# Antibacterial and Cytotoxic Effects ofMoringa oleifera (Moringa) and Azadirachta indica (Neem) Methanolic Extracts against Strains of Enterococcus faecalis

Authors: Lucía Arévalo-Híjar; Miguel Ángel Aguilar-Luis; Stefany Caballero-García; Néstor Gonzáles-Soto; Juana Del Valle-Mendoza
Journal: International Journal of Dentistry
 (2018)

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## Abstract

Objective. To evaluate antibacterial and cytotoxic effect of 2 methanolic extracts of Azadirachta indica and Moringa oleifera against strains of Enterococcus faecalis (ATCC 29212) in vitro. Methods. The methanolic extracts of Azadirachta indica and Moringa oleifera were prepared in vitro. The antibacterial effect of the extracts against Enterococcus faecalis was evaluated using the agar diffusion technique. The minimum inhibitory concentration (MIC) was determined using the microdilution method and the cytotoxicity using the cellular line MDCK. Results. The methanolic extract with the most antibacterial effect during the first 24 and 48 hours against Enterococcus faecalis was Moringa oleifera, evidencing a growth inhibition zone of 35.5 ± 1.05 and 44.83 ± 0.98, respectively. The MIC for both extracts was 75 µg/ml. The bactericidal effect of the Azadirachta indica extract was found at a concentration of 25 µg/ml and a concentration of 75 µg/ml for Moringa extract. Conclusions. In conclusion, we demonstrated that the methanolic extract of Azadirachta indica and Moringa oleifera both have an antibacterial effect against Enterococcus faecalis strains during the first 24 and 48 hours. None of the extracts exhibited toxicity against the cell lines under low concentrations.


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## Body

## 1. Introduction

One of the main objectives of endodontics is root canal disinfection, mainly associated with the colonization of anaerobic bacteria. There are endodontic treatments that fail due to a complex root canal anatomy or antibiotic resistance of some bacterial families, withEnterococcus faecalis being the most resistant bacteria reported [1–6].Various antibacterial agents have been previously studied, and they can be obtained from natural sources or synthetic agents [7]. Furthermore, recent studies have reported the pharmacological properties and high medicinal value of a myriad of natural extracts. Some of these products, however, have been used empirically, leaving aside the study and determination of its properties [8].The contemporary field of phytotherapy focuses on the study of plants and its pharmacological properties to treat diseases.Azadirachta indica also known as neem and Moringa oleifera also known as moringa are both native Indian trees known for their high medicinal values due to their curative properties [9–15]. A. indica has analgesic, antifungal, and antibacterial properties and has therefore been used as treatment for gastrointestinal ailments, mouth hygiene, and certain chronic diseases such as diabetes, high blood pressure, and dyslipidemia [16–19]. On the other hand, M. oleifera has been reported to have antiviral, antioxidant, antisclerotic, antibacterial, and anti-inflammatory properties. It has been used as treatment for malaria, malnutrition, colon cancer, and myeloma [20].Even though these plants have been studied to treat the previously mentioned ailments, no research evaluates if time is directly correlated with the antibacterial effect ofA. indica against E. faecalis. On the other hand, there has yet to be any research on the antibacterial effect of M. oleifera or its cytotoxicity as a root canal disinfectant.The objective of this study was to evaluate the antibacterial and cytotoxic properties of methanolic extracts ofA. indica and M. oleifera against some Enterococcus faecalis strains (ATCC 29212) in vitro.


## 2. Materials and Methods

### 2.1. Sample

The sample size was determined with a formula of comparative means using the statistical software Stata® version 12.0. A confidence interval of 95% was considered, and a power of 80% was used. The mean and standard deviation parameters for a group were obtained using a pilot test that was previously run. Finally, a sample of 6 wells per group was established.


### 2.2. Extract Preparation

FreshA. indica leafs and pulverized M. oleifera leafs were obtained from a naturist shop. Both were free of impurities and had a sanitary registry. These products were placed in different containers and were treated with absolute methanol (1 : 2 weight/volume) to be later stored at room temperature for 10 days without sunlight exposure. The solutions were filtered through Whatman paper no. 4 and placed in labeled tubes. A rotatory evaporator was then used at 50°C to separate the methanol by distillation to finally obtain a pure extract. The extracts were stored at 4°C until use.


### 2.3. Bacterial Cultures

Enterococcus faecalis ATCC 29212 was obtained from GenLab Laboratory in Peru, a representative of MicroBiologics Laboratory (USA). The bacteria were cultured in agar BHI (brain heart infusion) in anaerobic conditions at 37°C for 72 hours. 3 to 4 colonies were then isolated and later inoculated in 3 mL of a BHI broth under the same conditions previously mentioned. The cultures were then diluted in a sterile saline solution to reach a McFarland scale density of 0.5, which approximately estimates a concentration of 1.5 × 108 CFU/mL.


### 2.4. Antimicrobial Activity Evaluation

Antimicrobial activity evaluation was achieved using the well diffusion method [21]. BHI agar was prepared and autoclaved at 121°C for 15 minutes. The agar was left to cool, and then the previously prepared bacterial suspension was inoculated. Finally, we proceeded to add the agar to sterile petri dishes. A sterile cork borer was used to punch 9 mm holes on the agar plate which were then filled with 1000 mL of each methanolic extract. In addition, 2% chlorhexidine was used as the positive control and 1× saline solution as the negative control. The agar plates were incubated at 37°C, and the diameters of the inhibition growth zones were measured during the first 24 and 48 hours in millimeters with a vernier caliper.


### 2.5. Determination of the Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration was determined using the microdilution method described by Gupta and Negi [22]. Each methanolic extract underwent serial dilutions (1/2), and each solution was added to the wells in the petri dishes with concentrations ranging from 1.56 to 75 µg/ml. In addition, 2% chlorhexidine was used as the positive control and 1× saline solution as the negative control. The plates were incubated under anaerobic conditions at 37°C for 24 hours. The minimum inhibitory concentration was considered as the minimum concentration of the extract that inhibited bacterial growth.


### 2.6. Determination of the Minimum Bactericidal Concentration (MBC)

The methanolic extract dilutions were added into each well of a 96-well microtiter plate, and then the bacterial suspension was inoculated. The plate was incubated at 37°C for 24 hours. In order to determine the minimum bactericidal concentration (MBC), aliquots were pipetted out of each well and seeded onto the agar. The sample petri dishes were incubated under anaerobic conditions at 37°C for 24 hours. The minimum bactericidal concentration is defined as the minimum concentration required to inhibit any bacterial growth.


### 2.7. Cytotoxicity Evaluation

Cytotoxicity was evaluated by means of a colorimetric assay based on the reduction of MTT by mitochondrial enzymes [23]. A 96-well microtiter plate was used to culture 1 × 104 cells per well. The plate was then incubated at 37°C in a humid atmosphere of 5% CO2 for 24 hours. Subsequently, we proceeded to pipette the methanolic extracts Azadirachta indica (neem) and M. oleifera (moringa) with concentrations ranging from 0 to 100 µg/mL onto the monocellular layer. Each concentration assay had a positive control for cellular viability (culture medium without any extracts). The plates were incubated at 37°C for 6 days, and the morphology of the cells was surveilled daily. The cytotoxic concentration 50 (CC50) is defined as the concentration of a substance required to decrease cellular viability by 50%.Twenty microliters of MTT solution (3 mg/ml in PBS 1X) was added into each well and then left incubating for 3 hours. The medium was carefully removed to obtain the formazan crystals which were then diluted with 200µL of DMSO (dimethyl sulfoxide). In order to determine cellular viability, a microplate photometer was used to measure and compare the absorbance of the treated cultures against the nontreated culture. The results were analysed with the computer software Pharm/PCS. The monolayers were observed under the microscope to evaluate any morphological changes.


### 2.8. Statistical Analysis

The antibacterial effect was registered in millimeters (mm). It was analysed by means of a nonparametric test, Kruskal–Wallis, with a statistical significance level of 5% for the comparison betweenA. indica, M. oleifera, and chlorhexidine. Data were analysed using the statistical package Stata® version 12.0.


## 2.1. Sample

The sample size was determined with a formula of comparative means using the statistical software Stata® version 12.0. A confidence interval of 95% was considered, and a power of 80% was used. The mean and standard deviation parameters for a group were obtained using a pilot test that was previously run. Finally, a sample of 6 wells per group was established.


## 2.2. Extract Preparation

FreshA. indica leafs and pulverized M. oleifera leafs were obtained from a naturist shop. Both were free of impurities and had a sanitary registry. These products were placed in different containers and were treated with absolute methanol (1 : 2 weight/volume) to be later stored at room temperature for 10 days without sunlight exposure. The solutions were filtered through Whatman paper no. 4 and placed in labeled tubes. A rotatory evaporator was then used at 50°C to separate the methanol by distillation to finally obtain a pure extract. The extracts were stored at 4°C until use.


## 2.3. Bacterial Cultures

Enterococcus faecalis ATCC 29212 was obtained from GenLab Laboratory in Peru, a representative of MicroBiologics Laboratory (USA). The bacteria were cultured in agar BHI (brain heart infusion) in anaerobic conditions at 37°C for 72 hours. 3 to 4 colonies were then isolated and later inoculated in 3 mL of a BHI broth under the same conditions previously mentioned. The cultures were then diluted in a sterile saline solution to reach a McFarland scale density of 0.5, which approximately estimates a concentration of 1.5 × 108 CFU/mL.


## 2.4. Antimicrobial Activity Evaluation

Antimicrobial activity evaluation was achieved using the well diffusion method [21]. BHI agar was prepared and autoclaved at 121°C for 15 minutes. The agar was left to cool, and then the previously prepared bacterial suspension was inoculated. Finally, we proceeded to add the agar to sterile petri dishes. A sterile cork borer was used to punch 9 mm holes on the agar plate which were then filled with 1000 mL of each methanolic extract. In addition, 2% chlorhexidine was used as the positive control and 1× saline solution as the negative control. The agar plates were incubated at 37°C, and the diameters of the inhibition growth zones were measured during the first 24 and 48 hours in millimeters with a vernier caliper.


## 2.5. Determination of the Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration was determined using the microdilution method described by Gupta and Negi [22]. Each methanolic extract underwent serial dilutions (1/2), and each solution was added to the wells in the petri dishes with concentrations ranging from 1.56 to 75 µg/ml. In addition, 2% chlorhexidine was used as the positive control and 1× saline solution as the negative control. The plates were incubated under anaerobic conditions at 37°C for 24 hours. The minimum inhibitory concentration was considered as the minimum concentration of the extract that inhibited bacterial growth.


## 2.6. Determination of the Minimum Bactericidal Concentration (MBC)

The methanolic extract dilutions were added into each well of a 96-well microtiter plate, and then the bacterial suspension was inoculated. The plate was incubated at 37°C for 24 hours. In order to determine the minimum bactericidal concentration (MBC), aliquots were pipetted out of each well and seeded onto the agar. The sample petri dishes were incubated under anaerobic conditions at 37°C for 24 hours. The minimum bactericidal concentration is defined as the minimum concentration required to inhibit any bacterial growth.


## 2.7. Cytotoxicity Evaluation

Cytotoxicity was evaluated by means of a colorimetric assay based on the reduction of MTT by mitochondrial enzymes [23]. A 96-well microtiter plate was used to culture 1 × 104 cells per well. The plate was then incubated at 37°C in a humid atmosphere of 5% CO2 for 24 hours. Subsequently, we proceeded to pipette the methanolic extracts Azadirachta indica (neem) and M. oleifera (moringa) with concentrations ranging from 0 to 100 µg/mL onto the monocellular layer. Each concentration assay had a positive control for cellular viability (culture medium without any extracts). The plates were incubated at 37°C for 6 days, and the morphology of the cells was surveilled daily. The cytotoxic concentration 50 (CC50) is defined as the concentration of a substance required to decrease cellular viability by 50%.Twenty microliters of MTT solution (3 mg/ml in PBS 1X) was added into each well and then left incubating for 3 hours. The medium was carefully removed to obtain the formazan crystals which were then diluted with 200µL of DMSO (dimethyl sulfoxide). In order to determine cellular viability, a microplate photometer was used to measure and compare the absorbance of the treated cultures against the nontreated culture. The results were analysed with the computer software Pharm/PCS. The monolayers were observed under the microscope to evaluate any morphological changes.


## 2.8. Statistical Analysis

The antibacterial effect was registered in millimeters (mm). It was analysed by means of a nonparametric test, Kruskal–Wallis, with a statistical significance level of 5% for the comparison betweenA. indica, M. oleifera, and chlorhexidine. Data were analysed using the statistical package Stata® version 12.0.


## 3. Results

### 3.1. Antimicrobial Activity of the Natural Extracts

After the first 24 hours, the methanolicA. indica extract generated a growth inhibition zone of 33.16 mm and the extract of M. oleifera generated a 35.5 mm zone. At 48 hours, the growth inhibition zones were 38.83 mm and 44.83 mm, respectively. The methanolic extract of M. oleifera had a greater antibacterial effect in comparison with chlorhexidine (control group) and the methanolic extract of A. indica. Furthermore, statistically significant differences were found for both groups at 24 and 48 hours (p<0.01) (Table 1).Table 1
In vitro antibacterial effect of 2% chlorhexidine and the methanolic extracts of Moringa oleifera and Azadirachta indica against Enterococcus faecalis during the first 24 and 48 hours.
Time
Extracts
Growth inhibition zone (mm)
Mean
Standard deviation
Median
Minimum
Maximum
p
∗
24 hours
Azadirachta indica
33.16
0.75
33.00
32.00
34.00
<0.01
Moringa oleifera
35.50
1.05
35.50
34.00
37.00
Chlorhexidine
19.16
1.33
20.00
17.00
20.00
48 hours
Azadirachta indica
39.83
3.37
42.00
35.00
42.00
<0.01
Moringa oleifera
44.83
0.98
44.50
44.00
46.00
Chlorhexidine
27.00
1.09
27.00
26.00
29.00
∗Kruskal–Wallis test.


### 3.2. MIC, MBC, and Bacteriostatic Effect ofA. indica and M. oleifera Extracts

The MIC of both the methanolic extracts,A. indica and M. oleifera, was 75 µg/ml, demonstrating a bacteriostatic concentration up to this value.Additionally, the bactericidal concentrations forA. indica and M. oleifera were 25 µg/ml and 75 µg/ml, respectively (Table 2).Table 2
Bacteriostatic (MIC) and bactericidal (MBC) of 2% chlorhexidine and methanolic extracts ofMoringa oleifera and Azadirachta indica against Enterococcus faecalis strains.
Concentrations (µg/ml)
Study groups
Azadirachta indica
Moringa oleifera
Chlorhexidine
100
—
—
—
75
MIC
MIC/MBC
—
50
—
—
—
25
MBC
—
—
12.5
—
—
MIC
6.25
—
—
—
3.125
—
—
1.56
—
—
MBC


### 3.3. Cytotoxicity of the Methanolic Extracts ofA. indica and M. oleifera

The results indicate that the methanolic extracts ofA. indica and M. oleifera inhibit 50% of cellular viability (CC50) at a concentration of 70 µg/ml. Furthermore, none of the extracts produced any adverse effects on the MDCK cellular lines at low concentrations. We observed an indirect correlation between the concentration and cellular viability (Figure 1).Figure 1
Cellular viability evaluation of Jurkat cells against the methanolic extracts ofMoringa oleifera and Azadirachta indica against Enterococcus faecalis strains.


## 3.1. Antimicrobial Activity of the Natural Extracts

After the first 24 hours, the methanolicA. indica extract generated a growth inhibition zone of 33.16 mm and the extract of M. oleifera generated a 35.5 mm zone. At 48 hours, the growth inhibition zones were 38.83 mm and 44.83 mm, respectively. The methanolic extract of M. oleifera had a greater antibacterial effect in comparison with chlorhexidine (control group) and the methanolic extract of A. indica. Furthermore, statistically significant differences were found for both groups at 24 and 48 hours (p<0.01) (Table 1).Table 1
In vitro antibacterial effect of 2% chlorhexidine and the methanolic extracts of Moringa oleifera and Azadirachta indica against Enterococcus faecalis during the first 24 and 48 hours.
Time
Extracts
Growth inhibition zone (mm)
Mean
Standard deviation
Median
Minimum
Maximum
p
∗
24 hours
Azadirachta indica
33.16
0.75
33.00
32.00
34.00
<0.01
Moringa oleifera
35.50
1.05
35.50
34.00
37.00
Chlorhexidine
19.16
1.33
20.00
17.00
20.00
48 hours
Azadirachta indica
39.83
3.37
42.00
35.00
42.00
<0.01
Moringa oleifera
44.83
0.98
44.50
44.00
46.00
Chlorhexidine
27.00
1.09
27.00
26.00
29.00
∗Kruskal–Wallis test.


## 3.2. MIC, MBC, and Bacteriostatic Effect ofA. indica and M. oleifera Extracts

The MIC of both the methanolic extracts,A. indica and M. oleifera, was 75 µg/ml, demonstrating a bacteriostatic concentration up to this value.Additionally, the bactericidal concentrations forA. indica and M. oleifera were 25 µg/ml and 75 µg/ml, respectively (Table 2).Table 2
Bacteriostatic (MIC) and bactericidal (MBC) of 2% chlorhexidine and methanolic extracts ofMoringa oleifera and Azadirachta indica against Enterococcus faecalis strains.
Concentrations (µg/ml)
Study groups
Azadirachta indica
Moringa oleifera
Chlorhexidine
100
—
—
—
75
MIC
MIC/MBC
—
50
—
—
—
25
MBC
—
—
12.5
—
—
MIC
6.25
—
—
—
3.125
—
—
1.56
—
—
MBC


## 3.3. Cytotoxicity of the Methanolic Extracts ofA. indica and M. oleifera

The results indicate that the methanolic extracts ofA. indica and M. oleifera inhibit 50% of cellular viability (CC50) at a concentration of 70 µg/ml. Furthermore, none of the extracts produced any adverse effects on the MDCK cellular lines at low concentrations. We observed an indirect correlation between the concentration and cellular viability (Figure 1).Figure 1
Cellular viability evaluation of Jurkat cells against the methanolic extracts ofMoringa oleifera and Azadirachta indica against Enterococcus faecalis strains.


## 4. Discussion

There are many different types of endodontic infections described. One of them is the persistent root canal infections for which the highly antibiotic-resistantEnterococcus faecalis is the main etiologic agent, even amongst patients previously treated for this condition [2]. The pathogenicity of these bacteria includes its capacity to penetrate dentinal tubules, its lack of need of other bacteria to survive, its ability to form biofilms in anaerobic and nutrient lacking conditions, and its resistance to acids and alkalis [5, 6, 12]. It is therefore necessary to study new antibacterial agents specific for this bacteria in order to achieve endodontic treatments with a higher success rate and a better prognosis for the tooth.As a result of the well diffusion method, we were able to determine that the methanolic extracts forA. indica and M. oleifera had an antibacterial effect against E. faecalis during the first 24 and 48 hours. According to Olson and Fahey, the antibacterial effect of M. oleifera is due to the chemical compound 4-(4'-O-acetyl-α-L-rhamnopyranosyloxy)-benzylisothiocyanate, whose mechanism of action involves inhibition of essential cellular membrane enzymes [24, 25]. Moreover, Lakshmi et al. mentioned that the active compound responsible for the antibacterial efficacy of A. indica is azadirachtin, a cellular membrane synthesis inhibitor [16, 26].We also found the MIC of the extracts and concluded that no antibacterial effect was evidenced at low concentrations. In 2013, Reyes and Fernández found the MIC of the foliar extract ofA. indica against Staphylococcus aureus to be 35 µg/ml [27]. Muhammad et al. also evaluated the MIC of an aqueous extract of M. oleifera against S. aureus and found it to be 6.25 µg/ml. This difference could be explained, given the fact that the process of maceration was 3 times the present study, extracting a greater quantity of chemical compounds of M. oleifera [28].The minimum bactericidal concentration for the methanolic extract ofA. indica was 25 µg/ml and 75 µg/ml for M. oleifera which leads us to conclude that at lower concentrations, A. indica has a greater capacity to cause cellular lysis of the bacteria in comparison with M. oleifera.The methanolic extracts ofAzadirachta indica (neem) and Moringa oleifera (moringa) did not show any cytotoxicity against the cellular line of MDCK under low concentrations. Jung evaluated the cytotoxicity of the extract of M. oleifera against a cellular line Cos-7 and demonstrated a lack of cytotoxicity even with concentrations of 600 mg/ml [20]. In 2015, Jumba et al. evaluated the cytotoxicity of the methanolic extract of A. indica against the cellular line Vero-E6 and determined a CC50 of 149 µg/ml. Despite the difference in these results, the study concludes that the extract stays within a manageable parameter [29].Additionally, anin vivo study with rats showed that none of the subjects suffered any ailment after treatment with A. indica extract.In conclusion, according to this study,A. indica and M. oleifera demonstrated an antibacterial effect against E. faecalis without any toxicity using low concentration. Therefore, they could be considered as alternative antimicrobial agents to use in the root canal therapy field in Odontology. More research is required regarding the cytotoxicity of each of its active components and also its adverse reactions.The values that were obtained from this study show a direct correlation between the time of exposure to the extract its antibacterial effect. These results are important to continue a line of investigation in relation to fitotherapy applied to Odontology. The purpose of studies in this field is to promote the future creation of treatments based on these natural herbs considering that their use in radicular conduct treatment would lead to a better prognosis.



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*Source: 1071676-2018-09-25.xml*