International Journal of Medicine and Biosciences

Int J Med Biosci. 2012; 1(3): 33-41 International Journal of Medicine and Biosciences www.ijmbonline.com Mosquito Larvicidal, Oviposition deterrent and Repellent properties of Acalypha indica L extracts against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus S. Vijaya kumar 1*, Panagal Mani 2, T.M.M. John Bastin 2 and G. Ravikumar 1 1 Department of Zoology and Biotechnology, A.V.V.M Sri Pushpam College, Poondi, Thanjavur-613503, Tamil Nadu, India 2 Department of Biotechnology, Tamilvel Umamaheswaranar Karanthai Arts College, Thanjavur- 613002, Tamil Nadu, India Received 12 June 2o12; accepted 20 July 2012; published online 31 July 2012 Research Article Parasitology Abstract The leaf extract of Acalypha indica with different solvents viz., acetone, chloroform, hexane, petroleum ether and ethanol were tested for larvicidal, oviposition deterrent and repellent activities against the mosquitoes Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus. Larval mortality was observed and recorded after 24 h exposure period. In the present study, bioassay-guided fraction of Acalypha indica led to the separation and identification of a saponin as a potential mosquito larvicidal compound, with LC50 and LC90 value of 128.24, 230.40 ppm against Aedes aegypti, and 248.44, 447.19 ppm against An. stephensi and 228.6, 411.48 ppm against Cx. quinquefasciatus respectively. In oviposition deterrent activity, the highest concentration of (0.1%) Acalypha indica produce (99.4%) against Aedes aegypti, 98.0% against Aedes aegypti and 97.5 % against Culex quinquefasciatus respectively. Skin repellent test at 0.02 ppm concentration of Acalypha indica gives the complete protection time ranges from 68.4 to 122 minutes. The plant exerted the highest protection time of 126.2 minutes. GC-MS analysis data confirmed the identification of the active compound. This study investigates the potential of crude extracts from commonly used medical herbs in India as an environmentally safe measure to control the vector of dengue, malaria, and lymphatic filariasis. Keywords: Acalypha indica, Larvicide, Repellent, Filariasis, Malaria Introduction Mosquitoes are one of the most medically significant vectors, and they transmit parasites and pathogens, which continue to have devastating impact on human beings. Its caused vector borne diseases are major health problems in many countries [14].. Mosquito borne diseases such as malaria, lymphatic filariasis, dengue, yellow fever and Japanese encephalitis contribute significantly to human disease burden and death, in addition to poverty and social delibility in tropical countries [11]. Every year at least 500 million people in *Corresponding Author S. Vijaya kumar Copyright 2012, International Journal of Medicine and Biosciences All Rights Reserved the world suffer from one or the other tropical diseases that include malaria, lymphatic filariasis, schistosomiasis, dengue, trypanosomiasis and leishmaniasis [13]. Ae. aegypti, a vector of dengue, is widely distributed in the tropical and sub-tropical zones of our country. In fact two-thirds of the world s population lives in areas infested with these vectors are now endemic in more than 100 countries and threaten the health of approximately 2.5 billion people [3]. An. stephensi is the primary vector of malaria which result 300-500 million new infections and 1-3 million deaths from malaria worldwide [18]. Cx. quinquefasciatus is an impor-tant vector of filariasis in tropical and sub-tropical regions. According to WHO, about 90 million people worldwide are infected with Wucheria bancrofti, the lymphathic dwelling parasite. In India alone 25 million

S. Vijaya kumar et al., / Int J Med Biosci. 2012; 1(3):33-41 34 people harbour microfilaria and 19 million people suffer from filarial disease manifestations [19,14]. DDT and malathion against adult mosquitoes in the last five decades demonstrated a successive move, but this success was short lived due to the development of resistant to many mosquito strains, ecological imbalance and harm to mammals [10]. Plants may be a source of alternative agents for control of mosquitoes, because they are rich in bioactive chemicals, are active against limited number of species including specific target-insects and are biodegradable, [24] because mosquitoes develop genetic resistance to synthetic insecticides [27] and even tobiopesticides [25]. The leaves of A.indica are antimalarial, parasiticide, plasmodicide,protisticide, antibacterial, antimutagenic, cancer preventive, pesticide, etc. A decoction of the leaves and the vapours are employed in baths for the treatment of febrile, catarrhal and rheumatic affections. Hence, the present study undertaken to examine the larvicidal, oviposition deterrent and repellent properties of A. indica leaves extracts against hazardous mosquitoes Cx.quinquefasciatus, An. stephensi and Ae. aegypti. Materials and Methods Sample collection and Extraction The leaves of A.indica were collected from plants growing in Thanjavur district of Tamil Nadu, India. After proper identification, the leaves were air dried under shade for 10-12 days at room temperature of 25 ±2 C. About 500 g of dry powders was extracted with acetone, hexane, petroleum ether, chloroform and ethanol using soxhlet apparatus. The extraction was continued for 24 h and then filtered and kept in hot air oven at 40 Cfor 24 h. The residue was kept separately in air tight containers and stored in a deep freezer. Phytochemical analysis The various extract of leaves of A.indica subjected to following test for the identification of its various active constitutions by standard method. Alkaloids were identified by Dragendroff s test, flavonoids and were identified by lead acetate test, carbohydrates were identified by Fehling s test, proteins were identified by Million s test, phenols were identified by Libermann s test and tannins were identified by Ferric chloride test. Gas Chromatography Mass Spectroscopy (GC-MS) analysis The A.indica powdered sample (20 g) were soaked and dissolved in 75 ml of hexane, petroleum ether and ethanol for 24 h. Then the filtrates were collected by evaporated under liquid nitrogen. The GC-MS analysis was carried out using a Clarus 500 Perkin- Elmer (Auto System XL) Gas Chromatograph equipped and coupled to a mass detector Turbo mass gold Perking Elmer Turbomas 5.2 spectrometer with an Elite-1 (100% Dimethyl ply siloxane), 300 m x 0.25 mm x 1 µ m df capillary column. The instrument was set to an initial temperature of 110 C, and maintained at this temperature for 2 min. At the end of this period, the oven temperature was raised upto 280 C, at the rate of an increase of 5 C/min, and maintained for 9 min. Injection port temperature was ensured as 250 C and Helium flow rate as 1 ml/min. The ionization voltage was 70 ev. The samples were injected in split mode as 10:1. Mass Spectral scan range was set at 45-450 (mhz). The chemical constituents were identified by GC-MS. The fragmentation patterns of mass spectra were compared with those stored in the spectrometer database using National Institute of Standards and Technology Mass Spectral database (NIST-MS). The percentage of each component was calculated from relative peak area of each component in the chromatogram. Mosquito culture Cx. quinquefasciatus, An. stephensi and Ae. aegypti were collected from stagnant water at various places within Thanjavur. They were maintained at 27±2 C; 70-80% relative humidity and a photoperiod of L:D 14:10 (light/dark). Larvae were fed with 3:1 mixture of dog biscuits and yeast powder. Pupae were transferred from the trays to a cup containing tap water and placed in screened cages (23x23x32 cm dimension) for adult emergence. Cx. Quinquefasciatus, An. stephensi and Ae. aegypti adult mosquitoes were reared in the each glass cages of 45x38x38 cm separately covered with a plastic screen, with a glass topand a muslin sleeve for access. The adult colony was provided with 10% sucrose was available at all times. After three days, ovitrap was kept inside the cages and the eggs were collected and transferred to the enamel trays. They were maintained at the same condition.

S. Vijaya kumar et al., / Int J Med Biosci. 2012; 1(3):33-41 35 Larvicidal Assay Larvicidal activity was evaluated using WHO method with slight modification. Twenty five number of early IV instar mosquito larvae of Ae. aegypti, An. stephensi and Cx. Quinquefasciatus were released separately in 500ml capacity of beaker containing 250 ml of water and to treat in various concentrations of plant extracts, and then were distributed in each of the replicates. The experiments were carried out at 25 ± 2 C. The test mosquitoes were a laboratory strain of Ae. aegypti, An. stephensi and Cx. quinquefasciatus. The mosquito colonies were maintained at 25-28 C and relative humidity 80-90% under a photoperiod of 14:10 h (light/night) without exposure to any insecticides and pathogen. During this course, larva were feed on finely ground dog biscuits and yeast powered (1:1) and the adult colony was provide with 10% glucose. Oviposition deterrent activity The oviposition deterrent test of ethanol extract of A.indica was performed using the Xue method [28]. Fifteen gravid female mosquitoes Ae. aegypti, An. stephensi and Cx. quinquefasciatus were (6 days old, 4 days after blood feeding) transferred to each mosquito cage (45x38x38 cm) and separately covered a plastic screen, with a glass top and a muslin sleeve for access. A 10% sucrose solution was available at all times. Serial dilutions of leaf extract of each test plant were made in ethanol. Enamel bowls containing 100 ml of water treated with leaf extract to obtain test solutions of 0.01, 0.025, 0.050, 0.075 and 1.0%. Two enamel bowls holding 100 ml of water were placed in the opposite corners of each cage, one treated with the test material and the other with a solvent control (1 ml). The positions of the bowls were alternated between the different replicates so as to nullify any effect on oviposition. Five replicates for each concentration were run, with cages placed side by side for each bio- assay. All experiments were run at ambient temperature (27 ± 2 C) with a relative humidity of 70-80%. After 24 h,the number of eggs laid in treated and control bowls were recorded. The per cent effective repellency for each plant leaf extract concentration was calculated using the following formula: Where, ER(%) = NC- NT 100 (%) NC ER = Per cent effective repellency NC = Number of eggs in control Skin repellent activity The duration of protection period from all the three mosquito bites provided by test plant leaf extracts were determined using the method of Fradin and Day [5].From the stock solution of ethanol extract of A.indica, each test plant leaf extract was diluted with ethanol to obtain test solutions of 0.001, 0.005, 0.01, 0.015 and 0.02%. For each test solution, 10 diseases free, laboratory reared unfed adult mosquitoes of test species that were between 8 and 14 days old were introduced into separate laboratory cages (45x38x38 cm). Before each test, the skin of volunteers was washed with unscented soap, and the leaf extract of each test plant being tested was applied from elbow to fingertips. After the application, the arm was not rubbed, touched, or wetted. An arm treated with ethanol was served as control. In each cage, one arm of the volunteer was inserted into the cage for one test solution. Both test solution and control were repeated five times in separate cages. In each replicate, different volunteers were used to nullify any effect of skin differences on repellency. Volunteers were asked to follow the testing protocol. Volunteers conducted their test of each concentration by inserting their treated arm and control arm into a separate cage for one minute at every five minutes. If they were not bitten within 20 min, then the arms were reinserted for one minute at every 15 min, until the first bite occurred. Statistical analysis The larvicidal bio-assays and per cent control mortality were calculated using Abbyy s transformation [1]. LC50 and LC90 (lethal concentration causing 50 and 90 per cent mortality) were calculated using Probit analysis [4]. Data from larval mortality was subjected to an analysis of variance. Statistical software SPSS 11.5 was used for data analysis. Results The Hexane leaf extract of A. indica(figure 1) was subjected to GC-MS and the isolated the active compounds such as Benzene, 1, 2, 3-trimethyl (1.08%), piperidine-2-5-dione 69(2.78%), 3, 7, 11, 15-Tetramethyl- 2-hexadecen-1-01 (9.89%), n-hexadecanoic acid (11.57%), phytol (12.94%), 9, 12, 15-octadecatrienoic acid (43.89%), octadecanoic acid (2.54%), 9, 12, octadecanoic acid(0.88%), heptacosane (1.75%) and squalene (12.69%). were reported to possess larvicidal activity and also analgesic, antidiabetic, antibacterial,antifungal properties (Table 1).

S. Vijaya kumar et al., / Int J Med Biosci. 2012; 1(3):33-41 36 Fig.1. GC-MS chromatogram of hexane extract of Acalypha indica S.No. RT Name of the Compound Molecular formula MW Peak Area % 1. 3.90 Benzene, 1,2,3-trimethyl C9H12 120 1.08 2. 5.81 Piperidine-2,5-dione C5H7NO2 113 2.78 3. 14.78 3,7,11,15-Tetramethyl-2- hexadecen-1-ol C20H40O 296 9.98 4. 16.56 n-hexadecanoic acid C16H32O2 256 11.57 5. 18.86 Phytol C20H40O 296 12.94 6. 19.36 9,12,15-Octadecatrienoic acid, (Z.Z.Z)- C18H30O2 278 43.89 7. 19.59 Octadecanoic acid C18H36O2 284 2.54 8. 20.30 9,12-Octadecadienoic acid(z,z)- C18H32O2 280 0.88 9. 26.03 Heptacosane C27H56 380 1.73 10. 29.55 Squalene C30H50 410 12.69 Table.1. Qualitative and quantitative determination of biochemical constituents in A.indica by GC-MS

S. Vijaya kumar et al., / Int J Med Biosci. 2012; 1(3):33-41 37 Hazardous Mosquitoes Solvent used for extraction LC50 (ppm) (LCL - UCL) Acetone 251.17 (249.08-253.26) Chloroform 588.76 (585.91-591.61) Ae. Aegypti Hexane 128.24 (125.24-131.24) (247.21 - ) Petroleum ether 220.42 (217.71-223.13) LC90 (ppm) (LCL - UCL) 462.17 (456.36-467.98) 1059.78 (1.054.84-1064.72) 230.40 (226.57-234.23) 396.75 (391.53-1.97) χ2 value (df = 4) 0.81 1.14 0.56 0.70 Ethanol 665.95 (663.54-668.36) 1198.85 (1194.57-1203.13) 2.36 Acetone 367.17 (364.39-369.95) Chloroform 672.74 (669.84-6575.64) An. Stephensi Hexane 298.24.24 (295.59-300.89) Petroleum ether 248.44 (246.29-250.59) 660.9 (656.18-665.62) 0.92 1210.9 (1206.55-1215.25) 536.83 (531.96-541.73) 447.19 (441.94-452.44) 1.24 0.96 1.14 Ethanol 988.42 (985.57-991.27) 1779.62 (1774.42-1784.82) 1.28 Acetone 228.6 (225.99-231.21) 411.48 (406.27-416.67) 1.64 1.64 Cx. quinquefasciatus Chloroform 386.4 (383.6-389.2) Hexane 422.9 (420.65-423.15) 695.55 (689.63-701.47) 760.38 (755.63-765.13) 0.98 1.72 Petroleum ether 528.2 (525.34-531.06) 950.80 (1294.82-1305.82) 0.98 Ethanol 656.4 (653.98-658.82) 1181.55 (1176.69-1186.41) 2.14 Table.2. Lethal concentration values of Acalypha indica leaf extract against the Ae. Aegypti, An. Stephensiand Cx. quinquefasciatus larvae Control-Nil mortality significant at p<0.05 level; Values were based on different concentrations and four replications with 25 larvae in each; LC50 lethal concentration that kills 50 per cent of the exposed larvae in 24 hrs; LC90 lethal concentration that kills 90 per cent of the exposed larvae in 24 hrs; χ2 Chi-Square; df - degrees of freedom; LCL - Lower confidential limit; UCL - Upper confidential limit.

S. Vijaya kumar et al., / Int J Med Biosci. 2012; 1(3):33-41 38 A. indica was effectively possessing larvicidal activity against these three mosquitoes. However, the highest larvicidal potency with LC50 and LC90 value of hexane extract depicted 128.24 ppm and 230.40 ppm against Ae. Aegypti. While, the petroleum ether extract indicated 248.44 ppm and 447.19 ppm of LC50 and LC90 value against An. Stephensi. Whereas, 228.6 ppm and 411.48 ppm of LC50 and LC90 value were observed acetone extract against Cx. Quinquefasciatus respectively (Table 2). In laboratory oviposition deterrent tests, the leaf extract of A.indica greatly reduced the number of eggs deposited by gravid Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus at several concentrations (Table 3). At the highest concentrations the extract reduced egg laying by (99.4%) against Aedes aegypti. Lower oviposition deterrent was recorded in Anopheles stephensi (98.0%) and Culex quinquefasciatus (97.5 %) respectively. The results from the skin repellent activity of A. indica leaf extract against three mosquitoes are summarized in Table 4. The highest protection time of 122 min was observed at the highest concentration of 0.02 ppm against Aedes aegypti followed by 119 min and 116 min observed in Anopheles stephensi and Culexquinquefasciatus respectively. The lowest concentration of 0.001 ppm provided the protection time of 68.4, 62.4 and 60.4 min against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus, respectively.

S. Vijaya kumar et al., / Int J Med Biosci. 2012; 1(3):33-41 39 Concentratio n (ppm) Aedes aegtpti Complete protection time (min) Anopheles stephensi Culex.quinquefasciatu s 0.02Z 122.6± 1.6 119.4± 1.8 a a 116.4±1.6 a b 0.015 102.4 ± 0.8 94.6 ± 0.8b 92.6 ± 0.8 b 0.01 92.4 ± 0.8 88.4 + 0.2 c c 86.4 ± 0.6 c 0.005 78.4 ± 0 6 76.4 ± 0.6 d d 77.3 + 0.6 d 0.001 68.4 ± 0.4 62.4 + 0.8 e e 60.4 ± 0.8 e f f Control 2.4 ± 0.4 2.2 + 0.4 2.1 ± 0.4 f Table.4. Repellent activity of hexane leaf extract of Acalypha indica leaves against Aedes aegtpti, Anopheles stephensi and Cx. quinquflasciatus Discussion The quantitative GC/MS phytochemical analysis of the Acalypha indica showed the presence of the compounds belonged to alkaloids, flavonoids, carbohydrates, saponins, tannins, phenol and terpenes. Now-a-days the vector control program with plant extract focused more on the elimination of mosquitoes in larval stage. The advantage of targeting larvae is that they cannot escape from their breeding sites until the adult stage and also reduce overall pesticides use in control of adult mosquitoes by aerial application of adulticidal chemicals[7]. Earlier study observed that phytochemicals have a major role in mosquito control programme [9,21]and observed the presence of carbohydrate, saponins, phytosterols, phenols, flavonoids and tannins in the plant extract having mosquito larvicidal activity. The pure compounds like thymol, carvacrol and d-pienene have been documented for larvicidal activities towards Culex pipiens mollestus [26]. Similarly mono-terpene hydrocarbons showed a marked larvicidal activity against Cx. pipiens which is obtained from the fresh leaves of Anthemis melampodina and Pluchea dioscoridis [15]. A piperidine alkaloid from Piper longum fruit was found to be active against the larvae of Cx. pipiens [12]. Similarly an alkaloid derived from tropical vine Triphyophylum peltatum was found to have larvicidal activity against malaria vector Anopheles stephensi[6]. In a very crucial observation and interpretations are made on spectral data of the complex structure of the constituents of the plants, without giving consideration to these facts will lead to confusion. Hence, it becomes essential to analyse the plant constituent s of pharmaceutical importance. In our study, the toxic effect of leaf extract against larvae was evident at higher concentrations particularly against An. stephensi. Similar differences in the mortalities in various larval instars of An. stephensi exposed to crude extract of various plants were also recorded by other workers[2,8,23]. While, the microscopic examination of dead larvae showed abnormal stretching of body especially the neck region was also observed symptoms suggest growth regulating and probably neurotoxic action of the leaf extract of A.indica However, the 100 min repellent activity is comparable to previously screened variable plants using different species of mosquitoes [16, 17]. The

S. Vijaya kumar et al., / Int J Med Biosci. 2012; 1(3):33-41 40 insect repellent that is widely available is DEET, which has been used worldwide since 1957 [22]. Although DEET has claimed a safe use against biting insects over 40 years it may still impose a risk to human health [20]. While, the leaf extract of A. indica did not cause any such of discomfort or skin irritation to the volunteers. Conclusion The present study revealed that A. indica contain active anti-larvicidal oviposition deterrent and repellent compound in its leaves. This makes it a more suitable candidate for the development of new potential eco-friendly against mosquitoes. Further investigation is needed to identify the active compounds of these extracts responsible for its activity. References [1] Abbott W.S. (1925). A method of computing the effectiveness of aninsecticide. J. Econ. Entomol. 18; 265-267. [2] Bartarya R, Srivastava A, Tonk S, Bhatnagar VP, srivastava SS and Kumari KM. (2009). 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