ISSN: 1579-4377

BIOACTIVITY OF XYLOPIA AETIOPICA (DUNAL) A. RICH ESSENTIAL OIL

CONSTITUENTS ON MAIZE WEEVIL SITOPHILUS ZEAMAIS
MOTSCHULSKY (COLEOPTERA: CURCULIONIDAE)

Asawalam, E. F.1, *, Emosairue, S.O.2 and Hassanali. A3

1
Crop Protection Department,Michael Okpara University of Agriculture Umudike. P.M.B.7267 Umuahia, Abia State, Nigeria.
2
Agronomy Department, Delta state University Asaba campus Asaba, Delta State, Nigeria
3
Behavioural and chemical Ecology Department, International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi,
Kenya.

KEYWORDS
Xylopia aetiopica, essential oil, S. zeamais, maize grains, bioactivity

ABSTRACT
A laboratory study was conducted at Behavioural and Chemical Ecology department of International
Centre of Insect Physiology and Ecology Nairobi, Kenya to determine the efficacy of Xylopia aetiopica
essential oil for Sitophilus zeamais Motschulsky control. The experimental design was a completely
randomized design with four replications. The bioactivity of essential oil extracted by hydro distillation
from leaves of Xylopia aetiopica was assessed under laboratory conditions at 30 0C, 65 ±2% and 12h: 12h
light: dark regimes for their biological activity against S. zeamais (Maize weevil) in maize grains. The
essential oils at three levels of application were mixed with 250 g of disinfested maize in one litre volume
of glass jar and replicated four times. The effect of treatments on insect mortality and progeny production
was assessed. The repellent action of the essential oils was also evaluated. The individual components of
the essential oils were identified through GC, GC-MS and GC- Co injection with the authentic standards.
The identity of a total of 15 constituent compounds of the essential oils of the plants was confirmed and
their relative proportion determined. Eucalyptol (1, 8 Cineole) had the highest percentage composition in
Xylopia aetiopica essential oil (26.63%). All the treatments with the essential oils showed significant
level of toxicity to the insects. The highest dosage (750 mg corresponding to 0.3%) of the essential oils of
the plant material tested, induced the highest mortality in the S. zeamais after 7 days. Toxicity of the
essential oils was dose dependent. There was no mortality of the S. zeamais in the untreated grains.
Similarly, the maize grains treated with the essential oil extracts significantly reduced the number of
progeny produced by S. zeamais. The essential oils of the plant material tested significantly evoked a high
repellent action against S. zeamais. In view of the activity of the essential oil, it is suggested that the plant
is suitable for possible exploitation in insect pest control.

INTRODUCTION
The use of plant derived insecticides has played important role in traditonal method of storage pest
control in Africa and Asia (Hassanali et al., 1990; Niber, 1994; Bekele and Hassanali, 2001). In view of
the potential of plant products as alternative eco-friendly insect pest control agents, in Africa, there has
been a growing interest in evaluating their efficacies and in elucidating the basis of their protective action

*
Corresponding author E-mail: easawalam@icipe.org, asawaldo@yahoo.com

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(Weaver et al., 1991. Ndungu et al., 1995; Ndungu et al., 1999; Bekele and Hassanali 2001; Bekele et al
1996, 1997; Obeng-Ofori et al., 1997).

Xylopia aetiopica (Annonaceae) is a tree of about 20 m high often with short prop roots, smooth grey
back, and scented when fresh. They are found in lowland rainforest and coastal brackish swamps. They
are also found in deciduous and fringing forest of the Guinea savanna zones and often cultivated near
villages in East Africa and southern part of Nigeria. It is used as a spice and flavoring for food. In Congo,
the bark is steeped in palm wine and used in treating asthma attacks, stomach aches and rheumatism. The
root is strongly aromatic and used as mouth wash to treat tooth ache. The leaves have a pungent smell.
The fruit is the most important part of the tree. They are narrow, dark-brown and about 5 cm long with
many separate carpels borne together.

The aim of this study was to analyze the chemical composition of X. aetiopica leaves and to identify the
active chemical constituents of the essential oils (Characterization) as well as to evaluate the effect of the
essential oils of X. aetiopica leaves on mortality, reproduction (on number of F1 progeny) and repellency
of S. zeamais. It is hoped that this information will be useful in selection of plant-derived insecticides for
S. zeamais control.

MATERIALS AND METHODS

Mass rearing of Sitophilus zeamais

Mass rearing of S. zeamais was carried out following the method of Santhoy and Rejesus (1975), Jembere
et al., (1995); Bekele and Hassanali, (2001). S. zeamais was obtained from stocks maintained at the
International Centre of Insect Physiology and Ecology (ICIPE) and Kenya Agricultural Research Institute
(KARI), Laboratories. Maize seeds were obtained from Dikomba, in Kenya. Prior to the experiments, the
seeds were disinfested by keeping them in an oven at 400C for 4 h (Santhoy and Rejesus, 1975). Twenty
pairs of S. zeamais sexed following the methods of Halstead (1963) and Stenley and Wilbur (1966), were
introduced into the I-litre glass jar containing 400g disinfested maize grains. The jar was covered with
nylon mesh held in place with rubber bands and kept in a room maintained at 60± 5% relative humidity
and 26 ±10C using heaters and water troughs.

Plant materials collection and isolation of their essential oils:

Xylopia aetiopica fruits were collected from Umuahia, Nigeria. The identity of the plant materials was
confirmed at the Michael Okpara University of Agriculture, Umudike, Nigeria herbarium, before using
them for the bioassays. The plant materials were then air dried in a well ventilated area for 5 days before
extraction procedure. Voucher specimens are kept at the University of Agriculture, Umudike herbarium.

The essential oil extract was isolated from these plant materials by steam distillation using Clavenger
apparatus (Guenter, 1949). The condensing oils were collected in n-hexane solvent (Aldrich HPLC grade)
and the solution was filtered filter paper containing anhydrous sodium sulphate in a funnel to remove any
remaining traces of water. Hexane was then removed by distillation at 60 0C from 'Contes' Short Path
distillation apparatus. When condensation stopped, the oil was collected and weighed into small amber-
coloured vials

Analysis of essential oils

Characterization, identification and determination of relative amounts of the components of the essential
oils from the selected plants were done through Gas Chromatography (GC), Gas Chromatography - Mass
Spectrometry (GC-MS), and GC Co injection of the essential oils with authentic standards.

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Gas - Liquid Chromatography (GC) of essential oil:

GC analyses were performed on a capillary Gas - Chromatograph Hewlett Packard (HP) 5890 Series II
equipped with a split-less capillary injector system, 50 m x 0.20 mm (i.d) cross-linked with HP - ultra
1methylsilicone 0.33 µm (film thickness) capillary column, and Flame Ionization Detector (FID) coupled
to HP 3393A Series II integrator. The integrator was used to calculate the peak area.

The carrier gas was nitrogen at a flow rate of 0.84 ml/min. The flow rates of air and hydrogen were 400
and 30.5 ml/min, respectively. The temperature programme comprized of an initial temperature of 40 oC
(0 min) to 90 oC at 7 oC/min for 5 mins, then to 115 at 3 oC/min for 5 mins and finally to 280 oC at 4
o
C/min where it was maintained for 20 mins.

Gas Chromatography - Mass Spectrometry (GC-MS), of essential oil:

GC - MS analysis were carried out on a HP 8060 Series II Gas Chromatograph coupled to VG Platform II
Mass Spectrometer in order to identify the essential oil constituents. The MS was operated in the Electron
impact mode (EI) mode at 70 eV and an emission current of 200μA. The temperature of source was held
at 180 oC and the multiplier voltage at 300V.

The pressure of the ion source and MS detector were held at 9.4x 10 -6 and 9.4x 10 -6 mbar respectively.
The MS had a scan cycle of 1.5 sec (scan duration of 1s and inter-scan delay, 0.5 sec). The mass and scan
range was set at m/z 1-1400 and 38-650, respectively. The instrument was calibrated using
heptacosafluorotributyl amine, [CF3(CF2)3]3N, (Apollo Scientific Ltd., UK). The column used for GC -
MS temperature programmed as in the case of GC analysis but the film thickness of GC - MS was 0.5
μm. All GC - MS analysis were made in the splitless mode with helium as the carrier gas.

The preliminary identification of the constituents was based on the computer matching of mass spectral
data of the components against the standard Wiley and NIST library spectra constituted from spectra of
pure substances and components of the known essential oils, and literature MS data. They were confirmed
by their GC retention time comparison with those of reference compounds, peak enhancement as well as
Co- injection /Co-elution with authentic standards. The standards used were obtained from Aldrich
Chemicals UK. The relative proportion of the essential oil was computed in each case from GC- MS peak
areas.

Mortality, Progeny development and damage assessment assays:

The oils was applied to the grains at the rate of 0.012, 0.06 and 0.3% (30, 150 and 750 mg/250 g of grain
dissolved in 10ml of 95% n-hexane and shaken thoroughly to ensure uniform distribution over grain
surface. The treated grains were kept for 24h to allow the hexane to evaporate completely before
bioassays were conducted.

The essential oil extracts of the plant materials were mixed separately with 250g of maize grains in glass
jars at the three different dosages indicated above. Two blank controls were run periodically consisting of
hexane treated grain and the untreated grain. Ten pairs of 5-7 day old S. zeamais adult were introduced
into the jars containing the different treated and untreated maize grains. The jars were covered with nylon
mesh held with rubber bands. The contents in the jars were then shaken gently for proper admixture. Each
treatment was replicated four times. The experiment was arranged in completely randomized design in the
laboratory.

The number of dead insects in each vial was counted after 1, 2, 3, 4, 5, 6, and 7 days to estimate maize
weevil mortality. Maize weevil mortality was assessed as:

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⎛ Number of dead in sec ts ⎞

⎜⎜ ⎟⎟ × 100
⎝ Total numbers in sec ts ⎠

Data on percentage adult weevil mortality were corrected using Abbott’s (1925) formula thus:

PT =
(P0 − Pc )
(100 − Pc )

where

• PT = Corrected mortality (%)

• PO = Observed mortality (%)

• PC = Control mortality (%)

In a similar experiment, 10 pairs of S. zeamais were introduced into treated and untreated grains and after
30 days oviposition period, the parent adults were removed. Insects subsequently emerging were counted
to estimate FI progeny production. Counting was stopped after 63 days to avoid overlapping of
generation.

Repellency

The repellent action of the essential oil extracts of the plant materials against S. zeamais was assessed in a
choice bioassay system consisting of two 1-l glass jars connected together at their rims by means of a
30x10 cm nylon mesh tube. A 5.0 cm diameter circular hole was cut at the middle of the mesh for the
introduction of the test insects. In this devise, it was possible to test large quantities of materials. 250 g of
maize was put into the glass jars. The grains in one of the jars were treated with essential oil extracts
while the other jar was untreated and acted as control. Twenty-five adults of S. zeamais were introduced
into the nylon mesh tube through the circular hole by means of a 5cm-diametre funnel. The number of
insects present on control (NC) and treated (NT) jars were recorded after 1 hour exposure. There were four
replications in the bioassays. All repellency assays were carried out in the laboratory at 27 ±20C and 60-
65% r.h. Percent repellency (PR) values were computed as

PR =
(N c − NT ) × 100
(N c + NT )

PR data were analysed using ANOVA after arcsine transforming them.

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Statistical Analysis: Data obtained were subjected to analysis of variance (ANOVA) using a general
linear model procedure (SAS, 2000) and significant difference (P>0.05), means were separated by using
Student Newman- Keuls (SNK) test.

RESULTS AND DISCUSSION

Essential oil composition

The GC profile of the oil sample of X. aetiopica is shown in Figure 1. The analysis of the oil revealed
complex mixture of constituents. A total of 15 compounds were identified in the essentials of X. aetiopica
by GC - MS and GC Co - injection with authentic standards (Table.1) Constituents present in >1% were
the ones identified. The oils represent mainly a mixture of monoterpenes and sesquiterpenes. Their
chemical structures are shown in Figure 2. ie 1- 1, 8 Cineole, 2- Beta pinene 3- Myrtenal.

INS: VG 12-250 UPGRADE Date: 28-Jan-2005 Time: 11:45:34

Sample XT. OIL Inj. 4µl (5µl:1ml DCM) Column: HP ULTRA 1(MeSil.) 50mX0.2mmX0.33µm Prog: 40(0)@7-90(5)@3-115(5)@4-280(20
EF28105A Sb (60,0.10 ) Scan EI+
TIC
100 4 1.08e7

7
11 12
8 10
2 9 13
6

5
14 15

0 Time(Min)
10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

Fig. 1. GC-MS Chromatogram for Xylopia aetiopica essential oil

1, 8 Cineole occurred in the largest quantity (26.63%). This is followed by Beta pinene (15.34%) and
Myrtenal (6.64%).

1, 8 Cineole Beta pinene Myrtenal

Figure 2. Chemical structures of major identified constituents of Xylopia aetiopica essential oil

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Adult mortality in grains

Figure 3 shows the cumulative mean percentage mortality of S. zeamais in maize grains treated with
different concentrations of X. aetiopica essential oil.

Table.1. Major identified constituents of X. aetiopica essential oil and their relative proportion in the oils
Peak area (%
GC peak number Component Retention time
composition)
1 Alpha pinene 5.14 16.55
2 Beta phellandrene 2.66 18.35
3 Beta pinene 15.34 18.73
4 1,8 Cineole 26.63 21.45
5 Gamma terpinene 1.53 23.30
6 Linalyl acetate 3.88 25.33
7 Pinanol 6.02 28.38
8 Verbenene 2.06 28.60
9 Pinocarvone 3.19 29.33
10 L- carveol 4.44 30.53
11 Terpinene-4-ol 5.24 30.85
12 Myrtenal 6.64 31.35
13 Myrtenol 2.81 31.88
14 Cuminal 1.79 33.78
15 Phellandral 1.13 35.83

30mg
100 150mg
90 750mg
Cum ulativ e % m ortality

80 R2 = 0.9396
70
60 R2 = 0.9542
50
40
30 R2 = 0.8599
20
10
0
0 2 4 6 8
Days after treatment

Fig. 3. Cumulative mean % mortality of S. zeamais in maize grains treated with different concentrations of X.
aetiopica essential oil

All the treatments with the essential oils showed significant level of toxicity to the insects. The highest
dosage (750 mg corresponding to 0.3%) of the essential oils of the plant material tested induced the
highest mortality in the S. zeamais at 7 days after treatment. Toxicity of the essential oils was dose

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dependent. There was no mortality of the S. zeamais in the untreated grains. The essential oil of X.
aetiopica evoked high mortality in the weevil (82%).

Toxicity is one of the various effects of plant terpenoids to insects (Metcalf and Metcalf, 1992). Thus, the
toxic effects of plant materials can be attributed to their essential oil composition. The toxic effect of
plant essential oils has been reported by various authors (Hassanali et al., 1990; Shaaya et al., 1991;
Weaver et al., 1991; Jembere et al., 1995; Bekele et al., 1996; Bekele et al., 1997; Bekele and Hassanali
2001; Renault - Roger et al., 1993; Bouda et al., 2001), who attributed their effect to different terpene
constituents of the essential oils.

Similarly, the toxicity of the essential oils of X. aetiopica against S. zeamais in the present study can be
attributed to their essential oil constituents. The essential oils contained a lot of terpenoids and induced
82% mortality with X. aetiopica essential oils at 750 mg/250g application rate after 7 days of treatment.

Effect of the essential oils on Progeny development

The number of progeny produced by S. zeamais in untreated grains and grains treated with different
concentrations of X. aetiopica essential oil is shown in Figure 4. Significantly higher number of F1
progenies was produced by S. zeamais in the untreated grains compared with the grains treated with the
essential oil extracts. All the levels of the essential oil extract significantly reduced the number of F1
progeny produced by the weevil. No progeny was produced in grains treated with the highest dose of 750
mg (0.3%), of the essential oil extracts tested.

180
160
M e a n n o o f FI p ro g e n y

140
120
100
80
60
40
20
0
30mg 150mg Hexane Untreated
Treatments

Fig. 4. Effect of X. aetiopica essential oil on the number of F1 progeny produced by S. zeamais in stored maize
grains

This study has shown that the essential oils gave good protection to the stored maize grains by
suppressing reproduction (F1 progeny emergence). Bekele et al., (1997) observed similar result, where
grains treated with essential oil extract of Ocimum kenyense significantly reduced the number of progeny
produced by S. zeamais.

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Repellency effect of the essential oils on S. zeamais

Figure 5 represents the mean repellency values of the essential oil extracts at different dose levels against
S. zeamais in stored maize grains. All the dosages were repellent to S. zeamais, with the 750 mg evoking
the highest repellent action. Analysis of variance indicated significant differences (P < 0.05) between the
responses of the three dosages tested. The repellent action of the essential oil extract of X. aetiopica was
dosage dependent. The untreated grains induced no repellency against S. zeamais.

80

70

60
Mean %repellency

50

40

30

20

10

0
30mg 150mg 750mg
Dosage /250g grains

Fig. 5. Mean percentage repellency for different dose levels of X. aetiopica essential oil against S. zeamais in stored
maize grains

The result obtained has shown that the essential oils were significantly (P < 0.05) repellent to the weevil
relative to the controls. This is similar to the findings of Hassanali et al., (1990) who demonstrated the
repellent effect of essential oils of Ocimum suave leaves and the dried unopened flower buds of Eugenia
caryophyllata cloves against S. zeamais in olfactometric assays. Eugenol was found to be highly repellent
to the four beetle species tested, with overall repellency in the range of 80 - 100% (Obeng - Ofori and
Reichmuth, 1997).

All the aliphatic ketones and aldehydes of Commiphora rostrata constituent have been demonstrated to
possess greater repellency against S. zeamais than the synthetic commercial insect repellent N, N - diethyl
toluamide (DEET) (Lwande et al., 1992).

Ndungu et al (1995) reported that 1 - alpha terpenol and 2 - dodecanone were the most repellent
components of Cleome monophylla essential oil against Rhipicephalus appendiculatus and S. zeamais.

Thus, the repellent effect of the essential oils in this study could be attributed to their major components.
This study demonstrates the potential of plant volatile essential oil for use against S. zeamais

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ACKNOWLEDGMENTS
The first author is grateful to the Third World Organization for Women in Science (TWOWS) Trieste,
Italy for the fellowship they provided her with to undertake this work.

We thank the International Centre for Insect Physiology and Ecology, Nairobi, Kenya for laboratory
space.

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