Julia A. H. Kaeswurm

Phenolic structures are of great interest due to their antioxidant properties and various postulated benefits on human health. However, the quantification of these structures in fruits and vegetables, as well as in vivo or in vitro experiments, is demanding, as relevant concentrations are often low, causing problems in exactly weighing the respective amounts. Nevertheless, the determination of used concentrations is often a prerequisite for accurate results. A possibility to quantify polyphenol is the use of UV/vis spectroscopy. Therefore, the absorption coefficients of selected phenolic structures were determined in three different solvents relevant for polyphenol research (water/methanol (50/50, v/v), water, and phosphate buffer at pH 7.5). To confirm the values based on weight and to avoid errors due to impurities, hygroscopic effects, and inadequate balance care, the mass concentrations were additionally determined by quantitative NMR (q-NMR). The coefficients presented in this article can help to quickly and easily determine accurate concentrations in a laboratory routine without wasting the often-precious standard compounds.

Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fast-protein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated.

An inhibitory effect on α-amylase and α-glucosidase is postulated for polyphenols. Thus, ingestion of those secondary plant metabolites might reduce postprandial blood glucose level (hyperglycemia), which is a major risk factor for diabetes mellitus type II. In addition to a previous study investigating structure−effect relationships of different phenolic structures, the effect of anthocyanins is studied in detail here, by applying an α-amylase activity assay, on the basis of the conversion of 2-chloro-4-nitrophenyl-4-O-ß-galactopyranosyl maltoside (GalG₂CNP) and detection of CNP release by UV/Vis spectroscopy and isothermal titration calorimetry (ITC). All anthocyanin-3-glucosides showed a mixed inhibition with a strong competitive proportion, K_ic < 134 µM and K_iu < 270 µM; however, the impact of the B-ring substitution was not statistically significant. UV/Vis detection failed to examine the inhibitory effect of acylated cyanidins isolated from black carrot (Daucus carota ssp. Sativus var. Autrorubens Alef.). However, ITC measurements reveal a much stronger inhibitory effect compared to the cyanidin-3-glucoside. Our results support the hypothesis that anthocyanins are efficient α-amylase inhibitors and an additional acylation with a cinnamic acid boosts the observed effect. Therefore, an increased consumption of vegetables containing acylated anthocyanin derivatives might help to prevent hyperglycemia.