A note on the larvicidal efficacy of saponin constituted crude extracts of plant and animal origin against Aedes aegypti L

S.N. Surendran1*, V. Kumaran1, R. Sivarajah2, S.R. Krishnarajah3, R. Srikaran2 and K. Raghavendra4 1 Department of Zoology, Faculty of Science, University of Jaffna, Jaffna. 2 Department of Chemistry, Faculty of Science, University of Jaffna, Jaffna. 3 Department of Zoology, Open University of Sri Lanka, Nawala, Nugegoda. 4 National Institute of Malaria Research, Indian Council of Medical Research, 22, Shamnath Marg, Delhi 110 054, India.


IntroDuCtIon
Mosquito-borne diseases such as malaria, dengue and chickungunya are of public health importance to Sri Lanka [1][2][3] .Even though there has been a drastic decline in reported malaria cases, severe epidemics of chickungunya and Dengue Fever (DF) / Dengue Hemorrhagic Fever (DHF) were reported from different parts of the country in recent times [2][3][4] .One option for management of insect vector-borne disease is to control the immature forms of vector mosquitoes.This method is more appropriate for the control of domestic and peri-domestic container breeding mosquitoes such as Aedes aegypti and A. albopictus, the vectors of dengue and chickungunya.At present, synthetic insecticides are heavily used for this purposes.The uses of chemical constituents of plant origin as a part of integrated vector control programme is encouraging due to their ecofriendly nature.
Natural products of plant origin have been tested for insecticidal properties to control disease vectors [5][6][7][8] , but extracts from animal origin have been rarely tested or reported.Sukumar et al. 9 reported plants belonging to 99 families, 276 genera and 346 species to have insecticidal properties.Among the families reported 3 species of family Sapindaceae, namely Koelreuteria paniculata (extracts of seeds and leaves), Poullinia fuscescens (extracts of seeds and fruits) and Sapindus saponaria (extracts of seeds and fruits) were found to be effective against mosquito larvae.The present study was carried out in a laboratory to assess the larvicidal efficacy of the fruit extract of Sapindus emarginatus Vahl (Sapindaceae), a medicinal plant widely distributed in the dry-zone of Sri Lanka, against A. aegypti larvae.In addition, an attempt was made to identify crude extracts of animal sources with mosquito larvicidal properties.As sea cucumbers are reported to have medicinal values 10 , extracts of dried skin of Holothuria atra Jaeger (Holothuroidea), a sea-cucumber widely present in the shallow waters of Sri Lanka with less consumer or economical demand were tested and the results are reported here.

MEtHoDS AnD MAtErIALS
The fruits of S. emarginatus were collected in mid 2006 from Vavuniya (a locality in the northern dry zone of Sri Lanka) and H. atra was collected in early 2007 from the east coast of Jaffna peninsula of northern Sri Lanka.The collected H. atra was washed in running water and the skin was removed.The fruits of S. emarginatus and skin of H. atra were exposed to direct sunlight for 6-8 h a day for 14 days.The sun dried materials were chopped into small pieces with a knife and were powdered using a table model grinder.The powdered materials were stored in desiccators covered with aluminum foil.In the case of H. atra, initially 74 g of skin material was defatted using 250 mL dichloromethane at 40-45 o C for 8 h.Five grams each of the stored materials of S. emarginatus and H. atra were used for extraction.The crude extraction of S. emarginatus was carried out in a Soxhlet apparatus using 200 mL of absolute ethanol at 78 o C for 8 h.The residue fat-free H. atra was also extracted in Soxhlet apparatus in 250 mL of methanol at 60 o C for 7 h.The crude extracts were concentrated and solvents were removed under reduced pressure using a rotary vacuum evaporator.A portion of both crude extracts was tested for the presence of saponin using a standard method 11 .Stock solutions of desired concentrations (0.45 g/20 mL) of S. emarginatus crude extract and (1 g/20 mL) of H. atra were prepared using distilled water.Larval bioassays were carried out as per WHO techniques with slight modifications under laboratory conditions (29 ± 2 o C).For each bioassay 15 healthy third instar larvae of laboratory reared A. aegypti were tested in plastic containers of 300 mL capacity containing 200 mL of dechlorinated water.In each case 5 different concentrations were prepared using the above stock solutions (ranging from 67.5 -157.5 ppm for S. emarginatus and 62.5 -175 ppm for H. atra) and bioassayed using 3 replicates of each concentrations.Controls were run simultaneously with each set of experiments.Mortality of larvae was recorded after 24 h.The dose-mortality response values were analysed using log-probit regression analysis using the statistical software package 'Statistical Package for the Social Sciences' (SPSS; SPSS 12.0.1 for Windows, Chicago, Illinois, USA) to determine lethal concentrations for the mortalities of 50% and 90% of the treated larvae along with 95% Fiducial Limits.

rESuLtS AnD DISCuSSIon
Bioassays with crude extract of S. emarginatus and H. atra against A. aegypti revealed the LC 50 values of 92.9 and 68.82 ppm respectively (Table 1).Both extracts were positive for the presence of saponin.
S. emarginatus and H. atra could be used as sources to extract active compounds to produce effective mosquito larvicidal agents to control mosquito vectors.Preliminary studies are encouraging and studies on the larvicidal efficacy with other solvents and against other vectors are in progress.

Table 1 :
Laboratory evaluation of crude extracts of S. emarginatus and H. atra against third instar larvae of A. Aegypti