|Year : 2014 | Volume
| Issue : 2 | Page : 120-124
The antimicrobial effectiveness of 25% propolis extract in root canal irrigation of primary teeth
Manjesh Kumar Verma1, Ramesh Kumar Pandey1, Richa Khanna1, Jyotsna Agarwal2
1 Department of Pediatric with Preventive Dentistry, King George's Medical University, Lucknow, Uttar Pradesh, India
2 Department of Microbiology, King George's Medical University, Lucknow, Uttar Pradesh, India
|Date of Web Publication||17-Apr-2014|
Ramesh Kumar Pandey
Department of Pediatric with Preventive Dentistry, King George's Medical University, Lucknow - 226 003, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: The choice of irrigating solution used in root canals of primary teeth is complicated by their complex morphology and paucity of associated literature. Propolis is a natural product that has gained interest in this context due to its antibacterial effectiveness against several endodontic pathogens. Aim: The present study was undertaken to assess the potential of water-soluble 25% propolis extract against microorganisms present in root canals of primary teeth during endodontic procedures. Settings and Design: The child patients in the age group of 4-7 years with radiographic evidence of carious pulp exposure were included in the study. Definitive selection was done after gaining access into the pulp chamber and root canals of the selected teeth. The clinical and radiographic evidence of pathosis was ruled out for inclusion in the study. Materials and Methods: The selected teeth were divided into two groups randomly. In Group A 0.9% isotonic saline and in Group B 25% extract water-soluble propolis were used as irrigating solution, respectively. The bacterial samples were collected both pre- and post-irrigation and were transferred for microbial assay. Staististical Analysis: Wilcoxon matched signed rank test was used to compare the pre-and post-irrigation bacterial counts. Mann-Whitney test was used to compare the mean change (pre-post) in bacterial colony counts of groups in the study. Results: Antimicrobial effectiveness of 25% water-soluble extract of propolis in the root canals of primary teeth was confirmed in the present study. The reduction in the mean bacterial colony counts of all the isolated bacteria was noticed higher in Group B than Group A. Conclusion: The results of the present study have confirmed that the antibacterial effectiveness of water-soluble extract of propolis in the root canals of primary teeth in vivo. Considering the low toxicity concerns and antibacterial effectiveness, water-soluble extract of 25% propolis can be advocated as a root canal irrigant in endodontic treatment of primary teeth.
Keywords: Irrigating solution, primary teeth root canal, propolis, root canal irrigant
|How to cite this article:|
Verma MK, Pandey RK, Khanna R, Agarwal J. The antimicrobial effectiveness of 25% propolis extract in root canal irrigation of primary teeth. J Indian Soc Pedod Prev Dent 2014;32:120-4
|How to cite this URL:|
Verma MK, Pandey RK, Khanna R, Agarwal J. The antimicrobial effectiveness of 25% propolis extract in root canal irrigation of primary teeth. J Indian Soc Pedod Prev Dent [serial online] 2014 [cited 2022 Jun 25];32:120-4. Available from: https://www.jisppd.com/text.asp?2014/32/2/120/130786
| Introduction|| |
The prime objective of pediatric endodontic therapy aims to maintain the primary teeth in their form and function and to facilitate the proper eruption of permanent successors. The role of clinician ascribed to maintain strict asepsis in vital pulp situation and to attain antisepsis for non-vital and necrosed pulps. 
The infected, devitalized, and necrosed pulp serves as potential inducer of apical periodontitis, hampering the success of endodontic treatment as advocated by Moller.  Hence, the microbial reduction or elimination from root canals of infected teeth leads to successful treatment.
The endodontic instrumentation cannot effectively eliminate the microflora from the root canals of primary teeth mechanically owing to their anatomical complexity. The dependence on irrigating solutions for endodontic success becomes more crucial, especially in primary teeth due to thin dentinal walls, complex morphology, and irregularity of root canal system. 
Several formulations have been attempted in root canals of permanent teeth with their respective advantages/disadvantages. However, there is paucity of literature related to efficacy of root canal irrigants used in primary teeth. A total of 5.25% sodium hypochlorite is a common irrigant of choice, used both in primary and permanent teeth. The antibacterial effectiveness and tissue dissolution capacity of aqueous 5.25% hypochlorite is directly proportional to concentration and toxicity.  A total of 5.25% sodium hypochlorite cannot differentiate between vital and necrotic tissues during dissolution. The periapical damage resulted due to extrusion of 5.25% sodium hypochlorite is also widely reported. 
Chlorhexidine is second choice of irrigant used both in primary and permanent teeth. It has antibacterial effects and lower cytotoxicity, but the associated disadvantage is that it lacks the ability to dissolve necrotic pulp tissue. 
Hence, an ideal irrigating solution for endodontic treatment in primary/permanent teeth is still under investigation. Natural products that were introduced long back for curing diseases are now again looked upon as an alternative to commercially available chemical formulations. Apitherapy is one such approach that is being explored in all fields of medicine including dentistry. It incorporates medicinal use of honey bee products like honey, propolis, pollen, royal jelly, and bee venom.
Propolis is a natural product that has gained increased interest due to its antimicrobial activity against a wide range of pathogenic microorganisms.  It is composed of resin and balsams (50-60%), pollen (5-10%), and other constituents like amino acids, minerals, vitamins A and B complex, and highly active biochemical substance known as bioflavonoids (vitamin P), phenols, and aromatic compounds.  Propolis has been used in dentistry as pulp capping agent,  as storage media for avulsed teeth,  for prevention of caries,  and dentine hypersensitivity.  The use of propolis as a root canal irrigant is yet to be explored in primary teeth.
Considering the immense potential of propolis as an antimicrobial agent, as evidenced in literature, the present study was undertaken to assess its usage as an intracanal irrigant in endodontic therapy of primary teeth.
| Materials and Methods|| |
The present study was conducted in the Department of Pediatric with Preventive Dentistry, in collaboration with the Post Graduate Department of Microbiology, King George's Medical University, Lucknow, UP, in an attempt to evaluate the effectiveness of propolis as a root canal irrigant in primary teeth. The patients in the age group of 4-7 years, having good general health, without history of antibiotic coverage and radiographic evidence of carious pulp exposure in primary teeth with restorable tooth structure were screened for inclusion in the present study. Definite selection was done after gaining access into the pulp chamber and root canal of selected teeth and conforming presence of necrotic pulp. The clinical and radiographic evidence of pathosis in periapical or furcation area and presence of internal/external resorption were ruled out for the selected patients. Physiological resorption if present was confirmed to be restricted upto only one-third of the root lengths for every selected tooth. A total of 70 primary teeth thereby selected were divided into two groups randomly, based on the irrigating solution used. Group A consisted of 35 subjects with 0.9% isotonic saline as an irrigating solution. Group B consisted of 35 subjects with 25% water-soluble propolis as an irrigating solution. The informed consent was recorded from the parent/guardian of each patient included in the present study.
The patients were instructed to rinse his/her oral cavity for 30 s with 5 ml povidine iodine. After local anesthesia, rubber dam was placed, operative field including the tooth, clamp, and surroundings were cleaned with povidine iodine. A high speed hand piece and sterilized round bur (number 2) under constant irrigation were used to remove all carious tissue and to access the root canals of primary teeth. In mandibular primary molar distal canal and in maxillary primary molar palatal canal were chosen for sampling procedure. Sterile barbed broach of number 15 was introduced into the canal to the predetermined working length from the initial preoperative radiograph. Pulp was extirpated with the help of barbed broach. Paper point was introduced into the canal after pulp extirpation to the predetermined working length from diagnostic radiograph. The paper point was allowed to rest in the canal for 1 min after which it was removed and placed immediately into the eppendorf containing 200 μl nutrient broth medium. The pre-irrigation sample was sealed and kept separately for transportation to microbiological laboratory.
The root canal after pre-irrigation sample collection was prepared using sterile H-files. The canal was irrigated with saline in group A and with water-soluble propolis in group B, between the sequential filings every time. After drying the canal, post-irrigation sample was taken and stored like pre-irrigation sample with the help of paper points.
The pre- and post-irrigation samples collected were transferred for microbial assay by culture method.
In the laboratory, the sample was mixed in a vortex mixer for 30 s, 0.01 ml of the sample was plated via calibrated loop on blood agar plates (semi-quantitative culture). The plates were then incubated at 37° C for 48-72 h and counted for the growth of bacterial isolates. Each isolate was identified using standard conventional techniques including Gram staining and biochemical tests as advocated by Collee et al.,  2003 and Forbes et al.,  2007.
| Results|| |
The prime bacteria were isolated from root canals of primary teeth selected in the present study are Streptococci, Staphylococcus, Enterococcus faecalis, and Escherichia More Details coli. The frequency (%) of bacterial culture positivity between the two groups is highest for Streptococci and least for E. coli. Comparing the frequency of bacterial culture positivity of two groups, χ2 test revealed similar (P > 0.05) proportion of culture positivity between the two groups (χ2 = 0.66; P = 0.883).
It was observed that the mean bacterial colony counts of all the isolated bacteria decreased after irrigation, and the decrease was higher in Group B than in Group A [Figure 1] and [Figure 2]. On comparing the pre- and post-mean colony counts of Streptococci and Staphylococci of Group A Wilcoxon matched signed rank test revealed significantly (P < 0.001) decreased colony counts at post-irrigation as compared with pre-irrigation (P < 0.001). Similarly, the mean colony counts of Streptococci and Staphylococcus of Group B also decreased significantly (P < 0.001) at post-irrigation as compared with pre-irrigation (P < 0.001). The comparison of the mean change (pre-post) of Streptococci and Staphylococcus bacterial colony counts in two groups, Mann-Whitney test revealed significant (P < 0.01) difference and higher decrease (2.5 fold or 59.2% and 3.1 fold or 68.2% respectively) in Streptococci and Staphylococcus colony count in Group B as compared with Group A (P < 0.001).
|Figure 1: Pre- and post-irrigation bacterial colony counts in two groups|
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Furthermore, comparing the pre- and post-mean colony count of E. faecalis and E. coli of Group A, Wilcoxon matched signed rank test revealed significantly (P < 0.01) decreased colony counts at post-irrigation as compared with pre-irrigation (P = 0.001). Similarly, the mean colony counts of E. faecalis and E. coli of Group B also decreased significantly (P < 0.001) at post-irrigation as compared with pre-irrigation (P < 0.001). In comparison of mean change (pre-post) of E. faecalis and E. coli bacterial colony count in two groups, Mann-Whitney test revealed (2.1 fold or 52.3% and 1.6 fold or 37.4%) higher decrease of E. faecalis and E. coli colony count in Group B as compared with Group A, but the data were not statistically significant (P = 0.077 and P = 0.170).
| Discussion|| |
Root canal irrigation is an integral part of non-vital pulp therapy for primary teeth. The choice of irrigating solution used in root canals of primary teeth is complicated by the complex morphology and irregularity.  The irritation of periapical tissues is an important factor to be considered in selection of root canal irrigant in primary teeth. A comparative study of anti-inflammatory effects of propolis, Casearia sylvestris, otosporin, and saline solution (control) was done by Silva et al., in Wistar rats.  They confirmed propolis to be the least irritating solution, even less than saline. Ramos et al., stated that propolis paste exhibit fewer inflammatory reactions in comparison with corticosteroids antibiotic preparation in periapical tissue so that it can be used as an alternative root canal medication after pulpectomy.  Therefore, the present study was investigated to assess the antibacterial potential of water-soluble propolis extract in endodontic treatment of primary teeth.
A few studies have associated propolis and its anti-inflammatory and antibacterial effects with endodontic treatment ,, due to the intrinsic difficulty of introducing propolis and removing from the internal side of the root canal as an intracanal medicament. The difficulty in removal and insertion is due to presence of resins, waxes, and so forth. Therefore, the water-soluble derivative of propolis is used in present study as a root canal irrigant.
Bactericidal or bacteriostatic characteristic of propolis depends on its concentration in the applied extract. In the present study, 25% water-soluble derivative of propolis containing most of the flavonoids, vitamins, amino acids, and other water-soluble compounds without wax and resin is used in present study that facilitates easy removal of the propolis extract from the canal after irrigation. Najafi et al.,  confirmed that the water extracts did not affect the biological activity of propolis as most of the flavanoid compounds are retained.
Many studies have evaluated propolis antimicrobial activity using serial dilution tests , and diffusion tests on agar , with diverse results. The present study was designed to be in vivo to examine the properties of propolis clinically due to paucity of literature.
Molander et al.,  reported that the propagation of microorganisms takes place throughout the entire root canal system of primary teeth, including the lumen, lateral, accessory, and secondary canals, dentinal tubules, ramifications of the apical delta, apical foramen, areas of apical cementum resorption, and periapical biofilm. The adsorbent paper points used for collection of microbiological samples in vivo, as in the present study, can collect bacteria present only inside the canal and not from the dentinal tubules or deeper areas. This accounts for the variability in quantity and type of bacteria isolated from the root canal system in vitro/in vivo studies.
Edward (1972)  have demonstrated that root canal infections of primary teeth are usually polymicrobial in nature. The literature shows the presence of streptococci in (70%),  in (82%),  and in (76%)  of the root canals of deciduous teeth with pulp necrosis. In the present study, the frequency of culture positivity for streptococci was observed highest in intercomparison of all the bacteria isolated from the root canals of selected teeth. The findings of present study showed less bacterial count than previous studies. The variations in different studies might be attributed to directly exposed root canals in oral cavity, thus, enhancing the prevalence of Streptococci.
In the present study, the other bacteria isolated from the root canals of selected primary teeth were Staphylococcus aureus, E. faecalis, and E. coli in the pre-irrigation samples collected. The polymicrobial nature of endodontic pathogens was consistent to previous studies.  Ruviere et al.,  found that root canals of human primary teeth exhibited a great bacterial diversity characterized by a polymicrobial endodontic infection, similar to permanent teeth. They affirmed presence of anaerobic and facultative bacteria, black-pigmented rods, and streptococci. The differences in bacteria isolated in various studies were due to the difference in method of identification used. Ruviere et al.,  used more specific checkerboard DNA-DNA hybridization to detect uncultivable and fastidious microorganisms, which is expensive on a large scale. Conventional standardized microbial culturing method was method of choice used in the present study. The main goal was to assess the quantitative antibacterial effects of propolis.
The antimicrobial property of propolis as observed in the present study was in concurrence of previous reported studies.  There was significant reduction in the colony counts of Streptococcus group and S. aureus after irrigation with water-soluble extract of propolis. The results were consistent with studies of Ikeno et al.,  Koo et al.,  and Park et al., The reduction in colony counts of Streptococcus group and S. aureus after irrigation with saline was also obtained significant but less than that in propolis group. The reduction was attributed to the mechanical action of saline as an irrigant that flushes bacteria from the canals. Propolis or any other irrigant with antibacterial properties remove bacteria with their mechanical and antibacterial actions.
E. faecalis is a well-recognized pathogen associated with persistent apical periodontitis in endodontically treated teeth and in treatment failures as advocated by Vahdaty et al.,  Therefore, it was not found to be present in large counts in the selected teeth, as the sample selection comprised in teeth having primary root canal infection without apical periodontitis. E. faecalis was not isolated from every tooth selected in the present study.
There is contradictory evidence from literature regarding the effects of different irrigating solutions against E. faecalis. Moliz et al.,  propounded sodium hypochlorite to be the most effective agent, capable of eradicating E. faecalis biofilms after 1 min at a concentration of 0.00625%. Chlorhexidine eradicated biofilm after 5 min at 2% concentration. Madhubala et al., found the ethanolic extract of propolis to be more effective than tri-antibiotic mixture at the end of 2 day time period against E. faecalis. 
The reduction in microbial counts of E. faecalis after irrigation with water-soluble extract of propolis as well as with normal saline was evident in the present study. The change in pre- and post-irrigation samples was higher in the propolis group than saline.
Root canal irrigation with propolis and saline was found to be effective in completely eliminating E. coli as suggested by Valera et al.,  In the present study, the results for E. faecalis are in agreement with finding of Valera et al., E. coli colony counts were reduced after irrigation with propolis but not totally eliminated. The reduction in the control group (saline) was also evident but less than the propolis group.
| References|| |
|1.||Zehnder M. Root canal irrigants. J Endod 2006;32:389-98. |
|2.||Moller AJ, Fabricius L, Dahlen G, Ohman AE, Heyden G. Influence on periapical tissue of indigenous oral bacteria and necrotic pulp tissue in monkey. Scand J Dent Res 1981;89:475-84. |
|3.||Thomas AM, Chandra S, Chandra S, Pandey RK. Elimination of infection in pulectomized deciduous teeth: A short-term study using iodoform paste. J. Endod 1994;20:233-5. |
|4.||Beckling AG. Complications in the use of sodium hypochlorite during endodontic treatment. Report of three cases. Oral surg Oral Med Oral Pathol 1991;71:346-8. |
|5.||Marley JT, Ferguson DB, Hartwell GR. Effects of chlorhexidine gluconate as an endodontic irrigant on the apical seal: Short-term results. J Endod 2001;27:775-8. |
|6.||Santos FA, Bastos EM, Maia AB, Uzeda M, Carvaiho MA, Farias LM, et al. Brazilian propolis: Physicochemical properties, plant origin and antibacterial activity on periodontopathogens. Phytother Res 2003;17:285-9. |
|7.||Rathod S, Brahmankar R, Kolte R, Propolis. A natural remedy. Indian J Dent Res Review 2011;50-2. |
|8.||Sabir A, Tabbu CR, Agustiono P, Sosroseno W. Histological analysis of rat dental pulp tissue capped with Propolis. J Oral Sci 2005;47:135-8. |
|9.||Martin MP, Pileggi R. A quantitative analysis of Propolis: A promising new storage media following avulsion. Dent Traumatol 2004;20:85-9. |
|10.||Duarte S, Rosalen PL, Hayacibara MF, Cury JA, Boen WH, Marquis RE, et al. The influence of novel propolis on mutans streptococci biofilms and caries development in rats. Arch Oral Biol 2006;51:15-22. |
|11.||Mahmoud AS, Almas K, Dahlan AA. The effect of propolis on dentinal hypersensitivity and level of satisfaction among patients from a university hospital Riyadh, Saudi Arabia. Indian J Dent Res 1999;10:130-7. |
|12.||Collee JG, Miles RS, Watt B. Identification of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Practical Medical Microbiology. 14 th ed., Vol 2. Longman Singapore: Churchill Livingstone Publishers; 2003. p. 131-49. |
|13.||Forbes BA, Sahm DF, Weissfeld AS. Principles of Identification. 12 th ed. St. Louis; Missouri. Mosby-Year Book; 2007. p. 105. Bailey and Scott's Diagnostic Microbiology. |
|14.||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. |
|15.||Ramos IF, Biz MT, Paulino N, Scremin A, Della Bona A, Barletta FB, et al. Histopathological analysis of corticosteroid-antibiotic preparation and propolis paste formulation as intracanal medication after pulpectomy: An in vivo study. J Appl Oral Sci 2012;20:50-6. |
|16.||Heys DR, Cox CF, Heys RJ, Avery JK. Histological considerations of direct pulp capping agents. J Dent Res 1980;60:1371-9. |
|17.||Pulp mummification by the use of Propolis. Revs Bras Odont 1989;46:44. |
|18.||Najafi MF, Vahedy F, Seyyedin M, Jomehzadeh HR, Bozary K. Effect of the water extracts of propolis on stimulation and inhibition of different cells. Cytotechnology 2007;54:49-56. |
|19.||Sonmez S, Kirilmaz L, Yucesoy M, Yucel B, Yilmaz B. The effect of bee propolis on oral pathogens and human gingival fibroblast. J. Ethnopharmacol 2005;102:371-6. |
|20.||Victorino FR, Franco SL, Svidzinski TI, Avila-Campos MJ, Cuman RK, Hidalgo MM, et al. Pharmacological evaluation of propolis solutions for endodontic use. Pharm Biol 2007;45:721-7. |
|21.||Kujumgiev A, Tsvetkova I, Serkedjieva Y, Bankova V, Christov R, Popov S. Antibacterial, antifungal and antiviral activity of propolis of different geographic origin. J Ethnopharmacol 1999;64:235-40. |
|22.||Bruschi ML, Lara EH, Martins CH, Vinholis AH, Casemiro LA, Panzeri H, et al. Preparation and antimicrobial activity of gelatin microparticles containing propolis against oral pathogens. Drug Dev Ind Pharm 2006;32:229-38. |
|23.||Molander A, Reit C, Dahlén G, Kvist T. Microbiological status of root filled teeth with apical periodontitis. Ýnt Endod J 1998;31:1-7. |
|24.||Edwards S, Nord CE. Identification and characterization of micro-organisms isolated from infected primary teeth. J Int Assoc Dent Child 1972;3:15-8. |
|25.||Cohen MM, Joress SM, Calisti LP. Bacteriologic study of infected deciduous molars. Oral Surg Oral Med Oral Pathol 1960;13:1382-6. |
|26.||Marsh SJ, Largent MD. A bacteriological study of the pulp canals of infected primary molars. J Dent Child 1967;34:460-70. |
|27.||Tomic-Karovic K, Jelinek E. Comparative study of the bacterial flora in the surroundings, the root canals and sockets of deciduous molars. Int Dent J 1971;21:375-88. |
|28.||Ruviére DB, Leonardo MR, da Silva LA, Ito IY, Nelson-Filho P. Assessment of the microbiota in root canals of human primary teeth by checkerboard DNA-DNA hybridization. J Dent Child (Chic) 2007;74:118-23. |
|29.||Arslan S, Ozbilge H, Kaya EG, Er O. In vitro antimicrobial activity of propolis, BioPure MTAD, sodium hypochlorite, and chlorhexidine on Enterococcus faecalis and Candida albicans.Saudi Med J 2011;32:479-83. |
|30.||Ikeno K, Ikeno T, Miyazawa C. Effects of propolis on dental caries in rats. Caries Res 1991;25:347-51. |
|31.||Koo H, Vacca Smith AM, Bowen WH, Rosalen PL, Cury JA, Park YK. Effects of Apis mellifera propolis on the activities of streptococcal glucosyltransferases in solution and adsorbed onto saliva-coated hydroxyapatite. Caries Res 2000;34:418-26. |
|32.||Park YK, Koo MH, Abreu JA, Ikegaki M, Cury JA, Rosalen PL. Antimicrobial activity of propolis on oral microorganisms. Curr Microbiol 1998;36:24-8. |
|33.||Vahdaty A, Pitt Ford TR, Wilson RF. Efficacy of chlorhexidine in disinfecting dentinal tubules in vitro. Endod Dent Traumatol 1993;9:243-8. |
|34.||Arias-Moliz MT, Ferrer-Luque CM, Espigares-Garcý´a M, Baca P. Enterococcus faecalis biofilms eradication by root canal irrigants. J Endod 2009;35:711-4. |
|35.||Madhubala MM, Srinivasan N, Ahamed S. Comparative evaluation of propolis and triantibiotic mixture as an intracanal medicament against Enterococcus faecalis. J Endod 2011;37:1287-9. |
|36.||Valera MC, da Rosa JA, Maekawa LE, de Oliveira LD, Carvalho CA, Koga-Ito CY, et al. Action of propolis and medications against Escherichia coli and endotoxin in root canals. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;110:e70-4 |
[Figure 1], [Figure 2]
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