Chemical composition and insecticidal properties of the ...

African Journal of Biotechnology Vol. 5 (10), pp. 901-906, 16 May 2006
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2006 Academic Journals










Ful Length Research Paper

Chemical composition and insecticidal properties of the
underutilized Jatropha curcas seed oil

Adebowale, K.O.1* and Adedire, C.O.

1Department of Chemistry, University of Ibadan, Ibadan, Nigeria.
2Department of Storage Technology, Federal University of Technology, Akure, Nigeria.

Accepted 11 January, 2006

The chemical composition and insecticidal activity of Jatropha curcas L. seed were evaluated using
standard techniques. The oil content of the seed is high (66.4%). Triacylglycenol was the dominant lipid
species, while the major triacyglycerol was 1,2-Dioleoyl-3-linoleoyl-rac-glycerol. Linolenic acid was the
dominant fatty acid in the oil. Physico-chemical properties of the oil indicated that the acid value, fee
fatty acids, peroxide value and iodine value were high. Ten seterols and thirteen tritepene alcohol was
identified in the unsaponifiable fraction of the oil. Jatropha seed oil at various serial dilution ranging
from 0% to 2% (v/w) at 0.5% intervals were evaluated for anti-ovipositional activity and long-term
protective ability of treated cowpeas against the seed beetle Callosobruchus maculatus. The oil
significantly (P< 0.05) reduced oviposition by C. maculatus in no-choice test in all the concentrations
tested. The number of eggs laid by the seed beetle reduced from an average of 54. 33 ± 3.53 in the
control to only 4.00 ± 1.53 in 2% oil-treated seeds. There was no adult emergency in all the oil. However
in choice-tested seed, 6.67 ± 1.33 eggs were laid in cultures treated with 2% oil while 21. 67 ± 1.45 were
laid in control cultures. In dual-choice tests, oviposition was significantly reduced at all the oil
concentration evaluated. J. Curcas oil also offers a 12-week protection for treated seeds since there
were neither seed damage nor adult emergency in treated cowpea seeds. The results of this study
suggest J. curcas has antioviposition and ovicidal effects on C. maculatus therefore making it a vaiable
candidate for incorporation into pest control program of gain legumes.

Key words: Natural insecticides, Jatropha curcas seed oil, unsaponifiable matter, Cal osobruchus maculates.


INTRODUCTION

Cowpea, Vigna unguiculata L. (Walp) (family fabaceae) is
particularly in Nigeria, is currently being hampered by its
a widely grown grain legume in the tropics and sub-
multitude of insect pests both on the field and at storage
tropics purposely for its edible seeds which are rich in
(Jackai and Adal a, 1997). In Nigeria, fresh cowpeas of
protein (Jackai and Daoust, 1986). It is a major source of
high quality and relatively un-infested are often available
dietary plant protein for both humans and livestock
around November-December while it becomes scarce
(Okigbo, 1978; Adedire and Lajide, 1999). Profitable and
and heavily infested by bruchids notably Cal osobruchus
economic production of cowpeas in developing countries,
maculatus during its off-season or barely 2-3 months

after harvest (Caswel , 1976; Sowunmi, 1982). Bruchid

infestation usual y affects seed quality, market value and

can reduce cowpea seed viability to 2% after 3 months of

storage (Caswel , 1980, 1981; Ofuya and Credland,
*Corresponding authors E-mail: adebowale2003@yahoo.com.
1991).

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902 Afr. J. Biotechnol.







Over the years, several methods of bruchid control
Analytical methods
have been employed by farmers and researchers have

identified some efficient control of the pests. Some of
The oil extracted with petroleum ether (40 – 60°C) using the soxhlet
these methods range from store hygiene, physical and
procedure for 6 h. Iodine value, saponification value, peroxide
value, free fatty acid and refractive index were determined using the
cultural control methods and use of invert materials.
method already described in the AOAC (1980).
Chemical control appeared to be the most effective and
The fatty acids in the oil were determined by gas
efficient control method (Jackai and Daoust, 1986) but it
chromatography. To 0.1 g of the oil, 5 ml methanol and 1 ml
has adverse effect on both man and environment
CH2C12 were added. The mixture was cooled in ice and CH3COCl
(Makanjuola, 1989; Adedire and Lajide, 1999, 2000).
(0.6 ml) was added. 1 ml of the solution was withdrawn into the
hydrolysis tube and heated for 1 h at 110°C. The mixture was
Against this background, a search for natural-product
cooled and discharged into a 10 ml NaCl solution (1%) in a
based agrochemicals which are biodegradable, eco-
separating funnel. The organics were extracted with 3 x 4 ml
friendly and safe to the environment has intensified
hexane and 4 ml CH2Cl2. The CH2Cl2 phase was separated on a
(Jadhau and Jadhau, 1984).
DB5 30 m x 0.25 mm, capil ary column (J and W Scientific, Koln,
Jatropha curcas, a potential anti-feedant candidate,
Germany) instal ed in a GC Chrompack CP 9001 equipped with a
belongs to the family, Euphorbiaccae. The seed which is
flame ionization detector and Mosaic integrator. The temperature
was programmed as fol ows: 35°C for 3 min; temperature increased
black and oval in shape is rich in fixed oil (Shukla et al,
at 20°C per min up to 120°C; 5°C per min up to 230°C and retained
1996). The plant is a native of North America but now
at 230°C for 5 min. Heptadecanoic acid was used as the internal
thrives wel in Africa and Asia. It is easy to establish as it
standard.
grows relatively quickly with high yields (Wal s, 1967).
Lipids were separated on 0.75 mm plates (20 X 20 cm) coated
Recently there was renewed interest on the utilisation of
silica gel (MERCK). Plates were developed vertical y in a 80/20/1
the seed oils in view of the relatively high oil content
volume mixture of petroleum ether: diethylether: acetic acid. Details
of the procedure have been described earlier (Esuoso et al., 1998).
(Sujatha and Mukia, 1996).
The triacylglcerol fraction was identified, scrapped off and eluted
In our earlier studies, we considered the composition
with CH2Cl2. The samples and standards was injected into Gas
and insecticidal properties of Monodora tenuifola seed oil
Chromatography (CHROMPACK CP 9000) using Chrompack TAP
(Adedire et al., 2003). Therefore in continuation of our
capil ary column 25m x 0.25mm (J and W Scientific, Köln,
studies on the sourcing of natural product agrochemicals,
Germany). The carrier gas was hydrogen maintained between 95-
96 kpa. The temperature was programmed as fol ows: 80°C for 2
this article considers the physicochemical properties, lipid
min; temperature increased to 280°C at 30°C per min; temperature
classes, fatty acids and triacylglycerols of the Jatropha
increased to 355°C at 3°C per min.
seed oil. Unsaponifiable content of the oil wil also be

analysed for the triterpene alcohols and sterols. The work

Isolation of unsaponifiables
also involved studies on the insecticidal activity of the oil

on the cowpea storage bruchid, C. maculatus.
Oil (10 g) dissolved in 200 ml of ethanol potassium hydroxide (2 M)

was refluxed for 1 h. The reaction mixture was diluted to 400 ml

with distil ed water and transferred to a separating funnel. The
MATERIALS AND METHODS
unsaponifiables were extracted three times with 100 ml of

dietheylether. The ether extracts was first washed with 100 ml of
Collection and preparation of samples
aqueous solution of potassium hydroxide (0.5 M) to remove any

residual free fatty acids. Further washing and cleaning was carried
out five times with 100 ml distil ed water, and the ether layer
The samples were obtained from markets in Ibadan, Akure, Ilorin,
removed in a rotary evaporator. The value was expressed in weight
Warri and Kaduna. They were identified at the Genetic Resources
percent.
unit of the International Institute of Tropical Agriculture, IITA,

Ibadan, Nigeria. They were subsequently ground in a Christy

laboratory mil and stored in a cel ophane bag at 4°C prior analysis.
Separation of the unsaponifiables



A chloroform solution (50%) of unsaponifiable material (30
Triacylglycerol standards
cm/plate) was when applied uniformly along a line from the edge of

a 20 x 20 cm plate coated with 0.55 mm layer of silica gel and
Al the standards used for the studies were purchased from SIGMA
developed three times with hexane/ethyl acetate (6:1, v/v) as
Chemical Company. They include: Tripalmitin PPP; 1, 2-
mobile phase. After development, the plate were sprayed with a
Dipalmitoyl – 3-oleoyl- rac- glycerol PPO; Dipalmitoyl – 2-oleoyl –
solution of Rhodamine-6G in ethanol (0.5%) and observed under
rac- glycerol POP; 1, 2- Dipalmitoyl – 3-oleoyl –rac-glycerol PPO; 1,
UV light. Three different zones were marked: RF 0.9-0, hydrocarbon
2- Distearoyl – 3 – palmitoyl – rac – glycerol SSP; 1- Stearoyl – 2-
(n-alkane); RF 0.02-0.04, sterols. Each zone were careful y scraped
Oleoyl –3-palmitoyl – rac-glycerol SOP; 1,3- Dioleyl – 3- palmitoyl –
from the plates and extracted thoroughly with diethylether. Sterols
rac- glycerol OPO; 1- Palmitoy – 2- 3 dioleoyl – rac- glycerol POO;
and triterpene alcohols were silanated with 10 µl of bis
1, 3 – Dioleoyl –1,3 stearoyl – rac– glycerol OSO; 1, 3 – Distearoyl
(trimethylsilane) – trifluoacetamide (BSTFA) at 60°C for 1 h. The
– 2- oleoyl – rac –glycerol SOS; 1,2- Distearoyl – oleoyl – rac –
residue obtained was with diethylether and the solvent was
glycerol SSO; Triolein OOO; 1,3 Dipalmitoyl – 2 – linoleoyl – rac-
removed at reduced pressure in a rotary film evaporator.

glycerol PLP; Trilinolein LLL; 1,2 - Dilinoleoyl – 3-oleoyl – rac-

glycerol LLO; 1- Palmitoyl – 2– oleoyl-3 linoleoyl-rac-glycerol POL;
Analysis of unsaponifiables
1,2 Dioleoyl- 3- linoleoyl – rac-glycerol OOL; 1- Behenoyl-2,2-

dioleoyl-rac-glycerol BOO.
For the determination of hydrocarbons, the fraction was injected

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Adebowale and Adediran 903






Table 1. Percentage oil composition and lipid classes of
procedure involves 5 treated cowpea seeds of the same
Jatropha curcas.
concentration and 5 solvent treated cowpeas arranged alternatively

in ring-like manner in a Petri-dish coated with paraffin wax (Adedire
Composition
Percentage
and Lajide, 1999). The multi-choice system is similar except that al
the treated cowpea seeds of al the concentrations and control were
Oil
66.4
arranged in the same manner in Petri-dish. Then two copulating
Unsaponifiable
3.8
pairs of C. maculatus (0-24 h old) were introduced into each Petri-
dish and covered. Three replication of each experiment was
Hydrocarbons/sytereo esters
4.8
prepared. The number of eggs laid on each heated and untreated
Triacycerols
88.2
(solvent control) cowpea seed was enumerated after the demise of
Free fatty acid
3.4
the female bruchids.

Diacylglycerols
2.5

Sterols
2.2
Growth performance at 12 weeks post-treatment

Monoacyglycerols
1.7
Serial dilution (0, 0.5, 1 1.5 and 2%, v/w) of Jatropha oil were used
Polar lipids
2.0
to surface-treat 50 g grams of susceptible IFE brown variety of

cowpea in a 100 ml volumetric flask. The uniform mixing of the oil
and the seeds was accomplished by manual agitation with the aid

of a glass rod. The seeds were then air-dired for 1 h before
into the Gas Chromatography without derivation using a capil ary
introducing five couples of teneral adult C. maculatus into each
column (SE-54, 20 m x 0.27 mm, J and W. Scientific, Köln,
conical flask. Each treatment was carried out in triplicates while the
Germany). The programming was a fol ows: 35°C for 3 min,
oil was replaced with petroleum ether (the control flask). Each flask
temperature increased at 5°C/min to 280°C for 5 min. Further
was covered with muslin cloth held firmly in place with a rubber
determination was carried out on a GC – MS. Varian MAT 112S
band to prevent escape of the bruchids or entry of some other
using an ionization voltage of 60 eV. For sterols and tritepene
insects and at the same time ensure adequate aeration. The flasks
alcohols, the determination was carried out on GC with an OV-17
were kept in a completely randomized manner and left for 12 weeks
glass capil ary column (30 m x 0.3 mm i.d). Relative retention times
in an open laboratory at ambient temperature (30±2ºC) and relative
(RRT) were expressed on silanated cholesterol ester (1.00). The
humidity. After 12 weeks, the number of damaged and unaffected
RRT values of authentic samples of sterols and triterpene alcohols
seeds were counted and recorded. Also the numbers of dead and
given in Table 5.
live bruchids were enumerated.




Insect culture and maintenance
Statistical analysis


Cal osobruchus maculatus used in this study were derived from
Al the data obtained were subjected to analysis of variance
field infested cowpeas bought from Oba Market in Akure, Nigeria
(ANOVA) and where there are significant differences, means were
and reared in the laboratory on clean uninfected kilner jars while
separated the range test (DMRT). Data on oviposition- deterrent
cultivars of cowpeas was carried out in kilner jars at fluctuating
activity of the oil in dual-choice chamber was analysed by students
ambient temperature and relative humidity. The kilner jars were
t- test.
covered with muslin cloth held tightly in place for adequate aeration

of the culture and precluded entry or exit of insects. New
generations of bruchids were derived from this stock culture by

infesting clean un-infested beans with 10 pairs of teneral adult
RESULTS AND DISCUSSIONS
bruchids.


Percentage oil composition and lipid classes are

presented in Table 1. The oil content is high (66.4%).
Oviposition and adult emergency
This percentage is much higher than those recorded for

Disinfested Ife brown variety cowpea seeds weighing 20 g were
most oil-rich seeds (Esuoso and Bayer, 1998; Esuoso et
serial y treated with Jatropha oil corresponding to 0, 0.5, 1.0, 1.5
al., 1998). This would therefore be an advantage in terms
and 2% (v/w). The cowpeas were thoroughly coated with the oil
of the exploitation of the oil. Triacylglycerol was the
using a glass rod and then al owed to air-dry for about 1 h prior to
dominant lipid specie (88.2%). The high unsaponifiable
the extraction solvent. Each experimental plate was infested with a
matter (3.8%) is an advantage for use as natural
copulating pairs of male and female C. maculatus of about 0-24 h
insecticide. This is because unsaponifiable matter
old.
Each treatment was prepared in triplicates. After 14 days when the
contains sterols and triterpene alcohols which is
insects have died, the number of eggs laid on the seeds were
responsible for the insecticidal properties of fixed oils
counted and recoded while the number of F1 progeny was
(Jalad et al., 1977; Haftmann, 1970). The composition of
determined at 42 days post commencement of the experiment.
the unsaponifiable matter is presented in Tables 2 and 3

Ten sterols and thirteen terpene alcohol were identified in

the oil. The dominant sterols were 24-ethylcholesterol
Oviposition deterrent activity
and β-sitosterol, while 24-methylene-24-dihydroparkeol

and taraxasterol were the major triterpene alcohol in the
Two approaches were used to accomplish the oviposition-deterrent
effect of Jatropha oil on female bruchids. The first procedure
oil. Shankaranarayana et al. (1980) have shown the
involved in a dual-choice system while the second approach was
insect growth-inhibiting properties and chemosterilant
accomplished in a multi-choice chamber. The dual-choice bioassay
activity of a pentacyclic triterpenoid(urs-12-ene-3β-

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904 Afr. J. Biotechnol.







Table 2. Fatty acid composition of Jatropha curcas.
Table 5. Retention times of Sterols and Triterpene Alochols

used as References in Gas Chromatography
Composition
Percentage

Palmitic acid (C16:0)
11.3
Code RRT Compound
Sterol
Stearic acid (C18:0)
17.0
1.
1.00 Cholesterol (Cholest-5-enol)
Oleic acid (C18:1)
12.8
2.
1.17 27. methylcholesta- 5, 22E – dienol
Linoleic cid (C18:2)
47.3
3.
1.34 Campesterol (24-methylcholesterol)
Arachidic acid (C20:0)
4.7
4.
1.46 Stigmasterol (24-ethylcholesta – 5, 22E-
dienol)
Arachidoleic acid (C20:1)
1.8
5.
1.47 24 –ethylcholesterol (24-cholesterol)
Behenic acid ( C22:0)
0.6
6
1.66 β- sitosterol (24- cholesterol)
C24:0
44
7
1.68 24- ethylcholesta- 7, 22E 25- trienol

8
1.84 Isofucosterol (24Z-ethylidne cholesterol)

9
1.95 24 –ethylcholesta – 7, 25-dienol

10. 2.17 Avenasterol (24Z-ethylcholesta-7,24-d, enol)
Table 3. Trialclglycerols of Jatropha curcas
11. 2.19 Peposterol (24-ethlcholesta – 7, 24-d, enol)

Terpene Alcohols
Composition
Percentage
12
1.30 Euphol (eupha – 8, 28-denol)
PPO/POP
2.7
13
1.33 24- dihydrolanosterol (24 – lanost- 8-enol
PLP
1.5
14
1.50 Tirucal ol (tirucal a – 8, dienol)
SOP
2.0
15
1.58 Teraxerol (D- firedoolean – 14 –enol)
OPO/POO
12.5
16
1.66 (3-amyrin (olean – 12- enol)
POL
10.0
17
1.72 Butrospermol (eupha – 7, 24 – dienol)
SOO/OSO
3.3
18
1.77 Isomultiflorenol (D:c – fiedoolean – 8-enol)
OOO
0.4
19
1.79 24 – methleneanost – 8- enol
OOL
31.2
20
1.87 ∝ -amyrin (urs – 12 –enol
LLO
19.8
LLL
10.7
21
1.89 24 – methylene – 24- dihydro parkerol
BOO
3.5
22
1.90 Cycloartenol (9β-; 19 – cycloartenol)
Unidentified
2.4
23
1.96 Lupeol

24
2. 10 24 – methylenecycloartenol

25
2.40 Taraxasterol
26
2.52 γ taraxasterol
Table 4. Physicochemical parameter Jatropha curcas oil.


Composition
Percentage

oil. In their studies Khan et al. (1983) reported that the
Colour
Golden Yel ow
insect repel ant activity of fixed oil of Annona Squamosa
Specific gravity
0.8601
and Polyalthes longifolia were due mainly to the presence
Refractive index
1.4735
of high molecular weight fatty acids among other active
Free fatty acids (%)
4.54
agents.
Acid value (mg. KOH. g-1)
4.24
The composition of triacylglcerols is presented in Table
Saponification value (mg.KOH.g-1) 169.9
5 OOL (1,2,-dioleoyl-3- linleoyl-rac - glycerol) and LLO
Iodine value (mg. I2. g-1)
111.6
(1,2-dilinoleoyl-3-oleoyl rac-glycerol) were the dominant
Peroxide value (mg reac.O2 g-1)
3.5
triacylglcerol species.

The physico-chemical parameters of J. curcas oil are

presented in Table 6. The high iodine value (116 mg.l2.g-
palmitate) extracted from the bark of Santalum album. In
1) indicates a preponderance of unsaturated fatty acid.
addition the anti-feedant activities of pentacyclic
The fatty acids, acid value and peroxide values
triterpene acids have been demonstrated (Shukla et al.,
arerelatively high compared with most seed oils.
1996; Jagdeesh at al., 1998; Uppuluri et al., 2003;
The insecticidal properties of the J. curcas oil are
Makonjuola, 1989). Some oils might be responsible for
presented in Tables 7-10. J. curcas has long being
their anti-oviposition activity.
implicated in traditional medicine and also used as an
Fatty acid composition is presented in Table 4 Linoleic
insect repel ent, a mol uscide and a rodenticide (Duke,
acid constitutes the dominant fatty acid (47.3%). High
1985). The oil significantly reduced the number of eggs
molecules weight fatty acid (C24:0) was identified on the
laid by C. maculatus in no-choice experiments. In choice-

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Adebowale and Adediran 905






Table 6. Composition Sterols and Trieterpene Alcohols in Jatropha
Table 9. Ovispostion–deterent effect of graded concentration
Seed Oil.
of Jatropha curcas oil on adult C. maculatus in a multi –

choice chamber.
Sterols
Terpene alcohols

1.17(2)a
4.5b
1.30(12)a
3.5b
Oil concentration (%)
Mean number of eggs laid
1.34(3)
3.7
1.33(13)
6.1
0.00
21. 67 ± 1.45a
1.46(4)
4.8
1.50(14)
5.2
0.50
16.67 ± 67b
1.47(5)
41.5
1.58(15)
4.3
1.00
14. 33 ± 0.33b
1.66(6)
18.5
1.66(16)
-
1.50
10.67 ± 0.67c
1.84(8)
5.9
1.72(17)
3.1
1.51
6.67 ± 1.33c
1.95(9)
2.2
1.79(18)
1.5

2.17(10)
3.8
1.78(20)
2.0


2.19(11)
3.5
1.89(21)
37.8
Table 10. Effect of Jatropha curcas oil on growth performance
Unidentified peaks
5.7
1.90(22)
-
of C. maculatus at 12 weeks post-treatment.


1.96(23)
8.8



2.10(24)
7.9
Oil concentration
Oil seed damages Mean number of


2.40(25)
10.8
(%)
F3 Progeny


2.52(26)
5.6
0.00
1.00
1177z


Unidentified peaks
3.4
0.50
0
0

1.00
0
0
arelative retention times ; bComponents add to 100% in
each, - Not detectable (below) detection limit.
1.50
0
0

2.00
0
0



Table 7. Effect of Jatropha curcas oil ovipoosition and adult

emergence of Cal osobruchus maculates.


probably due to suffocation and or lethal chemical
Oil conc. (%)
Mean number
Adult emergency (%)
poisoning of the immature forms by the oil. This
of eggs laid
observation is in agreement with that of Jadher and
0.00
54. 33a ± 33.53
100
Jadher (1984) who reported that 0.2% (v/w) of J. curcas
0.50
30.67b ± 1.86
0.00
oil reduced oviposition by C. maculatus and total y
1.00
21.33b ± 1.76
0.00
prevented egg hatch even after 33 days of treatment.
1.50
10.00c ± 1.53
0.00
Egg stage is one of the stages that is usual y very
2.00
4.00c ± 1.53
0.00
tolerant to chemical treatment (Giga and Smith, 1987).

Thus this present study shows that Jatropha oil was able
Mean fol owed by the same letters are not significantly different
to inhibit oviposition and egg development even at a
(P>0.05) by Duncan’s multiple Range test.
lower concentration (0.5% v/w) since no adult bruchid


emerged. This oil was also effective as a grain protectant

against bruchid attack during short-term storage since
Table 8. Oviposition – deterent effect of Jatropha curcas oil on
there was no bruchid development in oil treated grain
female Cal osobrucus maculatus in a dual choice chamber.
legumes stored for 12 weeks. The insecticidal activity of

this seed oil could be due to the presence of several
Oil conc.
Mean number of
Adult emergency
sterols and terpene alchohols which have been known to
(%)
eggs laid
(%)
exhibit insecticidal properties (Heftmann, 1970; Adolf et
0.00
54. 33a ± 33.53
100
al., 1985; Duke, 1985).
0.50
30.67b ± 1.86
0.00

1.00
21.33b ± 1.76
0.00

1.50
10.00c ± 1.53
0.00
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2.00
4.00c ± 1.53
0.00


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