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ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 2  |  Page : 191-197
 

A clinical trial comparing antimicrobial efficacy of “essential oil of Ocimum sanctum” with triple antibiotic paste as an intracanal medicament in primary molars


1 Department of Pedodontics and Preventive Dentistry, RKDF Dental College and Research Centre, Bhopal, Madhya Pradesh, India
2 Department of Pediatric and Preventive Dentistry, Faculty of Dental Sciences, Krishna Institute of Medical Sciences, Hyderabad, Telangana, India
3 Department of Pediatric and Orthodontic Sciences, College of Dentistry, King Khalid University, Abha, Saudi Arabia
4 Department of Pedodontics and Preventive Dentistry, Rishiraj College of Dental Sciences, Bhopal, Madhya Pradesh, India

Date of Web Publication2-Jul-2018

Correspondence Address:
Pratibha Ahirwar
F-6 Surendra Gardens, Hoshangabad Road, Bhopal, Madhya Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_237_17

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   Abstract 


Objectives: To evaluate the aerobic and anaerobic antimicrobial efficacy of Ocimum Sanctum (Tulsi) essential oil and compare it with that of triple antibiotic paste (TAP) by collecting microbiological samples from the root canals of primary molars. Study Design: Forty children were selected for the study and were randomly divided into two groups of twenty each, namely, TAP group and O. sanctum group (basil). Six intracanal samples were collected for every patient, comprising of two each after access opening, irrigation and after 3 days of intracanal medicament placement. These samples were cultured in aerobic and anaerobic environment and later colony-forming units (CFUs) were counted and intragroup as well as intergroup comparison was done. Results: Analysis of the results showed that there was a statistically significant reduction in CFUs after using essential oil of O. sanctum as an intracanal medicament. Saline use also leads to a statistically significant reduction in CFUs irrespective of the intracanal medicament used. TAP showed better antibiotic properties in comparison with that of O. sanctum. Conclusion: Antibiotic use is often associated with the adverse effects and development of resistance due to injudicious use. O. sanctum can be used in cases of long-standing infection owing to its antimicrobial efficacy and anti-inflammatory potential as an intracanal medicament in primary teeth.


Keywords: Aerobic, anaerobic, basil, herbal, intracanal medicament, Ocimum sanctum, pulpectomy, triple antibiotic paste, Tulsi


How to cite this article:
Ahirwar P, Shashikiran N D, Sundarraj RK, Singhla S, Thakur RA, Maran S. A clinical trial comparing antimicrobial efficacy of “essential oil of Ocimum sanctum” with triple antibiotic paste as an intracanal medicament in primary molars. J Indian Soc Pedod Prev Dent 2018;36:191-7

How to cite this URL:
Ahirwar P, Shashikiran N D, Sundarraj RK, Singhla S, Thakur RA, Maran S. A clinical trial comparing antimicrobial efficacy of “essential oil of Ocimum sanctum” with triple antibiotic paste as an intracanal medicament in primary molars. J Indian Soc Pedod Prev Dent [serial online] 2018 [cited 2019 Oct 21];36:191-7. Available from: http://www.jisppd.com/text.asp?2018/36/2/191/235684





   Introduction Top


Dental caries is a widespread disease, and higher pulp horns and thinner dentin and enamel in children result in faster involvement of pulp due to caries progression. Once involved, the infected pulp cannot be retained in the tooth and pulpectomy is the most intended treatment plan.[1] However, due to torturous and ribbon-shaped anatomy of the root canals in primary teeth, complete disinfection is not possible.[2]

The flora of the infected root canal mainly comprises predominantly of Gram-positive bacteria, polymicrobial infections, and obligate anaerobes.[3]

Unfortunately, organic and inorganic residues and bacteria cannot be completely removed by the mechanical preparation alone and often persist even after biomechanical preparation.[4],[5] Only reduction of colony-forming units (CFUs) is possible with antimicrobial solutions;[6] therefore, a dressing with chemical medicaments to maintain the sterility of root canal after mechanical preparation and before obturation is important.[7]

Various intracanal medicaments have been suggested which included but were not limited to essential oils, phenolic compounds (formaldehyde, paraformaldehyde, and glutaraldehyde), halogens (iodine), antibiotics (penicillin, metronidazole, cephalosporins, and combinations of various antibiotics), and calcium hydroxide.[5] In spite of the presence of many intracanal medicaments, newer materials are needed. Calcium hydroxide, one of the frequently used medicaments, has a long duration of action and multiple recall visits and formocresol has a cytotoxic and mutagenic effect limiting its use.[8],[9],[10],[11] To overcome this, recent medicaments including triple antibiotic paste (TAP) and herbal extracts are being advocated which can pave the way for better and safer treatment modalities.

India has a rich history of using plants for medicinal purposes and essential oils, and plant extracts have been used for the treatment of various injuries and inflammatory conditions for ages. Tulsi/Basil (Ocimum sanctum), a sacred plant in India, is grown throughout the country and several medicinal properties have long been attributed to it.[12] In one of the pioneering studies regarding O. sanctum, O. sanctum has shown specific aromatic odor because of the presence of the volatile oil which mainly contains phenols, terpenes, and aldehydes. O. sanctum has one-tenth antitubercular potency of streptomycin and one-fourth that of isoniazid.[13] Oil of O. sanctum also showed inhibitory effects on various microorganisms such as Bacillus subtilis, drug-resistant strains of Staphylococcus aureus,  Salmonella More Details paratyphi, Shigella dysenteriae, Candida albicans, Mycobacterium tuberculosis, and Micrococcus pyogenes in a number of studies.[14],[15],[16]O. sanctum also showed inhibitory effects on Bacillus megaterium,  Escherichia More Details coli, pseudomonas spp. S. aureus, Streptococcus albus, methicillin-resistant S. aureus, and Vibrio cholerae. Tulsi extracts also showed almost 100% inhibition of growth of E. coli.[17]

TAP substantially decreases the bacterial count but cannot eradicate it completely; however, in a combination, it is sufficiently potent to eradicate the infection from the root canals.[18] A combination of three antibiotics, namely, metronidazole, ciprofloxacin and minocycline, has also been found to be very effective in sterilizing the carious lesions, necrotic pulps, and infected root dentin of deciduous teeth.[19]

Intracanal medicaments that are already available are associated with irritation potential and inflammation of the periapical tissue on use;[4] therefore, validating the need for this study, where we aim to use and compare the antimicrobial efficiency of the TAP with that of essential oil of O. sanctum for disinfecting the root canal, alleviates the pain and induces repair periapically.


   Materials and Methods Top


The study was conducted in the Department of Pedodontics and Preventive Dentistry, People's College of Dental sciences and Research Center, Bhopal, Madhya Pradesh, in collaboration with the Microbiology Department of People's College of Medical Sciences and Research Center, Bhopal, after approval from the Ethics Committee of People's College of Dental Sciences and Research Center, Bhopal, and People's College of Medical sciences, Bhopal. Written consent was taken from the parents of the children appearing in the study according to the “WHO informed consent form prototype for clinical trial.” Furthermore, informed assent form for minors/children was obtained according to the Declaration of Helsinki.

Study design

The study was a longitudinal clinical trial.

Source of data

Patients reporting to the Department of Pedodontics, People's College of Dental Sciences and Research Center, with the following inclusion and exclusion criteria were selected for the study.

Inclusion criteria

  • Patients of 4–9 years of age
  • Teeth with evident clinical signs of furcal abscess/periapical abscess/draining sinus
  • Periapical changes visible in the radiograph
  • Strategically important tooth
  • At least 2/3rd of root length available.


Exclusion criteria

  • Patients with any other severe systemic diseases
  • Patient on long-term antibiotic therapy, steroid therapy, antiepileptic therapy, etc.,
  • Patients with a history of allergies
  • Any gross oral pathologies
  • Grossly decayed crown


Steps in sample collection

All forty patients participated in the study were divided into the following two groups randomly:

  • O. sanctum Group
  • TAP group.


Twenty patients were included in each group.

For each patient, six samples were collected, of which four were collected in the 1st appointment and two in the 2nd appointment [Figure 1].
Figure 1: Collection of samples on two consecutive appointments in a patient for aerobic and anaerobic culture

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Sample collection

Microbiological samples were obtained from the root canal at the different steps of pulpectomy. After rubber dam application, access opening was done and sterile absorbent paper points of size 20–25 were introduced into the distal root canal (for mandibular molar)/palatal root canal (for maxillary molar) and samples were collected from the apical third of the roots. Thereafter, the paper points were stored in a transport medium, i.e., in brain–heart infusion broth for aerobic and sodium thioglycollate broth for anaerobic culture.

The rubber dam was again disinfected and selected root canals were irrigated with saline irrigation. Only 5 ml of saline solution was used for irrigation. All canals were dried using sterile paper point. Two samples again were obtained in a similar manner and stored in a respective transport medium. One drop of essential oil of O. sanctum Linn., was used as a medicament by placing it directly in root canal. TAP is a combination of three antibiotics metronidazole, ciprofloxacin, and minocycline, pulverized, and mixed in a ratio of 1:1:1. TAP was used in the paste form mixed with propylene glycol for coating the walls of root canal.

The samples were then transferred for microbiological culture.

Three days later, on the 2nd appointment, the tooth was isolated, temporary dressing was removed, and microbiological samples from the apical third of the canals were collected using sterile paper points for aerobic and anaerobic culture.

Microbiological culture

Samples collected using sterile paper points were stored in thioglycollate broth (anaerobic) and brain–heart infusion broth (aerobic) and incubated for 24 h. Sheep blood agar plates were used for microbiological culture for aerobic as well as anaerobic culture as it allows detection of hemolysis. The anaerobic samples were streaked and then placed in an anaerobic jar. The samples from brain–heart infusion broth were streaked and incubated under aerobic condition. Both plates were incubated at 37°C for 48 h. The plates were then examined and the numbers of CFUs were counted [Figure 2] and [Figure 3].
Figure 2: Sheep blood agar plates with aerobic and anaerobic culture of samples collected after access opening (TAP 1), after saline irrigation (TAP 2), and 3 days after triple antibiotic paste placement within root canals (TAP 3)

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Figure 3: Sheep blood agar plates with aerobic and anaerobic culture of samples collected after access opening (B1), after saline irrigation (B2), and 3 days after Ocimum sanctum essential oil placement within root canals (B3)

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Statistical analysis

One-way ANOVA was used for level of significance in reduction of CFU and Tukey's post hoc test was used in case of significant P value from ANOVA test for group-wise comparison [Table 1], [Table 2], [Table 3], [Table 4].
Table 1: Level of significance in reduction of aerobic and anaerobic colony-forming units on using triple antibiotic paste and difference of averages for Tukey's post hoc test

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Table 2: Level of significance in reduction of aerobic and anaerobic colony-forming units on using Ocimum sanctum Linn. and difference of averages for Tukey's post hoc test

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Table 3: Intergroup comparison of proportion of reduction in colony-forming units by both the medicaments

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Table 4: Proportion of reduction in aerobic and anaerobic colony-forming units after complete therapy, i.e., after saline irrigation and medicament placement

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Paired t-test was used to compare reduction of CFUs in aerobic cultures compared to anaerobic cultures [Table 5].
Table 5: Comparison of reduction in colony-forming units in aerobic cultures versus anaerobic cultures for both medicament groups

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   Results Top


The mean value of CFUs in both aerobic and anaerobic cultures before treatment, after saline irrigation, and after medicament placement in TAP group and O. sanctum essential oil (BASIL group) shows that maximum reduction was seen in anaerobic culture after TAP placement [Chart 1].



A significant difference is seen in reduction of aerobic and anaerobic colony count on using TAP alone. The combined effect of therapy that is saline irrigation as well as intracanal medicament shows a highly significant reduction in CFUs (P = 1.79 × 10–27* for aerobic culture and P = 4.56 × 10–18* for anaerobic culture) [Table 1].

A honestly significant difference (HSD) is seen in reduction of aerobic and anaerobic colony count, with difference of averages being 3142.15 and 3031.45 on using O. sanctum essential oil alone. The combined effect of therapy that is saline irrigation as well as intracanal medicament shows a statistically significant difference in reduction of colony count [Table 2].

The Tukey's HSD value for difference of averages in reduction of CFUs between TAP group and OS group was more than Tukey's HSD criteria. Therefore, the number of CFUs reduced because of TAP was significantly more than those reduced due to O. sanctum essential oil alone [Table 3].

Saline and TAP also showed a statistically significant difference in antibacterial efficacy than saline and O. sanctum essential oil with average difference of proportion equal to 0.273596 and 0.248405 for aerobic and anaerobic cultures, respectively, and this difference was statistically significant [Table 4].

No significant difference was seen in reduction of aerobic CFUs versus anaerobic CFUs for both the medicament groups [Table 5].


   Discussion Top


The TAP contains ciprofloxacin and minocycline which act against aerobic bacteria and metronidazole against facultative anaerobes which are the main inhabitants of the root canal.[20] According to previous studies, combination of ciprofloxacin and metronidazole can result in 99% reduction of bacteria and 100% asepsis is seen on using minocycline or fosfomycin.[19] Propylene glycol was used as a vehicle for maximum penetration within the dentinal tubules due to its low-surface tension.[21]

The medicinal properties of essential oil of O. sanctum are mainly due to its constituents such as glycosides, phenols such as eugenol, carvacrol, methyl-eugenol, and tannins.[12],[22] The anti-inflammatory potential of O. sanctum oil is well stated in various in vitro and animal studies.[22] Along with anti-inflammatory action, the medicament showed a higher biocompatibility and no cytotoxic effects.[23] Its mode of action is similar to formocresol, that is, aromatic fumes of essential oil of O. sanctum had bactericidal action.[15],[24]

Using above-mentioned medicaments as an intracanal medicament is a relatively newer modality. Microbiological culture was done using CFUs at three stages in accordance to the studies by William et al.[25] and Sato et al.[19]

In our study, the mean baseline values of CFUs for TAP and O. sanctum were 4783.55 and 4703.3 and 5290.9 and 4523.5 for aerobic and anaerobic cultures, respectively. These values are similar to various other studies where the mean value of CFUs on access opening ranges from 103 to 108 CFUs.

In the present study, only saline was used as an irrigant and the bacterial load reduction was seen for aerobic as well as anaerobic bacteria. It is already established from previous studies that saline does not help in asepsis of canals and main objective of irrigation is tissue dissolution, tissue biocompatibility, flushing action, and high cleaning efficiency.[26],[27]

Within the groups, it was seen that the highest number of CFUs was present immediately after access opening which were reduced after saline irrigation. This reduction in CFU due to saline irrigation was highly significant in both the treatment groups, namely, TAP and O. sanctum for aerobic and anaerobic cultures. The reduction in CFUs was in accordance with the study by William et al. In TAP group, on comparing CFUs after saline irrigation and medicament placement, there was a reduction but not as statistically significant as reduction after medicament placement. Similar results were reported by Harrison,[28] Hori et al.,[29] Sato et al.,[19] and Willam et al.[25] who advocated that irrigation alone cannot suffice for absolute disinfection of root canal and medicaments play a more vital role in asepsis of root canals than merely using root canal irrigants. Combined effect of therapy showed a statistically significant difference between baseline and postmedicament CFU count. When aerobic CFUs were compared with anaerobic culture, no significant difference was present which is mostly due to broad-spectrum action of TAP and thereby equal efficiency against aerobic as well as anaerobic microorganisms.[18],[19] Complete disinfection of root canals by TAP was seen in our study and similar results were observed by Hori et al.[29]

With O. sanctum, a significant reduction was seen in aerobic and anaerobic CFUs. Ahmad and Beg,[12] Prabuseenivasan et al.,[16] and Dharmagadda et al.[17] reported its antibacterial action in various in vitro studies against various aerobic and anaerobic bacteria including S. aureus, Bacillus pumilus, pseudomonas aeruginosa, Klebsiella, and E. coli. However, in vivo studies regarding bacterial growth inhibition by O. sanctum are yet not documented in literature. On comparing the combined effect of therapy, greater reduction of CFUs was observed when compared to medicament alone.[30],[31]

The antibacterial efficacy of TAP was found to be more than O. sanctum for aerobic cultures as well as for anaerobic cultures in our study. On comparing the activity of both the medicaments against aerobic and anaerobic bacteria, no statistically significant difference was observed in either of the groups. It can be concluded that both the medicaments were equally effective against aerobic and anaerobic bacteria within their respective groups.


   Conclusion Top


In an overview of the results of this study, it can be concluded that O. sanctum showed statistically significant antimicrobial action against aerobic as well as anaerobic CFUs. Therefore, more in vivo studies should be undertaken with more concentrated formulations to establish its widespread use as antibiotics in spite of their potent action do come with possibility of development of antibiotic resistance and therefore their unjustified and imprudent use should be avoided. Use of herbal extract in potent formulations can help in curbing the menace of antibiotic resistance and adverse reactions on children.

Financial support and sponsorship

Equipment and technical support was provided by the Department of Microbiology and Pathology, People's College of Medical Sciences, Bhopal, Madhya Pradesh.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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