Canadian Journal on Computing in Mathematics, Natural Sciences, Engineering and Medicine Vol. 3 No.
7, December 2012
Volatile constituents, antibacterial and insecticidal
activities of essential oil from the leaves of Vitex
agnus-castus L. (Verbenaceae)
Oluwole Omikorede *, Oladipupo A. Lawal and Opeyemi A. Iresemowo
Department of Chemistry, Lagos State University, PMB 001, Lasu Post Office, Ojo, Lagos, Nigeria
*Corresponding author: omikorede@yahoo.com; oluwole.omikorede@lasu.edu.ng
Abstract — The essential oil isolated from the leaves of
Vitex agnus-castus by hydrodistillation was analyzed by
GC/MS. Thirty-two components representing 94.3% of
the total oil were identified. The major components were
α-terpineol (11.5%), trans-β-farnesene (7.7%), 2-amino-
6,7-dimethylpteridin-4-ol (6.5%), terpinen-4-ol (6.3%),
γ-cadinene (6.3%) and terpinolene (5.6%). The in vitro
antibacterial activity of the oil was studied against five
bacteria strains using disc diffusion and broth
microdilution methods. The oil exhibited moderate to
high activity against S. aureus, E. coli, B. Cereus and B.
subtilis. The insecticidal activity of the oil (LC50) at 250
mg/L air to adult Acanthoscelides obtectus, Sitophilus
zeamais and Tribolium castaneum were 121.67, 106.23
and 146.45mg/ml, respectively.
Key Words — Vitex agnus-castus, Verbenaceae, essential
oil composition, α-terpineol, trans-β-farnesene, antibacterial
activity.
I. INTRODUCTION
Vitex agnus-castus L. (Verbenaceae) is a perennial
shrub or tree (ca 5-6 m) tall with gray-puberalent and
tormentous young branches. It is native in the whole
Mediterranean regions and mostly found in dense coastal
areas and along rivers, but, now grows as ornamental
plant in tropical and subtropical regions of the world [1].
The leaves are dark green and opposite, with 5-7
lanceolated pinnulas and about 10 cm long with entire
margins. The inflorescences are branched and spiked.
The flowers (8-10 mm) are tall and aromatic. The fruits
(about 3-4 mm) are spherical and elongated, and vary in
color from reddish to black [1]. Vitex agnus-castus has
long been used in traditional medicine as a remedy for
menopausal and perimenopausal symptoms (PMS),
stomach-ache, headache, influenza, diarrhea and syphilis
[2]-[5]. In addition, several pharmaceutical and biological
activities including immunodulatory, antimicrobial and
antioxidant have also been reported for the plant [6]-[9].
Previous studies on the phytochemical analysis of V.
agnus-castus revealed the presence of glycosides,
flavonoids, diterpenoids, steroids and essential oils [10]-
[14].
Compositional studies on essential oils from different
organs of V. agnus-castus were available in the literature,
and the major constituents identified were mostly 1,8-
Cineole (2.7-50.9%), 1,8-Cineole+limonene (22.4-
36.4%), sabinene (1.9-31.2%), (E)-β-farnesene (4.8-
9.9%), α-pinene (0.4-13.9%), (Z)-β-farnesene (5.4-9.4%)
and β-caryophyllene (1.4-6.0%). Other predominant
components were cis-ocimene, terpinen-4-ol, β-farnesene,
terpinen-4-ol, α-terpineol and β-phellandrene [5], [6], [8],
[12], [14]-[28].
Literature search on chemical composition of essential
oils of V. agnus-castus from Nigeria showed two reports
[5], [6]. However, to the best of our knowledge, no study
has reported the insecticidal activity of V. agnus-castus
essential oil from Nigeria and no previous information on
insecticidal activity of V. agnus-castus is reported
anywhere. Thus, this paper reports the chemical
composition, antibacterial and insecticidal activities of
essential oil from the leaves of V. agnus-castus growing
wild in Lagos State, Nigeria.
.
II. Materials and Methods
Plant materials
Fresh leaves of Vitex agnus-castus were collected from
a private garden in Magodo town, Ikeja, Ikeja Local
Government Area, Lagos State, Nigeria. Identification of
the plant material was carried out at the Department of
Botany, University of Lagos. A voucher specimen (LUH
4350) had been deposited at the University Herbarium.
Oil isolation
The air-dried and crushed leaves of V. agnus-castus
(500g) were hydrodistilled in a Clevenger-type glass
apparatus for 3h in accordance with the British
Pharmacopoeia specification [29]. The distillate isolated
was preserved in a sealed sample tube and stored under
refrigeration until analysis.
Gas Chromatography-Mass Spectrometry
GC-MS analyses of the oil were performed on a
Hewlett Packard Gas Chromatography HP 6890
interfaced with Hewlett Packard 5973 mass spectrometer
system equipped with a HP 5-MS capillary column (30m
x 0.25 mm id, film thickness 0.25 µm). The oven
temperature was programmed from 70- 240 oC at the rate
of 5oC/min. The ion source was set at 240oC and electron
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Canadian Journal on Computing in Mathematics, Natural Sciences, Engineering and Medicine Vol. 3 No. 7, December
2012
ionization at 70eV. Helium was used as the carrier gas at
a flow rate of 1 ml/min. Scanning range was 35 to 425
amu. Diluted oil in n-hexane (1.0 µL) was injected into
the GC/MS. The components of the oils were identified
based on the comparison of their retention indices and
mass spectra with those standards, Wiley 275 library
mass spectra database of the GC/MS system and
published data [30]-[32].
Antibacterial assay
The essential oil was tested against five local bacteria
isolates (three Gram-positive and two Gram-negative
strains) obtained from the Department of Microbiology,
University of Fort Hare, South Africa: Bacillus cereus,
Bacillus subtilis, Staphylococcus aureus, Escherichia coli
and Pseudomonas aeruginosa. The stock cultures were
maintained at 4oC in Müeller-Hinton agar (Oxoid,
Germany).
Agar disk diffusion
The V. agnus-castus essential oil was tested for
antibacterial activity by the agar disc diffusion method
according to [33]. The microorganisms were grown
overnight at 37oC in 20 mL of Müeller-Hinton broth
(MHB). The cultures were adjusted with sterile saline
solution to obtain turbidity comparable to that of
McFarland no. 5 standard (1.0 x 108) CFU/mL. 90 mm
Petri dishes containing 12mL of sterilized Müeller-
Hinton agar were inoculated with the microbial
suspensions. Sterile Whatman No.1 (6 mm) disc papers
were individually placed on the surface of the seeded agar
plates and 10 µL of the oil in dimethylsulfoxide (DMSO)
was applied to the filter paper disk. The plates were
incubated at 37 oC for 24 h and the diameter of the
resulting zones of inhibition was measured. All tests were
performed in triplicates. Ciprofloxacin and nalidixic acid
were used as positive controls, while hexane and DMSO
served as negative controls.
Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) of the oil
was determined using 96-well microtitre dilution method
[34]. Bacterial cultures were incubated in Müller-Hinton
broth overnight at 37 oC and a 1:1 dilution of each culture
in fresh MHB was prepared prior to use in the micro
dilution assay. Sterile water (100 μL) was pipetted into
all wells of the microtitre plate, before transferring 100
μL of the oil in DMSO. Serial dilutions were made to
obtain concentrations ranging from 10 mg/mL to 0.078
mg/mL. One hundred μL of bacterial culture of an
approximate inoculum size of 1.0 x 108 CFU/mL was
added to all well and incubated at 37 oC for 24h. After
incubation, 40 μL of 0.2 mg/mL p-iodonitotetrazolium
257
violet (INT) solution was added to each well and
incubated at 37 oC. Plates were examined after about 30-
60 min. of incubation. Microbial growth is indicated by
the presence of a reddish colour which is produced when
INT, a dehydrogenase activity detecting reagent, is
reduced by metabolically active microorganism to the
corresponding intensely coloured formazan. MIC is
defined as the lowest concentration that produces an
almost complete inhibition of visible micro-organism
growth in liquid medium. Solvent controls (DMSO and
hexane) and the standard antibiotics ciprofloxacin and
nalidixic acid were included in the assay.
Insecticidal activity
Insects
Adult insects of mixed sex, 7-14 days old of
Acanthoscelides obtectus, Sitophilus zeamais and
Tribolium castaneum were separately reared on beans,
maize and wheat flour, respectively at 25 ± 1 oC and 65%
± 5% relative humidity (R.H.) were used for the bioassay.
Fumigant toxicity
The fumigant toxicity of V. agnus-castus essential oil
was assayed. [35]. Filter paper (Whatman No. 1, cut into
2-cm diameter pieces) was impregnated with V. agnus-
castus oil at doses calculated to give equivalent fumigant
concentrations of 5, 10, 20, 50, 100 and 250 mg/L air.
The impregnated filter paper was then attached to the
undersurface of the Petri dishes (90 mm) containing 10
adults each of A. obtectus, S. zeamais and T. castaneum
to different concentrations of the oil. Each concentration
and the control were replicated three times. Mortality was
determined after 24, 48 and 72 h from the commencement
of exposure. When no leg movement was observed,
insects were considered dead. The percentage insect
mortality was calculated using Abbott’s formula for
natural mortality in untreated controls [36].
Statistics
The mean and standard deviation of three experiments
were determined. Statistical analysis of the differences
between mean values obtained for experimental groups
were calculated as means  standard deviation (SD) of
three independent measurements using Microsoft excel
program, 2003 and Origin 6.0 for IC50. Data were
subjected to one way analysis of variance (ANOVA). P
values ≤ 0.05 were regarded as significant and P values ≤
0.01 as very significant.
III. Results and Discussion
The essential oil constituents of V. agnus-castus are
given in Table 1, where compounds are listed in order of
Canadian Journal on Computing in Mathematics, Natural Sciences, Engineering and Medicine Vol. 3 No. 7, December
2012

their elution from the DB-5 column. The oil yield was V. agnus-castus [6], [28], it appears that the oil exhibited
0.11% (w/w) based on the dry weight. Thirty-two good activity which could be attributed to the presence of
constituents were identified, accounting for 91.7% of the some major components such as α-terpineol, terpinen-4-
total oil. It is of interest to note that this oil had a ol and terpinolene along with other components in lower
relatively high concentration of monoterpenoids (40.2%), amounts such as α-pinene, β-pinene, myrcene, β-
of which the hydrocarbon fraction was relatively higher caryophyllene and α-terpinene, which were already
(25.5%) than the oxygenated fraction (11.5%), while, the known to exhibit antibacterial and bacteriostatic activities
sesquiterpenoids content was 26.8%. The sesquiterpene [33].
composition of the oil was dominated by trans-β- The insecticidal activity of V. agnus-castus essential
farnesene (7.7%), γ-cadinene (6.3%) and β-caryophyllene oil was determined using three storage-product beetles
(4.9%). α-Terpineol (11.5%), terpinen-4-ol (6.3%) and (A. obtectus, S. zeamais and T. castaneum). Table 11
terpinolene (5.6%) were the main monoterpenes. shows the results of the insecticidal activity of V. agnus-
Comparing the present data (Table 1) with those castus essential oil and the controls (permethrin and
previously reported in literature on the essential oils from allethrin) after 72 h, and was found to be directly
the leaves of V. agnus-castus, a large diversity was proportional to the different concentrations of the oil. The
observed, which suggests the existence of many varieties lethal concentration (LC50) of V. agnus-castus essential
within the V. agnus-castus species. Although, all the oils oil against A. obtectus, S. zeamais and T. castaneum were
121.67, 106.23 and 146.45) mg/L air, respectively. When
were dominated by monoterpenoids, they differ in
compared to the standards (permethrin and allethrin) with
chemical compositions [5], [6], [8], [12], [14]-[28].
(LC50 = 7.45 and 11.13) mg/L air. It is apparent that V.
Interestingly, 1,8-cineole and sabinene which were found
agnus-castus essential oil has a weak toxic activity.
to be major components in most of the previous studies, However, the findings of this study are in agreement with
were not detected in our work. In addition, the major other previous reports on insecticidal activities of some
constituent of our oil sample (α-terpineol) has not been essential oils against storage-product beetles [39], [40].
previously reported in dominant quantity among the In conclusion, the ability of the oil to exhibit high
major compounds from the leaf oils of V. agnus-castus. activity against some bacteria pathogens and the
Furthermore, 2-amino-6,7-dimethylpteridin-4-ol, γ- relatively weak toxic activity against storage-product
cadinene and other constituents in relatively smaller beetles may have contributed to its use in folk medicine.
amounts such as γ-gurjunene, 2-methyl-2-norbornene and
trans-Z-α-Bisabolene epoxide are being identified for the Table I Chemical composition of Vitex agnus-castus essential oil
first time from the leaf oils of V. agnus-castus. The Compound RIa % composition
observed compositional difference between the V. agnus- α-Pinene 4.73 2.5
castus species could be due to climactic and 2-Amino-5-methylbenzoic acid 4.83 1.9
environmental conditions, chemotypes, nutritional status
β-Pinene 5.62 0.6
of the plants, and other factors, which can influence
1-Ethyl-2-methyl-benzene 5.72 1.1
essential oil composition [37], [38]. The results obtained
in this study is in agreement with previous works from Myrcene 5.96 1.1
Nigeria [5], [6] and other reports on the chemical 1,3,5-Trimethylbenzene 6.05 1.3
composition of essential oil of V. agnus-castus from other Decane 6.11 0.8
countries [8], [12], [14]-[28]. Z-(β)-Ocimene 6.25 1.0
The antibacterial activity of V. agnus-castus essential α-Phellandrene 6.39 2.8
oil (Table II) showed that S. aureus, E. coli, B. Cereus
α-Terpinene 6.58 1.9
and B. subtilis are the most susceptible bacteria with
inhibition zones of 23.3, 21.0, 18.3 and 14.7 mm, while, Terpinolene 6.90 6.4

P. aeruginosa (9.7 mm) exhibited the smallest zone of γ-Terpinene 7.73 2.9
inhibition. In addition, the MIC values showed S. aureus Terpinen-4-ol 11.41 6.3
having the lowest MIC value (0.31 mg/mL) and the α-Terpineol 16.87 11.5
highest MIC values of 5 and 10.0 mg/mL was against B.
β-Caryophyllene 18.93 4.9
subtilis and P. aeruginosa, respectively. When compared
trans-β-Farnesene 20.18 7.7
with standard antibiotic (ciprofloxacin), the oil showed
moderate to high range of inhibition zones (9.7 ± 0.6 to γ-Muurolene 20.71 0.3
23.3 ± 1.5) mm against the standard antibiotics (12.0 ± Bicyclogermacrene 21.83 2.8
1.0 to 29.3 ± 1.5) mm. Comparing this result with the α-Bisabolene 23.35 0.7
literature data on antibacterial activity of essential oils of
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2012

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