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 Table of Contents  
Year : 2013  |  Volume : 3  |  Issue : 1  |  Page : 5-9

Comparative evaluation of Morinda citrifolia with chlorhexidine as antimicrobial endodontic irrigants and their effect on micro-hardness of root canal dentin: An in vitro study

Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere, Karnataka, India

Date of Web Publication26-Nov-2013

Correspondence Address:
A R Prabhakar
Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere - 577 004, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2231-6027.122086

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Objectives: The current study was intended to compare and to evaluate in vitro the antimicrobial efficacy of Morinda citrifolia juice (MCJ) with chlorhexidine (CHX) as endodontic irrigants and their effect on micro-hardness of root canal dentin. Methodology: The study was divided into two parts. Part I for antibacterial testing consisted of preparing 60 dentin blocks of 4 mm height. All the dentin blocks were infected with Enterococcus fecalis for a period of 21 days. The experimental groups were Group I: 0.2% CHX; Group II: 6% MCJ; Group III: 6% MCJ + 0.2% CHX; Group IV: Saline. After 28 days of medication with the irrigants, the dentin shavings from root canal dentin was harvested and colony forming units counted. Part II for micro-hardness testing consisted of preparing 32 root halves and mounting them on blocks of acrylic resin. 8 samples per group were randomly divided into the experimental groups. The samples were then medicated with the irrigants for a period of 15 min and micro-hardness values were recorded. Results: Data obtained was analyzed using one-way ANOVA, Kruskal-Wallis and Mann-Whitney U-test and Student's paired t-test. Part I: Group I showed highest antibacterial activity followed by Group III with Group II taking the third place and were statistically significant (P < 0.001). Part II: None of the groups showed any effect on micro-hardness of root canal dentin, which was not statistically significant (P > 0.4). Conclusion: Nearly 0.2% of CHX showed the highest antimicrobial activity even after 28 days and 6% of MCJ also showed antibacterial activity, but to a lesser degree than CHX. None of the irrigants tested had any effect on the micro-hardness of root canal dentin.

Keywords: Chlorhexidine, endodontic irrigants, Enterococcus fecalis, Morinda citrifolia juice

How to cite this article:
Prabhakar A R, Basavraj P, Basappa N. Comparative evaluation of Morinda citrifolia with chlorhexidine as antimicrobial endodontic irrigants and their effect on micro-hardness of root canal dentin: An in vitro study. Int J Oral Health Sci 2013;3:5-9

How to cite this URL:
Prabhakar A R, Basavraj P, Basappa N. Comparative evaluation of Morinda citrifolia with chlorhexidine as antimicrobial endodontic irrigants and their effect on micro-hardness of root canal dentin: An in vitro study. Int J Oral Health Sci [serial online] 2013 [cited 2023 Mar 31];3:5-9. Available from: https://www.ijohsjournal.org/text.asp?2013/3/1/5/122086

  Introduction Top

Microorganisms and their by-products are considered to be the major cause of pulp and periradicular pathosis. Hence, a major objective in root canal treatment is to disinfect the entire root canal system, which requires that all contents of the root canal system be eliminated as possible sources of infection. This goal may be accomplished by using mechanical instrumentation and chemical irrigation, with medication of the root canal system between treatment sessions. To reduce or eliminate bacteria, various irrigation solutions have been advocated. [1]

Chlorhexidine (CHX) is a potent antiseptic, which is widely used for chemical plaque control in the oral cavity. Despite its usefulness as a final irrigant, CHX cannot be advocated as the main irrigant in standard endodontic cases, because: (a) CHX is unable to dissolve necrotic tissue remnants and (b) CHX is less effective on Gram-negative than on Gram-positive bacteria. [2]

The constant increase in antibiotic-resistant strains and side-effects caused by synthetic drugs has prompted researchers to look for herbal alternatives. Healing potential in plants is an ancient idea, but in recent times, it has gained renewed interest and importance. The major advantages of using herbal alternatives are easy availability, cost-effectiveness, increased shelf life, low toxicity and lack of microbial resistance reported so far. [3]

This study aims to determine the antibacterial efficacy and the effect on micro-hardness of root dentin by one such herbal compound - Morinda citrifolia juice (MCJ) (6%) in comparison with CHX (0.2%) and their combination.

  Methodology Top

The 30 human permanent, non-carious, single rooted teeth, extracted for therapeutic reasons were selected for this study.

The study was divided into Part I (antibacterial testing) and Part II (micro-hardness assessment).

Part I: Antibacterial testing

All specimens were disinfected with 5% of sodium hypochlorite (NaOCl) for 15 min and then cleaned with a rubber cup and prophylaxis brush and pumice to remove surface debris. The mesio-distal and bucco-lingual diameters of the specimens were determined at the crown limit of the root using a dial caliper. [4]

The crowns were then amputated at their cervical limit perpendicular to the long axis of the teeth with a rotary diamond disc under constant water cooling. A 4 mm mark was made apical to the amputated end of each root specimen with a dial caliper and sectioned with diamond disc under constant water cooling. Another 4 mm mark was made apical to the previous amputation and using a diamond disc to eventually obtain two root cylinders (dentin blocks) of 4 mm height. The internal diameter was standardized using a No. 12 bur in a slow speed handpiece. 60 such dentin blocks were prepared. The outer surface of the specimens was covered with two layers of nail varnish, in order to prevent contact of the medicament with the external surface. [5]

Fresh Tryptone Soya Broth (TSB) was prepared and 2 ml of this broth was poured in 30 pre-sterilized test tubes to which two dentine blocks were added to each test tube. These specimens were then subjected to the second cycle of sterilization by autoclaving (at 121 o C, for 15 min) in TSB to allow optimal penetration of the nutrient broth into the dentinal tubules. The specimens were closed at the bottom with decontaminated sticky wax, which also obliterated the apical surface of the root canal. [6]

Enterococcus fecalis was used as the test organism in this study. Isolated overnight pure culture of E. fecalis (ATCC 29212) suspension was prepared on TSB separately. This suspension was adjusted to match the turbidity equivalent to 0.5 McFarland standards. The test tubes containing sterilized TSB and dentin blocks were infected with 100 μl of this inoculum of E. fecalis. The infected test tubes with dentin blocks were then kept in the incubator at 37 o C for 21 days during which the broth was regularly changed at 2 day intervals.After 21 days, the infected dentin blocks were taken and washed in sterile saline to remove any remnants of the incubation broth. [4]

The 60 dentin blocks were randomly divided into four experimental groups. The experimental groups were then medicated as follows:

Group I (positive control): 0.2% CHX solution
Group II: 6% MCJ (Unicorn Natural Products Pvt. Ltd., Hyderabad)
Group III: Combination of 6% MCJ and 0.2% CHX gluconate solution
Group IV (control): Saline.

At the end of 28 days, sticky wax was removed and subsequently the medication was carefully rinsed from the dentin blocks using sterile saline solution. Dentin shavings from each block were harvested by drilling inside the canal lumen of with a round bur. Dentin shavings were collected in 1 ml of broth. [4] These culture plates were incubated up to 24 h at 37 o C to detect any bacterial growth and the number of bacterial colonies formed was counted at the end of 28 days with the help of Digital Colony Counting Machine (HiMedia Lab Mumbai). The results were recorded, tabulated and statistically analyzed.

Part II: Root dentin micro-hardness assessment

A total of 16 human single rooted premolar teeth extracted for therapeutic purpose were selected. The crowns were removed at the CEJ using a high-speed bur under water-cooling.

Four roots were sectioned vertically with diamond disc to produce 8 root halves from each group, which were embedded in acrylic resin. The specimens were polished with abrasive papers to remove any surface scratches. [6]

The micro-hardness from each group was determined using Vickers hardness tester (Mitotoyo, Japan) at two points, at a distance of 500 μm from canal lumen and their average value was recorded. All the indentations were made with 300 g and dwell time of 10 s. Baseline data was obtained before immersing the specimens in the irrigants. [7] Then the samples of experimental groups (Group I, II, III and IV) were again treated with respective irrigants for 15 min and then tested for any change in micro-hardness.

The experimental groups were then medicated as follows:

Group I (positive control): 0.2% CHX solution
Group II: 6% MCJ
Group III: Combination of 6% MCJ and 0.2% CHX gluconate solution
Group IV (control): Saline.
  Results Top

All results were recorded, tabulated and statistically analyzed. For antibacterial activity, one-way ANOVA, Kruskal-Wallis test and Mann-Whitney U-test for intergroup comparison and group wise comparison were used. For micro-hardness test Student's paired t-test, one-way ANOVA for intergroup comparison was done.

[Table 1] and Graph 1 represent the mean, standard deviation and group wise significance of the bacterial colonies in all four groups at the end of 28 days. Group I showed the highest antibacterial activity against E. fecalis followed by Group III and Group II showed lesser antibacterial activity. Intergroup comparison was also found to be statistically significant (P < 0.01).
Table 1: Descriptive statistics showing the comparison of bacterial colonies at the end of 28 days

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[Table 2] and Graph 2 represent the mean difference and group-wise significance of Vickers hardness number at baseline and after 15 min. None of the irrigants had any effect on the micro-hardness of root dentin. All three groups showed values that were not statistically significant (P = 0.4).
Table 2: Descriptive statistics of inter group mean difference comparison of micro-hardness test among different groups at the end of 15 min

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  Discussion Top

The first steps in every endodontic treatment are diagnosis, decision on treatment and information to the patient. Following these, treatment consists of preparation of access cavity, location of the root canals, instrumentation, irrigation and disinfection, root filling and coronal restoration of the tooth. In endodontic discussions, much of the focus is on instrumentation; however, in addition to removing some of the root canal contents, the main goals of instrumentation are to allow effective disinfection and to make root filling technically possible.

Irrigation has a number of important functions

Mechanical washing effect, reduction of friction between the instrument and canal wall dentin, temperature control, dissolution of organic and inorganic tissue and antimicrobial effect. Traditionally, NaOCl and ethylene diamine tetraacetic acid (EDTA) have been the most commonly used root canal irrigants. NaOCl dissolves organic tissue effectively (less so vital pulp tissue) and kills bacteria and yeasts. EDTA dissolves inorganic tissue by chelation, but it has no antibacterial activity in the root canal. [8] Recent studies indicate that long-term exposure (1 h) to NaOCl can weaken the structural integrity of dentin. It has also been shown that NaOCl has toxic effects on vital tissues, resulting in hemolysis, skin ulceration and necrosis. It has a pH of approximately 11-12 and causes injury primarily by oxidation of proteins. [9]

Another option, with several benefits, would be to use CHX as the final rinse. CHX does not dissolve tissue (no erosion), but it kills microbes. It also improves the quality (i.e. strength) of long-term resin bonding to dentin by inhibiting dentin enzymes (e.g., matrix metalloproteinases), which otherwise degrade the bond over time. However, as CHX has no tissue-dissolving effect, its use as the main or only root canal irrigant cannot be recommended. [8] CHX is a cationic bisguanide that seems to act by adsorption onto the cell wall of the microorganism and causing leakage of intracellular components. At low concentrations, CHX has a bacteriostatic effect, causing the leaching of small molecular weight substances from microorganisms. At higher concentrations, CHX has a bactericidal effect due to cytoplasmic precipitation and/or coagulation, probably caused by protein cross-linking. [10]

The lowest concentration of CHX evaluated in the present study (0.2%) showed an in vitro antimicrobial activity equivalent to that of 5.25% NaOCl, in agreement with which has been reported by Mohammadi and Abbot. [1]

E. faecalis is a persistent organism that, despite making up a small proportion of the flora in untreated canals, plays a major role in the etiology of persistent periradicular lesions after root canal treatment. It is commonly found in a high percentage of root canal failures and it is able to survive in the root canal as a single organism or as a major component of the flora. [11]

In the recent years attention has been diverted toward the search for the new novel compounds from plants, animals and microbes. Due to the increasing trend of multidrug resistance the study has been concentrated on newer antimicrobial compounds of plant origin. A number of plants have been identified with the properties of antimicrobial activity. Research has also been carried out on various aspects of M. citrifolia L. The research on the use of different solvent extracts of M. citrifolia revealed broad spectrum antibacterial and antifungal activity. [12]

Herbal irrigation solutions are generally considered as safe and nontoxic for the host and some have proved to be strong antibacterial materials in vitro. Antibacterial effect of green tea, MCJ, Zataria multiflora Boiss extract in infected canals has been investigated. [13] The use of MCJ as an endodontic irrigant might be of interest to patients and endodontic professionals as a part of the growing trend to seek natural remedies as a part of dental treatment. [13]

The highest antibacterial activity was expressed by Group I where the substantivity of CHX lasted up to 28 days. This is in concurrence with a study done by Carrilho, regarding the substantivity of different concentrations of CHX to human dentin. He concluded that the substantivity was dose dependent and that 0.2% of CHX though low, was still found adherent to dentin wall after 56 days. [14] This could explain the significant antibacterial activity of Group I in our study, even after 28 days.

Group III showed the next highest antibacterial activity with a mean antibacterial activity of 277.93 ± 10.67 (×10 5 CFU). This could be explained because of the action of CHX existing in the mixture. The main drawback of pure extracts is their degradation over a period of time. Preservatives were not added to the MCJ as it could alter its chemical properties and alter its performance. The degradation of the MCJ can be quoted as a reason for the lower antimicrobial activity.

Group II showed a lower percentage of antibacterial activity though it was statistically significant. As mentioned earlier the antibacterial activity of MCJ is attributed to its contents such as alizarin, scopoletin, aucubin and asperuloside. Unfortunately, since it is a natural pure extract the products tend to degrade over time and this could explain the initial antibacterial activity and then a decline in its performance.

Group IV acted as a control. All the other three groups proved better antibacterial products than the control group.

Apart from the beneficial effects, irrigants may exhibit detrimental effects on dentin or on root canal filling materials. Evaluating the effect of irrigants on mechanical properties of dentin is of utmost importance as the understanding of the mechanical properties of dentin is the first step toward predicting the behavior of the dentin/restoration interface. Micro-hardness testing was done using Vickers Hardness tester.

Group I showed a mean of 51.59 at baseline and 53.15 at the end of 15 min irrigation with 0.2% of CHX. This change is micro-hardness is not significant. The result of this study was in contrary to result from the work by Oliveira et al. who found a statistically significant decrease in the micro-hardness of root dentin when 2.0% CHX. [15] Our study is in concurrence with the study done by Hale Ari et al., which showed no decrease in micro-hardness or dentin surface roughness when irrigated with 0.2% CHX for 15 min. [16]

Group II also showed no effect on micro-hardness of root dentin at the end of 15 min. Baseline value being 54.40 and after 15 min of irrigation it was 57.38. This study for the first time has tested the effect of micro-hardness of a plant extract on root dentin. 6% MCJ has been shown to have an antibacterial effect. More research has to be done on this front. Based on its antibacterial activity and its ineffectiveness to alter the micro-hardness, 6% MCJ promises to be a novel irrigant.

Group III showed 58.94 at baseline and 59.14 after 15 min of irrigation. The combination of 0.2% CHX and 6% MCJ also had no effect on the micro-hardness of root dentin.

  Conclusions Top

The following conclusions were drawn from this study:

  1. 0.2% CHX showed the maximum antibacterial activity against E. fecalis used as test organism after 28 days
  2. 6% MCJ showed some antibacterial activity but to a lesser extent than CHX after 28 days
  3. None of the irrigants affected the micro-hardness of root canal dentin.

  References Top

1.Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics. Int Endod J 2009;42:288-302.  Back to cited text no. 1
2.Zehnder M. Root canal irrigants. J Endod 2006;32:389-98.  Back to cited text no. 2
3.Prabhakar J, Senthilkumar M, Priya MS, Mahalakshmi K, Sehgal PK, Sukumaran VG. Evaluation of antimicrobial efficacy of herbal alternatives (Triphala and green tea polyphenols), MTAD, and 5% sodium hypochlorite against Enterococcus faecalis biofilm formed on tooth substrate: An in vitro study. J Endod 2010;36:83-6.  Back to cited text no. 3
4.Haapasalo M, Orstavik D. In vitro infection and disinfection of dentinal tubules. J Dent Res 1987;66:1375-9.  Back to cited text no. 4
5.Cwikla SJ, Bélanger M, Giguère S, Progulske-Fox A, Vertucci FJ. Dentinal tubule disinfection using three calcium hydroxide formulations. J Endod 2005;31:50-2.  Back to cited text no. 5
6.Yoldaþ O, Doðan C, Seydaoðlu G. The effect of two different calcium hydroxide combinations on root dentine microhardness. Int Endod J 2004;37:828-31.  Back to cited text no. 6
7.Slutzky-Goldberg I, Maree M, Liberman R, Heling I. Effect of sodium hypochlorite on dentin microhardness. J Endod 2004;30:880-2.  Back to cited text no. 7
8.Haapasalo M. Can I use chlorhexidine as the only irrigating solution in my endodontic treatments? J Can Dent Assoc 2011;77:b16.  Back to cited text no. 8
9.Hülsmann M, Hahn W. Complications during root canal irrigation - Literature review and case reports. Int Endod J 2000;33:186-93.  Back to cited text no. 9
10.Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC, de Souza-Filho FJ. In vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:79-84.  Back to cited text no. 10
11.Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: Its role in root canal treatment failure and current concepts in retreatment. J Endod 2006;32:93-8.  Back to cited text no. 11
12.Sunder J, Jeyakumar S, Kundu A, Srivastava RC, Kumar De A. Effect of Morinda citrifolia extracts on in-vitro growth of Ralstonia solanacearum. Arch Appl Sci Res 2011;3:394-402.  Back to cited text no. 12
13.Samadi N, Zaree R, Bakhtiar H, Salehnia A, Azimi S. Comparative antibacterial efficacy of endemic satureja khuzistanica jamzad essential oil, sodium hypochlorite and chlorhexidine gluconate solutions as root canal irrigations. Dent Res J (Isfahan) 2011;8:28-32.  Back to cited text no. 13
14.Carrilho MR, Carvalho RM, Sousa EN, Nicolau J, Breschi L, Mazzoni A, et al. Substantivity of chlorhexidine to human dentin. Dent Mater 2010;26:779-85.  Back to cited text no. 14
15.Oliveira LD, Carvalho CA, Nunes W, Valera MC, Camargo CH. Effects of chlorhexidine and sodium hypochlorite on the microhardness of root canal dentin. OOOE 2007;104:125-12.   Back to cited text no. 15
16.Ari H, Erdemir A, Belli S. Evaluation of the effect of endodontic irrigation solutions on the micro hardness and the roughness of root canal dentin. J Endod 2004;30:792-5.  Back to cited text no. 16


  [Table 1], [Table 2]

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