Regina Aparecida Correia Gonçalves

Structure and degree of polymerisation of fructooligosaccharides present in roots and leaves of Stevia rebaudiana (Bert.) Bertoni

Fructooligosaccharides 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 β-fructofuranosyl, with terminal α-glucopyranosyl and β-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.

Structure and antiviral activity of arabinogalactan with (1→6)-β-D-galactan core from Stevia rebaudiana leaves.

Cell wall polysaccharides from leaves of Stevia rebaudiana were extracted successively with water and with aq. 10% KOH. After the purification steps, homogeneous fractions (SFW-10RM and SSFK-10RM) were analyzed by sugar composition, HPSEC, methylation and (13)C NMR spectroscopy analysis. The results showed that SFW-10RM is a pectic arabinogalactan with an unusual β-(1→6)-linked D-Galp residues forming the main chain. Approximately 38% of the β-D-Galp units of the backbone carry branches on position O-3, consisting of single D-Galp units or arabinan side chains. Arabinose residues were found to occupy mostly the terminal positions in both furanose and pyranose forms and as 2-, 5- and 3,5-linked residues in these side chains. Fraction SSFK-10RM is a similar arabinogalactan, differing mainly in the relative proportions of arabinans attached to the galactan core and in the content of D-GalpA residues present in the pectic domain. The crude aqueous and alkaline extracts and homogeneous SSFK-10RM showed antiviral activity against Herpes Simplex Virus type-1 (HSV-1) in vitro.

Anti-leishmanial activity of alkaloidal extracts obtained from different organs of Aspidosperma ramiflorum.

The present study was designated to evaluate semi-quantitative antileishmanial activity of alkaloidal extracts that were obtained from 1g of different parts of Aspidosperma ramiflorum (leaves, roots, seeds, and stem barks). Alkaloidal extracts of barks and leaves presented a good activity against the extracellular form (promastigotes) of Leishmania (L.) amazonensis. It is known that compounds responsible for the antileishmanial activity in the alkaloidal extracts from A. ramiflorum are the monoterpenoid indole alkaloids ramiflorine A and ramiflorine B, therefore extracts obtained from different plant parts were analyzed by electrospray ionization mass spectrometry (ESI-MS) in order to evidence the presence of these bioactive alkaloids. Based on these findings, alkaloidal extract from leaves was fractionated on preparative thin-layer chromatography in a bioassay-guided fractionation affording individual purified ramiflorines A and B. Both ramiflorines A and B showed significant activity against Leishmania (L.) amazonensis (LD(50) values of 18.5±6.5μg/ml and 12.63±5.52μg/ml, respectively). Our results are showing that alkaloidal extract from leaves is a promising alternative to the use of stem barks from A. ramiflorum.

Production of plant secondary metabolites in plant cell and tissue culture: the example of Tabernaemontana and Aspidosperma genera

Os estudos dos metabolitos secundarios de plantas se desenvolveram aceleradamente nos ultimos 50 anos. Estes compostos sao conhecidos por desempenharem um papel importante na adaptacao das plantas aos seus ambientes e tambem representam uma fonte importante de substâncias farmacologicamente ativas. As tecnicas de cultura de celulas de plantas iniciaramse na decada de 1960 como uma possivel ferramenta para estudar e produzir os metabolitos secundarios de plantas. O uso de cultura de celulas de planta para a producao de substâncias de interesse contribuiu grandemente para avancos em diversas areas da fisiologia e bioquimica vegetal. Diferentes estrategias, usando sistemas de cultura in vitro, foram estudadas com o objetivo de aumentar a producao de metabolitos secundarios. As plantas dos generos Aspidosperma e Tabernaemontana sao importantes fontes de alcaloides indolicos biologicamente ativos, sendo que no Brasil existe um numero consideravel de especies destes generos. As culturas de celulas de Aspidosperma e Tabernaemontana foram iniciadas ha pelo menos 16 anos, as quais produzem um grande numero de alcaloides, o que estimulou o desenvolvimento de diversas tecnicas para sua producao, extracao e identificacao.

Biosynthesis of indole-3-acetic acid by new Klebsiella oxytoca free and immobilized cells on inorganic matrices.

While many natural and synthetic compounds exhibit auxin-like activity in bioassays, indole-3-acetic acid (IAA) is recognized as the key auxin in most plants. IAA has been implicated in almost all aspects of plant growth and development and a large array of bacteria have been reported to enhance plant growth. Cells of Klebsiella oxytoca isolated from the rhizosphere of Aspidosperma polyneuron and immobilized by adsorption on different inorganic matrices were used for IAA production. The matrices were prepared by the sol-gel method and the silica-titanium was the most suitable matrix for effective immobilization. In operational stability assays, IAA production was maintained after four cycles of production, obtaining 42.80 ± 2.03 μg mL−1 of IAA in the third cycle, which corresponds to a 54% increase in production in relation to the first cycle, whereas free cells began losing activity after the first cycle. After 90 days of storage at 4°C the immobilized cells showed the slight reduction of IAA production without significant loss of activity.

Floral nectar chemical composition of floral nectar in conventional and transgenic sweet orange, Citrus sinensis (L.) Osbeck, expressing an antibacterial peptide

Floral nectar chemical composition of conventional and transgenic sweet orange trees expressing the antibacterial peptide sarcotoxin IA (Stx IA), which has been shown to efficiently inhibit in vitro growth of the bacteria responsible for citrus canker (Xanthomonas axonopodis pv. citri), is reported. The nectar contents of primary metabolites (sugars, amino acids, proteins, triacylglycerides) and of the secondary metabolites (total purine alkaloids, phenolic compounds and phytosteroids) were determined by spectrophotometric analysis. The results showed small differences in the nectar primary and secondary metabolites composition. The biochemical and biological significance and the possible effects of these variations on key citrus pollinators are discussed.

Structural characterization of two novel potential anticholinesterasic agents

Abstract Two novel compounds with possible anticholinesterase activity have been synthesized containing a carbamate and a dimethylamine group in 1,2-positions of a cyclohexane ring ( cis and trans isomers). Conformer populations were established by a combination of NMR 1 H coupling constant analysis and DFT (B3LYP/6-311+G(d,p)) calculations. 13 C chemical shifts were calculated in order to confirm signal attributions. The cis isomer adopts a conformation in which the carbamate group lies at the axial position (>99%), whereas the trans isomer adopts a diequatorial arrangement (98%). These preferences have been explained in terms of syn -1,3-diaxial interactions of the individual groups.

Chemical characterization and prebiotic activity of fructo-oligosaccharides from Stevia rebaudiana (Bertoni) roots and in vitro adventitious root cultures.

Stevia rebaudiana (Bertoni) is widely studied because of its foliar steviol glycosides. Fructan-type polysaccharides were recently isolated from its roots. Fructans are reserve carbohydrates that have important positive health effects and technological applications in the food industry. The objective of the present study was to isolate and characterize fructo-oligosaccharides (FOSs) from S. rebaudiana roots and in vitro adventitious root cultures and evaluate the potential prebiotic effect of these molecules. The in vitro adventitious root cultures were obtained using a roller bottle system. Chemical analyses (gas chromatography-mass spectrometry, (1)H nuclear magnetic resonance, and off-line electrospray ionization-mass spectrometry) revealed similar chemical properties of FOSs that were obtained from the different sources. The potential prebiotic effects of FOSs that were isolated from S. rebaudiana roots enhanced the growth of both bifidobacteria and lactobacilli, with strains specificity in their fermentation ability.

A new natural source for obtainment of inulin and fructo-oligosaccharides from industrial waste of Stevia rebaudiana Bertoni

Fructan-type inulin and fructo-oligosaccharides (FOS) are reserve polysaccharides that offer an interesting combination of nutritional and technological properties for food industry. Stevia rebaudiana is used commercially in the sweetener industry due to the high content of steviol glycosides in its leaves. With the proposal of using industrial waste, the objective of the present study was to isolate, characterize and evaluate the prebiotic activity of inulin and FOS from S. rebaudiana stems. The chemical characterization of the samples by GC-MS, NMR and off-line ESI-MS showed that it was possible to obtain inulin molecules from the S. rebaudiana stems with a degree of polymerization (DP) of 12, and FOS with a DP<6. The in vitro prebiotic assay of these molecules indicates a strain specificity in fermentation capacity of fructans as substrate. FOS molecules with a low DP are preferably fermented by beneficial microbiota than inulin molecules with higher DP.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro.

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.