Bioprospecção de substâncias bioativas de extratos e óleos essenciais de Piper alatipetiolatum Yunck. e Piper purusanum C.DC (Piperaceae) para controle de Aedes aegypti (Linnaeus, 1762), Anopheles darlingi Root, 1926 e Culex quinquefasciatus Say, 1823 (Culicidae)
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Rosemary Aparecida Roque
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Instituto Nacional de Pesquisas da Amazônia - INPA
Resumo
Os mosquitos Aedes aegypti, Anopheles darlingi e Culex quinquefasciatus são vetores de arboviroses, malária e filariose, cujas populações têm mostrado resistência a piretróides, o que reforça a busca por alternativas naturais de controle. Nesta tese, investigou-se o potencial larvicida do óleo essencial (OE) de Piper alatipetiolatum, dos sesquiterpenos 6-ishwarone e ishwarol B, além da piplartina isolada de Piper purusanum, com foco nos mecanismos de ação e seletividade. O OE de Piper alatipetiolatum e o 6-ishwarone apresentaram toxicidade contra Ae. aegypti e An. darlingi (CL50 = 25,03–42,58 μg/mL), induzindo espécies reativas de oxigênio e nitrogênio (36,67–49,33%), aumento de GST (36,00–80,67 μmol/min/mg) e CAT (0,31–0,50 μmol H2O2/min/mg), além de inibição da AChE (25,33–36,00 μmol/min/mg) e redução do teor de tióis (13,33–30,00 μmol/mg). A toxicidade foi baixa para organismos não alvo (CL50 = 415,25–739,29 μg/mL), em contraste com a α-cipermetrina (CL50 = 0,23–0,35 μg/mL). O ishwarol B exibiu atividade larvicida contra as três espécies vetoras (CL50 = 19,57–26,23 μg/mL), com efeito residual inicial de ~50%. Esse composto promoveu elevação de H2O2 (24,3–41,0 μmol/g), peroxidação lipídica (11,00–22,67 ηmol MDA/g) e oxidação proteica (10,00–17,00 nM carbonilas/mg). Houve aumento de SOD (14,33–14,67 mU/mg), CAT (10,47–11,23 mmol H2O2/min/mg) e GPx (11,91–16,46 mmol NADPH/min/mL), além de inibição marcante da AChE (6,67–8,33 μmol/min/mg vs. 105,30–111,30 no DMSO), com IC50 de 3,46 μg/mL. Ensaios de docking confirmaram interação favorável com o sítio ativo da enzima (–8,4 kcal/mol; Ki = 0,695 μM). A piplartina foi ativa contra Ae. aegypti (CL50 = 14,56 μg/mL) e An. darlingi (CL50 = 26,44 μg/mL). Induziu RONS (66,67–86,33%), aumento de CAT (87–94,67 μmol H2O2/min/mg), GST (76–134 μmol/min/mg), oxidases de função mista (26,67–55,0 nmol cti/mg) e esterases (27,67–46,33 nmol cti/mg). Houve inibição da AChE (43,33–48,00 μmol/min/mg). Ensaios ecotoxicológicos revelaram 100% de sobrevivência de organismos aquáticos não alvo expostos a 264,4 μg/mL de piplartina, enquanto a α-cipermetrina reduziu a sobrevivência para 9,1%. Em síntese, o OE de P. alatipetiolatum, o 6-ishwarone, o ishwarol B e a piplartina de P. purusanum mostraram-se larvicidas naturais eficazes, seletivos e multifuncionais, atuando por indução de estresse oxidativo e inibição da acetilcolinesterase. Esses resultados apontam as espécies vegetais promissoras de bioinseticidas para o controle ambientalmente seguro das larvas dos mosquitos estudados.
Abstract:
Aedes aegypti, Anopheles darlingi and Culex quinquefasciatus are vectors of arboviruses, malaria and filariasis in Brazil, with populations showing increasing resistance to pyrethroids, reinforcing the need for natural alternatives. This thesis evaluated the larvicidal potential of the essential oil (EO) of Piper alatipetiolatum, its sesquiterpenes 6-ishwarone and ishwarol B, and piplartine isolated from Piper purusanum, focusing on mechanisms of action and selectivity. The EO of P. alatipetiolatum and 6-ishwarone showed toxicity against Ae. aegypti and An. darlingi (LC50 = 25.03–42.58 μg/mL), inducing overproduction of reactive oxygen and nitrogen species (36.67–49.33%), increased GST (36.00–80.67 μmol/min/mg) and CAT activities (0.31–0.50 μmol H2O2/min/mg), followed by AChE inhibition (25.33–36.00 μmol/min/mg) and reduced thiol content (13.33–30.00 μmol/mg). Both products exhibited low toxicity to non-target organisms (LC50= 415.25–739.29 μg/mL), in contrast to α-cypermethrin (LC50 = 0.23–0.35 μg/mL). Ishwarol B displayed larvicidal activity against the three vector species (LC50 = 19.57–26.23 μg/mL), with an initial residual effect of approximately 50%. It increased H2O2 (24.3–41.0 μmol/g), lipid peroxidation (11.00–22.67 ηmol MDA/g) and protein oxidation (10.00–17.00 nM carbonyls/mg). Enzymatic analyses revealed enhanced SOD (14.33–14.67 mU/mg), CAT (10.47–11.23 mmol H2O2/min/mg) and GPx (11.91–16.46 mmol NADPH/min/mL) activities. AChE was strongly inhibited (6.67–8.33 μmol/min/mg), with IC50 of 3.46 μg/mL. Docking studies confirmed favourable interaction with the enzyme active site (–8.4 kcal/mol; Ki = 0.695 μM). Piplartine showed strong activity against Ae. aegypti (LC50 = 14.56 μg/mL) and An. darlingi (LC50 = 26.44 μg/mL). It induced RONS (66.67–86.33%), increased CAT (87–94.67 μmol H2O2/min/mg), GST (76–134 μmol/min/mg), mixed function oxidases (26.67–55.0 nmol cti/mg) and esterases (27.67–46.33 nmol cti/mg). AChE activity was inhibited (43.33–48.00 μmol/min/mg). Ecotoxicological assays demonstrated 100% survival of non-target aquatic organisms at 264.4 μg/mL of piplartine, whereas α-cypermethrin reduced survival to 9.1%. In summary, the EO of P. alatipetiolatum, 6-ishwarone, ishwarol B and piplartine from P. purusanum proved to be effective, selective and multifunctional natural larvicides, acting through oxidative stress induction and acetylcholinesterase inhibition. These results highlight the plant species as promising sources of bioinsecticides for the environmentally safe control of the larvae of the studied mosquitoes.