The effect of calcium on flavanol production in cell suspension cultures of Polygonum hydropiper

Cultured Polygonum hydropiper cells maintained in Murashige and Skoog (MS) medium supplemented with 10^–6 m 2,4-D, 10^–6 m kinetin, 0.1% casamino acids and 3% sucrose were transferred to medium containing a higher concentration of calcium chloride (15 mm). The content of flavanols in the cells on the 6th day was approximately twice that of the control culture (31.9–60.7 mg/g dry wt). However, the contents of other secondary metabolites such as chlorogenic acid and gallic acid were not changed. The levels of flavanols in the culture medium remained unchanged throughout the 21-day culture period. Of the the inorganic components supplemented to the culture medium , only elevated levels of calcium chloride induced an increase in flavanol contents of the cells. The results indicated that the elevated concentration of calcium in the culture medium played an important role in activating the accumulation of flavanols. Received: 4 June 1998 / Revision received: 30 October 1998 / Accepted: 29 November 1998     

Geranylated flavanones from the secretion on the surface of the immature fruits of Paulownia tomentosa

Chemical investigation of the methanol extract of the viscous secretion on the surface of immature fruits of Paulownia tomentosa furnished nine geranylated flavanones, 6-geranyl-5,7-dihydroxy-3',4'-dimethoxyflavanone (1), 6-geranyl-3',5,7-trihydroxy-4'-methoxyflavanone (2), 6-geranyl-4',5,7-trihydroxy-3',5'-dimethoxyflavanone (3), 6-geranyl-4',5,5',7-tetrahydroxy-3'-methoxyflavanone (4), 6-geranyl-3,3',5,7-tetrahydroxy-4'-methoxyflavanone (5), 4',5,5',7-tetrahydroxy-6-[6-hydroxy-3,7-dimethyl-2(E),7-octadienyl]-3'-methoxyflavanone (6), 3,3',4',5,7-pentahydroxy-6-[6-hydroxy-3,7-dimethyl-2(E),7-octadienyl]flavanone (7), 3,3',4',5,7-pentahydroxy-6-[7-hydroxy-3,7-dimethyl-2(E)-octenyl]flavanone (8), and 3,4',5,5',7-pentahydroxy-3'-methoxy-6-(3-methyl-2-butenyl)flavanone (9), along with six known geranylated flavanones. Among these, compounds 4, 6-9 and the known 6-geranyl-3',4',5,7-tetraahydroxyflavanone (diplacone), 6-geranyl-3,3',4',5,7-pentahydroxyflavanone (diplacol) and 3',4',5,7-pentahydroxy-6-[7-hydroxy-3,7-dimethyl-2(E)-octenyl]flavanone showed potent radical scavenging effects towards DPPH radicals.     

(-)-Epicatechin elevates nitric oxide in endothelial cells via inhibition of NADPH oxidase

Dietary (-)-epicatechin is known to improve bioactivity of (*)NO in arterial endothelium of humans, but the mode of action is unclear. We used the fluorophore 4,5-diaminofluorescein diacetate to visualize the (*)NO level in living human umbilical vein endothelial cells (HUVEC). Untreated cells showed only a weak signal, whereas pretreatment with (-)-epicatechin (10 microM) or apocynin (100 microM) elevated the (*)NO level. The effects were more pronounced when the cells were treated with angiotensin II with or without preloading of the cells with (*)NO via PAPA-NONOate. While (-)-epicatechin scavenged O2(*-), its O-methylated metabolites prevented O2(*-) generation through inhibition of endothelial NADPH oxidase activity, even more strongly than apocynin. From the effect of 3,5-dinitrocatechol, an inhibitor of catechol-O-methyltransferase (COMT), on HUVEC it is concluded that (-)-epicatechin serves as 'prodrug' for conversion to apocynin-like NADPH oxidase inhibitors. These data indicate an (*)NO-preserving effect of (-)-epicatechin via suppression of O2(*-)-mediated loss of (*)NO.     

Effect of cocoa/chocolate ingestion on brachial artery flow-mediated dilation and its relevance to cardiovascular health and disease in humans

Prospective studies indicate that high intake of dietary flavanols, such as those contained in cocoa/chocolate, are associated with reduced rates of cardiovascular-related morbidity and mortality in humans. Numerous mechanisms may underlie these associations such as favorable effects of flavanols on blood pressure, platelet aggregation, thrombosis, inflammation, and the vascular endothelium. The brachial artery flow-mediated dilation (FMD) technique has emerged as a robust method to quantify endothelial function in humans. Collectively, the preponderance of evidence indicates that FMD is a powerful surrogate measure for firm cardiovascular endpoints, such as cardiovascular-related mortality, in humans. Thus, literally thousands of studies have utilized this technique to document group differences in FMD, as well as to assess the effects of various interventions on FMD. In regards to the latter, numerous studies indicate that both acute and chronic ingestion of cocoa/chocolate increases FMD in humans. Increases in FMD after cocoa/chocolate ingestion appear to be dose-dependent such that greater increases in FMD are observed after ingestion of larger quantities. The mechanisms underlying these responses are likely diverse, however most data suggest an effect of increased nitric oxide bioavailability. Thus, positive vascular effects of cocoa/chocolate on the endothelium may underlie (i.e., be linked mechanistically to) reductions in cardiovascular risk in humans.     

Monomeric and oligomeric flavanols are agonists of membrane androgen receptors

The present work reports a new mode of action of the naturally occurring flavanols catechin and epicatechin and their dimers B2 and B5, in the breast cancer T47D cell line, namely, their interaction with membrane androgen receptors. We show that monomeric and dimeric flavanols are complete (B2) or partial displacers of radiolabeled testosterone bound on T47D membranes, with affinities ranging from 1.7 (B5) to 82.2 nM (B2). In addition, they trigger the phosphorylation of the same signaling molecules (FAK, PI3K) as testosterone-BSA, minutes after binding to membrane receptors, leading to actin cytoskeleton polymerization and redistribution, with formation of filopodia and lamellipodia. The PI3K inhibitor wortmannin reverts the effect of polyphenols and testosterone-BSA, providing additional evidence about activation of a similar signaling cascade. Incubation of T47D cells for more than 2 h with polyphenols or testosterone-BSA induces apoptosis, which follows the same time-dependent pattern. We conclude that flavanols (monomers or dimers) are agonists of membrane androgen receptors and could be used as testosterone-protein conjugates for the management of tumors, in which, application of testosterone-BSA induces regression, providing additional data about the mechanism of their antiproliferative action.     

Epicatechin protects endothelial cells against oxidized LDL and maintains NO synthase

Intake of flavanol-rich food or beverages was previously shown to ameliorate endothelial function and to enhance bioactivity of nitric oxide with individuals at risk for cardiovascular disease. Here, we examined whether the major dietary flavanol, (-)-epicatechin, counteracts the action of oxidized LDL on endothelial cells, an action considered pivotal for endothelial dysfunction in the pathogenesis of atherosclerosis. Oxidation by myeloperoxidase plus nitrite rendered human LDL cytotoxic towards endothelial cells, more so than oxidation by Cu2+. Oxidized LDL also caused a marked loss of endothelial NO synthase protein which did not occur in the presence of a proteasome inhibitor, lactacystin. Both actions of oxidized LDL, which were not evoked by native LDL, were effectively counteracted by (-)-epicatechin. We conclude that dietary flavanols contribute to protection of the integrity of endothelial cells not only by scavenging free radicals but also by maintaining endothelial NO synthase.     

The Antioxidant Activity of Regularly Consumed Fruit and Vegetables Reflects their Phenolic and Vitamin C Composition

Recent studies are emphasising the importance and putative modes of action of specific flavonoids as bioactive components of the diet in in vivo and in vitro models. Thus, it is important to have a clear idea of the major phenolic families of which fruit and vegetables are comprised and the levels contained therein. Regularly consumed fruit and vegetables of mixed varieties available on the UK market were analysed for the composition of the major individual phenolic components. The total phenolic content (applying the Folin assay) and the vitamin C levels were also determined. The antioxidant capacities of aqueous/methanolic extracts were comparatively assessed using the TEAC (Trolox Equivalent Antioxidant Capacity), the FRAP (Ferric Reducing Ability of Plasma) and ORAC (Oxygen Radical Absorbance Capacity) assays, which comprise contributions from polyphenols, simple phenols and the ascorbate component. The results were calculated in terms of 100 &#117 g fresh weight (FW) uncooked portion sizes. Fruit and vegetables rich in anthocyanins (e.g. strawberry, raspberry and red plum) demonstrated the highest antioxidant activities, followed by those rich in flavanones (e.g. orange and grapefruit) and flavonols (e.g. onion, leek, spinach and green cabbage), while the hydroxycinnamate-rich fruit (e.g. apple, tomato, pear and peach) consistently elicited the lower antioxidant activities. The TEAC, FRAP and ORAC values for each extract were relatively similar and well-correlated with the total phenolic and vitamin C contents. The antioxidant activities (TEAC) in terms of 100 &#117 g FW uncooked portion size were in the order: strawberry &#100 raspberry=red plum &#100 red cabbage>>>grapefruit= orange>spinach>broccoli>green grape &#59 onion> green cabbage>pea>apple> cauliflower &#59 pear> tomato &#59 peach=leek>banana &#59 lettuce.