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Chemical composition and larvicidal activity of Elaeagnus indica Servett.

(Elaeagnaceae) plant leaf extracts against dengue and malaria vectors

Article in International Journal of Mosquito Research · December 2014

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 International Journal of Mosquito Research 2014; 1 (4): 66-71

ISSN: 2348-5906 Chemical composition and larvicidal activity of

CODEN: IJMRK2
IJMR 2014; 1 (4): 66-71 Elaeagnus indica Servett. (Elaeagnaceae) plant
© 2014 IJMR
Received: 14-07-2014 leaf extracts against dengue and malaria vectors
Accepted: 16-08-2014

Ramalingam Srinivasan Ramalingam Srinivasan, Devarajan Natarajan, Sengodan Karthi,

Natural Drug Research Muthugounder S Shivakumar
Laboratory, Department of
Biotechnology, Periyar University,
Abstract
Salem- 636 011, Tamil Nadu,
India.
Mosquito control is facing a threat due to the emergence of resistance to synthetic insecticides.
Insecticides of botanical origin may serve as suitable alternative biocontrol techniques in the future. The
Devarajan Natarajan acetone, chloroform, ethyl acetate, hexane, methanol and petroleum benzene leaf extracts of E. indica
Natural Drug Research were tested against fourth instar larvae of malaria vector, Anopheles stephensi and dengue vector, Aedes
Laboratory, Department of aegypti. The larval mortality was observed after 24 h of exposure. Highest larval mortality was found in
Biotechnology, Periyar University, acetone leaf extracts against A. aegypti (LC50 and LC90 values of 2.97027 and 5.9820 mg/ml) and A.
Salem- 636 011, Tamil Nadu, stephensi (LC50 and LC 90 values of 3.92501 and 68.3250 mg/ml) respectively. GC-MS analysis of plant
India extracts of acetone solvent revealed 19 compounds, of which the major compounds were -Thujone 1-
Isopropyl-4-Methylbicyclo(3.1.0)Hexan-3-One 1- (6.71%), 1,6- Cyclodecadiene, 1-Methyl-5-Methylene-
Sengodan Karthi 8-(1-Methylethyl)-, [S-(E,E)]-Germacra-1(10),4(15),5-Trie N (3.11%), L-(+)-Ascorbic Acid 2,6-
Molecular Entomology Lab, Dihexadecanoate (4.06%), 2-Cyclohexylcyclohexanol [1,1'-Bicyclohexyl]-2-Ol (3.16%), Dotriacontane
Department of Biotechnology,
N- Bicetyl (58.7%) and Tetrapentacontane (3.85%). E. indica offers promise as potential biocontrol agent
Periyar University, Salem- 636
against major dengue and malaria mosquitoes particularly in larvicidal effect. Our results shows acetone
011, Tamil Nadu, India
leaf extracts of E. indica have the potential to be used as an ideal eco-friendly approach for control of
Muthugounder S Shivakumar mosquito vectors.
Molecular Entomology Lab,
Department of Biotechnology, Keywords: Aedes aegypti; Anopheles stephensi; GC-MS analysis; Elaeagnus indica; Larvicides.
Periyar University, Salem- 636
011, Tamil Nadu, India 1. Introduction
Mosquito-borne diseases have an economic impact, including loss in commercial and labour
outputs, particularly in countries with tropical and subtropical climates. However, no part of
the world is free from vector-borne diseases [1]. Mosquitoes transmit a variety of diseases, such
as yellow fever, dengue fever, malaria, several forms of encephalitis and filariasis. Aedes
aegypti is generally known as a vector for an arbovirus responsible for dengue fever, which is
endemic to Southeast Asia, the Pacific island area, Africa and the Americas. This mosquito is
also the vector of yellow fever in Central and South America and West Africa. 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. Dengue is prevalent in more than 100 countries and threatens the health of
approximately 2.5 billion people. Around 80 million people are infected annually at an attack
rate of 4 % worldwide [2]. Anopheles stephensi Liston is the primary vector of malaria in India
and other West Asian countries. Malaria remains one of the most prevalent diseases in the
tropical world, with 200 million to 450 million infections annually worldwide, it causes up to
2.7 million deaths. In India, malaria is one of the most important causes of direct or indirect
infant, child and adult mortality. About 2 million confirmed malaria cases and 1,000 deaths are
reported annually, although 15 million cases and 20,000 deaths are estimated by WHO South
East Asia Regional Office. India contributes 77 % of the total malaria in Southeast Asia [3].
For Correspondence:
Muthugounder S Shivakumar The use of conventional pesticides in the water sources for mosquito control introduces many
Molecular Entomology Lab, risks to people and the environment. Natural products of plant origin with insecticidal
Department of Biotechnology, properties have been tried in the recent past control of variety of insect pests and vectors.
Periyar University, Salem- 636 Mosquitoes in the larval stage are attractive targets for pesticides because mosquitoes breed in
011, Tamil Nadu, India
water, and thus, it is easy to deal with them in this habitat. Many researchers have reported on
~ 66 ~
International Journal of Mosquito Research

the effectiveness of plant extract against mosquito larvae [4]. In Elaeagnus indica against mosquito vectors of Aedes aegypti
recent years, many studies on plant extracts against mosquito and Anopheles stephensi were analyzed.
larvae have been conducted around the world. The crude
hexane extract obtained from the leaf of Leucas aspera was 2. Materials and methods
tested for the larvicidal activity against Culex quinquefasciatus 2.1 Plants collection and solvent extraction
and Aedes aegypti [5]. The leaf methanol, benzene and acetone The leaves of Elaeagnus indica (Fig. 1) were collected from
extracts of Cassia fistula were studied for the larvicidal, Kolli Hills of the Eastern Ghats of India and rich in
ovicidal and repellent activities against Aedes aegypti [6]. The biodiversity and indigenous populations. It is located in the
leaf extract of Acalypha indica with different solvents, viz., Namakkal district of the South East of Tamil Nadu, India and
benzene, chloroform, ethyl acetate and methanol, were tested lies between (10°12' - 11°7'N, 76° - 77°56'E and Altitude 1300
for larvicidal, ovicidal activity and oviposition attractancy meters) and the initial taxonomic identification was made by
against Anopheles stephensi [7]. The leaf extract of Citrullus Dr. D. Natarajan, Department of Biotechnology, Periyar
vulgaris with different solvents viz. benzene, petroleum ether, University, Tamil Nadu, India and further confirmed by
ethyl acetate and methanol were tested for larvicidal, ovicidal, Botanical Survey of India, Southern Circle, Coimbatore (Ref.
repellent and insect growth regulatory activities against No. BSI/SRC/5/23/2013-14/Tech./1942). The voucher
Anopheles stephensi [8]. Although effective, repeated use of specimen was numbered and kept in our research laboratory
synthetic insecticides in agriculture and public health programs for further reference.
has caused multifarious problems, including insecticide
resistance, environmental pollution, destabilization of the
ecosystem and toxic hazards to human and non-target
organisms [9]. An obvious method for the control of mosquito-
borne diseases is the use of insecticides, and many synthetic
agents have been developed and employed in the field with
considerable success. It has also provoked undesirable effects,
including toxicity to non-target organisms, and fostered
environmental and human health concerns [10]. The
environmental toxicity, together with the growing incidence of
insect resistance, has called attention to the need for novel
insecticides [11] and for more detailed studies of naturally
occurring insecticides [12]. These problems have necessitated
the need for search and development of alternative strategies
using eco-friendly, environmentally safe, biodegradable and
low cost natural products such as mosquito larvicides.
Larvicides are applied to relatively small areas where larvae
are concentrated. Therefore, the quantity and number of
Fig 1: Elaeagnus indica
applications of natural products needed to control mosquito
population is reduced [13]. Extracts or essential oils from plants
2.2. Preparation of plant extracts
may be alternative sources of mosquito larval control agents,
The leaves were dried for 7–10 days in the shade at the
since they constitute a rich source of bioactive compounds that
environmental temperatures (27–37 ºC day time). The dried
are biodegradable into non-toxic products and are potentially
leaves were powdered mechanically using commercial
suitable for use in control of mosquito larvae. In fact, many
electrical stainless steel blender and the powdered leaves (700
researchers have reported on the effectiveness of plant extracts
g) were extracted with chloroform, ethyl acetate, acetone,
or essential oils against mosquito larvae [14].
petroleum benzene and methanol in a soxhlet apparatus
Phytochemicals derived from plant sources can act as
(boiling point range 60–80 ºC) for 8 h. The extract was
larvicides, insect growth regulators, repellents, and oviposition
concentrated under reduced pressure 22–26 mmHg at 45 ºC
attractants and can play an important role in the interruption of
the transmission of mosquito-borne diseases at the individual and the residue obtained was stored at 4 ºC. One gram of crude
as well as at the community level [15]. Natural products of plant extract was first dissolved in 100ml of acetone (stock
solution).
origin with insecticidal properties have been tried in the recent
past for control of variety of insect pests and vectors.
2.3. GC–MS analysis and identification of compounds
Elaeagnus indica Servett. (Elaeagnaceae) is a large, branched,
The plant extract of E. indica was analysed by gas liquid
woody climbing shrub, densely covered with silvery scales
chromatography (Polaris Q Ion Trap GC/FID) and mass
usually spread an over trees. The leaves are variable, broadly
spectrometry (Perkin Elmer Q-700 equipment). We followed
elliptic or elliptic-lanceolate, obtuse or acuminate apex, upper
the method [18] which used only one type of column (preferably
surface silvery white. Flowers usually many in cluster and its
perianth covered with silvery scales. The fruits are nearly 2 cm Polaris Q Ion Trap GC/FID). The column temperature
programme was 35 °C for 2 min, increased to 180 °C at 4
long. The plant is found in the high elevated region of the hills
°C/min, then increased to 280 °C at 20 °C/min. Helium was
from India (>1000M above MSL). Tribal communities of
used as the carrier gas at a 0.9 ml/min. The mass spectrum was
Eastern Ghats of Tamilnadu use the fruits as edible and
obtained at a 70 eV ionization voltage. The identification of
ornamental purpose. Recently, the plant has reported as
individual compounds was done using the Wiley/NBS
contain many biological properties namely anticancer,
Registry of mass spectral database, the NIST (version 3.0)
antibacterial and larvicidal agents [16, 17].
database. Furthermore, the Retention Time (RT) and Kovats
In this study larvicidal activity of the plant extracts of
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International Journal of Mosquito Research

Index (KI) values of several authentic reference compounds replicates. The LC50 (lethal concentration that kills 50 % of the
were compared with isolated compounds for identification. exposed larvae) and LC90 (lethal concentration that kills 90 %
of the exposed larvae) values were calculated after 24 h by
2.4. Mosquito’s culture probit analysis [20].
For the present study A. aegypti and A. stephensi mosquito
larvae were obtained from National Centre for Disease Control 2.6. Statistical analysis
(NCDC), Southern India branch field station located at The average larval mortality data were subjected to probit
Mettupalayam (Coimbatore District, Tamil Nadu, India), analysis for calculating LC50, LC 90 and other statistics at 95%
which have been successfully maintaining laboratory colonies fiducial limits of upper confidence limits (UCL) and lower
of various mosquito vector species, of public health confidence limits (LCL), and chi-square values were
importance. The larvae were kept in plastic trays containing calculated by [20] method. SPSS version 9.0 was used.
tap water, and were maintained in laboratory and all the
experiments were carried out at 27±2 °C, 70–80% relative 3. Results and discussion
humidity and 14:10 light and dark photo period cycle. Larvae The larvicidal activity was done using the plant extracts of E.
were fed with dog biscuit and yeast powder in the ratio of 3:1. indica against important mosquito vectors viz. A. aegypti and
They were maintained and reared in the laboratory. A. stephensi. The toxicity of five solvent crude extracts was
evaluated for the potential larvicidal activity. The acetone,
2.5. Larvicidal bioassay chloroform, ethyl acetate, methanol and petroleum benzene
The larvicidal activity of the plant crude extracts was extracts of E. indica were tested against the fourth instar larvae
evaluated as per the method recommended by World Health of A. aegypti and A. stephensi. Highest larval mortality was
Organization [19]. 25 fourth instar larvae were transferred to a observed in the acetone extract (Table 1) of E. indica in A.
small disposable paper cups, in 249 ml of water and 1.0 ml of aegypti (LC50 and LC90 values of 2.97027and 5.9820 mg/ml).
the desired plant extract concentration, an equal number of The highest toxicity was achieved with acetone extract where
controls were set up simultaneously using tap water. Starting the respective LC50 and LC90 values were 3.92501and 68.3250
with the lowest concentration (0.1 to 2 mg/ml). The numbers mg/ml for A. stephensi (Table 2).
of dead larvae were counted after 24 h of exposure, and the
percentage mortality was reported from the average of three

Table 1: Larvicidal activity of Elaeagnus indica leaf extracts against fourth instar larvae of A. aegypti

LC50 LCL-UCL LC 90 LCL-UCL

Sps Sample na (95% confidence limit) (95% confidence limit) χ2 df
mg/ml mg/ml
2.97027 5.9820
Acetone 375 0.11065 3
(1.81160-4.67086) (3.81160-7.8203)
7.32820 15.80016 0.5605
Methanol 375 3
(5.26180-13.2860) (13.2682-18.201)
7.82501 11.32501 0.2036
Chloroform 375 3
(5.6250-9.8260) (9.9963-13.0031)
Aedes aegypti 5.823201 72.8260 0.0015
Ethyl acetate 375 3
(4.826501-6.2261) (54.0034)
6.76320 13.2816 0.3065
Petroleum benzine 375 3
(5.93250-8.11063) (11.7280-15.00160)
significant at the P<0.05 level
LC50 : lethal concentration that kills 50% of the exposed larvae, LC90: lethal concentration that kills 90% of the exposed larvae, UCL: upper
confidence limit (95% fiducial limit), LCL: lower confidence limit (95% fiducial limit), χ2 : chi-square, df: degrees of freedom.

Table 2: Larvicidal activity of Elaeagnus indica leaf extracts against fourth instar larvae of A. stephensi

LC50 LCL-UCL LC90 LCL-UCL

Sps Sample na (95% confidence limit) (95% confidence limit) χ2 df
mg/ml mg/ml
3.92501 68.3250
Acetone 375 0.0165 3
(2.01682-5.0132) (59.8201)
5.62501 13.1620
Methanol 375 0.1560 3
(4.98260-7.3250) (7.9820-18.8260)
7.82601 28.3250
Chloroform 375 0.2005 3
(5.8260-8.9620) (23.0627-31.8260)
Anopheles stephensi 13.2860 42.8250
Ethyl acetate 375 0.5650 3
(11.32682-16.1601) (0.8620)
11.4270 38.1103
Petroleum benzine 375 0.5632 3
(9.9382-13.0032) (1.32016)
significant at the P<0.05 level
LC50 : lethal concentration that kills 50% of the exposed larvae, LC90: lethal concentration that kills 90% of the exposed larvae, UCL: upper
confidence limit (95% fiducial limit), LCL: lower confidence limit (95% fiducial limit), χ2 : chi-square, df: degrees of freedom.
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International Journal of Mosquito Research

The environment is an incomparable reservoir of natural Cymbopogon citrus and Ageratum conyzoides against Aedes
products, many of which exhibit structural features that have aegypti and achieved 100% mortality at 120, 200, and 300
not been found in terrestrial natural products [21]. Each year, ppm concentrations, respectively. The plant L. aspera leaf
an increasing number of novel plant metabolites are reported extracts were reported that larvicidal activity of different
in the literature, indicating that the environment is likely to solvents against A. aegypti larvae [27].
continue to be prolific sources of more natural products for Several studies have shown that sesquiterpenoid and
many years to come. The screening of medicinal plants for benzenoid compounds possess significant larvicidal activities,
mosquito larvicidal activity may eventually lead to their use in and that a range of essential oils exhibit bioactive properties
natural product-based mosquito abatement practices. In against the larvae of A. aegypti [28]. Plant based compounds
contrast, in the present study, acetone leaf extracts of E. indica can be used in mosquito larval control ensuring maximum
against A. aegypti and A. stephensi larvae showed high safety in any ecological condition. Furthermore, studies have
mortality compared with other extracts. Similarly, Susitra et al. shown that plant extracts are readily biodegradable [29] and are
(2014) reported that C. dentata plant acetone leaf extracts relatively non-toxic to other organisms.
showed high larvicidal activity and identified from different GC-MS analysis results of acetone solvent extracts of E.
compounds, which shows an insecticidal activity in three indica (Fig.2) showed 22 peaks which indicating the presence
important mosquito vectors [22]. of 22 phytochemical constituents. On the comparison of the
Bhagavan et al. (2008) reported that chloroform extract of mass spectra of the constituents with the NIST library, the 19
Citrus sinensis, ethyl acetate extracts of Ocimum canum and compounds were characterized and identified (Table 3). The
Ocimum sanctum and chloroform extract of Rhinacanthus major chemical compounds were identified based on the 6
nasutus against the larvae of Anopheles subpictus (LC50 58.25, highest peaks are -Thujone 1-Isopropyl-4-
88.15, 21.67 and 40.46 ppm; LC90 298.31, 528.70, 98.34 and Methylbicyclo(3.1.0)Hexan-3-One 1- (6.71%), 1,6-
267.20 ppm) respectively [23]. High larval mortality observed Cyclodecadiene, 1-Methyl-5-Methylene-8-(1-Methylethyl)-,
in methanol extracts Cryptocoryne auriculata and Solanum [S-(E,E)]-Germacra-1(10),4(15),5-Trie N (3.11%), L-(+)-
torvum against the larvae of A. subpictus [24]. The varying Ascorbic Acid 2,6-Dihexadecanoate (4.06%), 2-
results obtained in lethal concentration and lethal time are Cyclohexylcyclohexanol [1,1'-Bicyclohexyl]-2-Ol (3.16%),
probably due to the differences in levels of toxicity among the Dotriacontane N- Bicetyl (58.7%) and Tetrapentacontane
insecticidal ingredients of each plant [25]. [26] reported that (3.85%).
larvicidal activities of essential oils of Ocimum gratissium,

Fig 2: Chemical compounds identified in acetone extracts of Elaeagnus indica

The isolation of compounds from leaf extracts of E. indica, as lansium has an effect on the central nervous system in mice,
described here, could lead to the development of natural i.e. antinociceptive, thus it can be considered as a neuroleptic
mosquitocidal products to replace the synthetic insecticides. [30]. Both α- and β-santalol have also reported to affect some

The use of natural plant based products by individuals and tumors. Phellandrene, which is the main constituent of the seed
communities would generate local employment, reduce oil of C. lansium, can be used as an effective molecule to kill
dependence on expensive imported synthetic products and insects [31]. Berenbaum (1985) reported that the crude
stimulate local efforts to enhance public health. Recent studies medicinal plant extracts produce rich bioactive chemical
have indicated that β-santalol, which is the main constituent of compounds [32]. They are complex mixtures of chemicals and
the essential oils from leaves, flowers and sarcocarps of C. may constitute effective alternatives to synthetic insecticides.
~ 69 ~
International Journal of Mosquito Research

Our study also provides useful information for potential use of Tetrapentacontane. Results obtained from the larvicidal tests
E. indica leaf extracts against insecticidal activity. using leaf extracts from E. indica had excellent larvicidal
In conclusion, According to GC–MS analyses the major activity against A. stephensi and A. aegypti mosquito vectors.
constituents of the leaf extracts were -Thujone 1-Isopropyl-4- Our findings suggest that the leaf extracts from E. indica and
Methylbicyclo(3.1.0)Hexan-3-One 1-, 1,6-Cyclodecadiene, 1- its bioactive constituents may be explored as potential and
Methyl-5-Methylene-8-(1-Methylethyl)-, [S-(E,E)]-Germacra- environmentally benign larvicides for mosquito control. These
1(10),4(15),5-Trie N, L-(+)-Ascorbic Acid 2,6- results are useful in search of more selective, biodegradable
Dihexadecanoate, 2-Cyclohexylcyclohexanol [1,1'- and naturally produced for mosquito larval control.
Bicyclohexyl]-2-Ol , Dotriacontane N- Bicetyl and

Table 3: Chemical composition acetone leaf extract from E. indica

S.
RT Area % Compound Name Activity
No
3-Penten-2-One, 4 Methyl- 4-Methylpent-3-en-2-One (ch3)2c=chc(=o)ch3 1-
1 4.036 0.80 Insect repellent activity
methylpent-2-en-4-on
2-Pentanone, 4-Hydroxy-4-Methyl- (Ch3)2c(Oh)Ch2c(O)Ch3 2-Hydroxy-2- Insecticidal and Antioxidant
2 4.744 0.75
Methyl-4-Pentanone 2 activity
3 9.262 6.71 -Thujone 1-Isopropyl-4-Methylbicyclo(3.1.0)Hexan-3-One 1- Insecticidal Activity
Bicyclo [3.1.0] hexan-3-one, 4-methyl-1-(1-methylethyl)-, [1s-(1.alpha. 4. Alpha., Antioxidant and
4 9.430 1.26
5.alpha.)]- 4-methyl1 Antimicrobial activity
5 15.579 1.93 Tricyclo[5.4.0.0(2,8)]undec-9-ene, 2,6,6,9-tetramethyl Insecticidal Activity
1,6-cyclodecadiene, 1-methyl-5-methylene-8-(1-methylethyl)-, [s-(e,e)]-germacra- Antimicrobial and
6 15.745 3.11
1(10),4(15),5-trie N Insecticidal property
Insecticidal and Antioxidant
7 17.010 1.39 4-(3-Hydroxyprop-1-Enyl)-2-Methoxyphenol
activity
8 18.125 0.58 Methyl 3-O-Benzyl-Alpha-D-Glucofuranoside 5,6-Carbonate Insecticidal activity
9 18.706 0.60 2,3-Dimethylpentan-3-Ol Insecticidal activity
10 19.158 1.50 4-((1e)-3-Hydroxy-1-Propenyl)-2-Methoxyphenol None
11 19.534 2.36 D-Fructose, 3-O-Methyl- None
12 19.592 2.93 1-Butanamine, N-Methyl-N-Nitroso- 1-Butyl-1-Methyl-2-Oxohydrazine 1- --
Antibacterial and
13 21.027 4.06 L-(+)-Ascorbic Acid 2,6-Dihexadecanoate
antioxidant activity
14 21.119 0.59 Pyributicarb ; O 3-Tert-Butylphenyl 6-Methoxy 2-Pridyl (Methyl) Thiocarbamate Insecticidal activity
Propanoic acid, 3,3'-thiobis-, didodecyl ester 3, 3'-thiobispropionic acid,
15 21.951 0.40 None
didodecyl ester 3,3'-t H
16 22.383 3.16 2-Cyclohexylcyclohexanol [1,1'-Bicyclohexyl]-2-Ol Insecticidal activity
17 22.538 2.31 16-Heptadecenal Insecticidal activity
1h-purine-2,6-dione, 3,7-dihydro-1,3-dimethyl-7-[2-[(1-methyl-2-
18 26.717 0.93 None
phenylethyl)amino]ethyl]- fenethylline 7-[2-[(.alpha.-methyl- P
19 27.207 3.85 Tetrapentacontane None

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