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Mokhtar Mohamed Elamin et al., IJSID, 2012, 2 (6), 559-566
International Journal of Science Innovations and Discoveries , Volume 2, Issue 6, November-December 2012
559
INSECTICIDAL AND REPELLENT EFFECTS OF ARISTOLOCHIA BRACTEOLATA LAM. AGAINST TROGODERMA GRANARIUM
EVERTS
Mokhtar Mohamed Elamin and Abdalla Abdelrahim Satti*
Department of Alternatives to Pesticides and Biocontrol, Environment and Natural Resources Research Institute, Nati onal
Centre for Research, P.O. Box 6096, Khartoum, Sudan
INTRODUCTION
INTRODUCTION
ISSN:2249-5347
IJSID
International Journal of Science Innovations and Discoveries
An International peer
Review Journal for Science
Research Article Available online through www.ijsidonline.info
Received: 09.11.2012
Accepted: 16.12.2012
*Corresponding Author
Address:
Name:
Abdalla Abdelrahim Satti
Place:
National Center for Research,
Sudan
E-mail:
satisattisat@yahoo.com
ABSTRACT
In the course of studying environmentally safe alternatives pesticides, this
research was dealt with insecticidal potentialities of scorpion root (Aristolochia
bracteolata) against the third instar larvae of the khapra beetle ( Trogoderma granarium).
Organic and water extracts of three parts (fruits, leaves and roots) were tested at three
concentrations (1.25%, 2.5% and 5% w/v) in laboratory experiments, during 2010-2011,
at the Environment and Natural Resources Research Institute, Sudan. The main
parameters considered included, mortality and repellent effects of treatments on the pest,
and the consequent impact on sorghum grains damage. The results of bioassay
experiment revealed very poor mortality effects by all extracts of scorpion root without
significant differences from the untreated control, except the highest dose (5%) of leaves
ethyl acetate extract which showed the best significant result, though hardly exceeded
50% mortality after three weeks post treatments. But, the latter extract has attained the
next inferior chemical yield compared to the other extracts. Considering the larval
damage, the highest savings of grains was attained by the fruits hexane extract, followed
by the leaves ethyl acetate extract. Although, the latter extract exerted higher repellent
action than fruits hexane extract, no significant differences were found between them.
However, the superior saving of sorghum grains showed by the fruits hexane extract may
need further investigation particularly for the presence of antifeedant compounds in this
extract. Moreover, the higher extractive yields obtained by this extract as compared with
that of leaves ethyl acetate, may suggest the potentiality of such portion (fruits hexane
extract) which should be emphasized in future resea rch.
Keywords: Scorpion root; mortality; repellent; extracts; Trogoderma granarium; Sudan.
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INTRODUCTION
Scorpion root (Aristolochia bracteolata Lam.) (Family; Aristolochiaceae), locally known as “Um-Galagil”, is a common
medicinal herb in Sudan that grown spontaneously in different areas, especially in the central semi-arid and semi-humid zones
of the country where enough rainfalls is found [1,2]. The herb is also recorded in several African and Arab countries including
Chad, Djibouti, Ethiopia, Kenya, Tanzania, Uganda, Central Africa Republic, Mali, Nigeria, Oman, Saudi Arabia, United Arab
Emirates and Yemen. Likewise, it prevails in most Asian countries like India, Pakistan and Sri Lanka [3,4,5]. In folk medicine, A.
bracteolata is used as purgative and for treatment of amenorrhea, dysmenorrheal, foul ulcer, boils, syphilis, gonorrhea,
dyspepsia, colic, skin diseases, scorpion bite, snake bite, eczema, artherlgia and intermittent fever. Seeds is ground in water to
form a lotion and used for softening hair [6,7,8]. Moreover, the root is used to treat male sterility [9]. Accordingly, studies proved
various biological activities of A. bracteolata. It is effective as antibacterial and antifungal. The plant is even poisonous to man,
rats, goats and livestock, and can kills intestinal worms [6,10,11,12,13].
Various active chemical compounds were detected in the different parts of A. bracteolata, which seem to contribute to
its variable biological activities. Among the main secondary metabolites reported are; alkaloids, aristolochic acid, β -sistosterol,
aristolactam, phenols, flavonoids, glycosides, terpenoids, sterols and saponins [14,15,16,17]. However, studies in Sudan revealed
that A. bracteolata contains sterols, alkaloids tannins and aristolochic acid [18,19]. The plant is therefore proved to have
antioxidant and insecticidal properties [20]. These records are encouraging to study this plant for different uses especially as
natural insecticides.
Currently there is an increasing interest worldwide in botanical insecticides as a result of the various problems
initiated by the irrational uses of synthetic chemicals. In this respect, the Sudan is considered one of the richest African
countries in floral diversity that may represents a potential source for natural pesticides [21]. Among the different active plants
utilized traditionally for various purposes in the country, A. bracteolata is one of the important remedial herbs against multi
human diseases, as explained above. However, indigenous studies regarding insecticidal properties of this plant are nearly
missing. In order to bridge the gap in this field, the present work focu ses on investigating the insecticidal and repellent effects
of Aristolochia bracteolata, using the third instar larva of Trogoderma granarium as a test insect.
MATERIALS AND METHODS
Collection of plant materials
Mature naturally grown scorpion root ( Aristolochia bracteolata) plants were uprooted from Shambat area, Khartoum
North, during April 2010 and brought to the laboratory at the Environment and Natural Resources Research Institute (ENRRI).
Fruits, leaves and roots were separated and dried under shade c onditions. As usually done, one day before extraction, these
parts were made into fine powders using an electric blender (Moulinex®, Type MS -223). The powders were kept in dark
bottles.
PREPARATION OF BOTANICAL EXTRACTS
Organic extracts:
Three solvents (viz. hexane, ethyl acetate and methanol) were used successively for extracting the prepared plant
samples (fruits, leaves and roots) in a soxhlet apparatus, with six units and 500 ml flasks sizes. The sequence of extraction
started with the removal of oil (apolar compounds) from the fruits sample by using the hexane, and then followed by the ethyl
acetate and methanol to obtain the intermediate and polar compounds, respectively. The other samples (leaves and roots)
were only extracted with ethyl acetate and methanol. A weight of 20g powder from each plant sample was loaded in the
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International Journal of Science Innovations and Discoveries , Volume 2, Issue 6, November-December 2012
561
soxhlet thimble and extracted to 9 h. Three to five extraction rounds were performed with new samples to get the required
quantity from each extract. All extracts were dried from solvents under normal air current. Similarly, the filtrate was also air
dried and re-extracted successively with the other solvents, in the same way. Each extract was kept separately in a dark bottle
and placed in a refrigerator until used. However, both the filtrates and extractive materials obtained at the end of each
extraction step were re-weighed so as to determine the percentages of yielded extracts.
The above prepared organic extracts were firstly diluted with water to prepare the highest extract concentration
(5%). However in case of the oil extract, 0.1% liquid soap was added as an emulsifier. Consequently, the other concentrations
(2.5% and 1.25%) were prepared by serial dilutions with water.
Water extracts:
Water extraction was done for all the three plant samples. As mentioned before, plant powders were generally
prepared one day before the extraction process. Therefore, this was also considered when preparing water extraction which
started 24h before the commencement of the experiment. Accordingly, the plant powder needed was weighed in a 100ml
conical flask where half volume of water was added, mixed thoroughly with a glass rod and allowed to stand overnight. In the
next day the mixture was agitated manually for few seconds before filtration, usi ng fine mesh. The volume of the extract was
completed with water to attain the highest concentration (5%w/v) indicated for the study. Consequently, the other
concentrations (2.5% and 1.25% w/v) were prepared through serial water dilutions.
Biological assays
Two bioassay laboratory experiments were conducted consecutively to evaluate the insecticidal potentialities of the
prepared extracts. The first experiment was aimed to test the mortality effects of treatments against the third instar larvae of
the khapra beetle, Trogoderma granarium (Coleoptera: Dermestidae). Afterwards, the highest concentrations of all extracts
were selected for the next experiment to study their repellent effects on the same pest. The number of larvae allocated for
each experiment was segregated from a culture found reserved at the Laboratory of Botanical Pesticides, Department of
Alternatives to Pesticides and Biological Control, ENRRI. However, the daily readings of temperatures (maximum and
minimum) and relative humidity in the laboratory were recorded throughout the study period.
Evaluation of mortality effects:
The first experiment of mortality effect was conducted during June-July 2010, applying all the prepared botanical
concentrations in comparison with an untreated control. Gla ss Petri dishes occupied with clean sound sorghum (Sorghum
bicolor) grains were used to accommodate the experiment. Each 10g of grains were treated with one of the different extracts
concentrations, left for five minutes to dry, and then placed into a Petri dish. Ten 3rd instar larvae of T. granarium were
introduced in each Petri dish and covered. Four replications were used assigned in a Completely Randomized design.
Investigations of the dishes were carried out periodically on the 2 nd, 7th, 14th and 21st days following treatments. Hence, the
number of dead insects and other observations were recorded. Based on the adopted design, ANOVA analysis was computed
for the recorded data and compared according to Duncan's Multiple Range Test.
Weight loss in sorghum grains:
Weight loss of sorghum grains as a result of larval damage during the bioassay experiment were calculated for the
highest concentrations of fruits and leaves extracts, including the control check. Such evaluation of damage was carried out 45
days following the commencement of the experiment. Firstly, the insect larvae were removed from the Petri -dishes, and then
the infested grains were subjected to sieving to get rid of cast skins, grain dust, insect excretion and other debris occurre d due
Mokhtar Mohamed Elamin et al., IJSID, 2012, 2 (6), 559-566
International Journal of Science Innovations and Discoveries , Volume 2, Issue 6, November-December 2012
562
to insect feeding and development. Lastly, Petri-dishes contents (sorghum grains) were weighed again for calculating the
percentages of damaged portion based on the initial weight. The percents of gains lost, and the percents saved in relation to
the control and the amounts used in the experiment were computed.
Repellency tests:
The bioassay tests of treatments for their repellent effects were carried out during December 2011. In this
experiment, only the highest concentrations of the prepared extracts of scorp ion root were checked against the 3rd instar
larvae of the pest. Locally made repellency equipment was prepared according to Berndt (1963), with slight modification in
the central platform to accommodate a Petri dish 5 cm in diameter [22,23].
After being treated with the different extracts, sorghum grains were distributed randomly in the peripheral holes of
the repellent equipment, including one untreated control check. A number of 200 third instar larvae of T. granarium were
introduced in a Petri dish and located in the central hole of the platform. This Petri dish was closed by a glass cover provided
with a glass rod fitted into the top opening cover of the equipment. After a moment, the glass rod was pulled up to start the
experiment by releasing the insects. In the second day (24h) the number of insects detected in each peripheral hole was
recorded. Such experiment was repeated successively in the subsequent two days, as replicates, following the same
procedures. Hence, three counts were taken and analyz ed to calculate the attractancy or repellency effect of each treatment
according to Leonard and Ehrman (1976) formula [24]:
A =
Where; A = attractancy (+) or repellency (-); No = number of insects in the test hole;
Nb = number of insects in the control hole; Nt = the total number of insects in both holes.
The output of this equation ranges from +1 (100% attractant) to -1(100% repellent) when compared to the control.
RESULTS AND DISCUSSION
Extraction yields of plant samples
The result of extraction yields (mean quantity and percentage extracted) of the studied plant samples were explained
in Table (1). It is clear that hexane extract recorded moderate yield of oil (19.4%) from the scorpion root fruits. This was
attributed to the fact that the whole content of fruits was extracted. Since the oil is always concentrated in seeds, higher yield
is expected to be obtained if pure seeds are used. This need to be verified in future research. Generally, fruit extracts rec orded
higher yields than the other parts of the scorpion root. Regarding leaves and roots, methanol extracts revealed significantly
higher yields than those of the ethyl acetate extracts, while the fruits showed no significant differences between the two
extracts. No similar studies are available on this plant.
TABLE 1. Yields of materials extracted from different parts of scorpion root ( SR), using water and organic solvents.
Plant samples
Extracted materials per 20 g
Mean±S.E.
(%)
SR-Fruits hexane extract
3.9±0.0 d
19.4
SR-Fruits ethyl acetate extract
7.4±0.0 a
36.9
SR-Fruits methanol extract
7.3±0.1 a
36.4
SR-Leaves ethyl acetate extract
2.0±0.0 e
09.9
SR-Leaves methanol extract
6.7±0.1 b
33.4
SR-Roots ethyl acetate extract
1.3±0.1 f
06.5
SR-Roots methanol extract
5.5±0.1 c
27.4
C.V. %
2.07
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International Journal of Science Innovations and Discoveries , Volume 2, Issue 6, November-December 2012
563
Larvicidal effects of treatments
The mean (maximum and minimum) temperature and relative humidity recorded for the laboratory during the study
period were, (37.0±0.2C° and 36.4±0.2C°) and (33.1±4.4% and 29.3±4.7%), respectively. Table (2) shows the larval mortality
of T. granarium as a result of feeding on sorghum grains treated with the different organic and water extracts of the scorpion
root. After the 2nd day and 7th day intervals, almost all botanical concentrations showed no significant differences from the
control, except the 5% concentration of leaves ethyl acetate extract and fruits hexane extract. During the subsequent interva ls,
the 5% leaves ethyl acetate extract was significantly the best treatment, though hardly exceeded 50% mortality after three
weeks. Since the latter extract has attained the next lowest chemical yield (Table 1), this will lessen its value from commer cial
point of view. The other treatments of scorpion root showed very meager mortality re sults without significant differences
from the control. Accordingly, it can be concluded that the tested extracts of the scorpion root were not effective as stomac h
toxicants against the current pest. This could be attributed to either one of two factors; low toxic action of plant materials or
high repellent and/or antifeedant actions that might prevent acquiring of lethal doses. The latter suggestion was the most
probable factor that needs to be confirmed particularly for the antifeedant actions.
Weight loss of sorghum grains:
The results of sorghum grains lost as a result of larval feeding on certain treatments within 45 days were shown in
Table (3). All scorpion root treatments showed significant effects in reducing the grain damage as compared with the
untreated control. The savings in grain losses by these treatments ranged between 16.7% and 77.8% in relation to control
damage, and between 3% and 14% relative to the stock used. However, the fruits hexane extract showed significantly the
lowest grain damage (4%) by the larvae which reflected in the highest grain savings as compared to the other treatments. Next
in order came the 5% leaves ethyl acetate and 5% fruits methanol extracts.
Repellency effects:
Table (4) shows the repellent effects of the highest concentrations of scorpion root extracts on the 3 rd instar larvae of
T. granarium. All the tested treatments reflected highly significant repellent effects, ranged between 61.7% and 89.5%
repellency, as compared with the control. The highest repellency percentages were achieved by the fruits methanol extract
and leaves ethyl acetate extract (89.5%), however all fruits extracts were non significantly different from the latter two
treatments. Regarding roots organic extracts, methanol extract revealed the best significant repellent effect.
Considering the two bioassay experiments of the scorpion root extracts, it is obvious that leaves ethyl acetate extract
was the best treatment reflected the highest mortality and the highest repellent effects o n the pest. Nevertheless, the best
protection of sorghum grains against larval damage was attained by the fruits hexane extract, though it induced lower
mortality compared to the former extract. Moreover, the fruits hexane extract also exerted comparable r epellent effect as
compared with that of the previous treatment. Regarding literatures, such repellent action of fruits hexane extract is nearly
similar to that attained by the neem seeds hexane extract against the same pest [23]. In other way, this extract may contain
additional biological activities such as antifeedant effect that might contributed in the recorded superior saving of sorghum
grains, a factor which needs further investigations. Another credit which can be added here is that the fruits hexane extract
recorded higher extractive yields than the leaves ethyl acetate extract. This is important whenever commercial production is
needed. Thus, hexane extracts of fruits and seeds of scorpion root should be emphasized in coming research as the best
bioactive portion based on the current results.
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564
TABLE 2. Mortality levels of Trogoderma granarium 3rd instar larvae fed on sorghum grains treated with different extracts of
scorpion root (Aristolochia bracteolata), during June-July 2010.
SR= Scorpion root; ext.= extract.
TABLE 3. Sorghum grains damaged by the 3rd instar larvae of Trogoderma granarium, after 45 days following treatments with
different extracts of scorpion root (SR), Jan. –Feb. 2011.
Treatments
Mortality percent means (±S.E.) at different intervals
2 days
7 days
14 days
21 days
SR-Leaves ethyl acetate ext. 1.25%
00.0±0.0c
05.0±0.3bc
05.0±0.3b
05.0±0.3b
SR-Leaves ethyl acetate ext. 2.5%
02.5±0.3bc
07.5±0.3b
10.0±0.4b
10.0±0.4b
SR-Leaves ethyl acetate ext. 5%
12.5±0.5a
30.0±0.4a
40.0±0.6a
55.0±0.7a
SR-Leaves methanol ext. 1.25%
00.0±0.0c
00.0±0.0c
00.0±0.0b
00.0±0.0b
SR-Leaves methanol ext. 2.5%
00.0±0.0c
00.0±0.0c
00.0±0.0b
00.0±0.0b
SR-Leaves methanol ext. 5%
00.0±0.0c
00.0±0.0c
00.0±0.0b
00.0±0.0b
SR-Leaves water ext. 1.25%
00.0±0.0c
00.0±0.0c
00.0±0.0b
00.0±0.0b
SR-Leaves water ext. 2.5%
00.0±0.0c
00.0±0.0c
00.0±0.0b
00.0±0.0b
SR-Leaves water ext. 5%
02.5±0.3bc
02.5±0.3bc
02.5±0.3b
05.0±0.5b
SR-Roots ethyl acetate ext. 1.25%
00.0±0.0c
00.0±0.0c
00.0±0.0b
02.5±0.3b
SR-Roots ethyl acetate ext. 2.5%
00.0±0.0c
00.0±0.0c
02.5±0.3b
02.5±0.3b
SR-Roots ethyl acetate ext. 5%
00.0±0.0c
00.0±0.0c
05.0±0.3b
05.0±0.3b
SR-Roots methanol ext. 1.25%
00.0±0.0c
00.0±0.0c
00.0±0.0b
00.0±0.0b
SR-Roots methanol ext. 2.5%
00.0±0.0c
00.0±0.0c
05.0±0.3b
05.0±0.3b
SR-Roots methanol ext. 5%
00.0±0.0c
02.5±0.3bc
02.5±0.3b
07.5±0.3b
SR-Roots water ext. 1.25%
00.0±0.0c
00.0±0.0c
02.5±0.3b
02.5±0.3b
SR-Roots water ext. 2.5%
00.0±0.0c
00.0±0.0c
05.0±0.3b
05.0±0.3b
SR-Roots water ext. 5%
00.0±0.0c
00.0±0.0c
02.5±0.3b
02.5±0.3b
SR-Fuits ethyl acetate ext. 1.25%
00.0±0.0c
00.0±0.0c
02.5±0.3b
02.5±0.3b
SR-Fuits ethyl acetate ext. 2.5%
00.0±0.0c
00.0±0.0c
07.5±0.3b
07.5±0.3b
SR-Fuits ethyl acetate ext. 5%
00.0±0.0c
02.5±0.3bc
10.0±0.0b
12.5±0.3b
SR-Fuits methanol ext. 1.25%
00.0±0.0c
00.0±0.0c
00.0±0.0b
00.0±0.0b
SR-Fuits methanol ext. 2.5%
00.0±0.0c
00.0±0.0c
00.0±0.0b
05.0±0.3b
SR-Fuits methanol ext. 5%
00.0±0.0c
00.0±0.0c
05.0±0.3b
10.0±0.7b
SR-Fuits water ext. 1.25%
00.0±0.0c
00.0±0.0c
00.0±0.0b
00.0±0.0b
SR-Fuits water ext. 2.5%
00.0±0.0c
00.0±0.0c
02.5±0.3b
05.0±0.3b
SR-Fuits water ext. 5%
02.5±0.3bc
02.5±0.3bc
05.0±0.3b
05.0±0.3b
SR-Fuits hexane ext. 1.25%
00.0±0.0c
00.0±0.0c
00.0±0.0b
05.0±0.3b
SR-Fuits hexane ext. 2.5%
00.0±0.0c
00.0±0.0c
05.0±0.3b
07.5±0.5b
SR-Fuits hexane ext. 5%
05.0±0.3b
07.5±0.5b
10.0±0.5b
12.5±0.8b
Control
00.0±0.0c
00.0±0.0c
00.0±0.0b
00.0±0.0b
C.V.%
282.84
121.21
101.38
99.34
Treatments
Grains weight lost (g)
Grains weight saved (%)
Mean
(±S.E.)
(%)
(relative
to control)
(relative to
stock)
SR-Fruits hexane extract 5%
0.4±0.0 d
04.0
77.8
14.0
SR-Leaves ethyl acetate extract 5%
0.9±0.0 c
09.0
50.0
09.0
SR-Fruits methanol extract 5%
0.9±0.1 c
09.2
50.0
09.0
SR-Fruits ethyl acetate extract 5%
1.1±0.1bc
11.0
38.9
07.0
SR-Fruits water extract 5%
1.2±0.1bc
11.7
33.3
06.0
SR-Leaves water extract 5%
1.3±0.2bc
12.7
27.8
05.0
SR-Leaves methanol extract 5%
1.5±0.1 b
14.7
16.7
03.0
Control
1.8±0.2a
18.3
-
-
C.V. %
19.8
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565
TABLE 4. Percentage repellency of some extracts of scorpion root (Aristolochia bracteolata) against the 3r d instar larvae of
Trogoderma granarium, during December 2011.
SR= Scorpion root; eth.ac.= ethyl acetate; ext.=extract.
Very meager research data is available on biological properties of scorpion root extracts in general, but information
concerning the insecticidal action of this plant is nearly absent. However, the repellent effect of scorpion root has been
demonstrated against mosquitoes [25]. The plant also proved to have antioxidant and insecticidal properties [20]. Moreover, the
poisonous actions of the plant to man, rats, goats and livestock were also indicated by Barakat et al. (1983) and Jou et al.
(2004) [10,15]. On the other hand, the plant was reported to contain active compounds like aristolochic acid which have good
antibacterial activity [11,12,13]. Other active secondary metabolites which seem to contribute to such variable biological activities
of A. bracteolata included; phenols, flavonoids, glycosides, terpenoids, ste rols, saponins, alkaloids, β-sistosterol and
aristolactam [14,15,16,17]. Studies in Sudan also proved the richness of the plant with various compounds like sterols, alkaloids,
tannins and aristolochic acid [18,19]. These different chemical groups detected in A. bracteolata may suggest the occurrence of
potential biocidal properties which need to be evaluated against various aspects of pest control. The plant is therefore
recommended for further investigations concerning its active ingredients and their multi-bioactivities, especially antifeedant
and repellent effects, as natural insecticides.
CONCLUSION AND RECOMMENDATION
The study proved that the tested extracts of scorpion root ( Aristolochia bracteolata) had very poor mortality effects
on the 3rd instar larvae of the khapra beetle (Trogoderma granarium), but contrarily, all extracts manifested significantly high
repellent activities against the pest. The fruits hexane extract was the best treatment saved sorghum grains from larval
damage. Such result was thought to be related to various biological activities of certain secondary metabolites in seeds,
including mainly repellent and antifeedant effects which need to be ascertained in further studies.
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Treatments
Mean (±S.E.) insects/ treatment.
(%) Repellency
SR-Fruits methanol ext. 5%
3.5±0.3 d
89.5
SR-Leaves eth.ac. ext. 5%
3.5±0.3 d
89.5
SR-Leaves methanol ext. 5%
3.7±0.7 d
89.0
SR-Fruits water ext. 5%
3.7±0.3 d
89.0
SR-Roots methanol ext. 5%
6.5±2.0cd
82.0
SR-Fruits hexane ext. 5%
7.5±0.3cd
78.8
SR-Roots water ext. 5%
8.7±2.0bcd
75.9
SR-Fruits eth.ac. ext. 5%
10.0±1.2bcd
72.7
SR-Leaves water ext. 5%
12.0±2.3bc
68.1
SR-Roots eth.ac.ext. 5%
15.0±0.6 b
61.7
Control
63.3±5.7 a
-
C. V. %
28.63
Mokhtar Mohamed Elamin et al., IJSID, 2012, 2 (6), 559-566
International Journal of Science Innovations and Discoveries , Volume 2, Issue 6, November-December 2012
566
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