Insecticidal activity of plant extracts against Tribolium castaneum Herbst

Abstract

The insecticidal activity of n-hexane, methanol and water extracts of Tamarindus indica, Azadirachta indica, Cucumis sativus, Eucalyptus species, Switenia mahagoni, and Psidium guajava leaves were investigated by using the Film residue method against a red flour beetle Tribolium castaneum Herbst. The results showed that four plant extracts showed a strong to moderate toxicity at a different concentration on red flour beetle. Among them, Cucumus sativus leaves extract showed highest mortality (80%) whereas Psidium guajava extract showed lowest mortality (50%). Among the solvents, the hexane extracts showed more toxic effect than other extracts. The LC50 results revealed that the hexane extract of Cucumus sativus is the most toxic to the pest followed by the hexane extracts of Azadirachta indica and Tamarindus indica. Qualitative phytochemical analysis has also been performed.

Full text

M. Mostafa et al, J Adv Sci Res, 2012, 3(3): 80-84 80
Journal of Advanced Scientific Research, 2012, 3(3)
Journal of Advanced Scientific Research
Available online through http://www.sciensage.info/jasr
Insecticidal activity of plant extracts against Tribolium castaneum Herbst
M. Mostafa*, Hemayet Hossain, M Anwar Hossain, Pizush Kanti Biswas, M. Zahurul Haque
Chemical Research Division, BCSIR Laboratories, Dhaka- 1205, Bangladesh
*Corresponding author: mostafabcsir@yahoo.com
ABSTRACT
The insecticidal activity of n-hexane, methanol and water extracts of Tamarindus indica, Azadirachta indica, Cucumis sativus, Eucalyptus
species, Switenia mahagoni, and Psidium guajava leaves were investigated by using the Film residue method against a red flour beetle
Tribolium castaneum Herbst. The results showed that four plant extracts showed a strong to moderate toxicity at a different
concentration on red flour beetle. Among them, Cucumus sativus leaves extract showed highest mortality (80%) whereas Psidium
guajava extract showed lowest mortality (50%). Among the solvents, the hexane extracts showed more toxic effect than other
extracts. The LC50 results revealed that the hexane extract of Cucumus sativus is the most toxic to the pest followed by the hexane
extracts of Azadirachta indica and Tamarindus indica. Qualitative phytochemical analysis has also been performed.
Keywords: Azadirachta indica, Tamarindus indica, Cucumis sativus, Eucalyptus species, Switenia mahagoni, Psidium guajava, insecticidal
activity
1. INTRODUCTION
The protection of stored grain and seeds against insect
pests has been a major problem from the development of
agriculture. Plant products have been successfully exploited as
insecticides, insect repellents and insect antifeedants [1-3]
Higher plants are a rich source of novel natural substances that
can be used to develop environmental safe methods for insect
control [4]. Insecticidal activity of many plants against several
insect pests has been demonstrated [5-7]. The deleterious
effects of plant extracts or pure compounds on insects can be
manifested in several manners including toxicity, mortality,
antifeedant growth inhibitor, suppression of reproductive
behaviour and eduction of fecundity and fertility. Yang and
Tang [8] reviewed the plants used for pest insect control and
found that there is a strong connection between medicinal and
pesticidal plants.
To minimize use of synthetic pesticides and to avoid
pollution of the environment, natural antifeedant, deterrent
and repellent substances have been searched for pest control
during recent times [9-11]. However, there is an urgent need
to develop safe alternatives that are of low cost, convenient to
use and environmentally friendly. Considerable efforts have
been focused on plant derived materials, potentially useful as
commercial insecticides. The aim of our study is to evaluate the
insecticidal activity of the hexane, methanol and water extracts
of Tamarindus indica, Azadirachta indica, Cucumis sativusn, Lens
culinaris, Eucalyptus species, Switenia mahagoni, and Psidium
guajava leaves
2. MATERIAL AND METHODS
2.1. Plant materials
The Tamarindus indica leaves (Tentul), Azadirachta indica
leaves (Neem), Cucumis sativus leaves (Shasha) and Lens culinaris
(Masur) were collected from the Norshindi district,
Bangladesh. The Eucalyptus species (Eucalyptus) and Switenia
mahagoni (Mahogoni) were collected from the BCSIR campus,
Dhaka and the Psidium guajava leaves (Goam) was collected
from the BCSIR Laboratories, Rajshahi campus. The leaves
were dried under shade and finally dried in an oven at 450C for
48 hours before grinding. The dried plant materials were
ground into powder with an electrical blender.
2.2. Extraction of plant materials
The plant powders (100 g each plant sample) were
separately extracted in hexane, methanol and water for 24 h on
an orbital shaker. The extracts were filtered using a Buchner
funnel and Whatman no. 1 filter paper. The hexane, methanol
extracts were evaporated to dryness under reduced pressure at
400C using a vacuum rotary evaporator, while the water
extract was freeze-dried with Savant Refrigerated Vapor Trap.
Each extracts were kept in freeze for further work in future.
2.3. Insect bioassays
2.3.1. Test Insects
The red flour beetle, T. castaneum were collected from
the stock cultures maintained in the BCSIR Laboratories,
Rajshahi. Mass cultures were maintained in glass jars (1000ml)
ISSN
0976-9595
Research Article

M. Mostafa et al, J Adv Sci Res, 2012, 3(3): 80-84 81
Journal of Advanced Scientific Research, 2012, 3(3)
and subcultures were in beakers (500ml) with food medium
and kept in an incubator at 30±0.5°C. A standard mixture of
whole-wheat flour with powdered dry yeast in a ratio of 19:1
[12, 13] was used as food medium throughout the
experimental period.
2.4. Mortality tests
Film residue method [14] was used to test the mortality of
the adults of T. castaneum. The extracted materials were
weighed and dissolved in acetone for dosing. For testing
beetle, mortality four doses were used including control
(water). Ten to fifteen day-old adults of T. castaneum was used
at 372.95, 785.91 and 1571.83 μg/cm2 concentrations. The
doses were prepared by mixing the requisite quantities of the
product with 1 ml acetone/ water. After mixing properly the
liquid was dropped in a petri dish (9.5-cm diameter). After
drying by fanning and finally in an oven at 40 ºC, 20 adults of
each species were released in each Petri dish. For each dose
three replications were taken. The doses were calculated by
measuring the weight of prepared product (μg) in 01 ml of
water divided by the surface area of the petri dish and it was
converted into μg/cm2. Mortality was assessed after 24, 48 and
72 h of the treatment. The calculation of mortality rate was
corrected for control mortality according to Abbott’s formula
[15]:
Mc = (Mo Mc/100 Me) 100
Where, Mo = Observed mortality rate of treated adults (%),
Me = mortality rate of control (%), and Mc = corrected
mortality rate (%)
The LD50 values were determined by probit analysis [14].
The experiments were performed in the laboratory at 30ºC ±
0.5ºC.
2.5. Statistical analyses
The experiment results were statistically analyzed by the
mean of one-way analysis of variance ANOVA and when
results were significant at p =0.5, Ducan test was used.
2.6. Phytochemical screening
Phytochemical screening of the extracts was carried out
using the standard procedures described by Edeoga et al [16]
for alkaloids, saponins, tannins, flavonoids, anthraquinones and
steroids.
3. RESULTS AND DISCUSSION
The results of preliminary phytochemical analysis of
various extracts of Tamarindus indica, Azadirachta indica, Cucumis
sativus, Eucalyptus species, Switenia mahagoni and Psidium guajava
are presented in Table 4. The hexane extracts of Azadirachta
indica, Cucumis sativus and Tamarindus indica showed the
presence of steroids and saponins. The methanol extracts of
Azadirachta indica, Eucalyptus species, Psidium guajava, Switenia
mahagoni and Tamarindus indica showed the presence of
flavonoids, tannins and saponins, whereas the water extracts of
Azadirachta indica, Cucumis sativus, Psidium guajava, Switenia
mahagoni and Tamarindus indica contained flavonoids and
tannins.
The toxic effects of hexane, methanol and water extracts
of Tamarindus indica, Azadirachta indica, Cucumis sativus,
Eucalyptus species, Switenia mahagoni and Psidium guajava were
evaluated against red flour beetle, T. castaneum by using the
method of residual film technique. Six different extracts of
Tamarindus indica (TI-1), Azadirachta indica (AI-1 & AI-3),
Cucumis sativus (CS-1 & CS-2) and Psidium guajava (PG-1) at
different concentration revealed toxicity but any solvent
extracts of two plants, Eucalyptus species and Switenia mahagoni
did not showed any toxic effect to red flour beetle. The
numbers of dead red flour beetle were counted after 24, 48
and 72 hours at all doses 198.48, 392.95, 785.91 and 1571.83
μg/cm2 respectively. Then the percentages of corrected
mortality were calculated by using Abbott’s formula and the
results are shown in Table-1. The results showed that the
hexane extract of Cucumis sativus (CS-1) possessed the highest
toxicity at all doses but the hexane extracts of Azadirachta indica
(AI-1), Tamarindus indica (TI-1) and the methanol extract of
Cucumis sativus (CS-2) showed the moderate toxicity at
concentrations 785.91 and 1571.83 μg/cm2, whereas the
hexane extract of Psidium guajava leaf (PG-1) possessed the
lowest (50%) toxic effect at highest concentration1571.83
μg/cm2 against red flour beetle, T. castaneum. The order of
toxicity of the six different plant extracts on red flour beetle,
T. castaneum were: Cucumis sativus (CS-1) > Azadirachta indica
(AI-1) > Cucumis sativus (CS-2) > Tamarindus indica (TI-1) >
Azadirachta indica (AI-3) > Psidium guajava (PG-1). The
mortality percentage was directly proportional to the level of
concentration of plant extract.
The results of the probit analysis for the estimation of LC50
values, 95% confidence limits and regression equation at 24,
48 and 72h for the mortality of red flour beetle are presented
in Table 2. The LC50 values of hexane extracts of Cucumis sativus
(CS-1), Azadirachta indica (AI-1), Tamarindus indica (TI-1) and
Psidium guajava (PG-1) at 24 hours after treatment are 20.64,
234.57, 732.53 and 1944.40 μg/cm2, at 48 hours after
treatment are 24.43, 91.80, 178.74 and 1944.40 μg/cm2 and
at 78 hours after treatment are 10.74, 155.13, 58.36 and
774.22 μg/cm2 respectively. The LC50 values of methanol
extract of Cucumis sativus (CS-2) at 24 hours after treatment is
557.87 μg/cm2, at 48 hours after treatment is 153.32 μg/cm2
and at 78 hours after treatment is 20.64 μg/cm2 respectively.

M. Mostafa et al, J Adv Sci Res, 2012, 3(3): 80-84 82
Journal of Advanced Scientific Research, 2012, 3(3)
The LC50 values of water extract of Azadirachta indica (AI-3) at
24 hours after treatment is 990.26 μg/cm2, at 48 hours after
treatment is 38.02 μg/cm2 and at 78 hours after treatment is
38.02 μg/cm2 respectively. The results indicated that the
hexane extract of Cucumis sativus (CS-1) at 72 hours after
treatment was the most toxic (10.74µg/cm2) and the hexane
extract of Psidium guajava (PG-1) was the least toxic
(1944µg/cm2). The hexane extract of Cucumis sativus (CS-1)
also maintained its toxicity, when the LC50 values were
compared at 24 HAT (20.64%) and 48 HAT (24.43%). The
Chi-square values of different plant extracts at different HAT
were insignificant at 5% level of probability and did not show
any heterogeneity of the mortality data. The present study
results are in conformity with the results of Mamun et al [17]
who reported that T. castaneum adults were significantly more
susceptible to the toxicity of the hexane and water extract of
Azadirachta indica. Khalequzzaman and Sultana [18] also
reported the toxic effect of petroleum ether extract of Annona
squamosa seed on T. castaneum. The toxic and sterilizing effects
of A. calamus rhizome oil to certain stored grain insects have
also been reported by Saxena and Mathur [19]. The secondary
metabolites of plants are vast repository of compounds with
wide range of biological activity. It has been reported that the
steroids, phenolic compounds and tannins had great impact on
insecticidal acitivities. The different plants extracts in our
present study revealed the toxicity against store insect may be
due to the presence of different classes of bioactive
compounds.
Table 1: Mortality percentage of red flour beetle, T. castaneum treated with different plant extracts by Film residue method
Name
of the
Plants
Concentration
(µg/cm2)
No of
Insect
used
No of Insect dead
Total No of Insects dead
% of Average
Mortality
% Corrected Mortality
24 hrs
48 hrs
72 hrs
CS1
196.48
120
84
90
96
90
75
75
392.95
90
90
96
92
76.66
76.66
785.91
96
96
96
96
80
80
1571.83
96
96
96
96
80
80
Control
0
0
0
0
AI1
196.48
120
60
66
66
64
53.33
53.33
392.95
60
90
72
74
61.66
61.66
785.91
78
90
90
86
71.66
71.66
1571.83
78
96
96
90
75
75
Control
0
TI1
196.48
120
30
66
72
56
46.66
46.66
392.95
42
66
90
66
55
55
785.91
72
90
96
86
71.66
71.66
1571.83
72
96
96
88
73.33
73.33
Control
0
PG1
196.48
120
30
30
30
30
25
25
392.95
30
30
36
32
26.66
26.66
785.91
54
54
72
60
50
50
1571.83
54
54
72
60
50
50
Control
0
CS2
196.48
120
30
66
84
60
50
50
392.95
66
72
90
76
63.33
63.33
785.91
66
96
96
86
71.66
71.66
1571.83
78
96
96
90
75
75
Control
0
0
0
0
AI3
196.48
120
42
72
72
62
51.66
51.66
392.95
60
84
84
76
63.33
63.33
785.91
60
84
84
76
63.33
63.33
1571.83
60
90
90
80
66.66
66.66
Control
0
0
0
0

M. Mostafa et al, J Adv Sci Res, 2012, 3(3): 80-84 83
Journal of Advanced Scientific Research, 2012, 3(3)
Table 2. χ2-values, regression equations, LD50 and 95% confidence limits of some indigenous medicinal plant extracts against T.
castaneum, a stored grain insect pests after 24, 48 and 72 h of treatment
Plant
extracts
Hrs after
treatment
Test insect
χ2- values for
Heterogeneit*
Regression equations
LD50
(µg.cm-2)
95% Confidence limits
Lower
Upper
CS 1
24 h
T. castaneum
0.810
Y= 4.413+0.465X
20.64
0.00
89.05
48 h
T. castaneum
1.713
Y= 4.305+0.492X
24.43
0.00
97.72
72 h
T. castaneum
0.877
Y= 4.34+0.64X
10.74
0.00
77.92
AI1
24 h
T. castaneum
2.205
Y= 3.80+0.505X
234.578
36.99
405.10
48 h
T. castaneum
4.111
Y= 3.64+0.708X
91.807
16.51
174.99
72 h
T. castaneum
1.093
Y= 1.70+1.57X
155.13
59.75
242.74
TI1
24 h
T. castaneum
5.195
Y= 0.73+1.56X
732.538
579.51
974.10
48 h
T. castaneum
3.734
Y= 3.01+0.89X
178.741
80.14
267.11
72 h
T. castaneum
2.886
Y= 3.88+0.64X
58.368
3.95
135.43
PG1
24 h
T. castaneum
4.222
Y= 2.63+0.718X
1944.40
1182.78
6493.45
48 h
T. castaneum
4.222
Y= 2.63+0.718X
1944.40
1182.78
6493.45
72 h
T. castaneum
7.599
Y= 1.61+1.17X
774.22
617.66
1025.98
CS2
24 h
T. castaneum
9.380
Y= 2.12+1.05X
557.87
425.44
730.78
48 h
T. castaneum
3.683
Y= 3.03+0.90X
153.32
64.66
234.95
72 h
T. castaneum
0.810
Y= 4.413+0.465X
20.64
0.00
89.05
AI3
24 h
T. castaneum
3.352
Y= 3.87+0.38X
990.26
495.08
126631.03
48 h
T. castaneum
0.874
Y= 4.34+0.42X
38.02
0.00
137.92
72 h
T. castaneum
0.874
Y= 4.34+0.42X
38.02
0.00
137.92
*χ2 = Goodness of fit. The tabulated value of χ2 is 5.99 (df = 2, P<0.05)
Table 3: Results of phytochemical Tests
Plant
name
Extract
Alkaloid
Steroid
Flavonoid
Tannins
Saponins
AI
Hexane
-
+
-
-
-
Methanol
-
-
+
++
-
Water
-
-
++
++
-
CS
Hexane
-
++
-
-
+
Methanol
-
-
-
-
-
Water
-
-
+
++
-
ES
Hexane
-
-
-
-
-
Methanol
-
-
++
++
+
Water
-
-
-
-
-
PG
Hexane
-
-
-
-
-
Methanol
-
-
-
++
-
Water
-
-
-
-
-
SM
Hexane
-
-
-
-
+
Methanol
-
-
++
+++
+
Water
-
-
-
-
++
TI
Hexane
-
+
-
-
-
Methanol
-
-
+
-
++
Water
-
-
++
+
-
+ = presence, -= Absence
From the insecticidal activity results, it is observed that
different solvent extracts of four plants would be more or less
effective for controlling red flour beetle. The hexane extract of
Cucumis sativus showed the highest toxic effect followed by the
hexane extract of Azadirachta indica . Cucumis sativus is available
throughout the country and the farmers may use this plant in
their storehouses for the management of stored grain pests.
Further investigation on the identification of active ingredient
from the hexane extracts, which is more effective than other
extracts, is utmost needed.
4. ACKNOWLEDGEMENT
The authors are grateful to L.A. Muslima Khanam, PSO,
BCSIR Laboratories, Rajshahi for providing necessary

M. Mostafa et al, J Adv Sci Res, 2012, 3(3): 80-84 84
Journal of Advanced Scientific Research, 2012, 3(3)
laboratory facilities. The work was supported by a Special
Research Grant from the Bangladesh Ministry of Science and
Information & Communication Technology.
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