Propolis and Calcium Hydroxide Against Enterococcus Faecalis

Propolis and Calcium Hydroxide Against Enterococcus FaecalisTitle An in-vitro comparison of endodontic medicines Propolis and Calcium hydrated oxide alone and in combination with ciprofloxacin and Moxifloxacin against Enterococcus faecalis.AbstractAim To evaluate and compare the healthful properties of Propolis and Calcium hydroxide alone and in combination with ciprofloxacin and Moxifloxacin against Enterococcus faecalis (E. Faecalis). Materials and Methods The laboratory national was carried out to test the effectualness of propolis and atomic number 20 hydroxidealone as well as in combination with the established endodontic medicaments (Moxifloxacin and ciprofloxacin). The versatile combinations were class1 Propolis, sort 2 calcium hydroxide, idea 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 regularise of inhibition for the specific stretch out (ATCC 29212) of Enterococcus faecalis at different eon intervals i.e. 24hr, 48hr and 72hr. Results Mean zone of inhibition was maximum in Group V (21.944.26) followed by Group VI (18.801.93), Group I (18.714.26), Group VIII (15.882.59), Group III (14.911.00), Group VII (14.572.17), Group IV (13.911.00) and lower limit in Group II (12.892.14). 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 prove study it can be concluded that Propolis and Calcium hydroxide show interactive effect with Moxifloxacin and Ciprofloxacin against E. Faecalis. Propolis in combination with antibiotics and alone is more strong 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 provide medicament when compared to traditional calcium hydroxide.Key words Calcium Hydroxide, Ciprofloxacin, E. fecalis, Intra canal medicaments, Laboratory research, Moxifloxacin, Propolis.IntroductionFor 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, frankincense 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.1The most common species recovered in over one-third of the canals of root filled dentition 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.2Earlier strong intracanal antiseptics such as formacresol and camphorated parachlorophenol were use, besides 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 demand made it the preferred choice for intracanal book binding of the infected roots canals.1 Calcium hydroxide has been added to several endodontic sealers to correct their biologic properties and to augment th eir 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 yard-negative species, there by inactivating the membrane transport mechanisms.3Propolis 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 high-and-mighty 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.1Moxifloxacin and Ciprofloxacin are members of the quinolones. Among the drugs commonly used for endodontic infection, ciprofloxacin is indicated due to its efficient process 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 g/mL respectively. totally the species of streptococcus are sensitive to concentrations between 1.0 and 8.0 g/mL Other Streptococcal species like S. aureus and S. epidermidis are also sensitive to concentrations between 0.25 and 1.0 g/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, particularly the multi-resistant ones.3,6-10 Moxifloxacin has bee n 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.3,6,8,9,11The engagement 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.12The 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 ruleThe 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 step-up 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 shield 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 mixed antibiotics used in this study was Propali s 340 g/mL, Calcium hydroxide 2500 g/mL, Moxifloxacin 2 g/mL and Ciprofloxacin 8 g/mL.2,13Propolis, Calcium hydroxide and antibiotics were used as a paste in which saline acted as a vehicle. Propolis and Calcium hydroxide were assorted individually 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 quelling Zone Scale tm C (HiMedia Laboratories Pvt Limited, Mumbai) (Figure 2). The observations were subjected to descriptive and inferential (ANOVA) statistical analysis.ResultsTable 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 (21.944.26) followed by Group VI (18.801.93), Group I (18.714.26),Group VIII (15.882.59), Group III (14.911.00), Group VII (14.572.17), Group IV (13.911.00) and minimum in Group II (12.892.14). Table 3 shows the ANOVA apply in different groups. Statistically, intergroup differences were significant (p 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.DiscussionIntracanal 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. receivable 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.15,18,19Calcium hydroxide, a white, odorless powder is one of the main stay of the root canal medicaments. It has a low solvabili ty in water, insoluble in alcohol and a high pH. The extended clinical run 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 deal 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.21,22Ciprofloxacin, a second generation fluoroquinolone antibiotic, inhibits bacterial deoxyribonucleic acid 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 l eads 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.12,23Antimicrobial 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.27Although 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 bouncy tissues. Protein denaturation and damage to DNA and cytoplasmic membranes are responsible for fatality of calcium hydroxide on bacterial cells .28E 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 electric 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.29In 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 force 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.31-36Calcium hydroxide also showed synergistic effect with antibiotic at all time intervals, which was maximum with Moxifloxacin followed by Ciprofloxacin at all time intervals.ConclusionOn 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 univocal conclusions, a wider and more detailed study needs to be undertaken.Clinical significanceSi nce 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.