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Parasitol Res (2008) 102:981988 DOI 10.

1007/s00436-007-0864-5

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 five medicinal plants, Abutilon indicum, Aegle marmelos, Euphorbia thymifolia, Jatropha gossypifolia and Solanum torvum were assayed for their toxicity against the early fourth-instar larvae of Culex 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 of A. indicum. In the present study, bioassay-guided fractionation of A. 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
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

larvicidal activities indicated toxicity against the larvae of C. quinquefasciatus. This article reports the isolation and identification 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 be 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 aegypti is 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 because of the ever-increasing insecticide resistance (Georghiou and Lagunes-tejeda 1991; WHO 1992). Besides insecticidal resistance in arthropod vectors of tropical diseases, the increased costs of insecticides and increased public concern over environmental pollution have necessitated a continued search for alternative vector-control methods, which would be environmentally safer and specific in their action (Coats 1994; Khan and Selman 1996; 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 Nair 1996). A. aegypti, a vector of dengue, is widely distributed in the tropical and subtropical zones. About two-thirds of the

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worlds population lives in areas infested with dengue vectors, mainly A. aegypti. Currently, dengue is endemic in all continents except Europe, and epidemic dengue haemorrhagic 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 the alphavirus genus, is of considerable public health concern in Southeast Asian and African countries (Pastorino et al. 2005). A. stephensi is 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 Wernsdorfer 2003). 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 of Spilanthes 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 of Nerium indicum and Thuja orientelis (Sharma et al. 2005), Murraya koenigii, Coriandrum sativum, Ferula asafoetida, Trigonella foenum (Harve and Kamath 2004) 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 of Euphorbia 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 of Euphorbia 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 of Solanum 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, ulcer and 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 and Butea 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 Mehrotra 1993; 1995). Earlier authors reported that the -sitosterol was isolated from Croton cajucara (Maciel et al. 2000); Sitophilus granarius (Johannes et al. 2001) and leaves of Sebastiania adenophora (Rubalcava et al. 2007) were tested against the larvae of Diabrotica virgifera (Pleau et al. 2002); anticancer activities (Kim et al. 2002; Wahab et al. 1998) from Senecio lyratus showed antifungal and anti-bacterial activities (Kiprono et al. 2000) and were studied for antiatherosclerotic 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 (454540 cm) at 272C 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.) (Euphorbiaceae), 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, Department 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 stainless 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 nhexane and ethyl acetate 100:0 (5200 ml) 50:50 (22 200 ml) and 0:100 (16200 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 chromatography (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 for further column chromoatographic separation. The fractions collected were combined based on thin layer chromatography (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 SHIMADZU 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 chromatography (GC)-mass spectrometry (MS) instrument using a temperature programme 60250C over a period of 15 min. The injection volume was 2 l, as hexane solution 1 H1H 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, Puducherry. 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 Yadav 1983). 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 were counted 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 (SigmaAldrich, S-5753) confirmed the identity of compound B2C6 to be, indeed, -sitosterol. -sitosterol was isolated from the petroleum ether extract of leaf of A. 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 of A. indicum showed 100% larval mortality at 1,000 ppm. Promsiri et al. (2006) have reported that 80% ethanol roots extract of A. 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 Cinnamomum camphora, Boswellia carteri, Anethum graveolens and Myrtus communis showed 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 of Pavonia zeylanica and Acacia ferruginea (Malvaceae) were tested against the late third-instar larvae of C. quinquefasciatus, and their LC50 values were 2,214.7 and 5,362.6 ppm, respectively (Vahitha et al. 2002); ethanol root extract of A. indicum showed the LC90 value after 48 h <200 g/ml, 82.6130.3 g/ml (Promsiri et al. 2006); the LC50 values after a 12-h contact with 13 selected oils were shown against the third-instar larvae of A. aegypti and ranged from 1 to 101.4 ppm, and the LC50 values for C. quinquefasciatus larvae ranged between 1 and 50.2 ppm (Amer and Mehlhorn 2006a, b). The present result reveals that the petroleum ether extract of A. indicum was effective compared with the other Malvaceae species extracts. Larvicidal activity of -sitosterol which was effective against A. aegypti, A. stephensi and C. quinquefasciatus were 11.49, 3.58 and 26.67 ppm, respectively (Table 2 and Fig. 1b). Higher percentages of mortality were observed at higher concentrations of the isolated compound. Earlier

Table 1 Larvicidal activity of crude plant extracts against early fourth-instar larvae of Culex at 1,000 ppm Botanical name/Family (herbarium numbers) vernacular names Abutilon indicum (Linn.) Sweet/Malvaceae [LC/ZB/014-05] Perumtutti Aegle marmelos (Linn.) Correa ex Roxb/Rutaceae [LE/ZB/007-05] Vilvam Euphorbia thymifolia (Linn.)/Euphorbiaceae [LC/ZB/002-05] Sittrapaladi Jatropha gossypifolia (Linn.)/Euphorbiaceae [LC/ZB/022-05] Atalai Solanum torvum Sw/Solanaceae [LC/ZB/025-05] Sundaikai Parts used % Mortalitya (ppm) SD 1 Leaf Leaf Whole plant Leaf Leaf 663.0331 723.2863 142.2803 543.6331 363.0331 2 342.2803 363.0031 263.3466 623.2863 524.3358 3 1000.0000 621.6733 423.5777 902.000 782.9664 4 802.4494 882.1908 000.0000 941.7888 603.1622 5 522.9664 941.0954 041.0954 724.3351 143.3466

Controlnil mortality 1 Hexane; 2 ethyl acetate; 3 petroleum ether (6080o C); 4 acetone; 5 methanol a Mean value of five replicates

Parasitol Res (2008) 102:981988 Fig. 1 a Larvicidal activity of crude plant extracts against early fourth-instar larvae of Culex at 1,000 ppm. b Graph showing LC50 and LC90 values of mosquito vectors A. aegypti (A), A. stephensi (B), and C. quinquefasciatus (C)

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a
120
% Mortality (ppm) SD

Hexane

Ethyl acetate

Petroleum ether

Acetone

Methanol

100 80 60 40 20 0
A. indicum A. marmelos E. thymifolia J. gossypifolia S. torvum

b
140 120
Doses (ppm)

LC50SE

LC90SE

100 80 60 40 20 0 A B C

report showed that the compounds like diterpenoid furans 6alpha-hydroxyvouacapan-7beta, 17beta-lactone (1), 6alpha, 7beta-dihydroxyvouacapan-17beta-oic acid (2) and methyl 6alpha, 7beta-dihydroxyvouacapan-17beta-oate (3) from seeds of Pterodon spolygalaeflorus exhibited LC50

values of 50.08, 14.69 and 21.76 microg/ml against fourthinstar A. aegypti larvae, respectively (Omena et al. 2006). Siddiqui et al. (2004) have reported that the compounds spipnoohine (1) and pipyahyine (2) isolated from the petroleum ether extract of dried ground whole fruits of

Table 2 LC50, LC90 and regression analysis for the larvicidal activity of -sitosterol (B2C6) compound from A. indicum Species (IV instar) A. aegypti Description log LC50 log LC90 LC50 LC90 log LC50 log LC90 LC50 LC90 log LC50 log LC90 LC50 LC90 Value (ppm) 1.06 1.68 11.49 47.35 0.551 1.176 3.58 15.03 1.42 2.01 26.67 102.92 V 0.003 0.005 1.224 56.339 0.001 0.005 0.127 5.930 0.001 0.005 6.142 259.240 SE 0.042 0.069 1.106 7.506 0.043 0.070 0.356 2.435 0.040 0.068 2.478 16.100 LL 0.979 1.540 9.325 32.633 0.469 1.039 2.879 10.256 1.346 1.879 21.816 71.358 UL 1.142 1.810 13.661 62.056 0.638 1.314 4.275 19.802 1.505 2.146 31.531 134.474 Other statistics

A. stephensi

C. quinquefasciatus

C x =0.1044 C y =5.0916 x2 =3.4220 b =2.0845 bo =4.8740 C x =0.6168 C y =5.1298 x2 =2.4226 b =2.0561 bo =3.8619 C x =0.4362 C y =5.0222 x2 =6.7651 b =2.1855 bo =4.0689

V Variance of estimated log lethal dose; SE standard error; LC50 lethal concentration that kills 50% of the exposed larvae; LC90 lethal C concentration that kills 90% of the exposed larvae; LL lower limit (95% confidence limit); UL upper limit (95% confidence limit); x mean of C 2 x; y mean of y; x chi-square; b regression coefficient; bo constant of linearity

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Piper nigrum exhibited toxicity at 35.0 and 30.0 ppm, respectively, against fourth-instar larvae of A. aegypti. Park et al. (2002) have also reported the compounds retrofractamide 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 of P. nigrum against A. aegypti thirdinstar larvae. This has been observed earlier; two fractions with the hydrogenate part was isolated from Tagetes minuta floral extract and was exhibited to be 2030 times toxic to larvae of A. aegypti and 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 of Allium 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 of A. indicum that contain -sitosterol demonstrate 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 of A. 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 correlation 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 experimental 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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