Over the past years, natural products have been explored in order to find biological active substances to treat various diseases. Regarding their potential action against parasites such as trypanosomatids, specially Trypanosoma cruzi and Leishmania spp., much advance has been achieved. Extracts and purified molecules of several species from genera Piper, Tanacetum, Porophyllum, and Copaifera have been widely investigated by our research group and exhibited interesting antitrypanosomal and antileishmanial activities. These natural compounds affected different structures in parasites, and we believe that the mitochondrion is a strategic target to induce parasite death. Considering that these trypanosomatids have a unique mitochondrion, this cellular target has been extensively studied aiming to find more selective drugs, since the current treatment of these neglected tropical diseases has some challenges such as high toxicity and prolonged treatment time. Here, we summarise some results obtained with natural products from our research group and we further highlighted some strategies that must be considered to finally develop an effective chemotherapeutic agent against these parasites.

Abstract The therapeutic drugs to treat Herpes simplex virus (HSV) infections have toxic side effects and there has been an emergence of drug-resistant strains. Therefore, the search for new treatments for HSV infections is mounting. In the present study, semi-solid formulations containing a crude hydroethanolic extract (CHE) from Schinus terebinthifolia were developed. Skin irritation, cutaneous permeation, and in vivo therapeutic efficacy of the formulations were investigated. Treatment with the ointment formulations did not result in any signs of skin irritation while the emulsions increased the thickness of the epidermis in Swiss mice. The cutaneous permeation test indicated that the CHE incorporated in the formulations permeated through the skin layers and was present in the epidermis and dermis even 3 h after topical application. In vivo antiviral activity in BALB/c mice treated with the CHE ointments was better than those treated with the CHE emulsions and did not significantly differ from an acyclovir-treated group. Taken together, this suggests that the incorporation of CHE in the ointment may be a potential candidate for the alternative topical treatment of herpetic lesions.

Abstract Hesperidin is a natural compound which is found in citric fruits and presents antitumor and antimicrobial activities. However, the in vivo efficacy of Hesperidin is reduced due to its low oral bioavailability. Protein-based nanoparticles have been applied to improve biological parameters of drugs and natural compounds. Gliadin is a monomeric protein present in wheat. In this study, gliadin-based nanoparticles containing hesperidin were obtained by desolvation technique and a Taguchi orthogonal array design was employed to optimize the formulation. The independent variables were set as concentration of CaCl2 (0.5; 1 or 2%) and stabilizing agent (Pluronic F68, Tween 80 or sodium caseinate). The dependent variables consisted of mean diameter, polydispersity index, zeta potential, and encapsulation efficiency. The results showed significant effects on the dependent variables when 1% CaCl2 and Pluronic F68 were used. The optimized formulation was coated with chitosan to increase the physical stability of the nanoparticles. The final nanoparticles presented a mean diameter of 321 nm and polydispersity index of 0.217, and spherical shape. After coating, the Zeta potential was +21 mV, and the encapsulation efficiency was 73 %. The in vitro release assay showed that about 98% of the drug was released from the nanoparticles after 48 h. Moreover, the nanoparticles reduced hesperidin cytotoxicity on healthy cells (Vero cells) and improved the cytotoxicity on tumor cells (HeLa, PC-3 and Caco-2 cells). Results showed that the chitosan-coated gliadin nanoparticles are potential carriers for hesperidin delivery for cancer treatment.

RESUMO: Protozoários do gênero Phytomonas são parasitas prejudiciais a várias culturas agrícolas de importância econômica. Devido a sua atividade biológica reconhecida, extratos brutos de folhas de Piper aduncum, P. crassinervium, P. hispidum e P. amalago, foram testadas pela técnica de microdiluição em placa para avaliar o seu potencial antiparasitário contra Phytomonas serpens. Os resultados mostraram que o extrato bruto etanólico de P. crassinervium e P. amalago apresentaram as melhores concentrações inibitórias para 50% das células (IC50), 16,5 µg mL-1 na fase clorofórmio e 18 µg mL-1 na fase aquosa, respectivamente, após 48 h de tratamento. Análises de citotoxicidade foram realizadas através do método colorimétrico da sulforodamina-B, em células de mamíferos LLCMK2. A fase clorofórmio de P. crassinervium foi submetida ao processo de fracionamento, no qual as frações acetato de etila e diclorometano obtiveram melhores valores de IC50. Imagens de microscopia eletrônica de varredura (MEV) mostraram alterações na membrana celular dos parasitas tratados com fase aquosa de P. amalago. Os dados obtidos indicam potencial efeito antiparasitário das espécies de Piper analisadas contra P. serpens, sendo consideradas candidatas promissoras para formulações de bioprodutos para controle do parasito.

ABSTRACT: Protozoa of the genus Phytomonas are harmful parasites to several agricultural crops of economic importance. Due to their recognized biological activity, crude extracts of Piper aduncum, P. crassinervium, P. hispidum, and P. amalago leaves, were tested using the microdilution plate technique to assess the antiparasitic potential against Phytomonas serpens. Results showed that the ethanolic crude extract of P. crassinervium and P. amalago presented the best inhibitory concentration for 50% of the cells (IC50), 16.5 µg mL-1 in chloroform phase, and 18 µg mL-1 in aqueous phase, respectively, after 48 h treatment. Cytotoxicity analyses were performed using the colorimetric method of sulforhodamine-B in LLCMK2 mammalian cells. The chloroform phase of P. crassinervium was subjected to the fractionation process, in which the ethyl acetate and dichloromethane fractions obtained better IC50 values. Scanning electron microscopy (SEM) images showed alterations in the cell membrane of the treated parasites. The data obtained indicate a potential antiparasitic effect of the Piper species analyzed against P. serpens, being considered promising candidates for formulations of bioproducts to control the parasite.

Abstract Trichilia catigua A. Juss., Meliaceae, known as "catuaba" in Brazil, has been popularly used as a tonic for fatigue, impotence and memory deficits. Previously, we have demonstrated that T. catigua ethyl-acetate fraction exerted antidepressive-like effects in mice. Affective-like symptoms are also well recognized outcome of cerebral ischemia in clinical and preclinical settings. Therefore, here we evaluated the effects of ethyl-acetate fraction on the emotional outcomes and its relation with hippocampal neurogenesis in ischemic mice. Male Swiss mice were subject to the bilateral common carotid occlusion during 20 min. The animals received ethyl-acetate fraction (400 mg/kg, orally) 30 min before and once per day during 7 days after reperfusion. Emotional outcomes were assessed using the open field test, elevated zero maze, and the tail suspension test. After the behavioral testing, the animals were sacrificed and their brains were processed to immunohistochemistry and Nissl staining. Ischemic mice exhibited anxiogenic-like behaviors in the elevated zero maze, hippocampal neurodegeneration and decreased hippocampal neurogenesis. The anxiogenic-like effect was counteracted by ethyl-acetate fraction administration. Furthermore, ethyl-acetate fraction restored the number of newborn neurons in the dentate gyrus of hippocampus of ischemic mice. In conclusion, T. catigua ethyl-acetate fraction promoted functional recovery and restored hippocampal neurogenesis in ischemic mice.

ABSTRACT Leishmaniasis is a disease that affects millions of people and it is an important public health problem. The drugs currently used for the treatment of leishmaniasis present undesirable side effects and low efficacy. In this study, we evaluated the in vitro activity of Melampodium divaricatum (MD-EO) and Casearia sylvestris (CS-EO) essential oils (EO) against promastigote and amastigote forms of Leishmania amazonensis. Sesquiterpenes E-caryophyllene (56.0%), germacrene D (12.7%) and bicyclogermacrene (9.2%) were identified as the main components of MD-EO, whereas E-caryophyllene (22.2%), germacrene D (19.6%) and bicyclogermacrene (12.2%) were the main constituents of CS-EO. CS-EO and E-caryophyllene were active against promastigote forms of L. amazonensis (IC50 24.2, 29.8 and 49.9 µg/mL, respectively). However, MD-EO, CS-EO and E-caryophyllene were more active against amastigote forms, with IC50 values of 10.7, 14.0, and 10.7 µg/mL, respectively. E-caryophyllene presented lower cytotoxicity against macrophages J774-A1 (CC50 of 62.1 µg/mL) than the EO. The EOs and E-caryophyllene should be further studied for the development of new antileishmanial drugs.

The fungus Penicillium brasilianum LaBioMMi 136 was isolated as an endophyte from Melia azedarach and has shown to be a prominent producer of great diversity of secondary metabolites, although it does not express some biosynthetic routes to other natural compounds found in Penicillium genera. The present study aimed at the diversification of P. brasilianum secondary metabolism by varying the chemical composition used for its growth. Medium composition supplemented with CuSO4 and MnSO4 locked verruculogen biosynthesis and addressed proline to the production of a series of cyclodepsipeptides identified as JBIR 113, JBIR 114 and JBIR 115, never described for this species so far. The induced cyclodepsipeptide JBIR 113 was isolated by the use of combined chromatographic procedures and identified by spectroscopic methods. The unique structure with three neighboring cyclic amino acids proline and twice pipecolinic acid is rare as natural products and has been described for the first time in terrestrial organism. Verruculogen and JBIR 113 exhibited weak antiparasitary activity against Leishmania amazonensis.

RESUMO Genótipos de citros mostram uma gama de resistência à infecção de Xanthomonas citri subsp. citri (Xcc), agente causal do cancro cítrico asiático (ACC). O objetivo deste estudo foi avaliar um método de inoculação em folha destacada para comparar nove genótipos diferentes de citros que apresentam graus de resistência à ACC e, avaliar a infecção, colonização e desenvolvimento de sintomas das variedades utilizando Microscopia Eletrônica de Varredura (MEV). A inoculação de Xcc em folhas foi realizada através de ferimentos produzidos com agulhas. Aos 7, 10 e 13 dias após a inoculação (DAI), diâmetros de lesão foram mensurados. A variedade Pêra IAC foi a mais resistente ao patógeno e apresentou lesões com menor diâmetro e a variedade Washington foi a mais suscetível e apresentou as maiores lesões, indicando baixa resistência à Xcc. As imagens de MEV foram feitas no desenvolvimento da lesão, e secções foram cortadas a partir das lesões da doença nas variedades testadas. A variedade mais resistente, Pêra IAC, teve aberturas estomáticas menores e produziram uma maior quantidade de exsudados foliares em comparação com a variedade Washington. Nossa hipótese é que os exsudados podem conter compostos tóxicos de Xcc e, portanto, pode ser um mecanismo de defesa para a infecção por Xcc, embora novas pesquisas são necessárias para estabelecer quaisquer efeitos antibióticos de exsudato contra o cancro cítrico.

ABSTRACT Citrus genotypes show a broad range of resistance to infection with Xanthomonas citri subsp citri (Xcc), the causal agent of Asiatic citrus canker (ACC). The aim of this study was to evaluate a detached leaf inoculation method to compare nine different citrus genotypes that exhibit varied resistance levels to ACC, and to evaluate infection, colonization and symptom development of the varieties by using Scanning Electron Microscopy (SEM). Inoculation of Xcc in the leaves was carried out by producing injuries with needles. Lesion diameters were measured at 7, 10 and 13 days after inoculation (DAI). The variety Pera IAC was most resistant to the pathogen and had lesions with smaller diameters, while the variety Washington was most susceptible and had the largest lesions, indicating low resistance to Xcc. SEM images were taken from the lesion development, and sections were obtained from the disease lesions in the tested varieties. The most resistant variety, Pera IAC, had smaller stomatal openings and produced greater quantity of foliar exudates, compared with the variety Washington. We hypothesize that the exudates may contain Xcc toxic compounds and thus may be a defense mechanism against infection by Xcc, but further research is required to establish any antibiotic effects of the exudates against citrus canker.

Abstract Chagas' disease is a parasitic infection caused by protozoan Trypanosoma cruzi that affect millions of people worldwide. The available drugs for treatment of this infection cause serious side effects and have variable efficacy, especially in the chronic phase of the disease. In this context, natural compounds have shown great potential for the discovery of new chemotherapies for the treatment of this infection and various other diseases. In present study, we evaluated the in vitro antiprotozoal activity of five species of Brazilian and Spanish marine sponges (Condrosia reniformes, Tethya rubra, Tethya ignis, Mycale angulosa and Dysidea avara) against T. cruzi. By GC–MS data, we observed that in these extracts were present the major classes of the following compounds: hydrocarbons, terpenes, steroids and alcohols. The extracts showed activity against the three forms of this parasite and did not induce toxicity in mammalian cells. Better activities were observed with the extracts of marine sponges, C. reniformes (EC50 = 0.6 μg/ml), D. avara (EC50 = 1.1 μg/ml) and M. angulosa (EC50 = 3.8 μg/ml), against trypomastigote forms. In intracellular amastigote forms, the extract of T. ignis showed IC50 of 7.2 μg/ml and SI of 24.65. On this basis, our results indicate that these extracts can be promising chemotherapeutic agents against T. cruzi.

No Brasil, bem como em outras regiões do mundo, Pseudomonas aeruginosa e Acinetobacter spp. surgiram como importantes agentes de infecção nosocomial e são comumente envolvidos em surtos. O objetivo principal deste estudo foi descrever a relação genética de P. aeruginosa e Acinetobacter spp. isoladas de pacientes internados em hospital universitário público do noroeste do Paraná - Brasil e reportar o perfil de resistência dessas bactérias. Um total de 75 P. aeruginosa e 94 Acinetobacter spp. isolados foi fenotipicamente identificado e testado para a suscetibilidade aos antibióticos por metodologia automatizada. A polimixina B foi testada por difusão em disco para P. aeruginosa. Metalo-β-lactamase (MBL) foi detectada por disco-aproximação. Análise genotípica foi realizada por enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Aproximadamente 55% dos isolados de P. aeruginosa e 92% de Acinetobacter spp. isolados foram multirresistentes, mas nenhum foi produtor de MBL. Os resultados de ERIC-PCR revelaram pequenos grupamentos de Acinetobacter spp. resistentes aos carbapenêmicos, provavelmente pela produção de carbapenemases do tipo OXA. Além disso, alta diversidade genética entre os isolados de P. aeruginosa e Acinetobacter spp. foi observada, sugerindo que a transmissão cruzada destas espécies bacterianas não é muito frequente em nosso hospital.

In Brazil and other regions of the world, Pseudomonas aeruginosa and Acinetobacter spp. have emerged as important agents of nosocomial infection and are commonly involved in outbreaks. The main objective of the present study was to evaluate the genetic relationship among P. aeruginosa and Acinetobacter spp. isolated from patients in a public university hospital in northwestern Paraná, Brazil, and report their antimicrobial resistance profile. A total of 75 P. aeruginosa and 94 Acinetobacter spp. isolates were phenotypically identified and tested for antibiotic susceptibility using automated methodology. Polymyxin B was tested by disk diffusion for P. aeruginosa. Metallo-β-lactamase (MBL) was detected using a disk approximation test. Genotyping was performed using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Approximately 55% of the P. aeruginosa isolates and 92% of the Acinetobacter spp. isolates were multiresistant, but none were MBL-producers. ERIC-PCR revealed the presence of small clusters of carbapenem-resistant Acinetobacter spp., most likely OXA-type carbapenemase producers. Furthermore, high genetic diversity in P. aeruginosa and Acinetobacter spp. clinical isolates was observed, suggesting that cross-transmission is not very frequent in the studied hospital.

Leishmaniasis is a neglected tropical disease. According to the World Health Organization, there are approximately 1.5-two million new cases of cutaneous leishmaniasis each year worldwide. Chemotherapy against leishmaniasis is based on pentavalent antimonials, which were developed more than a century ago. The goals of this study were to investigate the antileishmanial activity of diterpene acids in copaiba oil, as well as some possible targets of their action against Leishmania amazonensis. Methyl copalate and agathic, hydroxycopalic, kaurenoic, pinifolic and polyaltic acids isolated from Copaifera officinales oleoresins were utilised. Ultrastructural changes and the specific organelle targets of diterpenes were investigated with electron microscopy and flow cytometry, respectively. All compounds had some level of activity against L. amazonensis. Hydroxycopalic acid and methyl copalate demonstrated the most activity against promastigotes and had 50% inhibitory concentration (IC50) values of 2.5 and 6.0 µg/mL, respectively. However, pinifolic and kaurenoic acid demonstrated the most activity against axenic amastigote and had IC50 values of 3.5 and 4.0 µg/mL, respectively. Agathic, kaurenoic and pinifolic acid caused significant increases in plasma membrane permeability and mitochondrial membrane depolarisation of the protozoan. In conclusion, copaiba oil and its diterpene acids should be explored for the development of new antileishmanial drugs.

In vitro activity of the essential oil from Piper diospyrifolium leaves was tested using disk diffusion techniques. The antifungal assay showed significant potencial antifungal activity: the oil was effective against several clinical fungal strains. The majority compounds in the essential oil were identified as sesquiterpenoids by GC-MS and GC-FID techniques.

Objetivou-se avaliar duas vias de administração de probiótico sobre o desempenho, o rendimento de carcaça e a população microbiana do intestino delgado de codornas de corte. Foram utilizadas 1.050 codornas machos e fêmeas de 1 dia de idade distribuídas em delineamento inteiramente casualizado com três tratamentos (controle, probiótico na água bebida e probiótico na ração) e cinco repetições de 70 aves. A cada sete dias, foram avaliados o ganho de peso, o consumo de ração, a conversão alimentar e a viabilidade criatória. Aos 7 e aos 14 dias, 15 aves foram abatidas para determinação da contagem de Lactobacillus sp., enterobactérias e Escherichia coli do intestino delgado e, aos 35 dias, 30 aves foram abatidas para avaliação do rendimento de carcaça e de cortes. Dos 7 aos 14 dias, as codornas que receberam probiótico na ração, em relação àquelas que receberam na água de bebida, apresentaram menor consumo de ração, porém essa diferença no consumo não afetou o ganho de peso, o que levou à melhora na conversão alimentar. O rendimento de carcaça, a contagem de Lactobacillus sp., de enterobactérias e de Escherichia coli não foram influenciados pela adição do probiótico. O fornecimento de probiótico não melhora o desempenho e o rendimento de carcaça e não altera a microbiota do intestino delgado de codornas de corte.

The objective of this study was to evaluate the effect of different ways of administering probiotic on performance, carcass yield and small intestine microbial population of meat quail. One thousand and fifty quails, one-day old males and females were used, distributed in a randomized completedesign with three treatments (control, probiotic in drinking water and probiotic in feed) and five replications of seventy quail each. Every seven days, the quails were weighed to evaluate weight gain (WG), feed intake (FI), feed conversion (FC) and growth viability. At 7 and 14 days, fifteen quails were slaughtered to perform the count of Lactobacillus sp., enterobacteria and Escherichia coli of the small intestine at 35 days, 30 quails were slaughtered to evaluate the carcass and cut yields. From 7 to 14 days of age, the quails fed probiotic in the ration, compared to those fed probiotic in the drinking water, showed lower feed intake, which did not affect weight gain and improved feed conversion. Carcass yield, count of Lactobacillus sp., enterobacteria and Escherichia coli were not influenced by probiotic addition.The probiotic do not improve performance, carcass yield and also do not alter small intestine microbiota of meat quail.

A etiologia do processo diarréico na AIDS pode ser causada por vírus, bactérias, fungos, protozoários e helmintos, assim como pelo próprio HIV. Este trabalho avaliou enteropatogenos relacionados à diarréia em pacientes HIV que fazem uso de terapia anti-retroviral. Os métodos parasitológicos utilizados foram Faust, Hoffmann e Kinyoun. O isolamento e cultura dos fungos foram realizados conforme metodologia recomendada por NCCLS M27-A standard. A identificação das espécies de leveduras foi realizada através da reação em cadeia da polimerase. O isolamento de bactérias, foi feito em agar Mac Conkey e agar SS, a identificação das espécies através do Enterokit B (Probac do Brasil) e métodos bioquímicos. Foram avaliados 49 pacientes, 44,9% apresentaram enteroparasitas, 48,1% Candida sp com 61,5% Candida albicans, 7,6% Candida sp e 30,7% Candida não- albicans. Foram isoladas bactérias de 72% dos pacientes, 49% Escherichia coli, 13% Salmonella parathyphi, Klebsiella sp ou Proteus e 6% Citrobacter freundii ou Yersinia sp. Houve alta prevalência de Candida sp nos pacientes HIV com diarréia e foram isoladas espécies não albicans cuja presença pode ser entendida como cúmplice ou causa da infecção.

The etiology of the diarrheic process in AIDS may be caused by viruses, bacteria, fungi, protozoa or helminths, as well as HIV itself. This study evaluated enteropathogens relating to diarrhea in HIV patients who were on antiretroviral therapy. The parasitological methods used were Faust, Hoffmann and Kinyoun. Isolation and culturing of fungi were carried out in accordance with the methodology recommended by the NCCLS M27-A standard. The yeast species were identified using the polymerase chain reaction (PCR). Bacteria were isolated on MacConkey and SS agar and the species were identified using Enterokit B (Probac do Brasil) and biochemical methods. Forty-nine patients were evaluated: 44.89% presented enteroparasites and 48.1% presented Candida sp, of which 61.5% were Candida albicans, 7.6% were Candida sp and 30.7% were Candida non-albicans. Bacteria were isolated from 72% of the patients, of which 49% were Escherichia coli, 13% Salmonella parathyphi, Klebsiella sp or Proteus and 6% Citrobacter freundii or Yersinia sp. There was high prevalence of Candida sp in HIV patients with diarrhea and non-albicans species were isolated. Their presence could be taken to mean that they were accomplices in or causes of the infection.

The phytochemical investigation of Coussarea platyphylla led to the isolation of triterpenes betulonic and betulinic acid, monoterpenes monotropein and monotropein salt, the diterpene trans-phytol and esteroids. The structures of the isolated compounds were assigned on the basis of spectroscopic data, including two-dimensional NMR methods. The antiproliferative properties against human cancer cell lines and molluscicidal activity against Biomphalaria glabrata of the crude methanolic extract and of its fractions were investigated.