Bioassay-guided fractionation of the bark extract of Annona foetida afforded a new antileishmanial pyrimidine-beta-carboline alkaloid, N-hydroxyannomontine (1), together with the previously reported annomontine (2), O-methylmoschatoline (3), and liriodenine (4). The structure of compound 1 was established on the basis of extensive 1D and 2D NMR and MS analyses. This is the third reported pyrimidine-beta-carboline-type alkaloid and is particularly important for Annona genus chemotaxonomy. In addition, all compounds exhibit in vitro antileishmanial activity against promastigote forms of Leishmania braziliensis. Compounds 2 and 4 showed better activity than compounds 1 and 3 against L. braziliensis. Compound 2 was not active against L. guyanensis.

Este trabalho compara pela primeira vez a composição química do óleo essencial das folhas de Licaria canella coletadas em duas estações climáticas. Os resultados foram comparados com os obtidos para o óleo essencial das folhas de outra espécie da família Lauraceae, Aniba canelilla, coletada no mesmo período. Ambos os óleos essenciais foram analisados por CG-DIC e CG-EM, e os resultados indicaram uma grande quantidade de benzenóides, sendo o principal constituinte em L. canella o benzoato de benzila e para A. canelilla, o 1-nitro-2-feniletano. A comparação das atividades biológicas mostrou que o óleo de L. canella (IC 50 19 µg mL-1) foi mais ativo contra as cepas de Leishmania amazonensis e menos citotóxico em cultura de macrófagos do que o de A. canelilla (IC 50 40 µg mL-1). Por outro lado, o óleo de L. canella exibiu uma maior citotoxicidade contra Artemia salina com uma concentração letal (CL 50) igual a 5,25 µg mL-1. This work compares the chemical composition of the essential oils from the leaves of Licaria canella collected in two different seasons. The results of this investigation were compared with the leaf essential oils of other species of the Lauraceae family, Aniba canelilla, collected at the same time. Both essential oils were analyzed by GC-FID and GC-MS. The results demonstrated a larger predominance of benzenoids, being the main constituent benzyl benzoate for L. canella and 1-nitro-2-phenylethane for A. canelilla. The comparison of the biological activities showed that L. canella (IC 50 19 µg mL-1) was more active against Leishmania amazonensis strains and less cytotoxic in macrophage cultures than A. canelilla (IC 50 40 µg mL-1). On the other hand, the L. canella oil displayed a higher cytotoxicity against Artemia salina with a lethal concentration (LC 50), equal to 5.25 µg mL-1 .

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Due to the diversity of its physiological and pathophysiological functions and general ubiquity, the study of nitric oxide (NO) has become of great interest. In this work, it was demonstrated that Leishmania amazonensis promastigotes produces NO, a free radical synthesized from L: -arginine by nitric oxide synthase (NOS). A soluble NOS was purified from L. amazonensis promastigotes by affinity chromatography (2', 5'-ADP-agarose) and on SDS-PAGE the enzyme migrates as a single protein band of 116.2 (+/-6) kDa. Furthermore, the presence of a constitutive NOS was detected through indirect immunofluorescence using anti-cNOS and in NADPH consumption assays. The present work show that NO production, detected as nitrite in culture supernatant, is prominent in promastigotes preparations with high number of metacyclic forms, suggesting an association with the differentiation and the infectivity of the parasite.

The essential oils obtained from leaves of Piper duckei and Piper demeraranum by hydrodistillation were analyzed by gas chromatography-mass spectrometry. The main constituents found in P. demeraranum oil were limonene (19.3%) and β-elemene (33.1%) and in P. duckei oil the major components found were germacrene D (14.7%) and trans-caryophyllene (27.1%). P. demeraranum and P. duckei oils exhibited biological activity, with IC50 values between 15 to 76 μg mL−1 against two Leishmania species, P. duckei oil being the most active. The cytotoxicity of the essential oils on mice peritoneal macrophage cells was insignificant, compared with the toxicity of pentamidine. The main mono- and sesquiterpene, limonene (IC50 = 278 μM) and caryophyllene (IC50 = 96 μM), were tested against the strains of Leishmania amazonensis, and the IC50 values of these compounds were lower than those found for the essential oils of the Piper species. The HET-CAM test was used to evaluate the irritation potential of these oils as topical products, showing that these oils can be used as auxiliary medication in cases of cutaneous leishmaniasis, with less side effects and lower costs.

Infections due to protozoa of the genusLeishmaniasis is a tropical disease caused by protozoa of the Leishmania genus. These protozoa cause a disease with different clinical forms, among them cutaneous, hyperergic, mucocutaneous, and anergic diffuse leishmaniasis (Leon et al. 1990a). The disease is endemic in some geographical areas of Brazil, where it constitutes a serious health problem (Lonardoni et al. 1999, Leon et al. 2002.The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials (Sb V ), but they present renal and cardiac toxicity. A second choice for the treatment of the disease is a diamidine (pentamidine isethionate), which also has serious side effects (Korolkovas & Burckhalter 1988). However already in some trials alternative pharmaceutical formulations have been used to reduce the toxicity of these drugs (Frezard et al. 2000) The lack of an effective anti-leishmanial drug led a renewed interest in the study of traditional remedies as sources for the development of new chemotherapeutic compounds with better activity and less toxicity. Several plants have been used for the treatment of parasitic diseases (Araujo et al. 1998). In order to find new drugs against leishmaniasis, we have studied alkaloidal extracts of Brazilian plants (Oliveira et al. 2002) as "guatambu", is a tree which grows from to 12-30 m in height and is native to the forests in Southeastern Brazil (Lorenzi 1992). Some alkaloids have been previously isolated from the stem bark (Reis et al. 1996), and the alkaloid extract of the bark showed antimicrobial activity against Gram positive and negative bacteria (Oliveira 1999). The aim of the present study is to investigate the anti-leishmanial activity of alkaloidal extracts from the stem bark of A. ramiflorum against L. (V.) braziliensis and L. (L.) amazonensis. MATERIALS AND METHODSPlant materials -A. ramiflorum Muell. Arg. was collected in the Horto Florestal de Maringá, July 2000, in Maringá, state of Paraná, Brazil. The plant was collected and identified by Prof. Dr Ismar Sebastião Moscheta and an exsiccatum deposited and authenticated at the Herbarium of the State University of Maringá, Maringá, Brazil.Extraction of plant materials -Air-dried stem bark (1 kg) was extracted with 70% ethanol at room temperature. After removal of the ethanol, the crude extract was added to a 10% acetic acid solution (v/v) and kept at 5 o overnight. After filtration, the aqueous phase was first extracted with chloroform (acid extract), then the pH raised to 10 and the resulting solution re-extracted with chloroform (basic extract). The two chloroform extracts were concentrated under reduced pressure, and then lyophilised yielding the acid (7.7 g) and basic fractions (11.6 g), which were both analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The main bulk of the alkaloids was in the basic fraction which was called the alkaloidal extract and which was used for the assays.

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Immunoblot analysis was used to investigate antigenic differences among clinical isolates of Leishmania amazonensis and their role in the etiology of the disease. Western blots of promastigote homogenates were analyzed with either monoclonal antibodies (MAbs) specific for the L. mexicana complex (M-4, M-6, M-9, and M-11) or polyclonal sera from L. amazonensis infected patients with the various forms of clinical disease. In the case of the MAbs, no significant variation was observed among the strains of L. amazonensis, isolated from cases of cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), visceral leishmaniasis (VL) or post kala-azar dermal leishmaniasis (PKDL), in either the relative mobility (Mr) or the quantitative amount (intensity) of the antigenic determinants. In the case of the sera of the infected patients, the patterns of antigenic reactivity of these strains revealed that, despite showing the presence of shared antigens, differences were observed between some of the antigenic components of the various isolates of L. amazonensis that were recognized by a single serum. Differences were also demonstrated between the antigenic determinants of a single isolate of L. amazonensis that were recognized by the different patients' sera. No apparent association was consistently found, however, between the Mr components identified in these isolates and the clinical form of the disease or the geographical area of isolation. In addition, the spectrum of antigens recognized by the sera from patients with the same clinical form were not identical; although in some instances, similar Mr antigens were shared.(ABSTRACT TRUNCATED AT 250 WORDS)