Antimicrobial Efficacy of Aloe vera, Lemon, Ricinus communis, and Calcium Hydroxide as Intracanal Medicament Against Enterococcus faecalis: A Confocal Microscopic Study.

INTRODUCTION: The aim of this study was to evaluate the antimicrobial efficacy of different intracanal medicaments against Enterococcus faecalis using confocal microscopy.
MATERIALS AND METHODS: Eighty extracted single-rooted mandibular premolar teeth were used in this study, which were decoronated and instrumented with K-files following which biomechanical preparation was performed up to ProTaper F3. The teeth were inoculated with E. faecalis and incubated for 21 days. The specimens were divided into five groups according to the medicaments used: saline, calcium hydroxide, Aloe vera, Ricinus communis, and lemon extract. After 7 days of placement of intracanal medicament, the efficacy of the intracanal medicament against E. faecalis was assessed using confocal microscopy. The data were analyzed using one-way analysis of variance and the Tukey HSD (post hoc) test for multiple comparisons.
RESULTS: Calcium hydroxide showed significantly greater antimicrobial efficacy than A. vera, lemon, and R. communis (P < 0.05).
CONCLUSION: Complete elimination of E. faecalis was not achieved in any of the experimental groups. Calcium hydroxide was more effective than the other herbal medicaments used.

INTRODUCTION

The success of endodontic treatment is directly influenced by the elimination of microorganisms in infected root canals. Intracanal medicaments supplement this to eliminate the persistent bacteria and prevent their regrowth, thereby making root canal environment conducive for periapical tissue repair.[1] Calcium hydroxide is one of the most commonly used intracanal medicaments having antibacterial activity on a wide range of microflora present in the root canal.

Aloe vera leaf extracts contain anthraquinones that have antibacterial properties.[2] Similarly Ricinus communis has shown good antimicrobial activity in the treatment of root canals,[3] and fresh lemon solution also has shown to have wide antibacterial efficiency.[4] Hence, the purpose of this study was to compare the antimicrobial efficacy of calcium hydroxide, A. vera, R. communis, and lemon as intracanal medicament against Enterococcus faecalis using confocal microscopy.

MATERIALS AND METHODS

Eighty single-rooted human mandibular premolars with closed apices, which were sectioned below the cementoenamel junction with a diamond disk to obtain a standardized tooth length of 13 mm, were used in this study. A size #10 stainless-steel K-file was inserted into the canal until the file tip was just visible at the apical foramen and the working length was kept 1 mm short of the apical foramen.

The root canals were prepared using ProTaper files (Dentsply Maillefer, Ballaigues, Switzerland) up to size F3. All the prepared teeth were packed in suitable autoclave pouches and autoclaved at 121°C. Each root canal was inoculated with bacterial solution of E. faecalis (ATCC 29212) using a sterile endodontic needle. After inoculation, the samples were kept in a closed Eppendorf tube and incubated at 37°C for 21 days under aseptic conditions and the inoculation was repeated every 3 days to ensure viability of bacteria. The canal contents were aspirated after 21 days of incubation, rinsed with 5-mL saline, and patted dry with sterile paper points. The specimens were then randomly divided into five groups (n = 16 each) for intracanal medicaments: Group I - normal saline (control); Group II - calcium hydroxide mixed with saline; Group III - A. vera; Group IV - R. communis (castor oil); and Group V - fresh lemon solution. In all the samples, the prepared medicaments of 5 µL were injected in the root canals and completely filled. The canals were then sealed with sticky wax (Pyrax, Uttarakhand, India), and incubated at 37°C for 7 days following which the canals were irrigated with normal saline for 2 minutes.

After embedding the specimens in acrylic resin, each root was sectioned to obtain the apical transverse sections, using a hard tissue microtome (Leica, Nussloch, Germany). All the samples were stained with the fluorescent dyes - Fluorescent diacetate diluted in Acetone (FDA) and Propidium iodide (PI; SIGMA ALDRICH, Bengaluru, Karnataka, India) and washed with saline to remove any excess stain and blotted dry. All the sections were observed under a confocal laser scanning microscope (Zeiss, LSM 510 META, Jena, Germany) with ×20 magnifications [Figure 1].

Figure 1

Bacterial viability in the apical region using confocal laser microscopy (green fluorescence—live bacteria, red fluorescence—dead bacteria)

Data were obtained and descriptive statistics were performed using one-way analysis of variation followed by Tukey HSD (post hoc) with levels of significance set at P < 0.05.

RESULTS

The results showed that complete elimination of E. faecalis was not achieved with any of the intracanal medicaments used. On the basis of the results of the study, calcium hydroxide had the least number of live bacteria followed by A. vera, lemon, R. communis, and saline (control), but there was no statistically significant difference between calcium hydroxide, A. vera, and lemon [Tables 1 and 2 and Graph 1].

Table 1

Difference in mean values in between the groups analysis of variation

	Groups	n	Mean	Standard deviation	F value	P value
Green	Saline	5	49849.60	14664.138	9.080	0.001
	Ca(OH)2	5	4717.20	1877.598
	A. vera	5	10528.60	11403.080
	R. communis	5	38771.40	22977.222
	Lemon	5	11574.40	14757.772
Red	Saline	5	5464.80	4932.181	2.502	0.075
	Ca(OH)2	5	41843.40	43835.567
	A. vera	5	36510.00	18817.170
	R. communis	5	12357.60	3425.508
	Lemon	5	27289.20	9588.161

Table 2

Difference in mean values between the control and test groups (post hoc)

	Test group	Comparison group	Mean difference	P value
Green	Saline	Calcium hydroxide	45132.400*	0.001
		A. vera	39321.000*	0.004
		R. communis	11078.200	0.760
		Lemon	38275.200*	0.005
Red	Saline	Ca(OH)2	−36378.600	0.104
		A. vera	−31045.200	0.206
		R. communis	−6892.800	0.987
		Lemon	−21824.400	0.530

* = Significant difference

Graph 1

Live and dead bacteria comparision

DISCUSSION

Calcium hydroxide is the commonly used intracanal medicament. Herbal or natural products have been used in dental practice and have gained popularity because of their high antimicrobial activity, cost effectiveness, low toxicity, biocompatibility, anti-inflammatory, and antioxidant properties.[5]

Aloe vera contains anthraquinones[2] that are responsible for the antimicrobial effect of A. vera against E. faecalis.[6] R. communis, or castor oil plant has high percentage of ricinoleic acid, which inhibits many microbes[7] and can be used as an intracanal medicament.[8] Lemon solution has antimicrobial properties because of the presence of limonoids. Fresh lemon solution was shown to have wide antibacterial efficiency against E. faecalis and hence can be used as an intracanal medicament.[4] Scanning confocal microscopy is useful in identification of viable and dead bacteria in infected dentin after staining with FDA and PI.[9]

In a study[4] evaluating the antibacterial activity of freshly minced garlic extract, fresh lemon solution, 10% citric acid, 5.25% sodium hypochlorite, and camphorated parachlorophenol against E. faecalis and Streptococci pyogens, fresh lemon solution showed good antimicrobial effect against E. faecalis, which is similar to the results obtained in this study.

The results in this study showed that calcium hydroxide is better in terms of bacterial reduction, which were similar to the earlier studies.[6] The results of our study are in variation from another study,[10] in which A. vera showed better antibacterial efficacy compared to calcium hydroxide against E. faecalis. This may be because after placement of intracanal medicaments, the antimicrobial efficacy was checked only till 5 days unlike the efficacy checked after 7 days in our study.

The nature of the endodontic microflora is polymicrobial but single-species biofilm model of E. faecalis was used in our study, which can be a limitation. Though biofilm model with E. faecalis was used on extracted teeth, this does not entirely simulate the oral environmental conditions.

CONCLUSION

Within the limitations of this study, it was concluded that complete elimination of E. faecalis was not achieved in any of the experimental groups. Calcium hydroxide was most effective in causing reduction of E. faecalis followed by A. vera, lemon, and R. communis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.