Journal of ~t~~opharm~~o~ogy, 12 (1984) 274-286 279

Elsevier Scientific Publishers Ireland Ltd.

ANTIBACTERIAL CONSTITUENTS IN THE ESSENTIAL OIL OF

CYMBOPUGON CITRATUS (DC.) STAPF.
GRACE 0. ONAWUNMIa, WOLDE-AB YISAKb and E.O. OGUNLANAa’*
aDepartment of Pharmaceutics, bDepartment of Pharmaceutical Chemistry, University
of Ife, Ile-lfe (Nigeria)
(Accepted August 28, 1984)

Cymbopogon ~1~~~~s (DC.) Stapf., co~o~ly known as lemon grass and

used, over many years, for medicinal purposes in West Africa, produces a
volatile oil on steam extraction of its leaves. The antibacterial properties of
the essential oil have been studied. These activities are shown in two of the
three main components of the oil identified through chromatographic and
mass spectrometric methods. While the arcitral (geranial) and p-citral (neral)
components indi~du~ly elicit antibacterial action on gram-negative and
~~-positive organisms, the third component, myrcene, did not show
observable antibacterial activity on its own. However, myrcene provided
enhanced activities when mixed with either of the other two main com-
ponents identified.

Introduction

Cymbopogon citrutus (DC.) Stapf, is commonly known as lemon grass,

and has been cultivated over many years for medicinal purposes in West
Africa. Information collected from Ghana, Liberia, Nigeria, Sierra Leone and
Guinea Bissau indicate its use in a variety of traditions prep~tions. The
fragrant leaves which contain the volatile oil are usually extracted in the
form of tea to serve as febrifuge while the roots are used as chewing sticks
or rubbed on the teeth for cleaning (Sawyerr, 1982). Earlier ethnomedical
reports by Oliver (1960) and Alves et al. (1960) have stated that the plant
possesses rubef~~ient, stimulant and antispasmodic properties.
Another species of lemon grass which has also been of interest in folklore
and commerce in Asia is Cymbopogon flexuosus. Various workers have re-
ported on this plant. Bose (1950) indicated the antibacterial property of
the essential oil. Maruzzela and Liguori (1958), Kokate and Varma (1971),
Agarwal et al. (1981) have since confirmed the activity of the oil on microbes.

*To whom all correspondence should be addressed.

O378-8741/84/$02.70 o 1984 Elsevier Scientific Publishers Ireland Ltd.

Published and Printed in Ireland
280

Similar studies by Gynae (1976) and Chiori et al. (1977) have shown the
antibacterial property of the essential oil from the leaves of Cymbopogon
citrutus. However, all these reports gave no detailed study of the antibacte-
rial components of the oil. This study was therefore carried out as part of
a programme to establish the main components of the oil of Cymbopogon
citratus that exhibit antibacterial activity.

Materials and methods

Extraction of the essential oil

Since lemon grass is cultivated as a common ornamental plant on the

University of Ife Campus, this source was used for the collection of plant
leaves during the wet season (April-August). The plant sample was authen-
ticated by comparing it with a herbarium specimen of Cymbopogon
citrutus deposited at the Forest Research Institute of Nigeria, Ibadan.
Fresh aerial parts of the plants were collected, usually in the morning, and
cut into pieces 3-4 cm in length before packing into an all-glass still
described in the British Pharmacopoeia (1980). The extractions were
carried out to exhaustion and the yield of oil was 0.80-0.98% for various
batches. The dried oil was kept in air-tight glass containers, covered with
aluminium foil, at 4°C until required for use.

Separation and identification of the main components of the oil

A Varian gas chromatograph model 3700 equipped with a flame ioniza-

tion detector and fitted with a column packed with 3% SE-30 on gas chrom
Q was used in the analysis of the oil. Chromatography was run with column
temperature 110°C injection temperature 225°C and detector temperature
250°C.
Three major peaks A, B and C, respectively were obtained (Fig. 1). Pre-
parative gas chromatography was run on a Varian Aerograph model 920A
equipped with a thermal conductivity detector and fitted with a 182.88 X
0.64 cm stainless steel column packed with 3% SE-30 on gas chrom Q. The
temperature conditions were as described above. Fractions B and C were
collected using this preparative gas chromatography method.
However, Fraction A was collected by column chromatography. The oil
was chromatographed on a column (2.5 X 60 cm) packed with silica gel
(mesh 70-230) and eluted with petroleum ether (40-6O”C) containing
varying amounts of diethyl ether. The first fraction was collected and
screened as Fraction A without further purification. The three separated
fractions were identified by their physical constants and by comparison
with authentic samples. In addition, mass spectrometric data were obtained
from a Kratos MS 25 with a DS 55 computer data output coupled by a jet
interface separator to a Perkin Elmer Sigma 3 gas chromatograph. Ioniza-
tion was by electron impact at 70 eV at 200°C.
 281

1 A BC

Fig. 1. Gas liquid chromatogram of lemon grass oil run on a Varian 3’700 gas ehromato-
graph, FID detector, glass column packed with 3% SE-30 on gas chrom Q.

Bat terial strains

The bacteria used were Staphylococcus aureus NCTC 5671, Bacillus

subtilis NCTC 8236, Escherichia coli NCTC 9001 and Pseudomonas
aeruginosa NCTC 6750.

Antibacterial activity study by agar-diffusion assay method

The test bacterial strains were prepared as follows: a loopful of cells from
the stock culture was inoculated into 100 ml nutrient broth contained in
250 ml Erlenmeyer flasks incubated on an orbital shaker at 37°C for 18 h.
The culture obtained was centrifuged in a refrigerated centrifuge at
10,000 X g for 15 min. The resulting pellet was resuspended in sterile cool
distilled water for washing and repeated centrifugation twice. After the
final washings the cells were carefully dispersed in sufficient cold sterile
distilled water to produce a homogenous suspension. This was then adjusted
to contain the desired number of organisms per millilitre using a previously
calibrated nephelometer. The suspension of organisms were adjusted to
contain 1 X 10’ organisms/ml and 0.1 ml of that suspension was spread on
over-dried agar plates.
The separated fractions of the lemon grass oil were put on filter paper
discs (Whatman No. 1) of 6 mm diameter. Approximately 20 ~1 was suf-
ficient to saturate each disc, These were left to dry before they were placed
on the surface of the seeded plates. A standard disc of streptomycin 25 pg
was also placed on each of the seeded plates. In order to facilitate diffusion
the plates were left for about 1 h at 4°C before incubation at 37°C for 24 h.
The diameters of zones of inhibition were then measured.
These were repeated for three replicate plates of each of the tested organ-
isms. Since Agarwal et al. (1980) and Zamureenko et al. (1981) have reported
on some of the major constituents of the oil, it was considered necessary to
test the analytical grade of some known standard constituents of the oil for
antibacterial activity using the same agar diffusion technique. The studies
were also extended to incorporate some mixtures of citral and other con-
stituents in such proportions as were reported to be found in the oil. In this
set of tests the reference standard antibiotic used was ampicillin 10 yg.

Antibacterial activity study on growing cells

The cells were prepared as earlier stated for the inoculum used in the agar-
diffusion studies. The washed suspension of each of the organisms (0.2 ml)
was transferred to Erlenmeyer flasks containing 100 ml of nutrient broth.
The nephelometer readings were taken for all flasks before placing them in
a reciprocal shaker adjusted to about 120 throws/min at 37°C. The effects
of linalool, myrcene and citral as well as lemon grass oil and combinations

1 2 3 L 5 1 2 3 1 5
Time Ihoursi. Tlme;hours).

Fig. 2. Nephelometric studies of the effects of known constituents of lemon grass oil
on the growth of E. coli and I?. subtilis cells respectively, in nutrient broth. o-3,
control; 01, lemon grass oil 0.05% (v/v); a-, citral 0.05% (v/v); A----A myrcene
0.05% (v/v); n - n, linalool 0.05% (v/v); x-n, citral 0.05% + myrcene 0.0’5%
(v/v); A----5, citral 0.05% + linalool 0.05% (v/v).
 283

of the named components were studied using growing cells of E. codi and
B. subtilis in nutrient broth. In order to correct for any turbidity effect of
the concentrations of these compounds, the turbidity effects of three con-
centrations of lemon grass oil and the compounds in nutrient broth using
the nephelometer were determined. The values obtained in these readings
were used to correct the values from the experimental flasks. After 1 h
of growing the cells in nutrient broth, the compounds were introduced and
readings were taken. Triplicate flasks were used for each of the compounds/
mixtures. Readings were corrected appropriately. These were repeated at
intervals up to 5 h of groivth. The mean corrected nephelometer readings
were then graphed as shown in Fig. 2 for each of the organisms,

Results and discussion

The essential oil of C~~&opogo~ citrates (DC.) Stapf. sample extracted

from the cultivated plant showed three major components (Fig. I) viz peaks
A, B and C with retention times 0.81, 4.25 and 5.02 min respectively. The
percentage purity of the separated fractions was determined using analytical
gas chromatography and was found to be as follows: fraction A, 99%;
fraction B, 92.9%; fraction C, 98.68%, which indicate fairly good separations
of the components of the oil through the system.
These components were identified to be myrcene, a-citral (geranial) and
&citral (neral), respectively, using physical constants and by comparison
with authentic samples. cy-Citral (geranial) semi-carbazone, m.p. 162-164°C
(literature m.p. 164°C; Guenther, 1949), &citral (neral) semi-earbazone
m.p. 170-172°C (literature m-p. 171°C; Guenther, 1949).
In the mass-spectral data, each of the components were also identified by
their molecular ions as well as by other diagnostic ions.
Myrcene (peak A) gave the molecular ion M” at 136(6)* consistent with
the molecular formula of CI,-,HL6, other diagnostic peaks appeared at m/z
41(80), 69(85), 93(100) and 121(5.8). a-Citral (geranial) peak B had M”
at 152(1.5) and a base peak at m/z 41. Other diagnostic peaks were obser-
ved at m/z 53(12), 59(13.8), 69(81), 84(23.7), 94(X6) and lOS(17.0).
p-C&al (neral) peak Chad M’ at 152(U) and a base peak at m/z 69 with
other diagnostic ions at m/z 41(62.3), 68(35.0), 9.3(16.8) and 121jlO.Z).
The observed antibacterial activity of the pure lemon grass oil and the
three major components separated and identified as myrcene, a-citral and
@-citral are shown in Table 1 along with the reference standard streptomy-
cin 25 pg. Myrcene showed no observable effects against E. coli and B.
subtilis but some effect was obtained against Staph. aureus. Both a-citral
and p-citral were active against the three organisms tested with comparatively
similar results as with the pure oil. The E. coli strain was the most resistant
and 3. subtilis was more sensitive than Staph. aureus.

*This figure in parentheses indicate the relative abundance of the ions.

284

TABLE 1

AGAR DIFFUSION ASSAYS OF LEMON GRASS OIL AND FRACTIONS

Organisms Diameter of zones of inhibition (mm)”

Oil Fraction A Fraction B Fraction C Streptomycin

25 wg

E. coli 15 6 15 12 15
B. su btilis 32 6 40 20 32
Staph. aureus 32 11 30 17 25
-
a Incorporating 6 mm diameter of disc (mean of 6 readings).

The results also suggest that the antibacterial activity resides mainly in the
(Y-and (3-citralcomponents of the oil. Myrcene, which is also a major con-
stituent of the oil, did not show any observable activity on its own. A
number of compounds known to be components of lemon grass oil were
also tested for their antibacterial activity (Table 2). Other substances or
components of the oil that showed no antibacterial activity were hepte-
none, dipentene limonene (+ ) and limonene (-). At 100% purity linalool,

TABLE 2

AGAR DIFFUSION ASSAYS OF VARIOUS KNOWN CONSTITUENTS OF LEMON

GRASS OIL

Compounds Diameter of inhibition (mm)a

Concentration of compounds

100% 50% 10%

A B C D A B C D A B C D

Lemon grassoil 11 64 32 - 7 22 12 - ‘7 16 11 -
Citral 13 48 20 - 7 15 9 - 7 11 9 -
Citronella1 7 13 10 - 7 12 9 - - 8 7 -
Citronellol 7 13 11 - 7 7 7- - ---
Dipentene ____--------
Myrcene --------
Heptenone _ _ _ _ - - -- - - --
Geraniol 9 14 12 - 7 7 7 - - - - -
Linalool 12 9 9 - a 7 8 - 7 - 7 -
Ljmonene(+) --------- ---
Limonene (-)
Ampicillin 1O~g 20 10 46 32 - - - - - - - -

a Incorporating diameter of filter disc (6 mm) (mean of 6 readings),

b Organisms: A, E. colt’; B, B. subtilis; C, Staph. aureus; D, Ps. aeruginosa.
citronellal, citronellol, geraniol showed some activity against the organisms,
E. coli, B. subtilis and Staph. aureus, but not on Pseudomonas aeruginosa
which was resistant to even the pure lemon grass oil and citral. However,
on dilution of these compounds to 50% (v/v) and 10% (v/v) with 20% (v/v)
dimethyl-sulphoxide (DMSO) the observed activities of the compounds
were greatly reduced. In fact, at the 10% (v/v) dilutions only citral showed
clearly observable antibacterial activity.
The studies in respect of mixtures of some components of the oil with
citral are shown in Table 3. It is noteworthy that these reference com-
pounds, in the various percentages of combination, show enhanced acti-
vity on the most susceptible organism, B. subtilis, while no greater effects
than that of the pure oil were observed in the more resistant organisms.
These effects with both B. subtilis and E. coli could be due to agar, dif-
fusion etc. consequently the effects of linalool, myrcene and citral on
growing cells of E. coli and B. subtilis in nutrient broth were studied.
The nephelometer readings as shown against time in Fig. 2 clearly show
the effects of these compounds and combinations against growing cells.
Both organisms grew rapidly in the presence of myrcene while the citral/
myrcene combination was more effective in growth reduction of B.
subtilis than with E. coli. In the case of linalool some reduction in the

TABLE 3

AGAR DIFFUSION ASSAYS OF COMBINATIONS OF SOME LEMON GRASS OIL

CONSTITUENTS

Combination@ Diameter of inhibition (mm)

E. coli B. subtilis Staph. nureus

Citral 77% 9.5 z!z0.53 22.0 + 8.43 25.83 f 3.95

Citral 77% 10.6 f 0.71 47.0 f 6.06 24.33 + 3.50
+
Linalool 1.2%
Citral 77% 9.7 + 1.07 40.2 + 7.41 23.00 + 2.53
+
Myrcene 20%
Citral 77% 9.0 + 1.17 34.8 f 4.34 26.00 f 4.06
+
Citronellol 1.5%
Citral 77% 10.2 f 0.80 33.6 f 7.53 28.00 f 8.48
+
Geraniol 1.8%
Lemon grass oil 10.8 + 0.88 30.0 f 10.24 24.40 -I 6.95

a All percentage concentrations were made up with 20% (v/v) DMSO.

286

growth of E. coli and B. subtilis were observed but total inhibition was
not achieved. Linalool did not appear to increase the activity of citral as
previously shown in Table 3. Generally the results confirm the inhibition
of growth by citral while combinations with other constituents did not
show increased activity against growing cells.

Conclusions

Three main components of the essential oil of Cymbopogon citratus

(DC.) Stapf were separated by chromatographic methods and identified
as myrcene, or-citral and p-citral. The antibacterial activity of the oil
resides mainly in 01-and p-citral. Myrcene did not show observable anti-
bacterial activity but appeared to enhance the activity of citral.
Other minor components of lemon grass oil elicited some activity, while
in pure form, against the test organisms although the proportions of such
compounds in the oil were considerably low.

Acknowledgement

We acknowledge the financial support from the University of Ife

Research Grant.

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