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Life Science Archives (LSA)

ISSN: 2454-1354
Volume 1; Issue - 1; Year 2015; Page: 66 - 71

Research Article
LARVICIDAL AND OVICIDAL ACTIVITIES OF Vitex negundo Linn.
(Family:Verbenaceae) PLANT EXTRACTS AGAINST DENGUE MOSQUITO, Aedes
aegypti (Linn.) (Diptera: Culicidae)
J. Gokulakrishnana*, M. Baranitharanb, R. Abiramia, R. Kavikuyila, Ramyaa, J. Deepaa and
Baluselvakumara
a

Department of Zoology, Annamalai University, Annamalainagar-608 002, Tamilnadu, India

Department of Zoology, Poompuhar College (Autonomous), Melaiyur-609 107, Tamilnadu, India

Abstract
The present study deals with the investigation of larvicidal and ovicidal activities of hexane,
methanol and ethyl acetate leaf extracts of Vitex negundo (V. negundo) against dengue vector, Aedes aegypti
(Ae. aegypti). Twenty five early III instar larvae of Ae. aegypti was exposed to various concentrations (50250 ppm) and was assayed in the laboratory by using the protocol of WHO 1996; the 24 hrs LC50 of the V.
negundo leaf extracts was determined by Probit analysis. For ovicidal activity, slightly modified method of
Su and Mulla was performed. The ovicidal activity was determined against Ae. aegypti to various
concentrations ranging from 60-300 ppm under the laboratory conditions. The egg hatch rates were assessed
48 hrs post treatment. The LC50 values of hexane, methanol and ethyl acetate extracts of V. negundo against
early third instar larvae of Ae. aegypti were 189.70, 139.70 and 145.29 ppm, respectively. Maximum
larvicidal activity was observed in the methanol extract followed by ethyl acetate and hexane extracts. No
mortality was observed in control. Among three solvent tested the methanol extracts exerted 100% mortality
(zero hatchability) at 240 and 300 ppm. From the results, it can be concluded the crude extracts of V.
negundo was potential for controlling Ae. aegypti mosquito.
Article History
Received : 03.03.2015
Revised : 15.03.2015
Accepted: 28.03.2015

Key words: Vitex negundo, Dengue Mosquito,

Aedes aegypti, Solvent extracts, Larvicidal activity
and Ovicidal activity.

1. Introduction

A recent estimate shows that more than

50 million people are at risk of dengue virus
exposure worldwide. Annually, there are 2 million
infections, 500,000 cases of dengue hemorrhagic
fever, and 12,000 deaths (Guha-Sapir and
Schimme, 2005). Aedes aegypti is generally
known as a vector for an arbo-virus responsible
* Corresponding author: J. Gokulakrishnan
Tel.: +91-9443985831
E-mail: gokulagalya@gmail.com

for dengue fever, which is endemic to Southeast

Asia, the Pacic island area, Africa, and the
Americas. This mosquito also acts as a vector of
yellow fever in Central and South America and
West Africa. However, Dengue fever has become
an important public health problem as the number
of reported cases continues to increase, especially
with more severe forms of the disease, dengue
hemorrhagic fever, and dengue shock syndrome,
or with unusual manifestations such as central
nervous system involvement (Pancharoen et al.,
2002). A. aegypti is a cosmotropical species that

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Mathalaimuthu Baranitharan /Life Science Archives (LSA), Volume 1, Issue 1, Page 66 to 71, 2015

proliferates in water containers in and around

houses. Secondary vectors include Aedes
albopictus, an important vector in Southeast Asia
and that has spread to the Americas, western
Africa and the Mediterranean rim, Ae.
mediovittatus in the Caribbean, and Ae.
polynesiensis and Ae. scutellaris in the western
Pacic region. Ae. aegypti breeds in many types of
household containers, such as water storage jars,
drums, tanks, and plant or ower containers (Muir
and Kay, 1998; Honorio et al., 2003; Harrington et
al., 2005; Murugan et al., 2011).
Mosquito control, in view of their medical
importance, assumes global importance. In the
context of ever increasing trend to use more
powerful synthetic insecticides to achieve
immediate results in the control of mosquitoes, an
alarming increase of physiological resistance in
the vectors, its increased toxicity to non-target
organism and high costs are noteworthy (WHO,
1975). Most of synthetic chemicals are expensive
and destructive to the environment and also toxic
to humans, animals and other non-target
organisms. Besides, they are nonselective and
harmful to other benecial organisms. Some of the
insecticides act as carcinogenic agents and are
even carried through food chain which in turn
affects the non-target organism. Therefore
alternative vector control strategies, especially
effective and low cost are extremely imperative
(Piyarat et al., 1974; Kalyanasundaram and Das,
1985).
The use of different parts of locally available
plants and their various products in the control of
mosquitoes has been well established globally by
numerous researchers. The larvicidal and ovicidal
properties of indigenous plants have also been
documented in Tamilnadu, India (Baranitharan
and Dhanasekaran, 2014). Based on their
traditional experience for hundreds of years, these
plants are being subjected to research this results
would be utilized to replace the classical drugs.
Thus depended upon the ethanobotanical
information Vitex negundo was selected to screen
for their larvicidal potential against three different,
public health significant mosquito vectors. V.
negundo is a large aromatic shrub up to 4-5 meters
in height is found throughout the greater part of

67

India (Sharma, 2001). In homeopathy, it is being

used for the treatment of snake bite (Alam and
Gomes, 2003). It is used for its anti inflammatory
and analgesic activity (Dharmasiri, 2003). The
plant is also known to posses liver protective
effects (Avadhoot and Rana, 1991).
2. Methodology
2.1. Sample collection
Fresh, mature seeds of are collected from
the Yercaudu of Nilgiris hills in India. Plant and
seeds are properly authenticated in the Department
of Botany, Annamalai University. Fresh, mature
seeds are rinsed with distilled water and dried in a
shed before powered.
2.2. Extraction method
The dried powder of seeds was subjected
for oil extraction with hexane, methanol and ethyl
acetate using Soxhlet apparatus. After collecting
the crude oil, it was then allowed to condense in
Rotary Vacuum Evaporator. The condensed crude
oil extract was stored in refrigerator until required
for investigation for larvicidal, ovicidal and
repellent activity.
2.3. Larvicidal activity
Standard WHO protocol with slight
modifications was adopted for the study (WHO
1996). From the stock solution, the concentration
of 50, 100, 150, 200 and 250 was prepared. Early
third instar larvae were introduced in 250 ml
plastic cups containing 200 ml of water with each
concentration. A control was prepared by the
addition of acetone to water. Mortality was
recorded after 24 hours. For each experiment, four
replicates were maintained at a time. The observed
percentage mortality was corrected by Abbotts
Formula (Abbott, 1925).
2.4. Ovicidal activity
Evaluation of the V. negundo extracts for
ovicidal activity was carried out by following the
method of Su and Mulla (1998). Eggs were
exposed to different concentrations ranging from
70 to 350 ppm. The desired concentrations of the
test solutions were achieved by adding 1.0 ml of
an appropriate stock solution to 99 ml of tap
water. Each eggs raft containing 100 eggs of Ae.

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Mathalaimuthu Baranitharan /Life Science Archives (LSA), Volume 1, Issue 1, Page 66 to 71, 2015

aegypti, are exposed to each dose of extract for

48hr. counting of eggs was done under a
microscope. DSMO served as control. Four
replicates for each concentration were maintained.
After 24 hrs of incubation, the egg rafts or eggs
exposed to each concentration were transferred to
distilled water cups. The hatch rates were
calculated by the following formula.
Mortality at treatment-Mortality at control
% Mortality =
100-Mortality at control
3.1. Statistical analysis
The analysis program probit (Finney,
1971) was used for the determination of LC50,
LC90and other statistics at regression, chi-square,
slope, mean and standard deviation values were
calculated using the SPSS 12.0 software.

68

The toxicity of different solvent crude

extract of V. negundo was tested against larvae of
Ae. aegypti (Table - 1) and V. negundo extract
reported in the present study showed the larvicidal
and ovicidal activities in the plant signifying their
use in mosquito population control (Table - 2).
The data were recorded and statistical data
regarding LC50, LC90, LCL, UCL and chi-square
values were calculated. The LC50 and LC90 values
of hexane, methanol and ethyl acetate extract of V.
negundo against early third instar larvae of Ae.
aegypti
100were 189.70, 139.70 and 145.29 ppm and
284.09, 223.57 and 227.86 ppm respectively.
Maximum larvicidal activity was observed in the
methanol extract followed by ethyl acetate and
hexane extract. No mortality was observed in
control. The percentage of egg hatchability of Ae.
aegypti with the leaf extract of V. negundo was
presented in Table - 3. The methanol extracts
exerted 100% mortality (zero hatchability) at 240
and 300 ppm.

3. Results
Table - 1: Larvicidal activity of different solvent leaf extracts V. negundo against instar larvae of
Ae. aegypti
Species

Solvent

Ae. Aegypti

Hexane
Methanol
Ethyl acetate

50
4.20.44
12.21.48
10.41.67

% mortalitySD
100
150
200
10.41.14 26.82.16 56.22.48
26.41.81 50.22.04 78.81.78
22.42.30 48.62.60 76.82.28

250
80.42.50
99.80.44
98.81.78

Significant at P0.05 level, SD- Standard Deviation, PPM- Parts Per Million.
Table - 2: Probit analysis of larvicidal activity of V. negundo extract against Ae. aegypti
Species

Solvent

LC50 (LCL-UCL) ppm

LC90 (LCL-UCL) ppm

Slope

x2

Ae. aegypti

Hexane
Methanol
Ethyl acetate

189.70 (179.90-200.50)
139.70 (113.57-165.20)
145.29 (136.62-153.97)

284.09 (266.01-308.16)
223.57 (192.30-286.42)
227.86 (214.58-244.73)

3.598886
4.991848
4.542066

1.099*
8.430*
6.721*

LC50= Lethal concentration that kills 50% of the exposed parasite, LC 90= Lethal concentration that kills 90%
of the exposed parasite. LCL- Lower Confident Limit, UCL- Upper Confident Limit.

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Mathalaimuthu Baranitharan /Life Science Archives (LSA), Volume 1, Issue 1, Page 66 to 71, 2015

69

Table - 3: Ovicidal activity of the V. negundo extract against Ae. aegypti

Solvents
Hexane
Methanol
Ethyl acetate

Control
100.00.0
100.00.0
100.00.0

Percentage of egg hatch ability

Concentration (ppm)
60
120
180
240
96.81.30 82.42.60 62.22.86 44.62.30
NH
64.62.50 36.21.78
9.81.48
81.43.28 60.62.60 39.23.19 17.42.19

300
27.42.50
NH
NH

NH-No hatch ability

4. Discussion
Recently, bio-pesticides with plant origins
are given for use against several insect species,
especially disease transmitting vectors, based on
the fact compounds of plant origin are safer to use,
without phototoxic properties and leave no scum
in the environment (Schmutterer 1990). Ruskin
(1992) has reported that in mosquitoes, the
compounds extracted from Azadirachta indica
showed mortality for fourth instar larvae of An.
stephensi with LC50 values of 60 and 43 ppm. The
LC50 and LC90 values for C. caudata ethyl acetate
extract were 94.76 and 103.10 mg/L for Cx.
quinquefasciatus. Rajkumar and Jebanesan (2005)
reported that the leaf extract of Solanum
trilobatum reduced egg laying by gravid females
of An. stephensi from 18% to 99% compared with
ethanol treatment controls at 0.01%, 0.025%,
0.05%, 0.075 and 0.1%. The bioactive compound
azadirachtin (A. indica) showed complete ovicidal
activity in the eggs of Cx. Tarsalis and Cx.
quinquefasciatus exposed to 10 ppm concentration
(Su and Mulla, 1998). Broadbent and Pree (1984)
reported that when eggs were directly exposed to
high concentrations of the compounds, more
chemicals entered the egg shell, which affected the
embryogenesis; similarly, longer exposure periods
also facilitated the increased penetration of the
compounds into the shells, thus increasing their
effectiveness. The larvicidal and ovicidal efficacy
of different solvent leaf extract of A. indica
against An. stephensi. The hatch rates were
assessed 48 h after treatment. The LC50 and LC90
values of acetone, benzene, chloroform, hexane
and methanol extracts of A. indica against An.
stephensi larvae in 24 h were 76.29, 58.82, 53.59,
65.84, 51.78 and 205.85, 193.23, 185.16, 196.72
and 181.00 ppm, respectively (Gokulakrishnan et

al., 2012). The results that the LC50 and LC90

values of ethyl acetate followed by hexane,
chloroform and acetone of A. adenophora against
Cx. quinquefasciatus larvae I-instar in 24 h were
136.75, 145.69, 139.49 and 143.64 mg/L; 149.07,
158.24, 151.95 and 156.14 mg/L, respectively
(Baranitharan and Dhanasekaran, 2014). Elumalai
et al., (2012) reported that the E. roseum acetone
and methanol extracts of LC50 values of 121.65
and 139.86 ppm, it was that 100% mortality was
noted from the acetone and methanol extracts of
100 ppm.
5. Conclusion
This result clearly reveals that the leaf
extract of V. negundo potential larvicidal and
ovicidal agents against the dengue vector A.
aegypti and they have demonstrated a synergist act
too. Therefore, the present strategy should be
promoted in the dengue vector control program.
The larvicidal and ovicidal properties of the V.
negundo extract under the eld conditions should
be scrutinized and determined. Besides, further
investigation regarding the effect on non-target
organism is extremely important and imperative in
the near future.
Acknowledgements
Authors are thankful to the Professor &
Head, Department of Zoology and Botany,
Poompuhar College, Melaiyur.
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