Inulin extraction from Stevia rebaudiana roots in an autoclave.

Inulin is a polymer of d-fructose, characterized by the presence of a terminal glucose, and are a major component of Stevia rebaudiana roots. This type of polymer has nutritional properties and technological applications, such as fat substitutes in low-calorie foods and as the coating of pharmaceuticals. The aim of this study was to evaluate an alternative method for inulin extraction, in terms of extraction time and yield, since the traditional method of extraction under reflux is both time and energy consuming. Using the response surface methodology (RSM) with Box-Behnken design it was observed that the alternative extraction method using autoclave presented similar yields to the reflux-based method, but with a shorter extraction time, 121 °C by 17.41 min 1H Nuclear Magnetic Resonance and Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-ToF-MS) analysis showed that inulin crude extract from S. rebaudiana roots obtained by autoclave extraction had a higher degree of polymerization when compared to those obtained by the traditional method. Thus, it is concluded that the proposed method using an autoclave is a faster alternative for the extraction of inulin.

Antimicrobial and antioxidant activities of secondary metabolites from endophytic fungus Botryosphaeria fabicerciana (MGN23-3) associated to Morus nigra L.

This study was to evaluate the biological activity of the extract of Botryosphaeria fabicerciana isolated from leaves of Morus nigra. The volatile compounds from the crude extract were analysed by GC-MS which demonstrate that mellein and ß-orcinaldehyde were are the major compounds. The best minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract was observed against Gram-positive bacteria, with a MIC of 15.6 µg/mL towards B. cereus and MIC of 62.5 µg/mL towards S. aureus and B. subtilis. MBC values of 31.25 µg/mL, 62.5 µg/mL, and 250 µg/mL were observed towards B. cereus, B. subtilis, and S. aureus, respectively. The cytotoxicity analyses showed CC50 of 115 µg/mL. The crude extract showed antioxidant activity by the DPPH, ABTS, and FRAP assays. Therefore, the extract of the endophytic fungus presented biotechnological potential as an antibacterial and antioxidant agent.

Exopolysaccharides from Klebsiella oxytoca: anti-inflammatory activity

Abstract Exopolysaccharides (EPS) produced by Klebsiella oxytoca are of environmental, pharmaceutical, and medicinal interest. However, studies about the anti-inflammatory activity of EPS produced by this microorganism still remain limited. The aim of this study was to produce, characterize, and evaluate the anti-inflammatory activity of EPS from K. oxytoca in a pleurisy model. Colorimetric analysis revealed that precipitated crude exopolysaccharides (KEPSC) and deproteinated exopolysaccharides (KEPS) present high levels of total carbohydrates (65.57% and 62.82%, respectively). Analyses of uronic acid (7.90% in KEPSC and 6.21% in KEPS) and pyruvic acid (3.01% in KEPSC and 1.68% in KEPS) confirm that the EPS are acidic. Gas chromatography-mass spectrometry analyses demonstrated that the EPS consisted of rhamnose (29.83%), glucose (11.21%), galactose (52.45%), and mannose (6.50%). The treatment of an experimental pleurisy model in rats through subcutaneous administration of 50, 100, 200, and 400 mg/kg of KEPS decreased both the volume of inflammatory exudate and the number of leukocytes recruited to the pleural cavity. The present data showed that EPS production by K. oxytoca using the method described is easy to perform and results in a good yield. In addition, we show that KEPS exhibit anti-inflammatory activity when administered subcutaneously in rats.

Limonoid detection and profile in callus culture of sweet orange

Plant tissue culture has emerged as an important tool to produce bioactive compounds from various plant species, including the sustainable production of limonoids that are receiving considerable attention due to the benefits associated with human health such as anticancer activities. The purpose of the present study was to evaluate the capacity of limonoids aglycone production from callus culture from sweet orange cv. Pera (Citrus sinensis) seeds and identify the compounds produced in this cell line. Callus induction occurred in Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic (2,4-D), malt extract, agar and coconut water. For the analysis and identification of the limonoids, CG-MS-EI ion-positive mode and UPLC-QTOF-ESI were used operating in positive and negative mode. An intense peak corresponding to limonin appeared in the callus extracts at a retention time of 58.1 min. in CG-MS-EI and four major limonoids aglycone by positive ion mode UPLC-QTOF-ESI: limonin, nomilin, deacetylnomilin, and nomilinic acid. The culture medium was efficient at the bioproduction of limonoids aglycone in callus cultures of C. sinensis seeds. Therefore, data obtained from UPLC-QTOF-ESI proved its importance at identifying new compounds that benefit human health, and may assist future work in the identification of known or new limonoids in Citrus species and related genera.

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.

Evaluation of limonoid production in suspension cell culture of Citrus sinensis

ABSTRACTThe use of cell and plant tissue culture techniques to produce economically important active metabolites has been growing. Among these substances are total limonoid aglycones, which are produced by "pera" orange (Citrus sinensis (L.) Osbeck, Rutaceae) and have received considerable attention because of their anticancer actions. The main objective of the present study was to analyze and compare the levels of limonoid aglycones in seeds, callus cultures (originating from seeds), callus cultures (originating from hypocotyls), cell suspensions from hypocotyls cells, and cell suspensions from cotyledons. The cell cultures or C. sinensis were obtained by inoculating two strains of callus in MS medium supplemented with 2.0 µM 2,4-dichlorophenoxyacetic acid, 7.0 µM benzyl aminopurine, and 3% (w/v) sucrose in the dark. The highest concentrations of limonoid aglycone that were obtained were observed in cotyledon cell lines (240 mg/100 g dry weight) that were produced on day 21 of culture and hypocotyl cell lines on day 7 (210 mg/100 g dry weight). Explants of different origins under the same culture conditions had different limonoid aglycone content. The present results may suggest strategies for enhancing the productivity of biologically important limonoid aglycones and investigating the complex pathways of these secondary metabolites in plant tissue cultures.

Produção de metabólitos secundários em cultura de células e tecidos de plantas: o exemplo dos gêneros Tabernaemontana e Aspidosperma: [revisão] / Production of plant secondary metabolites in plant cell and tissue culture: the example of Tabernaemontana and Aspidosperma genera: [revision]

Os estudos dos metabólitos secundários de plantas se desenvolveram aceleradamente nos últimos 50 anos. Estes compostos são conhecidos por desempenharem um papel importante na adaptação das plantas aos seus ambientes e também representam uma fonte importante de substâncias farmacologicamente ativas. As técnicas de cultura de células de plantas iniciaramse na década de 1960 como uma possível ferramenta para estudar e produzir os metabólitos secundários de plantas. O uso de cultura de células de planta para a produção de substâncias de interesse contribuiu grandemente para avanços em diversas áreas da fisiologia e bioquímica vegetal. Diferentes estratégias, usando sistemas de cultura in vitro, foram estudadas com o objetivo de aumentar a produção de metabólitos secundários. As plantas dos gêneros Aspidosperma e Tabernaemontana são importantes fontes de alcalóides indólicos biologicamente ativos, sendo que no Brasil existe um número considerável de espécies destes gêneros. As culturas de células de Aspidosperma e Tabernaemontana foram iniciadas há pelo menos 16 anos, as quais produzem um grande número de alcalóides, o que estimulou o desenvolvimento de diversas técnicas para sua produção, extração e identificação.

Studies on plant secondary metabolites have been increasing over the last 50 years. These compounds are known to play a major role in the adaptation of plants to their environment and an important source of active pharmaceuticals. Plant cell culture technologies were introduced at the end of the 1960s as a possible tool for both studying and producing plant secondary metabolites. Different strategies, using in vitro systems, have been extensively studied with the objective of improving the production of secondary plant compounds. The Aspidosperma and Tabernaemontana genera are an important source of biologically active alkaloids and in Brazil there is a considerable number of species of these genera. About 16 years ago cell cultures of Tabernemontana and Aspidosperma were initiated. These cell cultures did produce a number of alkaloids of pharmaceutical interest that stimulated the development of several techniques to production, extraction and identification.

Comparação de diferentes metodologias para obtenção de cultura de calos de Stevia rebaudiana (Bertoni) Bertoni / Comparison of different methodologies for obtaining callus cultures from Stevia rebaudiana (Bertoni) Bertoni

As folhas de Stevia rebaudiana (Bertoni) Bertoni (Asteraceae) contêm glicosídeos diterpenóides (GDS), que são cerca de 300 vezes mais doce que a sacarose a 4%. O objetivo deste estudo foi avaliar a formação de calos, a partir de folhas obtidas in vivo e in vitro de S. rebaudiana em dois meios já descritos na literatura: Murashige e Skoog (MS), suplementado com 3 mg L-1 de ácido 2,4-diclorofenóxiacético (2,4-D), e o MS suplementado com 1 mg L-1 de ácido naftalenoacético (ANA) e 0,5 mg L-1 de 6-benzilaminopurina 6-BAP) e um desenvolvido em nosso laboratório o Woody Plant Medium (WPM), suplementado com 6 mg L-1 de ANA e 4 mg L-1 de cinetina (CIN). Os explantes obtidos in vitro iniciaram a formação de calos um pouco mais rapidamente que os das folhas de plantas advindas da natureza. A utilização dos nutrientes do meio WPM, associada a uma combinação de fitorreguladores adequada, proporcionou velocidade de indução e multiplicação de calos bem maiores que as apresentadas nos meios que empregaram os nutrientes do MS. Novos experimentos serão realizados, depois de alcançada a estabilidade genética dos calos, visando avaliar a capacidade destes em biossintetizar os GDS

The leaves from Stevia rebaudiana (Bertoni) Bertoni (Asteraceae) contain diterpenoid glycosides (GDS), which are almost 300 times sweeter than sucrose at 4%. The subject of this study was to evaluate the callus-formation from in vivo and in vitro leaves of Stevia rebaudiana in two already described in literature: Murashige and Skoog (MS) supplemented with 3 mg L-1 of 2,4-dichlorophenoxyacetic acid (2,4-D); MS supplemented with 1 mg L-1 of naphthaleneacetic acid (NAA) and 0.5 mg L-1 of 6-benzylaminopurine (6-BAP) and other developed in our laboratory the Woody Plant Medium (WPM) with 6 mg L-1 of NAA and 4 mg L-1 of Kinetin (KIN). The explants obtained in vitro initiated callus formation faster than leaves from natural plants. The utilization of WPM nutrients, associated with an adequate combination of phytoregulators, provided greater callus induction velocity and multiplication than the media that using MS nutrients. New experiments will be conducted after reaching genetic stability of the calluses, seeking to evaluate the capacity of these calluses to biosynthesize GDS

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

Este experimento teve como objetivo avaliar a polinização realizada pelas abelhas na produção e qualidade das sementes da soja (Glycine max L. Merril) na região de Maringá-PR. Os tratamentos constituíram de áreas demarcadas de livre visitação por insetos, áreas cobertas por gaiolas com uma colônia de abelhas (Apis mellifera) e plantas também cobertas por gaiolas que impediam a visitação por insetos. Todas as áreas possuíam 24 m2 (4 m x 6 m), com cinco repetições cada. A produção de sementes foi maior (P=0,0001) nas áreas cobertas com abelhas e de livre visitação com um incremento na produtividade de 50,64% e 57,73%, respectivamente, em relação à área coberta sem abelhas. Pode-se considerar que as abelhas A. mellifera foram responsáveis por 95,5% da polinização realizada pelos insetos no tratamento livre. O número de vagens no tratamento coberto com abelhas foi 61,38% maior (P=0,0002) do que no coberto sem abelhas. Onde as abelhas A. mellifera foram responsáveis pela polinização cruzada, houve um aumento de 58,86% no número de sementes em relação ao tratamento onde não foi permitida a polinização realizada por insetos. Entretanto, o peso médio de 100 sementes foi maior (P=0,0001) na área coberta sem abelhas, atingiu um peso médio de 17,80 g, mostrando que plantas com menor produção formaram sementes maiores. No tratamento livre, o peso médio de 100 sementes foi de 15,26 g e no coberto com abelhas foi de 15,37 g. O teor médio de proteína bruta no grão foi de 36,69% e a média do teor de óleo foi de 20,24%. O teste de germinação não mostrou diferenças entre as sementes nos diferentes tratamentos. Pode-se concluir que as abelhas A. mellifera foram eficientes no trabalho de polinização na soja, proporcionando um aumento considerável na produção de grãos e estes resultados reforçam a necessidade do uso das abelhas A. mellifera para elevar a produtividade da soja.

Anti-leishmanial activity of alkaloidal extract from Aspidosperma ramiflorum

Infections due to protozoa of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials (SbV), which present renal and cardiac toxicity. Besides, the precise chemical structure and mechanism of action of these drugs are unknown up to date. In order to find new drugs against leishmaniasis, we have been studying extracts of Brazilian trees. In the present study, we have evaluated the effectiveness of an alkaloid extract of Aspidosperma ramiflorum Muell. Arg. (Apocynaceae), against the extracellular forms promastigotes of L. (L.) amazonensis and L. (V.) braziliensis. The alkaloid extract of A. ramiflorum was much more effective against L. (L.) amazonensis (LD50 < 47 µg/ml) than L. (V.) braziliensis. Based on these in vitro results against L. (L.) amazonensis new studies should be made to find the compounds with anti-leishmanial activity.

Uma síntese alternativa para o (ñ)-1,2,3,4-tetraidro-{9H-pirido-[3, 4-b]}-indol / An alternative synthesis of 1,2,3,4-tetrahydro-9H-pyrido-{3, 4-b]-indole

O 1,2,3,4,-tetraidro-9H-pirido-[3,4-b]-indol, um composto com propriedades tranqüilizantes, foi preparado a partir do 3-indol-acetaldeido e triptamina através de uma reação de condensação de Pictet-Spengler. Os dados físicos e espectrais do composto são apresentados.