GI C AL
LO S
Proc Zool Soc (July-Sept 2020) 73(3):227–234
https://doi.org/10.1007/s12595-020-00323-9
RESEARCH ARTICLE
Larvicidal Activity of Neolamarckia cadamba Against
the Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus
Sofi Imtiyaz Ali1 • V. Venkatesalu1
Received: 21 November 2018 / Revised: 27 January 2020 / Accepted: 13 February 2020 / Published online: 21 February 2020
Ó Zoological Society, Kolkata, India 2020
Abstract The excessive use of conventional insecticides to
Culicidae leads to the emergence of mosquitoe resistant,
adverse environmental and human effects. Therefore, the
present study was carried out to explore the larvicidal
efficacy of petroleum ether, chloroform, ethyl acetate and
methanol extracts of roots, leaves, and bark of Neolamar-
ckia cadamba against the early 4th instar larvae of
Anopheles stephensi, Aedes aegypti and Culex quinque-
fasciatus at concentrations ranging from 12.5 to 200 ppm
under laboratory conditions. The root extracts showed the
highest larvicidal potential than the leaf and bark extracts.
After 24 h of exposure period, the highest larvicidal
activities were observed in the methanol extract of roots
with LC50 value of 43.29 and LC90 value of 202.85 ppm
against An. stephensi; LC50 value of 70.82 and LC90 value
of 253.73 ppm against Ae. Aegypti and LC50 value of 99.51
and LC90 value of 311.02 ppm against the Cx. Quinque-
fasciatus, respectively. Thus, the plant extracts of N.
cadamba showed considerable mosquito larvicidal activi-
ties and can be considered for further investigation.
Keywords Medicinal plants
Anopheles stephensi
Aedes
aegypti
Culex quinquefasciatus
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s12595-020-00323-9) contains sup-
plementary material, which is available to authorized users.
& V. Venkatesalu
venkatesalu@yahoo.com
1
Department of Botany, Annamalai University,
Annamalainagar, Tamil Nadu 608 002, India
O
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TH E Z
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Introduction
Mosquitoes are the most important agents in the trans-
mission of several diseases such as dengue fever, malaria,
filariasis, chikungunya, yellow fever and certain types of
encephalitis such as West Nile Fever which compromise
the human health (WHO 2010; Aziz et al. 2014). There are
approximately 3500 species of mosquitoes that belong to
Culicidae family, among which find the genera Anopheles,
Aedes and Culex act as vectors of important illness
worldwide (Benelli et al. 2016; Benelli and Mehlhorn
2016; Moulin et al. 2016; Mayer et al. 2017). Mosquito-
borne diseases are endemic in over 100 countries, causing
mortality to nearly two million people and at least one
million children each year, leaving approximately 2100
million people at risk all over the world (Klempner et al.
2007; WHO 2014). Anopheles stephensi is the most
important vector of malaria fever in the urban districts of
India and other West Asian countries. Malaria afflicts 36%
of the world people i.e. 2020 million in 107 countries and
territories situated in the tropical and subtropical regions
(Panneerselvam et al. 2013). The viruses of dengue, zika
and chikungunya fever are examples of pathogens trans-
mitted by female mosquitoes of the genus Aedes, occurring
mainly in tropical and subtropical urban regions of the
world. Approximately, 2500 million people are now at the
risk of dengue fever i.e., two-fifth of the world’s population
and there may be 50 million cases of dengue fever infection
worldwide every year (Moulin et al. 2016). Lymphatic
filariasis, is caused by the parasitic filarial nematodes
(roundworms—Family Filariodidea) Wuchereria bancrofti
(90% infections), Brugia malayi (9% infections) or Brugia
timori (1% infections) and the vector is Culex quinque-
fasciatus. There are approximately 120 million prevalent
infections with the filarial worms that cause lymphatic
123
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filariasis, most of which are W. bancrofti (Gyapong et al.
2010). They have, therefore, become a challenging prob-
lem for public health worldwide and have a severe com-
munal and economic impact mainly in tropical and
subtropical countries (Bossche and Coetzer 2008).
As there is no specific drug available for the treatment of
these diseases, vector control is the best strategy (WHO
2010). To solve this problem, one of the obvious methods
used to control these mosquito-borne diseases are the
insecticides and many industrialized synthetic agents
which are active in the fields with fair success (Nayak and
Mohan 2015). However, in recent past, need for developing
new, effective and environmentally friendly alternative
products for mosquito control has increased due to the
development of mosquito resistance to these conventional
insecticides and the hazardous effects of these insecticides
on public health and the environment worldwide (Benli
et al. 2009; Santos et al. 2011). Therefore, significant
efforts have been focused on finding an alternative form of
mosquito control, as insecticide resistance intimidates
towards the reintroduction of vector-borne diseases in
many parts of the world. Plants may be alternative sources
of mosquito larvicidal agents because they constitute a rich
source of bioactive compounds, which are effective, envi-
ronment-friendly, biodegradable and target-specific insect
control agents (Hoel et al. 2010; Cantrell et al. 2011;
Benelli 2015).
Neolamarckia cadamba is a member of the tribe Neo-
lamarckia in the family Rubiaceae and is distributed widely
in South Asia. It is a large, deciduous tree found all over
India (Chopra et al. 1956). N. cadamba is commonly
known as wild cinchona (English), kadamba (Hindi,
Gujarati, and Marathi), kadam (Bengali) and kadawala
(Kannada). In traditional and folk medicine systems, N.
cadamba is used for the treatment of fever, anemia, tumor,
skin diseases, uterine complaints, dysentery, leprosy,
syphilis, as an analgesic and for improvement of semen
quality (Umachigi et al. 2007; Alam et al. 2008; Acharyya
et al. 2010). The major phytoconstituents reported in the
plant are triterpenes, triterpenoid glycosides, saponins,
flavonoids, indole alkaloids, 3a-dihydro-cadambine, cada-
mine, isocadamine and isodihydrocadambine (Khare 2008;
Kumar et al. 2015; Jeyalalitha et al. 2015). Moreover,
antioxidant, wound healing and antimicrobial activities
(Umachigi et al. 2007; Sanadhya and Durve 2014), anti-
diarrheal (Alam et al. 2008), analgesic (Ambujakshi et al.
2009), anti-inflammatory (Kodangula et al. 2010), antidi-
abetic (Acharyya et al. 2010; Sanadhya and Durve 2014),
antifungal (Patel et al. 2011) and antioxidant (Islam et al.
2015) are the various pharmacological studies reported for
different parts of N. cadamba.
Against this background, the objective of this study was
to investigate whether the leaf, bark and root extracts of N.
123
Proc Zool Soc (July-Sept 2020) 73(3):227–234
cadamba elicit toxicity against early 4th instar larvae of
An. stephensi, Ae. aegypti and Cx. quinquefasciatus. To the
best of our knowledge, the present study provides the first
combined report on the larvicidal potential of different
parts of N. cadamba.
Materials and Methods
Plant Materials
The different parts viz., roots, leaves, and bark of N.
cadamba were collected from Institute of Forest Genetics
and Tree Breeding (IFGTB), Coimbatore, India (11.0168°
N, 76.9558° E) during August 2016 and the voucher
specimen (AUBOT#350) is deposited at the herbarium,
Department of Botany, Annamalai University. The col-
lected plant samples were instantly taken to the laboratory
in polythene bags and were washed with water to remove
all the unwanted impurities. The plant materials were then
surface sterilized in 10% sodium hypochlorite to prevent
contamination of any microbes and finally, they were
thoroughly rinsed with sterile distilled water. Then, the
plant materials were shade dried under room temperature
followed by oven drying at 60 °C for half an hour. The
oven dried plant materials were ground using an electric
blender and the powdered materials were packed in air-
lock zip pouches.
Preparation of Extracts
Five hundred gram of plant material was packed inside the
Soxhlet apparatus and successive extraction was carried
out using various solvent systems viz., petroleum ether,
chloroform, ethyl acetate and methanol for 72 h. The sol-
vent was evaporated under vacuum in a rotary evaporator
(Heidolph, Germany) and the crude extracts were stored at
4°C until further assay.
Mosquito Larvicidal Bioassay
The mosquitoes samples i.e., eggs of An. stephensi, Ae.
aegypti and the egg rafts of Cx. quinquefasciatus were
procured from Center for Research in Medical Entomology
(ICMR-Government of India), Madurai and reared in the
laboratory at a temperature of 29 ± 3 °C and relative
humidity of 75 to 85% by feeding with Brewer’s yeast/dog
biscuit (1:3). The larvae at the early 4th instar stage were
used for larvicidal bioassay. The standard procedures rec-
ommended by the WHO (2005) were used for exploring
the larvicidal potential of roots, leaves and bark extracts of
N. cadamba against the collected mosquitoes species. The
different concentrations of plant extracts viz., 12.5, 25, 50,
Proc Zool Soc (July-Sept 2020) 73(3):227–234
100 and 200 ppm were prepared with distilled water after
dissolving the plant extracts with 1 mL of DMSO. During
the assay, five replicates were maintained for each con-
centration and twenty larvae (in a 100-mL beaker) of the
early 4th instar stage for each concentration were used.
Also, no food was offered to the larvae during the exper-
iment and the larval mortality was calculated 24 h of the
exposure periods and all moribund mosquito larvae were
considered as dead. The larval mortality was also checked
for water and DMSO individually.
Statistical Analysis
The probit analysis (SPSS, version 21.0) was used for
calculating the lethal concentrations, LC50 and LC90 and
their 95% confidence limit of upper and lower confidence
levels (Finney 1971).
Results
Larvicidal Activity of Root, Leaf and Bark Extracts
of N. cadamba Against the An. stephensi, Ae. aegypti
and Cx. quinquefasciatus
The larvicidal efficacy of petroleum ether, chloroform,
ethyl acetate and methanol extracts of roots, leaves, and
bark of N. cadamba were explored against the early 4th
instar larvae of An. stephensi, Ae. aegypti and Cx. quin-
quefasciatus (Tables 1, 2). All the extracts tested in the
present study showed varying levels of larvicidal activities
against the three mosquitoe species after 24 h of exposure
period. However, no mortality was observed in the control
group and none of the larvae pupated throughout the assay.
The methanol extract of roots of N. cadamba showed more
toxicity against An. stephensi, Ae. aegypti and Cx. quin-
quefasciatus, when compared to leaf, bark, and other root
extracts tested in the present study. After 24 h of an
exposure period, the methanol extract showed LC50 values
of 43.29, 70.82, 99.51 ppm and LC90 value of 202.85,
253.73, 311.02 ppm against the An. stephensi, Ae. aegypti
and Cx. quinquefasciatus, respectively. The larvicidal
activity of methanol extract was followed by petroleum
ether and chloroform extracts. The ethyl acetate extract of
N. cadamba was found less potent after 24 h exposure
period (Tables 1, 2).
The petroleum ether extract of N. cadamba leaves
showed the highest larvicidal potential when compared to
other leaf extracts with LC50 values of 61.36, 82.66,
124.75 ppm and LC90 values of 262.12, 277.44,
368.88 ppm against the An. stephensi, Ae. aegypti and Cx.
quinquefasciatus, respectively. The larvicidal activity of
petroleum ether extract was followed by methanol,
229
chloroform and ethyl acetate extracts of N. cadamba after
24 h of exposure period (Tables 1, 2).The different extracts
of bark of N. cadamba also showed significant toxicity
against tested mosquito species. After 24 h exposure per-
iod, the highest larvicidal activity was recorded in metha-
nol extract with the LC50 value of 100.16 and LC90 value
of 326.35 ppm for An. stephensi; the LC50 value of 139.61
and LC90 value of 374.87 ppm for Ae. aegypti and LC50
value of 156.83 and LC90 value of 367.25 ppm for Cx.
quinquefasciatus. The larvicidal activity of methanol
extract was followed by petroleum ether, chloroform and
ethyl acetate extracts of barks against An. stephensi, Ae.
aegypti and Cx. quinquefasciatus after 24 h of exposure
period (Tables 1, 2).
Discussion
Larviciding is a fruitful way of diminishing mosquitoes
population in their breeding places before they came out
from the larval stage and emerge into adults. Larviciding
primarily depends on the use of synthetic insecticides.
Although they are effective, their frequent use has upset
natural biological control systems and resulted in the
extensive development of mosquitoes resistance (Kaushal
et al. 2011; Norris and Norris 2011; Addiss 2013). Various
studies on natural plant products against mosquito vectors
suggest the plant extracts as possible and potential alter-
native agents to combat the threat caused by vector born
diseases, due to a wide range of phytochemicals available
on plants which can act simultaneously on both behavioral
and physiological processes of mosquitoes. Moreover, due
to the natural synergism that constrains the development of
mosquito resistance, the plant crude extracts may be more
adequate than individual bioactive compounds (Maurya
et al. 2007). However, bioactivity of plant extracts against
mosquito larvae can vary notably depending on plant
species and part used, geographical origin of the plant,
solvent used in extraction, chemical nature, mode of action,
delivery methods, mosquito species and their develop-
mental stages against the particular extract (Shaalan et al.
2005; Ghosh et al. 2012).
The results of the present study are comparable with the
previous studies published with the larvicidal activity of
methanol extracts. Govindarajan and Sivakumar (2014)
assayed the toxicity of various extracts viz., hexane, ethyl
acetate, benzene, chloroform and methanol extracts of
roots of Asparagus racemosus against An. stephensi, Ae.
aegypti and Cx. quinquefasciatus. The methanol extract of
root of A. racemosus recorded the highest toxicity against
Cx. quinquefasciatus, Ae. aegypti and An. stephensi with
the LC50 and LC90 values of 115.13, 97.71 and 90.97 ppm
and 210.96, 179.92, and 168.82 ppm, respectively.
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 Table 1 A comparison of the larvicidal efficacies of different concentrations of root, leaf and bark extracts of Neolamarckia cadamba against three different species of mosquitoes
230

Plant parts Mosquito species Extract % Mortality ± SE at different concentrations after 12 h of exposure % Mortality ± SE at different concentrations after 24 h exposure
period period

123
200 100 50 25 12.5 200 100 50 25 12.5

Root An. stephensi Methanol 72 ± 0.50 58 ± 0.24 42 ± 0.37 30 ± 0.31 20 ± 0.00 88 ± 0.24 70 ± 0.31 55 ± 0.31 47 ± 0.24 34 ± 0.20
Ethyl acetate 45 ± 0.44 33 ± 0.24 24 ± 0.20 13 ± 0.24 6 ± 0.20 60 ± 0.47 44 ± 0.20 30 ± 0.00 18 ± 0.24 8 ± 0.40
Chloroform 50 ± 0.00 37 ± 0.24 25 ± 0.44 15 ± 0.31 9 ± 0.40 66 ± 0.20 45 ± 0.31 34 ± 0.37 25 ± 0.54 12 ± 0.24
Petroleum ether 68 ± 0.24 50 ± 0.63 35 ± 0.31 23 ± 0.50 14 ± 0.37 80 ± 0.00 65 ± 0.00 48 ± 0.24 37 ± 0.24 25 ± 0.00
Ae. aegypti Methanol 64 ± 0.37 51 ± 0.37 38 ± 0.50 24 ± 0.37 14 ± 0.37 79 ± 0.37 62 ± 0.24 50 ± 0.31 38 ± 0.24 27 ± 0.24
Ethyl acetate 39 ± 0.20 29 ± 0.50 16 ± 0.37 10 ± 0.44 4 ± 0.20 55 ± 0.31 38 ± 0.24 23 ± 0.24 15 ± 0.00 7 ± 0.24
Chloroform 45 ± 0.31 35 ± 0.54 20 ± 0.54 12 ± 0.24 6 ± 0.20 59 ± 0.20 43 ± 0.24 30 ± 0.00 18 ± 0.24 10 ± 0.00
Petroleum ether 60 ± 0.31 45 ± 0.54 29 ± 0.73 19 ± 0.37 12 ± 0.50 74 ± 0.20 58 ± 0.24 39 ± 0.20 30 ± 0.31 20 ± 0.00
Cx. quinquefasciatus Methanol 56 ± 0.37 44 ± 0.20 34 ± 0.37 20 ± 0.63 12 ± 0.50 70 ± 0.31 54 ± 0.20 44 ± 0.20 33 ± 0.24 23 ± 0.24
Ethyl acetate 33 ± 0.24 24 ± 0.58 13 ± 0.24 9 ± 0.37 5 ± 0.44 41 ± 0.37 29 ± 0.20 20 ± 0.00 12 ± 0.24 6 ± 0.20
Chloroform 39 ± 0.20 29 ± 0.20 17 ± 0.24 10 ± 0.00 6 ± 0.20 59 ± 0.00 35 ± 0.48 25 ± 0.31 15 ± 0.00 7 ± 0.24
Petroleum ether 50 ± 0.31 39 ± 0.37 25 ± 0.00 15 ± 0.31 10 ± 0.00 66 ± 0.37 49 ± 0.20 34 ± 0.20 25 ± 0.31 18 ± 0.24
Leaf An. stephensi Methanol 60 ± 0.63 50 ± 0.00 39 ± 0.20 23 ± 0.24 15 ± 0.44 78 ± 0.40 65 ± 0.54 53 ± 0.24 39 ± 0.20 32 ± 0.24
Ethyl acetate 40 ± 0.63 28 ± 0.50 17 ± 0.40 10 ± 0.54 6 ± 0.31 57 ± 0.40 39 ± 0.20 28 ± 0.40 17 ± 0.24 7 ± 0.24
Chloroform 47 ± 0.24 35 ± 0.00 22 ± 0.24 13 ± 0.24 8 ± 0.24 58 ± 0.67 45 ± 0.48 32 ± 0.37 20 ± 0.37 10 ± 0.24
Petroleum ether 67 ± 0.54 55 ± 0.54 40 ± 0.63 28 ± 0.40 18 ± 0.40 82 ± 0.24 65 ± 0.00 50 ± 0.00 37 ± 0.24 26 ± 0.20
Ae. aegypti Methanol 54 ± 0.37 45 ± 0.31 35 ± 0.54 20 ± 0.44 11 ± 0.37 69 ± 0.37 58 ± 0.24 46 ± 0.20 35 ± 0.54 21 ± 0.40
Ethyl acetate 34 ± 0.20 24 ± 0.20 13 ± 0.24 8 ± 0.24 4 ± 0.20 52 ± 0.50 35 ± 0.31 25 ± 0.31 15 ± 0.00 6 ± 0.20
Chloroform 39 ± 0.20 30 ± 0.31 19 ± 0.37 9 ± 0.20 5 ± 0.31 53 ± 0.40 40 ± 0.31 31 ± 0.37 17 ± 0.24 8 ± 0.24
Petroleum ether 61 ± 0.37 47 ± 0.24 38 ± 0.24 25 ± 0.31 13 ± 0.24 74 ± 0.37 60 ± 0.54 50 ± 0.00 36 ± 0.37 22 ± 0.40
Cx. quinquefasciatus Methanol 44 ± 0.37 36 ± 0.37 23 ± 0.58 13 ± 0.24 9 ± 0.37 62 ± 0.24 44 ± 0.20 36 ± 0.37 24 ± 0.20 13 ± 0.24
Ethyl acetate 26 ± 0.37 19 ± 0.37 9 ± ±0.37 5 ± 0.31 2 ± 0.24 44 ± 0.20 31 ± 0.37 20 ± 0.31 12 ± 0.24 5 ± 0.31
Chloroform 29 ± 0.20 19 ± 0.20 12 ± 0.24 6 ± 0.20 4 ± 0.20 45 ± 0.31 30 ± 0.54 20 ± 0.54 13 ± 0.24 7 ± 0.40
Petroleum ether 52 ± 0.50 37 ± 0.40 23 ± 0.40 13 ± 0.24 9 ± 0.20 62 ± 0.24 50 ± 0.54 38 ± 0.24 35 ± 0.54 18 ± 0.40
An. stephensi Methanol 52 ± 0.24 41 ± 0.20 30 ± 0.00 18 ± 0.24 10 ± 0.54 68 ± 0.40 55 ± 0.44 45 ± 0.54 33 ± 0.24 24 ± 0.20
Ethyl acetate 35 ± 0.89 22 ± 0.24 15 ± 0.31 8 ± 0.24 4 ± 0.20 49 ± 0.20 36 ± 0.00 25 ± 0.00 15 ± 0.00 6 ± 0.20
Chloroform 45 ± 1.00 31 ± 0.58 19 ± 0.37 10 ± 0.44 7 ± 0.40 50 ± 0.00 40 ± 0.00 28 ± 0.24 16 ± 0.40 8 ± 0.24
Petroleum ether 49 ± 0.37 35 ± 0.70 25 ± 0.83 13 ± 0.40 8 ± 0.50 60 ± 0.00 48 ± 0.24 37 ± 0.24 23 ± 0.20 17 ± 0.24
Ae. aegypti Methanol 46 ± 0.20 37 ± 0.24 25 ± 0.00 16 ± 0.20 7 ± 0.24 59 ± 0.54 47 ± 0.40 38 ± 0.50 26 ± 0.37 17 ± 0.24
Ethyl acetate 26 ± 0.37 16 ± 0.40 10 ± 0.54 5 ± 0.31 3 ± 0.24 42 ± 0.24 27 ± 0.24 18 ± 0.24 10 ± 0.00 5 ± 0.00
Bark Chloroform 40 ± 0.00 30 ± 0.37 15 ± 0.54 9 ± 0.58 4 ± 0.37 50 ± 0.00 35 ± 0.00 20 ± 0.00 10 ± 0.00 7 ± 0.24
Petroleum ether 42 ± 0.24 29 ± 0.20 18 ± 0.24 10 ± 0.00 6 ± 0.20 55 ± 0.77 37 ± 0.40 27 ± 0.58 19 ± 0.40 12 ± 0.24
Cx. quinquefasciatus Methanol 41 ± 0.37 31 ± 0.37 16 ± 0.20 9 ± 0.00 5 ± 0.00 56 ± 0.37 44 ± 0.20 30 ± 0.31 20 ± 0.00 13 ± 0.24
Ethyl acetate 24 ± 0.20 13 ± 0.24 7 ± 0.24 4 ± 0.20 2 ± 0.24 34 ± 0.20 25 ± 0.00 16 ± 0.20 9 ± 0.20 4 ± 0.00
Chloroform 35 ± 0.54 19 ± 0.37 12 ± 0.37 7 ± 0.40 3 ± 0.40 44 ± 0.58 29 ± 0.48 18 ± 0.24 8 ± 0.24 6 ± 0.20
Petroleum ether 34 ± 0.20 22 ± 0.24 14 ± 0.20 8 ± 0.24 5 ± 0.00 48 ± 0.24 34 ± 0.37 22 ± 0.24 14 ± 0.20 9 ± 0.20
Proc Zool Soc (July-Sept 2020) 73(3):227–234
 Table 2 LC50 and LC90 values of root, leaf and bark extracts of Neolamarckia cadamba against the Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus
Plant parts Mosquito species Extract After 12 h After 24 h
2 b
LC50 (ppm) LC90 (ppm) x (df = 3) LC50 (ppm) LC90 (ppm) x2 (df = 2)b
(LCL–UCL)a (LCL–UCL)a (LCL–UCL)a (LCL–UCL)a

Root An. stephensi Methanol 98.13 281.10 7.16 43.29 202.85 2.81
(54.72–168.24) (196.27–622.88) (25.87–57.92) (173.01–249.88)
Ethyl acetate 200.02 400.29 14.21 147.84 322.81 10.66
(123.71–6023.64) (239.62–16,983.10) (96.95–336.54) (215.94–957.83)
Chloroform 183.23 389.85 6.66 130.48 311.07 6.86
(128.45–397.55) (261.77–1024.78) (89.56–224.91) (219.52–655.85)
Petroleum ether 121.92 294.19 6.66 71.59 240.26 6.16
(83.73–198.30) (211.35–576.17) (29.14–113.82) (173.72–455.97)
Ae. aegypti Methanol 125.81 318.18 10.38 70.82 253.73 4.77
Proc Zool Soc (July-Sept 2020) 73(3):227–234

(72.76–312.37) (207.28–1138.71) (53.83–87.58) (214.16–318.37)

Ethyl acetate 226.75 432.04 7.82 168.27 344.96 6.84
(154.99–677.65) (279.90–1556.39) (122.21–300.02) (244.13–724.11)
Chloroform 200.01 400.31 8.89 150.93 335.19 8.09
(135.42–589.43) (259.39–1469.14) (103.55–299.92) (229.53–829.34)
Petroleum ether 146.90 332.64 6.38 97.53 269.76 5.34
(104.43–255.90) (234.98–695.89) (63.23–146.63) (198.85–476.76)
Cx. quinquefasciatus Methanol 154.83 367.99 9.78 99.51 311.02 4.72
(96.59–521.10) (232.50–1741.77) (80.73–122.15) (257.06–405.14)
Ethyl acetate 264.13 496.34 4.18 223.89 446.17 6.05
(216.49–355.36) (392.83–703.65) (155.63–566.13) (293.59–1335.32)
Chloroform 230.22 447.69 5.83 161.35 328.35 5.47
(161.56–554.40) (297.51–1260.99) (122.26–249.57) (243.11–576.60)
Petroleum ether 181.20 390.64 7.08 124.87 317.88 3.44
(125.21–419.63) (259.86–1113.52) (106.21–149.80) (266.90–402.46)
Leaf An. stephensi Methanol 134.47 348.04 11.43 61.36 262.12 4.75
(72.90–608.78) (214.64–2566.14) (41.65–79.28) (218.01–337.73)
Ethyl acetate 227.24 439.80 4.83 160.52 340.97 9.20
(191.00–289.87) (357.73–590.43) (109.49–349.13) (230.29–930.62)
Chloroform 194.98 401.03 6.50 149.76 344.10 10.50
(137.57–426.31) (270.56–1048.10) (94.60–419.71) (222.48–1323.33)
Petroleum ether 111.88 308.87 8.37 67.85 230.90 5.37
(62.58–228.60) (206.37–882.95) (29.78–103.95) (170.87–402.17)
Ae. aegypti Methanol 158.41 379.24 13.10 95.37 309.65 9.54
(89.54–8624.42) (224.39–33,985.17) (26.56–242.34) (196.69–1406.54)
Ethyl acetate 253.69 468.74 5.11 178.36 367.05 8.45
(211.11–330.95) (377.36–642.59) (123.40–406.66) (246.33–1030.96)
231

123
 Table 2 continued
232

Plant parts Mosquito species Extract After 12 h After 24 h

2 b
LC50 (ppm) LC90 (ppm) x (df = 3) LC50 (ppm) LC90 (ppm) x2 (df = 2)b

123
(LCL–UCL)a (LCL–UCL)a (LCL–UCL)a (LCL–UCL)a

Chloroform 226.40 439.56 9.17 168.53 372.09 11.98

(149.10–1027.54) (273.93–2495.92) (104.60–835.37) (230.47–2620.25)
Petroleum ether 135.95 344.26 10.43 82.66 277.44 9.92
(78.79–439.82) (217.57–1670.15) (11.64–177.86) (180.50–1003.64
Cx. quinquefasciatus Methanol 205.35 432.45 7.87 138.52 339.22 7.64
(136.32–724.67) (272.76–1925.06) (90.78–288.80) (228.02–920.11)
Ethyl acetate 291.85 509.54 6.07 207.77 409.85 7.54
(196.89–1049.38) (323.88–2121.92) (144.03–526.90) (270.78–1253.85)
Chloroform 287.04 518.91 3.50 208.16 417.69 4.31
(233.19–394.25) (407.25–749.91) (176.13–261.50) (342.25–552.79)
Petroleum ether 177.57 366.76 5.72 124.75 368.88 7.99
(130.40–313.26) (260.04–752.58) (66.25–406.33) (229.78–1937.94)
An. stephensi Methanol 171.21 387.17 9.48 100.16 326.35 5.48
(109.88–613.33) (243.82–1878.57) (53.65–180.98) (221.97–804.55)
Ethyl acetate 252.75 468.62 4.52 185.96 386.70 9.94
(210.28–329.76) (377.18–642.58) (122.85–614.04) (247.13–1652.93)
Chloroform 205.00 402.04 5.22 178.25 387.91 11.23
(175.09–253.18) (333.19–521.41) (112.33–910.08) (239.60–2745.42)
Petroleum ether 187.79 390.24 7.51 137.58 356.31 7.18
(130.07–443.52) (259.74–1127.75) (87.81–309.71) (234.95–1079.45)
Ae. aegypti Methanol 195.78 417.68 10.19 139.61 374.87 7.03
(124.62–1170.78) (255.19–3359.45) (86.98–356.54) (241.57–1336.39)
Ethyl acetate 303.77 533.48 2.96 219.04 419.99 5.25
(245.27–423.56) (416.27–781.65) (185.98–274.00) (345.71–551.62)
Bark Chloroform 220.18 414.64 8.33 184.64 362.97 6.79
(151.70–616.54) (271.67–1401.15) (134.61–341.40) (255.38–784.43)
Petroleum ether 217.64 422.47 5.30 169.27 377.98 3.24
(184.37–273.32) (346.77–557.71) (143.96–208.61) (312.04–493.13)
Cx. quinquefasciatus Methanol 216.53 413.02 7.91 156.87 367.25 6.58
(150.05–570.54) (272.35–1308.00) (107.75–324.62) (248.04–950.39)
Ethyl acetate 308.24 520.17 1.90 255.89 484.96 6.76
(251.03–422.95) (409.93–748.49) (171.24–948.37) (305.39–2140.60)
Chloroform 250.92 447.69 3.03 208.71 397.97 5.71
(211.63–319.08) (366.17–596.03) (153.22–392.53) (279.25–859.14)
Petroleum ether 260.23 484.72 3.21 194.05 402.92 4.45
(214.70–345.34) (386.54–676.82) (164.69–241.77) (331.30–529.70)

LCL lower confidence level, UCL upper confidence level

a
95% confidence interval
b
Proc Zool Soc (July-Sept 2020) 73(3):227–234

Degrees of freedom
Proc Zool Soc (July-Sept 2020) 73(3):227–234
Similarly, the methanol extract of aerial parts of Nepeta
menthoides showed more larvicidal activity than essential
oil with an LC50 value of 69.5 and LC90 value of 175.5,
while essential oil showed LC50 and LC90 values of 234.3
and 419.9 ppm against An. stephensi (Mahnaz et al. 2012).
The methanol leaf extract of Acalypha alnifolia was found
more toxic against early 4th instar larvae of An. stephensi
(LC50 = 125.73 and LC90 = 395.50 ppm), Ae. aegypti
(LC50 = 128.55 and LC90 = 381.67 ppm) and Cx. quin-
quefasciatus (LC50 = 127.98 and LC90 = 386.26 ppm),
then the other extracts tested after 24 h of the exposure
period (Kovendan et al. 2012). Similarly, the methanol
extract of leaves of N. cadamba showed remarkable lar-
vicidal potential against Cx. quinquefasciatus and the LC50
and LC90 values were 31.29 and 102.13 respectively
(Thirupathi et al. 2016).
The pronounced lethal toxicity was recorded in the leaf
extract of Gnetum ula against An. stephensi (LC50-
= 82.86 ppm), followed by Spermacoce hispida (LC50-
= 89.45 ppm). Similarly, Solena amplexicaulis showed
the LC50 value of 109.37 ppm against the larvae of Cx.
tritaeniorhynchus, followed by N. cadamba against An.
stephensi larvae (LC50 = 109.87 ppm) (Dhanasekaran
et al. 2013). The petroleum ether extract of leaves of Tridax
procumbens, Lantana camara and Datura stramonium
showed 100% mortality with least LC50 value of 219 lg/
mL in T. procumbens, followed by L. camara (251 lg/mL)
and D. stramonium (288 lg/mL) against Ae. aegypti (Ra-
jasekaran and Duraikannan 2012). Similarly, methanol
bark extract of Chloroxylon swietenia was found to be
more effective against An. stephensi, Ae. aegypti and Cx.
quinquefasciatus than that of aqueous bark extract. The
LC50 and the LC90 values of the methanol extract were
found to be 124.70 and 226.26 ppm (Ae. aegypti), 130.57
and 234.67 ppm (Cx. quinquefasciatus) and 137.55 and
246.09 ppm (An. stephensi) (Jayaprasad et al. 2015).
Conclusion
The data obtained in the present study have provided
valuable information on the larvicidal potential of different
extracts of N. cadamba against the early 4th instar larvae of
An. stephensi, Ae. aegypti and Cx. quinquefasciatus. The
findings of the present study suggested that all the tested
extracts showed varying levels of larvicidal activities
against the three mosquitoe species after 24 h of exposure
period. However, the highest larvicidal activities were
observed in the methanol extract of roots against the An.
stephensi. An assessment of the bioactivity of the single
Constituent of methanol extract of roots of N. cadamba on
Culicidae vectors is in progress.
233
Acknowledgements We are thankful to The Director, Centre for
Research in Medical Entomology (ICMR-Government of India),
Madurai for the supply of mosquito eggs/rafts.
Compliance with Ethical Standards
Conflict of interest We declare that we have no conflict of interest.
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