Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2009 Academic Journals
Full Length Research Paper
Inhibition of glutathione S-transferases (GSTs) activity
from cowpea storage bruchid,
maculatus Frabiricius by some plant extracts
Ayodele O. Kolawole
Accepted 13 March, 2008
Glutathione S-transferases, cowpea storage bruchids, plant extracts, inhibition.
L. (Walp) is an important
crop in tropical countries especially in West Africa where
it is a cheap source of dietary protein (Labeyire et al
1981). Cowpea storage bruchid (
) depredates stored cowpea (Jackai and Adalla,
1986). The huge post harvest losses and quality
detoriation caused by this insect pest are major problems
of assuring food security in developing countries like
Nigeria (Lale, 1992). Effective and efficient control of
storage insect pests are centred mainly on synthetic
insecticides. The use of these synthetic chemical is
hampered by many attendant problems: development of
resistant insect strains, toxic residues in foods and
humans; workers’ safety and high cost of procurements
(Adedire, 2003). These have necessisated research on
the use of alternative eco-friendly insect pest control
methods amongst which are the use of plant product.
Lale (1992) reported that plant materials and local
traditonal methods are much safer than insecticides and
suggested that their use needed exploitation.The effects
of some plants extracts on the biological parameters of
the herbivourous insects (e.g. oviposition, mortality rate,
adult emergence, developmental rate,mortality, fecudity
and egg viablity) has been reported. Boeke et al. (2001)
has undertaken a comprehensive review on the subject.
Studies by Adedire and Lajde (1999) and Adedire and
Akineye (2004) showed that powder and ethanolic extract
have insecticidal activity
against cowpea storage bruchid,
involved in the adaptation of some insects to their
allelopathic host plants: Myrosinase ( -thioglucosidase,
EC 18.104.22.168) and glutathione S-transferases. However
glutathione S-transferases play more important role in
detoxification (Francis et al, 2005). Glutathione S-
transferases (GSTs; EC 22.214.171.124) have attracted
attention in insects because of their involvement in the
defense towards insecticides mainly organophosphates,
organochlorines and cyclodienes (Clark et al., 1986;
Grant and Matsumura, 1989; Reidy et al., 1990; Fournier
1992). Fakae et al. (2000) has tested
against gastrointestinal helminthes of
animals and man. The nematodes glutathione S-
transferases are potential drug target and inhibitory
properties of these plant extracts against the GST may
contributed to the pharmacological basis of their efficacy.
Currently, comprehensive studies on glutathione S-
transferases from cowpea storage bruchid (
are lacking. This work is aimed at investigating the
against Cowpea storage bruchid (
Glutathione and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were from
purchased from Aldrich Chemicals, USA. Glutathione-Sepharose
was from Armersham Pharmacia, Upsalla, Sweden. Other reagents
used were of analytical grade and water used was Milli Q.
Fresh leaves of
Akure, Nigeria. The leaves were later taken to the Crop Production
Department of the Federal University of Technology for botanical
identification. The plant extracts were prepared as described by
Adedire and Akinneye (2003) with slight modification. Two hundred
gram (200 g) of each sample was air dried, pulverized and soaked
in the 200 ml of 95% absolute ethanol for 24 h and boiled at 60˚C
for 30 min on a heating mantle. The solution was then percolated
through Whatman No 1 filter paper and the resulting filtrate was
kept in a brown bottle and used as stock solution stored at 4˚C.
Solutions of lower concentrations were derived by diluting the stock
with ethanol prior to phytochemical screening. Aliquots for GST
inhibition studies was centrifuged at 30 000 X g for 15 min at 4˚C.
The clear supernatant was frozen dried and stored for 4˚C prior to
the toxicity bioassay on GST.
secondary metabolites such as saponins, tannins and flavonoids.
This was done according to the methods described by Sofowora
The concentrations of phenolic compounds in the ethanolic
extracts, expressed as tannin equivalents, were measured using a
modified method of Singleton et al
(1999) with some modifications.
Ethanolic extract (0.1 g) was dissolved in 5 ml of acetone for 10 min
on ice. To 0.5 ml of the solution, 0.5 ml of distilled water, 0.5 ml of
folin’s reagent (1:1) and 2.5 ml of 20% sodium carbonate were
added. The reaction mixtures were kept in the dark for 40 min, after
which the absorbance was read at 750 nm. Phenol contents were
extrapolated from standard tannin calibration curve.
The ability of ethanolic extracts to reduce ferric chloride was
The reducing property was expressed as tocopherol (Vitamin E)
equivalent. The ethanol extracts (5 g) was dissolved in 10 ml of
water and filtered. To 2.5 ml of the filtrate, 2.5 ml of phosphate
buffer (pH 6.6) and 2.5 ml of potassium ferrocyanide were added.
The mixtures were incubated at a temperature 40°C. 10% trichlo-
roacetic acid was added. The resulting mixtures were centrifuged
for 10 min. The supernatant (5 ml) was mixed with 5 ml of distilled
water and 1.0 ml of 0.1% ferric chloride. The absorbance of the
standard and the sample were read at 700 nm against reagent
blank made of ethanol instead of ethanolic extract.
Free radical scavenging
The scavenging activity of the Vitamin E and
ethanolic leaf extract on DPPH radicals
were determined according to the method of Chu et al
aliquot of 0.5 ml of 0.1 mM DPPH radical in ethanol was added to
test tubes containing 1 ml of different concentrations (0 – 5 mg/ml)
of the ethanolic extract. The reaction mixture was mixed at room
temperature and kept for 20 min. The absorbance was read at 520
nm against distilled water. Radical scavenging capacity of each
extract has been calculated as the percent DPPH radical
scavenging affect which is:
DPPH Scavenging Effect (%) = ([A
)] / A
is the absorbance of the control with ethanol and A
Preparation of cytosolic fraction and purification of GST
adults were obtained from naturally infested cowpea
seeds Sokoto white cultivar of cowpea from Oba Market in Akure,
Nigeria. The whole organism was homogenized in a buffer (35%
w/v, 10 mM Tris-HCl, 250 mM sucrose 1 mM phenylmetha-
nesulfonyl fluoride, 1 mM dithiothreitol pH 7.4) and centrifuged at
14,000 rpm for 40 min to remove cell debris using Eppendorf cold
centrifuge 5810 R. The supernatant was purified on GSH-
Sepharose column as described by Kolawole and Ajele (2004). The
glutathione removed by ultralfitration by Millipore Amicon ultralfitra-
tion and desalting column. The enzyme was stored at -70˚C until
Determination of protein concentration and glutathione S-
GST activity was determined according to Habig et al. (1974) as
modified by Ajele and Afolayan (1992). For a typical assay, the
reaction mixture of 3 ml containing a final concentration, 100 mM
potassium phosphate buffer pH 6.5 and 1 mM each of Glutathione
(GSH) and 1-chloro-2,4-dinitrobenzene (CDNB), together with an
appropriate amount of enzyme. Three replicates were used for
each measure. One unit of GSTs activity is defined as the amount
of enzyme producing l mol thioether per min. The extinction
coefficient for CDNB conjugate at 340 nm is 0.0096 M
(Habig et al
1974). The protein concentration was determined by
the method of Bradford Method (1976). Two replicates were used
for each experiments using bovine serum albumin as a standard. A
Shimadzu UV-Visible 1601 double beam digital spectrophotometer
was used for the assay. The kinetic analysis of inhibition and
inhibition interaction was done by adding known concentrations of
plant extracts to the assay buffer prior to assay.
. Plant used for insecticial activity and their phytochemical components.
Antioxidant activities of the plants.
0.26 ± 0.01
0.21 ± 0.007
0.040 ± 0.01
0.15 ± 0.00
The phytochemical screening of
revealed the presence of
alkaloids , saponin and tannins and flavonoids as shown
in Table 1.
The antioxidant properties of
as reveals by total phenol content was
0.26 ± 0.01, 0.21 ± 0.007 and 0.15 ± 0.01 mg/ml,
respectively as tannin equivqlents (Table 2).
properties level of 0.043, 0.040 and 0.034 mg/ml,
respectively. DPPH (1, 1–diphenyl–2-Picrylhydrazyl) per-
cent scavenging activities of the plants were measured in
different concentration ranging from 0 to 5 mg/ml. 5 mg/
showed 60% inhibtion;
44% inhibition as shown in
) was rapidly purified by affinity gel
of glutathione-agarose. The interested plant extracts
were screened for their effect on GST activity apart from
their antioxidant properties. The data were obtained in
the experiment to determine the effect of
glutathione S-transferases from cowpea storage bruchids
) and to establish the type of inhibition
mechanism. The straight line graph result of a series of
reciprocal of GST activity against inhibition concentration
of all ethanolic plant extracts was observed. The series of
line converged at a point away from the origin. The Ki
values are 84, 132 and 180 g/mL, respectively for
diversifolia , C. rotundus
(Figures 2, 3
The use of plant products to protect stored cowpea,
L. (Walp) against cowpea storage bruchids
) predation is an age long practice in
Nigeria (Lale, 1992). The ethanolic extract of
folia , C. rotundus
has been reported to
have potent insecticidal activity against cowpea storage
F. These plants have different
degree of insecticidal potentials on the insect (Adedire
and Akinneye, 2003; Adedire and Lajide, 1999)
In this present investigation our aim was to collect
information on the possible pharmacological basis for
the efficacy of these plant extracts on the effective
management of cowpea storage bruchids (
components that could be responsible for the efficacy.
Our result shows that alkaloids, saponin and tannins and
flavonoids are some of the major contituents of the plants
extracts. The presence of these secondary metabolites
might not be unconnected to their insecticidal activities.
These plant extracts also have high content of poly-
phenols and antioxidant activity. Phenolic constituents
have been studied extensively as important contributors
to the antioxidant activity in plants (Aruoma, 2003;
Skerget et al., 2005).There are reports in the literature
which correlate the total phenolics content of a plant
extract with its antioxidant activity (Coruh et al.
Skerget et al
2005) and this was also the case in the
present study, as shown in Table 1. The extracts with
high phenolics content, also has high DPPH radical
Our preliminary investigation shows that all the plant
extract used in this study bring about the inhibition of
crude GST extract from the cowpea storage bruchids (
). We therefore investigated the nature of the
inhibition on the purified sample of the GST. From the
Plant part used
DPPH scavenging activities abilities of the plant extracts.
Dixon Plot of the reciprocal of GST activity (1/V) versus
concentration utilizing 1.0 mM, 1.5 mM and 2.0 mM CDNB comcentrations. Lines of best fit
Dixon Plot of the reciprocal of GST activity (1/V) versus
concentration utilizing 1.0 mM, 1.5 mM and 2.0 mM CDNB comcentrations. Lines of best
Dixon Plot of the reciprocal of GST activity (1/V) versus
concentration utilizing 1.0 mM, 1.5 mM and 2.0 mM CDNB comcentrations. Lines of
results, the inhibition was found to be competitive as
showed by the Dixon plot. This suggests that the extract
binds to the active site of the enzyme and therefore
prevent detoxification role of the enzyme. The enzyme
has been reported to be involved in the adaptation of
insects to allelochemicals and insecticides (Francis et al.,
2005). Sigma class of GST is implicated to as protectors
against oxidative stress in insects (Enayati et al., 2005).
The observation of the
inhibition of cowpea
storage bruchids (
) glutathione S-
transferases by the extracts of
T. diversifolia, C. rotundus
is in good agreement with the
earlier report of Fakae et al.
(2000) that the GST of
parastic nematodes was inhibited by some Nigerian
medicinal plants. This is also supported by the reports of
Coruh et al. (2007a,b)
The inhibitory effects of naturally
occurring plant polyphenols such as tannic acid, ellagic
acid, ferulic acid, caffeic acid, stilbene, quercetin, curcu-
min and chlorogenic acid against GST have long been
reported by many researchers (Kawabata et al., 2000;
l., 2004). Quinines are also well-known exam-
ples of covalent inhibitors of GST enzymes (Zanden et
From our results it appears that the extracts with high
polyphenols and reducing properties have effective
inhibition on the GST from the cowpea storage bruchids
). This suggests that the phytochemicals
can exert insecticidal role by antioxidant activity. Francis
et al. (2005) reported that glucosinolates and isothio-
cynates from Brassicaceae plants are easily detoxified by
aphid and this is responsible
for its adaptation. However, Fakae et al. (2000) suggest
the combinatorial approach of some active allelochemi-
cals in the plant extracts on the effective inhibition of
GST. If this posture holds, the combinatorial approach
prevents either modification of the target site or to ampli-
fied production of this detoxification enzyme (Haubruge
and Amichot, 1995).
Spectrophotometric methods have succeeded in
answering the question of the pharmacological basis of
the insecticidal activity and the type of inhibition on the
GST, a dimeric enzyme. The question still remains if the
binding of this extracts brings about the cooperative
binding of the extracts on the other dimmers. This merits
our further investigation.
The authors gratefully acknowledge the material support
of Centre for Cellular and Molecular Biology (CCMB),
Adedire CO (2003). Use of nutmeg,
and oil for the control of cowpea storage bruchids,
(F.) J. Plant Dis. Prot. 109: 193-199.
Adedire CO, Akinneye JO (2003). Biological activity of three marigold,
, on cowpea seed bruchids,
(Coleoptra: Bruchidae) Ann. Appl. Biol. 144: 185-189.
Adedire CO, Lajide L (1999). Toxicity and Oviposition deterency of
some plants extracts on Cowpea storage bruchids
(F.) J. Plant Dis. Prot. 106: 647-653.
Ajele JO, Afolayan A (1992). Purification and characterization of
glutathione transferase from Giant African land snail
Comp. Biochem. Physiol. 103B: 47-55.
Aruoma OI (2003). Methodological considerations for characterizing
potential antioxidant actions of bioactive components in food plants.
Mutat. Res. pp. 523-524, 9-20.
Boeke SJ , Van Loon JJA, Van Huis A, Kossou DK , Dicke M (2001).
The use of plant materials to protect stored leguminous seeds
against seed bettles: a review. The Netherlands. Backhuys
Bradford KM (1976). A rapid and sensitive method for the quantification
of microgramme quantities of protein utilizing the principles of protein-
dye binding. Anal. Biochem, 72: 248-254.
YH, Chang CL, Hsu HF (2000). Flavonoid contents of several
vegetables and their antioxidant activity. J. Sci. Food Agric. 80:
Desf. from Apiaceae
family used as food in Eastern Anatolia and their inhibitory effects on
glutathione-S-transferase. Food Chem. 100: 1237-1242.
Coruh, N, Sagˇdıc¸ogˇlu Celep AG, Ozgokce FO, Iscan M (2007b).
Antioxidant capacities of
L. extracts and inhibi-
tion on glutathione-S-transferase activity. Food Chem. 100: 1249-
transferases in the adaptation to plant secondary metabolites in the
Myzus persicae Aphid. Archives of Insect Biochem. and Physiol. 58 :
S-transferases by thonningianin A, isolated from the African
medicinal herb, Thonningia sanguinea,
Food Chem. Toxicol.
WH, Pabst MJ, Jakoby WB (1974). Glutathione Stransferases the
first enzymatic step in mercapturic acid formation. J. Biol. Chem.
E, Amichot M (1995). Les mecanismes responsables de la
LEN, Daoust RA (1986). Insect pests of cowpea. Annu. Rev.
Kawabata K, Yamamoto T, Hara A, Shimizu M, Yamada Y, Matsunaga
K (2000). Modifying effects of ferulic acid on azoxymethane-induced
colon carcinogenesis in F344 rats. Cancer Lett. 157(1): 15-21.
AO, Ajele JO (2004). Substrate specifities and Inhibition
) liver glutathione s-
transferases. Glob. J. Pure Appl. Sci. 10(1): 179-182.
V (1981). Vaincre la carence proteique par le developpement
des legumineuses alimentaries et la protection de leurs recoltes
contres les bruches. Food Bull. 3: 24-38.
NES (1992). A laboratory study of the comparative toxicity of
Biol. Technol. 2: 612-664.
Pullido R, Bravo L, Saura-Calixto F (2000). Antioxidant activity of dietary
polyphenols as determined by a modified ferric reducing/
antioxidant power assay. J. Agric Food Chem. 48: 3396-3402.
VL, Orthofer R, Lamuela-Raventos RM (1999). Analysis of
means of Folin-Ciocalteu reagent. Methods Enzymol. 299: 152-178
Skerget M, Kotnik P, Hadolin M, Hras A, Simonic M, Knez Z (2005).
Phenols, proanthocyanidins, flavones and flavonols in some plant
materials and their antioxidant activities. Food Chem. 89(2): 191-
traditional medicine in Africa. 2nd Edition Spectrum Books Limited,
Nigeria, pp. 150-156
Zanden JJV, Geraets L, Wortelboer HM, Bladeren PJV, Rietjens IMCM,
Cnubben NHP (2004). Structural requirements for the flavonoid-
mediated modulation of glutathione Stransferase P1-1 and GS-X
pump activity in MCF7 breast cancer cells. Biochem. Pharmacol. 67: