|Year : 2014 | Volume
| Issue : 3 | Page : 122-127
Comparison of antimicrobial efficacy of propolis, Morinda citrifolia, Azadirachta indica, triphala, green tea polyphenols and 5.25% sodium hypochlorite against Enterococcus fecalis biofilm
Paridhi Garg1, Shashi Prabha Tyagi1, Dakshita Joy Sinha1, Udai Pratap Singh1, Vibha Malik2, Edgar Richard Maccune3
1 Department of Conservative Dentistry and Endodontics, Kothiwal Dental College and Research Centre, Moradabad, Uttar Pradesh, India
2 Pathologist, Dr. Vibha Pathology Laboratory, Moradabad, Uttar Pradesh, India
3 Pathologist;Senior Laboratory Technician, Dr. Vibha Pathology Laboratory, Moradabad, Uttar Pradesh, India
|Date of Web Publication||6-Aug-2014|
Department of Conservative Detistry and Endodontics, Kothiwal Dental College and Research Centre, Moradabad - 244 001, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
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 6 th 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.
Keywords: Antimicrobial efficacy, biofilms, enterococcus fecalis, herbal irrigants, sodium hypochlorite
|How to cite this article:|
Garg P, Tyagi SP, Sinha DJ, Singh UP, Malik V, Maccune ER. Comparison of antimicrobial efficacy of propolis, Morinda citrifolia, Azadirachta indica, triphala, green tea polyphenols and 5.25% sodium hypochlorite against Enterococcus fecalis biofilm. Saudi Endod J 2014;4:122-7
|How to cite this URL:|
Garg P, Tyagi SP, Sinha DJ, Singh UP, Malik V, Maccune ER. Comparison of antimicrobial efficacy of propolis, Morinda citrifolia, Azadirachta indica, triphala, green tea polyphenols and 5.25% sodium hypochlorite against Enterococcus fecalis biofilm. Saudi Endod J [serial online] 2014 [cited 2019 Nov 20];4:122-7. Available from: http://www.saudiendodj.com/text.asp?2014/4/3/122/138141
| Introduction|| |
Primary endodontic infection is caused by microorganisms colonizing the necrotic pulp tissue.  Endodontic infections are polymicrobial in nature dominated by obligate anaerobic bacteria. 
Although Enteroccocus fecalis makes up a small proportion of the flora in untreated canals, it is 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.  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.  The mature biofilm at the end of 6 weeks shows signs of mineralization.  Achieving predictable success of root canal treatment requires effective debridement and disinfection of root canal system and biofilm. 
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. 
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,  unpleasant taste, high toxicity, corrosiveness to instruments,  inability to remove smear layer,  reduction in elastic modulus and flexural strength of dentin.  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.  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. 
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.  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.  It contains anti-bacterial compounds L-asperuloside and alizarin. 
Azadirachtaindica A. Juss (Neem) is most commonly used traditional and medicinal plant of India. Each part of the neem tree has some medicinal property 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. 
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. 
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. 
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.  Six percent concentration of pure MCJ [Herbal Biosolutions, New Delhi] was taken. 
Aqueous extract of Azadirachtaindica (Neem) was prepared by mixing 15 g of dry powder of neem leaves [The Indian Neem Tree Company, Mumbai] with 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. 
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. 
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.  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. 
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/cm 2 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.  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.
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|| |
0Enterococcus fecalis is a saprophytic component of enteric flora and is the most common bacterium isolated from endodontic re-treatment of apical periodontitis, either as a single organism or as a major component of the flora.  Studies have shown that E. fecalis can colonize medicated root canals with biofilm formation. This is the reason why E. fecalis is used in studies regarding the efficiency of endodontic irrigation solutions in cleaning the root canal system.  E. fecalis is resistant to traditional antibiotics. When E. fecalis grows as a biofilm, the altered genetic and metabolic processes of bacteria along with its complex matrix prevent the entry and action of several antimicrobial agents. The antibiotic resistance has been found to increase up to 1,500 times when compared with planktonic cells.  Therefore, testing the effect of an antibacterial irrigation solution on planktonic cells will not fulfill its effectiveness in in-vivo conditions, thus a biofilm model has been used. Bacteria-induced dissolution of the dentin surface and the ability of E. fecalis to form calcified biofilm on root canal dentin may be another factor that contributes to their persistence after endodontic treatment.  It is established that the biofilm-forming capacity and its structural organization are influenced by the chemical nature of the substrate. Biofilm experiments conducted on polycarbonate or glass substrate did not provide a true indication of the bacteria-substrate interaction.  Hence, E. fecalis biofilm was formed on a tooth substrate in this study in accordance with the methodology done by Kishen et al. 
Sodium Hypochlorite is the most commonly used irrigating solution in clinical practice and has the most established anti-microbial activity. Giardino et al. demonstrated that 5.25% NaOCl eliminated E. fecalis biofilm in 30 seconds. Dunavant et al., have shown that only NaOCl is able to kill the whole bacteria population organized in a biofilm and that its activity is strictly correlated to its concentration.
Some components present in propolis extract, like flavonoids (quercetin, galangin, pinocembrin) and caffeic acid, benzoic acid, cinnamic acid, probably act on the microbial membrane or cell wall site, causing functional and structural damages.  Kujumgiev et al.,  reported the antimicrobial action of propolis to be due to flavonoids and esters of phenolic acids. These substances may act on eicosanoids production, suppressing prostaglandins and leukotrienes synthesis and inhibiting the release of free radicals and nitric oxide production.  Other compounds, such as steroids and salicylic acid found in propolis, may also act synergistically on the final antimicrobial and anti-inflammatory activity.  A lower concentration of the propolis solutions was more efficient in inhibiting the inflammatory reaction because the flavonoid titers were comparatively higher in low-concentration extracts. 
In our study, triphala has shown no statistically significant difference in comparison to NaOCl and Propolis. This may be attributed to its formulation, which contains three different medicinal plants in equal proportions, Terminalia bellerica, Terminalia chebula, Emblica officinalis. In such formulations, different compounds may be of help in enhancing the potency of the active compounds resulting in an additive or synergistic positive effect.  The strong antioxidant activity of triphala may be partially responsible for many of the biological properties.  T. belerica was the most active antioxidant followed by E. officinalis and T. chebula. The major ingredients of T. bellerica are ellagic and gallic-acid; E. officinalis has several gallic acid derivatives including epigallocatechingallate and in T. chebulagallic acid is the major ingredient.  The presence of these active ingredients of phenolic nature may be responsible to scavenge the free radicals generated by the bacteria. Tannic acid represents the major constituent of the ripe fruit of T. chebula, T. belerica and E. officinalis. Earlier studies have reported that tannic acid is bacteriostatic or bactericidal to some Gram positive and Gram negative pathogens.  Prabhakar et al., conducted a study on the antimicrobial efficacy of herbal alternatives (triphala and green tea polyphenols (GTP)), MTAD, and 5% NaOCl against E. fecalis biofilm formed on tooth substrate in which, triphala achieved 100% killing of E. fecalis at 6 min.  Dimethyl sulfoxide (DMSO) was used as a solvent for triphala and GTP, which is a clean, safe, highly polar, aprotic solvent that helps in bringing out the pure properties of all the components of the herb being dissolved.  Antibacterial inertness of 10% DMSO was confirmed with the disc diffusion test.
In this era, Neem is considered as a valuable source of unique natural products for development of medicines against various diseases. The antibacterial activity of Azadirachta indica (Neem) is attributed to the presence of active constituents such as nimbidin, nimbin, nimbolide, gedunin, azadirachtin, mahmoodin, margolone, and cyclictrisulphide.  These active constituents uncouple mitochondrial oxidative phosphorylation; thus, inhibiting the respiratory chain.  This results in its anti-adherence activity by altering the bacterial adhesion and the ability of the microorganism to colonize thereby causing maximum reduction in adherence of E. fecalis to dentin.  Bohora and co-workers  have concluded that neem leaf extract has a significant antimicrobial effect against E. fecalis, which is in support of our study.
Since last two decades, green tea has received much attention in regard to its beneficial effects on various human health problems.  Most of the biological activities of green tea, particularly its antibacterial properties, have been associated with the polyphenol catechin fractions which constitute up to 30% of solid green tea leaves.  There are four main types of catechins: Epigallocatechin-3-gallate (EGCG), epigallocatechin, epicatechin-3-gallate and epicatechin.  EGCG is the most abundant of these catechins, comprising about 50% of the catechin pool.  EGCG has been shown to cause irreversible membrane disruption in both Gram-positive and Gram-negative bacteria  and also to inhibit bacterial DNA gyrase preventing DNA supercoiling and leading to bacterial cell death. EGCG neutralizes toxic end metabolites such as collagenase, protein tyrosine phosphatase; alkaline phosphatase of pathogenic bacteria.  Prabhakar et al.,  showed that GTP can eradicate the E. fecalis biofilm formed on tooth substrate in 6 minutes. In our study, GTPs have shown significant activity against E. fecalis.
The beneficial antimicrobial effects of MCJ may be the result of acubin, L-asperuloside, alizarin, scopoletin and other anthraquinones. Murray et al., have concluded  that the efficacy of MCJ was similar to NaOCl as an intracanal irrigation solution. However, in our study, MCJ did not show as good an antimicrobial efficacy as expected. There have been no other studies conducted on E. fecalis till date. Banerjee et al.,  showed that MCJ had anti-candidal activity in vitro. Jayaraman et al.,  showed that MCJ showed no significant activity against C. albicans, which might be due to the fact stated by Jainkittivong et al.,  who showed that in case of Morinda citrifolia fruit longer the contact time, higher is the inhibitory effect.
| Conclusion|| |
Under the limitations of this study, it can be concluded ~
- Propolis is equally efficacious as NaOCl against E. fecalis biofilm
- Triphala also performed equally well as that of NaOCl and propolis
- GTPs also exhibited significant antimicrobial activity. Thus, from the results of the study, it can be suggested that all these three irrigation solutions, namely propolis, triphala and GTPs could be used as an alternative to NaOCl for endodontic infections although, further in-vivo studies are warranted.
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