|Year : 2016 | Volume
| Issue : 2 | Page : 165-171
A comparative evaluation of antibacterial effectiveness of sodium hypochlorite, Curcuma longa, and Camellia sinensis as irrigating solutions on isolated anaerobic bacteria from infected primary teeth
Neha Shashikant Dhariwal1, Shivayogi M Hugar1, Sheetal Harakuni2, Suma Sogi3, Harsha G Assudani1, Laresh Naresh Mistry4
1 Department of Pedodontics and Preventive Dentistry, KLE VK Institute of Dental Sciences, KLE University, Belagavi, Karnataka, India
2 Department of Microbiology, J. N. Medical College, KLE University, Belagavi, Karnataka, India
3 Department of Pedodontics and Preventive Dentistry, MM College of Dental Sciences and Research, MM University, Mullana, Ambala, Haryana, India
4 Department of Pedodontics and Preventive Dentistry, MGM College of Dentistry, Mumbai, Maharashtra, India
|Date of Web Publication||14-Apr-2016|
Dr. Neha Shashikant Dhariwal
Department of Pedodontics and Preventive Dentistry, KLE VK Institute of Dental Sciences, KLE University, Belagavi, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: In endodontics, most of the commercial intra-canal medicaments have cytotoxic reactions and because of their inability to eliminate bacteria from dentinal tubules, recent medicine has turned its attention to the usage of biologic medication prepared from natural plants. The literature to testify the efficacy of natural alternatives in primary teeth is meagre and its effects as irrigating solutions need to be evaluated. Aim: To evaluate the antibacterial effectiveness of sodium hypochlorite, ethanolic extracts of Curcuma longa (turmeric) and Camellia sinensis (green tea) as irrigating solutions against the anaerobic bacteria isolated from the root canals of infected primary teeth. Materials and Methods: Thirty patients were selected based on the selected inclusion and exclusion criteria. Preoperative radiographs were taken. Rubber dam isolation and working length estimation were done, following which thirty samples were taken from the root canals of infected primary teeth using sterile absorbent paper points and transferred to tubes containing thioglycolate transport medium. The bacteria were then isolated using standard microbiological protocols and were subjected to antibiotic sensitivity testing using the three test irrigants. Statistical Analysis: SPSS 18 software using Chi-square test was used for statistical analysis. Results: The most commonly isolated bacteria included Porphyromonas sp., Bacteroides fragilis, Peptostreptococcus, and Staphylococcus aureus. Sodium hypochlorite and C. longa (turmeric) showed good antibacterial effect and were effective against most of the isolated bacteria. There was statistically significant difference in the antibacterial effect among the three tested groups (P < 0.001). The least effective was C. sinensis (green tea). Conclusion: The infected primary teeth almost always present with a polymicrobial structure with a wide variety of anaerobic bacteria. The chemo-mechanical preparation plays an important role in eradicating the population of predominant micro-organisms in treating these teeth with promising effects with the use of newer test irrigants while avoiding the side effects of sodium hypochlorite.
Keywords: Anaerobic, bacteria, Camellia sinensis, Curcuma longa, irrigation, sodium hypochlorite
|How to cite this article:|
Dhariwal NS, Hugar SM, Harakuni S, Sogi S, Assudani HG, Mistry LN. A comparative evaluation of antibacterial effectiveness of sodium hypochlorite, Curcuma longa, and Camellia sinensis as irrigating solutions on isolated anaerobic bacteria from infected primary teeth. J Indian Soc Pedod Prev Dent 2016;34:165-71
|How to cite this URL:|
Dhariwal NS, Hugar SM, Harakuni S, Sogi S, Assudani HG, Mistry LN. A comparative evaluation of antibacterial effectiveness of sodium hypochlorite, Curcuma longa, and Camellia sinensis as irrigating solutions on isolated anaerobic bacteria from infected primary teeth. J Indian Soc Pedod Prev Dent [serial online] 2016 [cited 2019 Nov 15];34:165-71. Available from: http://www.jisppd.com/text.asp?2016/34/2/165/180447
| Introduction|| |
The unique histological characteristics of the primary teeth and its interactions with the oral cavity present an interesting challenge to treat its diseases and associated conditions, unlike the permanent teeth. The abundance of accessory and lateral canals, easy communication of the pulp with the furcal region due to highly porous inter-radicular dentin at the pulpal floor, ample of medullary bone spaces, and its association with the delicate developing permanent tooth bud are the critical areas in determining a successful pulpal treatment, especially in cases with necrotic pulps, teeth with abscesses, and/or with draining sinus tracts. ,
Belanger  mentioned that an intact primary tooth gets adequately disinfected and a restored natural tooth acts as a best space maintainer than any artificial appliance. Due to these reasons, much importance is given to chemical cleaning and disinfection of canals as opposed to thorough mechanical shaping of the canals. The pathway of progression of the infection and the ecology of infecting micro-organisms are different in the deciduous dentition as compared to the permanent dentition.  Hence, for complete elimination of infection, irrigation is an important step for the success of pulpectomy. 
In endodontics, most of the commercial intra-canal medicaments such as sodium hypochlorite and hydrogen peroxide cause cytotoxic reactions, have side effects, unpleasant taste, foul smell, allergic potential, and because of their inability to eliminate bacteria from dentinal tubules, recent medicine has turned its attention to the usage of biologic medication prepared from natural plants.  Hence, an impending need is upon the fraternity of dentistry to find alternatives to which it can best adapt to primary tooth physiology with maximum clinical benefit and minimum unwanted side effects.
The literature to testify the efficacy of various natural alternatives has been obtained for the permanent dentition. However, their use in primary teeth and its effects as irrigating solutions needs to be further evaluated. Hence, in this study, an attempt has been made to compare the antibacterial effect of turmeric and green tea extracts against the anaerobic bacteria isolated from infected primary teeth.
| Materials and Methods|| |
The following materials were used in the study:
Twenty percent ethanolic extract of Curcuma longa, 20% ethanolic extract of Camellia sinensis, 3% sodium hypochlorite, micro-organisms isolated from the root canals of the infected primary teeth, Whatman No. 1 filter paper sensitivity discs, thioglycolate transport media tubes (2 ml Borosil), 5% sheep blood agar, anaerobic jar, anaerobic catalyst, vacuum suction, platinum loop, etc.
The study was conducted in the Department of Pediatric and Preventive Dentistry. Ethical clearance for the study was obtained from the Ethical Committee of the institution prior to the start of the study. Thirty samples were taken from the root canals of infected primary teeth. Parental consent and child's assent were obtained after explaining them the entire procedure before taking the samples. The cases were selected based on standard inclusion and exclusion criteria. 
- Patient aged 3-10 years.
- Primary teeth with necrotic pulp, chronic abscess, and/or sinus tract.
- Compliant patients.
- Teeth with more than two-third loss of root structure/evidence of root resorption and/or mobility.
- Teeth with antibiotic usage for systemic diseases in past 4 weeks.
- Patient with systemic diseases.
- Unco-operative patients.
- Patients who were not willing to accept proposed treatment plan and/or participate in the study.
Preparation of ethanolic extract of Curcuma longa
Turmeric (C. longa) rhizomes were obtained and cut into irregular pieces and dried in an oven at a temperature of 45°C ± 5°C for a period of 24 h till completely moisture-free and then were ground to form a fine powder. Powdered spices were soaked in 200 ml of 95% ethanol and kept at room temperature, macerated for 7 days, and were filtered. The filtrate was heated at 40-50°C using water baths till the ethanol evaporated and until thick paste was formed. The thick paste was considered as 100% concentration of extract [Figure 1]. These extracts were stored at 4°C in refrigerator.
|Figure 1: Ethanolic extract of Curcuma longa (turmeric) and Camellia sinensis (Green tea)|
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Preparation of ethanolic extract of Camellia sinensis
Dried green tea leaves were obtained and ground into powder form. About 20 mg/ml concentration of ethanolic extract was obtained by boiling the leaves in 95% ethanol for 40-50 min following the similar procedure as mentioned above [Figure 1].
After administering local anesthesia using 2% lignocaine with 1:80,000 adrenaline, rubber dam isolation was done. A standard access cavity preparation was made and working length estimation was done. Three absorbent points were taken into the canal till the apex per canal [Figure 2] and were held there till 30 s, each one following another. They were then removed from the canals and transferred directly into a tube containing thioglycolate broth.  This tube was processed within 30 min of sample collection according to the standard microbiological protocol where growth was checked every 24 h [Figure 3]. Strains were identified based on Gram staining and classified by colony morphology and oxygen tolerance.
|Figure 3: Primary growth obtained after 48 h of incubation in anaerobic jar|
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All isolated microbial strains were subjected to antibiotic sensitivity testing [Figure 4] using disc diffusion method on agar plates. The isolated micro-organisms were then tested against the three irrigants used in the study [Figure 5]. The sensitivity or resistance of the isolated bacteria was determined.
|Figure 5: Determination of sensitivity of isolated bacteria against test chemicals|
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| Results|| |
There were ten different strains of bacteria isolated from the samples. These micro-organisms were divided into four different groups based on whether Gram-positive or Gram-negative, facultative or obligatory anaerobes, cocci or Bacilli.
Each isolated micro-organism was tested against all the three irrigants that were freshly dispensed. It was observed that sodium hypochlorite and C. longa (turmeric) showed 84.6% sensitivity whereas C. sinensis (green tea) was resistant in 82.05% cases [Table 1]. The observation of [Table 1] shows that there is statistically significant difference in the three groups (P < 0.001) using the Chi-square test. The intergroup comparisons done using Chi-square tests state that there is statistically significant difference between NaOCl and green tea, turmeric and green tea, but there was no significant difference between NaOCl and turmeric groups [Table 2], [Table 3], [Table 4].
|Table 1: Overall comparison of all three irrigants using Chi-square test |
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|Table 2: Comparison between sodium hypochlorite and turmeric irrigants using Chi-square test |
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|Table 3: Comparison between sodium hypochlorite and green tea irrigants using Chi-square test |
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|Table 4: Comparison between turmeric and green tea irrigants using Chi-square test |
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The most sensitive micro-organisms were the obligatory anaerobes such as Bacteroides fragilis, Porphyromonas gingivalis, and Prevotella intermedia, which were most commonly isolated. In addition, the facultative anaerobes such as Peptostreptoccci, Enterococcus faecalis, and Streptococci groups showed good antibacterial susceptibility against NaOCl and turmeric [Table 5].
|Table 5: The antimicrobial susceptibility of the individual isolated anaerobic micro-organisms against sodium hypochlorite, turmeric, and green tea |
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The Graphs 1 and 2 show the overall effect of the cocci and Bacilli against the test irrigants. Sodium hypochlorite and turmeric are equally effective while green tea showed resistance in maximum cases.
| Discussion|| |
Primary teeth serve an important function in the growth and development of a child because they prepare the child for assimilation of food. They also have an important role in the maintenance of space for the permanent teeth along with the stimulation of jaw growth.  Dental caries is one of the most common reasons for the early loss of tooth. In this process, the oral microflora plays a very crucial role. The colonization of the bacteria along with the other contributing factors is responsible for the progression of caries leading to pulpal inflammation, infection, swelling, abscesses, etc. For this reason, the identification of the oral microbiota found in the primary teeth is important.  If the collateral circulation is inadequate, in the necrotic pulpal area, the usual clearing process and the immune mechanisms of the body get hampered thus healing does not take place. The transformation of the root canal into a "refuge" for micro-organisms and their by-products occurs, along with the degeneration products of micro-organisms and the pulpal tissues. 
In the present study, it was observed that the most predominant organisms isolated from the infected, abscessed primary teeth were the black-pigmented colonies of obligatory anaerobic Bacilli such as P. intermedia, Porphyromonas species, Bacteroides species, and Fusobacterium species followed by the obligatory Gram-positive cocci Peptostreptococcus species. Facultative Gram-positive anaerobic cocci such as Streptococcus mutans, Streptococcus pyogenes, E. faecalis, and Staphylococcus aureus were also found but were comparatively less in number. Various studies have stated that in pulpitis and dentoalveolar abscesses, predominant organisms found were in accordance with this study. , In human deciduous root canals with necrotic pulp and periapical lesions, the infection is polymicrobial with a large number of micro-organisms and a predominance of streptococci and black-pigmented Bacilli.  The results obtained by Cogulu et al. were contrasting to our study where E. faecalis and Treponema denticola were isolated only in a limited number of cases. 
The method used for antibacterial susceptibility testing was the agar disk diffusion method. It is one of the most convenient, cost-effective, and practical methods, which is well standardized. 
The antimicrobial effectiveness of sodium hypochlorite has similarity to the mechanism of action of calcium hydroxide based on its high pH (hydroxyl ions action). The high pH causes an irreversible enzymatic inhibition by interfering with the cytoplasmic membrane integrity, biosynthetic alterations in cellular metabolism, and phospholipid degradation in lipidic peroxidation. The amino acid chloramination reaction forming chloramines interferes with cellular metabolism and oxidation that promotes irreversible bacterial enzymatic inhibition thus replacing hydrogen with chlorine. Thus, sodium hypochlorite presents antimicrobial activity with its action on essential enzymatic sites promoting irreversible inactivation originated by hydroxyl ions and chloramination action. The saponification reaction verifies the ability of sodium hypochlorite to dissolve organic tissue when sodium hypochlorite degrades fatty acids and lipids resulting in soap and glycerol formation. 
Sodium hypochlorite preparations are virucidal and sporicidal and show a far greater tissue dissolving effects on necrotic than on vital tissues.  Other than the effect on various tissues and allergic reactions caused, complications occur if it extrudes the root canal apex. A severe acute inflammatory reaction of the tissues can occur with a rapid tissue swelling both intraorally and extraorally extending to the skin and subcutaneous tissues. Edematous, hemorrhagic, or both kinds of swelling can occur and can extend in the region beyond than that expected with an acutely infected tooth. 
Exactly, the mechanism of NaOCl to destroy micro-organisms has never been demonstrated experimentally. The disinfecting efficiency of NaOCl depends on the concentration of undissociated hypochlorous acid (HOCl) in the solution. As essential enzymes are inhibited, important metabolic reactions are disrupted, resulting in the killing of the bacterial cells. However, some resistant strains of micro-organisms such as E. faecalis are resistant to NaOCl, especially at low concentrations.  For instance, 0.5% NaOCl took 30 min to destroy bacterial cells while the use of NaOCl at high concentrations is undesirable because it is an irritant to periapical tissues even though its antimicrobial action is proportional to its concentration.  Three percent sodium hypochlorite showed good antibacterial sensitivity against wide array of micro-organisms isolated in the present study [Table 1].
The search for alternatives to NaOCl has been evident since many years owing to its effects as stated earlier. Materials used on day-to-day basis for routine processes have been tested. One such area of interest is the herbal products which have been in the market for a wide range of products. Curcumin is the primary yellow bioactive component of turmeric and has a wide spectrum of actions such as anti-inflammatory, anti-oxidant, antibacterial, antifungal, antiprotozoal, and antiviral activities.  Curcumin, a polyphenolic compound, strongly inhibits bacterial cell proliferation by inhibiting the assembly dynamics of filamenting temperature-sensitive mutant-Z (FtsZ) in the Z-ring needed for bacterial cell division. It also increases the guanosine tri-phosphatase (GTPase) activity of FtsZ. The perturbation of the GTPase activity of FtsZ assembly is lethal to bacteria. Curcumin has a potent antibacterial activity against a number of pathogenic bacteria. 
Except for a few Gram-negative Bacilli obligate anaerobes and Gram-positive cocci facultative anaerobes, turmeric showed a wide spectrum of antibacterial effect against the organisms most commonly seen in the infected primary teeth such as Porphyromonas species, P. intermedia, Peptostreptococci, and E. faecalis that were isolated in this study.
The active ingredient curcumin does not lose its active potential when extracted in the ethanolic solvent. Various authors in their studies observed that compared to the aqueous extract, ethanolic extract showed better efficacy as the active component is not soluble in water and stated that ethanol is a good solvent for turmeric. , The present study also concluded turmeric to have a good antibacterial agent [Table 1] and was equally effective as NaOCl.
C. sinensis (green tea) is a commonly used beverage that can lower the risk of cardiovascular problems, periodontal disease, and has a good antibacterial effect. These effects are a result of large amount of polyphenols present within it such as the catechin, epicatechin, and epigallocatechin gallate (EGCG). The EGCG is the main component which acts as an anti-oxidant and scavenger of free radicals. The antibacterial activity is by disrupting the cell membrane and prevention of DNA supercoil formation, thus destroying the bacteria. Various studies have stated that polyphenols can inhibit or kill pathogens such as S. aureus, Streptococcus sobrinus, S. mutans, and P. gingivalis. 
Various studies have stated its anticaries effect, its effect against periodontal pathogens such as P. gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella species, resistant strains of S. aureus and Pseudomonas aeruginosa in nosocomial infections that can replace conventional antibiotics, but its effect against the endodontic microflora is less explored. ,
In the present study, only few strains of Gram-positive cocci facultative anaerobe and Gram-positive cocci obligatory anaerobe showed some antibacterial effect. The results of the present study were in accordance with other studies.  The results of the present study showed good but not 100% sensitivity of green tea against S. mutans, S. pyogenes, S. aureus, etc. Completely contrasting results (zero percentage sensitivity) were found in case of Porphyromonas sp., P. intermedia, B. fragilis, E. faecalis, etc., organisms isolated in the present study.
One remarkable finding is the high proportion of black-pigmented colonies of bacteria isolated in the study which correlates with many studies so far. The results of the present study can be justified by a larger sample size and use of some other antibacterial tests before its clinical application in dentistry.
| Conclusion|| |
In endodontic therapy, the main aim of treatment, especially in the primary teeth is eradication of micro-organisms. Hence, the antibacterial efficacy of the irrigants is an important consideration. From the present study, we conclude that:
- The micro-organisms prevalent in primary teeth with necrosis, abscess, and/or sinus tract include Porphyromonas species, P. intermedia, B. fragilis Peptostreptococcus, Fusobacterium, as obligate anaerobes. Streptococcus pyogenes, S. mutans, E. faecalis, and S. aureus, as facultative anaerobes.
- The antimicrobial sensitivity testing done with agar diffusion method on isolated micro-organisms showed sodium hypochlorite and C. longa (turmeric) to be equally effective whereas C. sinensis (green tea) had least antibacterial activity against the isolated microbes from primary teeth.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]