Biomedical Journal of Indonesia Vol 7 Issue 2 2021
Efficacy of Cyombopogon Citratus Extract Against Enterococcus Faecalis
Tessya Nitulo Permatasari Lahagu1*, Huynh Diem An1, Cindy Denhara Wijaya1, Mellisa Sim1
1Dental Education Study Program, Faculty of Dentistry, Prima Indonesia University
A R T I C L E I N F O Keywords:
Sodium Hypochlorite
Lemongrass (Cyombopogon citratus (DC).
Stapf)
Enterococcus faecalis
*Corresp onding author:
Tessya Nitulo Permatasari Lahagu
E-mail addre ss:
[email protected]
All authors have reviewed and approve d the final version of the manuscri pt.
https://doi.org/10.32539/BJI.v7i2.301
A B S T R A C T
Endodontic treatment refers to the act of removing infected pulp tissue and forming and reforming the root canals to prevent bacteria from entering the root canals.
Microbial infection in the root canal or in the periapical area is a factor that can le ad to failure of root canal treatments. Endodontic treatment failure can be caused the bacteria Enterococcus faecalis. The irrigation agent commonly used is sodium hypochlorite. Sodium hypochlorite in large concentrations can be toxic to tissues.
Lemongrass plant (Cymbopogon citratus) is a natural ingredient that can be used as an antibacterial agent. The aims of study to determine the comparison of the antibacterial effectiveness of lemongrass plant extracts (Cyombopogon citratus (DC).
Staph) concentrations of 25%, 50%, 75%, 100% with 2.5% sodium hypochlorite in inhibiting the growth of the bacterium Enterococcus faecalis. Lemongrass plant extracts Cyombopogon citratus (DC). Staph) made by maceration method and converted into 25%, 50%, 75%, and 100%. The bacterial test method used the agar diffusion test (Kirby-Bauer test) to see the effectiveness of antibacterialsThe result of the one way ANOVA statistical test resulted in significance value of p=0,001 (p<0,05) so that means there was differences in the ave rage diameter of inhibitory lemongrass plant extracts (Cyombopogon citratus (DC). Staph) concentrations of 25%, 50%, 75%, 100% with 2.5% sodium hypochlorite in inhibiting the growth of the bacterium the results are 13.68 ± 1.34 mm, 16.08 ± 0.87 mm, 16.33 ± 0.35 mm, 17.78 ± 0.93 mm and 7.83 ± 0.44 mm and reinforced by the posthoc Tukey HSD test. The conclusion from the study that there is a difference between the antibacterial effectiveness of lemongrass plant extract (Cyombopogon citratus (DC). Staph) concentrations of 25%, 50%, 75% and 100% with 2.5% sodium hypochlorite in inhibiting the growth of the bacterium Enterococcus faecalis.
1. Introduction
Endodontic treatment refers to the act of removing infected pulp tissue and forming and replenishing the root canals to prevent bacteria from entering the canal.1 Cleaning, formation and filling of three -dimensional root canals is an important step in endodontic treatment.3 Microbial infection of the root canal or the area around the root channel is an important factor leading to the failure of endodontic treatment.2
The failure of endodontic treatment can be caused by the bacteria Enterococcus faecalis.2 Enter-ococcus faecalis is included in gram- positive bacteria which are facultative anaerobi c bacteria which are the main cause of periradic ula r infections after root canal treatment.4 En- terococcus faecalis bacteria can be removed from
the root canal by using instruments and use of antibacterial irrigation agents.2
2.5% sodium hypochlorite is an irrigation agent that is often used in endodontic treatme nt.
Sodium hypochlorite has antibacterial propertie s and can destroy vital tissues, necrotic tissue and organic compone nts of dentin and biofilms. The sodium hypochlorite concentration of 2.5% is a very suitable concentration for root canal treatment because its cytotoxicity is quite low but always shows good bactericidal action and solution time when compared to concentrations of 0.5 and 1 percent.5
Lemongrass (Cymbopogon citratus) is a natural ingredient that can be used as an antibacte ri al
Biomedical Journal of Indonesia
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agent.3 Lemongrass (Cymbopogon citratus) contains flavonoids, triterpenoids / steroids, sap- onins, alkaloids, tannins, phenolics and essential oils.3,6,7
The antibacterial effectiveness test of Howart o et al (2015) was carried out using the disc diffusion method.8 This method uses filter pape r as a container for antimicrobial substances. Place the filter paper on agar media that has be e n inoculated with the bacteria to be tested, the n incubate for 24 hours at 37 0C. Results were obtaine d in the form of the presence or absence of a clear area surrounding the filter paper to indicate the inhibition zone for bacterial growth.9
Research by Howarto et al (2015) prove d that lemongrass essential oil can affect the inhibiti on of Enterococcus faecalis bacteria at a concentration of 25%, 50%, 75% and 100% where the diameter of the inhibition zone is 2.60 mm, 4.73 mm, 4.50 mm, and 5.34 mm.8 Howe ve r, there are no studies on the comparati ve effectiveness of lemongrass plant extracts and 2.5% sodium hypochlorite in inhibiting the growth of Enterococcus faecalis bacteria. Based on this background, the researchers are interested in conducting research on the comparison of the effectiveness of the antibacterial effect of citrone lla plant extract (Cyombopogon citratus (DC). Stapf) concentrations of 25%, 50%, 75% and 100% with 2.5% sodium hypochlorite in inhibiting bacterial growth Enterococcus faecal-is.
2. Method
This type of research used an experimental in - vitro study with a post-test only control grou p design. This research was conducte d at the Laboratory of Physiology and Plant Tissue Culture USU, USU Faculty of Biology Herbarium Medicine Laboratory, and the Labora tory of the Faculty of Medicine UNPRI from January to March 2021.
The sample size is calculated using the Federer formula (t-1) (r-1) ≥15. In this study, there were 6 treatment groups. Based on the calculation with this formula, the pengulanya is carried out 4
times. So that the number of samples used is 24 samples.
Plant determination
Plant determination was carried out at the USU Faculty of Biology Herbarium Medicine Laborat or y
Production of lemongrass extract and phytochem ical test
Provide ± 3 kg of washed lemongrass plants.
Then chop the lemongrass plant to make the drying process easier.3 The lemongrass slices are dried in the oven at 600C for 3 hours.10 When dry, use a blender to mash the lemongrass plant until it becomes powder The results obtained are ± 500 gr. Soak the powder of lemongrass in 2.5 L 96%
ethanol solvent for 72 hours.2,11 Filter with filter paper and then steam using a rotary evaporator at 45-50 0C to obtain a thick liquid extract.12 The extract that has been obtaine d is made into four concentrations by mixing into the DMSO solution , namely 25%, 50%, 75% and 100%. Perform phytoche mical screening which aims to determine the presence or absence of active compounds of lemongrass plant extract (Cyombopogon citratus (DC). Stapf). Phytoche mical screening include s phenolics, tannins, flavanoids, saponi ns , alkaloids, and triterpenoids.3,6
Preparation of bacterial suspension
Enterococcus faecalis bacteria culture on Mueller-Hinton Agar media and incubate d for 24 hours at 37 0C. Transfer the bacteria into a test tube with a sterile cotton swab and then mix it with 10 ml of sterile aquadest. After dissolvin g , take the bacteria and then spread it evenly on the Mueller-Hinton Agar media.8
Bacterial activity test
Testing for bacterial activity using the disc diffusion method was carried out by: Prepare 24 blank discs divided into six treatment grou ps ,
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namely extracts of lemongrass (Cyombopo- g o n citratus (DC). Stapf) 25%, 50%, 75% and 100%, sodium hypochlorite 2.5% and a blank disc is blank. Then dip the blank disc into the five treatment groups. Place the blank disc on the surface of the Mueller-Hinton Agar media with tweezers and then with tweezers then incubate for 48 hours with a temperature of 370C.13 Treat it 4 times. Invert the petri plate to measure the diameter of the drag zone.14 The diameter of the drag zone can be calculated using a caliper by measuring the clear area of horizontal, vertical and diagonal diameters. The result is the average of the three, expressed in millimeters.2
3. Result
Phytoche mical test results showed the presence of active compou nds in extracts of lemongrass (Cyombopogon citratus (DC). Stapf).
Lemongrass plant extract compo u n ds (Cyombopogon citratus (DC). Stapf) can be seen in the table below:
Inhibition test results on the growth of Enterococcus faecalis lemongrass plant extract concentrations of 25%, 50%, 75%, 100% and 2.5%
sodium hypochlorite indicated the formation of a clear area around the blank disc. Howe ve r,
negative control was not found in these clear areas. The presence of this clear area indicates an obstacle to the test material against bacteria. The results of the clear area measurement can be seen in the table below:
Based on table 2 above, the results prove that the average diameter of the inhibition zone of lemongrass (Cyombopogon citratus (DC). Stapf) concentrations of 25%, 50%, 75% and 100% are 13.68 ± 1.34 mm, 16, 08 ± 0.87 mm, 16.33 ± 0.35 mm, 17.78 ± 0.93 mm. The diameter of the inhibition zone for sodium hypochlorite (NaOCl) 2.5% was 7.83 ± 0.44 mm, while the blank disc had no inhibition zone diameter.
The results of research on differences in the diameter of the inhibition zone of all treatme nt groups in inhibiting the growth of the Enterococcus faecalis bacteria can be seen in the table below:
Based on table 3 above, the treatment group of citronella plant extract Cyombopogon image -tu s (DC). Stapf) with a concentration of 100% had the highest inhibition zone diameter value of 17.78 ± 0.93 mm and the resulting inhibition zone diameter of the 2.5% sodium hypochlorite grou p was 7.83 ± 0.44 mm.
Table 1 Phytoche mical test results of citronella plant extract (cyombopogon citratus (dc) stapf) Content of Active
Compounds Reagent Test results Observation result
Phenolic Methanol + FeCl3 +++ Black
Flavonoids Ethanol + Pb (CH3COO)2 ++ Yellow
Concentrated NaOH + HCL + Yellow
Alkaloids
Ethanol + Dilute HCL +
Mayer + Yellow sediment
Ethanol + Dilute HCL +
Dragendorff + Red sediment
Saponins Aquades + Foaming
Tannins FeCl3 + Blackish Green Color
Triterpenoids Chloroform + Lieberman-
Burchard's + A Chocolate Ring Is
Formed
(+) indicates less concentrate d, (++) indicates very concentrate d, and (+++) indicates very concentrate d.
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Table 2 Average values of inhibition zone values for citronella plant extract (Cyombpogon citratus (DC).
Stapf) concentrations of 25%, 50%, 75% and 100% with 2.5% sodium hypochlorite in i nhibiting the growth of Enterococcus faecalis bacteria.
Group
Inhibition Zone Diameter (mm)
Average SD Sample
1 2 3 4
25 % 15.6 13.5 12.6 13 13.68 1.34
50 % 15.3 17.1 16.5 15.4 16.08 0.87
75 % 16.5 16.7 15.9 16.2 16.33 0.35
100 % 17.6 18.8 18.1 16.6 17.78 0.93
NaOCl 2.5 % 8.3 8.1 7.5 7.4 7.83 0.44
Ctrl - 0 0 0 0 0.00 0.00
Table 3 Differences in inhibition zone diameter of all treatment groups in inhibiting the growth of Enterococcus faecalis bacteria
Group The average diameter of the zone of inhibition SD
25 % 13.68 1.34
50 % 16.08 0.87
75 % 16.33 0.35
100 % 17.78 0.93
Sodium Hipoklorit 2,5% 7.83 0.44
Ctrl - 0.00
0.004. Discussion
This study used lemongrass with the specie s Cyombopogon citratus (DC). Stapf. Phytoche mi c al test results obtaine d from extracts of lemongras s (Cyombopogon citratus (DC). Stapf) contain active compounds such as phenolics, tannins, flavonoids, saponins, alkaloids, and triterpenoi ds . This research is in accordance with that obtaine d by Soraya et al (2016) and Hendrik et al (2013) in which the active compound has been shown to have antibacterial benefits.3.6
Extract of lemongrass (Cyombopogon citratus (DC). Stapf) with a concentration of 25% has a significant difference with a concentration of 50%
in inhibiting the growth of Enterococcus faecalis anchovies (p <0.05). This result is in line with the research of Wahyuni et al (2018) which states that there is a significant difference between the
ethanol extract of lemongrass stalks (Cyombopogon image-tus (DC). Stapf) betwe e n 25% and 50% concentrations of Staphylococ c u s aureus bacteria. This is because there are differences in the components of the compo u n ds in each concentration, namely the higher the concentration, the more antibacterial properties it has15
The largest zone of inhibition against Enterococcus faecalis bacteria from extracts of lemongrass (Cyombopogon citratus (DC). Stapf) was found at a concentration of 100%. In this study, there were differences in the diameter of the inhibition zone at each concentration of citrone lla plant extract (Cyombopogon image -tus (DC).
Stapf) in inhibiting the growth of the bacte riu m Enterococcus faecalis, which prove d that there were differences in the active compo u n ds contained in it.16 However, the antibacte ri al
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effectiveness of citronella plant extract (Cyombopogon citratus (DC). Stapf) between 50%, 75% and 100% concentrations did not have a significant difference in inhibiting the growth of Enterococcus faecalis bacteria (p > 0.05). This result is inversely proportional to Marthinova et al (2016) which showed a significant difference between 50%, 75% and 100% concentrate d lemongrass essential oil extract in inhibitin g Escherichia coli and Stapylococcus aure us bacteria. Lemongrass essential oil in the study of Marthinova et al (2016) has a content similar to the extract of lemongrass (Cyombopogon citratus (DC). Stapf) in this study. The difference betwe e n these two results could be due to different types of bacteria used.17
This study showed that the extract of lemongrass (Cyombopogon citratus (DC). Stapf) with a concentration of 25% was more effective than 2.5% sodium hypochlorite. Howe ve r, research conducte d by Yuanita et al (2018) was inversely proportional where sodium hypochlorite was 2.5% more effective than 25% cocoa pee l extract (Theobroma cacao) against Por - phyrom onas gingivalis bacteria.18 Cocoa skin extract (Theobroma cacao) concentration 25% in Yuanita et al's (2018) study had active compo u n ds that were similar to the extracts in this study, but the results of the two studies were different. This is due to differences in the types of bacteria where Enterococcus faecalis is a gram-positive anaerobi c bacteria while Porphyromonas gingivalis is a gram-negative anaerobic bacteria.18.19
Extracts of lemongrass (Cyombopogon citratus (DC). Stapf) concentrations of 50%, 75% and 100%
prove that they can inhibit the growth of Enterococcus faecalis bacteria compared to 2.5%
sodium hypochlorite. This result is inverse ly proportional to Mozartha et al (2019), namely that sodium hypochlorite is 2.5% more effective than white turmeric extract (Cur-Saja zedoaria) with a concentration of 50% and 75% against Enterococcus faecalis. white turmeric (Curcu m a
zedoaria) rhizome extract where sen-yawa tannin can cause disrupte d bacterial cell growth and metabolism because it targets bacterial cell wall polype ptides.3 This result is also inconsiste nt with Rachmawati et al (2015) who showed that 2.5%
sodium hypochlorite was more effective than mangrove leaf extract (Acanthus ilicifolius) with a concentration of 100%.20 This is due to difference s in phytoche mical content in the tested plants.
where the mangrove leaf extract (Acanth u s ilicifolius) does not have phenolic and flavonoi d compounds where these two compou nds can cause protein denaturation so as to inhibit adenosine tripospate (ATP) bonds by burni n g bacterial cells so that they can damage the membrane.3.21
The level of antibacterial activity can be determined from the diameter of the inhibiti on zone. According to David Stout, the strength of antibacterial inhibition can be divided into 4 categories, namely the very strong category (≥20 mm), the strong category (10 mm – 20 mm), the moderate category (5 mm – 10 mm), and the we ak category (≤5 mm). .16 In this study extracts of lemongrass (Cyombopogon citratus (DC). Stapf) concentrations of 25%, 50%, 75% and 100% were in the strong category (10-20 mm), while 2.5%
sodium hypochlorite was in the moderate cate gory ( 5-10 mm). This study prove d that the extract of lemongrass (Cyom-bopogon citratus (DC). Stapf) has a higher inhibition zone diameter compare d to 2.5% sodium hypochlorite. Therefore, extracts of lemongrass (Cyombopogon citratus (DC). Stapf) can be used as a natural irrigation agent in endodontic treatments. However, it is necessary to test the cytotoxicity of extracts of lemongras s (Cyombopogon citratus (DC). Stapf) with a concentration of 25%, 50%, 75% and 100%.
5. Conclusion
There is a difference in the ratio of the antibacterial effectiveness of citronella plant extract (Cyombopogon citratus (DC). Stapf)
concentrations of 25%, 50%, 75% and 100% with sodium hypochlorite 2.5% in inhibiting the growth of the bacteria Enterococcus faecalis, where the citronella plant extract (Cyom- bopogon citratus (DC). Stapf) at 100% concentration was the most effective in inhibiting the growth of Enterococ c u s faecalis bacteria. Lemongrass extract (Cyombopogon citratus (DC). Stapf) was prove n to be more effective than sodium hypochlorite 2.5%
in inhibiting the growth of Enterococcus faecalis bacteria.
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