Isolation of mosquito larvicidal molecule from the leaves of Clausena anisata

Mosquito-borne diseases, such as dengue, chikungunya, filariasis, and malaria are the major public health problems in the tropical and subtropical countries due to their climatic conditions. In recent years, climate change is likely to expand the geographical distribution of vector and vector-borne diseases, and these have a significant social and economic impact. Culex quinquefasciatus, the vector of lymphatic filariasis is widely distributed in tropical and subtropical countries, with around 120 million people infected worldwide and 44 million people having common chronic manifestation (Bernhard et al., 2003). India alone contributes around 40% of global filariasis burden, and the estimated annual economic loss is about 720 crores (Hotez et al., 2004). The Aedes aegypti mosquito (Diptera: Culicidae) is the vector for the etiologic agents of yellow fever, chikungunya and dengue fever (Chhabra et al., 2008). Dengue fever is currently considered as the most important viral vector-borne disease. It is estimated that more than 2.5 billion people live in transmission risk areas (World Health Organization [WHO], 2013). Dengue fever was initially described during an epidemic in Philadelphia in 1780, and since then, intermittent pandemics have affected Asia, Africa and the Americas at 10-30 years intervals (Omena et al., 2007). Outbreaks of dengue fever have repeatedly occurred in Brazil since 1980s, after the resurgence of the dengue virus in the country (Garcez et al., 2009). Anopheles stephensi is the primary vector of malaria in India and other West Asian countries and improved methods of control are urgently needed (Burfield and Reekie 2005; Mittal et al., 2005).


INTRODUCTION
Mosquito-borne diseases, such as dengue, chikungunya, filariasis, and malaria are the major public health problems in the tropical and subtropical countries due to their climatic conditions.In recent years, climate change is likely to expand the geographical distribution of vector and vector-borne diseases, and these have a significant social and economic impact.Culex quinquefasciatus, the vector of lymphatic filariasis is widely distributed in tropical and subtropical countries, with around 120 million people infected worldwide and 44 million people having common chronic manifestation (Bernhard et al., 2003).India alone contributes around 40% of global filariasis burden, and the estimated annual economic loss is about 720 crores (Hotez et al., 2004).The Aedes aegypti mosquito (Diptera: Culicidae) is the vector for the etiologic agents of yellow fever, chikungunya and dengue fever (Chhabra et al., 2008).Dengue fever is currently considered as the most important viral vector-borne disease.It is estimated that more than 2.5 billion people live in transmission risk areas (World Health Organization [WHO], 2013).Dengue fever was initially described during an epidemic in Philadelphia in 1780, and since then, intermittent pandemics have affected Asia, Africa and the Americas at 10-30 years intervals (Omena et al., 2007).Outbreaks of dengue fever have repeatedly occurred in Brazil since 1980s, after the resurgence of the dengue virus in the country (Garcez et al., 2009).Anopheles stephensi is the primary vector of malaria in India and other West Asian countries and improved methods of control are urgently needed (Burfield and Reekie 2005;Mittal et al., 2005).
Conventional pesticides such as malathian, DDT and pyrethroides that are generally used for mosquito control are known to cause the problems such as environmental pollution, residual effects and resistance of mosquito species.Development of resistance in C. quinquefasciatus A. aegypti and A. stephensi have been noted by WHO (1989) and by other studies (Polson et al., 2011;Ocampo et al., 2011;Raghavendra et al., 2011).These problems forced to search for new, alternative and safer control measures, especially from a plant source.Because, plant-derived molecules are eco-friendly, biodegradable, and target specific (Nathan and Kalaivani, 2005).Moreover, the development of resistance by vectors against plantderived molecules has not been reported so far.Botanical and microbial insecticides have been increasingly used for mosquito control because of their efficacy and documented non-toxic effects on non-target organisms (Ascher et al., 1995).
The genus Clausena (Rutaceae) is represented by 20 species in India, and they are traditionally used for various diseases (Kirtikar and Basu, 1991).Various extracts, their isolated compounds and essential oil of Clausena anisata were already investigated for mosquito larvicidal activity (Govindarajan, 2010;Mavundza et al., 2013;Mukandiwa et al., 2015;Jayaraman et al., 2015).Based on our previous study (Jayaraman et al., 2015), the ethyl acetate extract of C. anisata was studied to isolate larvicidal molecule responsible for the activity.

Plant Material and Preparation of Crude Extract
Healthy and well grown leaves of C. anisata were collected from their natural habitats and leaves were immediately brought to the laboratory using polythene bags.The leaves were first washed with tap water, then surface sterilized in 10% sodium hypochlorite to prevent the contamination of any microbes.They were thoroughly rinsed with sterile distilled water and shade dried followed by oven drying (60°C) and milled in an electrical blender.Air-dried and powdered leaf material of C. anisata (2.25 kg-750 g × 3) was extracted in a soxhlet apparatus with threefold of ethyl acetate (w/v) until the complete extraction.The extracts were pooled, and the solvent was evaporated using a rotary evaporator(Heidolf-Germany) under reduced pressure at 40°C.The voucher specimen (Herbarium No. AUBOT#267) was deposited at the Herbarium, Department of Botany, Annamalai University.

Isolation of Larvicidal Molecule
50 g of ethyl acetate residue of C. anisata was taken, and slurry was prepared with the equal amount of silica gel (100-200 mesh, Hi-Media, Mumbai) and it was loaded on the glass column (5 cm × 60 cm) using hexane.The column was eluted with solvents of increasing polarity from hexane to chloroform (9:5-0:5).In all, 27 fractions were collected.Each fraction contained 100 ml.The fractions showing similar R f values in thin layer chromatography (TLC) were combined, and the fractions were dried under vacuum.In total, 16 fractions were obtained in hexane and chloroform combination with the ratio of 8.0:2.0.16 combined fractions were tested for larvicidal activity.Among the combined fractions tested, F2, F3, F4, and F5 showed the larvicidal activities.Based on the highest activity of F2 fraction, it was further subjected to separation and purification of the active compound.The active fraction F2 (5 g) was packed with 50 g of silica gel (100-200 mesh) in a glass column (2.0 cm × 50 cm)) using hexane.This was eluted with solvents of increasing polarity from hexane to ethyl acetate (7.0:3.0).In all, 40 fractions, each 50 ml were collected.The fractions showing similar R f values in TLC were combined, and 6 fractions were obtained in hexane and ethyl acetate combination with the ratio of 7.0:3.0.Again 6 combined fractions were tested against larvicidal activity.Among the combined fractions tested f 2 showed the larvicidal activities.Due to the highest activity of f 2 fraction, from that further subjected to separation and purification of the active compound.The active fraction f 2 (1.0 g) was further subjected to glass column (2.0 × 50 cm) packed with 40 g of silica gel (100-200 mesh) using hexane.This was eluted with solvents of increasing polarity from benzene to chloroform (9.90-0.10).In all, 6 fractions, each 20 ml were collected.The fractions showing similar R f values in TLC were combined.The combined fractions, 3-4 of benzene and chloroform (8.0:2.0)formed white powder substance.The compound was identified by Fourier transform-infrared (FT-IR), 1 H, 13 C NMR and GC-MS.

Larvicidal Assay
The eggs of A. stephensi and A. aegypti were received from the Field Station, Centre for Research in Medical Entomology (ICMR-Government of India), Viruthachalam, and the egg rafts of C. quinquefasciatus were collected from drainage of local residential area of Annamalai Nagar (11°23′17 N, 79°42′57 E) and reared in laboratory (29±3°C, 75-85% RH).The larvae were fed with Brewer's yeast:dog biscuit (1:3).The larvae at early fourth instar stage were used for the larvicidal assay.The larvicidal activity was analyzed as per the standard procedures recommended by WHO (1981).The compound 1, 2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester was dissolved in 2 ml of dimethyl sulfoxide and the concentrations of 40, 20, 10, 5, and 2.5 ppm were prepared with distilled water.20 larvae were taken in a glass beaker (250 ml) containing 199 ml of tap water and 1 ml of respective concentrations of the compound.Five replicates were maintained for each concentration and the dead larvae were counted after 24 h.

Statistical Analysis
All the data were analyzed using SPSS version 21.0.The LC 50 and LC 90 values of mosquito larvicidal activity were calculated by Probit analysis and their lower, and upper confidence levels were determined.The level of significance for the assay was P < 0.05.

DISCUSSION
Vector control is facing a threat due to the emergence of resistance to synthetic insecticides.Molecules from the botanical origin are suitable and alternatives to reduce or control the resistance problem.In the present study, the isolated compound exhibited 100% mortality of A. aegypti and A. stephensi at 40 ppm.The larvicidal activity of essential oil from C. anisata was already reported by Govindarajan (2010).The essential oil showed significant larvicidal activity against C. quinquefasciatus, A. aegypti, and A. stephensi with the LC 50 values of 140.96, 130.19, and 119.59 ppm, respectively after 24 h of exposure period.The larvicidal molecule isolated and identified from the ethyl acetate extract of the leaves of C. anisata is a phenolic derivative.Mukandiwa et al. (2015) reported larvicidal activity of leaf extracts and seselin from C. anisata against A. aegypti.The molecule isolated from the present study, 1,2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester exhibited cent per cent mortality at 40 ppm against  A. aegypti and A. stephensi which is similar to neotenone, an isoflavonoid isolated from Neorautaenia mitis tubers that resulted in 100% mortality of Anopheles gambia larva (Joseph et al. 2004).Pushpalatha and Muthukrishnan (1995) reported that the petroleum ether: ethyl acetate (3:1) fraction of V. negundo leaf extract showed LC 50 value of 8.21 ppm against the 2 nd instar larvae of C. quinquefasicatus.But the 2 nd instar larvae are more susceptible to larvicidal principles than the 4 th instar larvae.A new triterpene was isolated from the methanol extract of Coccinia indica and identified as an oleanolic acid derivative and the compound showed prominent larvicidal activity against C. quinquefasciatus, A. aegypti and A. stephensi with the LC 50 values of 5.6, 5.0, and 4.8 mg/L, respectively (Senthilkumar et al., 2012).A saponin isolated from Achyranthus aspera recorded the LC 50 value of 18.20 and 27.24 ppm against A. aegypti and C. quinquefasciatus, respectively (Bagavan et al., 2008).
The benzoic acid derivatives with a general structure C6-C1 are widely used as industrial chemicals and agrochemicals, and some of them are reported to have biological activity (Tomás-Barberánand Clifford, 2000).
Methyl-2-hydroxybenzoate, which is known as methyl-ohydroxybenzoate or methyl salicylate was found to exhibit high toxicity on adults and eggs of Pediculus humanus (Yang et al., 2003).It also possessed acaricide activity on Varroa jacobsoni (Lindberg et al., 2000).Unelius et al. (2006) reported that methyl-4-hydroxybenzoate (methyl-p-hydroxybenzoate) was better in the antifeedant activity against pine weevil, Hylobius abietis than methyl-2-hydroxybenzoate (methylo-hydroxybenzoate). p-Hydroxybenzoic acid is one of the wall-bound phenolic acids that play a major role in plant defense responses against pathogen attack (Dixon and Paiva, 1995).This supports the toxicity of the 1, 2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester towards mosquito larvae.Foliar polyphenols exhibited biocidal effects against mosquito larvae (David et al., 2000).Rey et al. (1999) reported that the toxicity of polyphenols is exerted against midgut epithelium of larvae.Kannadasan et al. (2011) reported that methyl-p-hydroxybenzoate was evaluated against early 4 th instar larvae of C. quinquefasciatus and A. aegypti.The compound exhibited 100% larval mortality of both the mosquitoes at 20 ppm with LC 50 values of 5.77 and 4.74 ppm against C. quinquefasciatus and A. aegypti, respectively.Although, several plants have been reported for mosquito larvicidal activity, only a few botanicals have been taken from the laboratory to field trials, because they are poorly characterized and in most cases active principles are not determined.Thus, the 1, 2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester could be used as a potential mosquito larvicidal compound.