Anthocyanidins decrease endothelin-1 production and increase endothelial nitric oxide synthase in human endothelial cells.

Epidemiological and intervention studies correlate anthocyanin-rich beverages and a low incidence of coronary heart diseases. Since endothelin-1 (ET-1) and nitric oxide (NO) produced by endothelial NO synthase (eNOS) are vascular tension regulators secreted by endothelial cells, we studied the influence of two anthocyanidins, namely cyanidin (CY) and delphinidin (DP), on the regulation of ET-1 and eNOS in cultured human umbilical vein endothelial cells (HUVECs). Aglycon anthocyanidin forms, such as CY and DP, may be present in vivo after the first deglycosylation step occurring in the jejunum and in the liver. DP showed a major action compared to CY inducing a significant dose-dependent inhibitory effect on both protein and mRNA levels of ET-1. CY and DP both increased the protein level of eNOS, but DP showed the major effect raising eNOS protein in a dose-dependent manner. To correlate the vasoprotective effect of CY and DP with their antioxidant activity, we analysed also the antioxidant effect of anthocyanidins both in vitro and in HUVECs. In particular, we examined the effect of anthocyanidins on endothelial heme oxygenase-1 (HO-1), an inducible stress protein. In all tests, DP showed a higher antioxidant activity than CY. Finally, the antiproliferative effect induced by DP was detected in HUVECs. DP and CY differ in the number and position of hydroxyl groups in their structure; therefore, the greater biological activity by DP, compared with CY, seems to be due to the presence of the three hydroxyl groups on the B ring in the molecular structure of DP.

Structure-activity relationship of resveratrol and its analogue, 4,4'-dihydroxy-trans-stilbene, toward the endothelin axis in human endothelial cells.

Resveratrol inhibits endothelin-1, a vascular tension regulator. We synthesized the resveratrol analogue 4,4'-dihydroxy-trans-stilbene with 2 hydroxyl groups in the 4 and 4' position to obtain a molecule more active than resveratrol (3,4',5-trihydroxy-trans-stilbene). The results demonstrate that 4,4'-dihydroxy-trans-stilbene led to a significant decrease in total endothelin-1 secretion and in endothelin-1 messenger RNA (mRNA) levels in human endothelial cells. In addition, resveratrol and its analogue decreased endothelin-converting enzyme-1 mRNA levels and further reduced the activity of the enzyme. 4,4'-dihydroxy-trans-stilbene was more active than resveratrol because the new molecule exerted greater activity at the level of endothelin synthesis and conversion, even at a lower concentration. Although 4,4'-dihydroxy-trans-stilbene and resveratrol inhibited formation of reactive oxygen species and lipid peroxidation, the treatment of cells with different oxidant agents did not modify the endothelin-1 release. This finding suggests that the inhibition of endothelin-1 secretion is independent of the antioxidant properties of the 2 compounds. On the basis of these results, the resveratrol analogue 4,4'-dihydroxy-trans-stilbene could be a promising chemopreventive agent against cardiovascular diseases.

Grape waste extract obtained by supercritical fluid extraction contains bioactive antioxidant molecules and induces antiproliferative effects in human colon adenocarcinoma cells.

Grape waste management is one of the main problems of winery industries, but, conversely, grape waste contains a high amount of polyphenols that might protect against human diseases related to oxidative stress, such as colorectal cancer. Therefore, the aim of this work was to investigate the antioxidant and antiproliferative activities of a grape waste extract obtained by supercritical fluid extraction. Because the beneficial effect of grape is related to its content of polyphenolic molecules, the extract was chemically characterized by high-performance liquid chromatography in order to assess its major bioactive components. The antioxidant activity of the grape extract was determined. The results showed that the grape extract presents a strong antiradical activity in the in vitro 2,2-diphenyl-1-picrylhydrazyl radical assay and protects against reactive oxygen species production in human colon adenocarcinoma cells (Caco-2). In contrast, the extract did not protect in the citronellal thermooxidation system and showed a weak protective action against lipid peroxidation in Caco-2 cells. The clonogenic assay and the cell cycle distribution analysis showed that the grape extract has a significant antiproliferative effect in a tumor cell line. These data indicate that grape extract is a promising product to be used as an anti-free radical agent and could exert a chemopreventive action.

Anthocyanins induce cell cycle perturbations and apoptosis in different human cell lines.

To investigate the mechanistic basis for the biological properties of anthocyanins, two aglycone anthocyanins [delphinidin (DY) and cyanidin (CY)] were used to examine their effects on cell cycle progression and on induction of apoptosis in human cancer cells (uterine carcinoma and colon adenocarcinoma cells) and in normal human fibroblasts. These compounds differ in the number and position of hydroxyl groups on the beta ring in the molecular structure. Cellular uptake of anthocyanins was confirmed by HPLC analysis and no metabolites were detected. The clonogenic assay showed that CY induces a dose-dependent growth inhibitory effect only in fibroblasts. This effect was confirmed by flow cytometric analysis, showing a significant reduction of cells in S phase. In contrast, DP inhibited cell growth in normal and tumour cell lines. This event is accompanied in fibroblasts by an accumulation of cells in the S phase suggesting a block in the transition from S to G2 phase. On the other hand, in tumour cell lines we observed a reduction of cells in G1 phase, paralleled by the appearance of a fraction of cells with a hypodiploid DNA content, thus demonstrating an apoptotic effect by DP. The occurrence of apoptosis induced by DP was confirmed by morphological and biochemical features, including nuclear condensation and fragmentation, annexin V staining, DNA laddering and poly(ADP-ribose) polymerase-1-proteolysis. Furthermore, the mitochondrial membrane potential of apoptotic cells after treatment with DP was significantly lost. The different effects exerted by DP as compared with CY suggest that the presence of the three hydroxyl groups on the beta ring in the molecular structure of DP may be important for its greater biological activity.