Metabolomic discrimination of the edible mushrooms Kuehneromyces mutabilis and Hypholoma capnoides (Strophariaceae, Agaricales) by NMR spectroscopy.

Two edible, cultivable mushroom species of the family Strophariaceae, Kuehneromyces mutabilis (sheathed woodtuft) and Hypholoma capnoides (conifer tuft), were studied using proton nuclear magnetic resonance metabolomic approach. The variation in the metabolites of the two species and their metabolic behaviour regarding caps and stipes and different collection sites were analysed by multivariate analysis methods. Altogether 169 cap and stipe samples of the mushrooms were investigated. The clearest difference between the species was in the sugar composition, which was more diverse in H. capnoides. When mushroom samples collected from different locations were compared, more variance was found in H. capnoides, whereas K. mutabilis appeared more homogeneous as a species. As far as the caps and stipes were concerned, in both species the amount of α-α-trehalose was clearly higher in the stipes, and the caps contained a larger proportion of the amino acids and organic acids.

Suitability of dried herbarium specimens for NMR metabolomics of mushrooms. A comparison of four species of the genera Kuehneromyces and Hypholoma (Strophariaceae).

Herbarium specimens are a treasure trove for biochemical studies. However, this implies understanding of the chemical changes during the drying and storage of the specimen. We compared herbarium specimens at different ages and fresh samples of four mushroom species (Kuehneromyces mutabilis, Hypholoma capnoides, Kuehneromyces lignicola, Hypholoma fasciculare) of two genera in the family Strophariaceae by using proton nuclear magnetic resonance (1H NMR) spectroscopy combined with principal component analysis (PCA). 25 metabolites were identified. No significant alterations were found between herbarium samples at different ages, suggesting that they are stable enough for comparative studies. The most dominant differences between fresh and herbarium samples was that sugars such as α-α-trehalose, and fumaric and malic acids were more abundant in fresh fungi. Total contents of fatty and amino acids, uracil and γ-aminobutyric acid (GABA) were higher in herbarium specimens. In addition, pyroglutamic acid was observed only in Kuehneromyces mutabilis and fasciculic acid E in Hypholomafasciculare. Hence, based on results of the studied taxa, we conclude that NMR metabolomics can be used for both fresh and dried mushrooms when such alterations are properly addressed.

Controlling of N-alkylpolyamine analogue metabolism by selective deuteration.

Replacing protium with deuterium is an efficient method to modulate drug metabolism. N-alkylated polyamine analogues are polyamine antimetabolites with proven anticancer efficacy. We have characterized earlier the preferred metabolic routes of N1,N12-diethylspermine (DESpm), N1-benzyl-N12-ethylspermine (BnEtSpm) and N1,N12-dibenzylspermine (DBSpm) by human recombinant spermine oxidase (SMOX) and acetylpolyamine oxidase (APAO). Here, we studied the above analogues, their variably deuterated counterparts and their metabolites as substrates and inhibitors of APAO, SMOX, semicarbazide-sensitive amine oxidase (SSAO), diamine oxidase (DAO) and monoamine oxidases. We found that targeted deuteration efficiently redirected the preferable cleavage site and suppressed reaction rate by APAO and SMOX in vitro We found a three- to six-fold decline in Vmax with moderate variable effect on Km when deuterium was located at the preferred hydrogen abstraction site of the analogue. We also found some of the metabolites to be potent inhibitors of DAO and SSAO. Surprisingly, analogue deuteration did not markedly alter the anti-proliferative efficacy of the drugs in DU145 prostate cancer cells, while in mouse embryonic fibroblasts, which had higher basal APAO and SMOX activities, moderate effect was observed. Interestingly, the anti-proliferative efficacy of the analogues did not correlate with their ability to suppress polyamine biosynthetic enzymes, induce spermidine/spermine-N1-acetyltransferase or deplete intracellular polyamine levels, but correlated with their ability to induce SMOX. Our data show that selective deuteration of N-alkyl polyamine analogues enables metabolic switching, offering the means for selective generation of bioactive metabolites inhibiting, e.g. SSAO and DAO, thus setting a novel basis for in vivo studies of this class of analogues.

Phenolic compounds extracted by acidic aqueous ethanol from berries and leaves of different berry plants.

Phenolic compounds of berries and leaves of thirteen various plant species were extracted with aqueous ethanol and analyzed with UPLC-DAD-ESI-MS, HPLC-DAD, and NMR. The total content of phenolics was consistently higher in leaves than in berries (25-7856 vs. 28-711mg/100g fresh weight). Sea buckthorn leaves were richest in phenolic compounds (7856mg/100g f.w.) with ellagitannins as the dominant compound class. Sea buckthorn berries contained mostly isorhamnetin glycosides, whereas quercetin glycosides were typically abundant in most samples investigated. Anthocyanins formed the dominating group of phenolics in most dark-colored berries but phenolic acid derivatives were equally abundant in saskatoon and chokeberry berries. Caffeoylquinic acids constituted 80% of the total phenolic content (1664mg/100g f.w.) in bilberry leaves. B-type procyanidins and caffeoylquinic acids were the major phenolic compounds in hawthorn and rowanberry, respectively. Use of leaves of some species with prunasin, tyramine and ß-p-arbutin, may be limited in food applications.

Stability of Hydroxycinnamic Acid Derivatives, Flavonol Glycosides, and Anthocyanins in Black Currant Juice.

The stability of phenolic compounds was followed in black currant juice at ambient temperatures (in light and in dark conditions) and at +4 °C for a year. Analyses were based on high-performance liquid chromatography-diode-array detection-electrospray ionization-mass spectrometry (or tandem mass spectrometry) and high-performance liquid chromatography-diode-array detection-electrospray ionization-quadrupole time-of-flight mass spectrometry methods supported by nuclear magnetic resonance after selective high-performance liquid chromatography isolation. Altogether, 43 metabolites were identified, of which 2-(Z)-p-coumaroyloxymethylene-4-ß-d-glucopyranosyloxy-2-(Z)-butenenitrile, 2-(E)-caffeoyloxymethylene-4-ß-d-glucopyranosyloxy-2-(Z)-butenenitrile, 1-O-(Z)-p-coumaroyl-ß-d-glucopyranose, (Z)-p-coumaric acid 4-O-ß-d-glucopyranoside, and (Z)-p-coumaric acid were novel findings in black currant juice. Hydroxycinnamic acid derivatives degraded 20-40% at room temperature during one year of storage, releasing free hydroxycinnamic acids. O-Glucosides of hydroxycinnamic acid compounds were the most stable, followed by O-acylquinic acids, acyloxymethyleneglucosyloxybutenenitriles, and O-acylglucoses. Light induced the isomerization of (E)-coumaric acid compounds into corresponding Z-isomers. Flavonol glycosides stayed fairly stable. Flavonol aglycones were derived mainly from malonylglucosides. Over 90% of anthocyanins were lost at room temperature in a year, practically independent of light. Storage at low temperatures, preferably excluding light, is necessary to retain the original composition of phenolic compounds.

Flavonol Glycosides in Currant Leaves and Variation with Growth Season, Growth Location, and Leaf Position.

Flavonol glycosides (FG) were analyzed in the leaves of six currant cultivars (Ribes spp.) with HPLC-DAD, HPLC-MS/MS, and NMR. The average amounts of the 12 major, identified FG constituted 86-93% (9.6-14.1 mg/g DW) of the total of 27 FG found. Quercetin and kaempferol were the major aglycones with trace amounts of myricetin. Quercetin-3-O-(2,6-α-dirhamnopyranosyl-ß-glucopyranoside), quercetin-3-O-(2-ß-xylopyranosyl-6-α-rhamnopyranosyl-ß-glucopyranoside), and kaempferol-3-O-(3,6-α-dirhamnopyranosyl-ß-glucopyranoside) were identified for the first time in currant leaves and existed in a white currant cultivar 'White Dutch' only. Kaempferol-3-O-ß-(6'-malonyl)glucopyranoside was also a new compound existing in abundance in five cultivars but not in the white one. The results show the primary importance of the genetic background of the cultivars. The content of malonylated FG of special importance in cardiovascular health decreased regularly during summer. Time of collection and leaf position were more prominent factors affecting the composition than were the year of harvest or the growth latitude. Randomly collected leaves differed in their FG profiles from those collected from the middle position of new branches.

A bisphosphonate increasing the shoot biomass of the metal hyperaccumulator Noccaea caerulescens.

The feasibility of using the hyperaccumulator plant, Noccaea caerulescens, to remove trace elements from contaminated soils has been studied extensively. However, this plant creates too low biomass and an inappropriately slow growth rate for actual use in the field. Soluble bisphosphonates (BPs) are well-known pharmaceutical compounds e.g. affecting the osteoclast function in body through metabolic pathways. We devised an insoluble aminoBP, hydroxyundecylidene-1,1,-bisphosphonic acid with a long alkyl chain to be extremely effective metal chelator, and its possible use in phytoremediation deserves more attention. This article examines the effects of seven BPs on the shoot biomass, shoot metal concentrations and removal (Cd, Ni, Zn and Pb) by N. caerulescens in a pot experiment. The soluble BPs were incorporated into the soil in the irrigation water and the insoluble BP as solid after which the soil was spiked with metals. The insoluble aminoBP was found to considerably increase the shoot yield of N. caerulescens, especially in Ni-spiked soil, thus showing enhanced shoot Ni removal.

First bisphosphonate hydrogelators: potential composers of biocompatible gels.

Recently, investigation of hydrogels has gained ever increasing attention mostly because of their biomedical and pharmaceutical properties, and novel hydrogelators are constantly studied to find functional applications. Bisphosphonates (BPs) are well-known compounds applicable in different fields but are mostly used in clinics as drugs for bone-related diseases. In this study, a novel class of BP-hydrogelators together with a BP-organogelator was found, and the gelating abilities of the compounds were studied. Several techniques to analyze the structure and the properties of the formed gels were used, including solid state 13C and 31P CPMAS and solution state NMR spectroscopy, IR spectroscopy, PXRD, thermoanalysis, as well as SEM.