Original Article
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Comparison of antimicrobial efficacy of propolis, Morinda citrifolia, Azadirachta indica, triphala, green tea polyphenols and 5.25% sodium hypochlorite against Enterococcus fecalis biofilm Paridhi Garg, Shashi Prabha Tyagi, Dakshita Joy Sinha, Udai Pratap Singh, Vibha Malik1, Edgar Richard Maccune1,2
Department of Conservative Dentistry and Endodontics, Kothiwal Dental College and Research Centre, Moradabad, 1Pathologist, 2Senior Laboratory Technician, Dr. Vibha Pathology Laboratory, Moradabad, Uttar Pradesh, India Key words: An timicrobial efficacy, biofilms, enterococcus fecalis, herbal irrigants, sodium hypochlorite
Address for correspondence: Dr. Paridhi Garg, Department of Conservative Detistry and Endodontics, Kothiwal Dental College and Research Centre, Moradabad - 244 001, Uttar Pradesh, India. E-mail:
[email protected]
ABSTRACT Introduction: Endodontic infections are polymicrobial in nature. Enterococcus fecalis is the most common micro‑organism isolated from failed endodontic cases. The constant increase in antibiotic resistant strains and side effects caused by synthetic drugs has prompted researchers to look for herbal alternatives since the gold standard for irrigation i.e., sodium hypochlorite (NaOCl) has many disadvantages. Objective: The present study was aimed to explore newer irrigation solutions, which would probably be as effective as NaOCl. Materials and Methods: Extracted human single rooted premolar teeth were biomechanically prepared, vertically sectioned, placed in tissue culture wells exposing the root canal surface to E. fecalis is grown on Mueller Hinton agar plates to form a biofilm for 6 weeks. At the end of 6th week, all seven groups were treated with 3 ml of test solutions and control for 10 minutes and evaluated for E. fecalis growth and number of colony forming units. Results: Propolis, NaOCl and triphala showed no statistically significant difference, whereas all the other inter‑group differences were statistically significant (Tukey’s honest significant difference (HSD)) (P < 0.001). Conclusion: Propolis and triphala were found to be as efficacious as NaOCl. The use of herbal alternatives as root canal irrigation solutions might prove to be advantageous considering several unfavorable properties of NaOCl.
INTRODUCTION
P
rimary endodontic infection is caused by microorganisms colonizing the necrotic pulp tissue.[1] Endodontic infections are polymicrobial in nature dominated by obligate anaerobic bacteria.[2] Although Enteroccocus fecalis makes up a small proportion of the flora in untreated canals, it is Access this article online Quick Response Code: Website: www.saudiendodj.com DOI: 10.4103/1658-5984.138141
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a persistent organism that plays a major role in the etiology of periradicular lesions after root canal treatment. It is commonly found in high percentages in root canal failure cases (22-77%) and it is able to survive in the root canal as a single organism or as a major component of the flora.[3] E. fecalis can survive harsh conditions due to biofilm formation and physicochemical properties of the organism that helps it to modify according to the prevailing environmental and nutritional conditions. Biofilm helps in resisting the destruction of the bacteria by making them thousand times more resistant to phagocytosis, antibodies and antimicrobial agents. This is attributed to the protective barrier provided by the extracellular matrix.[4] The mature biofilm at Saudi Endodontic Journal • Sep-Dec 2014 • Vol 4 • Issue 3
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Garg, et al.: Comparison of antimicrobial efficacy of herbal irrigants with sodium hypochlorite
the end of 6 weeks shows signs of mineralization.[5] Achieving predictable success of root canal treatment requires effective debridement and disinfection of root canal system and biofilm.[6] The choice of instrumentation and irrigating solutions that permit bacterial neutralization and toxin inactivation without negative interference with the healing process is fundamental to the success of the treatment.[7] Sodium hypochlorite has remained a popular root canal irrigation solution because of its antimicrobial potential and its ability to dissolve organic material. However, it is not only irritant to the periapical tissues but also inherently possesses certain disadvantages like staining of instruments, burning of surrounding tissues,[2] unpleasant taste, high toxicity, corrosiveness to instruments, [8] inability to remove smear layer, [9] reduction in elastic modulus and flexural strength of dentin. [10] These disadvantages have prompted researchers to look for other alternatives. The constant increase in antibiotic resistance strains and side effects caused by synthetic drugs has also led to the search for herbal alternatives.[11] Some products benefit from a greater attention due to their beneficial effects confirmed by research and because of the current worldwide “back to nature” trend.[12] Propolis is a brownish resinous substance collected by bees mainly from plants. It is a potent antimicrobial, antioxidant and anti‑inflammatory agent. The main chemical elements present in propolis are flavanoids, phenolics and various aromatic compounds.[13] Propolis (bee glue) is a natural resinous hive product. Recently, it has attracted much attention due to its antibacterial and antifungal activities. Morinda citrifolia juice (MCJ) has a broad range of therapeutic effects, including antibacterial, antiviral, antifungal, anti‑tumor, anti‑helminthic, analgesic, hypotensive, anti‑inflammatory and immune‑enhancing effects. Since more than 2000 years it is being used in folk medicine for the treatment of cancer, infection, arthritis, diabetes, asthma, hypertension and pain.[14] It contains anti‑bacterial compounds L‑asperuloside and alizarin.[15] Azadirachtaindica A. Juss (Neem) is most commonly used traditional and medicinal plant of India. Each part of the neem tree has some medicinal property Saudi Endodontic Journal • Sep-Dec 2014 • Vol 4 • Issue 3
and is thus commercially exploitable. Neem elaborates a vast array of biologically active compounds that are chemically diverse and structurally complex. More than 140 compounds have been isolated from different parts of neem. Neem leaf and its constituents have been demonstrated to exhibit immunomodulatory, anti‑inflammatory, antifungal, antibacterial, antiviral, antioxidant, antimutagenic and anticarcinogenic properties.[16] Triphala is an Indian ayurvedic herbal formulation consisting of dried and powdered fruits of 3 medicinal plants (Terminaliabellerica, Terminaliachebula, Emblicaofficinalis). It has a potential of antibacterial activity and anti‑inflammatory activity.[17] Green tea polyphenols (GTPs) is a traditional drink of Japan and China. It is prepared from the young shoots of tea plant Camellia sinensis. It is a widely used medicinal plant throughout India, China and popular in various indigenous system of medicine like ayurveda, unani and homoeopathy. The catechins and flavins are considered microbiologically active ingredients.[18] As, there is no study reported till date comparing the antimicrobial efficacy of the above mentioned herbal products with NaOCl, the purpose of this in‑vitro study was to evaluate the antimicrobial efficacy of propolis, Morinda citrifolia, Azadirachta indica (neem), triphala, green tea polyphenols and 5.25% NaOCl against E. fecalis biofilm formed on tooth substrate of extracted human teeth.
MATERIALS AND METHODS Enterococcus fecalis culture preparation
A pure culture of E. fecalis (ATCC 29212) [Himedia, Mumbai] was inoculated on Mueller‑Hinton agar plates [Himedia, Mumbai], incubated at 37°C overnight. Test solutions preparation
Propolis [HiTech natural products, New Delhi] was prepared using 11% alcoholic extract by diluting a 33% commercially available extract of propolis using warm saline in a ratio of 2:1.[2] Six percent concentration of pure MCJ [Herbal Biosolutions, New Delhi] was taken.[13] Aqueous extract of Azadirachtaindica (Neem) was prepared by mixing 15 g of dry powder of neem leaves [The Indian Neem Tree Company, Mumbai] with 123
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Garg, et al.: Comparison of antimicrobial efficacy of herbal irrigants with sodium hypochlorite
100 ml of sterile distilled water in a round bottom flask with occasional shaking. The extracts were then filtered through a muslin cloth for coarse residue and then through a Whatman no. 1 filter paper and kept in an airtight amber‑colored container.[19] Triphala and green tea polyphenols [Herbal Biosolutions, New Delhi] were made into solution by dissolving 60 mg/ml in 10% dimethyl sulphoxide [Himedia, Mumbai] each.[17] Tooth samples preparation
Seventy single rooted human premolar teeth with vertucci class 1 root canal configuration with fully formed apical third were taken. The specimens were cleaned of superficial debris, calculus, tissue tags and stored in normal saline.[17] The tooth specimens were sectioned below the cementoenamel junction with a diamond disc to obtain a standardized tooth length of 8 mm for uniform specimen. The root canals were then cleaned and shaped using the crown down technique and protaperrotary instruments to an apical size of F3. Two millilitres of 5.25% NaOCl was used between each instrument during the cleaning and shaping procedures. All teeth were then vertically sectioned along the mid‑sagittal plane into two halves. The concave tooth surface was minimally grounded to achieve flat surface.[11] Grouping and assessment protocol
The sectioned samples were divided into 5 test groups and 2 control groups namely: • Group A‑ Propolis • Group B‑ Morinda citrifolia • Group C‑ Azadirachta indica (Neem) • Group D‑ Triphala • Group E‑ Green Tea Polyphenols • Group F‑ 5.25% NaOCl (positive control) • Group G‑ Normal sterile saline (negative control). The samples were then sterilized by ultraviolet radiation with a dosage 300 kJ/cm2 for 10 minutes in the laminar air flow. The bacterium was then inoculated in 1 ml of brain heart infusion (BHI) broth in 140 tissue culture wells and the turbidity was adjusted to 1 on the densitometer with sterile BHI broth taken as baseline. The sectioned tooth specimens were then placed in the tissue culture wells and inoculated at 37°C for 6 weeks.[5] Each sample were taken with a sterile paper point and inoculated onto Mueller Hinton agar plates and incubated at 37°C for 24 hours to check for cell viability and purity of culture. 124
At the end of 6th week of inoculation, all specimens were placed in sterile petridisches and the test irrigation solutions was delivered onto them using a micropipette as follows: • Group A‑ 3 ml of Propolis (n = 20) • Group B‑ 3 ml of 6% Morinda citrifolia Juice (n = 20) • Group C‑ 3 ml of Azadirachta indica (neem) aqueous extract (n = 20) • Group D‑ 3 ml of 60 mg/ml of triphala in 10% Dimethyl Sulphoxide (n = 20) • Group E‑ 3 ml of 60 mg/ml of green tea polyphenolsin 10% Dimethyl sulphoxide (n = 20) • Group F‑ 3 ml of 5.25% NaOCl (n = 20) • Group G‑ 3 ml of sterile saline (n = 20). Each specimen remained immersed for 10 minutes. Then, the biofilm on root canal surface was taken with a sterile paper point and inoculated on Mueller Hinton agar plates and incubated for 24 hours at 37°C. The plates were then analyzed for colony forming units by a digital colony counter. The data collected was subjected to analysis of variance (ANOVA) and post‑hoc tukey’s tests.
RESULTS The results are summarized in Table 1 and Figure 1. Propolis, NaOCl and triphala showed no statistically significant difference, whereas all the other inter‑group differences were statistically significant (Tukey’s HSD). Propolis ~_ NaOCl ~_ Triphala > GTP > Neem > MCJ > Saline.
DISCUSSION Enterococcus fecalis is a saprophytic component of enteric flora and is the most common bacterium Table 1: The mean and standard deviations obtained for all groups Groups A. Propolis B. MCJ C. Neem D. Triphala E. Green Tea Polyphenols F. NaOCl G. Saline
N Mean Std. deviation Minimum Maximum 20 0.30 0.48 0.00 1.00 20 64.60 1.58 63.00 68.00 20 50.10 1.29 49.00 52.00 20 1.40 0.52 1.00 2.00 20 2.00 0.47 1.00 3.00 20 20
0.30 93.0
0.48 0.82
0.00 92.00
1.00 94.00
P
