Evaluation of leaf extracts of Vitex negundo L.(Family: Verbenaceae) against larvae of Culex tritaeniorhynchus and repellent activity on adult vector mosquitoes

Parasitol Res (2008) 103:545–550 DOI 10.1007/s00436-008-1005-5 ORIGINAL PAPER Evaluation of leaf extracts of Vitex negundo L. (Family: Verbenaceae) against larvae of Culex tritaeniorhynchus and repellent activity on adult vector mosquitoes Kaliyaperumal Karunamoorthi & Sayeenathan Ramanujam & Rajendran Rathinasamy Received: 14 March 2008 / Accepted: 16 April 2008 / Published online: 24 May 2008 # Springer-Verlag 2008 Abstract Petroleum ether (60–80°C) extracts of the leaves of Vitex negundo (Verbenaceae) were evaluated for larvicidal activity against larval stages of Culex tritaeniorhynchus in the laboratory. Larvae of C. tritaeniorhynchus were found more susceptible, with LC50 and LC90 values of 2.4883 and 5.1883 mg/l, respectively. Human volunteers wearing special terricot (68:32) fabrics, in the form of armbands, anklets, headbands, collar, and shoulder and pocket strips impregnated with V. negundo leaf extract were used, to test their repellent efficacy at two concentrations viz., 1.5 and 2.0 mg/cm2 under the field conditions. At 1.5-mg/cm2 concentration, more efficacies were found and 6-h complete protection against mosquito bites was provided. Complete protections for 8 h were found at 2.0 mg/cm2 against mosquitoes bites. These results clearly reveal that the V. negundo leaf extract served as a potential larvicidal agent against Japanese encephalitis vector C. tritaeniorhynchus and additionally acted as a promising repellent against various adult vector mosquitoes. Introduction It is estimated that every year at least 500 million people in the world suffer from one or other tropical diseases that K. Karunamoorthi (*) Unit of Vector Biology and Control, School of Environmental Health Science, Faculty of Public Health, Jimma University, P.O. Box No. 378, Jimma, Ethiopia e-mail: k_karunamoorthi@yahoo.com S. Ramanujam Department of Zoology, Annamalai University, Chidambaram, Tamil Nadu, India R. Rathinasamy Center for Research in Medical Entomology (ICMR), Madurai, Tamil Nadu, India include malaria, lymphatic filariasis, Japanese encephalitis schistosomiasis, dengue, trypanosomiasis, and leishmaniasis. One to two million deaths are reported annually due to malaria worldwide. Lymphatic filariasis affects at least 120 million people in 73 countries in Africa, India, Southeast Asia, and Pacific Islands. These diseases not only cause high levels of morbidity and mortality but also inflict great economic loss and social disruption on developing countries such as India, China, etc. India alone contributes around 40% of global filariasis burden and the estimated annual economic loss is about 720 (Hotez et al. 2004). Mosquitoes in the larval stage are attractive targets for pesticides because mosquitoes breed in water, and thus, it is easy to deal with them in this habitat. The use of conventional pesticides in the water sources, however, introduces many risks to people and/or the environment. Natural pesticides, especially those derived from plants, are more promising in this aspect (Amer and Mehlhorn 2006b). Many plant-based products are widely used for their insecticidal–repellent properties for control of mosquitoes and protection from mosquito bites (Jacobson and Crosby 1971; Sukumar et al. 1991). The repellent properties of plants to mosquitoes and other pest insects were well known before the advent of synthetic chemicals. In southern India, leaves of Vitex negundo are burned to repel mosquitoes from houses (Curtis et al. 1989). Since ancient times, plant products were used in various aspects. However, their use against pests decreased when chemical products became developed. Recently, concerns increased with respect to public health and environmental security requiring detection of natural products that may be used against insect pests (Amer and Mehlhorn 2006a). Currently, a large number of chemicals are available in the market for the control of pests, which are highly poisonous. These chemicals are not only costlier but also pose a high 546 residual toxicity which adds poisons not only to livestock but also to human beings. The problems of high cost and development of resistance in many vector mosquito species to several of the synthetic insecticides have revived interest in exploiting the pest control potential of plants (Grainge and Ahmed 1988). Therefore, herbal pesticides gained importance recently; plant products are considerably safer and ecofriendly for pest control even before the introduction of chemical pesticides; detailed knowledge concerning them is still lacking. As a result, it is the hour to launch extensive search to explore ecofriendly biological materials for control of insect pests. In the present communication, an attempt has been made to evaluate the repellent and larvicidal efficacy of leaf extracts of V. negundo against Culex tritaeniorhynchus, the vector of Japanese encephalitis. Objectives The objectives of this study are: to evaluate larvicidal activity of the leaf extracts of V. negundo against fourthinstar larvae of C. tritaeniorhynchus, under the laboratory conditions, and to evaluate repellent activity of the extract by conducting field trials with human volunteers wearing fabric strips (armbands, anklets, headband, collar, and shoulder and pocket strips) impregnated with V. negundo leaf extracts against adult mosquitoes biting at night. Materials and methods Collection of mosquito larvae The egg rafts of C. tritaeniorhynchus were obtained from the rearing and colonization division of the Center for Research in Medical Entomology, field station for JE studies, at Vridhachalam, Tamil Nadu, India. The egg rafts were floated on the water surface and the larvae were reared up to early fourth-instar stage, following the standard procedure. Parasitol Res (2008) 103:545–550 cloister pepper, Indrani, Nirgandhi, Nochi(Tamil) and nisinda, sambhalu (Hindi). Local rural population are widely using leaves of V. negundo to repel insects by mixing leaves with grain stores and fresh leaves are burnt with grass as a fumigant against mosquitoes. V. negundo leaves are available throughout the year, at the same time as seeds are available only in particular months. Therefore, the present study was undertaken to evaluate the leaf extracts of V. negundo against C. tritaeniorhynchus. Botanical description V. negundo is a much branched shrub with quadrangular tomentose, densely whitish tomentose branchlets, up to 5 m tall or sometimes a small, slender tree. Its bark is thin and gray; leaves are palmately compound, with three to five foliolate; leaflets are lanceolate, entirely or rarely crenate, terminal leaflets 5–10×1–3 cm, lateral leaflets smaller, all nearly glabrous above, whitish tomentose beneath. Flowers are bluish-purple and small, in peduncled cymes, forming large, terminal, often compound, pyramidal panicles. Fruit is a succulent drupe, black when ripe, 5–6 mm in diameter. Seed is 5–6 mm in diameter, invested at the base with enlarged calyx. The scented flowers are hermaphrodite (have both male and female organs) and are pollinated by insects. Physical characteristics The plant prefers light (sandy) and medium (loamy) soils, requires well-drained soil, and can grow in nutritionally poor soil. It is in flower from September to October. The scented flowers are hermaphrodite (have both male and female organs) and are pollinated by insects. We rate it 3 out of 5 for usefulness. The plant prefers light (sandy) and medium (loamy) soils, requires well-drained soil and can grow in nutritionally poor soil. The plant prefers acidic, neutral, and basic (alkaline) soils. It cannot grow in the shade. It requires dry or moist soil. General uses Plant—V. negundo Taxonomy Current name: V. negundo Authority: Linnaeus Family: Verbenaceae Leaves Root Fruit Dried fruit Antiparasitic, alterative, aromatic, vermifuge, pain reliever Tonic, febrifuge, expectorant, diuretic Nervine, cephalic, emmanagogue Vermifuge Collection and extraction of leaves Other names Five-Leafed Chaste Tree (English), Chasteberry, Nirgundi, Sambhalu, Vitex agnus castus, chaste tree, monk's pepper, Plants of V. negundo growing in and around Chidambaram town, Tamil Nadu, India and their fresh leaves were collected. The leaves were washed with water and cut into Parasitol Res (2008) 103:545–550 small pieces. These pieces were then shade-dried at room temperature (25±2°C) till they were complete dry. The dried leaves were then powdered with the help of Remi laboratory blender. The powdered leaves were weighed. Extraction were done using petroleum ether (boiling range 60–80°C) at room temperature. A total of 100 g of the powdered leaves of V. negundo were taken in extractors of 21-l capacity. Petroleum ether (1.0 l) was added to the extractor so that the materials were soaked completely. The extractor was shaken regularly at 24-h intervals and they were kept at room temperature for 10 days. After 10 days, the petroleum ether extract were transferred into roundbottomed flasks. The solvent were removed at reduced pressure to obtain dark residue. 547 LC50 value indicated the 50% mortality and LC90 value indicated the 90% mortality; they were calculated from a series of “exposure” concentration by comparing the percentage of mortality in the treated group to control by probit analysis (Finney 1971). The number of larva in both control and test were recorded after 24 h. When the control mortality was between 5 and 20, percentage effects were corrected by Abbott's formula (Abbott 1925). Corrected Observed mortality ð%Þ Control mortality ð%Þ mortality ¼  100 100 Control mortality ð%Þ Bioefficacy of plant extracts Evaluation of repellent activity of V. negundo leaf extracts against adult vector mosquitoes by using human volunteers—field study Testing of the leaf extracts for larvicidal activity was done at different concentrations ranging from 0.5 to 6.0 mg/l by preparing the required stock solutions for the different concentrations by following the standard procedure. The larvicidal activity of leaf extracts against fourth-instar larvae of C. tritaeniorhynchus were determined by using standard procedure (WHO 1975). One percent stock solution of leaf extracts were made, from which other lower concentrations were prepared in acetone. The desired concentrations of test solution were achieved by adding 1 ml of an appropriate stock solution to 249 ml of tap water taken in a 500-ml beaker. Twenty-five numbers of early fourth-instar larvae of C. tritaeniorhynchus were exposed to various concentrations of V. negundo leaf extracts. A pinch of larval food (125 mg) consisting of yeast powder and dog biscuit (1:1) were provided. Three replicates for each concentration and simultaneous control with same tap water were set up. Mortality in larvae was recorded 24 h posttreatment. The experiments were conducted at 28±2°C and 70–80% relative humidity. In recording the percentage effect for each concentration, moribund and dead larvae were considered as affected. a. Fabric strips: The arm-band, anklets, collar, headband, and shoulder and pocket strips were made from terry-cotton (68:32). Both ends were provided with press buttons. b. Impregnation procedure: Initially, each of the fabric strips was soaked in water and the quantity required to wet the fabric strips completely was measured by the standard procedure. The quantity of water thus determined for each fabric strips was used to mix the desired leaf extract of V. negundo at two different concentrations viz., 1.5 and 2 mg/cm2. The soaking of fabric strips was made in a nonabsorbent container. Uniform distribution of leaf extract of V. negundo was ensured by rubbing and squeezing of fabric strips. The fabric strips were then flattened on a polythene sheet for drying. After thorough drying, each set of fabric strips was kept in separate plastic bags, to avoid mixing with other concentration. c. Field evaluation method: Healthy male volunteers of Karmangudi village, Tamil Nadu, India were involved in these repellent testing. Two volunteers were provided each with a set of arm bands, anklets, headband, collar, and shoulder and pocket strips, one Fig. 1 Bioefficacy of V. negundo leaf extract against C. tritaeniorhynchus 548 Parasitol Res (2008) 103:545–550 Table 1 Larvicidal activity of leaf extract of V. negundo against C. tritaeniorhynchus Results Bioefficacy Value Laboratory evaluation of V. negundo leaf extract for larvicidal activity against C. tritaeniorhynchus LC50 (mg/l) LC50 LCL LC50 UCL LC90 (mg/l) LC90 LCL LC90 UCL Chi square (X2) 2.4883 2.3498 2.6350 5.1883 4.6500 5.7890 12.7788 LC Lethal concentration, LCL lower confidential limit, UCL upper confidential limit with leaf extract of V.-negundo-impregnated fabric strips and the other with the “control,” i.e., with untreated fabric strips. The mosquitoes landing on volunteers were collected by night (18.00–06.00). Informed and free consent were obtained from the volunteers for the present study. The volunteers (human baits) were interchanged between the control and test at an interval of 30 min. Mosquitoes landing on treated and untreated volunteers were collected throughout the night with the help of a suction tube and a flashlight by insect collectors. Insect collectors were rotated at an interval of 6 h to avoid bias collections. Mosquitoes collected every hour were kept separately and then brought to the laboratory on the morning of the next day. Mosquitoes collected on baits were counted and identified in the laboratory under a stereo zoom microscope. Percentage of protection from man–mosquito contact was calculated by using the following formula: ðNo: of mosquitoes control groupÞ ðNo: of mosquitoes in experimental groupÞ  100 % Protection ¼ No: of mosquitoes in control group The larvicidal effect of V. negundo leaf extracts was tested against C. tritaeniorhynchus at different concentrations viz., 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, and 6.0 ppm and various parameters for the said effects are presented below in the Table 1 and Fig. 1. The petroleum ether extraction of leaves of V. negundo was carried out under laboratory conditions. The larvicidal activity of V. negundo to vector mosquito showed that C. tritaeniorhynchus was susceptible. The LC50 and LC90 values for leaf extracts were 2.4883 and 5.1883 mg/l, respectively. From these values as well as from Fig. 1 and Table 1, it can be seen that C. tritaeniorhynchus is more susceptible to leaf extracts of V. negundo. The value of X2 is 12.7788 and the chi-square test showed that the test species is homogenous. Filed trial, using leaf extracts of V. negundo for repellent activity Leaf extract of V. negundo was tested for their repellent effects on human volunteers. It was evaluated at two different concentrations viz., 1.5 and 2.0 mg/cm2, against adult mosquitoes biting at night. The following Table 2 and Figs. 2 and 3 reveals that leaf extracts of V. negundo have strong repellent activity against adult vector mosquitoes biting at night. Discussion Vector control is facing a threat due to the emergence of resistance in vector mosquitoes to conventional synthetic Table 2 Protection offered by fabric strips impregnated with leaf extract of V. negundo at a concentration of 1.5 and 2.0 mg/cm2 against mosquitoes biting at night under the field conditions Period of observation (h) No. mosquitoes collected Control 18–19 19–20 20–21 21–22 22–23 23–00 00–01 01–02 02–03 03–04 04–05 05–06. 36 25 18 33 42 53 48 49 39 33 30 26 2 mg/cm2 0 0 0 0 0 0 0 0 1 3 5 6 % of reduction of man landing rate 100 100 100 100 100 100 100 100 97.43 91.00 76.00 76.00 No. mosquitoes collected 1.5 mg/cm2 0 0 0 0 0 0 3 5 6 8 9 9 % of reduction of man landing rate 100 100 100 100 100 100 93.00 89.00 84.61 75.75 70.00 65.38 Parasitol Res (2008) 103:545–550 549 Fig. 2 Proection offered by fabric strips impregnated with leaf extract of V. negundo insecticides, warranting either countermeasures or development of newer insecticides (Chandre et al. 1998). The petroleum ether extraction of leaves of V. negundo was carried out under laboratory conditions. The larvicidal activity of V. negundo to vector mosquito showed that C. tritaeniorhynchus was susceptible. The LC50 and LC90 values for leaf extracts were 2.4883 and 5.1883 mg/l, respectively. The larvicidal efficacy is comparable to fenthion, a commercially available organophosphorus larvicide (Amalraj and Das 1996). The larvicidal efficacy is also comparable with methanol leaf extracts of V. negundo, Vitex trifolia, Vitex peduncularis, and Vitex altissima against the early fourth-instar larvae of Culex quinquefasciatus (Kannathasan et al. 2007). Human volunteers, wearing armbands, anklets, headbands, collar, and shoulder and pocket strips impregnated with V. negundo leaf extracts, were used, to test their repellent efficacy at two different concentrations viz., 1.5 and 2.0 mg/ cm2, under the field conditions. At 1.5-mg/cm2 concentration, more efficacies were found and 6-h complete protections against mosquito bites was provided. It provided protection from mosquitoes bites, which varied between 76.5% and 100%. Complete protections from mosquitoes bites for 8 h were found at 2.0 mg/cm2. It provided protection from mosquitoes bites, which varied between Fig. 3 Field trial, using leaf extract of V. negundo for repellent activity 98.8% and 100%. In the field trial, we have found that the leaf extract of V. negundo has offered protection from the three major important vector mosquitoes such as Anopheles, Culex, and Aedes species. The final results clearly demonstrate that repellent activity of leaf extracts of V. negundo was dose dependent. The present investigation reveals that the repellent activity of leaf extracts of V. negundo is comparable with previously screened plants in the laboratory by using different species of mosquitoes (Rajkumar and Jebanesan 2004; Venkatachalam and Jebanesan 2001). A new natural mosquito repellent was isolated from fresh leaves of Vitex rotundifolia. Its structure was elucidated by an extensive nuclear magnetic resonance spectral analysis to be a cyclopentene dialdehyde named rotundial. This compound possessed potent repelling activity against Aedes aegypti (Watanabe et al. 1995). The repellent efficacy of leaf extract of V. negundo is also comparable with CO2 extract of the seeds of the Mediterranean plant Vitex agnus castus (monks pepper; Mehlhorn et al. 2005). The above-cited studies undoubtedly indicated that Vitex plants possess promising potent repelling activity against mosquitoes and other bloodsucking insects. In our present investigation, the leaf extracts did not cause any uneasiness or skin irritation to the human volunteers (Bowers et al. 1995). In view of the fact that local inhabitants are using V. negundo plant leaves for a number of 550 medicinal purposes, no side effects and discomfort have been found. Plants have been used since ancient times to repel or kill blood-sucking insects in the human history and, even now, in many parts of the world people are practicing plant substances to repel or kill the mosquitoes and other bloodsucking insects. We are all just around the corner to reinstate the chemical substances with plant-derived ones. In the present investigation, we have identified ecofriendly substances (leaf extract of V. negundo) for the control of vector mosquitoes. Plants can provide safer alternatives for modern deadly poisonous synthetic chemicals. Acknowledgement I have great pleasure in expressing my profound gratitude to Dr. PR. 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