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Acta Amazonica

Print version ISSN 0044-5967On-line version ISSN 1809-4392

Acta Amaz. vol.34 no.1 Manaus  2004 



Screening of plants found in the State of Amazonas, Brazil for activity against Aedes aegypti larvae


Triagem de plantas encontradas no Estado do Amazonas para atividade larvicida contra Aedes aegypti



Adrian Martin PohlitI,1; Etienne Louis Jacques QuinardI; Sergio Massayoshi NunomuraI; Wanderli Pedro TadeiII; Ari de Freitas HidalgoIII; Ana Cristina da Silva PintoIV; Elba Vieira Mustafa dos SantosIV; Sabrina Kelly Reis de MoraisIV; Rita De Cássia Guedes SaraivaV; Lin Chau MingVI; Alexandre Mascarenhas AlecrimI; Alfeu de Barros FerrazI; Andreza Cristiana da Silva PedrosoI; Elisangela Vieira DinizI; Ellen Kathryn FinneyI; Erika de Oliveira GomesI; Hercules Bezerra DiasI; Katiuscia dos Santos de SouzaI; Laura Cristina Pereira de OliveiraI; Luciana de Castro DonI; Maria Mireide Andrade QueirozI; Marycleuma Campos HenriqueI; Mirian dos SantosI; Orivaldo da Silva Lacerda JúniorI; Patrícia de Souza PintoI; Suniá Gomes SilvaI; Yara Rodrigues GraçaI

ICoordenação de Pesquisas em Produtos Naturais (CPPN)
IICoordenação de Pesquisas em Ciências da Saúde (CPCS), Instituto Nacional de Pesquisas da Amazônia (INPA), Av. André Araújo, 2936, 69083-000 Manaus, Amazonas
IIIFaculdade de Ciências Agrárias (FCA)
IVCurso de Pós-graduação em Química de Produtos Naturais, Universidade Federal do Amazonas (UFAM), Av. Gal. Rodrigo O. J. Ramos, 3000, 69077-000 Manaus, Amazonas
VCurso de Pós-graduação em Química, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Rio de Janeiro
VIUniversidade do Estado de São Paulo (UNESP), Fazenda Experimental Lageado, 237, 18603-970 Botucatu, São Paulo




Ethanol, methanol and water extracts representing mostly native plant species found in the Amazon region were prepared, respectively, by maceration, continuous liquid-solid extraction and infusion, followed by evaporation and freeze-drying. The freeze-dried extracts were tested for lethality toward Aedes aegypti larvae at test concentrations of 500 mg / mL. In general, methanol extracts exhibited the greatest larvicidal activity. The following 7 methanol extracts of (the parts of) the indicated plant species were the most active, resulting in 100% mortality in A. aegypti larvae: Tapura amazonica Poepp. (root), Piper aduncum L. (leaf and root), P. tuberculatum Jacq. (leaf, fruit and branch). and Simaba polyphylla (Cavalcante) W.W. Thomas (branch).

Key words: larvicide, Aedes aegypti, Tapura, Piper, Simaba, Amazonia.


Extratos aquosos, etanólicos e metanólicos, representando principalmente espécies vegetais nativas encontradas na região Amazônica, foram preparados, respectivamente, por infusão, maceração e extração contínua líquido-sólido, seguida de evaporação e liofilização. Os extratos liofilizados foram testados para atividade contra larvas de Aedes aegypti, na concentração única de 500 mg / mL. Os extratos metanólicos foram, em geral, os que apresentaram maior atividade larvicida. Os seguintes 7 extratos metanólicos das (partes das) espécies vegetais indicadas foram os mais ativos, provocando 100% de mortalidade em larvas de A. aegypti: Tapura amazonica Poepp. (raiz), Piper aduncum L. (folha e raiz), P. tuberculatum Jacq. (folha, fruto e galho) e Simaba polyphylla (Cavalcante) W.W. Thomas (galho).

Palavras-chave: larvicida, Aedes aegypti, Tapura, Piper, Simaba, Amazônia.




In response to strong selective pressures of herbivorous insects, toxic secondary metabolites have evolved in plants and often affect insect nerve function and behaviour (Sharma et al., 1998). Plant extracts show potential as sources of natural larvicides for the control of mosquito vectors and an early review has been published in which more than 300 larvicidal plant species were identified (Sukamar et al., 1991). In recent times, studies on the activity of plant extracts towards Aedes sp. larvae from different parts of the world, such as, North America (Bergeron et al., 1996), Argentina, Bolivia, Brasil and Peru (Chantraine et al., 1998; Ciccia et al., 2000; Macedo et al., 1997), Trinidade and Tobago (Chariandy et al., 1999), Mali (Diallo et al., 2001), Negev Desert (Sathiyamoorthy et al., 1997) and Africa (Marston et al., 1993), among others, have been published and have revealed numerous examples of active plant extracts representing diverse taxonomic groups. More systematic and directed studies have revealed a number of very active plant extracts, essential oils, and isolated larvicidal phytochemicals (Bandara et al., 2000; Bernard et al., 1995; Hostettmann & Potterat, 1997; Latha & Ammini, 2000; Lee, 2000; Oberlies et al., 1998; Park et al., 2002; Pushpalatha & Muthukrishnan, 1999; Rahuman et al., 2000; Sharma et al., 1998; Thorsell et al., 1998). In the present study, we have screened polar extracts of mostly native plants from the Brazilian Amazon for activity against larvae of the hemorrhagic dengue fever vector, Aedes aegypti (Diptera: Culicidae), at a single test concentration of 500 mg / mL. Data (species names, plant parts, extraction solvent) for both active and inactive extracts are presented.



Plant collecting, processing, and extract preparation.

Plants were collected from 1999 to 2002 in the State of Amazonas. Some genera (for example, Piper and Aspidosperma) and families (Euphorbiaceae, Simaroubaceae, Apocynaceae, Piperaceae, etc.) were better represented among the plants collected due to our specific interest in the bioactivity of these taxonomic groups. The plant materials were generally dried in the shade, ground, then stored in plastic bags under the protective warmth and luminosity of incandescent lighting. Where possible, fertile specimens of plant species were collected, used for identification and deposited at the INPA Herbarium. Many samples for study were collected from individual trees in INPA´s Ducke Reserve that had been catalogued and identified in recent systematic studies (Ribeiro et al., 1999). Ethanol, methanol and water extracts were prepared, respectively, by maceration, continuous liquid-solid extraction, and infusion, followed by rotary evaporation under vacuum with heating in a bath to ca. 40-45 ºC and freeze-drying. After this last step, extracts were presumed to be solvent-free. Dry extracts were deposited in our extract bank at the Coordenação de Pesquisas em Produtos Naturais (CPPN / INPA) where they were stored at –19 ºC. Many of these extracts were screened previously by some of us against larvae of the brine shrimp species Artemia franciscana (Quignard et al., 2003).

Assay for larvicidal activity against Aedes aegypti.

Aedes aegypti larvae were obtained by incubation of eggs from a laboratory colony at the Coordenação de Pesquisas em Ciências da Saúde (CPCS/INPA) in tap water. Stock solutions or suspensions of extracts in DMSO or deionized water (50 mg / mL) were prepared. Each stock solution (100 mL) was then transferred to plastic cups containing 10 third instar (three-day old) larvae in tap water having a final volume of 10 mL and a final extract concentration of 500 µg / mL. Negative controls were prepared analogously, substituting 100 mL of tap water or DMSO for sample solution in each cup, resulting in a final DMSO concentration of 1 %. Each experiment was run in triplicate and compared with a control set after 24h at 26 – 27 ºC. Dead larvae were counted and the larvicidal activity expressed as % mortality based on live larvae present initially. In general, no dead larvae were observed in the controls after 24 h.



Table 1 presents ethnobotanical and other information on the species investigated in this study. A number of these plants are traditionally used medicinal plants for the treatment of malaria, fevers, liver problems, and other symptoms caused by malaria infections, whose study is a major focus of our research group. Other plants studied were collected using a random sampling technique in INPA forest preserves in or near Manaus (Reserva Florestal Adolpho Ducke, Reserva da Campina / ZF-2). Still other species, from such genera as Aspidosperma (Apocynaceae) and Piper (Piperaceae), or from families such as Simaroubaceae, were studied here as part of a systematic approach to the investigation of the chemistry and biological activity of Amazon flora of these, and other taxonomic groups. Thus, the plants studied herein were not selected a priori for their known insecticidal, larvicidal or repellent properties. The discovery of larvicidal activity, as described below, serves as a demonstration of the power of bioprospection in the discovery of new larivicidal extracts and compounds based on many plants for which no such activity has been described or is known in the literature.

Tables 2 and 3 present, respectively, data for larvicidal and inactive plant extracts. Comparison of the results presented in Tables 2 and 3 reveals in a general way that extracts prepared from the less polar solvents ethanol and methanol were more active against A. aegypti larvae than the extracts prepared by extraction with the more polar water as solvent, which is probably an indication that toxicity to these larvae should be associated in general with substances of medium or even low polarity.

Our results confirmed the toxicity of several plant species which have been described as being toxic or are used traditionally for their toxicity. For example, methanol root extract of Tapura amazonica Poepp. was highly lethal to A. aegypti larvae which is interesting in light of the fact that it has been described by Colombian native indians as being a toxic species (Table 1; Schultes & Raffauf, 1990). In similar fashion, P. tuberculatum Jacq. presented highly lethal extracts in the assay and is, according to traditional use, an effective fish poison (Table 1). Lastly, Picrolemma sprucei Hook (caferana), which exhibited larvicidal root and stem extracts in the present study, is known for its toxic effects towards the human fetus, being used widely in the State of Amazonas and Manaus as an abortifacient tea (Pohlit et al., unpublished data).

Another Piper species, P. aduncum L., exhibited good activity against Aedes aegypti (Table 2) and is known to produce the larvicidal phytochemical compound, dillapiole, which has been shown by Bernard et al. (1995) to be active against another Aedes sp.. Given that P. aduncum is a fast-growing, invasive species, it is potentially useful as an industrial source of dillapiole and other active phytochemicals.

Another interesting observation is that larvicidal activity is definitely specific to certain parts of plants, such as P. aduncum, which exhibited very active methanol leaf and root extracts (Table 2), however, extracts of other parts of this plant were inactive (Table 3). Other examples of this specificity of insecticidal activity to certain plant parts is evidenced by comparing the data presented in Tables 2 and 3.

Finally, it is important to note that even the low mortality exhibited by some extracts during screening can be significant for the discovery of bioactive phytochemicals. An example of this is the weak activity of the ethanol root and leaf extracts of Pothomorphe peltata (L.) Miq. (each exhibiting only 7 % mortality at 500 mg / mL, Table 2). In further work, bioassay-guided fractionation of this ethanol root extract in our labs yielded very active intermediate fractions and isolation of the phytochemical larvicide, 4-nerolidylcatechol, which exhibited very significant lethality towards Aedes aegypti larvae and was shown by chromatographic analytical methods to be a major component of both root and leaf extracts (Pinto, 2002).



A number of larvicidal plants were identified during this work. Several of these are now under biomonitored phytochemical analysis and should yield further examples of isolated larvicidal phytochemicals in the future.



The authors proudly recognize the financial support provided by the Brazilian National Council for Scientific and Technological Development (CNPq; Grant nos. 520.354/99-0 and 550.260/01-3). The following people are thankful to these institutions for scholarships: ELJQ (DTI-MCT), SMN (DCR-CNPq), ACSP and EVMS (Mestr-CAPES), SKRM (AP-CNPq), MCH (IC-CNPq), KSS (AT-CNPq), AMA, ACSP, LCD, EOG, PSP and SGS (PIBIC-INPA-CNPq).



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Recebido em 30/12/2002
Aceito em 07/01/2004



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