ABSTRACT Synthetic chemical insecticides are widely used in population control of pests. Aedes aegypti is the primary vector of dengue, Zika, chikungunya and yellow fever to humans, and has proven resistance to chemical insecticides. As an alternative vector control method, the ethanolic extract of the leaves of Piper peltatum L. (Piperaceae) showed larvicidal activity against Ae. aegypti. Despite the wide medicinal use of this plant, the biological activity of its isolated constituents remains unexplored. In this sense, we isolated, identified and evaluated the larvicidal activity of 4-nerolidylcatechol (4-NC) from P. peltatum against Ae. aegypti, Culex quinquefasciatus and Anopheles darlingi, focusing on the larvicidal, adulticidal and genotoxic potential of 4-NC on Ae. aegypti. Larvae were captured in the city of Manaus, Amazonas state, Brazil. 4-NC was isolated from the extract of the leaves of P. peltatum via silica gel chromatography. This was identified using nuclear magnetic resonance spectroscopy and tested in Artemia franciscana (6.25, 12.5, 25, 50, 100 and 200 µg/mL). In the toxicity bioassay, Ae. aegypti larvae were exposed to 30, 50, 70, 90, and 110 µg/mL of 4-NC, while Cx. quinquefasciatus and An. darlingi were exposed to 6.25, 12.5, 25, 50 and 100 µg/mL. Ae. aegypti larvae were also subjected to 40 and 60 µg/mL of 4-NC (genotoxicity bioassay), and adult females to 62.5 to 1,000 µg/mL (adulticidal bioassay). The results of the 4-NC toxicity assays showed that there was 100% mortality in larvae of Ar. franciscana at the concentration of 200 µg/mL, with an LC50 of 8.0 μg/mL. In the larvae of Ae. aegypti, mortality was 100%, with an LC50 of 62 μg/mL. In larvae of Cx. quinquefasciatus, 97% mortality occurred, with an LC50 of 52.3 μg/mL, and in An. darlingi larvae there was an 83% mortality rate, with an LC50 of 55.8 μg/mL. In adults of Ae. aegypti, however, there was no adulticidal activity. In the larvae of Ae. aegypti, the genotoxic effect of 4-NC (40 and 60 µg/mL) showed significant frequency (p < 0.05) of cellular abnormalities (micronuclei, budding and nuclear bridges) of interphasic nuclei of neuroblasts and oocytes in relation to the negative control. This result may be associated with a decrease in oviposition of females, which was observed in two generations. We can confirm that 4-NC has larvicidal activity against Ae. aegypti, Cx. quinquefasciatus and An. darlingi. Although it does not present adulticidal activity in Ae. aegypti, it reduced the oviposition of females. Therefore, 4-NC seems to be a strong candidate for the development of an alternative method for the control of these mosquitoes in the immature phase. pests dengue Zika humans L Piperaceae (Piperaceae Ae plant unexplored sense 4nerolidylcatechol nerolidylcatechol 4 4NC NC (4-NC P Manaus state Brazil chromatography 625 6 25 (6.25 125 12 5 12.5 10 20 µgmL . µg mL bioassay 30 70 90 11 4NC, NC, Cx An 6.25 genotoxicity , bioassay) 62. 1000 1 000 1,00 bioassay. Ar LC LC5 80 8 0 8. μgmL μg μg/mL 97 occurred 523 52 3 52. 83 rate 558 55 55. however (4 p 0.05 005 05 micronuclei, micronuclei (micronuclei bridges generations Therefore phase 2 (6.2 12. 7 9 6.2 00 1,0 ( 0.0 (6. 6. 1, 0. (6

ABSTRACT The new molecules piperidyl dillapiole and propyl ether dillapiole were evaluated in Aedes aegypti as an alternative for the control of populations of this mosquito. A total of 1,690 samples, comprising 780 eggs, 780 larvae and 130 adults, were treated with both substances for 4 h, 24 h and 90 min, respectively. The dillapiole (80 µg/mL), temephos (0.012 µg/mL) as positive control, and the negative control (water + DMSO 0.05%). The 50% lethal concentrations (LC50) of propyl ether dillapiole and piperidyl dillapiole in eggs were 18.07 and 49.97 µg/mL and, in larvae, the LC50 of these substances were 29.15 and 72.93 µg/mL, which caused darkening of the cuticle and displacement of the head. In the adults, the LC50 of the two substances after 90 min was 148.25 and 263.26 µg/mL, respectively. The insertion of the propyl and piperidine radicals into the dillapiole molecule resulted in the substances propyl ether dillapiole and piperidyl dillapiole, both of which had a toxic effect on Ae. aegypti. However, propyl ether dillapiole, which has propylene in its side chain, showed greater ovicidal, larvicidal and adulticidal activity when compared to piperidyl dillapiole. These results are promising as an alternative form of control of Ae. aegypti, which is the primary vector of human arboviruses. mosquito 1690 1 690 1,69 samples 78 13 adults 2 9 respectively 80 (8 µgmL , µg mL 0.012 0012 0 012 (0.01 water 0.05%. 005 0.05% . 05 0.05%) 50 LC (LC50 1807 18 07 18.0 4997 49 97 49.9 LC5 2915 29 15 29.1 7293 72 93 72.9 head 14825 148 25 148.2 26326 263 26 263.2 Ae However chain ovicidal arboviruses 169 69 1,6 7 8 ( 0.01 001 01 (0.0 00 0.05 5 (LC5 180 18. 499 49. 291 29. 729 72. 1482 14 148. 2632 263. 16 6 1, 0.0 (0. (LC 0. (0

RESUMO Dentre as doenças negligenciadas, a leishmaniose cutânea americana (LCA) continua sendo altamente endêmica em algumas regiões tropicais. Os medicamentos disponíveis atualmente para o tratamento apresentam elevada toxicidade, o que torna a busca de novas opções terapêuticas necessária. O objetivo do estudo foi avaliar o potencial tóxico do éter n-butil dilapiol (EBD) contra Leishmania amazonensis e L. guyanensis e a sua toxicidade em células mononucleares do sangue periférico humano (CMSP) in vitro. Para a citotoxicidade celular, utilizou-se concentrações de 7,8 a 500 µM por 48 e 72 h. A atividade antileishmania do EBD foi testada em concentrações de 0,28 a 18 µM, por 24, 48 e 72 h. Para o ensaio em amastigotas usou-se um valor de 36 µM. O índice de seletividade (IS) foi determinado pela divisão CC50/CI50 (macrófagos/promastigotas). O EBD mostrou em células uma CC50 de 203,9 ± 0,5 µM em 72 h. O EBD inibiu as formas promastigotas com uma CI50 de 3,0 µM para ambas espécies de Leishmania por 72 h, já o padrão Pentacarinat® apresentou uma CI50 de 2,9 µM e 0,3 µM, respectivamente. O IS do EBD para as duas espécies foi de 67,9 por 72 h. O EBD inibiu as formas intracelulares de L. amazonensis com 65,5% por 48 h. Na modelagem molecular, o DLpOl-F aparece com duas ligações de hidrogênio (SER418 e 421). O EBD demostrou um potencial antileishmania promissor in vitro. negligenciadas LCA (LCA tropicais necessária nbutil n butil (EBD L CMSP (CMSP vitro celular utilizouse utilizou se 78 7 8 7, 50 4 h 028 0 28 0,2 1 24 usouse usou 3 (IS CC50CI50 CCCI CC CI CC50/CI5 macrófagos/promastigotas. macrófagospromastigotas macrófagos/promastigotas . macrófagos (macrófagos/promastigotas) CC5 2039 203 9 203, 05 5 0, CI5 30 3, Pentacarinat 29 2 2, 03 respectivamente 679 67 67, 655 65 65,5 molecular DLpOlF DLpOl F SER418 SER (SER41 421. 421 421) 02 CC50CI5 CC50/CI (macrófagos/promastigotas 20 6 65, SER41 (SER4 42 CC50CI SER4 (SER

ABSTRACT Among the neglected diseases, American cutaneous leishmaniasis (ACL) still remains highly endemic in some tropical regions. The currently available drugs for treatment are highly toxic, prompting the search for new therapeutic options. The aim of this study was to evaluate the toxic potential of dillapiole n-butyl ether (DBE) against Leishmania amazonensis and L. guyanensis, as well as its toxicity on human peripheral blood mononuclear cells (PBMCs) in vitro. For cell cytotoxicity, concentrations of DBE that ranged from 7.8 to 500 µM were used for 48 and 72 h. For the evaluation of the antileishmanial activity, DBE was tested at concentrations of 0.28 to 18 µM for 24, 48, and 72 h. A value of 36 µM was used for the amastigote assay. The selectivity index (SI) was determined by dividing the CC50/IC50 (macrophages/promastigotes). DBE exhibited a CC50 of 203.9 ± 0.5 µM in 72 h. DBE inhibited promastigote forms with an IC50 of 3.0 µM for both Leishmania species for 72 h. The standard, Pentacarinat®, showed an IC50 of 2.9 µM and 0.3 µM, respectively. The SI of DBE for both species was 67.9 for 72 h. DBE inhibited intracellular forms of L. amazonensis by 65.5% after 48 h. In molecular modeling, DLpOl-F showed two hydrogen bonds (SER418 and 421). DBE demonstrated promising in vitro antileishmanial potential. diseases ACL (ACL regions options nbutyl n butyl (DBE L guyanensis PBMCs (PBMCs cytotoxicity 78 7 8 7. 50 4 h activity 028 0 28 0.2 1 24 3 assay (SI CC50IC50 CCIC CC IC CC50/IC5 macrophages/promastigotes. macrophagespromastigotes macrophages/promastigotes . macrophages promastigotes (macrophages/promastigotes) CC5 2039 203 9 203. 05 5 0. IC5 30 3. standard Pentacarinat Pentacarinat® 29 2 2. 03 respectively 679 67 67. 655 65 65.5 modeling DLpOlF DLpOl F SER418 SER (SER41 421. 421 421) 02 CC50IC5 CC50/IC (macrophages/promastigotes 20 6 65. SER41 (SER4 42 CC50IC SER4 (SER

Abstract INTRODUCTION: Semi-synthetic dillapiole compounds derived from Piper aduncum essential oil are used as alternative insecticides to control insecticide-resistant Aedes aegypti. Thus, we aimed to evaluate the genotoxic effects of semi-synthetic isodillapiole on the nuclei of neuroblasts (larvae) and oocytes (females) and the mean oviposition rates of the females over four generations (G1, G2, G3, and G4) of Ae. aegypti. METHODS: Larvae were captured in the city of Manaus, Amazonas state, Brazil, and exposed to isodillapiole in bioassays (20, 40, and 60 µg/mL) and a negative control (0.05% DMSO in tap water) for 4 h. The cerebral ganglia were extracted from the larvae and oocytes from the adult females to prepare slides for cytogenetic analysis. Breeding pairs were established and eggs counts were quantified taken after the bioassays. RESULTS: The analysis of 20,000 interphase nuclei of neuroblasts and oocytes indicated significant genotoxicity (micronuclei, budding, polynucleated cells, and other malformations) compared to that of the control. Metaphasic and anaphasic nuclei presented chromosomal breaks; however, no significant variation and damage was observed in the negative control. A significant reduction in mean oviposition rates was also recorded following exposure to isodillapiole over the four generations (G1, G2, G3, and G4). CONCLUSIONS: The toxic and genotoxic effects of isodillapiole on Ae. aegypti were caused by reduced oviposition in the females and nuclear abnormalities over the four generations of the trials. Further studies are required, rather than our in vitro assays, to verify the efficacy of exposure to this compound for controlling Ae. aegypti.

Phytochemical investigation from the stems of Spathelia excelsa (Rutaceae) collected in Amazonas yielded deacetylspathelin (1), 7,8-dimethoxyflindersine (2), new glabretal-type triterpenoid 3β-angeloyl-21,24-epoxy-7α, 21α, 23α, 25-tetrahydroxy-4α, 4β, 8β, 10β-tetramethyl-25-dimethyl-14, 18-cyclo-5α, 13α, 14α, 17α-cholestane (3), in addition to the known steroids β-sitosterol and stigmasterol. Their structures were established on the basis of spectral data. The compounds 1 and 3 were assayed on Aedes aegypti (larvicidal and adulticidal activities and compound 3 exhibited larvicidal properties with LC50 of 4,8 µg/mL.

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).

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).

Foram testados 226 extratos metanólicos e aquosos de 74 espécies vegetais (a maioria nativas) encontradas no Estado do Amazonas, Brasil, para letalidade às larvas da espécie microcrustácea Artemia franciscana na concentração teste de 500 μg/mL. Várias espécies citotóxicas foram identificadas nesse estudo: Aspidosperma marcgravianum, A. nitidum, Citrus limetta, Croton cajucara, Geissospermum argenteum, Minquartia guianensis, Piper aduncum, P. amapense, P. capitarianum, P. tuberculatum e Protium aracouchini. Os resultados foram analisados levando em consideração as informações populares, quando disponíveis.

226 methanol and water extracts representing 74 mainly native plant species found in Amazonas State, Brazil, were tested at a standard concentration of 500 μg/mL for lethality towards larvae of the brine shrimp species Artemia franciscana. Several cytotoxic plant species were identified in this work: Aspidosperma marcgravianum, A. nitidum, Croton cajucara, Citrus limetta, Geissospermum argenteum, Minquartia guianensis, Piper aduncum, P. amapense, P. capitarianum, P. tuberculatum and Protium aracouchini. The results were analyzed within the context of the available traditional knowledge and uses for these plants.