Evaluation of antimicrobial effect of Malaysian geopropolis with Aloe vera against Enterococcus faecalis to be used as an intracanal medicament in endodontics.

INTRODUCTION: Enterococcus faecalis can be found in failed endodontic treatment (FET) even after performing primary endodontic treatment (PET). Calcium hydroxide (Ca(OH)2) cannot fully eliminate this microorganism during PET. Brazilian green propolis (bee glue) was found to be more effective against E. faecalis when compared to Ca(OH)2. A much less studied Malaysian geopropolis (MP) as well as Aloe vera (AV) is antibacterial but is unknown against E. faecalis.
OBJECTIVE: The objective of this study is to determine the antimicrobial effects of MP, AV, and MP + AV in comparison with Ca(OH)2 against E. faecalis, as an intracanal medicament.
MATERIALS AND METHODS: Antimicrobial activity of MP, AV, MP + AV, Ca(OH)2, and dimethyl sulfoxide was tested against E. faecalis using antimicrobial sensitivity testing, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The results were analyzed by Kruskal-Wallis test with Mann-Whitney post hoc test and repeated measures analysis of variance with Bonferroni post hoc test (P < 0.05).
RESULTS: For agar well-diffusion method, MP + AV gave maximum inhibition zone diameter (mean: 8.11 ± 0.015 mm), MP (mean: 6.21 ± 0.046 mm, Ca(OH)2 (mean: 5.5 ± 0.006), and AV (mean: 5.05 ± 0.012) with P < 0.05. MIC for MP + AV was 2 mg/ml, MP at 8 mg/ml, Ca(OH)2 at 8 mg/ml, and AV at 16 mg/ml. The MBC for MP + AV is at 4 mg/ml, MP at 16 mg/ml, Ca(OH)2 at 16 mg/ml, and AV at 32 mg/ml.
CONCLUSION: The combination of MP and AV consistently showed better antimicrobial activity compared to MP and AV alone against E. faecalis. The findings suggest that MP and AV used in combination may be an ideal intracanal medicament in FET and PET. Copyright:

INTRODUCTION

The cause of apical periodontitis is known to be because of the presence of bacteria within the root canal system.[1]Enterococcus faecalis is the most commonly found bacteria in the root canals of failed endodontically treated teeth causing root-filled teeth to have persistent periapical lesions.[234]

As part of the normal oral flora, E. faecalis is a Gram-positive facultative anaerobe[5] which has been shown to resist calcium hydroxide (Ca(OH)2) intracanal dressing.[6] They invade and colonize within the dentinal tubules to survive chemo-mechanical instrumentation as well as intra-canal medication.[7]

Ca(OH)2 is the most commonly used intracanal medicament, despite its possible toxicity, deficiency in anti-bacterial efficacy, and delayed effectiveness.[18] Furthermore, the buffering capability of dentin to reduce the high pH of Ca(OH)2 will decrease its antimicrobial property.[9] Ca(OH)2 did not eliminate E. faecalis even after a relatively extended period of use.[1011] The need to develop a different medicament with better properties at root canal cleaning and disinfection, therefore, seems reasonable.[12]

Propolis from honeybees is one of the bee-products,[13] whereas geopropolis is produced by stingless bees. Geopropolis contained wax, plant resin, and soil particles that are produced to seal the hives.[6] It is composed of very complex chemicals such as esters, phenolic compounds, and flavonoids and flavanones, and its properties include antimicrobial, inhibition of inflammation, reduction of osteoclastic activity, anti-quorum sensing, biofilm formation inhibition, and immunomodulatory capability.[1314] The antibacterial activity is contributed by the presence of esters, phenolic compounds, flavonoid, terpenes, and aromatic acids.[1516]

Previous studies have evaluated the antimicrobial efficacy of propolis against E. faecalis using in vitro models.[1718] They found some fascinating properties of propolis as an intracanal medicament. These are its capability to induce healing, reduce cytotoxicity, inhibit osteoclastic activity, influence inflammatory cytokine expression, and reduce cell death to enhance the proliferation of the periodontal ligament cells.[192021]

Malaysian geopropolis (MP) produced by stingless bees is now being considered as a potential medication. Heterotrigona itama and Geniotrigona thoracica, two of more than 30 stingless bees found in Malaysia, are known to produce geopropolis.[22]

Aloe vera (AV), a plant extract to serve both as a delivery vehicle and possessing is also being investigated. Aloe gel has been used traditionally to relieve thermal burns and sunburns to promote wound healing due to its antimicrobial activity and ability to stimulate the body's immune system.[23] AV gel and geopropolis are two separate naturally occurring substances that have been long used in the treatment of inflammation and infectious diseases of the mouth.[23]

There was inadequate research, regarding stingless bee in Malaysia although H. itama geopropolis have strong antioxidant activities.[24] Significantly, literature reports of antimicrobial activity for a combination of MP and AV against E. faecalis were not found. Although one study recommended combining both the concentrations of ethanolic extracts of MP with AV gel to disinfect the root canal.[23] To replace the existing Ca(OH)2, MP and AV in combination need to be investigated. Therefore, this research aimed to evaluate the antibacterial effects of MP and AV, individually and in combination, against E. faecalis.

MATERIALS AND METHODS

Sample preparation by the collection and extraction of propolis

Raw MP produced by H. itama bee was purchased, and five grams of raw MP were minced and 100 mL of 70% ethanol added. Sonication of the mixture was carried out and was filtered using Whatman paper no. 1. This was followed by rotary evaporating until appropriate viscosity is obtained. The evaporated filtrate then underwent lyophilization and was stored in a freezer (−25°C).

Purchasing of Aloe vera and Ca(OH)2

Calcium hydroxide (Ultra Cal XS®)[Ca(OH)2] was purchased from Ultradent Product Inc., and AV gel from Original Natural Center-Forever AV Gel®.

Contents of Forever Aloe vera Gel®

Aloe Vera Gel 100%

Sorbitol Not disclosed

Ascorbic acid (antioxidant) Not disclosed

Tocopherol (antioxidant) Not disclosed

Potassium sorbate (Protect flavor) Not disclosed

Sodium benzoate (Protect flavor) Not disclosed

Citric acid Not disclosed

Xanthan gum Not disclosed

Preparation of bacteria stock culture

E. faecalis (ATCC 29212) was purchased from SIGMA Aldrich and was rehydrated. Several drops of the media were taken and inoculated on brain–heart infusion (BHI) agar media to observe the colony morphology of the bacteria. The tube and agar media were then incubated at 37°C for 18–24 h. Pipette 0.5 mL of BHI broth into cryovial tubes, added with 0.5 mL of 50% glycerol, and followed by pipetting 0.1 mL of inoculated BHIB into each of the cryovial tubes, respectively.

Glycerol stock culture was prepared by pipetting 0.5 mL overnight grown culture and 0.5% sterilized glycerol in cryovial tubes. The stock cultures were stored at −80°C for further analysis.

Preparation of inoculum

McFarland standard (0.5) was used which is equivalent to 1.5 × 108 colony-forming unit (CFU) (A). E. faecalis was streaked out using a sterilized loop and mixed into 3 mL normal saline solution. The mixture was homogeneously mixed, and the turbidity of the suspension was determined using a densitometer (Biomerieux Densichek Plus). Glycerol stock was used for the preparation of the bacterial suspension. The bacterial suspensions were prepared in BHI broth, and the OD reading was adjusted to 1.5 × 108 CFU/mL according to the turbidity of 0.5 McFarland test standard.

Antimicrobial sensitivity testing

Three Mueller–Hinton agar (MHA) was prepared and divided into five portions labeled as MP, AV, and MP + AV. The most commonly used intracanal medicaments in endodontic treatment Ca(OH)2) and 1% dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. The extracts will be prepared in 1% DMSO. Bacterial suspension with 1.5 × 108 CFU was used for the preparation of the MHA plate. Five 6 mm wells were prepared to deposit the samples for MP, AV, MP + AV, Ca(OH)2 and 1% DMSO, respectively. Approximately 50 μL of the sample were added to their respective wells using a micropipette. The final concentration of MP, AV, MP + AV, and Ca(OH)2 in each well was 32 mg/mL. All plates in triplicate were incubated at 37°C for 18–24 h, and diameters of inhibition zone were measured in mm.

Minimum inhibitory concentration test

The minimum inhibitory concentration (MIC) test was carried out in the 96-well microplate, where 11 wells were allocated and labeled 1–9, while 10 and 11 were labeled as positive and negative controls, respectively. Pipetted 50 μL of Mueller Hinton broth was suspended into all microplate wells. Pipetted 50 μL (concentration of 32 mg/mL) of MP were added into wells no. 1 and no. 10, and then, the mixture was mix well. Next, double-fold dilution was performed by pipetting 50 μL of extract solution from well 1 to well 2 and is continued until well no. 9, but the remaining 50 μL from well 9 was discarded. The plates (done in triplicates) were incubated at 37°C for 18–24 h, and turbidity was measured at 620 nm using the microplate reader (Tecan M200 Infinite Pro Microplate Reader). The lowest concentration that inhibited visible growth of the tested organism was recorded as the MIC. Pipetted 5 μL of 108 CFU bacterial suspension was added in each tube except tube number 10. Well no. 10 contained broth and MP extracts only while well no. 11 contained only broth and bacteria. The same procedure was repeated for AV, MP + AV, and 1% DMSO (negative controls). The amount 50 μL was transferred from well no. 1 to well no. 2 and repeated to no 9. Well no. 10 served as positive control broth with 100% of MP, whereas well no. 11 served as a negative control with only broth and bacteria. The procedures were repeated for MP, but in 20 times larger in volume. Macrodilution was chosen (instead of microdilution) for the MIC of Ca(OH)2 because it tends to form sediment and harden. Turbidity was measured at 620 nm using the microplate reader (Tecan M200 Infinite Pro Microplate Reader). The lowest concentration that inhibited the visible growth of the tested organism was recorded as the MIC.

Minimum bactericidal concentration test

The minimum bactericidal concentration (MBC) was determined by sub-culturing all the contents from the wells and test tubes onto the MHA plate. The concentration of the sample that showed no visible growth of E. faecalis, examined after 18–24 h, was considered the MBC.

Data analysis

Data were analyzed using nonparametric tests, which are the Kruskal–Wallis test with Mann–Whitney U post hoc test and repeated-measures analysis of variance (ANOVA) with Bonferroni post hoc test from IBM® SPSS® Statistics Version 23, Perpustakaan Tun Abdul Razak, 40450 Shah Alam, Selangor Darul Ehsan, Malaysia. The significance level is set at 5% (α= 0.05, two-tailed). Thus, 95% confidence interval (CI) will be applied.

RESULTS

The antimicrobial sensitivity testing (AST) of the MP + AV produced a zone of inhibition that was significantly greater (mean = 8.11 mm ± 0.015) than those of other samples [Figure 1]. This is followed by MP (mean = 6.21 mm ± 0.046), Ca(OH)2 (mean = 5.51 mm ± 0.006), AV (mean = 5.05 mm ± 0.012), and 1% DMSO (mean = 0 mm). Table 1 shows the mean values of the diameter of the inhibition zone produced by each sample, and Table 2 shows the statistical significance between the zone of inhibition produced by the samples (P = 0.009).

Figure 1

Plate for antimicrobial assessment. Enterococcus faecalis was evenly streaked onto Mueller Hinton agar and 5 wells were prepared by using a cork borer of 6 mm in diameter

Table 1

Antimicrobial sensitivity testing results for the sample tested against Enterococcus faecalis

Sample of 50 μL in each well of 6 mm	Zone of inhibition (mm)			Mean (mm)±SD
	First group of agar plate	Second group of agar plate	Third group of agar plate
MP extract	6.25	6.16	6.22	6.21±0.046
AV	5.06	5.04	5.06	5.05±0.012
MP+AV	8.12	8.10	8.13	8.11±0.015
Ca(OH)2	5.51	5.52	5.51	5.51±0.006
Sdw	0.00	0.00	0.00	0.00±0.00

SD: Standard deviation

Table 2

Descriptive statistic zone of inhibition shown by each samples against E. faecalis, analyzed using Kruskal Wallis test

Variables	Sample	n	Median (IQR)	X2statistic (df)a	P
Zone inhibition (mm)	Calcium hydroxide	3	5.51 (0)	13.646 (4)	0.009
	Sterile distilled water	3	-
	Malaysian Propolis	3	6.22 (0)
	Aloe vera	3	5.06 (0)
	Malaysian Propolis + Aloe vera	3	8.12 (0)

Table 3 shows the MIC results, and it could be seen that the effect of MP + AV was higher than the control when E. faecalis was tested. The MIC for MP + AV was similar to the ones obtained by the corresponding positive control which was the Ca(OH)2. The mean values of MIC of MP + AV were 2.0 mg/mL, whereas the Ca(OH)2 was 8 mg/mL, respectively. For MP, the mean values of the MIC were mostly the same as the MIC values of the Ca(OH)2. For AV, however, the MIC was two times higher than the MIC of the Ca(OH)2. The MIC of all the tested samples showed higher reading in comparison with the negative controls (sterile distilled water). The results of the MBC assessment, as shown in Table 4 indicated that MP + AV (4 mg/mL) was more effective compared to Ca(OH)2 (16 mg/mL). For E. faecalis, the required mean MBC of MP + AV was four times higher than that of the Ca(OH)2. For MP (16 mg/mL), the mean values of the MBC were approximately the same as the MBCs of the positive control. For AV (32 mg/mL), however, the MBC showed the least mean value compared to all tested samples including Ca(OH)2.

Table 3

Minimum inhibitory concentration results of the sample tested against Enterococcus faecalis

Sample	Trial	Concentration (mg/mL)									Positive control	Negative control
		32.00	16.00	8.00	4.00	2.00	1.00	0.50	0.25	0.125
MP	1			+							Negative	Positive
	2			+							Negative	Positive
	3			+							Negative	Positive
AV	1		+								Negative	Positive
	2		+								Negative	Positive
	3		+								Negative	Positive
MP+AV	1					+					Negative	Positive
	2					+					Negative	Positive
	3					+					Negative	Positive
Ca(OH)2	1			+							Negative	Positive
	2			+							Negative	Positive
	3			+							Negative	Positive

Table 4

Minimum inhibitory concentration result of the tested samples against Enterococcus faecalis

Sample	Trial	Concentration (mg/mL)									Positive control	Negative control
		32.00	16.00	8.00	4.00	2.00	1.00	0.50	0.25	0.125
MP	1		+								Negative	Positive
	2		+								Negative	Positive
	3		+								Negative	Positive
AV	1	+									Negative	Positive
	2	+									Negative	Positive
	3	+									Negative	Positive
MP+AV	1				+						Negative	Positive
	2				+						Negative	Positive
	3				+						Negative	Positive
Ca(OH)2	1		+								Negative	Positive
	2		+								Negative	Positive
	3		+								Negative	Positive

Table 5 shows a comparison of the absorbance readings in each study group using the repeated-measures ANOVA. Pairwise comparison of absorbance at different sample was done in F-stat (df) = 79.254 (4), P < 0.05. Repeated-measures ANOVA between sample analyses was applied followed by the post hoc Bonferroni multiple comparisons. The level of significance was set at 0.05 (two-tailed). Table 5 also shows a comparison of the absorbance reading in each study group with each of the sample groups using the Bonferroni post hoc test. There was a statistically significant difference in mean absorbance within four samples (P < 0.05) for MP + AV between Ca(OH)2 and DMSO. The mean absorbance for each sample at different concentrations is depicted in Figure 2.

Table 5

The comparison of five samples against Enterococcus faecalis at different concentrations using repeated-measures ANOVA

Sample	Concentration (mg/ml)	Absorbance mean	SE	95% CI
				Lower bound	Upper bound
Malaysian propolis	32.00	0.255	0.058	0.125	0.385
	16.00	0.229	0.031	0.160	0.299
	8.000	0.256	0.028	0.195	0.317
	4.000	0.183	0.026	0.125	0.241
	2.000	0.142	0.025	0.087	0.197
	1.000	0.111	0.022	0.063	0.160
	0.500	0.085	0.012	0.059	0.111
	0.250	0.078	0.012	0.052	0.104
	0.125	0.065	0.012	0.038	0.092
Aloe vera	32.00	0.096	0.058	−0.034	0.226
	16.00	0.078	0.031	0.008	0.147
	8.000	0.081	0.028	0.019	0.142
	4.000	0.098	0.026	0.040	0.156
	2.000	0.094	0.025	0.038	0.149
	1.000	0.094	0.022	0.046	0.143
	0.500	0.087	0.012	0.061	0.113
	0.250	0.084	0.012	0.059	0.110
	0.125	0.077	0.012	0.050	0.104
Malaysian propolis+Aloe vera	32.00	0.846	0.058	0.716	0.976
	16.00	0.513	0.031	0.443	0.582
	8.000	0.310	0.028	0.249	0.371
	4.000	0.293	0.026	0.235	0.352
	2.000	0.212	0.025	0.157	0.267
	1.000	0.153	0.022	0.105	0.202
	0.500	0.099	0.012	0.073	0.125
	0.250	0.085	0.012	0.059	0.111
	0.125	0.064	0.012	0.037	0.091
Calcium hydroxide	32.00	0.094	0.058	−0.036	0.224
	16.00	0.087	0.031	0.017	0.156
	8.000	0.098	0.028	0.036	0.159
	4.000	0.101	0.026	0.043	0.160
	2.000	0.103	0.025	0.048	0.159
	1.000	0.107	0.022	0.058	0.155
	0.500	0.096	0.012	0.070	0.122
	0.250	0.092	0.012	0.067	0.118
	0.125	0.080	0.012	0.053	0.107
Sterile distilled water	32.00	0.066	0.058	−0.064	0.196
	16.00	0.062	0.031	−0.008	0.131
	8.000	0.060	0.028	−0.002	0.121
	4.000	0.074	0.026	0.016	0.133
	2.000	0.066	0.025	0.011	0.121
	1.000	0.076	0.022	0.027	0.125
	0.500	0.058	0.012	0.032	0.084
	0.250	0.091	0.012	0.066	0.117
	0.125	0.073	0.012	0.046	0.100

SE: Standard error, CI: Confidence interval

Figure 2

Represent plot graph of mean absorbance of minimum inhibitory concentration among nine different concentrations based on five samples using the repeated measures analysis of variance

Based on Table 6, the mean absorbance for MP + AV (0.212) does not overlap with MP (95% CI = 0.087, 0.197); AV (95% CI = 0.038, 0.149); Ca(OH)2 (95% CI = 0.048, 0.159) showed MP + AV was significant when compared to MP, AV, and Ca(OH)2 at 2.0 mg/mL. The mean absorbance for MP (0.256) does not overlap with AV (95% CI = 0.019, 0.142); Ca(OH)2 (95% CI = 0.036, 0.159) showed MP is significant in comparison with AV and Ca(OH)2 at 8.0 mg/mL. However, there was overlapping of mean absorbance of MP (0.256) with MP + AV (95% CI = 0.249, 0.371) indicated that there was no significant difference.

Table 6

Pairwise comparison of mean absorbance of minimum inhibitory concentration among nine different concentrations based on the different samples using Bonferroni hoc test

Sample	Comparison sample	Mean difference	SE	Significant	95% CI for differenceb
					Lower bound	Upper bound
Malaysian propolis	Aloe vera	0.069*	0.014	0.006	0.018	0.119
	Malaysian propolis+Aloe vera	−0.130*	0.014	0.000	−0.180	−0.080
	Calcium hydroxide	0.061*	0.014	0.015	0.011	0.111
	Sterile distilled water	0.087*	0.014	0.001	0.036	0.137
	Malaysian propolis	−0.069*	0.014	0.006	−0.119	−0.018
Aloe vera	Malaysian propolis+Aloe vera	−0.199*	0.014	0.000	−0.249	−0.148
	Calcium hydroxide	−0.008	0.014	1.000	−0.058	0.043
	Sterile distilled water	0.018	0.014	1.000	−0.032	0.068
Malaysian propolis+aloe vera	Malaysian propolis	0.130*	0.014	0.000	0.080	0.180
	Aloe vera	0.199*	0.014	0.000	0.148	0.249
	Calcium hydroxide	0.191*	0.014	0.000	0.141	0.241
	Sterile distilled water	0.217*	0.014	0.000	0.166	0.267
Calcium hydroxide	Malaysian propolis	−0.061*	0.014	0.015	−0.111	−0.011
	Aloe vera	0.008	0.014	1.000	−0.043	0.058
	Malaysian propolis+Aloe vera	−0.191*	0.014	0.000	−0.241	−0.141
	Sterile distilled water	0.026	0.014	0.961	−0.024	0.076
Sterile distilled water	Malaysian propolis	−0.087*	0.014	0.001	−0.137	−0.036
	Aloe vera	−0.018	0.014	1.000	−0.068	0.032
	Malaysian propolis+Aloe vera	−0.217*	0.014	0.000	−0.267	−0.166
	Calcium hydroxide	−0.026	0.014	0.961	−0.076	0.024

SE: Standard error, CI: Confidence interval

DISCUSSION

Geopropolis produced by H. itama has been selected for the current study for intracanal medicament in combination with AV gel was known to produce an antibacterial and anti-inflammatory effect. The result of this present study which used H. itama species against E. faecalis is in agreement with the study conducted by Ibrahim et al.[22]

AST, MIC, and MBC tests were conducted on AV gel, MPs, MPs + AV, Ca(OH)2, and DMSO (1%). We found that MP and AV alone showed that they were less effective against the tested bacteria compared to the combination of MP + AV. Previous studies reported that AV has the least antibacterial effect when compared to chlorhexidine, propolis, and triantibiotic mixture used, but it is more effective than Ca(OH)2.[25] However, their findings contradicted another study conducted by Bhardwaj et al. which showed that AV is more effective than Ca(OH)2.[26] It has a higher molecular weight (14 kDa) compared to Ca(OH)2 which is 0.074 kDa.[27] Substances with higher molecular weight will take longer to diffuse which results in reduced efficacy. This statement is supported by a study conducted by Swapna et al., which found that the gel form of AV takes longer to release their active ingredients into the canal due to its heavier molecular weight. This led to less penetration of AV into the dentinal tubules. Furthermore, the AV used in our study was not in pure form since it contains sorbitol, ascorbic acid, citric acid, potassium sorbate, sodium benzoate, xanthan gum, and tocopherol.

The MP demonstrated synergistic effects in combination with the AV, indicating that the active constituents of these samples were more effective against E. faecalis. The results of our current study agreed with the suggestion by Ehsani et al. to combine MP and AV. This synergism could be explained by the interaction of several components in both samples that elicit the effect.[23] Recent studies reported that the rich flavonoid and antioxidant properties contained by both samples synergistically gave the strongest antibacterial effect against E. faecalis.[2829] Moreover, MP combined with the anthraquinones component that was found mainly in AV has a structural analog of tetracycline which may increase antimicrobial activity. This study determined that flavonoids, antioxidants, and anthraquinones have a significant correlation between these constituents and antibacterial effects. These results are interesting because MP + AV when compared to AV alone shows intense synergism although the AV has other components added. These incorporated ingredients may interact with the MP and potentiate the antimicrobial effect used in MP + AV combination. There are currently no studies done on this type of AV and so we are unable to explain how the added ingredients influence the potentiating mechanism of action. Intriguingly, the effectiveness of AV gel as a medium of GP points storage by accelerating the decontamination of GP cones, adds to the promising future of AV gel to be incorporated in a root canal treatment.[30]

In our study, the antimicrobial effect of Ca(OH)2 against E. faecalis was better than AV but less antimicrobial effect when compared to other samples. Kurian et al. suggested that this could be due to the limited action of Ca(OH)2 against facultative anaerobes along with the buffering action of dentin by decreasing its capacity as an antimicrobial agent which is made worse by the arrangement of bacterial cells colonizing the root canal walls.[27] This could also be due to the E. faecalis tolerance of pH changes by the activation of specific proton pumps, specific enzymes, and/or buffering mechanisms, which help E. faecalis to neutralize the environmental pH. This relative inefficacy of Ca(OH)2 against E. faecalis was in accordance with previous studies.[31]

CONCLUSION

Within the limitations, this study showed that MP + AV has a higher antimicrobial activity when compared to MP or AV individually. Therefore, based on our findings, we suggest that a combination of MP and + AV may be suitable to be used as an intracanal medicament, particularly in preventing endodontic posttreatment failures. However, the AV used in this study needs further research on how the added materials affect the synergistic mechanism.

Financial support and sponsorship

This study was financially supported by LESTARI Research Grant: 600-IRMI/MyRA 5/3/LESTARI (035/2017).

Conflicts of interest

There are no conflicts of interest.