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Life Science Archives (LSA)


ISSN: 2454-1354
Volume 1; Issue - 1; Year 2015; Page: 46 - 52

Research Article
MOSQUITO LARVICIDAL PROPERTIES OF Ocimum sanctum Linn. (Lamiaceae)
AGAINST Aedes aegypti (Linn.), Anopheles stephensi (Liston),
Culex quinquefasciatus (Say)
J. Gokulakrishnana*, M. Baranitharanb, S. Dhanasekaranb, R. Kavikuyila, R. Abiramia, J. Deepaa,
Bakuselvakumara
a

Department of Zoology, Annamalai University, Annamalai nagar - 608 002, Tamil Nadu, India.
Department of Zoology, Poompuhar College (Autonomous), Melaiyur - 609 107, Tamil Nadu, India.

Abstract
The use of botanicals as an alternative to the chemical compounds is gaining tremendous momentum
because of its multifarious advantages. In view of its increasing interest, an attempt was made in the present
study to assess the larvicidal potential of important plant like Ocimum sanctum against three mosquito
species. The third instar larvae were exposed to different concentrations (i.e. 50, 100, 150, 200 and 250 ppm)
of methanol, ethyl acetate and hexane extracts of O. sanctum plant. The mortality was recorded after 24 hrs
exposure and LC50 and LC90 were determined. The present investigation revealed that the LC 50 and LC90
values methanol, ethyl acetate and hexane extracts of O. sanctum against Culex quinquefasciatus larvae were
101.32, 112.18 and 120.87 mg/L; 182.32, 193.96 and 202.61 mg/L, respectively. The results clearly show
that larvicidal activity was dose reliant. The highest larvicidal activity against Culex quinquefasciatus was
obtained with methanol extract of O. sanctum.
Article History:
Received : 05.02.2015
Revised : 18.02.2015
Accepted : 23.02.2015

Keywords: Ocimum sanctum, Aedes aegypti,


Anopheles stephensi, Culex quinquefasciatus,
Larvicidal and Ovicidal

1. Introduction

Mosquitoes represent a significant threat


to human health because of their ability to vector
pathogens that cause diseases that afflict millions
of people worldwide (WHO, 2010). Several
species belonging to genera Aedes, Anopheles and
Culex are vectors for the pathogen of various
diseases like dengue fever, dengue hemorrhagic
fever, malaria, Japanese encephalitis and filariasis
(Borah et al., 2010; Rahuman et al., 2009;
Samuel, 2010). Ae. aegypti is known to carry
* Corresponding author: J. Gokulakrishnan
Tel.: +91-9443985831
E-mail: gokulagalya@gmail.com

dengue and yellow fever; malaria is carried by An.


stephensi; and larial disease by Cx.
quinquefasciatus. The dengue fever incidence has
increased fourfold since 1970 and nearly half the
worlds population is now at risk. In 1990, almost
30% of the world population, 1.5 billion people,
lived in regions where the estimated risk of
dengue transmission was greater than 50% (Hales
et al., 2002). An outbreak of Chikungunya virus
disease emerged in the southwest Indian Ocean
islands in 2005, spread out to India, and resulted
in an ongoing outbreak that has involved 1.5
million patients, including travelers who have
visited these areas (Taubitz et al., 2007). An.

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J. Gokulakrishnan /Life Science Archives (LSA), Volume 1, Issue 1, Page 46 to 52, 2015

stephensi are major malaria vectors in India. With


an annual incidence of 300-500 million, malaria is
still one of the most important communicable
diseases. Currently, about 40% of the worlds
population live in areas where malaria is endemic
(Werndorfer, 2003). Culex quinquefasciatus, a
vector of lymphatic filariasis, is widely distributed
in tropical zones with around 120 million people
infected worldwide and 44 million people having
common chronic manifestation (Bernhard et al.,
2003). The present proliferation of these diseases
is not only due to higher number of breeding
places in urban agglomeration, but also due to
increasing resistance of mosquitoes to current
commercial insecticides such asorganochlorides,
organophosphates, carbamates and also to
biological insecticides (Goettel et al., 1992; Das
and Amalraj, 1997; Yadav et al., 1997).
The native people have exploited a variety
of herbal medicines for effective curing of various
diseases. The plants used and preparation and
administration of drugs varies from area to area.
Although the knowledge of herbal medicine is
gradually vanishing, some of the traditional
healers and aged tribals are still practicing plants
as a herbal medicine. Modernization has exposed
the human race to increased risk of bronchitis,
asthma, lung cancer and various skin diseases. The
faster pace of life and the need for rapid cure led
to the proliferation of synthetic drugs. However,
the use of synthetic drugs leads to the problems of
side effects, ill effects, and complications. This
has revived the herbal treatments for a large
number of diseases. In India, about 2,500 plant
species are used for medicinal purpose by
traditional healers (Aniwal et al., 2006; Bussmann
and Glenn, 2010; Chandel et al., 1996; Sankar et
al., 2007). Among them Holy Basil, Ocimum
sanctum has been well documented for its
therapeutic potential (Prakash and Gupta, 2005).
Tulsi is a fragrant bushy perennial growing up to
1.5 m in height with profusions of white blooms
and slightly purple tinted foliage. This herb has
been known from as the vedic period and is held
sacred by the Hindus and is often planted around
temples and used in rosaries. It is native to India,
reached Western Europe in the 16th century. In
several ancient system of medicine including

47

Ayrveda, Greek, Roman, Siddha and Unani, O.


sanctum has vast number of therapeutic
applications such as in cardiopathy, haemopathy,
leucoderma, asthma, bronchitis, catarrhal fever,
otalgia, hepatopathy, vomiting, lumbago, hiccups,
ophthalmia, gastropathy, genitourinary disorders,
ringworm, verminosis and skin diseases, etc. It is
commonly used in cough, cold, mild indigestion,
diminished appetite and malaise. The only side
effect reported is constipation (Subir Kumar Das
and Vasudevan, 2005). In this context, the purpose
of the present investigation is to explore the
larvicidal properties of O. sanctum leaf extract and
against Chikungunya vector, Ae. aegypti, An.
stephensi and Cx. quinquefaciatus under the
laboratory conditions.
2. Material and Methods
2.1. Collection and Identification of Plant
material
The medicinal plant, O. sanctum was
collected from Sirkazhi, Nagapattinam District, in
Tamil Nadu, India. Bulk samples were air-dried in
the shade. After drying, these were ground to fine
powder. At the time of collection, voucher
herbarium specimens were prepared and identified
with the help of Plant Taxonomist, Department of
Botany, Poompuhar College, Melaiyur.
2.2. Extraction method
The dried leaves (100 g) were powdered
mechanically using commercial stainless steel
Blender and extracted sequentially with methanol,
ethyl acetate and hexane (500 ml, Ranchem), in a
Soxhlet apparatus separately until exhaustion. The
extract was concentrated under reduced pressure
of 22 - 26 mm Hg at 45oC by Rotavapour and
the residue obtained was stored at 4oC in an amber
vial. Then, the vials were named and covered with
silver foil and transported to the laboratory. Until
use those vials were kept in cool and dark place at
4oC.
2.3. Larvicidal activity
The larvicidal activity of crude extract was
evaluated as per the protocol previously described
by WHO (2005). From the stock solution, six
different test concentrations (50, 100, 150, 200
and 250 mg/L) were prepared and tested against

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the freshly molted (0 6 hrs) III instar larvae of


Ae.
aegypti,
An.
stephensi
and
Cx.
quinquefaciatus. The test medium (500 ml plastic
cups) was prepared by adding 1 ml of appropriate
dilution of test concentrations and mixed with 249
ml of dechlorinated water to make up 250 ml of
test solution. The larvae were fed with dry yeast
powder on the water surface (50 mg/l). The
control (without plant extracts) experiments were
also run parallel with each replicate. For each
experiment, four replicates were maintained at a
time. A minimum of 25 larvae per concentration
was used for all the experiments. The larval
mortality was observed and recorded after 24 hrs
post-treatment. Per cent mortality was corrected
for control mortality using Abbotts formula
(Abbott, 1925).
2.4. Statistical analysis
The larval mortality data were subjected to
probit analysis (Finney, 1971) for calculating
LC50, LC90 and other statistics at 95% confidence
limits of upper confidence limit and lower
confidence limit values were calculated using the
SPSS software package 16.0 (2007). Results with
p 0.05 were considered to be statistically
significant.

48

3. Results
The result of the larvicidal activity of
crude methanol, ethyl acetate and hexane solvent
extracts of O. sanctum against Ae. aegypti, An.
stephensi and Cx. quinquefasciatus. The methanol
extract of O. sanctum reported in the present study
showed the larvicidal properties in the plant
signifying their use in mosquito population control
(Figure 1, 2 and 3). The data are presented in table
1.The methanol extracts of O. sanctum showed
larval mortality. Cx. quinquefasciatus was more
vulnerable followed by An. stephensi and Ae.
aegypti. The methanol extract of O. sanctum
exhibited the maximum larvicidal activity with
LC50and LC90 values of 101.32 and 182.32 mg/L
against the larvae of Cx. quinquefasciatus; The
LC50 and LC90 values of methanol, ethyl acetate
and hexane extract of O. sanctum against An.
stephensi were 115.32, 122.97 and 144.18 mg/L;
209.25, 212.37 and 249.41 mg/L, respectively.
The LC50 and LC90 values of hexane extract of O.
sanctum against Ae. aegypti were 134.73, 153.39
and 175.12 mg/L; 237.97, 265.67 and 290.07
mg/L, respectively (Table - 1).

Table - 1: Probit analysis of larvicidal activity of O. sanctum extracts against Ae. aegypti,
An. stephensi and Cx. quinquefaciatus
Species

Extract

Ae. Aegypti

Methanol
Ethyl acetate
Hexane
Methanol
Ethyl acetate
Hexane
Methanol
Ethyl acetate
Hexane

An. stephensi

Cx.
quinquefaciatus

LC50
(mg/L
)
134.73
153.39
175.12
115.32
122.97
144.18
101.32
112.18
120.87

95%Confidence
Limits
LCL UCL
124.23 144.85
142.53 164.37
164.03 187.12
104.91 124.92
113.30 132.14
133.72 154.47
91.57
110.21
102.85 120.92
111.82 129.51

LC90
(mg/L)
237.97
265.67
290.07
209.25
212.37
249.41
182.32
193.96
202.61

95%Confidence
Limits
LCL UCL
221.60 259.49
246.47 291.44
268.41 319.66
195.07 227.63
198.66 230.02
232.27 272.02
169.98 198.24
181.44 209.97
190.01 218.66

x2
4.165
0.489
0.087
5.761
6.233
1.283
4.317
3.660
3.631

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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49

4. Discussion

Fig - 1: Mortality of different solvent of S.


indicum against Ae. Aegypti

Fig - 2: Mortality of different solvent of S.


indicum against An. Stephensi

Fig - 3: Mortality of different solvent of S.


indicum against Cx. quinquefasciatus

The results of present study are comparable


with earlier reports Dhanasekaran et al., (2013)
have that the LC50 of ethanol crude extracts of
selected indigenous medicinal plants are G.ula
extract in the experimental larvae of An. stephensi
(LC50= 82.86ppm), followed by S. hispida (LC50=
89.45ppm). Baluselvakumar et al. (2012) reported
that theLC50 and LC90 values of methanol O.
esculentum leaf extract against An. stephensi were
63.84 and 122.48 ppm, respectively. Krishnappa et
al. (2012) reported that the LC50 and LC90 values of
methanol extract against An. stephensi were 78.18
and 155.42 mg/l, and A. digitata exerted 100% up
to 150 min at 4 and 6 mg/cm2. Cent percent
repellency of An. stephensi was noticed up to 180
min with chloroform extract at concentrations. But,
hexane extract showed 100% repellency up to
120min, whereas methanol extract showed strong
repellency up to 210 min except the minimal
concentration (2 mg/cm2). Baranitharan and
Dhanasekaran (2014) reported that the evident
larvicidal activity of ethyl acetate extract followed
by hexane, chloroform and acetone extracts of C.
caudata showed LC50 values of Ae. aegypti are
97.19, 112.85, 99.17 and 109.67 mg/L; An.
stephensi are 96.04, 104.16, 97.13 and 106.53
mg/L; Cx. quinquefasciatus are 94.76, 102.95,
95.98 and 105.09 mg/L, respectively. Krishnappa et
al. (2013) reported that the LC50 and LC90 values of
C. quadrangularis and C. ovalifolium methanol
extract against An. stephensi were 37.48 and 74.53
mg/L, respectively. Among two plant solvents
tested, C. quadrangularis extracts were found to be
most significant ovicidal activity 100% eggs
mortality observed at 50 ppm and 350 ppm for C.
quadrangularis. Gokulakrishnan et al. (2012)
reported that 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 hrs 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. The larvicidal activity of
different solvents extracts of C. sparciflrorus were
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tested against the three vector mosquitoes. Among


the different solvents the maximum larvicidal
activity was observed in ethyl acetate. The LC 50
and LC90 values of C. sparciflorus against three
vector mosquitoes of A. aegypti, An. stephensi and
Cx. quinquefasciatus were 34.02, 28.88 and 36.22
ppm, 74.57, 65.35 and 79.89 ppm, respectively
(Baranitharan et al., 2014).
Baluselvakumar et al. (2012) reported that
the methanol plant extract of M. maderaspatana
had ovicidal and repellency against Ae. aegypti
with the methanol extract of M. maderaspatana
exerted 100% egg mortality at 120, 160, 200 and
240 ppm for Ae. aegypti, and a higher
concentration of 3.0 mg/cm2 methanol extract of M.
maderaspatana provided 100% protection up to 80,
100,120 and 140 min. Baranitharan and
Dhanasekaran (2014) reported that the larvicidal
activity of diethyl ether followed by hexane,
benzene and acetone extracts of C. aromaticus
showed 73.49, 85.93. 76.03 and 80.56 mg/L,
respectively. 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. The leaf extract of
C. vulgaris with different solvents were tested for
repellent activities against An. stephensi and
showed that Skin repellent test at 1.0, 2.5 and 5.0
mg per cm2 concentration gave the mean complete
protection time ranged from 119.17 to 387.83
minutes with the four different extracts tested
(Mullai et al., 2008). Baranitharan and
Dhanasekaran (2014) 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.
These results could encourage the search for new
active natural compounds offering an alternative to
synthetic insecticides from other medicinal plants.
5. Conclusion
In general, it could be concluded that diethyl
ether extracts of O. sanctum used in the present
study act as larvicidal inhibiting against the
mosquito vectors, Ae. aegypti, An. stephensi and

50

Cx. quinquefaciatus. The high larvicidal activity


of methanol extract of O. sanctum and the
abundant availability of these plants in tropical
and subtropical countries may make them
economical for field use in mosquito control
programs.
Acknowledgements
Authors are thankful to the Professor &
Head, Department of Zoology and Botany,
Poompuhar College, Melaiyur.
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