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Larvicidal activity of selected plant extracts and their combination against the
mosquito vectors Culex quinquefasciatus and Aedes aegypti

Article  in  Environmental Science and Pollution Research · March 2018

DOI: 10.1007/s11356-018-1515-3

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Larvicidal activity of selected plant
extracts and their combination against the
mosquito vectors Culex quinquefasciatus
and Aedes aegypti

Irrusappan Hari & Nisha Mathew

Environmental Science and Pollution

Research

ISSN 0944-1344
Volume 25
Number 9

Environ Sci Pollut Res (2018)

25:9176-9185
DOI 10.1007/s11356-018-1515-3

1 23
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 Author's personal copy
Environmental Science and Pollution Research (2018) 25:9176–9185
https://doi.org/10.1007/s11356-018-1515-3

SHORT RESEARCH AND DISCUSSION ARTICLE

Larvicidal activity of selected plant extracts and their combination

against the mosquito vectors Culex quinquefasciatus
and Aedes aegypti
Irrusappan Hari 1 & Nisha Mathew 1

Received: 16 October 2017 / Accepted: 8 February 2018 / Published online: 19 February 2018
# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
In order to develop an environment-friendly botanical mosquito larvicide alternative to the chemical larvicides, extracts were
made from the leaves of Hyptis suaveolens, Lantana camara, Nerium oleander, and Tecoma stans with three organic solvents
such as methanol (ME), chloroform (CH), and petroleum ether (PE) using a Soxhlet extractor. The plant extracts were screened
for larvicidal activity individually and in combination against the larvae of Aedes aegypti and Culex quinquefasciatus as per
WHO protocol. Among the extracts, the maximum larvicidal activity was shown by the PE extract of L. camara (LC50 10.63 mg/
L) followed by the PE extract of T. stans (LC50 19.26 mg/L), ME extract of N. oleander (LC50 35.82 mg/L), and PE extract of
H. suaveolens (LC50 38.39 mg/L) against Cx. quinquefasciatus. In the case of Ae. aegypti, the PE extract of T. stans showed
maximum activity with LC50 value of 55.41 mg/L followed by H. suaveolens (LC50 64.49 mg/L), PE extract of L. camara (LC50
74.93 mg/L), and ME extract of N. oleander (LC50 84.09). A blend of these four extracts resulted in a combination with
corresponding LC50 values of 4.32 and 7.19 mg/L against Cx. quinquefasciatus and Ae. aegypti. The predator safety factors
were 12.55 and 20.88 for Gambusia affinis with respect to Aedes and Culex larvae for the extract combination. Chemical
constituents in extracts were also identified by FT-IR and GC-MS data. The present investigations suggest the possible use of
this blend of botanical extracts as an ideal ecofriendly, larvicide against Ae. aegypti and Cx. quinquefasciatus larvae.

Keywords Mosquito . Culex quinquefasciatus . Aedes aegypti . Larvicide . Plant extract . Non-target organism . Vector

Introduction Vector control is an indispensable part of the global strate-

The arboviral diseases dengue and chikungunya are transmit-
ted by the day biting mosquito Aedes aegypti. Culex
quinquefasiatus is the vector of lymphatic filariasis. Zika in-
fection is an emerging viral infection, and Ae. aegypti is con-
sidered to be the primary vector; however, there is indication
that other mosquito species, including Cx. quinquefasciatus,
transmit the virus (Benelli and Beier 2017; Benelli and
Romano 2017; Guedes et al. 2017; Benelli and Mehlhorn
2016).
Responsible editor: Philippe Garrigues
* Nisha Mathew
nishamathew@yahoo.com
1
ICMR-Vector Control Research Centre, Medical Complex, Indira
Nagar, Puducherry 605006, India
gy for managing mosquito-borne diseases, and insecticide ap-
plication is the most essential component in this. Larvicides
kill the mosquitoes in the juvenile stages before they emerge
in to haematophagous adults. Larvae are bound to their habi-
tats, and hence, the control operations are much easier with
larvicides. Larval control mainly relies on application of syn-
thetic chemical control agents. Although effective, repeated
use of chemical insecticides has resulted in resistant mosqui-
toes and environmental pollution (Liu 2015; Lawler et al.
2003, 2007) and the limited success of biocontrol programs
on Culicidae (Benelli 2015a) has spurred the need for search
for new insecticides (Liu 2015).
Plant products are coming up as a promising alternative for
synthetic chemical agents for insect management/control.
Phytochemicals belonging to different chemical classes such
as steroids, alkaloids, terpenes, and phenolic constituents were
investigated earlier for insect control potential and found
promising (Pavela 2015a; Pavela et al. 2017; Pavela and
 Author's personal copy
Environ Sci Pollut Res (2018) 25:9176–9185
Benelli 2016a; Nisha et al. 2009). The insect control ability
varies with age of the plant, species, part extracted, collection
site, and solvent used for extraction (Stevenson et al. 2017;
Isman 2015; Benelli 2015b). There is a need for an alternate
source with minimum risk to environment and human health,
and this study intends to identify potential plant-based mos-
quito larvicides against the mosquito vectors Ae. aegypti and
Cx. quinquefasciatus.
Several studies have shown the efficacy of essential oils
and different solvent extracts of plants as mosquito larvi-
cides, as repellents, and also as ovideterrents, without
causing harm to human health and the environment
(Lalthazuali and Nisha 2017; Benelli et al. 2017a; Pavela
and Benelli 2016a, Govindarajan et al. 2016a, b, c, d, e, f;
da Silva et al. 2016; Pavela 2015a). Plants for this study
were selected based on the earlier preliminary reports, the
local availability, and abundance. The extracts of Lantana
camara var. aculeata (L.) Moldenke (Verbenaceae) has
been reported for mosquito larvicidal, adulticidal, and re-
pellent properties (Pavela and Benelli 2016b; Sharma et al.
2016; Keziah et al. 2015; Kumar et al. 2012a; Dua et al.
2010). Various solvent extracts of leaves of Nerium
oleander syn. Nerium indicum Mill (Apocynaceae)
showed mosquitocidal activity (El-Sayed et al. 2016;
Roni et al. 2013; Kumar et al. 2012b; Lokesh et al. 2010;
Komalamisra et al. 2005). Mosquito larvicidal and repel-
lent properties of extracts and essential oil from Hyptis
suaveolens (L.) Poit (Lamiaceae) have been studied
(Yadav et al. 2015; Vongsombath et al. 2012; Kovendan
et al. 2012; Conti et al. 2012; Amusan et al. 2005). Only
limited reports on the insecticidal activity of extracts of
Tecoma stans (L.) Juss.ex Kunth (Bignoniaceae) against
mosquitoes. Navaneethan et al. (2016) studied the ovipo-
sitional deterrence and ovicidal properties of aqueous ex-
tracts of T. stans leaves against Ae. aegypti. The
mosquitocidal activity of the petroleum ether extracts of
T. stans flowers has been studied (Sakthivadivel and
Daniel 2008).
Earlier research (Pavela et al. 2015b; Benelli et al. 2017b)
had pointed out the potential of synergistic mixtures of essen-
tial oils and the components present in them in arriving at
plant-based mosquito larvicides. However, from the existing
knowledge, no study has been carried out to assess the effects
of the above mentioned four plant extracts in combination
against mosquito larvae. This prompted us to investigate the
combined effect of these effective plant extracts against the
larvae of Ae. aegypti and Cx. quinquefasciatus and analysis of
chemical constituents present in them. Hence, leaves of four
plants namely L. camara, N. oleander, H. suaveolens, and
T. stans were collected, and extractions were carried out with
an objective that the outcome of this research could provide a
perspective for the development of ecofriendly plant-based
alternatives for mosquito control.
9177
Materials and methods
Plant extraction
All the four selected plants viz., L. camara, N. oleander,
H. suaveolens, and T. stans growing in and around
Puducherry were collected, identified by a Botanist (Dr. N.
Ayyappan, Researcher, Department of Ecology, French
Institute of Pondicherry, Puducherry, India), and voucher
specimens were certified by the French Institute of
Pondicherry. All the plants after collection, leaves were sepa-
rated, cleaned from dust and other contaminant plants, and
shade dried at 27 + 2 °C. Completely dried parts were subject-
ed to grinding in a Remi mixie, and fine powder was obtained
by sieving.
Soxhlet extraction was carried out twice with 100 g fine
powder of each plant material which was put in a thimble
made up of Whatman cellulose chromatography paper grade
1 using three different solvents (750 mL) of varying polarity
viz., methanol (ME), chloroform (CH), and petroleum ether
(PE) (60–80 °C). All extracts were concentrated using rotary
vacuum evaporator.
Mosquito larvicidal screening
All the plant extract residues after weighing individually were
solubilized in ethanol/acetone to obtain a stock concentration
of 100,000 mg/L. This was further diluted in the same solvent
to get solutions corresponding to 5000, 10,000, 15,000,
20,000, 25,000, and 30,000 mg/L. Test concentrations of 50,
100, 150, 200, 250, and 300 mg/L (cutoff value for screening)
were made by transferring 1 mL of above respective concen-
trations to 99 mL of water kept in disposable cups. Larvicidal
activity was studied as per WHO method (WHO 2005).
Twenty-five early fourth instar or late third stage larvae of test
mosquito species were kept in four replicates for every test
concentration. The test species were Ae. aegypti and Cx.
quinquefasciatus. Control batches were set with 1 mL of
alcohol/acetone without the extract. Moquito larval diet was
supplied for extended duration of testing. Larval mortality was
investigated at 24 and 48 h, and percentage mortality was
calculated (Abbott 1925). Assay was performed at room tem-
perature (27 ± 2 °C) and 70–80% relative humidity and
14:10-h dark and light cycles. Bioassays were repeated three
times on different days with different batches of larvae each
time. After determining the mortality of larvae in this wide
range of concentrations, a narrower range of four to five con-
centrations, yielding between 10 and 95% was used to deter-
mine LC50 and LC90 values.
The combination was made from the best four extracts
selected from 12 extracts of four plants with three solvents
based on the LC50 values. Here, the residues from PE extracts
of L. camara, T. stans, and H. suaveolens and ME extract of
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9178
N. oleander were mixed in the ratio of 1:1:1:1 (w/w basis), and
the combination (blend) was dissolved in ethanol/acetone to
make the stock solution. The larvicidal activity of the combi-
nation was done at different dilutions ranging from 1 to 25 mg/
L as mentioned above to get the LC50 and LC90 values.
Screening against non-target organisms
The plant extract combination was screened against the non-
target organism Gambusia affinis the mosquito fish as per stan-
dard procedure to find out the suitability index. The predatory
mosquito fish was released in two numbers to 500 mL water
contained in four beakers. The fishes were exposed to concen-
trations of 50, 60, 70, 80, 90, 100, 110, and 120 mg/L of the
plant extract combination. Four replicates of control were also
kept without adding the extract combination. Mosquito larvae
were given as food for the fishes. The predators were observed
for mortality and other abnormalities such as sluggishness and
reduced swimming activity after 24 and 48-h exposure. LC50
and LC90 values were obtained by probit analysis. Suitability
index (SI) or predator safety factor (PSF) was calculated using
the following formula (Kumar et al. 2016):
SI or PSF = LC50 of non-target organism / LC50 of vector
species.
Data analysis Probit analysis was applied to get LC50 and
LC90 readings using SPSS statistical data analytical tool.
Chemical composition analysis
FT-IR analysis
The effective plant extracts were characterized by FT-IR
spectra (KBr) by a Shimadzu Fourier-transform infrared spec-
troscope model 8300.
GC/MS analysis The effective plant extract samples were ana-
lyzed by GC-MS for identification of different compounds.
Bruker Scion 436-gas chromatograph with BR-5MS column
(30 m × 0.25 mm ID × 0.25 μm) with carrier gas 1 mL/min
was used. TQ detector with Software MS Work Station 8,
injection volume 2 μL, oven-110 °C hold for 3.50 min till
200 °C (at 10 °C/min), zero hold till 280 °C (at 5 °C/min)—
12 min. Hold (injector temp. 280 °C), GC run time 40–50 min.
MS Program: NIST Library (Version-11), temp. 290 °C (inlet
line), temp. 250 °C (source), 70 eV, m/z (mass scan) 50–
500 amu, delay in solvent 0–3.5 min, MS run time 40–50 min.
Results and discussion
The larvicidal effect was observed at 48 h illustrating the slow
poisoning of the mosquito larvae by the phytochemicals. The
lethal concentrations demonstrating the 50% (LC50) and 90%
Environ Sci Pollut Res (2018) 25:9176–9185
(LC90) larval mortality, chi-squared (χ2) data, and 95% confi-
dence limits of extracts of four plants with petroleum ether,
chloroform, and methanol as solvents and the extract combi-
nations (1:1:1:1) for Cx. quinquefasciatus and Ae. aegypti
larvae are given in Table 1. PE extracts (leaf) of L. camara
caused mosquito larval mortality with LC50 and LC90 values
of 10.63 and 92.98 mg/L; 74.93 and 149.4 mg/L, respectively,
for Culex and Aedes larvae. The chloroform extract of
L. camara was effective with respective 50 and 90% lethal
concentrations of 55.92 and 146.61 mg/L for Cx.
quinquefasciatus. The mosquito larvicidal action of the
L. camara leaf extract supports the studies conducted by other
researchers. Kumar et al. (2012a) had illustrated the larvicidal
potential of hexane extract of L. camara against larvae of
Aedes (LC50 30.71 mg/L). Sharma et al. (2016) reported that
the petroleum ether extract of L. camara was more effective
than the ethanol extract to Ae. aegypti larvae. Our study shows
that the mosquitocidal components present in L. camara
leaves are lipophilic in nature as evidenced by the higher ac-
tivity of PE and CH extracts. The larvae of Cx.
quinquefasciatus were more susceptible to the extracts of
L. camara than that of Ae. aegypti. ME extract of L. camara
up to 300 mg/L (cutoff value for initial screening) could not
affect the mosquito larvae in our experiments.
The ME, CH, and PE extracts of the leaves of N. oleander
demonstrated lethal effect against Culex larvae (LC50 35.82,
46.51, and 66.67 mg/L), while only the ME extract was effec-
tive with LC50 value of 84.09 mg/L against Ae. aegypti. The
mosquito larvicidal activity shown in this study by the differ-
ent solvent extracts of N. oleander is supported by earlier
reports (Komalamisra et al. 2005; El-Sayed et al. 2016).
Here, the mosquitocidal principle may be polar in nature as
the mehanolic extract was more effective than the non-polar
one. Previous study also showed reduced activity for N.
oleander diethyl ether extract of leaf (LC50 10,500 mg/L)
against Culex pipiens (El-Sayed et al. 2016) while the
ethanolic extract of the leaves was effective against Ae.
aegypti with LC50 of 197.97 mg/L (Komalamisra et al.
2005). According to Roni et al. (2013), the aqueous extract
of N. oleander was effective against Anopheles stephensi lar-
vae. In this study, the PE and CH extracts of N. oleander up to
300 mg/L (cutoff value for initial screening) did not exhibit
reasonable mortality in Aedes larvae. El-Sayed et al. (2016)
also support the same observation.
Our study showed that the PE and CH extracts of
H. suaveolens (leaves) were effective against Cx.
quinquefasciatus with and Ae. aegypti with LC50 38.39 and
41.93 mg/L, respectively. Only the PE extract of
H. suaveolens was effective against Ae. aegypti, and the
LC50 was 64.49 mg/L. The ME extract of H. suaveolens was
not effective up to 300 mg/L against both species tested. The
respective LC50 values of 213.09, 217.64, and 86.93 mg/L
were obtained on exposure of third instar larvae of Cx.
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Environ Sci Pollut Res (2018) 25:9176–9185 9179

Table 1 Larvicidal activity of plant extracts against Cx. quinquefasciatus and Ae. aegypti

S. no Plant name and part used Effective extract LC50 (LCL-UCL) (mg/L) LC90 (LCL-UCL) (mg/L) Chi-squared (χ2)

Cx. quinquefasciatus
1 L. camara leaves PE 10.63 (7.24–13.89) 92.98 (65.06–163.67) 0.92
CH 55.92 (48.17–63.48) 146.61 (133.38–163.76) 5.22
ME > 300 > 300 –
2 N. oleander leaves PE 66.67 (58.35–74.26) 149.78 (137.50–166.09) 2.85
CH 46.51 (40.82–51.55) 99.09 (91.53–109.09) 1.66
ME 35.82 (30.97–39.97) 73.22 (67.78–80.35) 3.82
3 H. suaveolens leaves PE 38.39 (33.46–42.65) 78.11 (72.38–85.59) 0.93
CH 41.93 (36.45–46.63) 81.68 (75.40–90.11) 0.13
ME > 300 > 300 –
4 T. stans leaves PE 19.26 (11.50–24.85) 56.58 (51.36–63.58) 0.44
CH > 300 > 300 –
ME > 300 > 300 –
5 Plant extract combination 4.32 (3.15–5.35) 14.54 (12.71–17.26) 4.89
Ae. aegypti
1 L. camara leaves PE 74.93 (67.76–81.17) 149.45 (140.68–160.54) 4.17
CH > 300 > 300 –
ME > 300 > 300 –
2 N. oleander leaves PE > 300 > 300 –
CH > 300 > 300 –
ME 84.09 (75.26–92.02) 163.92 (151.31–181.10) 1.03
3 H. suaveolens leaves PE 64.49 (55.87–72.46) 139.29 (127.53–154.59) 2.84
CH > 300 > 300 –
ME > 300 > 300 –
4 T. stans leaves PE 55.41 (40.84–67.19) 109.33 (92.97–141.81) 11.93
CH > 300 > 300 –
ME > 300 > 300 –
5 Plant extract combination 7.19 (3.05–11.14) 20.15 (14.93–34.96) 8.79

Degrees of freedom (3) and χ2 > 7 show heterogeneity

PE petroleum ether, CH chloroform, ME methanol, LCL lower confidence limit, UCL upper confidence limit

quinquefasciatus to hexane, CH, and ME extracts of
H. suaveolens (Kovendan et al. 2012). Compared to the stud-
ies reported earlier, our data showed a better activity for PE
extract of H. suaveolens leaves against mosquito larvae.
The PE extract of T. stans leaves displayed lethal activity to
Cx. quinquefasciatus (LC50 19.26 mg/L) and Ae. aegypti
(LC50 55.41 mg/L). Earlier researchers reported ovicidal ac-
tivity and ovipositional deterrence efficiency of aqueous leaf
extracts of T. stans against Ae. aegypti (Navaneethan et al.
2016). The PE extract of T. stans flowers exhibited
mosquitocidal activity (LC50 100–200 mg/L) against larvae
of Cx. quinquefasciatus, Ae. aegypti, and An. stephensi. Our
studies have shown promising activity for the PE extract of the
leaves of T. stans.
Among 12 extracts of four plants, 8 extracts were found to
be effective against the Cx. quinquefasciatus larvae at <
100 mg/L and 4 extracts were effective against the Ae. aegypti
at < 100 mg/L. Among these, the highest activity was shown
by PE extract of L. camara, followed by the PE extract of
T. stans, ME extract of N. oleander, and PE extract of
H. suaveolens against Cx. quinquefasciatus and Ae. aegypti
larvae.
Pavela et al. (2017) have reported the mosquito larvicidal
potential of essential oil combinations from Clausena anisata
and Dysphania ambrosioides and highlighted the application
of ethanomedicines as commercial insecticides. Individual
chemical entities present in plant extracts can exhibit various
synergistic as well as antagonistic effects. This phenomenon
has been documented by systematic studies with binary com-
binations of the chemical components as well as plant extracts
(Pavela 2015b; Benelli et al. 2017b, c). In this study, we have
prepared a quaternary mixture of plant extracts from metha-
nolic extract of N. oleander and petroleum ether extracts of
L. camara, T. stans, and H. suaveolens. The combination was
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9180 Environ Sci Pollut Res (2018) 25:9176–9185

Table 2 Chemical constituents

identified in PE extract of No. RT Name of the compound Molecular Molecular Peak
L. camara by GC/MS analysis formula weight area %

1 6.22 (−)-Carvone C10H14O 150 0.88

2 13.97 3,7,11,15-Tetramethyl-2-hexadecen-1-ol C20H40O 296 0.34
3 14.28 Phytol, acetate C22H42O2 338 0.07
4 15.60 n-Hexadecanoic acid C16H32O2 256 0.33
5 17.64 Phytol C20H40O 296 2.07
6 18.09 Isopulegol C10H18O 154 0.27
7 18.44 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- C18H30O2 278 0.13
8 27.79 Squalene C30H50 410 72.13
9 28.97 Eicosane, 7-hexyl- C26H54 366 1.64
10 29.22 1,6,10,14-Hexadecatetraen-3-ol, C20H34O 290 0.41
3,7,11,15-tetramethyl-, (E,E)-
11 32.36 Tetracosane, 11-decyl- C34H70 478 1.65
12 32.85 α-Tocopherol C29H50O2 430 19.94
13 37.31 γ-Sitosterol C29H50O 414 0.12

effective in killing the larvae of Cx. quinquefasciatus and Ae. LC90 14.54 mg/L and to Ae. aegypti LC50 7.19 mg/L and
aegypti with lower LC50 values than that of the individual LC90 20.15 mg/L.
plant extracts. The combination exhibited lethal activity to To find out the toxicity of this plant extract combination
larvae of Cx. quinquefasciatus with LC50 4.32 mg/L and on the non-target organism, studies were conducted by

Table 3 Chemical constituents

identified in ME extract of No. RT Name of the compound Molecular Molecular Peak
N. oleander by GC/MS analysis formula weight area %

1 4.64 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- C6H8O4 144 0.33

2 5.89 5-Hydroxymethylfurfural C6H6O3 126 11.21
3 7.30 4-Hydroxy-2-methylacetophenone C9H10O2 150 1.91
4 9.31 1,5,5-Trimethyl-6-methylene-cyclohexene C10H16 136 0.15
5 10.70 3′,5′-Dimethoxyacetophenone C10H12O3 180 0.99
6 12.59 3-O-methyl-d-glucose C7H14O6 194 58.19
7 13.35 Myo-inositol, 4-C-methyl- C7H14O6 194 8.74
8 13.98 3,7,11,15-Tetramethyl-2-hexadecen-1-ol C20H40O 296 2.07
9 14.28 Phytol, acetate C22H42O2 338 0.35
10 14.51 Dodeca-1,6-dien-12-ol, 6,10-dimethyl- C14H26O 210 0.64
11 15.66 n-Hexadecanoic acid C16H32O2 256 3.55
12 17.66 Phytol C20H40O 296 0.55
13 18.13 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- C18H30O2 278 2.80
14 18.44 Octadecanoic acid C18H36O2 284 0.32
15 23.53 Glycerol 1-palmitate C19H38O4 330 0.23
16 25.60 Butorphanol C21H29NO2 327 0.21
17 26.17 2H-pyran, 2-(7-heptadecynyloxy) tetrahydro- C22H40O2 336 0.46
18 27.76 Squalene C30H50 410 1.38
19 31.00 δ-Tocopherol, O-methyl- C28H48O2 416 0.12
20 32.85 α-Tocopherol C29H50O2 430 4.18
21 35.62 Stigmasterol C29H48O 412 0.08
22 37.31 γ-Sitosterol C29H50O 414 0.28
23 39.33 Betulin C30H50O2 442 1.10
24 39.80 α-Amyrin C30H50O 426 0.15
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Environ Sci Pollut Res (2018) 25:9176–9185 9181

Table 4 Chemical constituents identified in PE extract of H. suaveolens by GC/MS analysis

No. RT Name of the compound Molecular Molecular Peak

formula weight area %

1 5.78 exo-2-Hydroxycineole C10H18O2 170 0.17

2 5.94 2-Oxabicyclo[2.2.2]octan-6-ol, 1,3,3-trimethyl- C10H18O2 170 0.24
3 6.18 2,6,10,10-Tetramethyl-1-oxaspiro[4.5]decan-6-ol C13H24O2 212 0.16
4 7.06 Thymol C10H14O 150 0.87
5 8.26 Copaene C15H24 204 0.18
6 8.93 Caryophyllene C15H24 204 4.96
7 9.05 trans-α-Bergamotene C15H24 204 0.39
8 9.43 Humulene C15H24 204 0.25
9 9.77 1,6-Cyclodecadiene, 1-methyl-5-methylene-8-(1-methyl ethyl)-, [S-(E,E)]- C15H24 204 0.33
10 11.12 Caryophyllene oxide C15H24O 220 0.42
11 12.33 Ledene oxide-(II) C15H24O 220 0.20
12 13.96 3,7,11,15-Tetramethyl-2-hexadecen-1-ol C20H40O 296 0.29
13 15.61 n-Hexadecanoic acid C16H32O2 256 1.18
14 16.51 Phenanthrene, 7-ethenyl-1,2,3,4,4a,4b,5,6,7, 8, 8a, 9-dodeca hydro-1,1,4b,7-tetramethyl-, C20H32 272 1.88
[4aS-(4aα,4bβ,7α,8aα)]-
15 17.06 7-Isopropyl-1,1,4a–trimethyl-1,2,3,4,4a,9,10,10a–octahydro phenanthrene C20H30 270 2.65
16 17.55 Kaur-16-ene, (8β,13β)- C20H32 272 1.97
17 20.90 Androst-2-en-4-one, 17-hydroxy- C19H28O2 288 15.37
18 21.55 1-Phenanthrenemethanol, 1,2,3,4,4a,9,10,10a-octahy dro-1,4a-dimethyl-7-(1-methylethyl)-, C20H30O 286 12.18
[1S-(1α,4aα, 10aβ)]-
19 24.11 Agatadiol or 1-naphthalenemethanol, C20H34O2 306 17.67
decahydro-5-(5-hydroxy-3-methyl-3-pentenyl)-1,4a-dimethyl-6-methylene-, [1S-[1α,4aα,5α
(E),8aβ]]-
20 25.36 2-[4-methyl-6-(2,6,6-trimethylcyclohex-1-enyl) C23H32O 324 9.94
hexa-1,3,5-trienyl]cyclohex-1-en-1-carboxaldehyde
21 27.76 Squalene C30H50 410 17.06
22 29.40 4,8,13-Cyclotetradecatriene-1,3-diol, 1,5,9-trimethyl-12-(1-methylethyl)- C20H34O2 306 1.55
23 32.86 α-Tocopherol C29H50O2 430 0.08
24 34.25 Hexacosyl acetate C28H56O2 424 5.02
25 37.30 γ-Sitosterol C29H50O 414 3.90
26 39.81 α-Amyrin C30H50O 426 1.09

exposing the larvivorus fish G. affinis to various concen-
trations of the extract combination ranging from 50 to
120 mg/L. No mortality was observed for the G. affinis
when they were exposed to 50 and 60 mg/L for 48 h.
However, above 60 mg/L, a dose-dependent response was
observed. The observed lethal activity in terms of LC50
was 90.21 mg/L (LCL-UCL 87.26–93.29), LC 90 was
128.49 mg/L (LCL-UCL 120.39–140.97), and chi-
squared value of 3.16. The SI or PSF was calculated from
the LC50 values of the non-target organism and that of the
vector mosquitoes. The predator safety factors were found
to be 12.55 and 20.88 for G. affinis with respect to Aedes
and Culex mosquito larval habitats for the plant extract
combination. Most of the chemical insecticides used for
controlling the mosquito larvae exhibit negative impact
on the natural enemies or predators of mosquito larvae
(Lawler et al. 2003, 2007). In this study, the identified
plant extract combination is showing less harm to non-
target organism G. affinis than to the mosquito larvae at
lower concentrations.
Here, the plant extract combination made from ME extract
of N. oleander and PE extracts of L. camara, T. stans, and
H. suaveolens was effective in killing the larvae of Cx.
quinquefasciatus and Ae. aegypti with lower LC50 values than
that of the individual plant extracts. This combination of four
extracts showed promising larvicidal potential with LC50
4.32 mg/L for Cx. quinquefasciatus and 7.19 mg/L for Ae.
aegypti.
FT-IR analysis of L. camara (PE extract of leaf) displayed
2954, 2927, 2854, and 1464 cm−1 characteristic absorptions
corresponding to alkyl (C-H) stretching and 1713 cm−1 for
carbonyl (C=O) stretching showing the presence of aliphatic
acids and ketones. N. oleander (ME extract of leaf) illustrated
3373 cm−1 characteristic absorptions matching O-H stretching
 Author's personal copy
9182 Environ Sci Pollut Res (2018) 25:9176–9185

Table 5 Chemical constituents identified in PE extract of T. stans by GC/MS analysis

No. RT Name of the compound Molecular Molecular Peak area

formula weight %

1 7.88 Eugenol C10H12O2 164 0.52

2 8.28 Copaene C15H24 204 0.26
3 8.51 Methyleugenol C11H14O2 178 1.21
4 8.95 Caryophyllene C15H24 204 4.29
5 9.45 cis-β-Farnesene C15H24 204 3.53
6 9.77 1,6-Cyclodecadiene, 1-methyl-5-methylene-8-(1-methylethyl)-, [S-(E,E)]- C15H24 204 0.49
7 9.97 Azulene, 1,2,3,3a,4,5,6,7-octahydro-1,4-dimethyl-7-(1-methylethenyl)-, C15H24 204 0.63
[1R-(1α,3aβ,4α,7β)]-
8 10.22 4-Epi-cubedol C15H26O 222 0.55
9 11.13 Caryophyllene oxide C15H24O 220 0.33
10 13.97 3,7,11,15-Tetramethyl-2-hexadecen-1-ol C20H40O 296 0.90
11 15.62 n-Hexadecanoic acid C16H32O2 256 1.90
12 17.66 Phytol C20H40O 296 7.64
13 18.14 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- C18H30O2 278 0.33
14 18.45 Butyl 9,12,15-octadecatrienoate C22H38O2 334 0.38
15 27.77 Squalene C30H50 410 32.98
16 28.97 Eicosane, 7-hexyl- C26H54 366 0.54
17 32.85 α-Tocopherol C29H50O2 430 12.54
18 33.98 Octadecane, 3-ethyl-5-(2-ethylbutyl)- C26H54 366 1.48
19 36.06 Stigmastan-6,22-dien, 3,5-dedihydro- C29H46 394 13.73
20 37.27 γ-Sitosterol C29H50O 414 15.78

frequency, 2927, 1451 cm−1 alkyl (C-H) stretching and
1741 cm−1 carbonyl (C=O) stretching showing the presence
of esters. T. stans (PE extract of leaves) displayed 2918, 2849,
and 1456 cm−1 characteristic absorptions corresponding to
alkyl (C-H) stretching and 1738 cm−1 for carbonyl (C=O)
stretching illustrating the presence of esters. H. suaveolens
(PE extract of leaf) displayed 2923, 2849, and 1466 cm−1
characteristic absorptions corresponding to alkyl (C-H) and
1734 cm−1 carbonyl (C=O) stretching indicating esters.
PE extracts of L. camara, T. stans, and H. suaveolens and
ME extract of N. oleander were subjected to GC-MS analysis.
Thirteen chemical constituents including aliphatic acids and
ketones as evidenced by FT-IR spectrum were found in PE
extract of L. camara (Table 2). ME extract of N. oleander
contained 24 chemical constituents including esters and hy-
droxy compounds as observed in IR spectrum (Table 3). PE
extract of H. suaveolens (Table 4) contained 26 and PE extract
of T. stans contained (Table 5) 20 chemical constituents includ-
ing esters and aliphatic acids as observed in IR spectrum. Here,
the ethanol-soluble fraction of the extract residues used for the
bioassay was only subjected to GC/MS analysis. Hence, the
number of components was found to be less compared to orig-
inal extract residue analysis. Many of the chemical components
present in the promising extracts were known for mosquitocidal
and other biological activities which strongly support the
mosquitocidal potential illustrated by the results of this study.
Govindarajan et al. (2012) recently reported mosquito lar-
vicidal activity of carvone. (−)-Isopulegol exhibited mosquito
larvicidal activity against An. gambiae (Santos et al. 2008).
Kweka et al. (2016) has reported the mosquito larvicidal ac-
tivity of (−)-isopulegol and other monoterpenes against
Anopheles gambiae. The mosquito larvicidal potential of β-
sitosterol was illustrated by Rahuman et al. (2008). Two
monoterpenes carvone and (−)-isopulegol and β-sitosterol
are among the chemical constituents identified by GC-MS
analysis of PE extract of L. camara and have contributed
towards the larvicidal activity of this extract. The mosquito
larvicidal activity of carvone and its synergistic combination
with various aromatic compounds found in plant essential oils
were reported (Pavela 2015b).
Betulin has been investigated for mosquitocidal activities
(Nsonde et al. 2010). α-Amyrin acetate was evaluated for its
activity against larvae and pupae of An. stephensi (Kweka
et al. 2016; Kuppusamy et al. 2009). GC-MS analysis of
ME extract of N. oleander showed the presence of betulin
and amyrin. These constituents may be contributing towards
the larvicidal activity of ME extract of N. oleander.
The mosquito larvicidal activity of β-caryophyllene, euge-
nol, and thymol against Ae. aegypti larvae have been reported
(Pavela 2015b; da Silva et al. 2016). Lethal activity of
humulene and β-caryophyllene was reported against Cx.
pipiens pallens and Ae. aegypti (Wang et al. 2016). GC/MS
 Author's personal copy
Environ Sci Pollut Res (2018) 25:9176–9185
analysis showed the presence of insecticidal principles thy-
mol, caryophyllene, and amyrin in H. suaveolens and eugenol,
caryophyllene, and amyrin in PE extract of T. stans.
Bergamotene, a sesquiterpene known for its insecticidal prop-
erties (Pinto et al. 2016) is present in the PE extract of
H. suaveolens. Caryophyllene oxide is present in PE extract
of T. stans which has been reported for insecticidal activity
(Liu et al. 2012). The insecticidal component Z,Z,Z-9,12,15-
octadecatrienoic acid reported by Zhou et al. (2006) is present
in PE extracts of L. camara and T. stans and ME extract of
N. oleander.
The phytochemical components of the leaves extracted
with those of other plants of the same family showed similar-
ity in the case of some of the molecules. The essential oils
from the leaves of Ocimum gratissimum L. of the family of
H. suaveolens viz., Lamiaceae was reported for activity
against Ae. albopictus showed the presence of caryophyllene,
copaene, bergamotene, caryophyllene oxide, gamma-
sitosterol in GC/MS analysis as observed in H. suaveolens
extract (Sumitha and John 2015). The essential oils from the
leaves of Lippia turbinata and L. polystachya of the family of
L. camara viz., Verbenaceae showed insecticidal activity
against larvae of Cx. quinquefasciatus, and these oils are re-
ported for the presence of carvone as that of L. camara
(Gleiser and Zygadlo 2007). The acetone extract of the leaves
of Tecomella undulate belonging to the family of T. stans viz.,
Bignoniaceae was reported for the presence of n-
hexadecanoic acid, phytol, 9,12,15-octadecatrienoic acid,
(Z,Z,Z)-, squalene in GC/MS analysis as observed in
T. stans extract (Yadav and Roy 2017). The GC/MS analysis
of methanolic extract of the leaves of Catharanthus roseus
belonging to the family of N. oleander viz., Apocynaceae
showed the presence of phytochemical compounds
octadecanoic acid, squalene, and hexadecanoic acid as ob-
served in methanolic extract of N. oleander (Thanwar et al.
2017). Phytochemical screening of N. oleander leaves have
shown tannins and saponins in the ethanolic extract of the
leaves (Kumar and Yadav 2013; Santhi et al. 2011).
For chemical component analysis of plant extracts by GC/
MS alone is not sufficient to identify all the components pres-
ent in the extracts. Special attention should be given in the
case of polar extracts where some of the components may
not be gasified to analyze by GC. In such cases, LC/MS will
be a better option along with NMR data. Also, in addition to
NIST library comparison, the RI value also should be calcu-
lated and compared with literature for identification of com-
ponents. The bioassay-guided fractionationation and identifi-
cation of the biologically active components will add more
value to phytochemical research. Here, we have analyzed
the extracts by GC/MS for their chemical constituents; how-
ever, some more components would have been identified in
the methanolic extracts of N. oleander if simultaneous LC/MS
and NMR studies were planned along with this.
9183
The presence of the reported insecticidal components along
with other components present in all the four plant extracts
supports the larvicidal activity of the novel plant extract com-
bination against Cx. quinquefasciatus and Ae. aegypti. From
the available literature search, the presently reported four plant
extract combination is the first study of that kind showing
promising potential lethal activity against both Cx.
quinquefasciatus and Ae. aegypti larvae. Local community
can easily utilize these extracts in the locality for the control
of mosquito larvae of Ae. aegypti and Cx. quinquefasciatus.
Conclusions
In conclusion, this study has identified one herbal combina-
tion with lethal effect against Cx. quinquefasciatus and Ae.
aegypti larvae. Further studies on bioassay-guided fraction-
ation, identification of the active principle, and enriching the
combination with that will yield a more effective botanical
insecticide.
Acknowledgements The authors gratefully acknowledge Dr. P.
Jambulingam, Director of ICMR-Vector Control Research Centre,
Puducherry, for reviewing the manuscript. Mr. H. Irrussappan is thankful
to Dr. A.M. Manonmani, Chief, HRD, ICMR-VCRC for her support
during the study, Dr. N. Ayyappan, Researcher, Department of Ecology,
French Institute of Pondicherry for identifying the plants, and Shri S.
Kumaravel, IICPT, Thanjavur, for taking the GC/MS spectra. The support
by staff of HRD and Unit of Chemistry are also acknowledged.
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