Life Science Archives (LSA)

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(Family:Verbenaceae) PLANT EXTRACTS AGAINST DENGUE MOSQUITO, Aedes aegypti (Linn.) (Diptera: Culicidae) Abstract J. Gokulakrishnan a*, M. Baranitharan b, R. Abirami a, R. Kavikuyil a, Ramya a, J.

1 Available online at Life Science Archives (LSA) ISSN: Volume 1; Issue - 1; Year 2015; Page: Research Article LARVICIDAL AND OVICIDAL ACTIVITIES OF Vitex negundo Linn. (Family:Verbenaceae) PLANT EXTRACTS AGAINST DENGUE MOSQUITO, Aedes aegypti (Linn.) (Diptera: Culicidae) Abstract J. Gokulakrishnan a*, M. Baranitharan b, R. Abirami a, R. Kavikuyil a, Ramya a, J. Deepa a and Baluselvakumar a a Department of Zoology, Annamalai University, Annamalainagar , Tamilnadu, India b Department of Zoology, Poompuhar College (Autonomous), Melaiyur , Tamilnadu, India The present study deals with the investigation of larvicidal and ovicidal activities of hexane, methanol and ethyl acetate leaf extracts of Vitex negundo (V. negundo) against dengue vector, Aedes aegypti (Ae. aegypti). Twenty five early III instar larvae of Ae. aegypti was exposed to various concentrations ( ppm) and was assayed in the laboratory by using the protocol of WHO 1996; the 24 hrs LC 50 of the V. negundo leaf extracts was determined by Probit analysis. For ovicidal activity, slightly modified method of Su and Mulla was performed. The ovicidal activity was determined against Ae. aegypti to various concentrations ranging from ppm under the laboratory conditions. The egg hatch rates were assessed 48 hrs post treatment. The LC 50 values of hexane, methanol and ethyl acetate extracts of V. negundo against early third instar larvae of Ae. aegypti were , and ppm, respectively. Maximum larvicidal activity was observed in the methanol extract followed by ethyl acetate and hexane extracts. No mortality was observed in control. Among three solvent tested the methanol extracts exerted 100% mortality (zero hatchability) at 240 and 300 ppm. From the results, it can be concluded the crude extracts of V. negundo was potential for controlling Ae. aegypti mosquito. Article History Received : Revised : Accepted: Key words: Vitex negundo, Dengue Mosquito, Aedes aegypti, Solvent extracts, Larvicidal activity and Ovicidal activity. 1. Introduction A recent estimate shows that more than 50 million people are at risk of dengue virus exposure worldwide. Annually, there are 2 million infections, 500,000 cases of dengue hemorrhagic fever, and 12,000 deaths (Guha-Sapir and Schimme, 2005). Aedes aegypti is generally known as a vector for an arbo-virus responsible * Corresponding author: J. Gokulakrishnan Tel.: gokulagalya@gmail.com for dengue fever, which is endemic to Southeast Asia, the Pacific island area, Africa, and the Americas. This mosquito also acts as a vector of yellow fever in Central and South America and West Africa. However, Dengue fever has become an important public health problem as the number of reported cases continues to increase, especially with more severe forms of the disease, dengue hemorrhagic fever, and dengue shock syndrome, or with unusual manifestations such as central nervous system involvement (Pancharoen et al., 2002). A. aegypti is a cosmotropical species that

2 Mathalaimuthu Baranitharan /Life Science Archives (LSA), Volume 1, Issue 1, Page 66 to 71, proliferates in water containers in and around houses. Secondary vectors include Aedes albopictus, an important vector in Southeast Asia and that has spread to the Americas, western Africa and the Mediterranean rim, Ae. mediovittatus in the Caribbean, and Ae. polynesiensis and Ae. scutellaris in the western Pacific region. Ae. aegypti breeds in many types of household containers, such as water storage jars, drums, tanks, and plant or flower containers (Muir and Kay, 1998; Honorio et al., 2003; Harrington et al., 2005; Murugan et al., 2011). Mosquito control, in view of their medical importance, assumes global importance. In the context of ever increasing trend to use more powerful synthetic insecticides to achieve immediate results in the control of mosquitoes, an alarming increase of physiological resistance in the vectors, its increased toxicity to non-target organism and high costs are noteworthy (WHO, 1975). Most of synthetic chemicals are expensive and destructive to the environment and also toxic to humans, animals and other non-target organisms. Besides, they are nonselective and harmful to other beneficial organisms. Some of the insecticides act as carcinogenic agents and are even carried through food chain which in turn affects the non-target organism. Therefore alternative vector control strategies, especially effective and low cost are extremely imperative (Piyarat et al., 1974; Kalyanasundaram and Das, 1985). The use of different parts of locally available plants and their various products in the control of mosquitoes has been well established globally by numerous researchers. The larvicidal and ovicidal properties of indigenous plants have also been documented in Tamilnadu, India (Baranitharan and Dhanasekaran, 2014). Based on their traditional experience for hundreds of years, these plants are being subjected to research this results would be utilized to replace the classical drugs. Thus depended upon the ethanobotanical information Vitex negundo was selected to screen for their larvicidal potential against three different, public health significant mosquito vectors. V. negundo is a large aromatic shrub up to 4-5 meters in height is found throughout the greater part of India (Sharma, 2001). In homeopathy, it is being used for the treatment of snake bite (Alam and Gomes, 2003). It is used for its anti inflammatory and analgesic activity (Dharmasiri, 2003). The plant is also known to posses liver protective effects (Avadhoot and Rana, 1991). 2. Methodology 2.1. Sample collection Fresh, mature seeds of are collected from the Yercaudu of Nilgiris hills in India. Plant and seeds are properly authenticated in the Department of Botany, Annamalai University. Fresh, mature seeds are rinsed with distilled water and dried in a shed before powered Extraction method The dried powder of seeds was subjected for oil extraction with hexane, methanol and ethyl acetate using Soxhlet apparatus. After collecting the crude oil, it was then allowed to condense in Rotary Vacuum Evaporator. The condensed crude oil extract was stored in refrigerator until required for investigation for larvicidal, ovicidal and repellent activity Larvicidal activity Standard WHO protocol with slight modifications was adopted for the study (WHO 1996). From the stock solution, the concentration of 50, 100, 150, 200 and 250 was prepared. Early third instar larvae were introduced in 250 ml plastic cups containing 200 ml of water with each concentration. A control was prepared by the addition of acetone to water. Mortality was recorded after 24 hours. For each experiment, four replicates were maintained at a time. The observed percentage mortality was corrected by Abbott s Formula (Abbott, 1925) Ovicidal activity Evaluation of the V. negundo extracts for ovicidal activity was carried out by following the method of Su and Mulla (1998). Eggs were exposed to different concentrations ranging from 70 to 350 ppm. The desired concentrations of the test solutions were achieved by adding 1.0 ml of an appropriate stock solution to 99 ml of tap water. Each eggs raft containing 100 eggs of Ae.

3 Mathalaimuthu Baranitharan /Life Science Archives (LSA), Volume 1, Issue 1, Page 66 to 71, aegypti, are exposed to each dose of extract for The toxicity of different solvent crude 48hr. counting of eggs was done under a extract of V. negundo was tested against larvae of microscope. DSMO served as control. Four Ae. aegypti (Table - 1) and V. negundo extract replicates for each concentration were maintained. reported in the present study showed the larvicidal After 24 hrs of incubation, the egg rafts or eggs and ovicidal activities in the plant signifying their exposed to each concentration were transferred to use in mosquito population control (Table - 2). distilled water cups. The hatch rates were The data were recorded and statistical data calculated by the following formula. regarding LC 50, LC 90, LCL, UCL and chi-square values were calculated. The LC 50 and LC 90 values of hexane, methanol and ethyl acetate extract of V. Mortality at treatment-mortality at control negundo against early third instar larvae of Ae. % Mortality = aegypti 100 were , and ppm and 3.1. Statistical analysis 100-Mortality at control The analysis program probit (Finney, 1971) was used for the determination of LC 50, LC 90 and other statistics at regression, chi-square, slope, mean and standard deviation values were calculated using the SPSS 12.0 software. 3. Results , and ppm respectively. Maximum larvicidal activity was observed in the methanol extract followed by ethyl acetate and hexane extract. No mortality was observed in control. The percentage of egg hatchability of Ae. aegypti with the leaf extract of V. negundo was presented in Table - 3. The methanol extracts exerted 100% mortality (zero hatchability) at 240 and 300 ppm. Table - 1: Larvicidal activity of different solvent leaf extracts V. negundo against instar larvae of Ae. aegypti Species Solvent % mortality±sd Hexane 4.2± ± ± ± ±2.50 Ae. Aegypti Methanol 12.2± ± ± ± ±0.44 Ethyl acetate 10.4± ± ± ± ±1.78 Significant at P 0.05 level, SD- Standard Deviation, PPM- Parts Per Million. Table - 2: Probit analysis of larvicidal activity of V. negundo extract against Ae. aegypti Species Solvent LC 50 (LCL-UCL) ppm LC 90 (LCL-UCL) ppm Slope x 2 Ae. aegypti Hexane ( ) ( ) * Methanol ( ) ( ) * Ethyl acetate ( ) ( ) * LC 50 = Lethal concentration that kills 50% of the exposed parasite, LC 90 = Lethal concentration that kills 90% of the exposed parasite. LCL- Lower Confident Limit, UCL- Upper Confident Limit.

4 Mathalaimuthu Baranitharan /Life Science Archives (LSA), Volume 1, Issue 1, Page 66 to 71, Table - 3: Ovicidal activity of the V. negundo extract against Ae. aegypti Percentage of egg hatch ability Solvents Concentration (ppm) Control Hexane 100.0± ± ± ± ± ±2.50 Methanol 100.0± ± ± ±1.48 NH NH Ethyl acetate 100.0± ± ± ± ±2.19 NH 4. Discussion NH-No hatch ability Recently, bio-pesticides with plant origins are given for use against several insect species, especially disease transmitting vectors, based on the fact compounds of plant origin are safer to use, without phototoxic properties and leave no scum in the environment (Schmutterer 1990). Ruskin (1992) has reported that in mosquitoes, the compounds extracted from Azadirachta indica showed mortality for fourth instar larvae of An. stephensi with LC 50 values of 60 and 43 ppm. The LC 50 and LC 90 values for C. caudata ethyl acetate extract were and mg/l for Cx. quinquefasciatus. Rajkumar and Jebanesan (2005) reported that the leaf extract of Solanum trilobatum reduced egg laying by gravid females of An. stephensi from 18% to 99% compared with ethanol treatment controls at 0.01%, 0.025%, 0.05%, and 0.1%. The bioactive compound azadirachtin (A. indica) showed complete ovicidal activity in the eggs of Cx. Tarsalis and Cx. quinquefasciatus exposed to 10 ppm concentration (Su and Mulla, 1998). Broadbent and Pree (1984) reported that when eggs were directly exposed to high concentrations of the compounds, more chemicals entered the egg shell, which affected the embryogenesis; similarly, longer exposure periods also facilitated the increased penetration of the compounds into the shells, thus increasing their effectiveness. The larvicidal and ovicidal efficacy of different solvent leaf extract of A. indica against An. stephensi. The hatch rates were assessed 48 h after treatment. The LC 50 and LC 90 values of acetone, benzene, chloroform, hexane and methanol extracts of A. indica against An. stephensi larvae in 24 h were 76.29, 58.82, 53.59, 65.84, and , , , and ppm, respectively (Gokulakrishnan et al., 2012). The results that the LC 50 and LC 90 values of ethyl acetate followed by hexane, chloroform and acetone of A. adenophora against Cx. quinquefasciatus larvae I-instar in 24 h were , , and mg/l; , , and mg/l, respectively (Baranitharan and Dhanasekaran, 2014). Elumalai et al., (2012) reported that the E. roseum acetone and methanol extracts of LC 50 values of and ppm, it was that 100% mortality was noted from the acetone and methanol extracts of 100 ppm. 5. Conclusion This result clearly reveals that the leaf extract of V. negundo potential larvicidal and ovicidal agents against the dengue vector A. aegypti and they have demonstrated a synergist act too. Therefore, the present strategy should be promoted in the dengue vector control program. The larvicidal and ovicidal properties of the V. negundo extract under the field conditions should be scrutinized and determined. Besides, further investigation regarding the effect on non-target organism is extremely important and imperative in the near future. Acknowledgements Authors are thankful to the Professor & Head, Department of Zoology and Botany, Poompuhar College, Melaiyur. 6. Reference 1) Abbott WS (1925). A method of computing the effectiveness of an insecticide. J Eco Entomol 18: ) Alam MI and Gomes A (2003). Snake venom neutralization by Indian medicinal plants

5 Mathalaimuthu Baranitharan /Life Science Archives (LSA), Volume 1, Issue 1, Page 66 to 71, (Vitex negundo and Emblica officinalis) root extracts. J Ethnopharmacol 86: ) Avadhoot Y and Rana AC (1991). Hepatoprotective effect of Vitex negundo against carbon tetrachloride-induced liver damage. Arch Pharm Res 14: ) Baranitharan M and Dhanasekaran (2014). Mosquito larvicidal properties of Commiphora caudata (Wight &Arn.) (Bursaceae) against Aedes aegypti (Linn.), Anopheles stephensi (Liston), Culex quinquefasciatus (Say). Int J Curr Microbiol App Sci 3(6) ) Baranitharan M and Dhanasekaran S (2014). Evaluation of larvicidal activity of Ageratina adenophora (Spreng.) King &H.Rob against Culex quinquefasciatus (Say). Int J Pharma Bio Archives 5(2): ) Baranitharan M and Dhanasekaran S (2014). Mosquitocidal efficacies of medicinal plant of Coleus aromaticus Benth (Lamiaceae) leaf extracts Chikungunya vector, Aedes aegypti (Linn.) (Diptera: Culicidae). Int J Curr Res Chemi Pharma Scien 1(5): ) Broadbent AB, Pree DJ (1984). Effects of diflubenzuron and bay SIT 8514 on the oriental fruit moth and Oblique banded leaf roller. J Econ Ent 77: ) Dharmasiri MG, Jayakody JR, Galhena G, Liyanage SS, Ratnasooriya WD (2003). Antiinflammatory and analgesic activities of mature fresh leaves of Vitex negundo. J Ethnopharmacol 87: ) Finney DJ (1971). A ststistical treatment of the sigmoid response curve. In: Probit analysis. Cambridge University Press, London 1971.pp ) Gokulakrishnan J, BaluSelvakumar, Elumalai K and Krishnappa K (2012). Mosquito larvicidal and ovicidal efficacy of Ariitolochia indica Linn (Aristolochiaceae) leaf extracts against malarial vector Anopheles stephensi Liston (Diptera: Culicidae). Int J Curr Lif Sci 2(10): ) Guha-Sapir D and Schimme B (2005). Dengue fever: new paradigms for a changing epidemiology. Emerg Themes Epidemiol 12, ) Harrington LC, Scott TW, Lerdthusnee K, Coleman RC, Costero A, Clark GG, Jones JJ, Kitthawee S, Kittayapong P, Sithiprasasna R, Edman JD (2005). Dispersal of the dengue vector Aedes aegypti within and between rural communities, part 1. Am J Trop Med Hyg 72, ) Honorio NA, Silva WC, Leite PJ, Gonc alves JM, Lounibosn LP, Lourenc o-de-oliveira R (2003). Dispersal of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in an urban endemic dengue area in the state of Rio de Janeiro, Brazil. Mem Inst Oswaldo Cruz 98, ) Kalyanasundaram M, Das PK (1985). Larvicidal and synergistic activity of plant extracts for mosquito control. Indian J Med Res 82, ) Muir LE and Kay BH (1998). Aedes aegypti survival and dispersal estimated by markrelease-recapture in northern Australia. Am J Trop Med Hyg 58, ) Murugan K, Hwang JS, Kovendan K, Prasanna Kumar K, Vasugi C, Naresh Kumar A (2011). Use of plant products and copepods for control of the dengue vector, Aedes aegypti. Hydrobiologia 666, ) Pancharoen C, Kulwichit W, Tantawichien T, Thisyakorn U, Thisyakorn C (2002). Dengue infection: a global concern. J Med Assoc Thai 85, ) Piyarat SWK, Freed M, Roy S (1974). Biologically active plant extract for the control of mosquito larvae. Mosq News 34, ) Rajkumar S, Jebanesan A (2005). Oviposition deterrent and skin repellent activities of Solanum trilobatum leaf extract against the malarial vector Anopheles stephensi. J Insect Sci 5:15 20) Ruskin FR (1992). Neem: a tree for solving global problems. Washington: National Academy Press; p ) Schmutterer H (1990). Properties and potential of natural pesticides from the neem tree Azadirachta indica. Annu Rev Entomol 35: ) Sharma (2001). Insecticidal, antifeedent and growth Inhibitory activities of essential oils of some medicinal plants. Journal of Medicinal and Aromatic Plant Science

6 Mathalaimuthu Baranitharan /Life Science Archives (LSA), Volume 1, Issue 1, Page 66 to 71, ) Su T and Mulla MS (1998). Ovicidal activity of neem products (Azadirachtin) against Culex tarsalis and Culex quinquefasciatus (Diptera: Culicidae). J Am Mosq Control Assoc 14: ) WHO (1996). Report of the WHO informal consultation on the evaluation and testing of insecticides. CTD/WHOPES/IC/ Giveaway: Control of Tropical Diseases Division. 25) World Health Organization (1975). Manual on Practical Entomology in Malaria Part I. Who division of malaria and other parasitic diseases, pp. 160.

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