PUB Inhibitory Activity Lemongrass

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support of the American people through the United
States Agency for Internatonal Development (USAID).
The contents are the responsibility of Texas A&M University
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the views of USAID or the United States Government.
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Artikel disampaikan pada Seminar Nasional Patpi 2013. Jember, 26-29 Agustus 2013.

INHIBITORY ACTIVITY OF LEMONGRASS ESSENTIAL OIL
AGAINST Eschericia Coli, Staphylococcus Aureus, AND Vibrio Cholera
Nyoman Semadi Antara*, Dwi Ayu Kirani Paramita, Anak Agung Duwipayana,
Ida Bagus Wayan Gunam
Laboratory of Bioindustry,

Department of Agroindustrial Technology, Faculty of
Agricultural Technology, Udayana University, Kampus Bukit Jimbaran, Bali.
*e-mail: ns_antara@yahoo.com
ABSTRACT
The lemongrass essential oil (LEO) extracted from the leaf of lemongrass
(Cymbopogon citratus) was tested for its inhibitory effects against three
pathogenic bacteria (Escherichia coli, Staphylococcus aureus, and Vibrio
cholerae). This research used a Completely Randomized Design with 5 treatments
of LEO concentration in 1% tween 80, namely 1%, 2%, 3%, 4%, 5% (v/v). By
using of GC-MS to determine LEO chemical composition, the LEO was
composed of several compounds which citral, a monoterpene compound, was the
dominant compound. This LEO could inhibit significantly the growth of E. coli, S.
aureus and V. cholerae. The most sensitive of the bacteria against the LEO was
Vibrio cholerae. In addition, the minimum inhibitory concentration (MIC) of LEO
was 1.0%, 0.8%, and 0.6% respectively against E. coli, S. aureus, and V. cholera.
Keyword : inhibitory activity, lemongrass essential oil, Escherichia coli, Staphylococcus
aureus, Vibrio cholera
Abstrak
Minyak atsiri daun sereh (MADS) yang diekstrak dari daun sereh dapur
(Cymbopogon citratus) diuji daya hambatnya terhadap tiga bakteri pathogen
(Escherichia coli, Staphylococcus aureus, dan Vibrio cholera). Penelitian ini
dirancang menggunakan Rancangan Acak Lengkap dengan 5 perlakuan
konsentrasi minyak atsiri daun sereh dapur di dalam 1% tween 80, yaitu 1%, 2%,
3%, 4%, 5% (v/v). Komposisi kimia MADS yang dianalisis dengan GC-MS terdiri
dari beberapa senyawa dengan citral, senyawa monoterpene, merupakan
senyawa yang paling dominan. MADS ini dapat menghambat secara nyata
pertumbuhan E. coli, S. aureus, dan V. cholera. Konsentrasi penghambatan
minimal (KPM) MADS terhadap E. coli, S. aureus, dan V. cholera adalah
berturut-turut 1,0%, 0,8%, dan 0,6%.
Kata kunci: daya hambat, minyak atsiri daun sereh, Escherichia coli,
Staphylococcus aureus, Vibrio cholerae.

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Introduction
Essential oil produced from plant extraction process is composed of volatile
secondary metabolites. Essential oils are found in all parts of the plant, such as
flowers, fruit, leaves, stems, and roots. Although essential oils are spread in all
parts of the plant, but generally concentrated in the leaves, bark, or fruit. Essential
oils from different parts of the plant have a different chemical composition (Hili et
al., 1997). Lemongrass (Cymbopogon citratus) concentrates their essential oil
content in the leaves. Lemongrass essential oil (LEO) contained in the leaves has
a different chemical composition compared to that extracted from the stem (Putra
et al., 2012).
Plants have a natural defense system. The content of natural bioactive compounds
such as the compounds of terpenoids, alkaloids, and phenolic compounds that are
generally used for self-defense of plants against invading microorganisms (Shukla
et al., 2013). In addition, such compounds are also easily decomposed,
environmentally friendly, and does not leave harmful residues (Bishop and
Thornton, 1997). These characteristics will be helpful to explore and develop food
bio-preservative recognized as safe. Among plant products, essential oils received
great attention because of its high antimicrobial activity. Essential oils can be used
as natural food additives and commercially produced through encapsulation
technology (Burt, 2004; Arana-Sanchez et al., 2010). Many studies have been
done related to the antibacterial and antifungal activity of essential oils. Former
studies indicate a higher antibacterial effect of essential oils against Gram-positive
than Gram-negative bacteria (Lang and Buchbauer, 2012). Garlic extract has
bactericidal efficacy against Helicobacter pylori (Cellini et al., 1996). Some
pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, and
Salmonella typhi are also sensitive to garlic extract (Arora and Kaur, 1999). Garlic
extract also showed anti-Candida activity. Extract of garlic and clove has efficacy
as bacteriostatic and bacteriocidal against E. coli O157 and Sal. enteric serovar
Enteritidis at concentrations between 0.25 to 1.0% (Leuschner and Zamparini,
2002). Oregano and thyme hydrosol also shown to kill pathogenic bacteria such as
E. coli, S. aureus, and Yersinia enterocolitica (Sadi, 2003). In addition to
bacterial pathogens, essential oils are also effective to inhibit spoilage bacteria.
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Essential oil of lemongrass could inhibit the growth of genus Aspergillus
(Matasyoh et al., 2010; Ella et al., 2013) and detected as antifungal within wider
spectrum (Mishra and Dubey, 1994). The oil was also found effective against
several pathogenic bacteria, so it would be helpful in the treatment of infections
caused by multidrug resistant organisms. Several essential oils showed efficacy to
eradicate biofilm-forming bacteria with higher efficiency than using standard
antimicrobial treatment (Kavanaugh and Ribbeck, 2012).
In present study we investigated and confirmed the inhibition activity of
essential oil extracted from lemongrass leaf against three important pathogenic
bacteria related to food safety. The study also determined the minimum inhibitory
concentration (MIC) of LEO to inhibit E. coli, Staph. aureus, and Vibrio cholera.

Materials and Methods
Materials
The main material of the study was LEO obtained from the extraction of
lemongrass leaves Pelaga village origin, Regency of Badung, Bali, Indonesia.
Bacterial cultures of E. coli ATCC 25922, Staphylococcus aureus ATCC 25923,
Vibrio cholerae were obtained from the Laboratory of Microbiology, Faculty of
Medicine, University of Udayana. Another necessary reagents were an emulsifier
tween 80, media Nutrient Agar (CM Oxoid 3), Nutrient Broth (CM Oxoid 1),
Thiosulfate Citrate Bile-salt Sucrose Agar (TCBSA) (Oxoid CM 333), Eosin
Methylen Blue Agar (EMBA) (Oxoid CM 69), NaCl (JT. Baker) obtained in UPT.
Integrated Bioscien and Biotechnology Laboratory, University of Udayana.

Design of Experiment
This research was a laboratory experiment designed by completely randomized
design (CRD). Five treatment concentrations of LEO in 1% tween 80
experimented in this study are 1%, 2%, 3%, 4%, and 5% (v / v). Each treatment
was tested against three pathogenic bacteria namely E. coli, S. aureus, and V.
cholerae. Each experiment be repeated 3 times, so that the research was
conducted in 45 experimental units. The data were analyzed with analysis of
variance and followed by Duncan's test (Steel and Torrie, 1993). When the LEO
concentration of 1% still has the inhibitory activity then do the treatment by
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lowering concentrations of LEO which will be tried on the concentration of 0.2%,
0.4%, 0.6%, and 0.8% in 1% tween 80 to determine the MIC.
Determination of Chemical Composition of LEO
Chemical composition of LEO was analyzed by using GC-MS (Agilent
Technologies, USA). Gas chromatography was run on HP 5MS column with
internal diameter and column length was 0.25 mm and 30 m. Helium was used as
carrier gas at a flow rate of 1 ml/min. Injector temperature was set up at 250C.
The oven temperature program began at 70C for 5 min and ramped to final
temperature (270C) at 10C/min, the total run time was 25 min. Identification of
compounds was done by using software Wiley 229, NIST 12, and NIST 62
Library.
LEO Sterilization and Emulsion Preparation
LEO was sterilized by filtering through 0.22 m filter (Millex-Gv) sterile. The
LEO was then collected in dark bottle and subsequently stored in a refrigerator at
4C until used for experiments. LEO emulsion was made by mixing LEO into a
1% solution of tween 80 (v/v). And then the mixture was shaken until LEO
emulsion uniform and stable.
Preparation of the Bacteria
Preparation of bacterial culture started with that stock cultures of bacteria E.coli
cultured on EMBA, S. aureus cultured on agar MSA and V. cholerae on TCBSA
and incubated at 37C for 24 hours. Furthermore, each culture was grown in agar
slant for stock that will be used to test inhibition. A total of one loop of each
bacterial culture was inoculated into 5 ml Nutrient Broth for E. coli and 5 ml
Nutrient Broth containing 1.5% NaCl for S. aureus and V. cholerae, and then
subsequent cultures were incubated at 37C for 8 hours. Further suspension of
bacterial culture was used in the inhibitory activity assay.
I nhibitory Activity Assay
Inhibitory effect of LEO on the growth of indicator bacteria was carried by the
agar diffusion method. The LEO was tested of its inhibitory activity against E.
coli, S. aureus, and V. cholerae. The media used were Nutrient Agar, the steril
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agar medium was poured in petri dishes 2-3 mm thick (15 ml) and allowed to
solidify. Indicator bacteria in Nutrient Broth were shaken using vortex to obtain a
suspension of the bacteria. Then 200 l suspension of bacteria were spread on the
surface of Nutrient Agar lawn by a bent glass rod and then allowed to dry for 30
minutes. Furthermore sterile paper discs with a diameter of 8 mm spotted by 50 l
LEO of suitable concentration treatment, then placed paper discs on agar plates
that have inoculated indicator bacteria suspension. Further, the agar plates were
incubated for 24 hours at 37C. After 24 hours the plates were observed for the
presence of a clear zone around the paper disc which is the inhibitory activity
formation and the diameter of the inhibition areas in mm were measured using a
caliper. Inhibition area is the average total diameter of inhibition reduced by paper
disc diameter. Inhibition activity is positive if formed of at least 2 mm clear zone
around the paper disc that showed inhibition of the growth of pathogenic bacteria
test.
Determination of Minimum I nhibitory Concentration
In the process of determining the MIC, the medium used was sterile NA. Agar
medium was poured in petri dishes 2-3 mm thick (15 ml) and allowed to solidify.
Bacterial suspension of about 200 l was then inoculated and spreaded on agar
lawn and then allowed to dry for 30 minutes. Furthermore sterile paper discs with
a diameter of 8 mm to 50 mL spotted by LEO of 0.2%, 0.4%, 0.6%, and 0.8% and
held for 5 minutes for dispersing LEO into the paper disc, then placed them on
agar plates that have inoculated by bacterial suspension test. Further agar plates
were incubated for 24 hours at 37C. After 24 hours the plates were observed in
the presence of a clear zone around the paper disc. The MIC was determined by
the minimal concentration of LEO showed inhibitory activity.
Results and Discussion
Chemical Composition of LEO
Chemical composition of LEO determined by GC-MS showed that several
components were detected as illustrated in Figure 1. By using the library three
major compounds were identified as two isomers of citral and 3-carene, which
citral were the dominant compounds contained in LEO (Table 1). This result of
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determination was in agreement with the obtained that the components of LEO
were dominated by neral and geranial, which were two geometric isomers of
aldehyde citral (Hanaa et al., 2012). Paranagama et al. (2003) confirmed by using
thin layer chromatography (TLC), citral a and b are constituents LEO that has
fungicidal activity.

Figure 1. GC-MS Chromatogram of essential oil of lemongrass leaf.
Table 1. Bioactive compounds identified in essential oil of lemongrass leaves
Peak
No.
Retention
time (min)
Compounds Molecular
formula
Relative
conct. (%)
Class of
compound
6 11.61 Z-Citral C
10
H
16
O 32.93 monoterpene
7 11.78 3,7,7-trimethyl Bicyclo hept-
3-ene (3-Carene)
C
10
H
16
3.82 monoterpene
8 12.09 E-Citral C
10
H
16
O 37,85 monoterpene

3-Carene
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I nhibitory Activity
This study used sterile distilled water as a negative control and positive control
used amoxicillin and tetracycline. The test results showed that the concentration
of 30 g/100 ml amoxicillin with volume of 50 l was able to inhibit E. coli with
diameter of 23.3 mm and 22.5 mm for S. aureus, and tetracycline concentration of
30 g/100 ml with a volume of 50 l was able to inhibit V. cholerae with diameter
of 21.3 mm. Amoxicillin and tetracycline are a broad-spectrum antibiotic that
inhibits bacterial Gram-positive and Gram-negative. The diameter of the resulting
inhibition confirmed that E. coli, S. aureus, and V. cholerae were sensitive to
amoxicillin and tetracycline.
The results also showed that concentration of LEO highly significant affected the
growth of E. coli, S. aureus and V. cholerae. The LEO inhibition activities against
E. coli, S. aureus and V. cholerae are given in Table 2.
Table 2. Inhibition diameter (mm) of LEO against E.coli, S. aureus, and V.
cholerae
LEO Conct. E. coli S. aureus V. cholera
1% 6,030,5 e 4,970,5 e 9,675,7 d
2% 7,030,5 d 5,970,5 d 10,675,7 c
3% 8,030,5 c 6,970,5 c 11,675,7 b
4% 9,030,5 b 7,970,5 b 12,835,7 a
5% 9,930,5 a 8,870,5 a 13,675,7 a
Note: same letter behind the average value showed no significant difference (p<0.05)

All levels of treatment showed inhibition against test bacteria. LEO 5%
concentration treatments resulted in inhibition diameter against E. coli, S. aureus,
and V. cholerae was about of 9.93 mm, 8.87 mm, and 13.67 mm, respectiovely.
LEO still gave inhibition at a concentration of 1% which was of 6.03 mm, 4.97
mm, and 9.67 mm, respectively. The results were consistent with those expressed
by Eddy (2009) which states that the essential oil extracts from plants can
suppress the growth of microorganisms. The higher concentration of the volatile
oil content a higher of antimicrobial compounds, so the inhibition of the growth of
the bacteria will become larger. Figure 1 shows the inhibitory effects of LEO
against pathogenic bacteria E. coli, S. aureus, and V. cholerae.

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Figure 2. Inhibition of LEO of lemongrass against the growth of E. coli, S. aureus, and V.
cholerae. A: 1% oil concentration; B: 5% oil concentration.

According Arswendiyumna (2010), compounds that have antimicrobial properties
contained in lemongrass is compounds group of terpene which determined in the
essential oil fraction. Derivative terpenes contained in the lemongrass oil are
geranial ( citral) and neral (citral ). Inhibition of leaf oil of lemongrass against
bacteria S. aureus is suspected as phenolic compounds, which are present in the
essential oil of lemongrass leaves. The compounds contained in LEO inhibits the
microbes by damaging the structure of peptidoglycan (protein) present in the cell
wall and protein denaturation, which can damage cell walls or membranes and
inactivate enzymes. In general, the inhibition activity can be caused by
interference constituent compounds of the cell wall, increased permeability of the
cell membrane that can lead to loss of the components of cells, inactivate
enzymes, and the destruction or damage to the genetic material.
Minimum inhibitory concentration (MIC) of LEO against E. col , S. aureus, and
V. cholerae was of 1 %, 0.8 % and 0.6 %, respectively, with successive inhibition
was equal to 6.03 mm , 4.77 mm and 3.70 mm (Table 2) . The results of this study
indicated that V. cholerae was more sensitive than E. coli and S. aureus to the
LEO. Figure 2 shows the inhibition activity of LEO against E. coli, S. aureus, and
S. aureus E. coli V. cholerae
A
B
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V. cholerae at MIC by agar diffusion method . The results are consistent with
those disclosed in Nursini (2005) that the natural antimicrobial compounds are
highly effective in inhibiting or killing foodborne microbes, although it is used in
relatively small concentrations. Generally, Gram- negative bacteria is inhibited by
essential oils at lower concentrations than Gram-positive bacteria.
Table 2. Minimum inhibitory concentration (MIC) of LEO against Escherichia
coli, Staphylococcus aureus, and Vibrio cholerae.
LEO Conct.
Diameter Inhibition. (mm)
E.coli (mm) S.aureus (mm) V.cholerae (mm)
0,2% 0 0 0
0,4% 0 0 0
0,6% 0 0 3,700,51c
0,8% 0 4,770,05 b 4,430,98 b
1% 6,030,05 a 4,970,05 a 9,670,57 a
Keterangan : huruf yang sama dibelakang nilai rata-rata menunjukkan perbedaan
tidaknyata (P > 0,05)

E
Figure 2. Minimum inhibitory concentration (MIC) of LEO on the growth of E.
coli, S. aureus, and V. cholerae.

Conclusion
Essential oil extracted from leaves of lemongrass (Cymbopogon citratus) could
significantly inhibit the growth of E. coli, S. aureus and V. cholerae. Vibrio
cholerae was more sensitive than E. coli and S. aureus. The minimum inhibitory
concentration of the essential oils to the inhibition of V. cholerae, S. aureus, and
E. coli was of 0.6%, 0.8%, and 1%, respectively. Further research is being carried
out against spoilage bacteria and other pathogens and various types of fungi.

E. coli 1% S. aureus 0,8%, V. cholerae 0,6%
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Acknowledgement
We are grateful thank to USAID TPC project for funding the experiment and
coming to the national seminar of IAFT 2013.
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