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ORIGINAL ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 1  |  Page : 16-20

Antibacterial effects of moringa oleifera leaf extract against enterococcus faecalis in vitrio


1 Department of Conservative Dentistry, Faculty of Dentistry, Trisakti University, West Jakarta, Indonesia
2 Department of Biochemistry and Molecular Biology, Faculty of Dentistry, Trisakti University, West Jakarta, Indonesia

Date of Submission01-Oct-2019
Date of Acceptance11-Jan-2020
Date of Web Publication7-Feb-2020

Correspondence Address:
Dr. Piasti Sopandani
Department of Conservative Dentistry, Faculty of Dentistry, Trisakti University, West Jakarta
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/SDJ.SDJ_43_19

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  Abstract 


Background: The aim of endodontic treatment is to eliminate bacteria from the root canal. Bacterial removal from the root canal can be achieved with a mechanical approach using an instrument and a disinfecting irrigation agent. Enterococcus faecalis is the most prevalent bacteria found in root canals. Research studies have also been conducted to examine irrigation systems using herbal products such as drumstick tree leaf (Moringa oleifera) instead of NaOCl. Active compounds in M. oleifera, such as flavonoids, tannins, saponins, alkaloids, phenolics, and triterpenoids, possess antibacterial effects. Objectives: The aim of this study is to assess the antibacterial effect of drumstick tree extract (M. oleifera) in several concentrations (25%, 50%, 75%, and 100%) as an irrigation solution against E. faecalis through in the root canal ex vivo. Methods: This study used 24 mandibular premolars, divided into six category groups. Group 1 received 5.25% NaOCl as a positive control, Group 2 received 25% M. oleifera extract, Group 3 received 50% M. oleifera extract, Group 4 received 75% M. oleifera extract, Group 5 received 100% M. oleifera extract, and Group 6 received irrigation using phosphate-buffered saline as a negative control. Quantitative polymerase chain reaction methods used to analyze the E. faecalis number in the root canal after treatment with M. oleifera extract. Results: A one-way ANOVA showed significant differences (P = 0.05) between the three types of irrigation solutions against E. faecalis. Among the six study groups, the most prominent efficacy was found in Groups 1, 4, and 5. Conclusion: M. oleifera extract solution at concentrations of 75% and 100% is as effective as 5.25% NaOCl. This extract may be used as an alternative irrigation agent for root canal treatment. However, further studies are warranted to examine the toxicity effect.

Keywords: Antibacterial, Enterococcus faecalis, irrigation agent, Moringa oleifera


How to cite this article:
Sopandani P, Iskandar BO, Ariwibowo T, Djamil MS. Antibacterial effects of moringa oleifera leaf extract against enterococcus faecalis in vitrio. Sci Dent J 2020;4:16-20

How to cite this URL:
Sopandani P, Iskandar BO, Ariwibowo T, Djamil MS. Antibacterial effects of moringa oleifera leaf extract against enterococcus faecalis in vitrio. Sci Dent J [serial online] 2020 [cited 2020 Mar 28];4:16-20. Available from: http://www.scidentj.com/text.asp?2020/4/1/16/277876




  Background Top


The use of irrigation solutions during the cleaning and shaping process involved in root canal treatment can efficiently reduce the number of microorganisms present in the root canal. Mechanical preparation and irrigation of the root canal using a solution suitable for oral and periapical tissues is very important. The solution used for irrigation must be able to eliminate bacteria without damaging dental and supporting tissues.[1]

Sodium hypochlorite (5.25%) is widely used as a root canal irrigation solution because it has a bactericidal effect to Enterococcus faecalis; however, it is also toxic.[2] Several studies have shown that Moringa oleifera has biocompatible and antibacterial effects on tissues. M. oleifera leaf extract has antibacterial activity because it contains saponins, flavonoids, tannins, alkaloids, phenolics, and triterpenoids. Each compound has its own mechanism of destroying bacteria.[3] This study will examine the antibacterial effect of M. oleifera leaf extract, as a root canal irrigation solution, on E. faecalis.


  Materials and Methods Top


Phytochemical screening

Moringa leaf extract was subjected to phytochemical screening to detect plant compounds according to class. Alkaloid examination was conducted by means of 1 mL of extract added to a few drops of Wagner reagent and Meyer reagent. A positive reaction was indicated if brown sediment formed in Wagner reagent and white sediment formed in Meyer reagent. Flavonoid examination was carried out using three reagents, namely 10% NaOH reagent, Wilsatater reagent, and Smith-Metacalve reagent. As much as 1 ml of extract was added to a few drops of 10% NaOH, and a positive reaction was indicated if specific color changes occurred. Wilsatater reagent was created by mixing 1 ml of extract with a few drops of concentrated HCl with a magnesium powder (Mg); a positive reaction was indicated if there was a red-orange color change. The Smith-Metacalve reagent was created by mixing 1 ml of the extract with a few drops of concentrated HCl and then heating it; a positive reaction was indicated if it took on a white color. Saponin examination was conducted by mixing 1 mL of extract with hot water then shaking this mixture; a positive reaction was indicated if durable foam was formed. Polyphenol examination was conducted by mixing 1 ml of the extract with 1% FeCl3 reagent; a positive reaction was indicated if a blackish or dark blue color formed. Tannin examination was conducted by mixing 1 ml of 3% ferric chloride with 1 mL of the extract; brownish-green color development indicated the presence of tannins. Phytochemical tests were carried out in the laboratory of the Spice and Medicinal Plants Research Institute (BALITTRO), Bogor, Indonesia.

Bacterial culture

E. faecalis ATCC 29212 bacteria were taken from the stock and then, 50 μL of bacteria were cultured into 5 mL of brain heart infusion (BHI) broth and incubated at 37°C for 24 h. Then, the bacteria were diluted to reach McFarland 0.5 or the equivalent of a bacterial density of 1.5 × 108 CFU.

Teeth preparation

The selection criteria required that the teeth used in this study are lower premolar teeth with completely closed roots, no root caries, and no history of previous root canal treatment. Each tooth also had a single, straight root canal, which was confirmed by periapical photographs. The teeth crown were shortened with a round edge wheel bur (Mani, Japan) so that all tooth samples had the same root length of 13 mm. Root length was confirmed using file #10 until the tip was visible at the apex of each tooth.

The root canal of each tooth was prepared using the crown down technique with TF Adaptive files. The TFA small/medium (SM) shaping sequence consists of three NiTi instruments with the following tip sizes and tapers: SM1 (20.04), SM2 (25.06), and SM3 (35.04). Each root canal was prepared in the following order: File SM1, SM2, and SM3. Each file change was irrigated with 5.25% NaOCl (Cerkamed, Poland). The prepared teeth were then irrigated with 5.25% NaOCl, rinsed with distilled water, irrigated with 17% ethylenediaminetetraacetic acid, rinsed again with distilled water, irrigated with 2% chlorhexidine, and then dried with paper points. The outer surface of each tooth was coated with two layers of nail polish to avoid bacterial contamination. Then, the teeth were sterilized in an autoclave (TOMY ES-315, SEIKO Japan) at 121°C and a pressure level of 15 pounds per square inch for 15 min [Figure 1].
Figure 1: Lower maxillary premolar teeth, shortened the crown to a root length of 12 mm and prepared for a root canal treatment

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Sample preparation

Samples were obtained from the 5.25% NaOCl irrigation solution, and M. oleifera extract solutions (25%, 50%, 75%, and 100%) were injected into the prepared root canals. Each root canal contained a culture of up to 100 μL of E. faecalis in BHI broth, which was incubated for 48 h at 37°C. Twenty-four teeth were randomly divided into six groups, and each root canal was irrigated with 5 mL of the prepared irrigation solution. In Group 1, the root canals were irrigated with 5 mL of 5.25% NaOCl as the positive control. In Group 2, the root canals were irrigated with 5 mL of 25% M. oleifera extract solution. In Group 3, the root canals were irrigated with 5 mL of 50% M. oleifera extract solution. In Group 4, the root canals were irrigated with 5 mL of 75% M. oleifera extract solution. In Group 5, the root canals were irrigated with 5 mL of 100% M. oleifera extract solution. In Group 6, the root canals were irrigated with 5 mL of phosphate-buffered saline (PBS) as a negative control. Each solution tested was in contact with the root canal for 40 min in each group then, the stagnant solution in the root canals was collected using a paper point and rinsed with 5 mL of PBS. The remaining liquid, which was wasted during PBS rinsing, was placed in a centrifuge tube and mixed with the rinsed teeth. Then, put it into a vortex mixer for 10 s. Subsequently, the teeth were removed from the tube and proceeded with centrifugation at a speed of 4000 rpm for 5 min. The bacterial cell in the supernatant was calculated using plate count methods and confirmed with reverse transcription-polymerase chain reaction.

Plate count method

Dilution was carried out by mixing 10 μL of liquid in the centrifuge tube with 990 μL of PBS, transferring it to the microtube using a micropipette, and then transferring it to the vortex. A total of 50 μL of liquid was aspirated from the microtube and examined on a  Petri dish More Details. A triangle spreader was used to spread and then count the number of bacterial colonies that formed manually 24 h after incubation.

Quantitative polymerase chain reaction

DNA extraction was conducted using the heat shock method, in which 1 mL of the irrigation treatment results were centrifuged at a speed of 4500 rpm for 10 min. Then, the pellets in the microtube were rinsed with 1 mL of PBS and then placed in the vortex mixer. Centrifugation proceeded at 10,000 rpm for 10 min. Then, the pellets were resuspended with 100 μL of ddH2O. The pellets were heated in a 100°C water bath for 20 min, incubated in ice for 10 min, and then placed in vortex mixer to mix it uniformly. Centrifugation proceeded at 10,000 rpm for 2 min. The supernatant containing DNA was transferred to a new tube and stored at −20°C. The products of DNA extraction (2 μL) were mixed with a master mix containing 10 μL of SYBR Green as a coloring reagent, 0.8 μL of forward primers, 0.8 μL of reverse primers, and 6.4 μL of nuclease-free water. Overall, 24 samples were used and duplicated. Amplification began with an initial denaturation at 95°C for 5 min. One cycle included denaturation for 10 s at 95°C and a combination of annealing and extension cycles at 60°C for 30 s, respectively. QuantiFast SYBR Green was used as a fluorescent detector. Fluorescence data were measured using a real-time thermocycler.

Statistical analysis

The Shapiro–Wilk test was used to analyze whether the data are normally distributed. One-way analysis of variance test was applied to analyze significant differences of E. faecalis number after treatment with M. oleifera extract in different concentrations. Differences were considered statistically significant if P < 0.05. Statistical calculations were performed with SPSS Statistics for Windows software version 20 (IBM Corp. Released 2011. Armonk, NY: IBM Corp.).


  Results Top


The results of phytochemical tests stated that the ethanol extract of M. oleifera contained alkaloids, saponins, tannins, phenolics, flavonoids, triterpenoids, and glycosides [Table 1]. The results of identification of the twigs and leaves were done by Botanical Garden Plant Conservation Center (LIPI) showed that they were confirmed of the M. oleifera Lam., Moringaceae, Moringa group.
Table 1: Phytochemical results from ethanol extract Moringa oleifera

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The E. faecalis number was significantly decreased after irrigation with M. oleifera extract (P < 0.05). The most effective concentration was 100% M. oleifera extract [Figure 2], [Figure 3], [Figure 4]. The results for one-way ANOVA, which confirms that the differences were statistically significant in all concentrations to reduce the E. faecalis number compared to the negative control (P < 0.05).
Figure 2: Enterococcus faecalis number (CFU/mL) after treatment with Moringa oleifera extract with different concentrations (100%, 75%, 50%, and 25%) and the standard plate count method was carried out. Phosphate-buffered saline was used as a negative control and 5.25% NaOCl as a positive control. All treatments were repeated four times

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Figure 3: Enterococcus faecalis colonies after treatment with Moringa oleifera extract with different concentrations (100%, 75%, 50%, and 25%) in brain heart infusion agar. Phosphate-buffered saline was used as a negative control and 5.25% NaOCl as a positive control

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Figure 4: The numbers of Enterococcus faecalis after treatment with Moringa oleifera extract with different concentration (100%, 75%, 50%, and 25%) and quantitative polymerase chain reaction test was carried out. Phosphate-buffered saline was used as a negative control and 5.25% NaOCl as a positive control. All treatments were performed in duplicate

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  Discussion Top


According to Retamozo, a 40-min application, 5.25% sodium hypochlorite is very effective at killing E. faecalis.[4] Although sodium hypochlorite is widely used as an irrigation solution, it is also toxic.[2] Therefore, this study investigated natural materials as root canal treatment irrigation agents that could effectively kill E. faecalis and were biocompatible with dental tissue and its surroundings. Several studies have shown that M. oleifera, commonly referred to as Moringa leaves, have antibacterial, biocompatible effects on tissues.[3]

M. oleifera leaf extract has antibacterial activity because it contains saponins, flavonoids, tannins, alkaloids, phenolics, and triterpenoids.[3] The antibacterial effect of saponins works by interfering with the permeability of the bacterial cell wall.[5],[6] Flavonoid compounds can form complex compounds with proteins through hydrogen bonds so that the tertiary structure of the protein is disrupted, and the protein can no longer function, causing damage or denaturation of proteins and nucleic acids. This denaturation causes protein coagulation and interferes with the metabolism and physiological functions of bacteria.[7] Flavonoids also inhibit cell membrane synthesis and aggregate effects on all bacterial cells.[8] Tannin compounds can inhibit protein synthesis for cell wall formation and shrink cell walls, thereby disrupting cell permeability and leading to death.[9],[10] Alkaloids have an antibacterial, inhibitory mechanism in which they interfere with the constituent components of peptidoglycan in bacterial cells so that intact cell wall layers are not formed, thus causing cell death.[11] Terpenoid compounds can react with porin on the outer membrane of the bacterial cell wall and form strong polymeric bonds that cause damage to the pile; this results in the entry of compounds that reduce the permeability of the bacterial cell wall so that the bacterial cell lacks nutrients and bacterial growth is inhibited or dead.[12]

According to research conducted with mice, Tiloke stated that the use of 70 g of M. oleifera per day is relatively safe.[13] In the present study, M. oleifera extraction was completed using 60% ethanol solvent. Plant extraction with 60% ethanol solvent proved to be the best compared to extractions using other concentrations. This result is consistent with research conducted by Luginda (2018), which stated that the use of 60% ethanol as a solvent extract for plants containing flavonoids yields the highest total flavonoid levels compared to ethanol with concentrations of 70%, 80%, and 96%.[14] This is important because higher flavonoid levels correspond to higher antibacterial effectiveness.[15] Ethanol solvents have hydroxyl groups, which can bind polar compounds such as flavonoids and alkaloids.[16] Research conducted by Piexoto showed that M. oleifera extract with ethanol solvent is more effective in inhibiting the growth of E. faecalis compared to distilled water. M. oleifera extract solution contains compounds that can inhibit the growth of Gram-positive and Gram-negative bacteria.[17]

In the present study, the amount of E. faecalis in the root canal decreased with higher concentrations of M. oleifera extract solution. These results are in accordance with the results of a study, in which Shailemo examined the antibacterial effects of M. oleifera extract with concentrations of 10%, 20%, 35%, and 50%.[18]M. oleifera antibacterial test results at a concentration of 10%, 20%, and 35% showed only a slight turbidity in the diluted tube compared with distilled water that looks more turbid.[18] A study conducted by Arevaro et al. showed that 75% M. oleifera is the lowest concentration of M. oleifera extract that can kill E. faecalis.[19] Therefore, samples in the present study were treated using M. oleifera extract at concentrations of 25%, 50%, 75%, and 100%. The present study also showed that 75% and 100% M. oleifera extract solutions were as effective as 5.25% NaOCl in killing E. faecalis in root canals. However, the amount of E. faecalis was lower in root canals irrigated with 5.25% NaOCl than in root canals irrigated with the highest concentration of M. oleifera extract solution (100%).


  Conclusion Top


Based on the results of the present study, it can be concluded that M. oleifera extract solution has an antibacterial effect against E. faecalis at concentrations of 50%, 75%, and 100%. M. oleifera extract solution at concentrations of 75% and 100% is as effective as 5.25% NaOCl. This extract may be used as an alternative irrigation for the root canal treatment. However, further studies are warranted to examine the toxicity effect.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Hajimaghsoodi S, Zandi H, Bahrami M, Hakimian R. Laboratory comparison of the anti-bacterial effects of spearmint extract and hypochlorite sodium on Enterococcus faecalis bacteria. J Dent Biomater 2016;3:322-6.  Back to cited text no. 1
    
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Spencer HR, Ike V, Brennan PA. Review: The use of sodium hypochlorite in endodontics potential complications and their management. Br Dent J 2007;202:555-9.  Back to cited text no. 2
    
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Wang L, Chen X, Wu A. Mini review on antimicrobial activity and bioactive compounds of Moringa oleifera. Med Chem (Los Angeles) 2016;6:578-82.  Back to cited text no. 3
    
4.
Retamozo B, Shabahang S, Johnson N, Aprecio RM, Torabinejad M. Minimum contact time and concentration of sodium hypochlorite required to eliminate Enterococcus faecalis. J Endod 2010;36:520-3.  Back to cited text no. 4
    
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Podolak I, Galanty A, Sobolewska D. Saponins as cytotoxic agents: A review. Phytochem Rev 2010;9:425-74.  Back to cited text no. 5
    
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Arabski M, Węgierek-Ciuk A, Czerwonka G, Lankoff A, Kaca W. Effects of saponins against clinical E. coli strains and eukaryotic cell line. J Biomed Biotechnol 2012;2012:286216.  Back to cited text no. 6
    
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Xie Y, Yang W, Tang F, Chen X, Ren L. Antibacterial activities of flavonoids: Structure-activity relationship and mechanism. Curr Med Chem 2015;22:132-49.  Back to cited text no. 7
    
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Cushnie TP, Lamb AJ. Antimicrobial activity of flavonoids. Int J Antimicrob Agents 2005;26:343-56.  Back to cited text no. 8
    
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Length F.In vitro evaluation of the interaction between tea extracts and penicillin G against Staphylococcus aureus. Afr J Biotechnol 2006;5:1082-6.  Back to cited text no. 9
    
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Akiyama H, Fujii K, Yamasaki O, Oono T, Iwatsuki K. Antibacterial action of several tannins against Staphylococcus aureus. J Antimicrob Chemother 2001;48:487-91.  Back to cited text no. 10
    
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Cushnie TP, Cushnie B, Lamb AJ. Alkaloids: An overview of their antibacterial, antibiotic-enhancing and antivirulence activities. Int J Antimicrob Agents 2014;44:377-86.  Back to cited text no. 11
    
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Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev 1999;12:564-82.  Back to cited text no. 12
    
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Tiloke C, Anand K, Gengan RM, Chuturgoon AA. Moringa oleifera and their phytonanoparticles: Potential antiproliferative agents against cancer. Biomed Pharmacother 2018;108:457-66.  Back to cited text no. 13
    
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Luginda RA, Lohita B, Indriani L. Effect of variations in the concentration of ethanol solvents on total flavonoid levels of beluntas leaves by the microwave method. Online Student Journal (JOM) in the Pharmaceutical Field 2018;1:1-9.  Back to cited text no. 14
    
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Manik DF, Hahamani T, Anshory H. Analysis of the correlation between levels of flavonoids with the antibacterial activity of ethanol extract and fractions of cherry leaves (Muntingia calabura) against Staphylococcus aureus. Khazanah UII Student J 2014;6:1-11.  Back to cited text no. 15
    
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Nuraini N, Ilyas A, Novianty I. Identification and characterization of anticancer bioactive compounds from ethanol extracts of bitti bark (Vitex cofassus). Alchemy 2015;3:15-27.  Back to cited text no. 16
    
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Peixoto JR, Silva GC, Costa RA, de Sousa Fontenelle JR, Vieira GH, Filho AA, et al.In vitro antibacterial effect of aqueous and ethanolic Moringa leaf extracts. Asian Pac J Trop Med 2011;4:201-4.  Back to cited text no. 17
    
18.
Shailemo DH, Kwaambwa HM, Kandawa-Schulz M, Msagati TA. Antibacterial activity of Moringa ovalifolia and Moringa oleifera methanol, N-Hexane and water seeds and bark extracts against pathogens that are implicated in water borne diseases. Green Sustain Chem 2016;6:71-7.  Back to cited text no. 18
    
19.
Arévalo-Híjar L, Aguilar-Luis MÁ, Caballero-García S, Gonzáles-Soto N, Del Valle-Mendoza J. Antibacterial and cytotoxic effects of Moringa oleifera (Moringa) and Azadirachta indica (Neem) methanolic extracts against strains of Enterococcus faecalis. Int J Dent 2018;2018:1071676.  Back to cited text no. 19
    


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