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Int. J. Biosci.

2015 
International Journal of Biosciences | IJB | 
ISSN: 2220-6655 (Print), 2222-5234 (Online) http://www.innspub.net Vol. 6, No. 9, p. 9-17, 2015 

RESEARCH  PAPER  OPEN  ACCESS  Chemical  composition  and 


antibacterial  activity  of  essential  oil  of  Cymbopogon  citratus  and 
Cymbopogon nardus against Enterococcus faecalis 
Partiban Subramanian*, Che Wan Imanina Che Wan Takwa, Nurul Emelia Ahmad 
Zubair 
Department of Biomedical Sciences, Faculty of Biomedicine and Health, Asia Metropolitan 
University, Selangor, Malaysia 
Key words: Antibacterial, Essential oil, Cymbopogon citratus, Cymbopogon nardus, Enterococcus faecalis. 

http://dx.doi.org/10.12692/ijb/6.9.9-17 Article published on May 08, 2015 


Abstract 
Cymbopogon citratus and Cymbopogon nardus is a common medicinal plant that is widely used for various 
medicinal properties. The aim of this study is to determine the chemical composition and to assess the 
antibacterial activities of Cymbopogon citratus and Cymbopogon nardus. Chemical composition of C. citratus 
and C. nardus essential oils were analyzed by using GC and GCMS. The chemical analysis was carried out by 
using GC-MS for identification of components of the two essential oils. The percentage composition of the 
studied oil samples listed twenty four compounds identified in C. citratus oil sample that included geranial 
(32.4%), beta citral (23.8%) and geraniol (12.0%) as major components. Twenty three compounds were 
identified in C. nardus essential oil including Elemol (17.3%), Citronellol (12.5%) and Naphthalene (10.1%) as 
major components. C. citratus and C. nardus essential oils were studied for their antibacterial activity against 
Enterococcus faecalis. In vitro antibacterial activity was performed by disc diffusion method in MH agar. C. 
citratus essential oil showed maximum zone of inhibition against E. faecalis (6.00±1.73) whereas the highest 
zone of inhibition of C. nardus oils against E. faecalis (7.00±2.65). Minimum Inhibitory Concentration (MIC) 
was carried out. C. citratus essential oil extract showed MIC results at the concentration of 62.5 mg/ml, whereas 
C. nardus essential oil showed MIC results at the concentration of 31.25 mg/ml. As a conclusion, the current 
study found these oils have the potential to be used as anti bacterial properties. * Corresponding Author: Partiban Subramanian 
✉ partiban@amu.edu.my 

9 Subramanian et al. 
 
Int. J. Biosci. 2015 
Introduction 
Enterococcus faecalis, a natural inhabitant of the 
gastrointestinal tract and is a known cause of infective 
endocarditis since ca. 1900. It has recently emerged 
as a significant nosocomial pathogen. Interest in 
enterococci derives in part infections which are 
difficult to control or treat, their propensity for 
incorporation of mobile elements and their ability to 
transfer these resistance phenotypes to other 
pathogen including the apparent transfer of 
vancomycin resistance from E. faecalis to Methicillin 
Resistant Staphylococcus aureus in human 
(Nallapareddy et al., 2005). 
Bacteria hold the greatest importance in medical 
profession and living organism. Bacterial infection 
can give harmful effects if not treated. Enterococci is 
one of the primary causes of hospitalized-acquired 
infections, although enterococci can also cause 
human infections in the community (Roias et al., 
2013). The conventional treatments of bacterial 
disease are limited, and the reason is due to the 
limited spectrum of antibacterial drugs, expensive 
treatment and need of prolonged therapy (Gore et al., 
2010). Bacterial infection can also be treated by using 
herbal medicine or traditional medicine (Ubulom et 
al., 2011). The World Health Organization (WHO) has 
estimated that approximately 80% of the world’s 
population depends on traditional medicines (Gore et 
al., 2010). 
Current methods used to control E. faecalis infections 
are by using Ampicillin and Vancomycin. Ampicillin 
is the drug of choice for monotherapy of 
susceptible E. faecalis infection. For rare strains that 
are resistant to Ampicillin because of beta-lactamase 
production, Ampicillin plus sulbactam may be used. 
Vancomycin should be used on patients with 
Penicillin allergy or infections with strains that have 
high-level Penicillin resistance due to altered PBPs. 
Nitrofurantoin is effective in the treatment of 
enterococcal urinary tract infections, including those 
which are caused by vancomycin-resistant 
enterococci (VRE) strains. As more experience is 
gained with the use of linezolid, daptomycin, and 
tigecycline, these drugs may be used more commonly 
to treat VRE infections (Koba et al., 2009). 
Lemongrass is widely used as an ingredient in Asian 
cuisines due to its sharp lemon flavor. Lemongrass 
essential oil is applied for its medicinal value to cure 
acne, oily skin, scabies, flatulence headaches, blood 
circulation problem and excessive perspiration due to 
its antimicrobial and antibacterial activities. Previous 
studies have shown that lemongrass has antibacterial 
properties. Lemongrass oil’s antimicrobial properties 
make it an effective drug to treat bacterial infections. 
It can be used for food poisoning, staphylococcal 
infections and other common infections of the colon, 
stomach and urinary tract. Previous studies were 
conducted to elucidate the antibacterial activity of 
essential oil through testing it against E. faecalis 
(Singh et al., 2011). 
Cymbopogon citratus (lemongrass) is an aromatic 
perennial tall grass with rhizomes and densely tufted 
fibrous root. It has short underground stems with 
ringed segments, coarse, green slightly leathery leaves 
in dense clusters. The plant is a native herb from 
India and is cultivated in other tropical and 
subtropical countries. It is used as traditional folk 
medicine in the treatment of nervous, gastrointestinal 
disturbances, fevers and hypertension. Lemongrass is 
also a folk remedy for cough, consumption, 
elephantiasis, flu, gingivitis, headache, leprosy, 
malaria, ophthalmia, pneumonia and vascular 
disorders. It is principally taken in the form of tea as a 
remedy for digestive problems, diarrhea and stomach 
ache. Other than as a medicinal plant, lemongrass has 
been considered as carminative and insect repellant. 
Studies the on extract from C. citratus leaves have 
demonstrated to be anti-inflammatory, vasorelaxing, 
diuretic and valuable remedy in treating ringworm as 
local application (Dama, 2011). 
Cymbopogon nardus is a local medicinal plant, 
traditionally used for post partum bath. Scientific 
studies have proven C. nardus to possess several 
biological activities, such as antiviral, antibacterial 
and insect repellent. C. nardus, a Poaceae is a 

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Int. J. Biosci. 2015 
medicinal plant widely used in culinary and 
perfumery. The Chinese use the leaves more 
specifically for rheumatism and other uses in the 
treatment of fever, intestinal parasites, digestive and 
menstrual problems. C. nardus produced yellow 
essential oil which presents some pharmacological 
properties as antifungal, repellent against mosquito 
(Adibah et al., 2009). Literature is poor on the 
analgesic effect and African comparative study of 
chemical compounds of essential oil of this plant. 
Essential oils are known to possess antibacterial 
activity, which has been evaluated mainly in liquid 
medium (Inouye et al., 2001). The essential oil 
extracted from tulsi leaves contains eugenol, a 
phenolic compound which may be attributed to its 
antimicrobial, antidiabetic, and anticancer properties. 
Mangifera indica is commonly called as mango in 
English. Mangiferin, a major C-glucosylxanthone is 
found to occur in the M. indica stem bark, leaves, 
heartwood, roots, and fruits. The antibacterial activity 
of mango kernel may be attributed to the tannins 
present in them (Subbiya et al., 2013). 
Even though there are many green plants and their 
derivatives are used widely to treat bacterial and 
fungal infection, the search for more effective and 
potent antimicrobial drug still continue. In previous 
studies the essential oils from C. citrates and C. 
nardus were tested against E. faecalis for their 
antibacterial activity but they didn’t study the 
chemical composition in the same study. The 
chemical compositions study is very crucial to identify 
the active compound that responsible for antibacterial 
effect. The current investigation is aimed to 
determine the chemical composition and assess the 
antibacterial potential of essential oil from C. citrates 
and C. nardus against E. faecalis. This study also 
attempts to compare the effectiveness of essential oil 
of C. citrates and C. nardus against E. faecalis. 
Materials and method 
Plant material 
The plants were collected from a farm in Kemaman, 
Terengganu, Malaysia. Plant materials were 
taxonomically authenticated by Mr. Shamsul Khamis 
at the Institute of Bioscience, University Putra 
Malaysia. The voucher number for C. nardus is SK 
2157/13 and for C. citrates is SK 2158/13. 
Bacterial culture 
The strain of E. faecalis was purchased from Premier 
Diagnostic (M) Private Limited. The bacterium was 
cultured on Mac Conkey agar and blood agar by 
performing streak plate method. The plate was 
incubated at 37°C overnight. Prior to use, the bacteria 
was subcultured by taking one or two colonies of the 
bacterial from stock plate and inoculated onto 
nutrient agar to maintain its life and purity 
(Nallapareddy et al., 2005). 
Extraction of essential oils 
Hydrodistillation method used to obtain the essential 
oils is a similar method as used by Koba et al., 2009. 
The extraction of essential oils C. citratus and C. 
nardus was performed with 2 kg of fresh whole plant 
in the Toxicology Laboratory, University Malaya, 
Kuala Lumpur, Malaysia. 
Essential oils analysis using GC and GC-MS 
Gas chromatographic analysis was carried out on a 
Shimadzu GC Q2010 with FID detector using fused 
silica capillary column HP-5ms, 5% 
phenylethylsiloxane (30.0 m x 0.25 mm ID x 0.25 μm 
film thickness) with helium as carrier gas at a flow 
rate of 1 mL per minute. The column temperature was 
initially set at 60°C for 10 minutes, then increased 
3°C per minute to 230°C and was kept isothermally 
for 1 minute. The temperature of injector port and 
interface of mass spectrometry was 230°C and 250°C, 
respectively. 
The GC-MS analysis was performed by using an 
Agilent Technologies 6890N gas chromatograph 
equipped with 5975 inert mass selective detector (70 
eV direct inlet) on fused silica capillary column HP- 
5ms (30.0 m x 0.25 mm ID x 0.25 μm film thickness). 
The carrier gas was helium (99.999%) at a flow rate of 
1 mL per minute and a split ratio of 1:10. The column 
temperature was initially set at 60°C and was kept 

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Int. J. Biosci. 2015 
isothermally for 10 minutes, then increased at 3°C per 
minute to 230°C and held for 1 minute. 
Component identification 
Retention indices were determined from the gas 
chromatogram by logarithmic interpolarization 
between bracking alkanes by using a homologous 
series of n-alkanes as standards and in accordance 
with established method. The total ion chromatogram 
obtained as auto integrated by Chemstation software 
and the compounds were identified by comparison 
with Wiley 9th edition NIST 11 Mass Spectral Library. 
Preparation of inoculum 
Colonies were inoculated directly from the blood agar 
plate for E. faecalis. A loopful of isolated colonies was 
inoculated into 4 ml of Trypticase Soy Broth (TSB). 
The turbidity of actively growing bacterial suspension 
was adjusted to match the turbidity standard of 0.5 
McFarland. This was done by using 
spectrophotometer (625nm) and 1 cm path in which 
the absorbance is equivalent to 0.08 to 0.10). This 
suspension should be used within 15-30 minutes of 
preparation. 
Determination of Minimum Inhibitory 
Concentration (MIC) 
The minimum inhibitory concentration (MIC) of C. 
citratus and C. nardus against E. faecalis was 
determined by using two-fold macrodilution (tube) 
broth method. Twelve sterile and dry test tubes were 
used. Each of the tubes was labeled according to the 
different concentration of essential oil. First, 1.0 ml of 
Trypticase Soy Broth (TSB) was added into all of the 
tubes. Then 1.0 ml of essential oil was added into the 
first tube and 1.0 ml of Ethyl acetate was added 
respectively to dissolve between oil and broth. The 
content was mixed and 1.0 ml of the mixture was 
transferred into the second tube. The dilution was 
continued in the same manner for eleven tube. Then, 
1.0 ml from the eleventh was discarded. The twelfth 
tube was set to be as negative control. Next, 0.1 ml of 
prepared bacterial inoculum was added into all tubes. 
Then, the tube was incubated at 37°C overnight. After 
overnight incubation, the tubes were examined for 
visible sign of bacterial growth. 
Positive and Negative control 
Ampicillin (10 μg/disc) was used as positive control 
for E. faecalis and ethyl acetate was used as negative 
control. 
Antibacterial Susceptibility Test (AST) 
Antibacterial susceptibility test for E. faecalis was 
done by disc diffusion method. The concentration of 
C. citratus and C. nardus that show MIC were 
impregnated onto standard empty disc (6 mm). Then, 
the disc was placed onto Mueller Hinton agar with 
inoculated E. faecalis cultures. Ampicillin (10U) was 
used as positive control whereas Ethyl acetate as 
negative control. The plates were then incubated at 
37°C overnight. The antibacterial activity of the test 
agent was determined by measuring the diameter of 
the zone inhibition in millimeters. The zone of 
inhibition was compared to the positive control 
(Ampicillin) to categorize either C. citratus or C. 
nardus essential oils were susceptible, intermediate 
or resistance to be used as antibacterial agent. E. 
faecalis was considered as susceptible if the zone of 
diameter is more than 4mm (≥ 4mm). This method 
was carried out three times and the mean of reading 
was recorded. 
Results 
24 compounds were identified in the C. citratus 
essential oil, comprising 87.95 % of the total oil 
(Table 1). The highest peak was at retention time 
30.879 minutes. Geranial was identified as most 
abundant compound with 32.4% followed by beta 
citral (23.8%) and geraniol (12.0%) as major 
components. Whereas for C. nardus 23 compounds 
were identified comprising 70.12 % of the total oil 
(Table 2). The highest peak was at retention time 
42.538 minutes. Elemol was identified as most 
abundant compound with 17.3%. Detected 
constituents were Elemol (17.3%), Citronellol (12.5%) 
and Naphthalene (10.1%) as major components. 
The MIC of C. citratus against E. faecalis was 
determined due to 80% reduction in bacterial growth 

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Int. J. Biosci. 2015 
with 0.100 absorbance. Based on the result, MIC of 
C. citratus against E. faecalis was at concentration 
62.5 mg/ml. The MIC of C. nardus against E. faecalis 
was determined due to 80% reduction in bacterial 
growth with 0.171 absorbance. Based on the result, 
MIC of C. nardus against E. faecalis was at 
concentration 31.25 mg/ml. 
Table 1. Chemical composition of C. citratus essential oil. 
Chemical 
Constituents 
R.T. R.I Percentage (%) Method of 
C. citratus Adams, 2001 C. citratus Huynh et al., 2009 
Identification 
6-methyl-5-hepten- 
2-one 
14.188 982 986 4.94 0.40 MS, RI 
beta-Myrcene 14.271 983 991 2.09 10.20 MS, RI 
cis-Ocimene 17.133 1029 1050 0.33 - MS 
beta-z-Ocimene 17.752 1039 1037 0.13 0.32 MS, RI 
Linalool 20.951 1091 1097 1.37 0.58 MS, RI 
E-citral 23.450 1136 1341 0.22 - MS, RI 
Citronella 23.918 1145 1153 0.33 0.20 MS, RI 
Nerol 28.634 1236 1230 5.49 32.40 MS, RI 
beta-Citral 29.198 1248 1318 23.77 - MS 
Geraniol 30.306 1270 1253 12.05 5.50 MS 
Geranial 30.879 1282 1267 32.43 45.20 MS 
cis -beta-Farnesene 38.456 1449 1457 0.12 0.18 MS, RI 
beta-Chamigrene 39.385 1471 1478 0.12 - MS, RI 
Naphthalene 39.647 1477 1181 0.14 - MS, RI 
alpha-Muurolene 40.330 1493 1500 0.10 0.20 MS, RI 
beta-Bisabolene 40.822 1505 1506 0.09 - MS, RI 
delta-Cadinene 41.269 1516 1523 0.24 0.95 MS, RI 
Elemol 42.359 1543 1550 0.17 0.60 MS, RI 
Caryophylene oxide 43.754 1578 1583 0.46 0.18 MS, RI 
alpha-Gurjene 45.208 1616 1410 1.76 0.45 MS 
tau-Muurolol 46.006 1637 1642 0.30 - MS, RI 
beta-Eudesmol 46.348 1646 1651 0.18 0.20 MS, RI 
Germacrene B 46.509 1651 1561 0.55 0.31 MS, RI 
beta-Panasinsene 46.688 1656 1383 0.39 - MS 
MS = Mass Spectrometry, NIST 05 Mass Spectral Library. 
RI = Retention Indices. 
"-" = not reported. 
Table 4 shows the zone of inhibition in mm of C. 
citratus and C. nardus on E. faecalis. The results 
show that C. citratus oil at concentration of 62.5 
mg/ml is 6.00 mm. The oil of C. nardus at 
concentration of 31.25 mg/ml is 7.00 mm. This shows 
that C. nardus have a higher antibacterial activity 
against E. faecalis compared to C. citratus. 
Discussion and conclusion 
The highest percentages of compositions in the C. 
nardus essential oil in the present study are Elemol 
(17.3%), Citronellol (12.5%) and Naphthalene is 
(10.1%). The study conducted by Abena et al., shows 
that the C. nardus essential oil possesses higher 
percentage of Citronellol that is about 9.2% and 
followed by Geraniol (29.4%). As compared with the 
study of Koba et al., it shows that the highest 
percentage is Citronellal (35.5%), followed by 
Geraniol (27.9%) and Citronellol (10.7%). 

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Int. J. Biosci. 2015 
Chemical composition of Cymbopogon citratus in the 
study conducted by Koba et al., shows the highest 
percentage of Geranial (45.2%), followed by Neral 
(32.4%) and Mycrene (10.2%). Compared with the 
present study, the highest percentage of chemical 
composition of C. citratus are Nerol (32.4%), beta 
citral (23.8%) and geraniol (12.0%). 
Table 2. Chemical composition of C. nardus essential oil. 
Chemical Constituents R.T. R.I Percentage (%) Method of 
C. nardus Adams, 2001 C. nardus Koba et al., 2009 
Identification 
Mesitylene 12.600 952 996 0.33 - MS D-Limonene 16.440 1018 1029 1.42 0.50 MS, RI alpha-Terpinolene 20.893 1090 1089 
0.48 0.30 MS, RI cis-Rose Oxide 21.504 1100 1108 0.19 - MS, RI Isopulegol 23.447 1136 1150 1.18 0.10 MS Citronella 24.031 
1147 1153 8.70 35.50 MS, RI Citronellol 28.127 1226 1226 12.54 10.70 MS, RI E-Citral 30.034 1265 1341 0.32 - MS 
alpha-Humelene 33.896 1346 1455 0.14 - MS, RI beta-Selinene 38.321 1446 1490 0.30 - MS Naphthalene 38.732 1446 1181 
0.21 - MS Germacrene-D 39.327 1460 1485 0.48 3.30 MS cis-murola-3,5-diene 39.751 1470 1450 0.71 - MS alpha-Muurolene 
39.976 1475 1500 0.20 - MS Germacrene A 40.100 1478 1509 0.46 - MS alpha Amorphene 40.308 1483 1485 0.82 - MS, RI 
Aromadendrene 40.899 1507 1441 1.50 - MS delta-Cadinene 41.292 1517 1523 2.57 3.20 MS, RI aplha-Cadinene 41.830 1530 
1539 0.18 1.10 MS, RI Elemol 42.538 1530 1550 17.35 0.10 MS beta-Gurjene 45.149 1614 1434 0.52 - MS gama-Eudesmol 
45.643 1628 1632 4.77 0.30 MS, RI Azulene 47.673 1682 1298 0.56 - MS MS = Mass Spectrometry, NIST 05 Mass Spectral 
Library. 
RI = Retention Indices. 
"-" = not reported. 
Table 3. MIC values of essential oils against E. 
faecalis. 
MIC in mg/ml Cymbopogon citratus Cymbopogon nardus 62.5 31.25 
This study confirms that both species of Cymbopogon 
have antibacterial property and concludes that C. 
nardus is more effective against E. faecalis as 
compared to C. citratus because C. nardus essential 
oil have a greater zone of inhibition. In the past, oil of 
Cymbopogon species had been used widely as 
antibacterial agents for pathogenic bacteria such as 
Escherichia coli, Salmonella typhimurium, and 
Stapyhlococcus aureus. Previous study shows that 
Cymbopogon nardus and Cymbopogon flexuosus can 
be used against E. coli and S. aureus (Rocha et al., 
2011). Based on study carried out by Singh et al., 
(2011), C. citratus essential oil can be applied for its 
medicinal value to cure acne, oily skin, scabies, 
flatulence, headaches and blood circulation problem. 
Although, in a few preliminary antibacterial 
screenings, the oil had shown no activity against four 
Gram positive (Bacillus subtillis, Corynebacterium 
diphtheria, Streptococcus pyogenes and 
Stapyhlococcus aureus) and three Gram negative 
(Salmonella paratyphi A, Escherichia coli and 
Pseudomonas aeruginosa). Later on, several studies 
have shown that the lemongrass has antibacterial 
properties. 
In conclusion, the essential oils of Cymbopogon 
citratus and Cymbopogon nardus have been found to 
possess antibacterial activity against Enterococcus 
faecalis. Further studies should be conducted to 
determine the bioactive compound that elicits the anti 
bacterial activity. 

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Int. J. Biosci. 2015 
Table 4. Antibacterial activity of C. citratus and C. nardus against E. faecalis. 
Plants Inhibition zone diameter (mm) 
EO PC NC Cymbopogon citratus 6.00±1.73 30.00±0.00 0.00±0.00 Cymbopogon nardus 
7.00±2.65 30.00±0.00 0.00±0.00 
Values are expressed as mean ± standard deviation of the three replicates. 
EO: essential oils, 
PC: positive control, 
NC: negative control. 
Acknowledgement 
The authors wish to thank Asia Metropolitan 
University, Malaysia for providing funding and 
facilities to carry out this study. 
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