long term broad spectrum antibiotics. It has been called as "the disease of the diseased." C. albicans isolates may exhibit intrinsic (primary) resistance or secondary resistance to the drug during therapy.  Inclusion of C. albicans and E. faecalis in the study was based on the literature that relates these microorganisms to the pulp and periapical infections, mainly in recalcitrant infections after endodontic treatment. 
The use of a biocompatible endodontic irrigant having antimicrobial properties may reduce or eliminate bacteria in the root canal system and significantly increases the success of root canal treatment. Therefore, the use of an endodontic irrigant obtained from a natural source is of great significance.
Synthetic irrigants and medicaments including sodium hypochlorite, calcium hydroxide, povidone-iodine, Bio pure MTAD, citric acid, chlorhexidine, tetraclean, ozonated water, EDTA, photon-activated disinfection, and phosphoric acid have been tried against E. faecalis and C. albicans but have not been very efficient. , These irrigants are caustic, nonspecific, and have deleterious effects on dentin including reduction of elastic modulus and flexural strength.  Further, apical extrusion of the synthetic irrigants results in adverse periapical reactions. 
In the present in vitro study, herbal extracts of bitter gourd, neem, tulsi, and arka with medicinal values were screened as irrigants for evaluating their antimicrobial activity against E. faecalis and C. albicans. Bitter gourd showed the maximum antimicrobial activity with widest zone of inhibition against both C. albicans (21.53 mm) as well as E. faecalis (20.83 mm) followed by neem, tulsi, and arka. The difference was found to be very highly significant when compared statistically. Neem extract showed a greater zone of inhibition for E. faecalis (19.83 mm) as compared to C. albicans (18.86 mm) but the difference was not significant. Tulsi and arka extracts showed higher antimicrobial activity against C. albicans in comparison to E. faecalis with 17.0 mm, 16.5 mm and 15.66 mm, 13.76 mm zones of inhibition, respectively. C. albicans was strongly inhibited by the herbal extract of bitter gourd followed by neem, tulsi, and arka. The organic extracts of bitter gourd, tulsi, and arka also inhibited the growth of E. faecalis in a decreasing order, but not as strongly as C. albicans. The findings of the study suggest that plant extracts can be used to inhibit the growth of resistant endodontic pathogens and thus can be used to prevent root canal failures.
The antimicrobial effects of the tested plant extracts in the study may be attributed to certain chemical compounds present in them. Mahmood et al.  demonstrated the antibacterial activities of the phenolic extracts of M. charantia (bitter gourd) in vitro. Kight  reported two proteins, alpha and beta momorcharin in the seeds, fruit, and leaves of M. charantia with significant antimicrobial action. Similar antimicrobial activities were observed in our study.
In the study, A. indica was highly efficient in reducing E. faecalis and C. albicans within the root canals. Biswas et al.  reported that neem leaves contain several active constituents such as nimbidin, nimbin, nimbolide, gedunin, azadirachtin, mahmoodin, margolone, and cyclictrisulfide responsible for its antibacterial action. Polaquini et al.  studied the anti-adherence activity of extract and stated that neem alters the adhesion and ability of E. faecalis to colonize dentin. Aromdee and Sriubolmas  stated that the antifungal activity of neem against C. albicans was because of the presence of sulfur compounds. Several authors , suggested that neem leaf extract has significant antimicrobial efficacy against both E. faecalis and C. albicans; is biocompatible and antioxidant, suggesting its use as an endodontic irrigant. However, the bitter taste associated with neem can be altered by the addition of various sweetened formulations and flavors to increase compliance and acceptability.
In the present study, the herbal extract of tulsi showed significant zones of inhibition against E. faecalis and C. albicans. Nanasombat and Lohasupthawee  reported that leaf extract of tulsi has significant insecticidal properties. Opalchenova and Obreshkova  observed the antibacterial effect of tulsi oil against multidrug-resistant clinical isolates of Staphylococcus, Enterococcus, and Pseudomonas. It has been observed that O. sanctum has extensive therapeutic potential in various areas like immune-stimulation, anticancer, antioxidant, as an adjuvant to radiotherapy, antiulcer, analgesic, and anti-diabetic. But, the main focus of the present study is to make the dental professional aware regarding the use of holy tulsi as endodontic irrigant.
Regarding C. procera, to the best of author's knowledge, the present study pioneers its antimicrobial potential against endodontic pathogens.
The solvent, DMSO, used in the study was taken as a control and did not show any inhibitory activity against the experimental pathogens. DMSO 10% (v/v) is one of the most common solvents used experimentally for in vivo and in vitro purposes.  It is a clear, colorless, strong organic solvent with an ability to bring out the pure properties of all the components of herbs being dissolved. Further, it has low toxicity favoring its choice as a solvent for the present study.
The agar well diffusion technique involves impregnation of tested solutions into wells punched into agar plates seeded with the test bacterial strain. Antimicrobial component diffuses from these sources into the agar medium and leads to inhibition of bacterial growth in the vicinity of the source. The "clear zone" without bacterial lawn is called zone of inhibition. Greater the diameter of these zones, higher the anti-microbial activity. 
The present study provided a standardized experimental setup which allowed for validated comparison of different herbal irrigants. All aspects of the testing procedure are standardized to ensure consistent and accurate results. The pH level of blood agar was adjusted between 7.2 and 7.4. Further, all procedures were carried out in accordance with the local codes of safe practice.
The powdered extracts of the plants were used in our study instead of preparing extracts using raw methods. Nowadays, using latest techniques and equipment, pharmaceutical companies have been able to provide us with powdered extracts with nearly 100% purity. In the study, powdered extracts were taken from Ayush Pvt. Ltd, with 99.98% of powder purity. This in turn simplified the whole procedure and saved the valuable time consumed in the experiment. However, the active biocompound responsible for such antimicrobial activities in the herbal extracts has not been identified. This study generates keen interest for biochemical analysis and a future study for fractionation of potentially active components in studied plants for dental use is suggested.
| Conclusion|| |
The present study showed and compared the antimicrobial activity and therapeutic effects of native Indian plants suggesting their potential use as endodontic irrigants and added a valuable research data paving a stable and valid path for future herbal endodontic treatment. Within the limitations of this study, all the tested irrigants showed antimicrobial efficacy against E. faecalis and C. albicans, with bitter gourd showing maximum effect followed by neem, tulsi, and calotropis. However, preclinical and clinical trials are needed to evaluate their biocompatibility and safety before recommendation as intra-canal endodontic irrigants.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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