Lentinus crinitus: Traditional use, phytochemical and pharmacological activities, and industrial and biotechnological applications.

The saprophytic basidiomycete Lentinus crinitus (L.) Fr is a Brazilian native fungus with pantropical occurrence. L. crinitus produces edible fruiting bodies with medicinal, nutritional, and biotechnological applications. The compounds from fungal fruiting bodies can be applied to the preparation of products in the food, cosmetic, biomedical, and pharmaceutical industries. Our aim was to review the literature on L. crinitus concerning its botanical description, geographical distribution, phytochemistry, pharmacological properties, nutritional value, and biotechnology potential (in vitro cultivation and enzyme production). Scientific search engines, including ScienceDirect, CAPES Journals Portal, Google Scholar, PubMed, SciELO, MEDLINE, LILACS, and SciFinder, were consulted to gather data on L. crinitus. The present review is an up-to-date and comprehensive analysis of the phytochemical compounds, phytopharmacological activities, and biotechnological value of L. crinitus. Extracts from L. crinitus have been reported to exhibit numerous in vitro pharmacological activities such as antioxidant, antifungal, antibacterial, antiviral, and anticancer. The substances in these extracts belong to different classes of chemical compounds such as polysaccharides, fatty acids, terpenes, phenolic acids, and flavonoids. Reviews on Brazilian native fungi are of great importance for scientific knowledge, with great applicability as a mirror for species of the same family. The ethnobotanical, phytochemical, pharmacological, ethnomedicinal, and biotechnological properties of L. crinitus highlighted in this review provide information for future studies and commercial exploitation, and reveal that this fungus has enormous potential for pharmaceutical, nutraceutical, biotechnological, and ecological applications.

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.

Low-cost alternative for the bioproduction of bioactive phenolic compounds of callus cultures from Cereus hildmannianus (K.) Schum.

The aim of this study was to establish a sustainable alternative callus culture of Cereus hildmannianus for the production and bioactive determination of phenolic compounds from this species. The conventional callus was cultivated using agar and Murashige and Skoog (MS) medium, while for the alternative culture the agar was replaced with a cotton support covered with filter paper and MS medium (incubated at 32 °C with photoperiod of 16 h), and the morphological characteristics and growth index were assessed (8 weeks). Extracts were obtained by maceration followed by partition, characterized by nuclear magnetic resonance - NMR and ultra-high performance liquid chromatography - UHPLC, quantified (phenolic compounds) by UV-Vis methods, and their antioxidant, antitumor activities, as well as cytotoxicity, were evaluated. The establishment of an alternative callus culture was carried out successfully. Characteristic signals of phenolic compounds were determined by NMR, and 46 compounds with fragment ions were identified using UHPLC analysis. The highest concentrations of phenolic compounds, and greatest antioxidant and antitumor activities, were obtained with the dichloromethane fractions of both callus tissue cultures, which were not cytotoxic. The callus culture from C. hildmannianus has shown promise as a source for the sustainable production of phenolic compounds with antioxidant and antiproliferative activities and thus, has potential use as a natural antitumor product.

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.

Metabolic extract of the endophytic fungus Flavodon flavus isolated from Justicia brandegeana in the control of Alicyclobacillus acidoterrestris in commercial orange juice.

In this work, the antibacterial activity of a crude extract of the endophytic fungus Flavodon flavus (JB257), isolated from leaves of Justicia brandegeana, was evaluated against both the vegetative and sporulated forms of Alicyclobacillus acidoterrestris. The microdilution technique was performed in order to determine the antibacterial activity of the crude extract alone as well as in combination with the bacteriocin, nisin. The minimum inhibitory concentration (MIC) of the crude extract and nisin alone against A. acidoterrestris vegetative forms were 250 µg/mL and 31.5 µg/mL, respectively, while the minimum bactericidal concentrations (MBC) were 1000 µg/mL and 62.5 µg/mL,respectively. For A. acidoterrestris spores, treatment with the crude extract at a concentration of 500 µg/mL caused a 47% reduction in growth, while nisin at 62.5 µg/mL could reduce 100% of the growth. The in vitro evaluation of the crude extract combined with nisin against A. acidoterrestris by the Checkerboard method showed a synergistic interaction between the two compounds. In addition, greater selectivity towards bacterial cells over host cells, a human hepatocyte cell line, was achieved when the crude extract was combined with nisin, Using scanning electron microscopy, interferences in the cell membrane of A. acidoterrestris could be observed after treatment with the crude extract. The results presented in this study indicate that the crude extract of the endophyte F. flavus has biotechnological potential in the food industry, especially for the treatment of orange juices through the control of A. acidoterrestris.

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.

Effects of fructans and probiotics on the inhibition of Klebsiella oxytoca and the production of short-chain fatty acids assessed by NMR spectroscopy.

Generally, the selection of fructans prebiotics and probiotics for the formulation of a symbiotic has been based on arbitrary considerations and in vitro tests that fail to take into account competitiveness and other interactions with autochthonous members of the intestinal microbiota. However, such analyzes may be a valuable step in the development of the symbiotic. The present study, therefore, aims to investigate the effect of lactobacilli strains and fructans (prebiotic compounds) on the growth of the intestinal competitor Klebsiella oxytoca, and to assess the correlation with short-chain fatty acids production. The short-chain fatty acids formed in the fermentation of the probiotic/prebiotic combination were investigated using NMR spectroscopy, and the inhibitory activities were assessed by agar diffusion and co-culture methods. The results showed that Lactobacillus strains can inhibit K. oxytoca, and that this antagonism is influenced by the fructans source and probably associated with organic acid production.

Anti-herpes activity of polysaccharide fractions from Stevia rebaudiana leaves.

The antiviral potential of natural polysaccharide compounds has been demonstrated, especially against enveloped viruses and members of the Herpesviridae family. Two polysaccharide fractions obtained from Stevia rebaudiana (Bertoni) leaves, that were active against Herpes simplex virus type 1 (HSV-1) were studied to investigate their mode of action. Both polysaccharides - SFW (crude faction) and SSFK (homogeneous alkaline fraction) - exerted antiviral effects on the initial stages of HSV-1 infection by inhibiting viral adsorption and penetration. When added after virus internalization, both fractions decreased plaque size. The effect of the fractions was confirmed by investigating viral glycoprotein expression. Based on the mode of action of the polysaccharides demonstrated in the present work and on their selectivity index, the polysaccharides obtained from S. rebaudiana could be an alternative treatment of infections caused by HSV-1.

Biochemical composition of symplastic sap from sugarcane genetically modified to overproduce proline.

Global interest in sugarcane has increased significantly in recent years because of its economic impact on sustainable energy production. The purpose of the present study was to evaluate changes in the concentrations of total sugars, amino acids, free proline, and total proteins by colorimetric analyses and nuclear magnetic resonance (NMR) to perform a metabolic profiling of a water-soluble fraction of symplastic sap in response to the constitutive expression of a mutant Δ1-pyrroline-5-carboxylate synthetase (P5CS) gene from Vigna aconitifolia. However, there was not a significant increase in the free proline content in the sap of transgenic plants compared to the non-transformed control plants. The most noticeable difference between the two genotypes was an almost two-fold increase in the accumulation of sucrose in the stem internodes of P5CS transgenic sugarcane plants. The results presented in this work showed that transgenic sugarcane plants with increased levels of free proline accumulates high soluble sugar content and, therefore, may represent a novel genotype for improving sugarcane cultivars.

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.

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.

Isolation and prebiotic activity of inulin-type fructan extracted from Pfaffia glomerata (Spreng) Pedersen roots.

Pfaffia glomerata (Amaranthaceae) is popularly known as "Brazilian ginseng." Previous studies have shown that fructose is the major carbohydrate component present in its roots. Inulin-type fructans, polymers of fructose, are the most widespread and researched prebiotics. Here, we isolated and chemically characterized inulin extracted from P. glomerata roots and investigated its potential prebiotic effect. Fructans were isolated and their structures were determined using colorimetric, chromatography, polarimetry, and spectroscopic analysis. The degree of polymerization (DP) was determined, and an in vitro prebiotic test was performed. The structure of inulin was confirmed by chromatography and spectroscopic analysis and through comparison with existing data. Representatives from the genera Lactobacillus and Bifidobacterium utilized inulin from P. glomerata, because growth was significantly stimulated, while this ability is strain specific. The results indicated that inulin extracted from P. glomerata roots represents a promising new source of inulin-type prebiotics.

Structure and antiviral activity of arabinogalactan with (1→6)-ß-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 ß-(1→6)-linked D-Galp residues forming the main chain. Approximately 38% of the ß-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.

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 ß-fructofuranosyl, with terminal α-glucopyranosyl and ß-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.

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