Jeobp 13 (4) 2010 pp 426 - 431 426

ISSN 0972-060X

Chemical Composition and Antibacterial Evaluation of the

Essential Oil from Cymbopogon winterianus Jowitt (Gramineae)

Thially B. Gonçalves 1, Erlânio O. de Sousa 2,

Fabíola F.G. Rodrigues 2, José Galberto M. da Costa 2*

1
Faculdade de Ciências Aplicadas Dr. Leão Sampaio, Curso de Biomedicina,
Av. Leão Sampaio S/N, 63010-000, Juazeiro do Norte-CE, Brazil
2
Laboratório de Pesquisas de Produtos Naturais, Departamento de Química
Biológica, Universidade Regional do Cariri, Rua Cel. Antônio Luiz,
1161, 63105-000, Crato-CE, Brazil
Received 12 December 2009; accepted in revised form 19 April 2010

Abstract: This paper reports the chemical composition and verification of the antibacterial
potential of essential oil from fresh leaves of Cymbopogon winterianus collected in the region of
Cariri Ceará. The essential oil obtained by hydrodistillation showed yield of 0.5 %, and its chemical
composition analysis obtained by GC-MS confirmed the presence of citronellal (61.6 %) and other
constituents. Evaluation of antibacterial activity in vitro was performed by microdilution in BHI
broth. We tested six strains: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa,
Proteus vulgaris, Shigella flexneri, and Bacillus cereus. Trial data showed that the essential oil from
leaves of C. winterianus showed activity for all strains tested, and the best result with the strain of
Proteus vulgaris (ATCC 13315) (MIC 128 μg/mL).

Key words: Cymbopogon, essential oils, chemical composition, antibacterial activity.

Introduction: The resistance of pathogenic microorganisms to multiple drugs has

increased due to the indiscriminate use of antibiotics, commonly marketed and used in the
treatment of infectious diseases. In recent years, processes of synthesis of new molecules
and modification of natural and / or synthetic pharmacological properties have been defined
the methods of choice used to obtain new drugs 1.
New vegetal sources presenting antimicrobial activity and low toxicity could be a
viable alternative, with low cost and easily accessible to poor communities where the species
are found 2. The herbs in addition to supplying essential oils, has shown great potential
microbiological and volatile substances obtained has also been used as flavoring, flavoring

*Corresponding author (José Galberto M. da Costa)

E- mail: < galberto.martins@urca.br >
 José Galberto M. da Costa et al. / Jeobp 13 (4) 2010 pp 426 - 431 427

and therapeutic in food, pharmaceutical and cosmetic industries 3.

The family Gramineae comprises about 600 genera and 10,000 species, constitute a
family of great economic account 4. In Brazil, there are about 180 genera and 1500 species
distributed in the Southeast, North and Northeast 5. The genus Cymbopogon has about 100
described species, found in several tropical countries, and approximately 56 species of
herbs and some have medicinal amounts, pharmacological and industrial 3. Species of this
genus has presented several pharmacological and antimicrobial activities 6.
Cymbopogon winterianus Jowitt (Gramineae), hailing from India and popularly
known as citronella, is widespread in Brazil. It is a perennial herb which forms 1-meter
high compact and strong clumps. Citronella has economical importance in the production
of essential oils employed in the aromatic industry and as an insect repellant; furthermore,
it has antimicrobial and acaricide properties 7. More than 80 substances are comprised,
from which citronellal (40.0 %), citronellol, geraniol, limonene and esters have particular
importance 8.9.
This study aimed to verify the chemical composition and antibacterial effects of
essential oil of C. winterianus Jowitt (Gramineae) obtained in the region of Cariri, Ceará,
Northeast Brazil.

Experimental
Plant material: Fresh leaves of C. winterianus Jowitt (Gramineae) were collected
in the Medicinal and Aromatic Plant Garden at the Pimenta Campus of the Regional
University of Cariri (URCA), Crato, CE, Brazil, in May 2009. A representative voucher
sample was classified and deposited (no. 4680) in the Caririensis Dárdano de Andrade
Lima Herbarium of the Regional University of Cariri.

Extraction of essential oil: The fresh leaves (650 g) of C. winterianus Jowitt

(Gramineae) were triturated and extracted by hydrodistillation for two hours using a
Clevenger-type apparatus 10. The collected essential oil was dried over anhydrous sodium
sulfate (Na2SO4), and subsequently stored under low light conditions at < 10°C until analysis.

Essential oil analysis: Analysis of the volatile constituents of essential oil of C.

winterianus Jowitt (Gramineae) was carried out on a Hewlett-Packard Model 5971 GC-
MS using a non-polar DB-5 fused silica capillary column (30 m x 0.25 mm i.d., 0.25 μm film
thickness); carrier gas helium, flow rate 1 mL/min. and with split mode. The injector
temperature and detector temperature were 250°C and 200°C, respectively. The column
temperature was programmed from 35°C to 180°C at 4°C/min. and then 180°C to 250°C at
10°C/min. Mass spectra were recorded from 30-450 m/z, electronic impact 70 eV.
Individual components were identified by computer library MS searches using retention
indices as a pre-selection routine and visual inspection of the mass spectra from literature
for confirmation 11,12,13.

Antibacterial activity and Minimal Inhibitory Concentration (MIC): The

antibacterial activity of essential oil of C. winterianus Jowitt was investigated employing a
 José Galberto M. da Costa et al. / Jeobp 13 (4) 2010 pp 426 - 431 428

microdilution method, recommended by NCCLS M7-A6 14. In the assay six bacterial
species was used, obtained from Fundação Oswaldo Cruz-FIOCRUZ: Staphylococcus
aureus (ATCC 10390), Escherichia coli (ATCC 25922), Bacillus cereus (ATCC 33018),
Proteus vulgaris (ATCC 13315), Pseudomonas aeruginosa (ATCC 15442) and Shigella
flexneri (ATCC 12022).
Brain Hear Infusion Broth (BHI 3.8 %) was used for bacterial growth (24 h, 35 ±
2°C). The inoculum was an overnight culture of each bacterial species in BHI broth diluted
in the same media to a final concentration of approximately 1 x 108 CFU/mL (0.5
nephelometric turbidity units - McFarland scale). After this, the suspension was dilluted to 1
x 106 CFU/ mL in 10 % BHI. 100 μL of each dilution were distributed in 96-well plates plus
essential oils, achieving 5 x 105 CFU/mL as final concentration of the inoculum.
Essential oil of C. winterianus Jowitt was dissolved in distilled water and dimethyl
sulfoxide (DMSO) to a concentration of 1024 μg/mL. Further serial dilutions were per-
formed by addition of BHI broth to reach a final concentration in the range of 8 to 512 μg/
mL. All experiments were performed in triplicate and the microdilution trays were incubated
at 35 ± 2°C for 24 h. Antibacterial activity was detected using a colorimetric method by
adding 25 μL of resauzurin staining (0.01%) aqueous solution in each well at the end of the
incubation period. The minimal inhibitory concentration (MIC) was defined as the lowest
essential oil of C. winterianus concentration able to inhibit the bacteria growth, as indicated
by resauzurin staining (bacteria died cells are not able to change the staining color by visual
observation-blue to red).

Results and discussion: The oil isolated from fresh leaves of C. winterianus
represented 0.5 % of their total fresh weight. Chromatographic analysis permitted the
identification and quantification of nine components, representing a total of 97.6 %. Citronelal
(61.0 %), geraniol (19.0 %) and citronelol (11.5 %) were identified as majority components,
as presented in Table I.
The citronelal, a major component of C. winterianus is an excellent flavoring
environment and insect repellent, also has antimicrobial and acaricide activity against mites
micro air are responsible for allergic respiratory processes, common in environments with
carpet and air-conditioning 8,15.
In the analysis of antibacterial activity of essential oil of C. winterianus by microdilution,
we observed a similar inhibitory activity for the strains S. aureus and P. aeruginosa (MIC
256 μg/mL) and E. coli and S. flexineri (MIC 512 μg/mL). The essential oil of C. winterianus
showed antibacterial activity against all strains is more effective for gram-negative P.
vulgaris (MIC 128 μg/mL).
These results confirm and complement previous studies in which essential oil of C.
winterianus effective against strains of gram-negative and gram-positive 16, 17. In trials
with antibacterial essential oil of C. citratus, it was found effective activity to E. coli with
89.9 % inhibition 18. Antibacterial activity was also observed for bacteria S. aureus 16.
Studies with the essential oil of C. winterianus showed the same major components,
but with variations in the percentages as citronellal (34.1 %), geraniol (21.5 %) and citronellol
(11.5 %) and high antibacterial activity against the Pseudomonas putida associated with
 José Galberto M. da Costa et al. / Jeobp 13 (4) 2010 pp 426 - 431 429

deterioration of meat 19. The wide range of major constituent citronellal evidenced in this
study can be associated with various seasonal factors such as temperature, climate and
time of collection, which therefore affect microbial activity 20.
A recent study on the essential oil of C. winterianus showed activity against strains
of enterotoxigenic. Escherichia coli as a potential inhibitor of growth of this microorganism.
This same study revealed the analysis of the essential oil of Cymbopogon martinii showing
the presence of compounds with known antimicrobial activity, including geraniol isolated as
active antimicrobial compound 21.
Essential oils may interact with and affect the plasma membrane, interfering with
respiratory chain activity and energy production 22.
The inhibition of gram-negatives bacteria growth here demonstrated is a significant
result, as the essential oils, in general, are more able to have antibacterial activity against
gram-positives (more sensitive to antibiotics). Gram-negatives bacteria present structural
particularities that difficult the antibiotics penetration of some antibiotics, as the
lipopolysaccharide structures containing polysaccharides of different length that largely
contribute to cell surface properties, such as membrane permeability and antibiotic suscepti-
bility 23,24.
In conclusion, this study demonstrated that C. winterianus leaves essential oils have
antibacterial activities. This essential oil is beneficial to human health, they have the potential
to be used for therapeutic purposes and to be utilized as antibacterial additives.

Acknowledgments: The authors would like to acknowledge financial support from

CAPES, CNPq and FUNCAP, UFPI for the chromatograms and FIOCRUZ for the microbial
lines.

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Table 1. Chemical composition of the essential oil of Cymbopogon winterianus

Component RT (min) KIexp* Leaf oil (%)

Limonene 7.9 1029 2.6

Linalool 10.2 1095 0.5
Isopulegol 11.6 1142 0.6
Citronellal 12.1 1152 61.0
Citronellol 14.6 1224 11.5
Geraniol 15.5 1225 19.0
Geranial 15.8 1263 0.9
Citronellyl acetate 18.4 1348 0.7
Neryl acetate 19.4 1359 0.8
Total 97.6

* retention indices experimental.

* n-Alkanes were used as reference points in the calculation of relative retention
indices.

Table 2. Values of the Minimal Inhibitory Concentration (MIC)

of Cymbopogon winterianus leaves essential oil

Strains μg/mL)
MIC (μ

E. coli (ATCC 25922) 512

S. aureus (ATCC 10390) 256
P. aeruginosa (ATCC 15442) 256
P. vulgaris (ATCC 13315) 128
S. flexineri (ATCC 12022) 512