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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 5  |  Issue : 3  |  Page : 138-143

The effect of garlic extract (Allium sativum L.) (Amaryllidaceae) to eradicate the Porphyromonas endodontalis biofilm: An in-vitro research


1 Department of Conservative Dentistry, Trisakti University, Jakarta, Indonesia
2 Department of Oral Biology, Faculty of Dentistry, Trisakti University, Jakarta, Indonesia

Date of Submission24-Mar-2021
Date of Decision03-Aug-2021
Date of Acceptance29-Aug-2021
Date of Web Publication18-Oct-2021

Correspondence Address:
Adi Dharma Widjaya
Department of Conservative Dentistry, Faculty of Dentistry, Trisakti University, Jakarta.
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/SDJ.SDJ_79_21

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  Abstract 

Background: The Gram-negative bacterium Porphyromonas endodontalis is one of the causes of endodontic infections. Irrigation in root canal treatment was intended to eliminate bacteria. The garlic extract (Alium sativum Linnaeus) (Amaryllidaceae) is a natural material believed to have antimicrobial effects and may be used as an alternative irrigation solution. Objectives: The aim of this article is to evaluate the effect of garlic extract on the eradication of P. endodontalis biofilms. Methods: The A. sativum extract with concentrations of 100%, 50%, and 25%, NaOCl 5.25%, CHX 2% as a positive control, and sterile Aquadest as a negative control were used as irrigation solutions. A random sample of six groups was tested for P. endodontalis ATCC® 35406D-5TM biofilm eradication by irrigation of the A. sativum extract. Results: It was shown with a one-way analysis of variance that there was a significant difference in optical density (OD) between the results of biofilm eradication using A. sativum with concentrations of 100%, 50%, and 25% and NaOCl 5.25%, CHX 2%, and Aquadest (P < 0.05). With Tukey′s post hoc test, it was shown that the OD value from the eradication of A. sativum with concentrations of 100%, 50%, and 25% did not differ significantly compared with NaOCl 5.25% or CHX 2% (P > 0.05). Conclusion: The garlic extract (A. sativum) is effective in eradicating the biofilm of P. endodontalis in vitro.

Keywords: Allium sativum, biofilm, eradication, Porphyromonas endodontalis


How to cite this article:
Widjaya AD, Amin MF, Aryadi, Roeslan BO. The effect of garlic extract (Allium sativum L.) (Amaryllidaceae) to eradicate the Porphyromonas endodontalis biofilm: An in-vitro research. Sci Dent J 2021;5:138-43

How to cite this URL:
Widjaya AD, Amin MF, Aryadi, Roeslan BO. The effect of garlic extract (Allium sativum L.) (Amaryllidaceae) to eradicate the Porphyromonas endodontalis biofilm: An in-vitro research. Sci Dent J [serial online] 2021 [cited 2021 Nov 27];5:138-43. Available from: https://www.scidentj.com/text.asp?2021/5/3/138/328425




  Background Top


Based on the Indonesian Health Data (2011), pulp and periapical diseases in Indonesia are the seventh outpatient care disease in the country.[1] Pulp disease requires immediate attention to root canal treatment.[2] The pulp tissue is functioned by odontoblasts, which form a tertiary dentin layer to respond to various stimuli such as microbial, mechanical, and chemical irritants.[3] Microbial irritants are the primary irritants in the dental pulp tissue, containing bacteria from dental caries. Caries will continue to widen and reach the pulp, which may cause pulp disease.[4]

Root canal infection (pulp disease) is classified into two types, i.e., primary and secondary infections. Bacteria such as Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas endodontalis, Fusobacterium nucleatum, Dialister invisus, and Treponema denticola are usually involved in primary root canal infection. P. endodontalis can invade the endothelial cells of the heart arteries and can reactivate the Epstein—Barr virus (EBV).[5],[6] EBV was recently suggested to be involved in the pathogenesis of other chronic inflammatory infections occurring in the periodontium such as peri-implantitis and periapical periodontitis. In contrast to unexplained peri-implantitis, some interesting mechanisms are reported with periapical periodontitis, i.e., butyric acid excreted by P. endodontalis and F. nucleatum in addition to EBV is involved in the progression of periapical periodontitis. An underlying mechanism may involve reactive oxygen species released from EBV-infected host cells that induce local imbalance between receptor activator NF-κB and osteoprotegerin, which consequently promotes alveolar bone resorption.[6]

P. endodontalis is a Gram-negative stem bacterium found in the pulp disease. It also has a black stain derived from its primary pigments, protoheme, and protoporphyrin.[7],[8] The morphological characteristics of P. endodontalis have a smooth and shiny convex colony and have a black color diameter of 1—2 mm.[9]P. endodontalis was found in root canals and was always associated with endodontic infection, gingivitis, and pulp necrosis. Cao et al.[10] showed that P. endodontalis could be found in non-vital teeth.

In endodontic treatment, cleaning the root canals is usually done by irrigation to kill bacteria. The standard irrigation solution used for root canal treatment is still toxic to the surrounding soft tissue. The most commonly used irrigation solutions are sodium hypochlorite (NaOCl) and chlorhexidine (CHX). When exposed to the gingiva, NaOCl will cause a burning feeling and may cause necrosis in the periapical tissue if the irrigation solution goes out through the root apex.[11] Although the use of 2% CHX as an irrigation solution is acceptable to the soft tissue, it is still unsafe if accidentally swallowed.[12]

One of the natural ingredients believed to be an antimicrobial agent is garlic (A. sativum Linnaeus) (Amaryllidaceae). Historically, garlic has been used as a medicine.[13] Garlic has four configurations: essential garlic oil, macerated oil, garlic powder, and garlic extract. Garlic has two compounds (e.g., allicin and ajoene) that function as antibacterial. The two compounds have been shown to inhibit the growth of Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli). In the preliminary study of the bacterial test, it was found that the garlic extract had antibacterial properties against Pseudomonas aeruginosa at a concentration of 30%, whereas in another study with a concentration of 25—100%, it could inhibit the growth of E. coli and S. aureus.[14],[15] The allicin compound can modify and inhibit sulfhydryl. Using a 10% garlic solution in mouthwash may reduce the amount of bacteria in the mouth.[16] Based on these studies, it is still necessary to investigate the ability of garlic to eradicate P. endodontalis bacteria. This study was aimed to determine the effect of garlic on biofilm formation by P. endodontalis.


  Materials and Methods Top


Quantitative test for active compounds

Active compound identification using liquid chromatography—mass spectrometry (LC/MS) was performed on preparation of A. sativum after centrifugation at 4500 rpm for 15 min. The supernatants were transferred into 2 mL vials and placed in the LC/MS system. The quantitative test was performed at the Regional Health Laboratory (Lembaga Kesehatan Daerah), Jakarta, Indonesia.

Phytochemical tests

Steroid test

Drops of the test substance were placed in a measuring flask as much as 0.5 mL along with anhydrous acetic acid and then eluted with eluent. Concentrated sulfuric acid (H2SO4) was then added. Under normal light, color changes to blue/deep green confirming the presence of steroids.

Tannin test

The tested substance was added as much as 1 mL in drops into a measuring flask and a few drops of 0.1% ferric chloride (FeCl3) was added. It was eluted with eluent and observed if a bluish-green color is observed, which confirmed the presence of tannin.

Flavanoid test

The stock solution (1 mL) was taken in a test tube and a few drops of dilute NaOH solution was added. An intense yellow color that appeared in the test tube would become colorless when a few drops of dilute acid that indicated the presence of flavonoids was added.

Saponin test

Hydrochloric acid (HCl) amounting to 2 mL was added to 2 mL of the sample. It was mixed for 5 min and then observed. If the bubbles do not dissipate in 10 min, this confirmed the presence of saponin.

Terpenoid test

Chloroform amounting to 2 mL was added to 1 ml of the sample and then eluted with the eluent. Concentrated H2SO4 (sulfuric acid) amounting to 3 mL was then added. If the color changed to reddish-brown, then this confirmed the presence of terpenoid.

Bacterial culture of P. endodontalis ATCC® 35406D-5TM

Prior to opening the vial freeze-dried P. endodontalis ATCC® 35406D-5TM, a laminar is prepared which is filled with CO2 gas. ATCC® 35406D-5TM P. endodontalis was opened and then partly cultured in the liquid medium. An aliquot of 1 mL of enriched tryptic soy broth with 0.005% hemin, 0.05% L-cysteine HCl, 0.5% yeast extract, and 0.001% menadione was added into the vial to rehydrate the contents. The liquids with the bacteria were mixed with 5 mL of liquid medium in the tube and inoculated into an enriched blood agar medium and partly freeze-dried. All procedures were performed in an anaerobic atmosphere (5% CO2, 20% N2, 75% H2). Bacteria in the liquid medium as well as those in the blood agar are put into an anaerobic jar with addition of GasPack (AnaeroGen™, Thermo Fisher, Waltham, MA, USA) CO2-generating sachet and incubated in a CO2 incubator for 7—14 days. After 7 and 14 days, pure colonies of black-pigmented P. endodontalis in a blood agar medium were obtained, respectively. All bacterial suspensions were prepared according to the 0.5 McFarland standard and then incubated at 37°C for 24 h. The suspensions of P. endodontalis were then re-diluted in a brain heart infusion liquid medium with the concentration of 1.5 ×  108 CFU/mL.

Biofilm assay

Single root premolar teeth were used, and root canal preparation was done using a universal ProTaper rotary system up to the F3 file to eradicate the biofilm of P. endodontalis and approach the actual state in the mouth. The crown was cut off from the teeth, and the apical part was sealed with nail polish. There were 24 teeth used, and each treatment was carried out in duplo. Then, the teeth were sterilized. After that, the teeth were put into suspensions P. endodontalis that were re-diluted in a liquid brain heart infusion medium cultured for 3 weeks in which the P. endodontalis bacteria had grown.

The formed biofilm in the root canal was eradicated using the A. sativum extract with concentrations of 100%, 50%, 25%, 5.25% sodium hypochlorite (NaOCl) along with 2% CHX as the positive control and sterile distilled water as the negative control. At the time of irrigation using the A. sativum extract, it was collected in a Petri dish and the tooth is allowed to sit for 40 min. The teeth were then rinsed with phosphate-buffered saline (PBS), and the teeth contained in the tube were later rinsed along with water. Then, vortexing was then used to release the bacteria in the root canal into PBS. The teeth were then removed and the residue was centrifuged at 5000 rpm for 10 min at 4oC. The supernatant was discarded. The pellet which contained the bacteria was resuspended and spread in blood agar after diluting the pellet in 1:10,000 ratio. The blood agar was incubated at 37oC for 24 h in an anaerobic condition (5% CO2, 20% N2, 75% H2), and the formed colony was counted to analyze the colony-forming unit (CFU/mL).

Statistical analysis

All data acquired were statistically analyzed with the Kolmogorov—Smirnov normality test with P > 0.05, followed by a one-way analysis of variance (ANOVA) test with the significance of P < 0.05. All statistical analyses were done using SPSS version 23 (IBM, Armonk, NY, USA).


  Results Top


In the LC/MS test of A. sativum extract, several flavonoids were identified [Figure 1].
Figure 1: Chromatography test of A. sativum

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According to the phytochemical tests, steroids, tannins, flavonoids, and saponins compounds were found in the A. sativum extract [Table 1].
Table 1: Phytochemical test results, showing that all compounds were found in the A. sativum extract

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All obtained data from each group had a normal distribution (P > 0.05). Therefore, hypothesis testing was performed using a one-way ANOVA test. There was a significant difference in optical density (OD) values between the six groups tested (P < 0.05). According to Tukey′s post hoc test, the OD value for the groups with 5.25% NaOCl and 2% CHX as a positive control did not significantly differ from that of the A. sativum group 100%, 50%, or 25% (P > 0.05). Still, significant differences in OD values were found between the Aquadest group as a negative control compared with the A. sativum group 100%, 50%, and 25% and the positive control groups of NaOCl 5.25% and CHX 2% (P < 0.05).

The results of P. endodontalis biofilm eradication by A. sativum are presented in a graph, based on the OD values with CFU/1 mL of bacteria. In the results, it is observed that NaOCl reached the lowest OD values of biofilm eradication with a concentration of 5.25% as a positive control (0.5), CHX 2% as a positive control (0.81), and A. sativum extract preparation with a concentration of 100% (1.14). Meanwhile, the highest CFU/mL was obtained with Aquadest (3.91) [Figure 2].
Figure 2: Mean of biofilm mass on the eradication of A. sativum preparations against P. endodontalis biofilms. The vertical axis of panels indicates the P. endodontalis biofilm mass OD. The horizontal axis indicates the concentration of A. sativum (100%, 50%, and 25%) and NaOCl 5.25% and CHX gluconate (2%) as a positive control **P < 0.01

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  Discussion Top


The process of root canal infection is a dynamic process in which each stage involves different bacteria species. In the early stages of the process, pulp inflammation is caused by facultative anaerobic bacteria. After oxygen is depleted because it was consumed by facultative bacteria, which caused tissue necrosis, the anaerobic environment develops in which obligate anaerobic bacteria begin to grow.[17]

Bacterial colonization in the root canal is formed through biofilms attached to the wall of the root canals. Biofilms were found on the walls of the primary root canals and in the apical ramifications, lateral root canals, and isthmus. Biofilms are found in endodontic infections with apical periodontitis. Endodontic infection may lead to apical periodontitis because of the bacteria in the root canals, which grow in numbers and cause further damage.[18]

In this study, P. endodontalis was used because research on this species is not of the kind found in intraarticular primary infections, which can cause endodontic infection, as it occurs with other bacterial species.[7]P. endodontalis is also a known cause of meningitis and endocarditis.[19] Li et al.[5] stated that P. endodontalis found in the oral cavity might have a role in causing systemic disease. This result is confirmed by Dorn et al.,[20] who reported that the pathogenic bacteria P. endodontalis occurring in the oral cavity were able to invade the endothelial cells of the arteries in the human heart.

In our study, we tested the eradication ability of garlic extract (A. sativum) against P. endodontalis biofilms. From the obtained results, it is known that the eradication ability of A. sativum with a concentration of 100%, 50%, or 25% of the P. endodontalis biofilm was not significantly different from that of 5.25% NaOCl and 2% CHX. The NaOCl and CHX as positive controls were considered more toxic against gingival fibroblasts when compared with A. sativum. This result can be observed from the OD value, which had no statistically significant difference (P > 0.05) between them. The eradication ability of A. sativum with a concentration of 100%, 50%, or 25% against the biofilm was higher than that of Aquadest. It can be observed that the OD value of the Aquadest test group was significantly higher than that of the A. sativum group (P < 0.05).

Garlic has several characteristics, including antibacterial, antiviral, antifungal, and antiprotozoal properties. The antibacterial activity in the garlic extract has a broad spectrum, and it is effective against both Gram-positive and Gram-negative bacteria.[21] Furthermore, the rupture may occur due to this exchange reaction owing to the enzyme alliinase, located at the cytoplasm and vacuole, is produced when tissue damage occurs. Allicin, produced by this enzyme, may stop cell metabolism and bacterial growth, protecting against bacteria and fungi.[22] The results of the phytochemical test in this study are indications that the A. sativum extract contains flavonoid compounds, as shown by Bozin et al., who argued that the A. sativum extract contains phenolics and flavonoids functioning as antioxidants. The 2,2-diphenyl-1-picrylhidrazyl compound contained in the A. sativum extract acts as a free radical binding agent.[23] In the research by Salima,[24] the statement that phenol compounds may inhibit bacterial enzyme activity, interfering with metabolism and the survival of the bacteria, is supported.

As a solvent for producing the A. sativum extract, 96% of ethanol is used because it is volatile, has a low boiling point, and has a high polarity. With the properties of 96% ethanol as a solvent, it is expected that the active substance needed will be fully extracted. In addition, 96% ethanol used in the A. sativum extract has been shown to produce a more significant inhibition zone than other solvents, such as methanol and butanol.[25]

The treatment in each test group in this study was repeated twice for 40 min at the same time and incubated for 3 weeks, after which the blood agar was checked. We found that bacteria grew under these conditions but did not have a black pigment image. Similar results were reported by Suzuki et al.,[26] who showed that P. endodontalis colonies isolated from infected root canals gave beige colony images on blood agar media. This colony was confirmed to be P. endodontalis through phenotype identification. The pigmentation that is formed is probably related to the outer membrane system of P. endodontalis. The unidentified black pigment on the blood agar was not identified in this study because it was challenging to maintain the work in an anaerobic state or due to bacterial contamination in the air because of the lack of incubation time.

Based on this research, it was shown that the eradication ability of the A. sativum extract was almost similar to 5.25% NaOCl and 2% CHX. Still, further in-vivo animal research is necessary to assess the effectiveness of its eradication ability. The drawback of this study lies in the difficulties in isolating the bacteria, for instance, during the incubation time for culture, which takes between 7 and 14 days. If the bacteria failed to grow during their culture, they must be regenerated and re-incubated for the required time, making development of the research more difficult.


  Conclusion Top


Based on the results of this study, it can be concluded that the garlic extract (A. sativum) can inhibit P. endodontalis biofilm at concentrations of 100%, 50%, and 25%. Adequate time and specific medium were required to isolate samples of P. endodontalis from the patients, which must be done anaerobically to prevent contamination from other bacteria. Further in-vivo animal tests and experiments are required to determine the ability of garlic to eradicate biofilms in the actual conditions of the oral cavity. Moreover, further research is needed to evaluate the antibacterial mechanism of garlic (A. sativum) to determine how long it takes to dissolve the bacterial biofilm structure in the root canal.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Hargreaves KM, Berman LH. Cohen’s Pathways of the Pulp. 11th ed. Missouri: Elsevier; 2016. p. 599-629.  Back to cited text no. 12
    
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