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Year : 2021  |  Volume : 5  |  Issue : 1  |  Page : 37-41

Differential effectiveness of calcium hydroxide with 2% chlorhexidine digluconate and 25% propolis as a root canal medicament against Enterococcus faecalis (In vitro)

Departments of Conservative Dentistry Education Study Program, Faculty of Medicine and Health Sciences, Universitas Muhammadiyah Yogyakarta, Yogyakarta, Indonesia

Date of Submission02-Sep-2020
Date of Decision15-Sep-2020
Date of Acceptance19-Jan-2021
Date of Web Publication16-Feb-2021

Correspondence Address:
Erma Sofiani
Departement of Conservative Dentistry, Faculty of Medicine and Health Sciences, Universitas Muhammadiyah Yogyakarta, Yogyakarta
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/SDJ.SDJ_45_20

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Background: One of the most common microorganisms found in infected teeth after root canal treatment is Enterococcus faecalis (E. faecalis). This bacteria can cause failure in root canal treatment, so it is necessary to provide intracanal medicament, such as calcium hydroxide, which can increase antibacterial effectiveness by adding a mixing agent. One of the calcium hydroxide mixing ingredients is chlorhexidine (CHX) digluconate gel 2%, which can increase the activity of hydroxyl ions in calcium hydroxide. A natural mixing ingredient is 25% propolis, which contains flavonoids with an antibacterial effect. Objective: The aim is to determine the difference in antibacterial effectiveness between a combination of calcium hydroxide with 2% CHX digluconate or 25% propolis as an intracanal medicament against E. faecalis (in vitro) bacteria. Methods: This research used an experimental in vitro laboratory test with 18 teeth that had a single root canal. Teeth were sterilized, had root canal treatment, and cut off at the crown. Antibacterial efficacy was calculated by measuring optical density (OD) values with spectrophotometry. The data were analyzed using the Kruskal–Wallis test followed by the Mann–Whitney test. Results: The largest OD difference group was in the calcium hydroxide mixture 25% propolis than calcium hydroxide mixture 2% CHX gel. Conclusion: There was a difference in the antibacterial power of the intracanal medicament, calcium hydroxide combination with 2% CHX digluconate gel or 25% propolis. The combination of calcium hydroxide with propolis 25% more effective than the mixture of calcium hydroxide with 2% CHX digluconate as root canal medicament against E. faecalis bacteria.

Keywords: Calcium hydroxide, chlorhexidine digluconate 2%, Enterococcus faecalis, propolis 25%

How to cite this article:
Sofiani E, Wahyuningrum H. Differential effectiveness of calcium hydroxide with 2% chlorhexidine digluconate and 25% propolis as a root canal medicament against Enterococcus faecalis (In vitro). Sci Dent J 2021;5:37-41

How to cite this URL:
Sofiani E, Wahyuningrum H. Differential effectiveness of calcium hydroxide with 2% chlorhexidine digluconate and 25% propolis as a root canal medicament against Enterococcus faecalis (In vitro). Sci Dent J [serial online] 2021 [cited 2022 Dec 7];5:37-41. Available from: https://www.scidentj.com/text.asp?2021/5/1/37/309545

  Background Top

The presence of microorganisms in the oral cavity is closely related to the causes of pulp and periradicular disease. One of the diseases of the pulp is pulp necrosis, which is the condition of the death of the pulp tissue, partly or wholly, that can occur due to inflammation or trauma. Pulp necrosis can be caused by bacteria, trauma, and irritation from the restoration material, or continued pulp inflammation. Dental treatment for pulp necrosis is a root canal treatment. The purpose of root canal treatment is to maintain nonvital teeth in the dental arch to last as long as possible in the oral cavity by cleaning and disinfecting the root canal system, thereby reducing bacterial growth.[1]

Microorganisms that remain in the root canal or that grow after obturation of the root canal are the leading cause of root canal treatment failure.[2] One of the most common microorganisms found in teeth that is present during infection after root canal treatment is Enterococcus faecalis (E. faecalis)[3] This bacterium is found in a high percentage (up to 77%) of cases regarding root canal treatment failure.[4] Hence, it is necessary to provide medicinal intracanal treatment to eliminate microorganisms that are still left after preparation or to prevent recurrent infections after root canal treatment.[5] Root canal medicaments between visits are the most commonly used treatment. To date, calcium hydroxide (Ca(OH)2) has become the “gold standard” because it can eliminate bacteria, as shown by the activity of releasing a constant hydroxyl ion that dissolves the remaining necrotic tissue, inactivating lipopolysaccharides in bacteria and reducing the inflammatory process.[6]

Research has shown that direct contact between Ca(OH)2 and the root canal influences changes in the physical properties of dentin, which are caused by reduced dentinal moisture, both coronal and radicular, by as much as 9%. The decrease in humidity is a result of pulp tissue loss.[7] Therefore, Ca(OH)2 powder needs to be added to a mixing material to obtain a paste preparation to facilitate application and adhesion with minimal damage to the root canal. The addition of other substances to calcium hydroxide affects the rate of release of Ca[2]+ ions and improves antibacterial properties, radiopacity, and consistency.[8],[9]

One example of a calcium hydroxide mixing agent is chlorhexidine digluconate (CHX). The combination of calcium hydroxide with 2% CHX digluconate increases the release of hydroxyl ions, provides better healing of periradicular lesions, and effectively eliminates E. faecalis bacteria in infected root canals.[10] In addition to CHX digluconate, there is a natural ingredient currently being developed as an alternative antimicrobial ingredient to address dental health problems, namely propolis. One of the constituents in propolis is flavonoids. These flavonoids have antibacterial, antifungal, antiviral, and anti-inflammatory properties. Flavonoids can inhibit bacterial growth by disrupting bacterial cell wall permeability, causing lysis of the cell wall.[11] Propolis is more effective in eliminating E. faecalis bacteria than calcium hydroxide, so propolis combined with calcium hydroxide is expected to have a more effective antibacterial ability.[12] Considering this hypothesis, therefore, this study is aimed to assess and compare the efficacy between the combination of calcium hydroxide with 2% CHX gluconate or 25% propolis as an intracanal medicament against E. faecalis in vitro.

  Materials and Methods Top


This research used an in vitro laboratory test to determine the differences in the effectiveness of calcium hydroxide (Ca(OH)2) combined with 2% CHX digluconate and combination of Ca(OH)2 with 25% propolis as a root canal medicament against E. faecalis (in vitro) bacteria. Each treatment of calcium hydroxide combination with 2% CHX digluconate gel and 25% propolis, 2% CHX digluconate gel (positive control) had six repetitions. In each treatment, there were six pieces of test tubes containing brain heart infusion broth (BHIB) CM 1135 (Oxoid Ltd., Wade Road, Basingstoke, Hants, UK) broth each group. Group A was prepared by mixing calcium hydroxide powder (≤1.5 g) and 2% CHX digluconate gel (≤1.5 g) in conicle tubes until homogeneous. Group B was prepared by mixing calcium hydroxide powder (≤1.5 g) and 25% propolis (≤1.5 ml) in conicle tubes. Mixtures were stirred using a stainless-steel spatula until it formed a paste and then put into a syringe. Group C (positive control) contained 2% CHX digluconate gel (1.5 g), thus, the total number of the test tube is 18.

In contrast, this study's affected variable was the growth of E. faecalis bacteria in the root canal. Controlled variables were the length of the root canal (20 mm), Step Back preparation technique, length of bacterial incorporation on acrylic (24 h), incubator temperature 37°C, length of incubation of medical intracanal (24 h), number of BHI in the test tube (2 ml), and 20 ml root canal irrigation solution.

Bacterial culture and materials

The intracanal tests used in this study included E. faecalis clinical bacteria obtained from necrotic root canals. Pure calcium hydroxide powder, 2% CHX digluconate gel, 25% propolis, and BHIB media were used in this study.

Preparation of the teeth

This research was conducted at the molecular and medical therapy Laboratory in the Asri Medical Center from November 2019 to February 2020. Implementation was started with the preparation of 18 teeth with a single root canal. Teeth were prepared as follows: open access with a round and fissure bur, extirpation of the pulp with a barber broach, measure working length of the teeth using a sliding caliper, root canal preparation with step-back technique (K-file number 55) while being irrigated using a syringe containing 0.9% saline solution, and cut 18 permanent incisors that were prepared using a diamond disc in the cementoenamel junction section. After that, the teeth were implanted in an acrylic mold. Dental sterilization was performed by inserting teeth that had been planted in acrylic into a beaker containing 90% ethanol solution and covered with plastic wrap. Teeth were left to stand at room temperature for 12 h and inserting a beaker containing a tooth sample (solid material) into the HVE 50 autoclave (Hirayama, Tokyo, Japan) with a temperature of 121°C for 15 min of sterilization with 1 atm pressure, before being removed and cooled down.

Cultivation of Enterococcus faecalis and root canal inoculation

Suspensions of E. faecalis were made in BHIB. The suspension was made by placing 2 ml of BHIB and 1 ml suspension of E. faecalis bacteria into a test tube. The tube was covered with cotton and incubated for 24 h at 37°C. Teeth were incorporated by inserting a suspension of E. faecalis bacteria into the root canal using a syringe and incubated at 37°C for 24 h.

Measurement of the number of bacteria was recorded before being given an intracanal medicament. The medium BHIB was poured into the test tube and the incorporated teeth was removed from the incubator until the optimal moist condition was achieved. After that, a sterile paper point of the same size as K-file number 55 was used. A root canal swab was done using the paper point and placed in a test tube containing BHIB, along with a blank (test tube) that only contained BHIB without paper points as the control. All tubes were vortexed and incubated at 37°C for 24 h.

Preparation of intracanal medicament

Intracanal medicament was mixed in accordance with its combination. Using a micropipette, the controls were placed directly into a syringe. The medicament was applied to the root canals in all treatment groups using plastic syringe and combined with paste filler (lentulo). All groups were incubated for 3 days (72 h) at 37°C in an incubator (Labtech, Gyeonggi-do, South Korea).

Measurement of optical density

Measurement of optical density (OD) was done in the test tubes containing the root canal swabs (paper point number 55) before intracanal medicament treatment using a spectrophotometer (Scilogex, Rocky Hill, USA). The root canal was irrigated with an irrigation syringe using 0.9% saline solution (≤20 ml), making sure that it was spotless and no intracanal medicament remained. The canal was dried by swabbing with sterile paper points consistently from coronal to apical. The paper point was inserted into a test tube containing BHIB, then vortexed and incubated with an incubator at 37°C for 24 h. In the final step, OD was measured in the test tube containing the swab paper point after intracanal medicament treatment to obtain the turbidity value. The percentage of bacterial reduction was calculated using OD before and after treatment.

Statistical analysis

The data obtained from the research results were ratio data obtained by calculating the zone of inhibition against E. faecalis bacteria, which were grouped and tabulated. The grouped data were tested for normality and homogeneity. When the resulting data were homogeneous, it was combined and a parametric statistical test of one-way analysis of variance with P ≤ 0.05 was used. If the resulting data were abnormal and not homogeneous, a Kruskal–Wallis test was used to determine the difference in the effectiveness of calcium hydroxide combination with 2% CHX digluconate gel and 25% propolis against E. faecalis bacteria. If the results of the Kruskal–-Wallis statistical test showed significance, then the Mann–Whitney test was carried out to determine the significance level of differences in antibacterial activity.

  Results Top

The study results can be seen in the level of turbidity of BHIB after being given root canal medicament treatment in [Figure 1]. Result showed that Ca(OH)2 + Propolis 25% has the highest antibacterial effect compare to other group (P < 0.05) [Figure 2]. The data obtained were tested for normality using the Shapiro–Wilk test. The result showed the data was not normally distributed. Data were further analyzed using the nonparametric Kruskal Wallis test and the Mann–Whitney U-test.
Figure 1: BHI turbidity level after treatment with root canal medicament material: A. Calcium Hydroxide combined with Chlorhexidine digluconat gel 2%. B. Calcium Hydroxide combined Propolis 25% C. Chlorhexidine digluconate gel 2%. D. Glycerine 100%. (from the left the right : group A,B,C and D)

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Figure 2: The percentage of bacterial reduction in the root canal before and after treatment with calcium hydroxide with 2% chlorhexidine, calcium hydroxide with 25% propolis, Chlorhexidine gel 2% as an intracanal medicament against Enterococcus faecalis. All treatment were done in six repetition. *: P < 0.05 was considered statistically significant

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Based on the results of the statistical test, a significance value of 0.032 was obtained [Table 1]. The Mann–Whitney U-test result showed groups A (Ca(OH)2 + 2% CHX gel) and B (Ca(OH)2 + Propolis 25%) had a significance value (P < 0.05) [Table 2]. This result showed that there was significant difference in the effectiveness of the antibacterial effect between these groups. However, there was no significant difference between Groups A (Ca(OH)2 + 2% CHX gel) and C (2% CHX) (P > 0.05).
Table 1: Result of the Kruskall-Wallis test analysis

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Table 2: Result of Mann-Whitney post hoc test analysis

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  Discussion Top

Root canal failure is closely related to the presence of anaerobic, facultative bacteria, especially E. faecalis. The provision of root canal medicament is a significant consideration to help reduce the number of bacteria that remain. Some medicament materials tested in this study have antibacterial power against E. faecalis bacteria, which can be seen in the OD difference, meaning that there was a decrease in the number of E. faecalis bacterial cells after incubation for 72 h.

This study showed results in accordance with the hypothesis that there is a difference in the antibacterial power of root canal treatment with a combination of calcium hydroxide with 2% CHX digluconate gel and 25% propolis. Calcium hydroxide has a deficiency in eliminating microbes from the root canal and requires a long time to achieve its antimicrobial activity, so a mixing agent is needed to increase its effectiveness.[13] Calcium hydroxide combined with propolis forms a calcium salt compound with an active antibacterial compound derived from propolis, namely flavonoids.[14]

Flavonoids can form complex compounds with proteins through hydrogen bonds so that the tertiary structure of the protein is disrupted, and the protein cannot function anymore, so denaturation of proteins and nucleic acids occurs. Protein denaturation causes protein coagulation and interferes with the metabolism and physiological function of bacteria. Disrupted metabolism causes permanent cell damage due to insufficient energy needs and ultimately causes bacterial cell lysis.[11] A straightforward solution characterizing the decrease in the number of E. faecalis colonies after 25% propolis calcium hydroxide paste treatment also proves that the research group, coupled with propolis extract, showed more significant inhibition in inhibiting growth compared to the group without propolis.[14]

The medicament material that had sufficient antibacterial property was 2% CHX digluconate gel, which can inhibit bacterial cell wall synthesis, inhibit bacterial activity in the anaerobic process, and remain effective at killing bacteria for 48–72 h after root canal treatment, lasting up to 12 weeks. According to Gomes et al.,[15] 2% CHX is useful as a root canal disinfection agent due to its antibacterial power obtained by damaging the integrity of bacterial cell membranes and causing changes in the permeability of the cytoplasmic membrane. This can increase the deposition of cytoplasmic proteins, change cellular osmotic balance, and interfere with metabolism, growth, and division of bacterial cells, so that the cell wall of E. faecalis is damaged, lyses, and eventually dies. The antibacterial effect of combination calcium hydroxide with CHX may prove to benefit in the treatment of certain types of persistent infections in primary and particularly in retreatment cases where E. faecalis is the most common isolate.[16]

Calcium hydroxide mixed with 2% CHX digluconate had a lower bacterial population decline than the 25% propolis combination calcium hydroxide group. The antibacterial power found in the combination of calcium hydroxide with 2% CHX digluconate gel occurred due to chemical reactions between the Ca(OH)2 powder and 2% CHX Gel. Ca(OH)2 releases hydroxyl ions, increasing pH, it can damage the bacterial cytoplasmic membrane to cause denaturation of proteins, which will inhibit bacterial DNA replication so that its growth is inhibited.[17] Schafer and Bossman showed that in human dentin, 2% CHX digluconate gel had more significant activity against E. faecalis, followed by CHX digluconate gel combined with calcium hydroxide, than calcium hydroxide used alone; these results are in accordance with this study.[18]

In this study, there were suboptimal results in each medicament group in eliminating root canal microorganisms caused by many factors affecting the road, and the study results due to limitations. One of the limitations in this study lies in the research tool, namely, the tool used to mix the medicament material made it difficult to stir until it was entirely homogeneous, and the unavailability of suitable tools to apply the medicament material according to the consistency of the material.

  Conclusion Top

Based on this research, it can be concluded that the combination of calcium hydroxide with 25% propolis was more effective than the combination of calcium hydroxide with 2% CHX digluconate as an intracanal medicament against E. faecalis bacteria. Future studies are still needed to confirm this result of this study, such as another in vitro test with other endodontic bacteria, along with in vivo cytotoxicity test.


We gratefully thank Molecular Medicine and Therapy Research Laboratory, Faculty of Medicine and Health Science, Universitas Muhammadiyah Yogyakarta.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Nisa UAD, Darjono A. Analysis Of Certain Essential Oil (Cymbopogon Citratus) As An Alternative Root Irrigation Materials Using The Growth Of Enterococcus faecalis. Majalah Ilmiah Sultan Agung 2020;49:59-68.  Back to cited text no. 1
Widiadnyani NK, Mulyawati E, Dayinah HS. The Effect Of Contact time Of Calcium Hydroxide With 2% Chlorhexidine Digluconate, saline water, and 2% HCl Lidocaine As A Sterilizing Material To Dentine pH In One Third Apical Root. J Ked Gigi 2014;5:176-88.  Back to cited text no. 2
Bolla N, Kavuri SR, Tanniru HI, Vemuri S, Shenoy A. Comparative evaluation of antimicrobial efficacy of odontopaste, chlorhexidine and propolis as root canal medicaments against Enterococcus faecalis and Candida albicans. J Int Dent Med Res 2012;5:14.  Back to cited text no. 3
Karale R, Thakore A, Shetty V. An evaluation of antibacterial efficacy of 3% sodium hypochlorite, high-frequency alternating current and 2% chlorhexidine on Enterococcus faecalis: An in vitro study. J Conserv Dent 2011;14:2-5.  Back to cited text no. 4
[PUBMED]  [Full text]  
Cogulu D, Uzel A, Oncag O, Aksoy SC, Eronat C. Detection of Enterococcus faecalis in necrotic teeth root canals by culture and polymerase chain reaction methods. Eur J Dent 2007;1:216-21.  Back to cited text no. 5
Athanassiadis B, Abbott PV, Walsh LJ. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. Aust Dent J 2007;52:S64-82.  Back to cited text no. 6
Cohen S. Cohen's pathways of the pulp. 11th ed. St. Louis, Missouri (MO): Elsevier; 2016. p. 907.  Back to cited text no. 7
Ariani NG, Hadriyanto W, Kristanti Y. The effect of calcium hydroxide sterilizer with a mixture of saline, 2% chlorhexidine digluconate and 2% lidocaine HCl on the micro hardness of dentin in the cervical two-thirds segment of the root canal. J Ked Gigi 2013;5:169-75.  Back to cited text no. 8
Gautam S, Rajkumar B, Landge SP, Dubey S, Nehete P, Boruah LC. Antimicrobial efficacy of metapex (calcium hydroxide with iodoform formulation) at different concentrations against selected microorganisms – An in vitro study. Nepal Med Coll J 2011;13:297-300.  Back to cited text no. 9
Rusdiana Puspa Dewi S, Agung Santoso R, Sujatmiko B, Seto Wibowo I. Antibacterial activity of various calcium hydroxide solvents against Fusobacterium nucleatum and Enterococcus faecalis. J Phys Conf Ser 2019;1246:1-7.  Back to cited text no. 10
Awawdeh L, AL-Beitawi M, Hammad M. Effectiveness of propolis and calcium hydroxide as a short-term intracanal medicament against Enterococcus faecalis: A laboratory study. Aust Endod J 2009;35:52-8.  Back to cited text no. 11
Sabir A. Aktivitas antibakteri flavonoid propolis Trigona sp. terhadap bakteri Streptococcus mutans (in vitro) (in vitro antibacterial activity of flavonoids Trigona sp. propolis against Streptococcus mutans). Dent J (Maj Ked Gigi) 2005;38:135.  Back to cited text no. 12
Walton RE, Torabinejad M. Endodontics: Principles and Practice. 5th ed.. St. Louis, Missouri (MO): Elsevier; 2015. p. 482.  Back to cited text no. 13
Effendy R, Ariesta V, Saraswati W. Antibacterial potency of calcium hydroxide and propolis mixture on mixed bacteria of carious dentin. Conserv Dent J 2019;9:5-12.  Back to cited text no. 14
Gomes BP, Vianna ME, Zaia AA, Almeida JF, Souza-Filho FJ, Ferraz CC. Chlorhexidine in endodontics. Braz Dent J 2013;24:89-102.  Back to cited text no. 15
Sirén EK, Haapasalo MP, Waltimo TM, Ørstavik D. In vitro antibacterial effect of calcium hydroxide combined with chlorhexidine or iodine potassium iodide on Enterococcus faecalis. Eur J Oral Sci 2004;112:326-31.  Back to cited text no. 16
Delgado RJ, Gasparoto TH, Sipert CR, Pinheiro CR, Moraes IG, Garcia RB, et al. Antimicrobial effects of calcium hydroxide and chlorhexidine on Enterococcus faecalis. J Endod 2010;36:1389-93.  Back to cited text no. 17
Schäfer E, Bössmann K. Antimicrobial efficacy of chlorhexidine and two calcium hydroxide formulations against Enterococcus faecalis. J Endod 2005;31:53-6.  Back to cited text no. 18


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]

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