International Journal of Environmental Biology

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1 Available online at International Journal of Environmental Biology ` Universal Research Publications. All rights reserved ISSN X Original Article LARVICIDAL ACTIVITY OF PEDILANTHUS TITHYMALOIDES (L.) POIT (EUPHORBIACEAE) LEAF AGAINST THE DENGUE VECTOR AEDES AEGYPTI (L.) (DIPTERA: CULICIDAE) R. Raveen 1, P. Dhayanidhi 1, K. Dhinamala 1, S. Arivoli 2 and Samuel Tennyson 1 * 1 Department of Zoology, Madras Christian College, Chennai , Tamilnadu, India 2 Department of Advanced Zoology and Biotechnology, Loyola College, Chennai , Tamilnadu, India *Corresponding author: Dr. Samuel Tennyson, Assistant Professor, Department of Zoology Madras Christian College, Chennai , Tamilnadu, India E.mail: samtennyson@gmail.com Received 21 March 2012; accepted 09 April 2012 Abstract The crude ethanolic leaf extract of Pedillanthus tithymaloides was evaluated for larvicidal activity against the dengue vector, Aedes aegypti at concentrations of 0.01, 0.02, 0.05, 0.1, 0.2 and 0.4 per cent. Mortality was observed for 24 and 48 hours. The crude ethanolic leaf extract of Pedilanthus tithymaloides exhibited a moderate level of larvicidal property with a LC 50 value of 0.32 and 0.12 per cent after 24 and 48 hours. Further investigations are needed to elucidate this activity against a wide range of all stages of mosquito species and also the active ingredients of the extract responsible for larvicidal activity should be identified Universal Research Publications. All rights reserved Key words: Pedillanthus tithymaloides, ethanolic leaf extract, Aedes aegypti, larvicidal activity INTRODUCTION Mosquitoes are vectors of serious human diseases and are the single most important group of insects in terms of public health importance (WHO, 2010). Over two billion people, primarily in tropical countries are at risk from mosquito-borne diseases (Service, 1993). Several mosquito species belonging to the genera Anopheles, Culex and Aedes are vectors for the pathogens of various diseases like Malaria, Filariasis, Japanese encephalitis, Dengue fever, Dengue haemorrhagic fever and Chikungunya causing millions of deaths every year (Rahuman et al., 2009; Borah et al., 2010). Aedes aegypti (L.) is one of the most important disease transmitting and nuisance causing mosquito, which transmits Dengue and Chikungunya is domestic in its habit and is found breeding almost entirely in the vicinity of human dwellings (Jayakumar et al., 2007). Chemical measures in public health programs were initially considered likely to decrease mosquito population, but have failed since constant use of chemical insecticides led to the disruption of natural biological control system. Moreover, problems created by using synthetic insecticides included the development of mosquito resistance, environmental pollution, and undesirable effects on humans, mammals and non-target organisms (Brown, 1986). In an attempt to resolve these problems, attention to insecticides of natural origin, particularly plant derived product, has been recently revived (Chaithong, 2006). In recent years interest in plant-based product has been revived because of the development of resistance and possible toxicity hazards associated with synthetic insecticides. Phytochemicals obtained from the huge diversity of plant species are important source for safe and biodegradable chemicals, which can be screened for mosquitoicidal activities (Mittal et al., 2003). More than 2,000 plant species have been known to produce chemical factors and secondary metabolites of value in pest control programs (Ahmed et al., 1984). Sukumar et al. (1991) reported 99 families, 276 genera and 346 species to have insecticidal properties and a review by Shaalan et al. (2005) treated this aspect in detail. 36

2 Pedilanthus tithymaloides (L.) Poit belongs to the family Euphorbiaceae and its leaves are used for treating headache and leaf decoction in abdominal disorder. The latex is used in the treatment of venereal diseases and to relieve sprain while the stem is used as an abortifacient, as antivenom for snake bite and also in treating rheumatism (Jain and Srivastava, 2005). The plant possess anticancer (Bunyapraphatsara and Chokchaichareonporn, 2000), anti-inflammatory and antifungal (Abreu et al., 2006), antimycobacterial (Mongkolvisut et al., 2007) and antioxidant properties (Abreu et al., 2008). The phytochemical constituents present in the leaf of the plant include terpenes, terpenoids, quercitin, isoquercitrin, flavonoids, steroids and phenols. Besides these, the latex of the plant is reported for mosquito larvicidal property (Parkash et al., 2009) and the leaf extract against Aedes aegypti and Culex quinquefasciatus (Rahuman et al., 2008; Kamalakannan et al., 2010). Therefore, the present study was carried out to evaluate the larvicidal activity of Pedilanthus tithymaloides leaf extracts against the dengue vector, Aedes aegypti (L.). MATERIALS AND METHODS Plant collection and extraction Mature fresh leaves of Pedilanthus tithymaloides collected from places in and around Chennai, Tamilnadu, India was shade dried at room temperature and powdered. Dried and powdered leaves (1 kg) was then macerated with 3 L of ethanol for a period of 96 hours and filtered. The filtrate was then concentrated at reduced temperature on a rotary evaporator yielding 11.11g of a dark greenish brown, gummy extract with a pungent odour. The crude extract thus obtained was lyophilized and a stock solution of 1,00,000 ppm prepared by adding adequate volume of acetone was refrigerated at 4 0 C until testing for bioassay. Test mosquitoes Aedes immatures collected from stagnant water at various places in Chennai, Tamilnadu, India were transported to the laboratory in plastic containers. In the laboratory, the immature mosquitoes were transferred to enamel larval trays until adult emergence. After emergence, the adult mosquitoes were identified and species confirmed before rearing. Cyclic generations of Aedes aegypti were maintained separately in two feet mosquito cages in an insectary. Mean room temperature of 27 ±2 0 C and a relative humidity of per cent were maintained in the insectary. The adult mosquitoes were fed on 10 per cent glucose solution. For continuous maintenance of mosquito colony, the adult female mosquitoes were blood fed with laboratory reared albino mice. Ovitraps were placed inside the cages for egg laying. The eggs laid were then transferred to enamel larval trays maintained in the larval rearing chamber. The larvae were fed with larval food (dog biscuits and yeast in the ratio 3:1). The larvae on becoming pupae were collected, transferred to plastic bowls and kept inside mosquito cage for adult emergence. Larvicidal bioassay Standard WHO (1996) protocol with slight modifications was adopted for the study. The tests were conducted in glass beakers. Mosquito immature particularly third instar larvae were obtained from laboratory colonized mosquitoes of F 1 generation. From the stock solutions, concentrations of 0.01, 0.02, 0.05, 0.1, 0.2 and 0.4% was prepared. Twenty healthy larvae were released into each 250 ml glass beaker containing 200 ml of water and test concentration. Mortality was observed for 24 and 48 hours after treatment. A total of three trials were carried out. Three replicates for each concentration and a control were run during each trial. Controls were run simultaneously. Control was prepared by the addition of acetone to water. However, when the control mortality ranged from five to twenty per cent, the observed percentage mortality was corrected by Abbott s (1925) formula. Statistical analysis SPSS 11.5 version package was used for determination of LC 50 and LC 90 (SPSS, 2007). Data from mortality and effect of concentrations were subjected to analysis of variance and Chi-square analysis. Differences between the treatments was determined by Tukey s test (P<0.05). RESULTS AND DISCUSSION The results of phytochemical screening of Pedilanthus tithymaloides ethanolic leaf extract indicated the presence of flavonoids, phenols, alkaloids, steroids, tannins, saponins, anthraquinones, proteins, carbohydrates, glycosides and steroids (Table 1). Results of the larvicidal effects of ethanolic leaf extract of Pedilanthus tithymaloides against Aedes aegypti reported in the present study exhibit the presence of larvicidal properties present in the plant suggesting their use in mosquito larval population control (Tablei2,i3).iThe ethanolic extract of Pedilanthus tithymaloides leaves showed LC 50 value of 0.32 and 0.12 per cent after 24 and 48 hours. Man has struggled hard to eradicate mosquitoes by adopting various techniques. Since the discovery of DDT, control of mosquitoes has been mostly through synthetic chemicals ilke malathion and synthetic pyrethroids which was used conventionally. However, these non-biodegradable and hazardous chemicals have created environmental pollution during the last five decades and non-target organisms and man have been severely affected by these toxic pesticides (Devine and Furlong, 2007). This has necessitated the need for environmental friendly, 37

3 Table 1: Phytochemical screening of Pedilanthus tithymaloides ethanolic leaf extract S. No. Phytochemical constituents Results 1 Alkaloids + 2 Triterpenoides - 3 Coumarins - 4 Steroids + 5 Tannins ++ 6 Saponins + 7 Flavones + 8 Quinones - 9 Flavanones - 10 Anthocyanins - 11 Anthraquinones + 12 Phenols Proteins + 14 Carbohydrates Glycosides Aminoacids present in high quantity ++ - moderately present + - present in low quantity - - absent Table 2: Per cent larval mortality of Aedes aegypti against the leaf extracts of Pedilanthus tithymaloides Concentration (%) Extract h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h Ethanol 5.33 ± ± (26.66) (40.00) (33.33) (53.33) (40.00) (55.00) (41.66) (56.66) (46.66) (61.66) (48.33) (71.66) Control 0.0 ±0 0.0 ±0 0.0 ±0 0.0 ±0 0.0 ±0 0.0 ±0 0.0 ±0 0.0 ±0 0.0 ±0 0.0 ±0 0.0 ±0 0.0 ±0 Values are mean (%) of the three-replication of three trials ±standard deviation. Figures in paranthesis denote per cent larval mortality Table 3: Probit analysis of larvicidal efficacy of ethanolic leaf extracts of Pedilanthus tithymaloides against Aedes aegypti LC 50 (%) LC 90 (%) F value Chi-square 24h 48h 24h 48h 24h 48h 24h 48h * 40.50* 25.14* 26.82* *Significant at P < level biodegradable methods for vector control. In recent years, the focus is mainly on the use of environmental friendly and biodegradable materials as pesticides. Several phytochemicals extracted from various parts of the plant have been found to be effective in vector control (Sukumar et al., 1991). Numerous workers have tested the efficacy of several plant extract using different mosquito species (Sukumar et al., 1991; Shaalan et al., 2005; Sakthivadivel and Daniel, 2008; Samuel et al., 2012). A variety of plant species have been identified to have medicinal properties from ancient times. Some of these medicinal plants have been discovered to have insecticidal properties also. The screening of local medicinal plants for mosquito larvicidal activity may eventually lead to their use in natural product-based mosquito abatement practices because they provide an alternative to synthetic insecticides as they are considered safe, biodegradable and can often be obtained from local sources (Prabakar and Jebanesan, 2004; Shaalan et al., 2005; Arivoli and Samuel, 2011a, b). 38

4 The ethanolic extract of Apium graveolens exhibited larvicidal effect against the immatures of Aedes aegypti (Choochote et al., 2004). Gu et al. (2009) reported that among the ethanolic extracts of wood, bark and leaf extract of Cryptomeria japonica against Aedes aegypti and Aedes albopictus, wood extract exhibited the best larvicidal activity. Quershi et al. (1986) assayed extracts of Erva tamia coronaria against fourth instar larvae of Aedes aegypti and found the alcoholic extracts to be highly toxic whereas the petroleum ether extract had no mortality. The crude chloroform extract of seeds of Millettia dura showed high activity against second instar larvae of Aedes aegypti (Yenesew et al., 2003). The methanol and ethanol flower extracts of Lantana camara was found to have a higher larvicidal rate against Aedes aegypti (Kumar and Maneemegalai, 2008). Supavarn et al. (1974) reported that methanol extracts of whole plants of Anethum graveolens demonstrated low toxicity to the fourth instar larvae of Aedes aegypti In the present study, ethanolic leaf extract of Pedilanthus tithymaloides was reported to exhibit moderate level of larvicidal property. This biological activity is attributed to the phytochemical compounds/constituents in the leaves, including flavonoids, phenols, and steroids that together or independently produce mortality effects in Aedes aegypti. In this regard, Pedilanthus tithymaloides phytochemicals warrant further study for use as natural product based compounds for mosquito control and as an adjunct and/or alternative to the use of synthetic pesticides for vector control. Further investigations are needed to elucidate this activity against a wide range of all stages of mosquito species and also the active ingredients of the extract responsible for larvicidal activity should be identified. REFERENCES Abbott, W.S. (1925). A method of computing the effectiveness of an insecticide. Journal of Economic Entomology, 18: Abreu, P., Matthew, S., Gonzalez, T., Costa, D., Segunda, M.A. and Fernandes, E. (2006). Anti-inflammatory and antioxidant activity of a medicinal tincture from Pedilanthus tithymaloides. Life Sciences, 78(14): Abreu, P., Matthew, S., Gonzalez, T., Vanickova, L., Costa, D., Gomes, A., Segundo, M.A. and Fernandes, E. (2008). Isolation and Identification of antioxidants from Pedilanthus tithymaloides. Journal of Natural Medicine, 62(1): Ahmed, S., Graivge, M., Hylin, J.W., Mitchell, W.C. and Listinger, J.A. (1984). Some promising plant species for use as pest control agents under traditional farming system. In: Schmutterer, H. and Ascher, K. R. S., (eds) Proc. 2 nd International Neem Conference, Rauischolzhausen, Germany, 1983 pp GTZ, Eschborn, Germany. Arivoli, S. and Samuel, T. (2011a). Larvicidal efficacy of Cleistanthus collinus (Roxb.) (Euphorbiaceae) leaf extracts against vector mosquitoes. Asian Pacific Journal of Tropical Biomedicine, S275-S277. Arivoli, S. and Samuel, T. (2011b). Effects of Leucas aspera (Willd.) Spreng (Lamiaceae) leaf extracts against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus (Diptera: Culicidae). World Applied Sciences Journal, 14(4): Borah, R., Kalita, M.C., Kar, A. and Talukdar, A.K. (2010). Larvicidal efficacy of Toddalia asiatica (Linn.) Lam against two mosquito vectors Aedes aegypti and Culex quinquefasciatus. African Journal of Biotechnology, 9(16): Brown, A.W.A. (1986). Insecticide resistance in mosquitoes: pragmatic review. Journal of the American Mosquito Control Association, 2: 123:140. Chaithong, U., Choochote, W., Kamsuk, K., Jitpakdi, A., Tippawangkosol, P., Chaiyasit, D., Champakaew, D., Tuetun, B. and Pitasawat, B. (2006). Larvicidal effect of pepper plants on Aedes aegypti (L.) (Diptera: Culicidae). Journal of Vector Ecology, 31(1): Choochote, W., Tuetun, B., Kanjanapoothi, D., Rattanachanpichai, E., Tippawangkosol, P., Riyong D. and Pitasawat, B. (2004). Potential of crude seed extract of celery, Apium graveolens L., against the mosquito Aedes aegypti (L.) (Diptera: Culicidae). Journal of Vector Ecology, 29(2): Devine, G.J. and Furlong, M.J. (2007). Insecticide use: Contexts and ecological successions. Agriculture and Human Values, 24: Gu, H.J., Cheng, S.S., Huang, C.G., Chen, W.J. and Chang, S.T. (2009). Mosquito larvicidal activities of extractives from black heartwood type Cryptomeria japonica. Parasitology Research, 105: Jayakumar, M., Samuel, T. and William, S.J. (2007). Aedes: An ever dangerous enemy in our society. In: Defeating the public enemy, the mosquito: A real challenge. (Ed.) William, S.J., Loyola Publications, Chennai, PP Kamalakannan, S., Madhiyazhagan, P., Dhandapani, A., Murugan, K. and Barnard, D. (2010). Pedilanthus tithymaloides (Euphorbiaceae) leaf extract phytochemicals: Toxicity to the filariasis vector Culex quinquefasiatus 39

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