|Year : 2016 | Volume
| Issue : 2 | Page : 61-66
Ethanolic extracts of Aloe vera and propolis as cavity disinfectants: An in vitro study
Karuna Yarmunja Mahabala1, Suprabha Baranya Shrikrishna1, Srikant Natarajan2, Anupama P Nayak1
1 Department of Pedodontics and Preventive Dentistry, Manipal College of Dental Sciences, Manipal University, Mangalore, Karnataka, India
2 Department of Oral Pathology, Manipal College of Dental Sciences, Manipal University, Mangalore, Karnataka, India
|Date of Web Publication||9-Jun-2016|
Karuna Yarmunja Mahabala
Departments of Pedodontics and Preventive Dentistry, Manipal College of Dental Sciences, Manipal University, Light House Hill, Mangalore - 575 001, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: Though a large number of agents including chlorhexidine have been used for cavity disinfection during restorative procedures, till date none has proved to be "ideal." Thus, there is a need for an alternative cavity disinfectant, which is safe, effective, and economic. Herbal extracts of Aloe vera and propolis have shown the potential to be used as cavity disinfectants but not much is known about them. The aim of this study was to evaluate and compare the antibacterial activity of ethanolic extracts of Aloe vera and propolis against S. mutans and L. acidophilus, along with assessing the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Materials and Methods: The study included three groups: 2% chlorhexidine solution (group I), ethanolic extract of Aloe vera (group II), and ethanolic extract of propolis (group III). An MIC test was carried out using tissue culture microplates and serial dilution technique and later to MBC, an aliquot of each incubated well with concentrations higher than MIC was subcultured on brain heart infusion (BHI) medium. Antibacterial assay was carried out using agar disk diffusion technique and zones of inhibition were determined. One-way analysis of variance (ANOVA) was used for intergroup comparison followed by post hoc Tukey's test for groupwise comparison. Results: Group II and Group III showed bacteriostatic effect but no bactericidal effect against both S. mutans and L. acidophilus. There was a significant difference in the antibacterial activity against both the tested microorganisms among all the groups, the highest being in group I followed by group II and group III. Conclusions: Ethanolic extracts of Aloe vera and propolis are only bacteriostatic and their antibacterial efficacy is inferior to chlorhexidine against both S. mutans and L. acidophilus.
Keywords: Aloe vera , antibacterial, cavity disinfection, chlorhexidine, dental caries, disinfection, minimum bactericidal concentration (MBC), propolis
|How to cite this article:|
Mahabala KY, Shrikrishna SB, Natarajan S, Nayak AP. Ethanolic extracts of Aloe vera and propolis as cavity disinfectants: An in vitro study. Dent Hypotheses 2016;7:61-6
|How to cite this URL:|
Mahabala KY, Shrikrishna SB, Natarajan S, Nayak AP. Ethanolic extracts of Aloe vera and propolis as cavity disinfectants: An in vitro study. Dent Hypotheses [serial online] 2016 [cited 2019 Jun 17];7:61-6. Available from: http://www.dentalhypotheses.com/text.asp?2016/7/2/61/183769
| Introduction|| |
Though dentistry has magically developed with newer materials and newer techniques, dental caries still remains a disease of great prevalence, more so in children.  While the goal of restorative treatments for dental caries is to remove the infected dentin and fill the area with a suitable restorative material, ,, failure to totally remove the infected tooth structure and achieve complete sterilization of the cavity can lead to microleakage, increased pulp sensitivity and pulpal inflammation, and secondary caries, necessitating replacement of the restoration. ,
Therefore, after removal of the carious dentin it is important to eliminate any remaining bacteria that may be present on the cavity walls, in the smear layer, at the enamel-dentin junction, or in the dentinal tubules. 
But unfortunately no definitive and reliable criteria are available to ensure the complete removal of carious tooth structure.  Results of many investigations have shown the presence of bacteria in the dentin even after removal of dye-stainable dentin. ,,, It has been confirmed histologically that only a portion of the tooth is sterile after termination of routine cavity preparation and fermentative organisms remain viable for as long as 139 days under nonantispetic restorations. , Furthermore, bacteria present in the smear layer can multiply, allowing their toxins and degradation products to diffuse to the pulp. ,
One solution to overcome this problem is to use cavity disinfectants in order to achieve near-total elimination of bacteria before restoring a tooth. Various cavity disinfectants used till date include chlorhexidine, silver nitrate precipitated with eugenol, thymol, potassium ferrocyanide sodium hypochlorite, fluoride solutions, benzalkonium chloride, hyaluronic acid, iodine/copper sulfate, antibiotic-based solutions, disodium ethylene diamine tetra acetic acid dehydrate (EDTA), cetyl pyridinium chloride, ozone, and lasers. ,,, However, a few of the above listed chemicals are not biocompatible with the pulp.  Also, there exists a concern about the effect of use of a few cavity disinfectants on subsequent bonding of adhesive restorative materials.  Though chlorhexidine has been accepted as a positive control for future studies on cavity disinfectants,  the available dental literature projects the association of cavity disinfection with chlorhexidine and bonding of subsequently applied adhesive system to be a controversial issue. ,, To overcome these limitations an alternate cavity disinfectant, which is safe, effective, and economic is need of the hour. With the growing popularity of phytotherapy, ethanolic extract of Aloe vera and ethanolic extract of propolis have been tried for cavity disinfection and found to be efficient.  However, little is known about these herbal extracts as cavity disinfectants and more exploration are required. Therefore, this study was conducted to evaluate and compare the antibacterial activity of ethanolic extracts of Aloe vera and propolis, along with assessing the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The null hypothesis was set as there is no difference in the antibacterial activity of ethanolic extracts of Aloe vera or propolis against S. mutans and L. acidophilus when compared to chlorhexidine.
| Materials and Methods|| |
This microbiological in vitro study was initiated after approval from the institutional ethics committee (protocol reference no. 15133). The obtained sample size per group was 12 to achieve 83% power to detect differences among the means at 0.05 significance level.
The study consisted of three groups: Group I (control) −2% chlorhexidine solution (CHX) - Consepsis, Ultradent, South Jordan, Utah, USA; group II - ethanolic extract of Aloe vera (EEA); group III - ethanolic extract of propolis (EEP). EEA and EEP were prepared in Bapuji Pharmacy College, Davangere, Karnataka, India.
The microorganisms against which the above cavity disinfectants were tested included: S. mutans (MTCC 497, Institute of Microbial Technology, Chandigarh, India) and L. acidophilus (MTCC 10307, Institute of Microbial Technology, Chandigarh, India).
Preparation of ethanolic extract of Aloe vera (EEA)
The leaves of the Aloe vera plant (Aloe barbadensis Mill) were washed with distilled water, cut opened, and the fresh pulp was collected. The gel was dried in an oven (Dongguan Yuanyao Electronics Technology Co., Ltd, Guangdong, Mainland China) at 800°C for 48 h and then powdered. Later, 20 g of the powder was dissolved in in 200 mL of ethanol (Farrel Ltd., Noida, India). The contents were filtered using Whatman filter paper no. 1 (Advance International, New Delhi, India), and the filtrate was evaporated for dryness.  A working concentration (100 mg/mL) of ethanolic extract of Aloe vera was thus obtained.
Preparation of ethanolic extract of propolis (EEP)
Ethanolic extract of Brazilian green propolis was prepared according to Wojtyczka et al.  The propolis samples (Hi-Tech Natural Product Ltd., New Delhi, India) were ground mechanically and bottled in 10-g portions. These 10-g portions were put into a flask and 100 g of 70% (w/v) ethanol (Farrel Ltd., Noida, Uttar Paradesh, India) was added. The flask was placed on a rotary shaker in a dark, closed room for 2 weeks at room temperature. After this period, the extract was cooled at 4°C for 24 h in order to precipitate all insoluble particles, which were removed from the extract by filtration through filter paper (Whatman number 1). Later, the filtrate was evaporated at 40°C using a rotary vacuum evaporator (Nantong Purui Technical Instrument Co., Ltd, Natong, China). Following evaporation, the obtained brown colored viscous substance was dissolved in 70% ethanol to obtain a 100 mg/mL working concentration. 
Minimum inhibitory concentration and minimum bactericidal concentration
MIC tests for ethanolic extracts of Aloe vera and propolis were carried out against both S. mutans and L. acidophilus, using tissue culture microplates containing 100 mL/well brain heart infusion (BHI) medium (Titan Biotech Ltd., Delhi, India). After transferring the extracts to the first well, serial dilutions were performed to obtain concentrations ranging 100-0.2 mg/mL (100 mg/mL, 50 mg/mL, 25 mg/mL, 12.5 mg/mL, 6.25 mg/mL, 3.12 mg/mL, 1.6 mg/mL, 0.8 mg/mL, 0.4 mg/mL, and 0.2 mg/mL). The inoculums of S. mutans and L. Acidophilus (1 × 10 6 CFU/mL) were added to the wells, and the plates were incubated at 37°C in 5% CO 2 for 24 h. MIC was defined as the lowest concentration of the propolis and Aloe vera extracts that inhibited microorganism visible growth indicated by resazurin (0.01%) (Karan Laborates, Mumbai, Maharashtra, India). Later, to determine MBC an aliquot of each incubated well with concentrations higher than MIC was subcultured on the BHI medium. MBC was defined as the lowest concentration of the Aloe vera and propolis extracts that allowed no visible growth on the test medium. 
Bacterial sensitivity or resistance to cavity disinfectants was detected by the disk diffusion assay, also known as the Kirby-Bauer method. Aliquots of S. mutans and L. acidophilus containing 1.0 × 10 8 CFU/mL were subcultured in Mueller-Hinton agar supplemented with 5% of dextrose and in blood agar medium. Sterile filter papers soaked with 20 mL of 2% chlorhexidine (acted as a positive control), propolis extract, and Aloe vera extract each were placed onto the agar. The diameter of inhibition zone around the filter paper formed at 37°C was measured in mm after 48 h and recorded. Any inhibition zone around the filter paper measuring ≤7 mm was considered to be a negative result. 
The obtained data were analyzed by SPSS software (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY) using one-way analysis of variance (ANOVA) and with Tukey's Post Hoc for groupswise comparison.
| Results|| |
MIC and MBC
The tested cavity disinfectants (EEA and EEP) stopped the growth of S. mutans and L. acidophilus (bacteriostatic) but did not have bactericidal effects [Table 1]. The MICs of EEA and EEP for S. mutans were 25 mg/mL and 100 mg/mL, respectively, whereas for L. acidophilus the MIC of both EEA and EEP was 100 mg/mL.
[Table 2] and Graph 1 show comparison of the inhibition zones seen during disk diffusion test against S. mutans, as well as L. acidophilus while using EEA/EEP. A statistically significant difference was seen among three groups, the highest diameter of zones of inhibition being shown by chlorhexidine followed by propolis and then Aloe vera against both S. mutans and L. acidophilus (P < 0.001) [Figure 1]. When groupwise comparison was done using post hoc Tukey's test [Table 3], differences in the zones of inhibition against S. mutans as well as L. acidophilus were significant in both group II (EEA) and group III (EEP) when compared to the control group (CHX) (P < 0.001). However, when the zones of inhibition were compared against S. mutans between group II (EEA) and group III (EEP), there was no statistically significant difference (P = 0.098) but a significant difference against L. acidophilus was found (P < 0.001).
|Figure 1: (a) Zones of inhibition shown by group II (EEA) as compared to the control group against S. mutans (b) Zones of inhibition shown by group III (EEP) as compared to the control group against S. mutans (c) Zones of inhibition shown by group II (EEA) as compared to the control group against L. acidophilus (d) Zones of inhibition shown by group III (EEP) as compared to the control group against L. acidophilus|
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|Table 2: Comparison of antimicrobial activity of three cavity disinfectants against S. mutans and L. acidophilus |
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|Table 3: Groupwise comparison of antibacterial activity of cavity disinfectants against S. mutans and L. acidophilus |
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| Discussion|| |
The results of our study showed that EEA was able to inhibit the growth of S. mutans at a concentration of 25 mg/mL while EEP could inhibit S. mutans growth only at its highest concentration, i.e., at 100 mg/mL. Also, both EEA and EEP could inhibit the growth of L. acidophilus only at the maximum concentration (100 mg/mL). When MBC was evaluated, it was found that these herbal extracts have only bacteriostatic activity and are not bactericides. However, this does not limit their use as cavity disinfectants; even bacteriostatic effect is a great benefit to prevent secondary caries.
The method used for testing antibacterial activity against S. mutans and L. acidophilus by each of the extracts was agar disk diffusion assay. It is the standard method used in many clinical microbiology laboratories for routine antimicrobial susceptibility testing and offers many advantages over other methods such as simplicity, low cost, the ability to test enormous numbers of microorganisms and antimicrobial agents, and the ease to interpret results. Moreover, it demonstrates good clinical correlation. Due to these advantages, it is commonly used for the antimicrobial screening of plant extracts, essential oils, and other drugs. However, since the bacterial growth inhibition does not mean bacterial death, this method cannot distinguish bactericidal and bacteriostatic effects. 
In this study, bacterial inhibition was shown by both EEA and EEP against tested microorganisms (S. mutans and L. acidophilus). The mechanism of activity of propolis against microorganisms is very complex. Some components present in propolis extracts such as flavonoids (quercetin, galangin, and pinocembrin) and caffeic acid, benzoic acid, and cinnamic acid probably act on the microbial cytoplasmic membrane or cell wall site, causing functional and structural damages. , The antibacterial activity could also be related to the synergistic effect of all components than an individual compound. 
In the present study, the antibacterial activity shown by EEP against S. mutans as well as L. acidophilus is partially in agreement with the study conducted by Akca et al.  However, in the latter study EEP was found to have bactericidal effect against both the abovementioned bacteria, which was greater or equal to that of chlorhexidine. In another study conducted by Liberio et al., inhibitory activity was displayed by geopropolis against S. mutans, as previously observed in extracts and fractions obtained from Apis propolis but none of the geopropolis extracts tested in the study were seen to have antimicrobial activity against L. acidophilus.,,,
These differences in MIC/MBC values and the antibacterial activity were possibly owing to the differences in the strains and/or to the diverse origins of the propolis samples since the composition of propolis depends on the regional vegetation.  In addition, bacterial cell wall and its biofilm properties were concluded as adjunct factors, which determine the type of antibacterial activity exerted by propolis against a particular bacterium.  Thus, propolis can act against each microorganism in different ways.
The antibacterial effects of Aloe vera have been attributed to various pharmacologically active constituents such as aloe emodin, aloetic acid, aloin, anthracine, anthranol, barbaloin, chrysophanic acid, ethereal oil, ester of cinnamonic acid, isobarbaloin, and resistannol.  The bacterial inhibition by EEA in our study is in agreement with the study conducted by George et al., which concluded that Aloe vera tooth gel is effective against both S. mutans and L. acidophilus.  In another study conducted by Subramaniam et al., hydroalcoholic extract of Aloe vera had significant antibacterial effect on S. mutans but only at 100% concentration.  However, in our study EEA could inhibit bacteria at a concentration of 25 mg/mL. This could be due to the use of plants from different geographical locations with variations in their chemical composition and also because of different isolation techniques that were used to extract compounds from the Aloe leaf pulp.  Few of the previous researchers have used Aloe vera-based oral products, , whereas in the present study only the Aloe vera extract was used.
The antibacterial effect of CHX varies with different concentrations, 2% being the most efficient;  thus, we used 2% chlorhexidine solution in our study as control. The disinfectant efficiency of CHX depends on the mechanism by which the adsorption of CHX occurs onto the cell wall of microorganisms causing the leakage of intracellular components. At a low concentration, CHX is bacteriostatic and causes the leaking of a small molecular weight substance from microorganisms. At a higher concentration, CHX is bactericidal as it diffuses into bacteria, causing irreversible damage including precipitation and coagulation of cytoplasmic content due to protein cross-linking.  In the present study, the antibacterial effect exerted by both the herbal cavity disinfectants was less compared to that of chlorhexidine. This could be due to either the inherently strong antibacterial efficacy of CHX or the difference in the physical form of the agents; chlorhexidine 2% being a solution could diffuse more readily in agar medium compared to EEA and EEP, which are in semisolid form. When the in vivo use of EEA and EEP are considered, the semisolid nature might prevent these cavity disinfectants from penetrating deep into dentinal tubules. It has been demonstrated that bacteria are capable of invading the dentinal tubules up to a depth of 1 mm.  The difference in depth of penetration between the invading bacteria and the cavity can be responsible for secondary caries in a few of the cases in spite using cavity disinfectant. Thus, it would be better if these extracts can be used in solution form, which requires further exploration. Also, studies need to be conducted to assess the effect of cavity disinfection with these herbal extracts on subsequent bonding of adhesive restorative materials before considering them as potential cavity disinfectants during restorative procedures.
| Conclusions|| |
Financial support and sponsorship
- Ethanolic extracts of Aloe vera and propolis have bacteriostatic activity against S. mutans and L. acidophilus but do not possess bactericidal effect.
- Both tested herbal extracts show some antibacterial activity against both S. mutans and L. acidophilus, the ethanolic extract of propolis being more efficient but inferior to chlorhexidine.
The authors do not have any financial support.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]