Uptake and bioavailability of anthocyanins and phenolic acids from grape/blueberry juice and smoothie in vitro and in vivo.

The goal of eating five servings of fruits and vegetables a day has not yet been achieved. The intake of polyphenols such as anthocyanins (ACN) could be improved by consuming smoothies and juices that are increasingly popular, especially in children; however, bioavailability data concerning food matrix effects are scarce. Thus, we conducted a randomised, cross-over, bioavailability study (n 10) to determine the bioavailability of ACN and their metabolites from an ACN-rich grape/blueberry juice (841 mg ACN/litre) and smoothie (983 mg ACN/litre) in vivo, and the uptake of a corresponding grape/blueberry extract in vitro. After the intake of beverage (0·33 litres), plasma and fractionated urine samples were collected and analysed by ultra-performance liquid chromatography coupled to MS. The most abundant ACN found in plasma and urine were malvidin and peonidin as native ACN and as glucuronidated metabolites as well as 3,4-dihydroxybenzoic acid (3,4-DHB); minor ACN (delphinidin, cyanidin and petunidin) were only detected as native glycosides. Plasma pharmacokinetics and recoveries of urinary metabolites of ACN were not different for juice or smoothie intake; however, the phenolic acid 3,4-DHB was significantly better bioavailable from juice in comparison to smoothie. In vitro data with absorptive intestinal cells indicated that despite their weak chemical stability, ACN and 3,4-DHB could be detected at the basal side in their native forms. Whether smoothies as well as juices should be recommended to increase the intake of potentially health-promoting ACN and other polyphenols requires the consideration of other ingredients such as their relatively high sugar content.

A comprehensive high-resolution mass spectrometry approach for characterization of metabolites by combination of ambient ionization, chromatography and imaging methods.

RATIONALE: An ideal method for bioanalytical applications would deliver spatially resolved quantitative information in real time and without sample preparation. In reality these requirements can typically not be met by a single analytical technique. Therefore, we combine different mass spectrometry approaches: chromatographic separation, ambient ionization and imaging techniques, in order to obtain comprehensive information about metabolites in complex biological samples. METHODS: Samples were analyzed by laser desorption followed by electrospray ionization (LD-ESI) as an ambient ionization technique, by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging for spatial distribution analysis and by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) for quantitation and validation of compound identification. All MS data were acquired with high mass resolution and accurate mass (using orbital trapping and ion cyclotron resonance mass spectrometers). Grape berries were analyzed and evaluated in detail, whereas wheat seeds and mouse brain tissue were analyzed in proof-of-concept experiments. RESULTS: In situ measurements by LD-ESI without any sample preparation allowed for fast screening of plant metabolites on the grape surface. MALDI imaging of grape cross sections at 20 µm pixel size revealed the detailed distribution of metabolites which were in accordance with their biological function. HPLC/ESI-MS was used to quantify 13 anthocyanin species as well as to separate and identify isomeric compounds. A total of 41 metabolites (amino acids, carbohydrates, anthocyanins) were identified with all three approaches. Mass accuracy for all MS measurements was better than 2 ppm (root mean square error). CONCLUSIONS: The combined approach provides fast screening capabilities, spatial distribution information and the possibility to quantify metabolites. Accurate mass measurements proved to be critical in order to reliably combine data from different MS techniques. Initial results on the mycotoxin deoxynivalenol (DON) in wheat seed and phospholipids in mouse brain as a model for mammalian tissue indicate a broad applicability of the presented workflow.

Inhibition of low-grade inflammation by anthocyanins from grape extract in an in vitro epithelial-endothelial co-culture model.

BACKGROUND: Anthocyanins (ACNs) are the most prevalent flavonoids in berries and their health promoting effects on vascular functions are still discussed. The aim of the present study was to identify the anti-inflammatory effect of ACNs on activated human umbilical vein endothelial cells (HUVECs) after their transport across an epithelial monolayer. STUDY DESIGN: We established a transwell epithelial-endothelial co-culture system with Caco-2/HT29-B6 cells mimicking the intestinal layer and HUVECs as endothelial cells mimicking the vascular layer. Caco-2 were seeded alone (100%) or together with HT29-B6 cells (10 and 20%) on transwell inserts in order to simulate different metabolization sides of the gut. ACNs as well as malvidin-3-glucoside (M3G) were applied to the luminal compartment of the transwell-system. Transport and degradation rates were determined by high performance liquid chromatography with ultraviolet detection (HPLC-UV) or by ultra-PLC coupled to mass spectrometry (UPLC-MS). After 4 hours incubation time, co-cultured HUVECs were used immediately (short-term incubation) or after 20 hours (long-term incubation). Thereafter, HUVECs were stimulated for 3 hours with 1 ng mL(-1) TNF-α to mimic a low-grade or 10 ng mL(-1) to mimic a high-grade inflammation. Afterwards, (1.) leukocyte adhesion, (2.) expression of cell adhesion molecules (ICAM-1, VCAM-1 and E-selectin) and (3.) cytokine expression and secretion (IL-6 and IL-8) were determined using flow cytometry and real-time PCR. RESULTS: Degradation and incubation studies revealed that ACNs were differently degraded depending on the ACN structure and the seeding densities. Incubation of ACNs and M3G to Caco-2 cells (100%) led to a fast decrease, which was not observed when HT29-B6 cells were co-cultured (10 and 20%). Concomitantly, anti-inflammatory effects were only observed using 100% Caco-2 cells, whereas mixtures of Caco-2 and HT29-B6 cells failed to induce an effect. ACN extract and M3G significantly attenuated TNF-α-stimulated low-grade leukocyte adhesion, expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 and cytokine expression and secretion (IL-8 and IL-6) as well as NF-κB mRNA expression. No effects were observed with high TNF-α (10 ng mL(-1)) or after short-term incubation (4 hours). CONCLUSIONS: ACNs in physiological concentrations reached the serosal compartment and reduced inflammation-related parameters, which were related to the initial steps during the pathogenesis of atherosclerosis.

Anthocyanin-rich juice does not affect gut-associated immunity in Fischer rats.

SCOPE: Most studies on immunomodulatory effects of anthocyanins are concentrated on their anti-inflammatory potential. In vitro studies suggest that anthocyanins possess anti-inflammatory potential, but results from in vivo studies are rare and inconclusive. Sparse information is available about the immune tissues that are affected by anthocyanins. As systemic bioavailability of anthocyanins is rather low, predominantly luminal anthocyanins could influence the gut-associated lymphoid tissue (GALT). Therefore, the present study investigated the immunomodulatory effects of an anthocyanin-rich grape-bilberry juice (ARJ) on the systemic immune system, GALT, and mesenteric adipose tissue (MAT). METHODS AND RESULTS: Fischer rats (n = 24/group) received ARJ or anthocyanin-depleted grape-bilberry juice (control) for 10 wk. Lymphocytes were isolated from blood, spleen, Peyer's Patches, and mesenteric lymph nodes. Anthocyanin intake was 15 mg/day and concentrations were determined in plasma and intestinal tract. Number of T and natural killer cells, natural killer cell activity, cytokine secretion from lymphocytes (IL-10, IFN-γ, and TNF-α) and MAT (IL-6, IL-10, and MCP-1), inflammation markers in serum (sICAM, IFN-γ, and MCP-1), and activation status of NF-κB were not influenced by ARJ. CONCLUSION: This in vivo study suggests that anthocyanins at physiological doses affect neither the systemic immune system, nor GALT, or MAT in healthy, unchallenged rats.