Title: An in-vitro comparison of endodontic medicaments Propolis and Calcium hydroxide alone and in combination with Ciprofloxacin and Moxifloxacin against Enterococcus faecalis.
Aim: To evaluate and compare the antimicrobial properties of Propolis and Calcium hydroxide alone and in combination with Ciprofloxacin and Moxifloxacin against Enterococcus faecalis (E. Faecalis). Materials and Methods – The laboratory study was carried out to test the effectiveness of propolis and calcium hydroxidealone as well as in combination with the established endodontic medicaments (Moxifloxacin and Ciprofloxacin). The various combinations were : Group1: Propolis, Group 2: calcium hydroxide, Group 3: Moxifloxacin, Group 4: Ciprofloxacin, Group 5: Propolis + Moxifloxacin, Group 6: Propolis + Ciprofloxacin, Group 7: Calcium hydroxide + Ciprofloxacin, Group 8: Calcium hydroxide + Moxifloxacin. The efficacy of these medicaments was tested by checking for the zone of inhibition for the specific strain (ATCC 29212) of Enterococcus faecalis at different time intervals i.e. 24hr, 48hr and 72hr. Results – Mean zone of inhibition was maximum in Group V (21.94±4.26) followed by Group VI (18.80±1.93), Group I (18.71±4.26), Group VIII (15.88±2.59), Group III (14.91±1.00), Group VII (14.57±2.17), Group IV (13.91±1.00) and minimum in Group II (12.89±2.14). Mean zone of inhibition were found to be maximum at 72 hr and minimum at 24 hr. At all time intervals, the combination of Propalis with Moxiflocacin showed the maximum antimicrobial efficacy. Conclusion: On the basis of the results of the present study it can be concluded that Propolis and Calcium hydroxide show synergistic effect with Moxifloxacin and Ciprofloxacin against E. Faecalis. Propolis in combination with antibiotics and alone is more effective than calcium hydroxide. Clinical significance: Since propolis alone and in combination with antibiotics was observed to be more effective than calcium hydroxide, propolis can be considered as an intra canal medicament when compared to traditional calcium hydroxide.
Key words: Calcium Hydroxide, Ciprofloxacin, E. fecalis, Intra canal medicaments, Laboratory research, Moxifloxacin, Propolis.
For successful endodontic treatment, proper diagnosis, thorough chemo-mechanical preparation, and three dimensional obturation of root canal space are paramount. Regardless of these treatment protocols, bacteria can still persist in the complex anatomy of root canal space, thus the ability of intra canal medicament to restrain or eliminate residual bacteria and prevent re-infection may play an increasingly important role in achieving and maintaining a higher success rate of root canal treatment.1
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The most common species recovered in over one-third of the canals of root filled teeth with persisting periapical lesion is the Enterococcus faecalis (E. Faecalis). Enterococcus faecalis is a gram positive, facultative anaerobic, coccoid bacteria. Medicaments are recommended to eliminate remaining microbes in the root canal, dentinal tubules, accessory canals, canal irregularities (such as fins, transverse anastomoses, apical deltas and other ramifications) and in the periapical/periodontal tissue to reduce periapical inflammation, encourage periapical healing, eliminate apical exudates, control inflammatory root resorption and avert contamination of the canal between appointment.2
Earlier strong intracanal antiseptics such as formacresol and camphorated parachlorophenol were used, but due to their detrimental effects on the connective tissue, their use is limited, now-a-days. The excellent biologic and antimicrobial properties of calcium hydroxide have made it the preferred choice for intracanal dressing of the infected roots canals.1 Calcium hydroxide has been added to several endodontic sealers to improve their biological properties and to augment their antibacterial activity. When used as an intracanal medicament, the most important property of calcium hydroxide is its strong antimicrobial activity. Its high pH alters the biological lipopolysaccharides in the cell walls of Gram-negative species, there by inactivating the membrane transport mechanisms.3
Propolis is a resinous product rich in flavanoid. It has been used as in a number of ways; an anticaries agent, for dentinal hypersensitivity as sealant, storage medium for avulsed tooth, for pulp capping. Propolis is ten times less cytotoxic than calcium hydroxide and has a distinguished antibacterial, antifungal, antiviral, immunomodulatry, antioxidant effect. Recent studies have reported that propolis is more effective against resistant microorganisms and is biocompatible. In endodontic procedures antibiotics can be used as an important aide but their ineffectiveness through systemic route of administration has led to the intra canal application, in order to increase their efficacy.1
Moxifloxacin and Ciprofloxacin are members of the quinolones. Among the drugs commonly used for endodontic infection, ciprofloxacin is indicated due to its efficient action against oral anaerobes, gram +ve aerobic microorganisms (Staphylococcus aureus, S. epidermidis, Sptreptococcus spp) and gram -ve enterobacteria (Escherichia coli, Enterobacter spp and Pseudomonas), which show MIC90 between 0.015 and 2 μg/mL respectively. All the species of streptococcus are sensitive to concentrations between 1.0 and 8.0 μg/mL; Other Streptococcal species like S. aureus and S. epidermidis are also sensitive to concentrations between 0.25 and 1.0 μg/mL.4,5 Ciprofloxacin has antimicrobial activity against most Gram-negative bacilli and cocci but limited activity against most Gram-positive organisms. Moxifloxacin is a new fluoroquinolone with expended spectrum of activity, including anaerobes and Gram positive organisms, especially the multi-resistant ones.3,6-10 Moxifloxacin has been found to be one of the most active antibiotics against E. faecalis with the lowest MIC50 and MIC90. From the previous literature it has been proved that ciprofloxacin to be more active other antibiotics.3,6,8,9,11
The usage of local antibiotics in endodontic treatment was advocated by Grossman in 1951. Since then numerous antibiotics have been used during endodontic treatment. The antibiotic combination has proved advantageous with the usage of triantibiotic paste.12
The laboratory study was carried out to test the effectiveness of propolis and calcium hydroxidealone as well as in combination with the established endodontic medicaments (Moxifloxacin and Ciprofloxacin).
Materials and method:
The study was carried out in the Department of Conservative Dentistry and Endodontics and Department of Microbiology at Saraswati Dental College and Hospital, Lucknow, India. Propolis (Hi-Tech Natural product India Ltd), Calcium Hydroxide (Vishaldentocare), Moxifloxacin (Avelox), Ciprofloxacin (Ciplox-500) were used in this study. They were distributed in different groups as presented in table no 1.
The efficacy of these medicaments was tested by checking for the zone of inhibition for the specific strain (ATCC 29212) of Enterococcus faecalis at different time intervals i.e. 24hr, 48hr and 72hr. Specific stain (ATCC 29212) of Enterococcus Faecalis was procured and grown on Brain Heart Infusion Broth (BHI) under favorable temperature overnight and the growth was checked by changes in the turbidity at 24 hours. Blood Agar Media Plate was inoculated with Enterococcus faecalis (ATCC 29212) by spreading the culture over the surface of the plate with a swab. Intermittent scraping of the culture was done at different sites to make wells for the placement of experimental intracanal medicament. The experimental groups were used in their minimum inhibitory concentration (MIC) against Enterocaccus faecalis (ATCC 29212). MIC of the various antibiotics used in this study was – Propalis – 340 µg/mL, Calcium hydroxide 2500 µg/mL, Moxifloxacin 2 µg/mL and Ciprofloxacin 8 µg/mL.2,13
Propolis, Calcium hydroxide and antibiotics were used as a paste in which saline acted as a vehicle. Propolis and Calcium hydroxide were mixed separately with saline on a glass slab, with the help of cement spatula, to prepare the paste. For obtaining the antibiotic paste, tablets of Ciprofloxacin and Moxifloxacin were crushed in mortar and pestle and then mixed with saline on glass slab with cement spatula. Plates were subjected to incubation at 30 °c for 24hrs, 48 hrs and 72 hrs (Figure 1) and the diameter of the zones of inhibition were measured in millimeters with HiAntimicrobial Inhibition Zone Scale tm C (HiMedia Laboratories Pvt Limited, Mumbai) (Figure 2). The observations were subjected to descriptive and inferential (ANOVA) statistical analysis.
Table 2 shows the mean zones of inhibition in different groups irrespective of time. Zones of inhibitions ranged from 10 mm (Group II) to 28 mm (Group VI). Mean zone of inhibition was maximum in Group V (21.94±4.26) followed by Group VI (18.80±1.93), Group I (18.71±4.26),Group VIII (15.88±2.59), Group III (14.91±1.00), Group VII (14.57±2.17), Group IV (13.91±1.00) and minimum in Group II (12.89±2.14). Table 3 shows the ANOVA applied in different groups. Statistically, intergroup differences were significant (p<0.001). The following order of antimicrobial efficacy of different groups was observed: Group V > Group I ~ Group VI > Group VIII ~ Group III ~ Group VII ~ Group IV ~ Group II. Table 4 shows the mean zones of inhibition at different time intervals in different groups. Mean zone of inhibition were maximum at 72 hr and minimum at 24 hr, and the intergroup differences were significant. At all the time intervals, group V showed the maximum antimicrobial efficacy.
Intracanal medicaments are used to eliminate remnant microbial flora after a thorough chemo-mechanical preparation of root canal and hasten the healing process. Propolis which is a very effective natural product and has been a part of popular medicine since thousands of years 14, 15. The color of Propolis is dark. Made from material collected from plants by bees, It is used against pathogenic microorganisms. Due to its anti-inflammatory properties it is effective against infection, rheumatism, muscular and articular diseases, and other types of inflammation.15-17 The chemical composition of propolis varies widely. Over 200 substances have been identified in the various varieties of propolis extracted from various geographical regions, which includes phenolic acids, esters, flavonoids, alcohols, aromatic aldehydes, fatty acids, amino acids, vitamins and minerals. Special emphasis is given to the flavonoids and phenolic acids mainly responsible for its biological activity. Propolis exhibits antimicrobial and anti-inflammatory properties, that why it has been suggested as a root canal medicament.15,18,19
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Calcium hydroxide, a white, odorless powder is one of the main stay of the root canal medicaments. It has a low solubility in water, insoluble in alcohol and a high pH. The extended clinical action of calcium hydroxide is well known. It is biocompatible, and has antimicrobial and anti-inflammatory action; it also activates the alkaline phosphatase enzyme, which in turn induces mineralized tissue formation and aid in the repair process. Chemically it is classified as a strong base, and when it is associated with a suitable vehicle produces an alkaline paste. Calcium hydroxide paste dissociates into calcium and the hydroxyl ions, the resulting environment will turn alkaline because of hydroxyl ions, due to this action it is a very good root canal dressing material. For the destruction of pathogenic bacteria of root canal and dentinal tubules , these hydroxyl ions should be capable to diffuse in dentin and persist in pulp tissues in a maximum concentration to produce the pH level required 15,20. Moreover various other studies reported by Molander A. and Tirali et al found CaOH to have synergistic effect with other endodontic medicaments.21,22
Ciprofloxacin, a second generation fluoroquinolone antibiotic, inhibits bacterial DNA synthesis. In dentistry, it is used as a broad spectrum antimicrobial and is effective against gram negative bacteria (staphylococcus and pseudomonas). One of the important drugs of fourth-generation syntheticfluoroquinolone is Moxifloxacin. It has superior activity against Gram+ve bacteria and anaerobes as compared to levofloxacin and ciprofloxacin.Moxifloxacin is abroad-spectrum antibioticthat is active against bothGram-positiveandGram-negativebacteria. It inhibitsDNA gyrase, enzyme which inhibit cell replication by separating bacterial DNA 12.
First and second generation fluoroquinolones selectively inhibit topoisomerase II ligase domain, which leaves the two nuclease domains intact. This particular modification, coupled with the stable action of the topoisomerase II within the cell of bacteria through nucleasic activity of the intact enzyme leads to DNA fragmentation. The further generations of fluoroquinolones i.e third and fourth acts selectively for topoisomerase IV ligase domain, which makes them more effective against gram positive coverage.12,23
Antimicrobial property of propolis is due to inhibition of bacterial DNA-dependant RNA polymerases’. Synergism amid antibacterial agents and propolis has been observed by Madarova L in 1980. Grange JM and Davey RW found that there was synergism between antimicrobial drugs and propolis against S. aureus particularly those which interfere with bacterial protein synthesis.24-26 Noori Al-Wahi found propalis to have synergistic properties with honey.27
Although calcium hydroxide does not bond to dentin, it does have antibacterial property. Its mechanism of actions is through the ionic break down of Ca(+) and OH(-) ions and corresponding effects on vital tissues. Protein denaturation and damage to DNA and cytoplasmic membranes are responsible for fatality of calcium hydroxide on bacterial cells .28
E fecalis appears to be highly resistant to the antimicrobial effect of calcium hydroxide. Evans et al. reported that at pH of 11.1, E. faecalis was resistant to calcium hydroxide but unable to survive at higher pH. After intracanal dressing of calcium hydroxide in radicular dentin, due to its buffering effect, the alkalinity may only reach a ph of 10.3. This one factor may be responsible to the resistance of E faecalis to Calcium hydroxide. The presence of proton pump in the cell wall of E faecalis, could be another reason for its resistance towards Calcium hydroxide.29
In the present study Propolis alone and with antibiotic was found to be more effective than calcium hydroxide and calcium hydroxide with antibiotics. Rezende Ceps R et al and Jahromi MZ et al have also found propolis to be more effective than CaOH. Propolis showed the synergistic effect with ciprofloxacin and moxifloxacin at all time intervals.13,30 In this study Propolis showed maximum efficiency against Enterococcus faecalis in combination with Moxifloxacin followed by Ciprofloxacin, which is similar to the results obtained by Krolet et al, Fernandis A et al, Ricardo Oliveria et al, Gheda Helaly et al, Kracko M et al and Stepanovic S et al who found propolis to have synergistic effect with various antibiotics.31-36
Calcium hydroxide also showed synergistic effect with antibiotic at all time intervals, which was maximum with Moxifloxacin followed by Ciprofloxacin at all time intervals.
On the basis of the results of the present study it can be concluded that Propolis and Calcium hydroxide show synergistic effect with Moxifloxacin and Ciprofloxacin against E. Faecalis. Propolis in combination with antibiotics and alone is more effective than calcium hydroxide. The use of Moxifloxacin as an intracanal medicament should be seriously explored. In order to draw more definitive conclusions, a wider and more detailed study needs to be undertaken.
Since propolis alone and in combination with antibiotics was observed to be more effective than calcium hydroxide, propolis can be considered as an intracanal medicament when compared to traditional calcium hydroxide.
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