Abstract Around the world, the main problems of livestock are caused by ectoparasites, however, commercial acaracide are toxic to the environment and detrimental to One Health. Therefore, research has increasingly focused on development of natural products as alternatives for tick control. The purpose of this study was to evaluate the larvicidal effect on Rhipicephalus (Boophilus) microplus, through use of essential oils (EOs) extracted from the leaves, flower buds and stems of Tetradenia riparia. The chemical composition of these EOs was determined through gas chromatography coupled to mass spectrometry (GC-MS). They were tested on larvae at concentrations of 100.000 to 40 µg/mL, using the larval packet test and under semi-natural conditions. The main class of compounds in the chemical composition was sesquiterpenes (both oxygenates and hydrocarbons), whereas the predominant compounds in the leaves, flower buds and stems were 14-hydroxy-9-epi-caryophyllene, T-cadinol and 6-7-dehydroroyleanone, respectively. The leaves proved to be the most effective, with highest larvicidal activity (LC99.9 = 83.53 µg/mL). When tested under semi-natural conditions, the oils obtained efficiency above 98% in all compound tests. The results indicated that these EOs were effective against R. (B.) microplus larvae in vitro and ex-situ, proving that this plant has bioactive molecules with significant larvicidal activity.

Resumo Os principais problemas para a pecuária estão relacionados às ectoparasitoses, e ao fato dos carrapaticidas apresentarem elevada toxicidade ao meio ambiente e à saúde única. Surgem, então, demandas na busca por inovações e desenvolvimento de produtos naturais, como alternativas para o controle dos carrapatos. O objetivo deste trabalho foi avaliar o potencial da atividade larvicida sobre Rhipicephalus (Boophilus) microplus a partir dos óleos essenciais de Tetradenia riparia (TrOEs) extraídos das folhas, botões florais e caules. A composição química foi determinada por cromatografia gasosa acoplada à espectrometria de massa (GC/MS). Os TrOEs foram testados sobre larvas nas concentrações de 100.000 a 40 µg/mL pelo teste de pacote de larvas e em condições seminaturais. Na composição química, a classe majoritária foi os sesquiterpenos (oxigenados e hidrocarbonetos); já os compostos em destaques foram 14-hidroxy-9-epi-caryophyllene, T-cadinol e 6-7-dehidroroyleanone para folhas, botões florais e caules, respectivamente. As folhas demonstraram ser mais eficientes e com maior poder larvicida (CL99.9 = 83.53 µg/mL). Quando testado em condições seminaturais os óleos obtiveram eficiência acima de 98% em todos os compostos testados. Os resultados indicaram que os TrOEs, foram eficazes sobre as larvas de R. (B.) microplus in vitro e ex-situ, evidenciando que esta planta possui moléculas bioativas com ação larvicidas significativas.

Abstract Tetradenia riparia (Hochst.) Codd (Lamiaceae) is a species native to the African continent and used as an insect repellent. The objective of the study was to evaluate the larvicidal potential of essential oils (EOs) from the leaves, flower buds, and stem of T. riparia, collected in winter against Aedes aegypti larvae. The EOs were extracted by hydrodistillation (3 h) and identified by GC/MS. The EOs were tested against larvae of A. aegypti at concentrations ranging from 12500 to 1.5 µg/mL for 24 h. The insecticide activity was evaluated by probit analysis, and the anticholinesterase activity was determined by bioautographic method. The results of the class projection indicated sesquiterpenes as the majority class, corresponding to 60.66% (leaves), 64.70% (flower buds) and 83.99% (stem), and the bioassays on A. aegypti larvae indicated LC50 of 1590, 675 and 665 µg/mL, respectively. The anticholinesterase activity indicated that the EO of the leaves inhibited the enzyme at a concentration of 780 µg/mL, and those from the flower buds and stem inhibited up to 1560 µg/mL. The results indicated weak activity of essential oils against A. aegypti larvae.

Resumen Introducción: Los fitoinsecticidas son alternativas para el control de insectos en diferentes etapas, Gallesia integrifolia (Spreng.) Harms, familia Phytolacaceae, conocida popularmente como pau d’alho, árbol de ajo y guararema en Brasil, es conocida por su fuerte olor aliáceo debido a la presencia de moléculas de azufre en la planta. Esta especie presenta actividad biológica y potencial efecto insecticida que aún no está explotado. Objetivo: El objetivo de este estudio fue evaluar la actividad biológica del extracto crudo etanólico de las hojas, flores y frutos de G. integrifolia en el control de las larvas y pupas de la tercera etapa de Aedes aegypti. Métodos: El material botánico fue recolectado en la ciudad de Umuarama, Paraná, Brasil (23º46’16” S & 53º19’38” W), a una altitud de 442 m y los frutos de G. integrifolia fueron recolectados en mayo de 2017 y las hojas y flores en diciembre del mismo año. Los extractos crudos de hojas, flores y frutos de G. integrifolia se prepararon por técnica de maceración dinámica. La composición química de los extractos se determinó por cromatografía de gases acoplada a espectrometría de masas. La actividad insecticida de los extractos crudos de G. integrifolia fue evaluada en larvas y pupas de A. aegypti en concentraciones entre 0.001 a 25 000 mg/mL, y las concentraciones letales que matan 50 % (CL50) y 99.9 % (CL99.9) de larvas fueron determinados por análisis de probit. La actividad anticolinesterasa fue evaluada por método bioautográfico en concentraciones de 0.000095 a 50 mg/mL. Resultados: El rendimiento de los extractos crudos de G. integrifolia fue de 8.2, 9.1 y 17.3 % para flores, frutos y hojas, respectivamente. La composición química de los extractos de G. integrifolia se caracterizó por la presencia de ésteres de ácidos grasos, fitosteroles, vitaminas, diterpenos oxigenados y compuestos organosulfurados. El extracto de las flores presentó alta cantidad de compuestos de azufre (20.2 %) como disulfuro, bis(2-sulfhidril etilo) (11.9 %), 2,3,5-tritiahexano (6.2 %), 1,2,4-tritiolano (1.1 %) y 2,4-ditiapentano (1.1 %). En relación con la actividad insecticida, el extracto de las flores mostró una gran actividad con CL99.9 de 0.032 mg/mL y CL99.9 de 0.969 mg/mL en larvas y pupas de A. aegypti, respectivamente, y la inhibición más alta de la enzima acetilcolinesterasa (0.00019 mg/mL) ex situ. El extracto de las flores presentó actividad anticolinesterasa y larvicida, 12.8 y 35.6 %, respectivamente, mayor que el control temephos. Conclusiones: Este estudio abre nuevas perspectivas sobre el uso de extractos de G. integrifolia como alternativa bioinsecticida para el control de larvas y pupas de A. aegypti.

Abstract Introduction: Phytoinsecticides are alternatives to control insects in different stages, Gallesia integrifolia (Spreng.) Harms, Phytolacaceae family, popularly known as pau d’alho, garlic tree, and guararema in Brazil, is known due to its strong alliaceous odor because of the presence of sulfur molecules in the plant. This species presents biological activity and potential insecticide effect that is still unexploited. Objective: This study aimed to evaluate the biological activity of the ethanolic crude extract from G. integrifolia leaves, flowers, and fruits on the control of Aedes aegypti third-stage larvae and pupae. Methods: The botanical material was collected in city Umuarama, Paraná, Brazil at the coordinates (23º46’16” S & 53º19’38” WO), and altitude of 442 m, the fruits of G. integrifolia were collected in May and the leaves and flowers in December 2017. The crude extracts of G. integrifolia leaves, flowers, and fruits were prepared by dynamic maceration technique. The chemical composition of the extracts was determined by gas chromatography coupled to a mass spectrometry. The insecticidal activity of the crude extracts of G. integrifolia were carried out on larvae and pupae of A. aegypti in concentrations between 0.001 to 25 000 mg/mL, and afterwards the lethal concentrations that kill 50 % (LC50) and 99.9 % (LC99.9) were determined by probit analysis. Anticholinesterase activity was determined by bioautographic method at concentrations from 0.000095 to 50 mg/mL. Results: The yield of G. integrifolia crude extracts were 8.2, 9.1, and 17.3 % for flowers, fruits, and leaves, respectively. The chemical composition of G. integrifolia extracts was characterized by presence of fatty acid esters, phytosterols, vitamins, oxygenated diterpenes and organosulfur compounds. The flower extract presented the high amount of sulfur compounds (20.2 %) such as disulfide, bis (2-sulfhydryl ethyl) (11.9 %), 2,3,5-trithiahexane (6.2 %), 1,2,4-trithiolane (1.1 %), and 2,4-dithiapentane (1.1 %). Regarding the insecticidal activity, flower extract showed highly active with LC99.9 of 0.032 mg/mL and LC99.9 of 0.969 mg/mL on A. aegypti larvae and pupae, respectively, and the highest inhibition of acetylcholinesterase enzyme (0.00019 mg/mL) ex situ. The flower extract presented anticholinesterase and larvicide activity, respectively, 12.8 % and 35.6 % greater than the control temephos. Conclusions: This study opens new perspectives on the use of extracts from G. integrifolia as a bioinsecticide alternative for the control of A. aegypti larvae and pupae.

RESUMO: O Brasil dispõe de um dos maiores rebanhos bovinos comerciais do mundo, sendo natural que junto a esse rebanho, coexista uma enorme quantidade de espécies parasitárias; dentre estes o carrapato Rhipicephalus (Boophilus) microplus que interfere na produtividade animal, causando prejuízos à pecuária de corte e leite. A utilização de acaricidas químicos no controle deste ácaro tem causado o surgimento de populações resistentes e neste sentido, controles alternativos utilizando plantas como fontes de acaricidas botânicos têm se mostrado eficazes. Eugenia pyriformis Cambess é uma planta brasileira com atividades antioxidante e antimicrobiana, entretanto não há relatos da atividade acaricida. O objetivo do presente estudo consistiu na avaliação do potencial acaricida e larvicida do óleo essencial das folhas de E. pyriformis sobre o carrapato bovino nos diferentes estágios do ciclo reprodutivo. As folhas foram coletadas, secas e o OE extraído por hidrodestilação (3 horas) em aparelho Clevenger modificado. A análise química foi realizada por cromatografia gasosa acoplada à espectrometria de massas (CG/EM) sendo identificados 32 compostos, pertencentes à classe sesquiterpenos: hidrocarbonetos (17.98%) e oxigenados (81.96%); tendo como majoritários o spathulenol (43,65%) e caryophylene oxide (12,17%). O OE foi avaliado pelo teste de imersão de adultos nas concentrações (500,00 a 3,12mg/mL) onde foram mensurados os parâmetros: mortalidade das fêmeas (%), eclodibilidade dos ovos (%) e eficiência do produto (%). As larvas foram avaliadas pelo teste de imersão larval (Larval Packet Test) nas concentrações que variaram de 25,00 a 0,00004mg/mL. Foram determinadas as concentrações letais (CLs) necessárias para matar 50 e 99,9% das fêmeas adultas e das larvas utilizando a análise de Probitos. As CL50 e CL99,9 do OE foram (0,06 e 24,60mg/mL) para as larvas e (1.208,80 e 2.538mg/mL) para as fêmeas adultas, respectivamente. Outro parâmetro avaliado em nossa pesquisa foi mensurar a ação do OE no ciclo de vida livre das larvas de R.(B.) microplus; onde as larvas foram depositadas em vasos com Brachiaria decumbens e após migração destas para o ápice das folhas, foi aspergido solução contendo a CL99,9 (24,60mg/mL) do OE. Após 24 horas, 72,25% das larvas morreram indicando que houve estabilidade do OE quando submetido a condições de temperatura e umidade não controladas. O mecanismo de ação pelo qual o OE matou as larvas e fêmeas adultas foi investigado pelo método bioautográfico, indicando uma inibição de 3,15mg/mL do óleo essencial sobre a enzima acetilcolinesterase (AChE). Os resultados encontrados no presente experimento indicaram que o óleo essencial de E. pyriformis é uma alternativa no controle do carrapato bovino no estágio larval (parasitário) e no ciclo de vida livre (estágio não parasitário) em condições de campo.

ABSTRACT: Brazil has one of the largest commercial cattle herds in the world, which naturally coexist with an enormous number of parasitic species. Southern cattle tick, Rhipicephalus (Boophilus) microplus, is among these species, interfering with animal productivity and causing losses to the beef and dairy cattle sector. The use of chemical acaricides in the control of this mite has resulted in the emergence of resistant populations. In this sense, alternative control measures using plants as sources of botanical acaricides have shown to be effective. Eugenia pyriformis Cambess is a Brazilian plant with antioxidant and antimicrobial activity; however, there are no reports on its acaricidal activity in the literature. The present study aimed to evaluate the acaricidal and larvicidal potential of E. pyriformis leaf essential oil (EO) on southern cattle tick at different stages of the reproductive cycle. E. pyriformis leaves were collected and dried, and had their EO extracted by hydrodistillation (3h) using a modified Clevenger apparatus. Chemical analysis was performed by gas chromatography coupled with mass spectrometry (GC/MS), and 32 compounds belonging to the sesquiterpene class were identified: hydrocarbons (17.98%) and oxygenated forms (81.96%), with spathulenol (43.65%) and caryophyllene oxide (12.17%) as the most common. The EO was evaluated by the Adult Immersion Test at the concentrations (500.00 to 3.12mg/mL) in which the following parameters were measured: mortality of females (%), hatchability of eggs (%), and product efficiency (%). Larvae were assessed by the Larval Packet Test at concentrations ranging from 25.00 to 0.00004mg/mL. Lethal concentrations (LC) required for killing 50 and 99.9% of adult females and larvae were determined using Probit analysis. LC50 and LC99.9 of EO were 0.06 and 24.60mg/mL and 1,208.80 and 2,538mg/mL for larvae and adult females, respectively. Action of the EO in the free-living cycle of R. (B.) microplus larvae was another parameter assessed. To this end, the larvae were deposited in pots containing Brachiaria decumbens and, after migration to the leaf apex, a solution containing LC99.9 (24.60mg/mL) of the EO was sprayed. After 24h, 72.25% of the larvae had died, indicating stability of the EO when subjected to uncontrolled temperature and humidity conditions. The mechanism of action through which the EO killed the larvae and adult females was investigated by the Bioautographic Method, which showed inhibition of 3.15mg/mL of the EO on the acetylcholinesterase (AChE) enzyme. The results found in the present experiment indicate that E. pyriformis essential oil is an alternative in the control of southern cattle tick in the larval (parasitic) and free-living cycle (non-parasitic) stages under field conditions.

ABSTRACT Gallesia integrifolia (Phytolaccaceae) is native to Brazil and has a strong alliaceous odor. The objective of this study was to identify the chemical composition of G. integrifolia fruit essential oil and evaluate fungicidal activity against the main food-borne diseases and food spoilage fungi. The essential oil was extracted by hydrodistillation and identified by GC–MS. From 35 identified compounds, 68% belonged to the organosulfur class. The major compounds were dimethyl trisulfide (15.49%), 2,8-dithianonane (52.63%) and lenthionine (14.69%). The utilized fungi were Aspergillus fumigatus, Aspergillus niger, Aspergillus ochraceus, Aspergillus versicolor, Penicillium funiculosum, Penicillium ochrochloron, Penicillium verrucosum var. cyclopium, and Trichoderma viride. Minimal fungicidal concentration for the essential oil varied from 0.02 to 0.18 mg/mL and bifonazole and ketoconazole controls ranged from 0.20 to 3.50 mg/mL. The lower concentration of the essential oil was able to control P. ochrochloron, A. fumigatus, A. versicolor, A. ochraceus and T. viride. This study shows a high fungicidal activity of G. integrifolia fruit essential oil and can support future applications by reducing the use of synthetic fungicides.

The herbaceous shrub Tetradenia riparia has been traditionally used to treat inflammatory and infectious diseases. Recently, a study showed that T. riparia essential oil (TrEO) obtained in summer has antileishmanial effects, although these results could be influenced by seasonal variation. This study evaluated the activity of the TrEO obtained in different seasons against Leishmania (Leishmania) amazonensis, in vitro and in vivo. The compounds in the TrEO were analysed by gas chromatography-mass spectrometry; terpenoids were present and oxygenated sesquiterpenes were the majority compounds (55.28%). The cytotoxicity and nitric oxide (NO) production were also tested after TrEO treatment. The TrEO from all seasons showed a 50% growth inhibitory concentration for promastigotes of about 15 ng/mL; at 30 ng/mL and 3 ng/mL, the TrEO reduced intracellular amastigote infection, independently of season. The TrEO from plants harvested in summer had the highest 50% cytotoxic concentration, 1,476 ng/mL for J774.A1 macrophages, and in spring (90.94 ng/mL) for murine macrophages. NO production did not change in samples of the TrEO from different seasons. The antileishmanial effect in vivo consisted of a reduction of the parasite load in the spleen. These results suggest that the TrEO has potential effects on L. (L.) amazonensis, consonant with its traditional use to treat parasitic diseases.

A Calendula officinalis L., pertence à família Asteraceae, é originária da Europa e está aclimatada na região Sul do Brasil, onde vem crescendo o seu cultivo comercial para fins farmacêuticos e cosméticos. Suas inúmeras propriedades terapêuticas entre as quais antiinflamatória, cicatrizante e anti-séptica, justificam a grande procura desta planta pela população. O presente trabalho compara a composição química da fração volátil da cultura de calêndula aclimatada na região Noroeste do Paraná, com os dados da literatura da cultura na França. As substancias químicas majoritários identificadas por cromatografia gasosa acoplada à espectrometria de massas (CG/MS), nas duas culturas, foram sesquiterpenos hidrocarbonetos e álcoois; d-cadineno (22,53%) e a-cadinol (20,40%) para a cultura de calêndula produzida na região Noroeste do Paraná, Brasil, e d-cadineno (12,10%) e a-cadinol (25,50%) distinto do registrado para a cultura de calêndula produzida na região de Massif Central, França. Adicionalmente, para garantir a qualidade da planta na comercialização, foram realizadas análises físico-químicas (perda por secagem = 85,0% e perda por dessecação = 11,6%).

Calendula officinalis L., belongs to the Asteraceae family from Europe and is acclimatized in the south region of Brazil where its commercial cultivation has been increasing for pharmaceutical and cosmetic purposes. Its several therapeutic properties (anti-inflammatory, healing, antiseptic) justify the great demand for this plant by the population. The present study compares the chemical composition of the volatile fraction of two cultivations of calendula, one from Massif Central, France, and the other acclimatized in the Northeast region of Paraná State, Brazil. Most compounds identified by Gas Chromatography-mass spectrometry (GC-MS) in both cultivations included the sesquiterpenes hydrocarbons and alcohols; d-cadinene (22.53%) and á-cadinol (20.40%) for the cultivation of the calendula produced in the Northeast region of Paraná State, and d-cadinene (12.10%) and a-cadinol(25.50%) for the cultivation of the calendula produced in Massif Central, France. In addition, physical-chemical analyses (determination of water at room temperature = 85.0% and loss on drying = 11.6%), were carried out in order to assure the quality of the plant for commercialization.