|Year : 2015 | Volume
| Issue : 1 | Page : 13-16
Comparative evaluation of the antimicrobial efficacy of neem, green tea, triphala and sodium hypochlorite: An in vitro study
Karan Bhargava1, Tanaya Kumar1, Shalini Aggarwal1, Smita Zinzarde2, Anita Sanap1, Priyanka Patil1
1 Department of Endodontics, Dr. D Y Patil Dental College and Hospital, Pune, India
2 Institute of Bioinformatics and Biotechnology, University of Pune, Pune, India
|Date of Web Publication||8-Apr-2015|
Department of Endodontics, Dr. D Y Patil Dental College and Hospital, Pune
Source of Support: None, Conflict of Interest: None
Introduction: The main objective of root canal treatment is to disinfect the entire root canal system. Although cleaning and shaping and use of antimicrobial medicaments are effective in reducing the bacterial load some bacteria do remain behind and multiply, causing reinfection of the canal. Considering the ineffectiveness, potential side effects and safety concerns of synthetic drugs, the herbal alternatives for endodontic usage might prove to be advantageous. Aim: To check the antimicrobial efficacy of Neem, Green Tea, Triphala and sodium hypochlorite against the endodontic microflora. Materials and Methods: Mixed cultures of bacteria were isolated from patients, with carious teeth and associated with periapical radiolucencies. A paper point was inserted into the canal to obtain a sample of a mixed culture of micro organisms. These paper points were cultured and agar diffusion test was done for the different irrigants. The irrigants were divided into Group I: Neem (60mg/ml in 10% DMSO),Group II: Green Tea Polyphenols (60mg/ml in 10% DMSO), Group III: Triphala (60 mg/ml in 10% DMSO), Group IV: 3% NaOCI, Group V: Sterile saline. Results: Sodium hypochlorite showed the maximum antimicrobial activity, followed by Neem. There was no statistical difference between the activity of sodium hypochlorite and Neem. This was followed by Triphala and Green Tea, respectively. Conclusion: Neem is as effective as Sodium Hypochlorite against endodontic microflora.
Keywords: Anti-microbial efficacy, green tea, neem, sodium hypochlorite, triphala
|How to cite this article:|
Bhargava K, Kumar T, Aggarwal S, Zinzarde S, Sanap A, Patil P. Comparative evaluation of the antimicrobial efficacy of neem, green tea, triphala and sodium hypochlorite: An in vitro study. J Dent Res Rev 2015;2:13-6
|How to cite this URL:|
Bhargava K, Kumar T, Aggarwal S, Zinzarde S, Sanap A, Patil P. Comparative evaluation of the antimicrobial efficacy of neem, green tea, triphala and sodium hypochlorite: An in vitro study. J Dent Res Rev [serial online] 2015 [cited 2019 Aug 20];2:13-6. Available from: http://www.jdrr.org/text.asp?2015/2/1/13/154638
| Introduction|| |
Primary root canal infection is due to microorganisms colonizing in the pulp tissue.  There is an abundant number of bacterial species that have been observed as inhabitants of the oral microflora, because of high nutrient availability, low oxygen potentials and bacterial interactions in root canals but the number of bacterial species present in endodontic infections is limited.  Primary endodontic infections are poly-microbial in nature and dominated by Gram-negative anaerobic rods. 
Although bio-mechanical preparation of root canal reduces the number of bacteria, irrigants and intracanal medicament with antibacterial action are necessary for the disinfection of root canal system in such cases.  Various therapeutic and chemicals solutions are used in disinfection of the root canal. The gold standard among those are sodium hypochlorite (NaOCl), 2% solution of chlorhexidine and calcium hydroxide, which vary in their antibacterial activity. 
Sodium hypochlorite is the most commonly used irrigating solution since its introduction in 1936 by Walker. Its superior properties of tissue dissolution and antibacterial efficacy make it the irrigating solution of choice for treatment of teeth with pulp necrosis. However, it has a few shortcomings of which the main disadvantages of NaOCl are high toxicity, reduction in elastic modulus as well as flexural strength of dentin, corrosive to instruments,  unpleasant taste and inability to remove smear layer.  There is a constant increase in antibiotic resistant strains and in the side effects caused by synthetic drugs.
All these factors have led to the search for herbal alternatives like Neem, green tea polyphenols (GTPs), Triphala, Morinda citrifolia, etc. The advantageous reasons of using herbal irrigants are low toxicity and lack of microbial resistance and easy availability. 
An herbal medicine is defined as a plant-derived substance which contains raw ingredients from one or multiple plants with therapeutic uses (WHO). 
Neem or Azadirachta indica has shown to be effective against various microorganisms found in the oral microflora such as Enterococcus faecalis and Candida albicans. Its excellent antibacterial properties make it a material of choice for root canal irrigation and also an alternative to harmful chemical irrigants such as NaOCl. ,
Green tea polyphenols are the traditional and most widely consumed beverage of China and Japan, obtained from Camellia sinensis.  Cathechins and the flavins are present in GTPs, and they are considered as microbiologically active ingredients. 
Triphala consist of Terminalia chebula Terminalia bellerica and Emblica officinalis, the three medicinal plants which are dried and powdered to get an Indian Ayurvedic herbal formulation.  It has a potential of antibacterial activity against enteric pathogens,  and also have anti-inflammatory activity. 
The aim was to assess the antimicrobial efficacy of conventional irrigants that is, NaOCl and three alternative irrigants of herbal origin Triphala, Neem and GTPs against endodontic microflora in in-vitro condition.
| Materials and Methods|| |
Collection of samples
Cultures of bacteria were isolated from patients reporting to Dr. D. Y. Patil Dental College, Pune with carious teeth associated with per apical radiolucencies. This was thus a mixed culture which simulated the flora at the periapex of the tooth.
Coronal access was gained to the root canals of the involved teeth under rubber dam isolation. A paper point was inserted into the canal and held there for 3-5 min to obtain a sample of the mixed culture of microorganisms. This paper point was then immediately transferred to the sterile agar plates. These agar plates were taken to the IBB Department at Pune University for the further processing. Bacteria were grown overnight at 37°C on agar plates for 24 h.
Preparation of herbal extracts
120 g of pure neem powder (Himalaya, India), green tea (K Patel Pvt Ltd, India) and triphala (Himalaya, India) were mixed with 2 ml of 10% dimethyl sulfoxide (DMSO) (MERCK, Specialty Pvt. Ltd.,) liquid. DMSO is added to improve the efficiency of herbal products. About 10% DMSO did not interfere with antibacterial properties was confirmed with the disc diffusion method. This was stirred for 2 min and then passed through fast filter paper. The strained liquid was collected and used samples were prepared.
To check the antimicrobial efficacy of herbs and 3% NaOCl: Agar well diffusion method was performed [Figure 1].
The irrigants were divided into five groups:
- Group I: Neem (60 mg/ml in 10% DMSO)
- Group II: GTPs (60 mg/ml in 10% DMSO)
- Group III: Triphala (60 mg/ml in 10% DMSO)
- Group IV: 3% NaOCl
- Group V: Sterile saline.
Agar plates were prepared, and cultures (200 μl) were spread on the agar plates [Figure 2] and [Figure 3]. Wells of 6 mm diameter were punch cut on the agar surfaces. Depending upon the group, irrigants were pipetted into the agar wells. Three subgroups (50 μl, 100 μl and 150 μl) were created within each group depending upon the quantity of the irrigant that had been pipetted. These plates were incubated for 24 h at 37°C in an incubator. After incubation, plates were assessed for zones of inhibition.
For each volume of the irrigant, 5 wells were observed for antimicrobial activity, and the average value was recorded for the graphical representation.
Statistical analysis was done by one-way analysis of variable and compared by the post-hoc Tukey test. The criterion for statistical significance was defined as P < 0.05.
| Results|| |
Sodium hypochlorite showed the maximum antimicrobial activity, followed by neem [Figure 4]. There was no statistical difference between NaOCl and neem. This was followed by triphala and green tea. There was statistical difference between the efficacy of NaOCl and green tea and triphala. The efficacy of neem was also significantly more than that of green tea and triphala.
(The units depict the diameter of zones of inhibition in millimeters).
| Discussion|| |
Herbal products have been used in dental practice and have become more common now due to their high antibacterial activity, biocompatibility, and antioxidant properties.  Herbal and alternative medicine are gaining popularity among the public but as dental practitioners we have to understand these products and imply them in our clinical practice. 
In this study, all the tested irrigants were shown to inhibit and eliminate the tested strains. NaOCl showed the best antimicrobial followed by Neem, Triphala and Green Tea. Saline showed no antibacterial activity. About 3% NaOCl was best among all the groups. It exhibited excellent antibacterial activity on the bacteria. Neem was also as efficient as NaOCl against the endodontic microflora. There was no significant difference between the two.
Neem extract has been shown to have a wide spectrum of antibacterial efficacy against Gram-positive, as well as Gram-negative microorganisms. These effects have been seen against Streptococcus mutans and Enterococcus feacalis. High levels of anti mycotic activity have been reported with extracts from different parts of neem. It is extremely effective against Candida. 
Nimbidin a major crude bitter principle extract of neem has been shown to be the key reason for its antibacterial activity. Nimbidin is also responsible for the anti-inflammatory action of neem. 
In this study green tea and triphala also showed activity against the bacteria however it was not statistically significant.
Triphala showed more potency on the endodontic microorganisms. This is due to its formulation, which has three different ayurvedic plants in equal amounts. Also, different compounds may be of help in increasing the efficacy of the active compounds and helping in an additive effect. 
Green tea extracts (GTEs) contain catechin, which is one of the polyphenols from green tea.  It has been reported that GTEs have remarkable anti-inflammatory, antioxidant, and anti-carcinogenic effects in a number of animal tumors, cell culture systems, and epidemiological studies. 
Dimethyl sulphoxide was used as a solvent for Neem, Triphala, and GTP because it is safe, bacterially inert highly polar solvent that helps in bringing out the true properties of the herb being used in the study. , Neem has proven to be as effective as NaOCl in the elimination of endodontic microflora.
Sodium hypochlorite is very caustic, and a nonspecific agent and also has deleterious effects of dentine like reduction of the elastic modulus and the flexural strength.  Neem, Triphala, and GTPs have good chelating properties. , Out of which Triphala consists of medicinal plants that have citric acid which may help in smear layer removal. Thus, these irrigants show effectiveness in various endodontic applications and should be considered as alternative agents. The purpose of using herbal alternatives are low toxicity, lack of microbial resistance, easy availability, cost-effectiveness and increased shelf life. 
| Conclusion|| |
Within the limitations of this study, 3% NaOCl and Neem showed maximum antibacterial activity against the endodontic microflora. Triphala and Green Tea also showed antimicrobial efficacy. Herbal alternatives used for root canal irrigantion will prove to be preferable considering the various unenviable properties of NaOCl. Further research is required to recommend the use of herbal alternatives as a root canal irrigant.
| Acknowledgement|| |
Special thanks to Dr. Shobha Bhargava, Dr. Lotika Beri, Dr. Rajesh Shetty, Dr. Kalpana Pai, Dr. Narhari and Dr. Fasila Shaikh.
| References|| |
Tronstad L, Sunde PT. The evolving new understanding of endodontic infections. Endod Topics 2003;6:57-77.
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.
Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis
: Its role in root canal treatment failure and current concepts in retreatment. J Endod 2006;32:93-8.
Gomes BP, Souza SF, Ferraz CC, Teixeira FB, Zaia AA, Valdrighi L, et al
. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis
in bovine root dentine in vitro
. Int Endod J 2003;36:267-75.
Silva FB, Almeida JM, Sousa SM. Natural medicaments in endodontics - A comparative study of the anti-inflammatory action. Braz Oral Res 2004;18:174-9.
McComb D, Smith DC. A preliminary scanning electron microscopic study of root canals after endodontic procedures. J Endod 1975;1:238-42.
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.
Parle M, Bansal N. Herbal medicines: Are they safe? Nat Prod Radiance 2006;5:6-14.
Karimi K. The impact of current alternative herbal remedies on dental patient management. Gen Dent 1999;47:264-6.
Bohora A, Hegde V, Kokate S. Comparison of antibacterial efficacy of neem leaf extract and 2% sodium hypochlorite against E. faecalis
, C. albicans
and mixed culture - An in vitro
study. Endodontology 2010;22:8-12.
Hamilton-Miller JM. Anti-cariogenic properties of tea (Camellia sinensis
). J Med Microbiol 2001;50:299-302.
Smullen J, Koutsou GA, Foster HA, Zumbé A, Storey DM. The antibacterial activity of plant extracts containing polyphenols against Streptococcus mutans
. Caries Res 2007;41:342-9.
Jagetia GC, Baliga MS, Malagi KJ, Sethukumar Kamath M. The evaluation of the radioprotective effect of Triphala (an ayurvedic rejuvenating drug) in the mice exposed to gamma-radiation. Phytomedicine 2002;9:99-108.
Tambekar DH, Dahikar SB, Lahare MD. Antibacterial potentials of some herbal preparations available in India. Res J Med Med Sci 2009;4:224-7.
Sumantran VN, Kulkarni AA, Harsulkar A, Wele A, Koppikar SJ, Chandwaskar R, et al.
Hyaluronidase and collagenase inhibitory activities of the herbal formulation Triphala guggulu. J Biosci 2007;32:755-61.
Pujar M, Makandar S. Herbal usage in endodontics - A review. Int J Contemp Dent 2011;2:34-7.
Kamat S, Rajeev K, Saraf P. Role of herbs in endodontics: An update. Endodontology 2011;23:96-100.
Biswas K, Chattopadhyay I. Biological activites and medicinal properties of neem. Curr Sci 2102;82:1336-245.
Siddiqui S. Curr Sci 1942;11:278-9.
Dreosti IE. Bioactive ingredients: Antioxidants and polyphenols in tea. Nutr Rev 1996;54:S51-8.
Elbling L, Weiss RM, Teufelhofer O, Uhl M, Knasmueller S, Schulte-Hermann R, et al.
Green tea extract and (-)-epigallocatechin-3-gallate, the major tea catechin, exert oxidant but lack antioxidant activities. FASEB J 2005;19:807-9.
Serafini M, Ghiselli A, Ferro-Luzzi A. In vivo
antioxidant effect of green and black tea in man. Eur J Clin Nutr 1996;50:28-32.
de la Torre JC. Biological actions and medical applications of dimethyl sulfoxide. Ann N Y Acad Sci 1983;411:1-403.
Sim TP, Knowles JC, Ng YL, Shelton J, Gulabivala K. Effect of sodium hypochlorite on mechanical properties of dentine and tooth surface strain. Int Endod J 2001;34:120-32.
Jagetia GC, Malagi KJ, Baliga MS, Venkatesh P, Veruva RR. Triphala, an ayurvedic rasayana drug, protects mice against radiation-induced lethality by free-radical scavenging. J Altern Complement Med 2004;10:971-8.
Zhao B. Antioxidant effects of green tea polyphenols. Chin Sci Bull 2003;48:315-9.
Abascal K, Yarnell E. Herbs and drug resistance. Part 2 - Clinical implications of research on microbial resistance to antibiotics. Altern Complement Ther 2002;8:284-90.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]