In vitro and in vivo biotransformation of glucosinolates from mashua (Tropaeolum tuberosum) by lactic acid bacteria.

Mashua (Tropaeolum tuberosum) is an Andean tuber with a high content of glucosinolates (GLSs). GLSs subjected to biotransformation by plant enzymes or enzymes of the gastrointestinal microbiota give rise to biologically active compounds, to which chemo preventive properties are attributed. In this work, the biotransformation of mashua GLSs was evaluated in vitro by six strains of lactic acid bacteria (LAB) and in vivo using rats with and without previous LAB dosing. The results showed that L. rhamnosus GG utilized the totality of glucosinalbin and glucotropaeolin, and 46.7 % of glucoaubrietin. Four GLSs derivatives were detected. The GLSs were absorbed and metabolized by the rats with low contents in feces (0.02 %) and urine (0.59 %) and were detected up to 3 h after consumption in plasma. The results showed that probiotic bacteria play an important role in transforming GLSs into beneficial compounds for the health of consumers.

Jasmonate regulates plant resistance to Pectobacterium brasiliense by inducing indole glucosinolate biosynthesis.

Pectobacterium brasiliense (P. brasiliense) is a necrotrophic bacterium that causes the soft rot disease in Brassica rapa. However, the mechanisms underlying plant immune responses against necrotrophic bacterial pathogens with a broad host range are still not well understood. Using a flg22-triggered seedling growth inhibition (SGI) assay with 455 Brassica rapa inbred lines, we selected six B. rapa flagellin-insensitive lines (Brfin2-7) and three B. rapa flagellin-sensitive lines (Brfs1-3). Brfin lines showed compromised flg22-induced immune responses (oxidative burst, mitogen-activated protein kinase (MAPK) activation, and seedling growth inhibition) compared to the control line R-o-18; nevertheless, they were resistant to P. brasiliense. To explain this, we analyzed the phytohormone content and found that most Brfin lines had higher P. brasiliense-induced jasmonic acid (JA) than Brfs lines. Moreover, MeJA pretreatment enhanced the resistance of B. rapa to P. brasiliense. To explain the correlation between the resistance of Brfin lines to P. brasiliense and activated JA signaling, we analyzed pathogen-induced glucosinolate (GS) content in B. rapa. Notably, in Brfin7, the neoglucobrassicin (NGBS) content among indole glucosinolates (IGS) was significantly higher than that in Brfs2 following P. brasiliense inoculation, and genes involved in IGSs biosynthesis were also highly expressed. Furthermore, almost all Brfin lines with high JA levels and resistance to P. brasiliense had higher P. brasiliense-induced NGBS levels than Brfs lines. Thus, our results show that activated JA-mediated signaling attenuates flg22-triggered immunity but enhances resistance to P. brasiliense by inducing indole glucosinolate biosynthesis in Brassica rapa. This study provides novel insights into the role of JA-mediated defense against necrotrophic bacterial pathogens within a broad host range.

Chemical Analysis of Eruca sativa Ethanolic Extract and Its Effects on Hyperuricaemia.

In vivo assays and chemical analyses were performed on the ethanolic extract from leaves of Eruca sativa. UHPLC-ESI-QTOF analysis confirmed the presence of glucosinolates and flavonol glucosides. The major flavonoid of the ethanolic extract, kaempferol-3,4'-di-O-ß-glucoside, was isolated, a HPLC-DAD method developed and validated to quantify its content in the extract. In vivo experiments were carried out on Wistar rats with hyperuricaemia induced by potassium oxonate and uric acid. A hypouricaemic effect was observed in hyperuricaemic Wistar rats treated with ethanolic extract at dose of 125 mg/kg and kaempferol-3,4'-di-O-ß-glucoside at dose of 10 mg/kg. The main anti-hyperuricaemic mechanism observed in the extract was uricosuric. Kaempferol-3,4'-di-O-ß-glucoside was identified as an important component responsible for the total activity of the ethanolic extract and was considered as a good chemical and biological marker of the ethanolic extract of E. sativa. The obtained results indicated the potential of E. sativa in the treatment of hyperuricaemia and its comorbidities.

Chemical Genetics Applied to Elucidate the Physiological Role of Stress-Signaling Molecules on the Wound-Induced Accumulation of Glucosinolates in Broccoli.

Wounding stress is an effective strategy to induce glucosinolate (GS) biosynthesis in broccoli. However, there is insufficient knowledge on the physiological and molecular mechanisms underlying this stress response. Herein, a chemical-genetic approach was applied to elucidate the role of jasmonic acid (JA), ethylene (ET), and reactive oxygen species (ROS) on the wound-induced biosynthesis of GS. Broccoli was processed into chops to induce wounding stress. Broccoli chops were treated with phenidone (PHEN) and diphenyleneiodonium chloride (DPI) as inhibitors of JA and ROS biosynthesis, respectively, whereas 1-methylcyclopropene (1-MCP) was applied as an inhibitor of ET action. Wounding stress induced the expression of genes related to the biosynthesis of indolic and aliphatic GS, which was correlated with the accumulation of GS and modulated by the inhibitors of signaling molecules applied. Results of gene expression analysis indicated that JA played a key role in the activation of most genes, followed by ROS. Furthermore, except for the CYP79B2 gene, PHEN and 1-MCP synergistically downregulated the expression of GS biosynthetic genes evaluated, showing that the interaction between JA and ET was fundamental to modulate GS biosynthesis. Results presented herein increased our knowledge of the physiological and molecular mechanisms governing the wound-induced biosynthesis of GS in broccoli.

Glucosinolate catabolism during postharvest drying determines the ratio of bioactive macamides to deaminated benzenoids in Lepidium meyenii (maca) root flour.

Postharvest processing of maca (Lepidium meyenii Walp., Brassicaceae), a traditional high-altitude Andean root crop, involves slow field drying prior to milling into flour. The progressive tissue dehydration and release of hydrolytic enzymes and substrates from cellular compartments results in the slow accumulation of free monosaccharides, fatty acids and amino acids. A more complex, and faster, kinetic profile is that of glucosinolate breakdown. A number of reactive transient and stable accumulation products are generated during drying, some of which have noteworthy bioactive properties. Among these are macamides, inhibitors of endocannabinoid neurotransmitter degradation in mammalian nervous systems. They result from the condensation of benzyl amine, a glucosinolate hydrolysis product, with free fatty acids released from lipid hydrolysis. Recent research has focused on developing drying processes under controlled conditions that can modulate the biochemistry of glucosinolate hydrolysis to optimize the content of bioactive compounds in the root flour. Low temperature (35 °C) oven-drying of shredded maca roots under controlled air flow generates benzyl amine as primary accumulation product, accounting for up to 94% of hydrolyzed glucosinolate in the flour. Kinetic evidence suggests that both deaminated benzenoids and macamides are allocated from the benzylamine pool through amine oxidase activity or condensation with free fatty acids, accounting for the remaining hydrolyzed glucosinolate (<5%). These activities determine the allocation to either one of these pathways. Later stages of dehydration result in shifts in the molar ratios of deaminated benzenoids, the accumulation of benzoic acid esters and benzyl alcohol. We propose that these are the result of changes in the rates of the reductive and oxidative half-reactions of endogenous aldehyde dehydrogenases. It is the ratio of benzylamine deamination to amide formation that determines the eventual yields of macamides in relation to benzenoids and their esters in maca flour.

Bioactive Compounds and Antioxidant Activity in Leaves of Endemic and Native Isatis spp in Turkey

Abstract This study was undertaken to evaluate the health-promoting potentials of Isatis aucherii, Isatis buschiana, Isatis candolleana, Isatis tinctoria subsp. corymbosa and Isatis tinctoria. I. aucherii and I. candolleana are endemic, I. buschiana, I. tinctoria subsp. corymbosa are native in Turkey. While I. tinctoria is a well studied species, there is insufficient information about other endemic and native species. Therefore, this study is focused to reveal the bioactive compounds of poorly studied endemic and native species. In this context, protein, ash, glucosinolates, fatty acids, total phenolic and flavonoid content, and antioxidant activities were determined in leaf extracts. The highest protein and fatty oil contents were observed in I. tinctoria and I. buschiana. Arachidic acid was predominant inI. tinctoria subsp. corymbosa, I. buschiana and I. aucherii, while predominant fatty acids were arachidonic and oleic acids inI. candolleana and I. tinctoria. Glucobrassicin was the main glucosinolate in I. tinctoria, while the others contained gluconapin as the main glucosinolate. Antioxidant activities were correlated with phenolic and flavonoid content, the highest and lowest antioxidant activities were observed in I. buschiana and I. aucherii, respectively. According to results, I. buschiana leaves were high in contents of bioactive compounds; it could be a promising plant with its health- promoting effects.

UVA, UVB Light Doses and Harvesting Time Differentially Tailor Glucosinolate and Phenolic Profiles in Broccoli Sprouts.

Broccoli sprouts contain health-promoting glucosinolate and phenolic compounds that can be enhanced by applying ultraviolet light (UV). Here, the effect of UVA or UVB radiation on glucosinolate and phenolic profiles was assessed in broccoli sprouts. Sprouts were exposed for 120 min to low intensity and high intensity UVA (UVAL, UVAH) or UVB (UVBL, UVBH) with UV intensity values of 3.16, 4.05, 2.28 and 3.34 W/m², respectively. Harvest occurred 2 or 24 h post-treatment; and methanol/water or ethanol/water (70%, v/v) extracts were prepared. Seven glucosinolates and 22 phenolics were identified. Ethanol extracts showed higher levels of certain glucosinolates such as glucoraphanin, whereas methanol extracts showed slight higher levels of phenolics. The highest glucosinolate accumulation occurred 24 h after UVBH treatment, increasing 4-methoxy-glucobrassicin, glucobrassicin and glucoraphanin by ~170, 78 and 73%, respectively. Furthermore, UVAL radiation and harvest 2 h afterwards accumulated gallic acid hexoside I (~14%), 4-O-caffeoylquinic acid (~42%), gallic acid derivative (~48%) and 1-sinapoyl-2,2-diferulolyl-gentiobiose (~61%). Increases in sinapoyl malate (~12%), gallotannic acid (~48%) and 5-sinapoyl-quinic acid (~121%) were observed with UVBH Results indicate that UV-irradiated broccoli sprouts could be exploited as a functional food for fresh consumption or as a source of bioactive phytochemicals with potential industrial applications.

Glucosinolate composition of Colombian accessions of mashua (Tropaeolum tuberosum Ruíz & Pavón), structural elucidation of the predominant glucosinolate and assessment of its antifungal activity.

BACKGROUND: The content of individual and total glucosinolates in 65 mashua tuber accessions (Tropaeolum tuberosum) from the germplasm bank at Universidad Nacional de Colombia was determined by reverse phase high-performance liquid chromatography on enzymatically desulfated extracts. The predominant glucosinolate was identified and the possible structure of the glucosinolate present in lower proportion was postulated from evidence obtained by high-performance liquid chromatography/mass spectrometry, 1 H and 13 C nuclear magnetic resonance and bi-dimensional experiments. The biological action of the hydrolysis products generated from the glucosinolates in the accessions that showed a higher content of these compounds was assessed in the presence of Fusarium oxysporum f. sp. dianthi, Rhizoctonia solani and Phytophthora infestans. RESULTS: The total content of glucosinolates ranged between >3.00 × 10-1 and 25.8 µmol g-1 dry matter. p-Methoxybenzyl glucosinolate was identified as the predominant glucosinolate in Colombian mashua accessions; besides, the possible presence of p-hydroxybenzyl glucosinolate was postulated. In vitro assays established an important fungal growth inhibition of the potato pathogen P. infestans. CONCLUSION: The biological action from p-methoxybenzyl glucosinolate and p-hydroxybenzyl glucosinolate found in Colombian mashua accessions depends on their concentration, with the Tt30 accession, characterized for showing the highest content of glucosinolates, being the most promising to control the assessed pathogens. © 2016 Society of Chemical Industry.

Nematicidal activity of crambe extracts on Meloidogyne spp. / Atividade nematicida de extratos de crambe sobre Meloidogyne spp.

Alternative methods for the control of nematodes, such as the use of plant secondary metabolites, can be explored for integrated pest management systems. The objective of this work was to assess the best solvent for obtaining allyl isothiocyanate from Crambe abyssinica leaves, and the effects of this extract on Meloidogyne incognita and M. javanica. Dry leaves of C. abyssinica at 200 mg L-1 were used to prepare extracts by using water (by infusion and grinding), acetone, water + ethanol (hydroalcoholic extraction), methanol, hexane, and chloroform as solvents. Following the evaporation of the solvents, the residue was resuspended in water for use in the experiments. Distilled water and chemical nematicide were used as control treatments. Once the most effective extracts were defined, the following dosages of dried crambe leaves were used: 0, 200, 300, 400, and 500 mg L-1. High performance liquid chromatography (HPLC) was used to quantify the allyl isothiocyanate present in the extracts. After the solvents evaporated, the residues were eluted with water and used in assays with 200 eggs for the hatching test or 200 second stage juveniles (J2) for mobility and mortality tests. The hydroalcoholic extract was the most effective in reducing the hatching of M. incognita and M. javanica juveniles, by 71.6 and 74.4 percentage points, respectively. The mortality of M. incognita and M. ja

Medidas alternativas de controle de fitonematoides, como o uso de metabólitos secundários produzidos pelas plantas, podem ser exploradas no contexto do manejo integrado em fitossanidade. O objetivo deste trabalho foi verificar quais os melhores solventes para obtenção de alil isotiocianato de folhas de Crambe abyssinica, e o efeito dos mesmos sobre Meloidogyne incognita e Meloidogyne javanica. Folhas secas de crambe na concentração de 200 mg L-1 foram utilizadas para o preparo dos extratos com os solventes água (para os extratos obtidos por infusão e trituração), acetona (cetônico), água + álcool etílico (hidroalcoólico), álcool metílico (metanólico), hexano (hexânico) e clorofórmio (clorofórmico). Após a rotoevaporação dos solventes o resíduo foi ressuspenso em água para utilização nos ensaios. Água destilada e nematicida foram usados como tratamentos controle. Após a seleção dos melhores extratos, os selecionados foram avaliados nas seguintes doses: 0, 200, 300, 400 e 500 mg L-1 de folhas secas de crambe. Para quantificar o alil isotiocianato presente nos extratos foi utilizada cromatografia líquida de alta pressão (HPLC). Para avaliar a eclosão foram utilizados 200 ovos e para motilidade e mortalidade, 200 juvenis de segundo estádio (J2). O extrato hidroalcoólico foi o mais efetivo em reduzir a eclosão de juvenis de M. incognita e M. javanica, com redução de 71.6 e 74.4 pont

Nematicidal activity of crambe extracts on Meloidogyne spp / Atividade nematicida de extratos de crambe sobre Meloidogyne spp

Alternative methods for the control of nematodes, such as the use of plant secondary metabolites, can be explored for integrated pest management systems. The objective of this work was to assess the best solvent for obtaining allyl isothiocyanate from Crambe abyssinica leaves, and the effects of this extract on Meloidogyne incognita and M. javanica. Dry leaves of C. abyssinica at 200 mg L-1 were used to prepare extracts by using water (by infusion and grinding), acetone, water + ethanol (hydroalcoholic extraction), methanol, hexane, and chloroform as solvents. Following the evaporation of the solvents, the residue was resuspended in water for use in the experiments. Distilled water and chemical nematicide were used as control treatments. Once the most effective extracts were defined, the following dosages of dried crambe leaves were used: 0, 200, 300, 400, and 500 mg L-1. High performance liquid chromatography (HPLC) was used to quantify the allyl isothiocyanate present in the extracts. After the solvents evaporated, the residues were eluted with water and used in assays with 200 eggs for the hatching test or 200 second stage juveniles (J2) for mobility and mortality tests. The hydroalcoholic extract was the most effective in reducing the hatching of M. incognita and M. javanica juveniles, by 71.6 and 74.4 percentage points, respectively. The mortality of M. incognita and M. ja

Medidas alternativas de controle de fitonematoides, como o uso de metabólitos secundários produzidos pelas plantas, podem ser exploradas no contexto do manejo integrado em fitossanidade. O objetivo deste trabalho foi verificar quais os melhores solventes para obtenção de alil isotiocianato de folhas de Crambe abyssinica, e o efeito dos mesmos sobre Meloidogyne incognita e Meloidogyne javanica. Folhas secas de crambe na concentração de 200 mg L-1 foram utilizadas para o preparo dos extratos com os solventes água (para os extratos obtidos por infusão e trituração), acetona (cetônico), água + álcool etílico (hidroalcoólico), álcool metílico (metanólico), hexano (hexânico) e clorofórmio (clorofórmico). Após a rotoevaporação dos solventes o resíduo foi ressuspenso em água para utilização nos ensaios. Água destilada e nematicida foram usados como tratamentos controle. Após a seleção dos melhores extratos, os selecionados foram avaliados nas seguintes doses: 0, 200, 300, 400 e 500 mg L-1 de folhas secas de crambe. Para quantificar o alil isotiocianato presente nos extratos foi utilizada cromatografia líquida de alta pressão (HPLC). Para avaliar a eclosão foram utilizados 200 ovos e para motilidade e mortalidade, 200 juvenis de segundo estádio (J2). O extrato hidroalcoólico foi o mais efetivo em reduzir a eclosão de juvenis de M. incognita e M. javanica, com redução de 71.6 e 74.4 pont

Linking phytochrome to plant immunity: low red : far-red ratios increase Arabidopsis susceptibility to Botrytis cinerea by reducing the biosynthesis of indolic glucosinolates and camalexin.

Shade-intolerant plants respond to low red : far-red (R : FR) ratios, which signal the proximity of potential competitors, by down-regulating immune responses. Here we investigated the mechanisms underlying this immune suppression in Arabidopsis. We used genetic, transcriptomic and metabolomic approaches to examine the functional connections between R : FR ratio and Arabidopsis resistance to the fungus Botrytis cinerea. Low R : FR ratios reduced the concentration of indol-3-ylmethyl glucosinolate (I3M) (an indolic glucosinolate, iGS) and camalexin in plants inoculated with B. cinerea, and attenuated the I3M response triggered by jasmonate elicitation. These effects on metabolite abundance correlated with reduced expression of iGS and camalexin biosynthetic genes. Furthermore, the effect of low R : FR increasing Arabidopsis susceptibility to B. cinerea was not present in mutants deficient in the biosynthesis of camalexin (pad3) or metabolism of iGS (pen2). Finally, in a mutant deficient in the JASMONATE ZIM DOMAIN-10 (JAZ10) protein, which does not respond to low R : FR with increased susceptibility to B. cinerea, supplemental FR failed to down-regulate iGS production. These results indicate that suppression of Arabidopsis immunity against B. cinerea by low R : FR ratios is mediated by reduced levels of Trp-derived defenses, and provide further evidence for a functional role of JAZ10 in the link between phytochrome and jasmonate signaling.

Screening of filamentous fungi for production of myrosinase

Aspergillus sp. NR46F13, isolated from soil via sinigrin-barium sulphate agar technique, was tested for myrosinase production. The fungus degraded glucosinate and produced 3.19 U mL-1 of myrosinase after 48h cultivation. due to the high myrosinase production, this new isolate is a potential candidate for industrial applications.

A linhagem Aspergillus sp. NR46FB, isolada de solo através da técnica do ágar sulfato de bário-sinigrina, foi testada quanto à produção de mirosinase. O fungo degradou completamente o glicosinolato e produziu 3,19 U.mL-1 de mirosinase, após 48 h de cultivo. Devido à alta produção de mirosinase, esse novo isolado é um potente candidato para aplicações industriais.