Sulfated Metabolites of Flavonolignans and 2,3-Dehydroflavonolignans: Preparation and Properties.

Silymarin, an extract from milk thistle (Silybum marianum) fruits, is consumed in various food supplements. The metabolism of silymarin flavonolignans in mammals is complex, the exact structure of their metabolites still remains partly unclear and standards are not commercially available. This work is focused on the preparation of sulfated metabolites of silymarin flavonolignans. Sulfated flavonolignans were prepared using aryl sulfotransferase from Desulfitobacterium hafniense and p-nitrophenyl sulfate as a sulfate donor and characterized by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR). Their 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and N,N-dimethyl-p-phenylenediamine (DMPD) radical scavenging; ferric (FRAP) and Folin⁻Ciocalteu reagent (FCR) reducing activity; anti-lipoperoxidant potential; and effect on the nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway were examined. Pure silybin A 20-O-sulfate, silybin B 20-O-sulfate, 2,3-dehydrosilybin-20-O-sulfate, 2,3-dehydrosilybin-7,20-di-O-sulfate, silychristin-19-O-sulfate, 2,3-dehydrosilychristin-19-O-sulfate, and silydianin-19-O-sulfate were prepared and fully characterized. Sulfated 2,3-dehydroderivatives were more active in FCR and FRAP assays than the parent compounds, and remaining sulfates were less active chemoprotectants. The sulfated flavonolignans obtained can be now used as authentic standards for in vivo metabolic experiments and for further research on their biological activity.

Preparation and extensive characterization of hyaluronan with narrow molecular weight distribution.

The physicochemical properties and biological functions of hyaluronan (HA) are closely related to its molecular weight (MW) and molecular weight distribution (MWD). Therefore, it is crucially important to provide a reliable characterization of these parameters for proper use of HA and its degradation products in both chemical and clinical fields. In this study, we present a novel method for the preparation of HA fragments of defined size with narrow molecular weight distribution. The HA fractionation was performed using an anion-exchange chromatography and is applicable either after enzymatic or chemical hydrolysis of polymeric HA. Isolated fractions with a molecular weight ranging from 3000-420,000gmol-1 were analyzed by size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS). Hundred-milligram scale HA fragments were obtained from 5g hyaluronan starting material. Independently on weight-average molecular weight (Mw), the polydispersity index (PDI) of the HA fractions was less than 1.23. The fractionation methodology can be easily up-scaled and is applicable on any negatively charged polymers. We have also found that PDI is insufficient to characterize almost monodisperse fractions and for proper material characterization we proposed a new characteristic termed "distribution angle", calculated from the slope of the cumulative molecular weight distribution curve. Compared to PDI, the distribution angle reflects more efficiently changes in size distribution and thus is highly recommended to be used along with Mw determination of any polymer.

One-pot synthesis of α,ß-unsaturated polyaldehyde of chondroitin sulfate.

Chondroitin sulfate (CS) was chemoselectively oxidized by Tempo/NaClO to C-6 aldehyde of a D-galactosamine subunit (GalNAc). The subsequent, spontaneous desulfatation of oxidized CS gave rise to α,ß-unsaturated aldehyde. A new derivative of CS was fully characterized and a degree of oxidation was determined by spectroscopic analysis. The optimization of reaction conditions showed a proportional degree of oxidation to an amount of sodium hypochlorite. The utility of α,ß-unsaturated aldehyde for crosslinking and conjugation was demonstrated by a seamless condensation with various N-nucleophiles. We also demonstrated pH-dependent release of biologically active agents from oxidized CS. A live-dead assay in the presence of α,ß-unsaturated aldehyde revealed unaffected viability of NIH 3T3 fibroblasts, which made this precursor promising for several biomedical applications including drug delivery and tissue engineering.

Enhanced multiparametric hyaluronan degradation for production of molar-mass-defined fragments.

Hyaluronic acid (HA) is known to serve as a dynamic mediator intervening in many physiological functions. Its specific effect has been repeatedly confirmed to be strongly influenced by the molecular size of hyaluronan fragments. However common technological approaches of HA fragments production have their limitations. In many cases, the final products do not meet the strict pharmaceutical requirements, specifically due to size polydispersity and reaction contaminants. We present novel methodology based on combination of unique incidental ability of the plant-derived protease papain to split the glycosidic bonds and an indispensable advantages of biocompatible macroporous material with incorporated ferrous ions serving as carrier for covalent papain fixation. This atypical and yet unpublished highly efficient multiparametric approach allows enhanced HA fragmentation for easily and safely producing molar-mass-defined HA fragments with narrow size distribution. Native polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography/multi-angle light scattering (SEC-MALS) confirmed the effectiveness of our multiparametric approach.

Cryptococcus laurentii extracellular biopolymer production for application in wound management.

Cryptococcus laurentii growth and extracellular polysaccharide (EPS) production in bioreactor were studied. Biomass yield 14.3 g/L and EPS synthesis 4.3 g/L in 144 h of submerged cultivation were achieved. EPS synthesis and cell growth had different optima. For EPS formation, pH 3, 25 °C and low aeration (1 % < pO2 < 10 %) were advantageous, while cell growth optimum was at pH 6, 20 °C, and high aeration (pO2 > 30 %). As medium pH changed from pH 3 to pH 6, glucuronic acid (GluAc) content in EPS increased, while galactose, xylose, and glucose decreased. Twenty-five degrees Celsius was optimal for GluAc containing polysaccharide synthesis, while lower temperature (15 °C) increased glucose content in EPS. Aeration intensity and time of cultivation had little effect on EPS composition. Molecular mass distribution of raw C. laurentii EPS was determined by SEC-MALS as 1.352. The row EPS was composed of acidic glucuronoxylomannan for more than 85 %. In the in vivo experiments, EPS significantly improved excisional wound healing in healthy rats. The results suggest that C. laurentii EPS is a promising biotechnological product and an advanced material for application in wound management.