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ORIGINAL PAPER

Isolation and identification of mosquito larvicidal compound

from Abutilon indicum (Linn.) Sweet

A. Abdul Rahuman &Geetha Gopalakrishnan &

P. Venkatesan &Kannappan Geetha

Received: 11 December 2007 /Accepted: 14 December 2007 /Published online: 3 January 2008# Springer-Verlag 2007

Abstract Larvicidal activity of crude hexane, ethyl acetate,

petroleum ether, acetone and methanol extracts of fivemedicinal plants, Abutilon indicum, Aegle marmelos,

Euphorbia thymifolia, Jatropha gossypifolia and Solanum

torvum were assayed for their toxicity against the early

fourth-instar larvae ofCulex quinquefasciatus. The larval

mortality was observed after 24 h exposure. All extracts

showed moderate larvicidal effects; however, the highest

larval mortality was found in petroleum ether extract ofA.

indicum. In the present study, bioassay-guided fractionation

ofA. indicum led to the separation and identification of a -

sitosterol as a potential new mosquito larvicidal compound

with LC50 value of 11.49, 3.58 and 26.67 ppm against

Aedes aegypti L, Anopheles stephensi Liston and C.

quinquefasciatus Say (Diptera: Culicidae), respectively. 1H

NMR, 13C NMR and mass spectral data confirmed the

identification of the active compound.-sitosterol has been

recognized as the active ingredient of many medicinal plant

extracts. All the crude extracts when screened for their

larvicidal activities indicated toxicity against the larvae of

C. quinquefasciatus. This article reports the isolation andidentification of the-sitosterol as well as bioassay data for

the crude extracts. There are no reports of-sitosterol in the

genus A. indicum, and their larvicidal activities are being

evaluated for the first time. Results of this study show that

the petroleum ether extract of A. indicum may b e

considered as a potent source and -sitosterol as a new

natural mosquito larvicidal agent.

Introduction

Mosquitoes are known vectors of several disease-causing

pathogens, which affect many millions of people all over the

world.Aedes aegyptiis known to carry dengue, yellow fever

and Chikungunya; malaria is carried by Anopheles stephensi;

and filarial disease by Culex quinquefasciatus. To prevent

mosquito-borne diseases and improve public health, it is

necessary to control them. But in recent years, mosquito

control programmes have been suffering from failures

beca use of the ever-increasing insecticide resistance

(Georghiou and Lagunes-tejeda1991; WHO1992). Besides

insecticidal resistance in arthropod vectors of tropical

diseases, the increased costs of insecticides and increased

public concern over environmental pollution have necessi-

tated a continued search for alternative vector-control

methods, which would be environmentally safer and specific

in their action (Coats1994; Khan and Selman1996; Peng et

al. 1998). Plant products or plant-derived compounds are

promising alternatives to synthetic insecticides in controlling

insect pests of medical importance (Mwangi and Mukiama

1988; Green et al.1991; Kelm and Nair1996).

A. aegypti, a vector of dengue, is widely distributed in

the tropical and subtropical zones. About two-thirds of the

Parasitol Res (2008) 102:981988

DOI 10.1007/s00436-007-0864-5

A. Abdul Rahuman (*)

Unit of Bioactive Natural Products, Department of Zoology,

C.Abdul Hakeem College,

Melvisharam 632 509, India

e-mail: abdulrahuman6@hotmail.com

G. Gopalakrishnan

Center for Natural Products, SPIC Science Foundation,

64, Mount Road,

Chennai 600 032, India

P. Venkatesan

Department of Zoology, Loyola College,

Chennai 600 032, India

K. Geetha

Department of Chemistry, Muthurangam Govt.Arts College,

Vellore 632 002, India

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worlds population lives in areas infested with dengue

vectors, mainlyA. aegypti. Currently, dengue is endemic in

all continents except Europe, and epidemic dengue hae-

morrhagic fever occurs in Asia, the Americas and some

Pacific islands. Dengue viruses, causative agents of dengue

fever and more severe dengue hemorrhagic fever/dengue

shock syndrome, infect over 100 million people every year

(Hahn et al. 2001); Chikungunya virus, a member of thealphavirus genus, is of considerable public health concern

in Southeast Asian and African countries (Pastorino et al.

2005). A. stephensiis a major malaria vector in India. With

an annual incidence of 300500 million clinically manifest

cases and a death toll of 1.12.7 million, malaria is still one

of the most important communicable diseases. Currently,

about 40% of the worlds population lives in areas where

malaria is endemic (Wernsdorfer and Wernsdorfer2003).C.

quinquefasciatus is one of the potential vectors of

Wuchereria bancrofti, the causative agent of human

lymphatic filariasis infecting over 120 million people all

over the world (Terranella et al. 2006).Many studies on plant extracts against mosquito larvae

have been conducted around the world. The crude hexane

extracts obtained from flower heads ofSpilanthes acmella,

Spilanthes calva and Spilanthes paniculata (Pandey et al.

2007), seed extract of Sterculia guttata (Katade et al.

2006a, b); the ethyl acetate extract of fruit mesocarp of

Balanites aegyptiaca (Wiesman and Chapagain 2006);

partially purified extracts of leaves of Vitex negundo,

Nerium oleander and seeds of Syzygium jambolanum

(Pushpalatha and Muthukrishnan 1995), the petroleum

ether root extract of Solanum xanthocarpum (Mohan et al.

2007), leaves of Artemisia annua and Azadirachta indica

(Tonk et al. 2006), A. annua (Sharma et al. 2006), the

acetone crude extract of Fagonia indica and Arachis

hypogaea(Chaubal et al.2005), extracts ofNerium indicum

and Thuja orientelis (Sharma et al. 2005), Murraya

koenigii, Coriandrum sativum, Ferula asafoetida, Trigo-

nella foenum (Harve and Kamath2004) were tested against

mosquito larvae. Rongsriyam et al (2006) have reported

that the methanol extracts of dried root powder of

Rhinacanthus nasutus was tested against A. aegypti and

C. quinquefasciatus larvae. The crude methanol extract of

Chamaecyparis obtusa (Jang et al. 2005), extract of leaves

from the Indian white cedar Dysoxylum malabaricum

(Nathan et al. 2006), extracts ofEuphorbia tirucalli latex

and stem bark were evaluated for larvicidal activity (Yadav

et al. 2002) against A. aegypti, A. stephensi and C.

quinquefasciatus.

The ethyl acetate extracts of the stem bark of Aegle

marmelos exhibit moderate insecticidal activity against

Phaedon cochleariae and Musca domestica (Samarasekera

et al. 2004), the EtOAc fraction ofEuphorbia thymifolia

showed anti-HSV-2 activity (Lin et al. 2002), Jatropha

gossypifolia tested phytotoxic disorders upon feeding of

Bemisia tabaci (Brown et al. 2000), and the methanolic

extracts ofSolanum torvum tested for molluscicidal activity

against Biomphalaria glabrata (Silva et al. 2005).

Abutilon indicum (Malvaceae), known commonly as

Thuthi, is distributed throughout the hotter parts of India

(Chopra et al. 1992). It has been reputed in the Siddha

system of medicine as a remedy for jaundice, piles, ulcerand leprosy (Yoganarasimhan 2000). The plant is also

reported to possess analgesic activity (Ahmed et al. 2000).

Approximately 80% ethanol root extract of A. indicum

showed toxic effect against A. aegypti fourth-instar larvae

and guppy fish (Promsiri et al. 2006). The aqueous extract

of A. indicum was tested for hepatoprotective activity

against carbon tetrachloride- and paracetamol-induced

hepatotoxicities in rats (Porchezhian and Ansari 2005).

Seven flavonoid compounds were isolated and identified

from the flowers of A. indicum (Matawska and Sikorska

2002); clomiphene citrate, centchroman, and embelin were

isolated from the methanolic extracts of A. indicum andButea monosperma and studied on uterotropic and uterine

peroxidase activities in ovariectomized rats (Johri et al.

1991); Gossypetin 8 and 7 glucosides, cyanidin-3-rutinoside,

-pipene, cincole, farnesol, borneol from oil, eudesmol,

geramiol, caryophyllene from flower extract, gallic acid,

allantolactone and isoalantolactone were isolated from A.

indicum (Rastogi and Mehrotra1993;1995).

Earlier authors reported that the-sitosterol was isolated

from Croton cajucara (Maciel et al. 2000); Sitophilus

granarius (Johannes et al.2001) and leaves ofSebastiania

adenophora(Rubalcava et al.2007) were tested against the

larvae of Diabrotica virgifera (Pleau et al. 2002); anti-

cancer activities (Kim et al. 2002; Wahab et al. 1998) from

Senecio lyratus showed antifungal and anti-bacterial activ-

ities (Kiprono et al. 2000) and were studied for antiathero-

sclerotic effect (Wang et al. 2006); and insecticidal activity

(Zolotar et al. 2002a) against the larvae of Leptinotarsa

decemlineata (Zolotar et al.2002b) was studied against the

larval parasitoid Lariophagus distinguendus (Johannes et

al. 2001). This is the first report of compound -sitosterol

being isolated from A. indicum and tested against the

mosquito larvae.

Materials and methods

Mosquito culture

A. aegypti, A. stephensi and C. quinquefasciatus colonies

were maintained in our insectary (45 45 40 cm) at 27 2C

and 802% relative humidity (RH) with a photoperiod of

14:10 h light and dark cycles as per the procedure of Sharma

and Saxena (1994). The egg strips were obtained from Zonal

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Entomological Research Centre, Vellore (125548N, 797

48E) to start the colony. The strips were immersed in

dechlorinated tap water for hatching. Larvae were fed with a

diet of finely ground brewer yeast and dog biscuits (3:1).

The adults were given a blood meal from a pigeon (Reuben

1987). Glass petri dishes with 50 ml of tapwater lined with

filter paper was kept inside the cage for oviposition.

Plant materials

The leaf of A. indicum (Linn.) Sweet (Malvaceae), A.

marmelos (Linn.) Correa ex Roxb (Rutaceae), E. thymifolia

(Linn.) (Euphorbiaceae), J. gossypifolia (Linn.) (Euphor-

biaceae), and S. torvum Sw (Solanaceae), were collected

from Chitheri Hills, Dharmapuri district (115328N

0783026E, altitude 959), Tamil Nadu, India in November

2005 and was authenticated by Dr. B. Annadurai, Depart-

ment of Plant Biology and Biotechnology, C. Abdul

Hakeem College, Melvisharam, Vellore, India.The voucher

specimens have been deposited in the zoology laboratory.

Preparation of plant extracts

The dried leaf (800 g) and the whole plant (600 g) were

powdered mechanically using commercial electrical stain-

less steel blender and extracted with hexane (Qualigens),

ethyl acetate (Fine), petroleum ether (6080C, Qualigens),

acetone (Qualigens) and methanol (Qualigens) in a soxhlet

apparatus (3,000 ml) separately until exhaustion (Irungu

and Mwangi 1995). The extract was concentrated under

reduced pressure 2226 mgHg at 45C, and the residue

obtained was stored at 4C.

Purification of active principle

The petroleum ether crude extract (23.124 g) was subjected

to a column chromatography (505 cm, gravity, 1:2

charcoal/Si gel, 60120 mesh, 400 g) to obtain three

fractions A, B and C by increasing polarity of eluents n-

hexane and ethyl acetate 100:0 (5200 ml) 50:50 (22

200 ml) and 0:100 (16 200 ml), respectively. Further

elution of the column with different proportions of

chloroform and methanol yielded three more fractions

namely D, E and F with the elutions of 100:0 (9

200 ml), 50:50 (12200 ml) and 0:100 (14200 ml),

respectively. Each fraction (AE) obtained was tested

against fourth-instar larvae of Culex at the concentration

of 1,000 ppm, and those fractions showing 100% mortality

in 24 h alone were selected for further separation by

column chromatography.

Fractions B (9.468 g), B2 (2.741 g) and B2C (1.816 g)

were subjected to a subsequent repeated column chromatog-

raphy (gravity) using different Si gel mesh (70320 mesh,

220 g and 230400 mesh, 140 g) with varying proportions of

n-hexane and ethylacetate as eluents to collect different

subfractions. Bioassay-guided fractionation was carried out,

and a pure compound B2C6 (0.941 g) was obtained from IV

column with the elution of 88:12 (3220 ml).

Each fraction was tested against fourth-instar larvae of

Culex at the concentration of 1,000 ppm. Those fractions

showing 100% mortality in 24 h alone were selected forfurther column chromoatographic separation. The fractions

collected were combined based on thin layer chromatogra-

phy (TLC) results. After 24 h of exposure, the percentage

mortality of larvae is reported from the average of five

replicates.

All fractions were monitored by TLC (precoated plate,

0.02 mm thick, E. merck, Germany 60 F254) until a single

spot was obtained. The pure fractions were carefully

evaporated to dryness and subsequently characterized by

spectral analysis.

Gas chromatographymass spectrometry analysis

The pure compounds were subjected to infrared (IR),

ultraviolet (UV), 1H and 13C NMR and mass spectral

analysis. IR spectra were recorded on a BRUKER FT-IR

instrument and UV spectra were recorded on a SHI-

MADZU instrument. The 1H and 13C NMR were recorded

in BRUKER 200 MHz DPX instrument using CDCI3 with

tetramethylsilane as internal standard. The mass spectra

were recorded in SHIMADZU QP 5000 gas chromatogra-

phy (GC)-mass spectrometry (MS) instrument using a

temperature programme 60250C over a period of

15 min. The injection volume was 2 l, as hexane solution1H1H COSY, 13C1H HETCOR and 13C1H COLOC

were performed using Bruker standard microprograms.

Larvicidal bioassay

During preliminary screening with the laboratory trial, the

larvae of Culex were collected from the stagnant water in

the premises of hostel, Loyola College Campus, Chennai

and identified in Vector Control Research Centre, Pudu-

cherry. One gram of crude extract was first dissolved in

100 ml of respective solvent (stock solution). From the

stock solution, 1,000 ppm was prepared with dechlorinated

tap water. Polysorbate 80 (Qualigens) was used as an

emulsifier at the concentration of 0.05% in the final test

solution. Early fourth-instar larvae were used for bioassay

test. A total of 100 larvae were exposed in five replicates of

20 larvae each. Experiments were conducted for 24 h at

room temperature (282C). The control was set up with

solvent and polysorbate 80 (Saxena and Yadav1983). The

experimental media, in which 100% mortality of larvae

occurs alone, were selected for isolation and purification.

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The different fractions isolated were tested against the

fourth-instar larvae of mosquitoes by the procedure of

WHO (1996) with some modification and as per the

method of Rahuman et al. (2000). For Bioassay test, larvae

were taken in five batches of 20 in 249 ml of water and

1.0 ml of plant extract concentration. From the stock

solution, different concentrations ranging from 0.94 to

120 ppm were prepared. The numbers of dead larvae werecounted after 24 h of exposure, and the percentage

mortality was reported from the average of five replicates.

Statistical analysis

The average larval mortality data were subjected to probit

analysis for calculating LC50, LC90 and other statistics at

95% fiducial limits of upper confidence limit and lower

confidence limit, and chi-square values were calculated by

using the software developed by Reddy et al. (1992).

Results and discussion

A literature survey of the plant sterols revealed that the

compound under investigation could be -sitosterol. The

physical and spectral data of the present compound were in

agreement with those of the values reported in the literature

(Hong et al. 1999; Deepak and Handa 2000). However, a

TLC analysis with the standard -sitosterol (Sigma-

Aldrich, S-5753) confirmed the identity of compound

B2C6 to be, indeed, -sitosterol.

-sitosterol was isolated from the petroleum ether

extract of leaf ofA. indicum. However, it is the first report

of isolated active fractions tested for mosquito larvicidal

activity. All extracts showed moderate larvicidal effects;

however, the highest larval mortality was found in

petroleum ether extract of A. indicum (Table 1 and

Fig. 1a). Among the crude extracts tested, the petroleum

ether extract ofA. indicum showed 100% larval mortality at

1,000 ppm. Promsiri et al. (2006) have reported that 80%

ethanol roots extract ofA. indicum showed 57% mortality

at the concentration of 100 g/ml. In the present observation,

the petroleum ether extract was more toxic than the ethanol

extract. The larvicidal activity of oil from Cinnamomumcamphora, Boswellia carteri, Anethum graveolens and

Myrtus communisshowed 100% mortality at 50 ppm within

3 h against third-instar larvae of A. aegypti (Amer and

Mehlhorn 2006a, b). Mean median lethal concentration

values of the aqueous extract from the roots of Hibiscus

abelmoschus (Malvaceae) against the larvae of Anopheles

culicifacies, A. stephensi and C. quinquefasciatus were

52.3, 52.6 and 43.8 ppm, respectively (Dua et al. 2006);

larvicidal efficacy of leaf extracts ofPavonia zeylanicaand

Acacia ferruginea (Malvaceae) were tested against the late

third-instar larvae ofC. quinquefasciatus, and their LC50

values were 2,214.7 and 5,362.6 ppm, respectively (Vahithaet al.2002); ethanol root extract ofA. indicum showed the

LC90 value after 48 h

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Piper nigrum exhibited toxicity at 35.0 and 30.0 ppm,

respectively, against fourth-instar larvae ofA. aegypti. Park

et al. (2002) have also reported the compounds retrofracta-

mide A (0.039 ppm), pipercide (0.1 ppm), guineensine

(0.89 ppm), pellitorine (0.92 ppm) and Piperine (5.1 ppm)

derived from the fruits ofP. nigrumagainstA. aegyptithird-

instar larvae.

This has been observed earlier; two fractions with thehydrogenate part was isolated from Tagetes minuta floral

extract and was exhibited to be 2030 times toxic to larvae

ofA. aegyptiand A. stephensi (Perich et al.1995). Fraction

A1 of ethanol from S. guttata seed extract was found to be

most promising; its LC50 was 21.552 and 35.520 ppm

against C. quinquefasciatus and A. aegypti, respectively

(Katade et al.2006a,b); the larvicidal principles ofAllium

sativum have been isolated and identified as diallyl

disulfide and diallyl trisulfide, which were fatal at 5 ppm

to Culex pipiens quinquefasciatus Say (Amonkar and

Banerji 1971). The results of this study clearly show that

extract and fraction ofA. indicum that contain -sitosteroldemonstrate a high larval mortality.

The failure to discover a significant new class of

insecticides has led many researchers back to biodiscovery

studies in the search for new and economically viable

alternatives. A considerable number of plant derivatives has

shown to be effective against mosquito with a safe manner;

however, due to the dramatic increase in resistance of

mosquitoes to familiar chemicals, better alternative means

of control are sought. The use of the active fraction was

shown to be sufficient to inhibit the emergence of the larvae

population; this will certainly help reduce the mosquito

population drastically. Since a large proportion of the

human population, living in areas where mosquito disease

infection is a serious problem, suffers from varying degrees

of poverty, the discovery of plant-derived compounds that

could control the mosquito population would be of great

value. In this context, the highly bioactive compounds ofA.

indicum, which is being grown widely in most areas of

India, offer an opportunity for developing alternatives to

rather expensive and environmentally hazardous organic

insecticides. Furthermore, the findings of the high correla-

tion between -sitosterol content and larval mortality

would also open the door for using -sitosterol as natural

larvicidal agent.

Acknowledgements The authors are grateful to C. Abdul Hakeem

College Management, Prof. U. Peer, Principal and Dr.Ahmed Najib,

HOD of Zoology Department for their help and suggestion. We wish

to thank The Principal and HOD of Zoology Department, Loyola

College, Chennai for providing necessary facilities for our experi-

mental work. We are thankful to Dr. S. Narasimhan, Associate

Director, SPIC Science Foundation, Chennai for his help and

encouragement. AR is indebted to University Grants Commission,

New Delhi for award of fellowship.

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