Diterpenos de Copaifera reticulata Ducke com atividade larvicida contra Aedes aegypti (L.) (Diptera, Culicidae)

The objective of this study was to evaluate the larvicidal activity of diterpenoids obtained from the oil-resin of Copaifera reticulata against Aedes aegypti larvae, the principal vector of dengue and urban yellow fever. Four diterpenes were obtained from oil-resin extraction with organic solvents and subsequent chromatographic and spectroscopic procedures allowed to isolation and identification of these compounds as 3-b-acetoxylabdan-8(17)-13-dien-15-oic acid (1), alepterolic acid (2), 3-b-hidroxylabdan-8(17)-en-15-oic acid (3), and ent-agatic acid (4). Each compound was previously dissolved in dimethylsulphoxide, and distilled water was added to obtain the desired concentrations. Twenty larvae of third instars were placed into plastic beckers, containing the solution test (25 mL), in a five repetitions scheme, and their mortality, indicated by torpor and darkening of the cephalic capsule, was recorded after 48h. Probit analyses were used to determine lethal concentrations (LC50 and LC90) and their respective 95% confidence intervals. This study showed that only diterpenoids 1 and 2 exhibited larvicidal properties with LC50 of 0.8 ppm and 87.3 ppm, respectively, revealing the former as the most toxic compound against third instars of Ae. aegypti. Therefore, this compound seems to be an interesting source for new metabolite to be exploited.

Atividade larvicida de taninos isolados de Magonia pubescens St. Hil. (Sapindaceae) sobre Aedes aegypti (Diptera, Culicidae)

Apresenta-se, pela primeira vez, o estudo fitoquímico das frações larvicidas, isoladas da Magonia pubescens, monitorado pelo estudo de eficácia sobre larvas de 3º estádio de Aedes aegypti, na busca de alternativas para o controle desse mosquito e obtenção de estruturas químicas passíveis de aprimoramento da atividade pela via sintética de outros derivados. As frações bioativas foram monitoradas quimicamente através de cromatografia de camada delgada, utilizando como revelador uma solução ácida de vanilina, e analisadas por ressonância magnética nuclear de hidrogênio e espectrometria de massas. Os bioensaios com as frações foram realizados em quintuplicata, à temperatura de 28±1ºC, 80±5% de umidade relativa e fotofase de 12 horas. As concentrações letais encontradas da fração MP-9, que apresentou o maior potencial larvicida, CL50 e CL90, foram de 3,1 e 36,6ppm, respectivamente. Todos os experimentos foram acompanhados por uma série controle, contendo o mesmo número de larvas.

Atividade larvicida do óleo de Anacardium humile Saint Hill sobre Aedes aegypti (Linnaeus, 1762) (Diptera, Culicidae)

O objetivo deste estudo foi avaliar o potencial do cajuzinho do cerrado (Anacardium humile) sobre larvas de Aedes aegypti. Os extratos hexânico, etanólico, aquoso e o óleo das folhas foram obtidos do material vegetal coletado em fragmento de cerrado. Estes foram testados nas concentrações 1%; 0,5%, 0,25%, 0,125%, 0,05% e 0,0125% diluídas em dimetil sulfóxido 1%. A contagem das larvas mortas foi realizada após 24 horas. Utilizou-se o método Probit de análise para obtenção das CL50 e respectivos intervalos de confiança. Conclui-se que apenas o óleo extraído de folhas de Anacardium humile causa 100% de mortalidade em larvas de 4º estádio de Aedes aegypti nas concentrações até 0,125%, o que parece indicar que os ingredientes ativos estão na fase mais apolar. O que indica a potencialidade de uso da planta como larvicida de Aedes aegypti, entretanto, novos testes deverão ser conduzidos utilizando outros órgãos vegetais, assim como outros métodos e solventes utilizados na extração.

Atividade larvicida do extrato bruto enzimático do fungo Duddingtonia flagras sobre larvas de primeiro estádio de Angiostrongylus vasorum

INTRODUÇÃO: Angiostrongylus vasorum é um nematóide que parasita cães domésticos e eventualmente o homem. MÉTODOS: O objetivo deste trabalho foi observar a atividade predatória in vitro do extrato bruto enzimático do fungo Duddingtonia flagrans sobre larvas de primeiro estádio A. vasorum em condições laboratoriais no meio ágar-água 2%. RESULTADOS: Ao final do experimento, os percentuais de redução das L1 de A. vasorum observados foram de: 53,5% (24h) e 71,3% (48h) CONCLUSÕES: O extrato bruto enzimático do fungo D. flagrans destruiu in vitro as L1, podendo ser utilizado como controle biológico desse nematóide.

Produtos naturais para o controle da transmissão da dengue: atividade larvicida de Myroxylon balsamum (óleo vermelho) e de terpenóides e fenilpropanóides

The bioassay-guided fractionation of the hexane extract obtained from the medicinal plant Myroxylon balsamum (red oil) was conducted in preparative thin layer chromatography on silica gel. The obtained fractions and some terpenoids and phenylpropanoids were assayed as larvicidal on third instar Aedes aegypti larvae, NPPN colony. The results indicate that the sesquiterpene nerolidol was the active constituent in the extract and that the sesquiterpenes were more active than the monoterpenes and phenylpropanoids utilized in this study. Lipophilicity seems to be an important property for the activity since the compounds with hydroxyl, carbonyl and methoxyl groups were less active. The results confirm also that essential oils can be a good tool for the control of dengue.

Constituintes químicos de Capraria biflora (Scrophulariaceae) e atividade larvicida de seu óleo essencial

Analysis of essential oil from fresh leaves of Capraria biflora allowed identification of fourteen essential oil constituents among which thirteen are sesquiterpene compounds, and α-humulene (43.0%) the major constituent. The essential oil was tested for larvicidal activity against Aedes aegypti showing good activity, with LC50 73.39 µg/mL (2.27 g/mL). Chromatographic studies of extracts from roots and stems allowed the isolation of five compounds: naphthoquinone biflorin, sesquiterpene caprariolide B, the steroid β-sitosterol, the carbohydrate D-mannitol and iridoid myopochlorin first reported in the species C. biflora. The structures of compounds were characterized by spectroscopic data, IR, MS, NMR13C, NMR¹H, NOE, HSQC and HMBC.

Atividade larvicida dos óleos essenciais de Syzygium aromaticum e Citrus sinensis em populações de Aedes aegypti

Pós-graduação em Biociências - FCLAS

Isolamento de uma quitinase extraída do cefalotórax do camarão marinho Litopenaeus schmitti (BURKENROAD, 1936) : avaliação de suas atividades antimicrobiana e larvicida

Chitinases are enzymes involved in degradation of chitin and are present in a range of organisms, including those that do not contain chitin, such as bacteria, viruses, plants and animals, and play important physiological and ecological roles. Chitin is hydrolyzed by a chitinolytic system classified as: endo-chitinases, exo-chitinases and N-acetyl-b-D-glucosaminidases. In this study a Litochitinase1 extracted from the cephalotorax of the shrimp Litopenaeus Schmitt was purified 987.32 times using ionexchange chromatography DEAE-Biogel and molecular exclusion Sephacryl S-200. These enzyme presented a molecular mass of about 28.5 kDa. The results, after kinetic assay with the Litochitinase1 using as substrate p-nitrophenyl-N-acetyl-b-Dglucosaminideo, showed apparent Km of 0.51 mM, optimal activity at pH ranging from 5.0 to 6.0, optimum temperature at 55°C and stability when pre-incubated at temperatures of 25, 37, 45, 50 and 55°C. The enzyme showed a range of stability at pH 4.0 to 5.5. HgCl2 inhibited Litochitinase1 while MgCl2 enhances its activity. Antimicrobial tests showed that Litochitinase1 present activity against gram-negative bacterium Escherichia coli in the 800 μg/mL concentration. The larvicidal activity against Aedes aegypti was investigated using crude extracts, F-III (50-80%) and Litochitinase1 at 24 and 48 hours. The results showed larvicidal activity in all these samples with EC50 values of 6.59 mg/mL for crude extract, 5.36 mg/mL for F-III and 0.71 mg/mL for Litochitinase1 at 24 hours and 3.22 and 0.49 mg/mL for the F-III and Litochitinase1 at 48 hours, respectively. Other experiments confirmed the presence of chitin in the midgut of Aedes aegypti larvae, which may be suffering the action of Litochitinase1 killing the larvae, but also the absence of contaminating proteins as serine proteinase inhibitors and lectins in the crude extract, F-III and Litochitinase1, indicating that the death of the larvae is by action of the Litochitinase1. We also observed that the enzymes extracted from intestinal homogenate of the larvae no have activity on Litochitinase1. These results indicate that the enzyme can be used as an alternative to control of infections caused by Escherichia coli and reducing the infestation of the mosquito vector of dengue.

Isolamento de uma quitinase extraída do cefalotórax do camarão marinho Litopenaeus schmitti (BURKENROAD, 1936) : avaliação de suas atividades antimicrobiana e larvicida

Chitinases are enzymes involved in degradation of chitin and are present in a range of organisms, including those that do not contain chitin, such as bacteria, viruses, plants and animals, and play important physiological and ecological roles. Chitin is hydrolyzed by a chitinolytic system classified as: endo-chitinases, exo-chitinases and N-acetyl-b-D-glucosaminidases. In this study a Litochitinase1 extracted from the cephalotorax of the shrimp Litopenaeus Schmitt was purified 987.32 times using ionexchange chromatography DEAE-Biogel and molecular exclusion Sephacryl S-200. These enzyme presented a molecular mass of about 28.5 kDa. The results, after kinetic assay with the Litochitinase1 using as substrate p-nitrophenyl-N-acetyl-b-Dglucosaminideo, showed apparent Km of 0.51 mM, optimal activity at pH ranging from 5.0 to 6.0, optimum temperature at 55°C and stability when pre-incubated at temperatures of 25, 37, 45, 50 and 55°C. The enzyme showed a range of stability at pH 4.0 to 5.5. HgCl2 inhibited Litochitinase1 while MgCl2 enhances its activity. Antimicrobial tests showed that Litochitinase1 present activity against gram-negative bacterium Escherichia coli in the 800 μg/mL concentration. The larvicidal activity against Aedes aegypti was investigated using crude extracts, F-III (50-80%) and Litochitinase1 at 24 and 48 hours. The results showed larvicidal activity in all these samples with EC50 values of 6.59 mg/mL for crude extract, 5.36 mg/mL for F-III and 0.71 mg/mL for Litochitinase1 at 24 hours and 3.22 and 0.49 mg/mL for the F-III and Litochitinase1 at 48 hours, respectively. Other experiments confirmed the presence of chitin in the midgut of Aedes aegypti larvae, which may be suffering the action of Litochitinase1 killing the larvae, but also the absence of contaminating proteins as serine proteinase inhibitors and lectins in the crude extract, F-III and Litochitinase1, indicating that the death of the larvae is by action of the Litochitinase1. We also observed that the enzymes extracted from intestinal homogenate of the larvae no have activity on Litochitinase1. These results indicate that the enzyme can be used as an alternative to control of infections caused by Escherichia coli and reducing the infestation of the mosquito vector of dengue.

Potencial larvicida de extratos de plantas regionais no controle de larvas de Aedes Aegypti (Diptera:Culicidae)

Dengue, amongst the virus illnesses one can get by vectorial transmission, is the one that causes more impact in the morbidity and mortality of world s population. The resistance to the insecticides has caused difficulties to control of vector insect (Aedes aegypti) and has stimulated a search for vegetables with larvicidal activity. The biodiversity of Caatinga is barely known and it is potential of use even less. Some plants of this biome are commercialized in free fairs northeast of Brazil, because of its phytotherapics properties. The vegetables in this study had been selected by means of a questionnaire applied between grass salesmen and natives of the Serido region from Rio Grande do Norte state; culicids eggs had been acquired with traps and placed in container with water for the larva birth. Thirty larvae had been used in each group (a group control and five experimental groups), with four repetitions four times. The vegetables had been submitted to the processes of decoction, infusion and maceration in the standard concentration of 100g of the vegetable of study in 1l of H2O and analyzed after ½, 1, 2, 4, 8, 12, 24 and 48 hours for verification of the average lethal dose (LD50) from the groups with thirty larva. The LD50 was analyzed in different concentrations (50g/l, 100g/l, 150g/l, 200g/l e 300g/l) of Aspidosperma pyrifolium Mart. 48 extracts of rind, leaf and stem of the seven vegetal species: Aspidosperma pyrifolium Mart., Mimosa verrucosa Benth, Mimosa hostilis (Mart.) Benth., Myracrodruon urundeuva Allemão, Ximenia americana L, Bumelia sartorum Mart Zizyphus joazeiro Mart, had been analyzed. The extracts proceeding from the three methods were submitted to the freezedrying, to evaluate and to quantify substances extracted in each process. The results had shown that Aspidosperma pyrifolium Mart. and Myracrodruon urundeuva Allemão are the species that are more distinguished as larvicidal after 24 hours of experiment, in all used processes of extraction in the assays. The Zizyphus joazeiro Mart species has not shown larvicidal activity in none of the assays. In relation to the extraction method, the decoction was the most efficient method in the mortality tax of the A. aegypti larvae

Avaliação da α-glicosidase Cqm1 de Culex quinquefasciatus e o impacto de suas mutações na fisiologia de larvas

Lysinibacillus sphaericus (Lsp) é uma bactéria entomopatógena que produz a toxina Bináriav(Bin) com atividade larvicida para culicídeos. A sua ação em Culex quinquefasciatus depende da ligação da toxina Bin à α-glicosidase (Aglu) Cqm1, que atua como receptor no epitélio intestinal de larvas. Na colônia R2362, foram caracterizados dois alelos de resistência ao Lsp: cqm1REC e cqm1REC-2, cujas mutações impedem a expressão da Aglu Cqm1. O objetivo deste trabalho foi avaliar a atividade catalítica da Cqm1 e comparar a atividade α-glicosidase e o desenvolvimento pré-imaginal de larvas de indivíduos susceptíveis (S) e resistentes (R) para cada alelo. Para isto, foram avaliados os seguintes parâmetros: atividade catalítica da Cqm1 recombinante; padrão de transcrição de outras Aglus parálogas à Cqm1; atividade de Aglus nativas em larvas; sobrevivência de indivíduos frente a diferentes dietas. A Aglu Cqm1 mostrou atividade enzimática ótima à 37o C, pH 7,5-8,0 e utilizando o substrato sintético pNαG. A atividade α-glicosidase total em larvas S e R foi similar, apesar da ausência de expressão da Cqm1 nas larvas R. A investigação in silico revelou 18 proteínas parálogas à Cqm1 e, dentre 11 investigadas, nove são expressas em larvas S e R. A análise quantitativa de três parálogas demonstrou que duas tem um padrão de transcrição mais elevado em larvas resistentes, sugerindo a existência de um mecanismo de compensação de expressão de α-glicosidases. O desenvolvimento pré-imaginal de larvas S foi decrescente nas seguintes dietas: ração de gatos, ração de peixes, leite desnatado, extrato de levedura e sacarose. De uma forma global, a taxa de sobrevivência de larvas R foi inferior à S em todas as dietas testadas. Os dados obtidos mostram que as mutações ligadas aos alelos cqm1REC e cqm1REC-2 não parecem impactar a atividade Aglu nas larvas e que o custo biológico observado poderia estar relacionado a outros genes e vias metabólicas.

Potencial larvicida de extratos de plantas regionais no controle de larvas de Aedes aegypti

Dengue, amongst the virus illnesses one can get by vectorial transmission, is the one that causes more impact in the morbidity and mortality of world s population. The resistance to the insecticides has caused difficulties to control of vector insect (Aedes aegypti) and has stimulated a search for vegetables with larvicidal activity. The biodiversity of Caatinga is barely known and it is potential of use even less. Some plants of this biome are commercialized in free fairs northeast of Brazil, because of its phytotherapics properties. The vegetables in this study had been selected by means of a questionnaire applied between grass salesmen and natives of the Serido region from Rio Grande do Norte state; culicids eggs had been acquired with traps and placed in container with water for the larva birth. Thirty larvae had been used in each group (a group control and five experimental groups), with four repetitions four times. The vegetables had been submitted to the processes of decoction, infusion and maceration in the standard concentration of 100g of the vegetable of study in 1l of H2O and analyzed after ½, 1, 2, 4, 8, 12, 24 and 48 hours for verification of the average lethal dose (LD50) from the groups with thirty larva. The LD50 was analyzed in different concentrations (50g/l, 100g/l, 150g/l, 200g/l e 300g/l) of Aspidosperma pyrifolium Mart. 48 extracts of rind, leaf and stem of the seven vegetal species: Aspidosperma pyrifolium Mart., Mimosa verrucosa Benth, Mimosa hostilis (Mart.) Benth., Myracrodruon urundeuva Allemão, Ximenia americana L, Bumelia sartorum Mart Zizyphus joazeiro Mart, had been analyzed. The extracts proceeding from the three methods were submitted to the freezedrying, to evaluate and to quantify substances extracted in each process. The results had shown that Aspidosperma pyrifolium Mart. and Myracrodruon urundeuva Allemão are the species that are more distinguished as larvicidal after 24 hours of experiment, in all used processes of extraction in the assays. The Zizyphus joazeiro Mart species has not shown larvicidal activity in none of the assays. In relation to the extraction method, the decoction was the most efficient method in the mortality tax of the A. aegypti larvae

Larvicidal activity of the water extract of Moringa oleifera seeds against Aedes aegypti and its toxicity upon laboratory animals

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Essential oil composition of Croton palanostigma Klotzsch from north Brazil

Os óleos essenciais das folhas, ramos finos, galhos, cascas do caule e frutos de Croton palanostigma foram analisados por CG e CG-EM. Os componentes principais determinados no óleo das folhas foram linalol (25,4%), (E)-cariofileno (21,0%), metileugenol (17,2%) e β-elemeno (6,0%); no óleo dos ramos finos foram α-pineno (41,4%), limoneno (29,0%), sabineno (11,5%) e β-pineno (5,7%); no óleo dos galhos foram metileugenol (24,1%), (E)-metilisoeugenol (15,3%), α-pineno (11,2%) e (E)-cariofileno (8,5%); no óleo das cascas do caule foram a-pineno (31,6%), metileugenol (25,6%) e (E)-metilisoeugenol (23,7%); e no óleo dos frutos foram linalol (42,7%), metileugenol (16,3%) e β-elemeno (6,4%). Análise estatística mostrou que as folhas e os frutos apresentam significante similaridade entre si, assim como os galhos e as cascas do caule. Adicionalmente, o óleo obtido das cascas do caule possui elevada atividade larvicida sobre Artemia salina (CL₅₀, 3,71 ± 0,01 mg mL^-1).

Antioxidant capacity and larvicidal activity of essential oil and extracts from Lippia grandis

The leaves and thin branches of Lippia grandis Schauer, Verbenaceae, are used for flavoring of food in the Brazilian Amazon, as substitute for oregano. In this study the constituents of the essential oil were identified and the antioxidant capacity and larvicidal activity of the oil and methanol extract and its sub-fractions were evaluated. A sensory evaluation was determined in view of absence of toxicity. The oil showed a yield of 2.1% and its main constituents were thymol (45.8%), p-cymene (14.3%), γ-terpinene (10.5%), carvacrol (9.9%) and thymol methyl ether (4.8%), totalizing 85%. The DPPH radical scavenging activity showed values for the EC50 between 9.0 and 130.5 µg mL^-1 and the TEAC/ABTS values varied from 131.1 to 336.0 mg TE/g, indicating significant antioxidant activity for the plant. The total phenolic content ranged from 223.0 to 761.4 mg GAE/g, contributing to the antioxidant activity observed. The crude extracts inhibited the bleaching of β-carotene and the oil showed the greatest inhibition (42.5%). The oil (LgO, 7.6±2.4 µg mL^-1) showed strong larvicidal activity against the brine shrimp bioassay. The sensory evaluation was highly satisfactory in comparison to oregano. The results are very promising for the use of L. grandis in seasoning and antioxidant products.