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Screening of Asteraceae (Compositae) plant extracts for larvicidal activity against Aedes fluviatilis (Diptera: Culicidae) Maria E MacEdo, Rotraut AGB Consoli, Telma SM Grandi**, Antonio MG dos Anjos**, Alaide B de Oliveira***, Nelymar M Mendes*, Rogerio O Queiroz*, Carlos L Zani*/^+ Laboratorio de Biologia e Sistematica de Culicideos Received 16 August 1996
Code Number: OC97103
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Ethanol extracts of 83 plants species belonging to the Asteraceae (Compositae) family, collected in the State of Minas Gerais, Brazil, were tested for larvicidal activity against the mosquito Aedes fluviatilis - Diptera: Culicidae). The extract from Tagetes minuta was the most active with a LC90 of 1.5 mg/l and LC50 of 1.0 mg/l. This plant has been the object of several studies by other groups and its active components have already been identified as thiophene derivatives, a class of compounds present in many Asteraceae species. The extract of Eclipta paniculata was also significantly active, with a LC90 of 17.2 mg/l and LC50 of 3.3 mg/l and no previous studies on its larvicidal activity or chemical composition could be found in the literature. Extracts of Achryrocline satureoides, Gnaphalium spicatum, Senecio brasiliensis, Trixis vauthieri, Tagetes patula and Vernonia ammophila were less active, killing more than 50% of the larvae only at the higher dose tested (100 mg/l). Key words: mosquitoes - larvicidal - Aedes fluviatilis - Asteraceae - plant extracts The selective pressure of conventional insecticides is enhancing resistance of mosquito populations at an alarming rate (Brown 1986), increasing the demand for new products that are environmentally safe, target-specific and degradable. Co-evolution has equipped plants with a plethora of chemical defenses against insect predators. Aware of this effect, mankind has used plant parts or extracts to control insects since ancient times. Plant derived products have received increased attention from scientists and more than 2000 plant species are already known to have insecticide properties (Balandrin 1985, Rawls 1986, Sukamar et al. 1991). Natural insecticides such as pyrethrum, rotenone and nicotine, among others, have been extensively used until recently for insect control (Balandrin 1985). Limonoids such as azadirachtin and gedunin, present in species from the Meliaceae and Rutaceae are recognized for their toxic effects on insects and are used in several insecticide formulations in many parts of the world (Dua et al. 1995, Nagpal et al. 1996). Recently, the discovery of insecticide activity of phototoxins present in Asteraceae species has stimulated the interest in this plant family as part of the search for new plant derived insecticides (Rawls 1986). In Brazil, the resurgence of several mosquito transmitted diseases such as malaria, dengue and yellow fever, together with the appearance of insect resistance to conventional insecticides, stresses the necessity for the search for new insecticides. Aiming for the discovery of cost effective alternatives for the control of disease vector insects, we decided to evaluate the toxicity of crude ethanol extracts of 83 Asteraceae species from our local flora against the larvae of Aedes (Ochlerotatus) fluviatilis (Lutz, 1904). This mosquito shares many biological characteristics with Ae. aegypti, the vector of yellow fever, and has been shown to be an useful model in biological studies of experimental infections and insecticide susceptibility tests (Consoli & Williams 1978, 1981, Camargo et al. 1983). MATERIALS AND METHODS Plant collection - The plants (Table I a) and Table I b were collected in Belo Horizonte and its vicinities, in the State of Minas Gerais, Brazil. After botanical identification, voucher specimens were deposited in the BHCB Herbarium at the Department of Botany, Federal University of Minas Gerais. Extracts preparation - The aerial parts of the plants were dried in the shade, ground in a knife mill or in a homogenizer and extracted twice (24 hr) with ethanol (95%) at room temperature. The solvent was removed by rotary evaporation under reduced pressure at temperature below 45 C. The resulting crude extracts were stored in a freezer at -20 C until assayed. Immediately before running the bioassay, sufficient amounts of extract were transferred to a vial and the residual solvent removed under high vacuum for at least 24 hr. Bioassay - Stock solutions of each extract were prepared at 1000 mg/l by sonicating them in a ultrasound bath (45 kHz, 100W) for 5-10 min. Test solutions of 100, 10 and 1 mg/l were then prepared by diluting the stock solution in tap water. The extracts were tested against young fourth instar Ae.(Ochlerotatus) fluviatilis (Lutz, 1904) larvae from a colony maintained at the Centro de Pesquisas Rene Rachou (Consoli & Lourenco-de-Oliveira 1994). Each dilution was placed in sterile glass dishes (9 cm diam./150 ml capacity) and 30 larvae were added. After 24 hr contact at room temperature, the number of dead larvae in each dish was counted. The larvae were considered dead if they were immobile and unable to reach the water surface. Previous experiments showed no significant differences in mortality when the assay was extended to 48 hr (Consoli et al. 1988). The ambient temperature during all experiments ranged between 23-28 C. Control experiments without extract were run in parallel and the mortality was always bellow 4.5%. All experiments were run in triplicate. Statistical evaluation - Mortality means were compared using Duncan's Test (Edwards 1960) at the alpha significance level of 0.05; LC50 and LC90 were calculated for the most active extracts using probit analysis (Armitage & Berry 1987). RESULTS AND DISCUSSION Eighty-three species, belonging to 48 genera of the Asteraceae family were collected for this survey. Table I lists all plants in alphabetical order and includes their habitats, BHCB herbarium codes and common name when available (Correa 1984). The genera Baccharis, Eupatorium, Mikania and Vernonia were the best represented, with at least five species each. Table II summarizes the results of the bioassays for those species that promoted statistically significant mortality, using Duncan's significance test (Edwards 1960), for at least one concentration when compared to the control. Larvicidal activities higher than 50% at any tested concentration were highlighted. The crude extract from the aerial parts of T. minuta ( Table II, entry 22) displayed an LC90 and an LC50 of 1.5 and 1.0 mg/l, respectively, making it the most active of all extracts tested. 5-E-ocimenone was initially described as the active component of Tagetes minuta (Maradufu et al. 1978) but Green et al. (1991) suggested that further compounds, not identified by them, were also responsible for the observed toxicity towards mosquito larvae. More recently, four thiophene derivatives were identified from a larvicidal floral extract fraction of this plant (Perich et al. 1995). This fraction displayed an LC50 of 3.9 against Ae. aegypti and Anopheles stephensi 3rd instar larvae, i. e., four times less potent than the crude extract tested here. T. patula extract (Table II, entry 23) which is also known to contain thiophene derivatives (Bicchi et al. 1992) was, on the other hand, much less active than T. minuta, a result that is in agreement with other published works (Green et al. 1991, Wells et al. 1993, Perich et al. 1994). The extract of Eclipta paniculata was the second most active of the 83 tested in this screening. It promoted the death of 83% of the larvae at 10 mg/l and presented LC90 and LC50 values of 17.2 and 3.3 mg/l, respectively. No studies describing its insecticide activity or chemical fractionation has been found in the literature. However, considering the chemistry of the genus Eclipta (Singh 1988), it is conceivable that thiophene and polyacetylene derivatives are also present in E. paniculata and could account for its larvicidal properties. A bioassay-guided fractionation of E. paniculata extract will be necessary to confirm this hypothesis. The extracts of Achryrocline satureoides, Gnaphalium spicatum, Senecio brasiliensis, Trixis vauthieri and Vernonia ammophila were much less active than those discussed above. Concentrations of 100 mg/l for each extract were necessary to kill more than 50% of the larvae (Table II). The extract of V. ammophila, for example, showed LC90 and LC50 values of 87.8 and 40 mg/l, respectively. To the best of our knowledge this is the first time the larvicidal activity of these species has been described. The extract of T. vauthieri has already been the object of phytochemical studies (Bohlmann et al. 1981, Ribeiro et al. 1994) and has been shown to contain 7-methoxyaroma-dendrin, a larvicidal flavonoid (Echeverry et al. 1992) that could account for its activity. In addition to these studies, a bioassay-guided chemical fractionation protocol should be conducted in order to identify further larvicidal components in this extract. Except for the extracts discussed above, all others listed in Table II were unable to kill more than 50% of the larvae at the highest concentration tested (100 mg/l) and were considered weakly active. Concerning these species, comparison of our results to those found in the literature yielded the following observations: a) the extract of Ageratum conyzoides, reported to be larvicidal in a previous work (Sujatha et al. 1988), was devoid of activity in the present trial; b) the genera Bidens, Mikania and Verbesina, known to contain species with pronounced insecticide activity (Heal et al. 1950, Consoli et al. l988), afforded no larvicidal extract under our experimental conditions; c) extracts of A. australe, A. conyzoides, B. pilosa, E. bonariensis, J. floribunda, P. ruderale, P. alupecuroides and V. claussenii showed no effect over Ae. fluviatilis larvae in this study but have, according to previous works (MacEdo 1995), interfered with oviposition behavior in this species suggesting that different components in the extracts are responsible for these effects; d) while Heal et al. (1950) described the activity of five Baccharis species against Ae. aegypti and An. quadri-maculatus larvae in his survey (Heal et al. 1950), none of the six Baccharis species tested here were larvicidal. These inconsistencies in activities may be attributable to seasonal fluctuations in the biosynthesis of the active components, differences in extraction methods, bioassay protocols or difficulties in species authentication (Farnsworth 1966). In conclusion, from this screening several larvicidal extracts were detected among local Asteraceae species, some of them already described by other research groups. T. minuta was the most active and thiophene derivatives were identified as its larvicidal components (Perich et al. 1995). The larvicidal flavonoid 7-metho-xyaromadendrin is present in T. vauthieri extracts (Bohlmann et al. 1981, Ribeiro et al. 1994) and could account, at least in part, for its larvicidal activity. Finally, the extract of E. paniculata showed strong activity and, as it has not yet been subjected to any phytochemical investigation, it is a good candidate for a bioassay-guided fractionation to identify its larvicidal constituents. It is conceivable that its active components are also thiophene or polyacetylene derivatives, compounds very common in the genus Eclipta. Studies to confirm this hypothesis are underway. ACKNOWLEDGMENTS To Prof. Julio A Lombardi, Curator of the Dept. of BotAnica Herbarium, ICB/UFMG, for his kind help. Supported by CNPq/FIOCRUZ REFERENCES
Copyright 1997 Fundacao Oswaldo Cruz The following images related to this document are available:Photo images[oc97103b.jpg] [oc97103c.jpg] [oc97103a.jpg] |
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