Arildo José Braz de Oliveira

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.

Establishment of adventitious root culture of Stevia rebaudiana Bertoni in a roller bottle system

Cultures of adventitious roots of Stevia rebaudiana (Bert.) Bertoni were performed in a roller bottle system for the production of both primary and secondary metabolites. Adventitious roots were induced from 1-cm-long root tip explants derived from in vitro regenerated plantlets on solid Murashige and Skoog (MS 1962) media supplemented with 10.7xa0μM of α-naphthaleneacetic acid. These cultures were successfully maintained in the same medium for 6xa0months with regular subcultures after 4xa0weeks. Thereafter, the roots were cut into 1.0- to 1.5-cm-long segments and transferred to the roller bottle system containing a fresh root tissue culture on liquid MS medium supplemented with 10.7xa0μM NAA. The apparatus consisted of a flask rolling system adjusted to 4g, and 3° of flask inclination. The roots were allowed to grow in the absence of light for adaptation and adventitious root formation. The best conditions for cultivation were investigated, considering culture volume (25xa0ml), culture period (4xa0weeks), salt concentrations in the nutrient medium (33%) and optimal initial inoculum (0.2xa0g) of S. rebaudiana roots. These results could give important information on how to improve the development of adventitious roots of S. rebaudiana for the production of primary and secondary metabolites.

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.

Pollination of soybean (Glycine max L. Merril) by honeybees (Apis mellifera L.)

This experiment was carried out to evaluate the effect of the honeybee pollination in the production and quality of soybean seeds (Glycine max L. Merril). Seed production was higher (P=0.0001) in covered areas with honeybee colonies (50.64%) and uncovered areas (57.73%) than in covered areas without honeybee colonies. It could be concluded that honeybees were responsible for 95.5% of the pollination accomplished by insects. The pod number in covered treatment with honeybees was 61.38% higher (P=0.0002) than in the covered treatment without honeybees. The average weight of 100 seeds was larger (P=0.0001) in the area covered without honeybees, and reached 17.8 g. The medium content of crude protein in grains was 36.7% and the average oil content was 20.2%. The germination test did not show differences (P>0.05) among the seeds in different treatments. It was concluded that the honeybee pollination in the soybean increased the seeds production.

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.

Absence of antidiabetic and hypolipidemic effect of Gymnema sylvestre in non-diabetic and alloxan-diabetic rats

ABSTRACT In this study we investigated the antidiabetic and hypolipidemic potential of dried powdered leaves of Gymnema sylvestre (GS). The acute effect of GS administered by oral gavage on glucose blood level of and lipids in non-diabetic and alloxan-diabetic rats were investigated in the following conditions: a) after a balanced meal; b) after the ingestion of 1000 mg/kg amylose or 1000 mg/kgglucose; c) after the ingestion of a mixture of 12 mL/kg soybean oil + 1% cholesterol (SOC). In addition, the effect of the treatment with GS during two (sub-acute) or four weeks (chronic) on body weight, food and water ingestion, glucose blood level and lipids in non-diabetic and alloxan-diabetic rats were measured. The dose of GS utilized in the majority of the experiments, i.e ., 30 mg/kg,corresponds to that given to treat diabetes in Brazil. GS acutely did not influence the elevation of glycemia promoted by a balanced meal or by the administration of amylose or glucose; but promoted more intense (P<0.05) elevation of serum lipids after the administration of SOC. Moreover, the sub-acute and chronic treatment with GS in non-diabetic and alloxan-diabetic rats did not change: a) the body weight gain; b) food and water ingestion; c) the blood level of glucose and lipids. Thus we concluded that GS, at least in the form commercialized in the Brazil, i.e

Immunocapture-RT-PCR detection of Cassava common mosaic virus in cassava obtained from meristem-tip culture in Paraná state

A virus survey conducted in the northwest region of Parana, the main cassava-producing region of that state, showed Cassava common mosaic virus (CsCMV) to be widespread, infecting more than 90% of plants from all cassava cultivars. A CsCMV isolate was purified and used to raise a high-titer (1/1.000) polyclonal antiserum for indexing plants produced from meristem-tip culture, using PTA-ELISA. From an initial production of 110 plants of cultivar Olho Junto, 31 remained infected as indicated by PTA-ELISA. To improve the sensitivity of virus detection, an immunocapture-RT-PCR (IC-RT-PCR) protocol was established. Virus-specific IgG, purified and combined with a primer set for the genus Potexvirus, could readily detect CsCMV in cassava crude leaf extracts. IC-RT-PCR products of 750 bp were amplified from six out of 35 plants previously tested as virus-negative by PTA-ELISA. Sequence analysis of cloned IC-RT-PCR products confirmed they were part of the CsCMV replicase gene, indicating that the virus was still present after thermotherapy and meristem-tip culture. PTA-ELISA enabled initial screening of virus-positive cassava, reducing the number of plants to be further analyzed by IC-RT-PCR. Though CsCMV has been considered of minor importance, its widespread nature, as noticed in Parana, indicates the need for adoption of effective control measures.

Preliminary studies on the antibacterial activity of crude extracts and alkaloids from species of Aspidosperma

Screening tests of hydroethanolic crude extracts of six species of Aspidosperma (Apocynaceae) against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa were performed. Aspidosperma ramiflorum Muell. Arg. showed good activity against Bacillus subtilis with MIC and MBC of 15.7 and 125u2009μg/mL, moderate activity against Staphylococcus aureus with MIC and MBC of 250 and 500u2009μg/mL, and weak activity against Escherichia coli with MIC and MBC of 1000u2009μg/mL. Aspidosperma pyricolum Muell. Arg. (MIC/MBC 125/250u2009μg/mL) and Aspidosperma olivaceum Muell. Arg. (MIC/MBC 250/u2009>u20091000u2009μg/mL) displayed moderate antibacterial activity against Bacillus subtilis. Separation of the crude extract of Aspidosperma ramiflorum was performed according to the usual acid–base process, which produces alkaloid mixtures and closely related metabolites. The basic fraction was active against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, with MICs of 31.2, 62.5, and 250u2009μg/mL, respectively. The basic fractions were more active than the acid fractions, probably because they contained some active alkaloids and/or closely related metabolites absent from the other fractions, or they contained a higher concentration of these active compounds.

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.