A targeted prenylation analysis by a combination of IT-MS and HR-MS: Identification of prenyl number, configuration, and position in different subclasses of (iso)flavonoids.

Prenylated (iso)flavonoids are potent bioactive compounds found in the Fabaceae family. Analysis and quantification of this type of phytochemicals is challenging due to their large structural diversity. In this study, the fragmentation of prenylated (iso)flavonoids was investigated using electrospray ionization ion trap mass spectrometry (ESI-IT-MSn) with fragmentation by collision induced dissociation (CID) in combination and Orbitrap-MS (ESI-FT-MS2) with fragmentation by higher energy C-trap dissociation (HCD). With this combination of IT-MSn and high resolution MS (FT-MSn), it was possible to determine the fragmentation pathways and characteristic spectral features of different subclasses of prenylated (iso)flavonoid standards, as well as characteristic fragmentations and neutral losses of different prenyl configurations. Based on our findings, a decision guideline was developed to (i) identify (iso)flavonoid backbones, (ii) annotate prenyl number, (iii) configuration, and (iv) position of unknown prenylated (iso)flavonoids, in complex plant extracts. In this guideline, structural characteristics were identified based on: (i) UV absorbance of the compound, (ii) mass-to-charge (m/z) ratio of the parent compound; (iii) ratio of relative abundances between neutral losses 42 and 56 u in MSn; (iv) retro-Diels-Alder (RDA) fragments, neutral losses 54 and 68 u, and the ratio [M+H-C4H8]+/[M+H]+. Using this guideline, 196 prenylated (iso)flavonoids were annotated in a Glycyrrhiza glabra root extract. In total, 75 skeletons were single prenylated, 104 were double prenylated, and for merely 17 skeletons prenyl number could not unambiguously be annotated. Our prenylation guideline allows rapid screening for identification of prenylated (iso)flavonoids, including prenyl number, configuration, and position, in complex plant extracts. This guideline supports research on these bioactive compounds in the areas of plant metabolomics and natural products.

Procyanidin dimers A1, A2, and B2 are absorbed without conjugation or methylation from the small intestine of rats.

Intervention studies with procyanidin (PC)-rich extracts and products such as cocoa and wine suggest protective effects of PC against cardiovascular diseases. However, there is no consensus on the absorption and metabolism of PC dimers. Interestingly, nothing is known about the absorption of A-type PC. In this study, the absorption and metabolism of purified PC dimers A1 [epicatechin-(2-O-7, 4-8)-catechin], A2 [epicatechin-(2-O-7, 4-8)-epicatechin], and B2 [epicatechin-(4-8)-epicatechin], A-type trimers, a mixture of A1, B2, and a tetrameric A-type, and monomeric epicatechin were compared by in situ perfusion of the small intestine of rats for 0-30 min. The rats had their bile duct, portal vein, and small intestine cannulated. Unmodified and methylated metabolites were distinguished from their conjugates by differential beta-glucuronidase treatment. A1 and A2 dimers were absorbed from the small intestine of rats and they were better absorbed than dimer B2. Absorption of the A-type dimers was only 5-10% of that of monomeric epicatechin. Dimers were not conjugated or methylated in contrast to epicatechin, which was partly methylated and 100% conjugated. A-type trimers were not absorbed. Furthermore, the presence of tetrameric PC enhanced the absorption of B2 but not that of A1. Epicatechin, methylated epicatechin, and their conjugates were not found as metabolites of the PC tested. In conclusion, dimers A1, A2, and B2 are slightly absorbed but are not conjugated or methylated, thus conserving their biological activity after absorption. Because PC contents of foods are relatively high, dimers may contribute to systemic effects of PC.

Modulation of the cellulose content of tuber cell walls by antisense expression of different potato (Solanum tuberosum L.) CesA clones.

Four potato cellulose synthase (CesA) homologs (StCesA1, 2, 3 and 4) were isolated by screening a cDNA library made from developing tubers. Based on sequence comparisons and the fact that all four potato cDNAs were isolated from this single cDNA-library, all four StCesA clones are likely to play a role in primary cell wall biosynthesis. Several constructs were generated to modulate cellulose levels in potato plants in which the granule-bound starch synthase promoter was used to target the modification to the tubers. The StCesA3 was used for up- and down-regulation of the cellulose levels by sense (SE-StCesA3) and antisense (AS-StCesA3) expression of the complete cDNA. Additionally, the class-specific regions (CSR) of all four potato cellulose synthase genes were used for specific down-regulation (antisense) of the corresponding CesA genes (csr1, 2, 3 and 4). None of the transformants showed an overt developmental phenotype. Sections of tubers were screened for altered cell wall structure by Fourier Transform Infrared microspectroscopy (FTIR) and exploratory Principal Component Analysis (PCA), and those plants discriminating from WT plants were analysed for cellulose content and monosaccharide composition. Several transgenic lines were obtained with mainly decreased levels of cellulose. These results show that the cellulose content in potato tubers can be reduced down to 40% of the WT level without affecting normal plant development, and that constructs based on the CSR alone are specific and sufficient to down-regulate cellulose biosynthesis.

Combined normal-phase and reversed-phase liquid chromatography/ESI-MS as a tool to determine the molecular diversity of A-type procyanidins in peanut skins.

Peanut skins, a byproduct of the peanut butter industry, are a rich source of proanthocyanidins, which might be used in food supplements. Data on the molecular diversity of proanthocyanidins in peanut skins is limited and conflicting with respect to the ratio of double- (A-type) versus single-linked (B-type) flavan-3-ol units. NP- and RP-HPLC-MS were used as tools to analyze the molecular diversity of proanthocyanidins in a 20% (v/v) methanol extract of peanut skins. NP-HPLC was used to prepurify monomeric to pentameric fractions, which were further separated and characterized by RP-HPLC-MS. With this method, 83 different proanthocyanidin molecular species were characterized and quantified. Furthermore, it was possible to determine that A-type procyanidin oligomers were predominant and represented 95.0% (w/w) of the extract. In addition, the position of the A-linkages in 16 trimers and 27 tetramers could be determined, which in this case appeared to occur at all possible positions. The majority of trimers and tetramers with one or more A-linkage always had an A-linkage at the terminal unit.

Promiscuous, non-catalytic, tandem carbohydrate-binding modules modulate the cell-wall structure and development of transgenic tobacco (Nicotiana tabacum) plants.

We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth.