Ultra(high)-pressure liquid chromatography-electrospray ionization-time-of-flight-ion mobility-high definition mass spectrometry for the rapid identification and structural characterization of flavonoid glycosides from cauliflower waste.

In this paper, a strategy for the detection and structural elucidation of flavonoid glycosides from a complex matrix in a single chromatographic run using U(H)PLC-ESI-IMS-HDMS/MS(E) is presented. This system operates using alternative low and high energy voltages that is able to perform the task of conventional MS/MS in a data-independent way without re-injection of the sample, which saves analytical time. Also, ion mobility separation (IMS) was employed as an additional separation technique for compounds that are co-eluting after U(H)PLC separation. First, the fragmentation of flavonoid standards were analyzed and criteria was set for structural elucidation of flavonoids in a plant extract. Based on retention times, UV spectra, exact mass, and MS fragment characteristics, such as abundances of daughter ions and the presence of radical ions ([Y0-H](-)), a total 19 flavonoid glycosides, of which 8 non-acylated and 11 acylated, were detected and structurally characterized in a cauliflower waste extract. Kaempferol and quercetin were the main aglycones detected while sinapic and ferulic acid were the main phenolic acids. C-glycosides were also found although their structure could not be elucidated. The proposed method can be used as a rapid screening test for flavonoid identification and for routine analysis of plant extracts, such as these derived from cauliflower waste. The study also confirms that agroindustrial wastes, such as cauliflower leaves, could be seen as a valuable source of different bioactive phenolic compounds.

Angiotensin-converting enzyme inhibitory effects by plant phenolic compounds: a study of structure activity relationships.

In this study, 22 phenolic compounds were investigated to inhibit the angiotensin-converting enzyme (ACE). Tannic acid showed the highest activity (IC50 = 230 µM). The IC50 values obtained for phenolic acids and flavonoids ranged between 0.41 and 9.3 mM. QSAR analysis confirmed that the numbers of hydroxyl groups on the benzene ring play an important role for activity of phenolic compounds and that substitution of hydroxyl groups by methoxy groups decreased activity. Docking studies indicated that phenolic acids and flavonoids inhibit ACE via interaction with the zinc ion and this interaction is stabilized by other interactions with amino acids in the active site. Other compounds, such as resveratrol and pyrogallol, may inhibit ACE via interactions with amino acids at the active site, thereby blocking the catalytic activity of ACE. These structure-function relationships are useful for designing new ACE inhibitors and potential blood-pressure-lowering compounds based on phenolic compounds.

Anthocyanin determination in blueberry extracts from various cultivars and their antiproliferative and apoptotic properties in B16-F10 metastatic murine melanoma cells.

Blueberry consumption is associated with health benefits contributing to a reduced risk for cardiovascular disease, diabetes and cancer. The aim of this study was to determine the anthocyanin profile of blueberry extracts and to evaluate their effects on B16-F10 metastatic melanoma murine cells. Seven blueberry cultivars cultivated in Romania were used. The blueberry extracts were purified over an Amberlite XAD-7 resin and a Sephadex LH-20 column, in order to obtain the anthocyanin rich fractions (ARF). The antioxidant activity of the ARF of all cultivars was evaluated by ABTS, CUPRAC and ORAC assays. High performance liquid chromatography followed by electrospray ionization mass spectrometry (HPLC-ESI-MS) was used to identify and quantify individual anthocyanins. The anthocyanin content of tested cultivars ranged from 101.88 to 195.01 mg malvidin-3-glucoside/100g fresh weight. The anthocyanin rich-fraction obtained from cultivar Torro (ARF-T) was shown to have the highest anthocyanin content and antioxidant activity, and inhibited B16-F10 melanoma murine cells proliferation at concentrations higher than 500 µg/ml. In addition, ARF-T stimulated apoptosis and increased total LDH activity in metastatic B16-F10 melanoma murine cells. These results indicate that the anthocyanins from blueberry cultivar could be used as a chemopreventive or adjuvant treatment for metastasis control.

Evaluation of the anti-adhesive effect of milk fat globule membrane glycoproteins on Helicobacter pylori in the human NCI-N87 cell line and C57BL/6 mouse model.

BACKGROUND: The interest in non-antibiotic therapies for Helicobacter pylori infections in man has considerably grown because increasing numbers of antibiotic-resistant strains are being reported. Intervention at the stage of bacterial attachment to the gastric mucosa could be an approach to improve the control/eradication rate of this infection. MATERIALS AND METHODS: Fractions of purified milk fat globule membrane glycoproteins were tested in vitro for their cytotoxic and direct antibacterial effect. The anti-adhesive effect on H. pylori was determined first in a cell model using the mucus-producing gastric epithelial cell line NCI-N87 and next in the C57BL/6 mouse model after dosing at 400 mg/kg protein once or twice daily from day -2 to day 4 post-infection. Bacterial loads were determined by using quantitative real-time PCR and the standard plate count method. RESULTS: The milk fat globule membrane fractions did not show in vitro cytotoxicity, and a marginal antibacterial effect was demonstrated for defatted milk fat globule membrane at 256 µg/mL. In the anti-adhesion assay, the results varied from 56.0 ± 5.3% inhibition for 0.3% crude milk fat globule membrane to 79.3 ± 3.5% for defatted milk fat globule membrane. Quite surprisingly, in vivo administration of the same milk fat globule membrane fractions did not confirm the anti-adhesive effects and even caused an increase in bacterial load in the stomach. CONCLUSIONS: The promising anti-adhesion in vitro results could not be confirmed in the mouse model, even after the highest attainable exposure. It is concluded that raw or defatted milk fat globule membrane fractions do not have any prophylactic or therapeutic potential against Helicobacter infection.

Metabolism of ferulic acid during growth of Lactobacillus plantarum and Lactobacillus collinoides.

BACKGROUND: Food-isolated lactic acid bacteria can transform ferulic acid (FA) into several products. Since quantification of these metabolites during the different bacterial growth phases is lacking, the aim of this study was to identify and quantify conversion products of FA and to follow the kinetics of FA metabolism during growth of Lactobacillus plantarum and Lactobacillus collinoides. RESULTS: Lactobacillus plantarum and Lactobacillus collinoides were incubated in MRS broth, to which different amounts of FA were added (final concentrations of 0, 0.5, 1.5 and 3 mmol L⁻¹), at 30 °C until the late stationary phase. Lactobacillus plantarum metabolised FA into 4-vinylguaiacol (4-VG) and hydroferulic acid (HFA). Conversion to 4-VG started simultaneously with the degradation of FA, while formation of HFA started in the mid-exponential phase. Lactobacillus collinoides only formed 4-VG, mainly in the stationary phase. No significant effect of the different amounts of FA was seen on the growth and fermentation characteristics of both bacteria. CONCLUSION: The results demonstrate that both bacteria are able to convert FA. However, start of conversion differs between the two strains. The different amounts of FA had no influence on the growth and fermentation characteristics of both bacteria.

The chain length of lignan macromolecule from flaxseed hulls is determined by the incorporation of coumaric acid glucosides and ferulic acid glucosides.

Lignan macromolecule from flaxseed hulls is composed of secoisolariciresinol diglucoside (SDG) and herbacetin diglucoside (HDG) moieties ester-linked by 3-hydroxy-3-methylglutaric acid (HMGA), and of p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) moieties ester-linked directly to SDG. The linker molecule HMGA was found to account for 11% (w/w) of the lignan macromolecule. Based on the extinction coefficients and RP-HPLC data, it was determined that SDG contributes for 62.0% (w/w) to the lignan macromolecule, while CouAG, FeAG, and HDG contribute for 12.2, 9.0, and 5.7% (w/w), respectively. Analysis of fractions of lignan macromolecule showed that the higher the molecular mass, the higher the proportion of SDG was. An inverse relation between the molecular mass and the proportion (%) CouAG+FeAG was found. Together with the structural information of oligomers of lignan macromolecule obtained after partial saponification, it is hypothesized that the amount of CouAG+FeAG present during biosynthesis determines the chain length of lignan macromolecule. Furthermore, the chain length was estimated from a model describing lignan macromolecule based on structural and compositional data. The average chain length of the lignan macromolceule was calculated to be three SDG moieties with CouAG or FeAG at each of the terminal positions, with a variation between one and seven SDG moieties.

Comparison of atmospheric pressure chemical ionization and electrospray ionization mass spectrometry for the detection of lignans from sesame seeds.

In sesame seeds, high concentrations of lignans are present. When these lignans are fermented in the human colon, a range of structurally different lignans is formed. A good liquid chromatography/mass spectrometry (LC/MS) protocol for the analysis of lignans in complex mixtures is lacking. In order to develop such a protocol, electrospray ionization (ESI)-MS and atmospheric pressure chemical ionization (APCI)-MS, both in the positive and negative ionization mode, were compared. An extract from defatted sesame meal was analyzed by APCI-MS and ESI-MS, before and after deglucosylation. APCI-MS was found to be a more generic method than ESI-MS because lignans, especially sesamolin, sesamin and pinoresinol, were better detected by APCI-MS than by ESI-MS. Positive and negative ionization modes had to be combined in order to detect all lignans in a bacterial culture grown on aglyconic, acid-treated lignans from sesame oil and defatted sesame meal. Lignans with methylenedioxy-bridged furanofuran structures mostly lack phenolic hydroxyl groups and were, therefore, optimally detected in positive ionization mode. Dibenzylbutadiene lignans, which were formed during fermentation, carry hydroxyl groups and were better detected in negative ionization mode.

Metabolism of the lignan macromolecule into enterolignans in the gastrointestinal lumen as determined in the simulator of the human intestinal microbial ecosystem.

Estrogenic plant compounds from the human diet such as the lignan secoisolariciresinol diglucoside (SDG, 1) can exert biological activity in the human body upon ingestion and bioactivation to enterodiol (END, 5) and enterolactone (ENL, 6). Bioavailability of lignans is influenced by the food matrix and gut microbial action, of which the latter is subject to a large interindividual variation. In this study, the fate of the lignan precursor SDG, present in the lignan macromolecule of flax seed ( Linum usitatissimum), was determined during an artificial stomach and small intestinal digestion and during metabolism by two different enterolignan phenotypes in a TWINSHIME environment (TWIN Simulator of the Human Intestinal Microbial Ecosystem). The lignan macromolecule acted as a delivery system of SDG in the large intestine. SDG was only hydrolyzed into secoisolariciresinol (SECO, 2) through microbial action in the ascending colon, after which it was bioactivated into enterolignans from the transverse colon onward. Single demethylation was a first step in the bioactivation, followed by dehydroxylation. Enterolignan phenotypes remained stable throughout the experimental period. The establishment of END and ENL production equilibria reflected the subdominance of ENL-producing bacteria in the gastrointestinal tract.

Hydroxycinnamic acids are ester-linked directly to glucosyl moieties within the lignan macromolecule from flaxseed hulls.

In flaxseed hulls, lignans are present in an oligomeric structure. Secoisolariciresinol diglucoside (SDG), ester-linked to hydroxy-methyl-glutaric acid (HMGA), forms the backbone of this lignan macromolecule. The hydroxycinnamic acids p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) are also part of the lignan macromolecule. However, their position and type of linkage are still unknown. The aim of this study was to investigate how CouAG and FeAG are linked within the lignan macromolecule from flaxseed hulls. Fragments of the lignan macromolecule were obtained by partial saponification. After isolation of the fragments by preparative RP-HPLC, several key structures were identified by MS and NMR. Within the lignan macromolecule, CouAG is attached to the C-6 position of a glucosyl moiety of SDG. FeA is linked to the C-2 position of a glucosyl moiety of SDG. FeAG is ester-linked within the lignan macromolecule with its carboxyl group, but it remains unclear whether FeAG links to the C-2 or C-6 position of SDG. Attachment of HMGA to the glucosyl moiety of CouAG or FeAG was not observed. The results clearly show that within the lignan macromolecule, the hydroxycinnamic acids are linked directly via an ester bond to the glucosyl moiety of SDG.

The flavonoid herbacetin diglucoside as a constituent of the lignan macromolecule from flaxseed hulls.

Lignans in flaxseed are known to be part of a macromolecule in which they are connected through the linker-molecule hydroxy-methyl-glutaric acid (HMGA). In this study, the lignan macromolecule was extracted from flaxseed hulls and degraded to its monomeric constituents by complete saponification. Besides secoisolariciresinol diglucoside (SDG), the phenolic compounds p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) were isolated, which was expected based on indications from the literature. Also the flavonoid herbacetin diglucoside (HDG) was found. The presence of HDG was confirmed by NMR following preparative RP-HPLC purification. Also the presence of the three other constituents (CouAG, FeAG and SDG) was confirmed by NMR. To prove that HDG is a substructure of the lignan macromolecule, the macromolecule was fragmented by partial saponification. A fragment consisting of HDG and HMGA was indicated. This fragment was isolated by preparative RP-HPLC and its identity was confirmed by NMR. It is concluded that the flavonoid HDG is a substructure of the lignan macromolecule from flaxseed hulls and that it is incorporated in the macromolecule via the same linker-molecule as SDG.

Transgenic overexpression of expansin influences particle size distribution and improves viscosity of tomato juice and paste.

Suppression of the expression of a ripening-related expansin gene, LeExp1, in tomato enhanced fruit firmness and overexpression of LeExp1 resulted in increased fruit softening. Because of the incompletely understood relationship between fresh fruit texture and the consistency of processed products, we examined the effects of LeExp1 overexpression on the processing characteristics of tomato fruit. As determined by Bostwick consistency and by controlled strain rheometry, juices and pastes prepared from transgenic tomatoes with suppressed LeExp1 expression had a higher viscosity than preparations from control fruits. However, the viscosity of juice and paste prepared from fruit overexpressing LeExp1 was significantly greater than products from controls or lines with reduced LeExp1. Bostwick consistency increased by 9% (juice) and 6% (paste) in lines with suppressed LeExp1 expression but increased by 27.5% (juice) and 19.5% (paste) in lines overexpressing LeExp1, relative to controls. Determined by laser diffraction, the particles in juice and paste prepared from transgenic fruits with reduced LeExp1 expression were smaller, and preparations from fruits overexpressing LeExp1 had a size distribution indicating more large particles. Analysis of cell wall polysaccharides size indicated that LeExp1 overexpression enhanced depolymerization of water soluble pectins as well as tightly bound matrix glycans. LeExp1 overexpression may allow increased cell wall hydration, resulting in expanded particle size and increased viscosity of products. Because either LeExp1 suppression or overexpression leads to improved consistency, the interactions that contribute to optimal product rheological properties are complex.

Biphasic modulation of cell proliferation by quercetin at concentrations physiologically relevant in humans.

Optimal in vitro conditions regarding quercetin solubility and stability were defined. Using these conditions, the effect of quercetin on proliferation of the colon carcinoma cell lines HCT-116 and HT29 and the mammary adenocarcinoma cell line MCF-7 was investigated. For the colon carcinoma cell lines, at relatively high concentrations, a significant decrease in cell proliferation was observed, providing a basis for claims on the anti-carcinogenic activity of quercetin. However, at lower concentrations, a subtle but significant stimulation of cell proliferation was observed for all cell lines tested. These results point at a dualistic influence of quercetin on cell proliferation that may affect present views on its supposed beneficial anti-proliferative effect.