We evaluated the antibacterial activities of the crude methanol extract, fractions (I-V) obtained after acid-base extraction and pure compounds from the stem bark of Aspidosperma ramiflorum. The minimum inhibitory concentration (MIC) was determined by the microdilution technique in Mueller-Hinton broth. Inoculates were prepared in this medium from 24-h broth cultures of bacteria (10 7 CFU/mL). Microtiter plates were incubated at 37ºC and the MICs were recorded after 24 h of incubation. Two susceptibility endpoints were recorded for each isolate. The crude methanol extract presented moderate activity against the Gram-positive bacteria B. subtilis (MIC = 250 µg/ mL) and S. aureus (MIC = 500 µg/mL), and was inactive against the Gram-negative bacteria E. coli and P. aeruginosa (MIC > 1000 µg/ mL). Fractions I and II were inactive against standard strains at concentrations of ≤1000 µg/mL and fraction III displayed moderate antibacterial activity against B. subtilis (MIC = 500 µg/mL) and S. aureus (MIC = 250 µg/mL). Fraction IV showed high activity against B. subtilis and S. aureus (MIC = 15.6 µg/mL) and moderate activity against E. coli and P. aeruginosa (MIC = 250 µg/mL). Fraction V presented high activity against B. subtilis (MIC = 15.6 µg/mL) and S. aureus (MIC = 31.3 µg/mL) and was inactive against Gram-negative bacteria (MIC > 1000 µg/mL). Fractions III, IV and V were then submitted to bioassay-guided fractionation by silica gel column chromatography, yielding individual purified ramiflorines A and B. Both ramiflorines showed significant activity against S. aureus (MIC = 25 µg/mL) and E. faecalis (MIC = 50 µg/mL), with EC 50 of 8 and 2.5 µg/ mL for ramiflorines A and B, respectively, against S. aureus. These results are promising, showing that these compounds are biologically active against Gram-positive bacteria.

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BackgroundSeveral species of Aspidosperma (Apocynaceae) are used as treatments for human diseases in the tropics. Aspidosperma olivaceum, which is used to treat fevers in some regions of Brazil, contains the monoterpenoid indole alkaloids (MIAs) aspidoscarpine, uleine, apparicine, and N-methyl-tetrahydrolivacine. Using bio-guided fractionation and cytotoxicity testing in a human hepatoma cell line, several plant fractions and compounds purified from the bark and leaves of the plant were characterized for specific therapeutic activity (and selectivity index, SI) in vitro against the blood forms of Plasmodium falciparum.MethodsThe activity of A. olivaceum extracts, fractions, and isolated compounds was evaluated against chloroquine (CQ)-resistant P. falciparum blood parasites by in vitro testing with radiolabelled [3H]-hypoxanthine and a monoclonal anti-histidine-rich protein (HRPII) antibody. The cytotoxicity of these fractions and compounds was evaluated in a human hepatoma cell line using a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, and the SI was calculated as the ratio between the toxicity and activity. Two leaf fractions were tested in mice with Plasmodium berghei.ResultsAll six fractions from the bark and leaf extracts were active in vitro at low doses (IC50 < 5.0 μg/mL) using the anti-HRPII test, and only two (the neutral and basic bark fractions) were toxic to a human cell line (HepG2). The most promising fractions were the crude leaf extract and its basic residue, which had SIs above 50. Among the four pure compounds evaluated, aspidoscarpine in the bark and leaf extracts showed the highest SI at 56; this compound, therefore, represents a possible anti-malarial drug that requires further study. The acidic leaf fraction administered by gavage to mice with blood-induced malaria was also active.ConclusionUsing a bio-monitoring approach, it was possible to attribute the anti-P. falciparum activity of A. olivaceum to aspidoscarpine and, to a lesser extent, N-methyl-tetrahydrolivacine; other isolated MIA molecules were active but had lower SIs due to their higher toxicities. These results stood in contrast to previous work in which the anti-malarial activity of other Aspidosperma species was attributed to uleine.

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Este trabalho descreve um procedimento rápido e eficiente para a separação e identificação de alcalóides indólicos do extrato etanólico de Tabernaemontana fuchsiaefolia (Apocynaceae). As frações alcaloídicas obtidas dos extratos etanólicos (das folhas, das cascas do caule e das cascas das raízes) foram fracionadas e analisadas por cromatografia em camada delgada (CCD) e por cromatografia gasosa acoplada a espectrometria de massa (CG-EM). Foram identificados os alcalóides indólicos ibogamina, coronaridina, pseudoindoxil ibogaina, hidroxiindolenina voacangina, pseudoindoxil voacangina, tabernantina, catarantina, voacangina, 19-oxovoacangina, 10-hidroxicoronaridina, afinisina, 16-epi-afinina, voachalotina, ibogalina e conofaringina. This paper describes a fast and efficient procedure to separate and identify indole alkaloids from the ethanolic extract of Tabernaemontana fuchsiaefolia (Apocynaceae). The alkaloidal fractions obtained from ethanolic extracts of leaves and stem barks and root barks were fractioned and analyzed by Thin-Layer Chromatography (TLC) and by Gas Chromatography coupled to Mass Spectrometry (GC-MS). The following indole alkaloids were identified: ibogamine, coronaridine, ibogaine pseudoindoxyl, voacangine hydroxyindolenine, voacangine pseudoindoxyl, tabernanthine, catharanthine, voacangine, 19-oxovoacangine, 10-hydroxycoronaridine, affinisine, 16-epi-affinine, voachalotine, ibogaline, and conopharyngine.

a b s t r a c tFructooligosaccharides have been isolated from roots and leaves of Stevia rebaudiana, by hot aqueous extraction, followed by precipitation with ethanol. Their structure has been determined using methylation and NMR analysis, MALDI-TOF, and ESI-MS. Fructooligosaccharides contained almost exclusively (2?1)-linked b-fructofuranosyl, with terminal a-glucopyranosyl and b-fructofuranosyl units. MALDI-TOF and ESI-MS analyses showed the wide range of degree of polymerisation (DP) present in various extracts. From roots and leaves, three different fractions gave profiles of homologous series, with DPs ranging up to 17 with MALDI-TOF and 19 using ESI-MS. These inulin-type fructooligosaccharides were the major component of extracts from S. rebaudiana roots and significant components from the leaves.

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.