Jess D. Reed

Anthocyanin- and hydrolyzable tannin-rich pomegranate fruit extract modulates MAPK and NF-κB pathways and inhibits skin tumorigenesis in CD-1 mice

Chemoprevention has come of age as an effective cancer control modality; however, the search for novel agent(s) for the armamentarium of cancer chemoprevention continues. We argue that agents capable of intervening at more than one critical pathway in the carcinogenesis process will have greater advantage over other single‐target agents. Pomegranate fruit extract (PFE) derived from the tree Punica granatum possesses strong antioxidant and antiinflammatory properties. Pomegranate fruit was extracted with acetone and analyzed based on matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and found to contain anthocyanins, ellagitannins and hydrolyzable tannins. We evaluated whether PFE possesses antitumor‐promoting effects. We first determined the effect of topical application of PFE to CD‐1 mice against 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced conventional markers and other novel markers of skin tumor promotion. We found that topical application of PFE (2 mg/mouse) 30 min prior to TPA (3.2 nmole/mouse) application on mouse skin afforded significant inhibition, in a time‐dependent manner, against TPA‐mediated increase in skin edema and hyperplasia, epidermal ornithine decarboxylase (ODC) activity and protein expression of ODC and cyclooxygenase‐2. We also found that topical application of PFE resulted in inhibition of TPA‐induced phosphorylation of ERK1/2, p38 and JNK1/2, as well as activation of NF‐κB and IKKα and phosphorylation and degradation of IκBα. We next assessed the effect of skin application of PFE on TPA‐induced skin tumor promotion in 7,12‐dimethylbenz(a)anthracene‐initiated CD‐1 mouse. The animals pretreated with PFE showed substantially reduced tumor incidence and lower tumor body burden when assessed as total number of tumors per group, percent of mice with tumors and number of tumors per animal as compared to animals that did not receive PFE. In TPA‐treated group, 100% of the mice developed tumors at 16 weeks on test, whereas at this time in PFE‐treated group, only 30% mice exhibited tumors. Skin application of PFE prior to TPA application also resulted in a significant delay in latency period from 9 to 14 weeks and afforded protection when tumor data were considered in terms of tumor incidence and tumor multiplicity. The results of our study provide clear evidence that PFE possesses antiskin‐tumor‐promoting effects in CD‐1 mouse. Because PFE is capable of inhibiting conventional as well as novel biomarkers of TPA‐induced tumor promotion, it may possess chemopreventive activity in a wide range of tumor models. Thus, an in‐depth study to define active agent(s) in PFE capable of affording antitumor‐promoting effect is warranted.

Bioavailability, bioactivity and impact on health of dietary flavonoids and related compounds: an update.

There is substantial interest in the role of plant secondary metabolites as protective dietary agents. In particular, the involvement of flavonoids and related compounds has become a major topic in human nutrition research. Evidence from epidemiological and human intervention studies is emerging regarding the protective effects of various (poly)phenol-rich foods against several chronic diseases, including neurodegeneration, cancer and cardiovascular diseases. In recent years, the use of HPLC–MS for the analysis of flavonoids and related compounds in foods and biological samples has significantly enhanced our understanding of (poly)phenol bioavailability. These advancements have also led to improvements in the available food composition and metabolomic databases, and consequently in the development of biomarkers of (poly)phenol intake to use in epidemiological studies. Efforts to create adequate standardised materials and well-matched controls to use in randomised controlled trials have also improved the quality of the available data. In vitro investigations using physiologically achievable concentrations of (poly)phenol metabolites and catabolites with appropriate model test systems have provided new and interesting insights on potential mechanisms of actions. This article will summarise recent findings on the bioavailability and biological activity of (poly)phenols, focusing on the epidemiological and clinical evidence of beneficial effects of flavonoids and related compounds on urinary tract infections, cognitive function and age-related cognitive decline, cancer and cardiovascular disease.

Multi-laboratory validation of a standard method for quantifying proanthocyanidins in cranberry powders.

BACKGROUND The objective of this study was to validate an improved 4-dimethylaminocinnamaldehyde (DMAC) colorimetric method using a commercially available standard (procyanidin A2), for the standard method for quantification of proanthocyanidins (PACs) in cranberry powders, in order to establish dosage guidelines for the uropathogenic bacterial anti-adhesion effect of cranberry. RESULTS Commercially available cranberry samples were obtained (five from U.S. sources and six from European sources) for PAC quantification in five different analytical laboratories. Each laboratory extracted and analyzed the samples using the improved DMAC method. Within-laboratory variation (mean +/- SD) was 4.1 +/- 1.7% RSD (range, 2.3-6.1% RSD) and the between laboratory variability was 16.9 +/- 8.5% RSD (range, 8-32% RSD). For comparative purposes, the cranberry samples were alternatively quantified using weights of extracted PACs (gravimetric). The correlation coefficient between the two methods was 0.989. CONCLUSION This improved DMAC method provides a simple, robust and relatively specific spectrophotometric assay for total PACs in cranberry samples using commercially available procyanidin A2 dimer as a standard. DMAC is most useful within a given type of food such as cranberries, but may not be appropriate for comparing concentrations across different food types, particularly in those cases where large differences exist among the relative amounts of each oligomer and polymer.

Cranberry Flavonoids, Atherosclerosis and Cardiovascular Health

Abstract Atherosclerosis is the deposition of plaques containing cholesterol and lipids in arterial walls. Atherosclerosis causes cardiovascular disease that lead to heart attacks and stroke. Mortality from these diseases is the leading cause of death in the U.S. Atherogenisis starts with the uptake of oxidized LDL by endothelial macrophages, the accumulation of foam cells in the intima of the artery and the formation of fatty streaks. Research indicates that consumption of flavonoids in foods and beverages may decrease the risk of atherosclerosis. In vitro and in vivo experiments with flavonoids demonstrate that flavonoids are dietary antioxidants and inhibit LDL oxidation, inhibit platelet aggregation and adhesion, inhibit enzymes involved in lipid and lipoprotein metabolism that affect the immune response to oxidized LDL and their uptake by endothelial macrophages, may induce endothelium–dependent vassorelaxation, and may increase reverse cholesterol transport and decrease total and LDL cholesterol. Cranberries contain both hydroxycinnamic acids and flavonoids. The cranberry flavonoids belong to three groups: anthocyanins, flavonols, and proanthocyanidins. This article reviews the literature on the effects of flavonoids on atherosclerosis with an emphasis on the potential effects of the flavonols and proanthocyanidins in cranberries.

Contrast-Enhanced Ultrasound Imaging of the Vasa Vasorum: From Early Atherosclerosis to the Identification of Unstable Plaques

Proliferation of the adventitial vasa vasorum (VV) is inherently linked with early atherosclerotic plaque development and vulnerability. Recently, direct visualization of arterial VV and intraplaque neovascularization has emerged as a new surrogate marker for the early detection of atherosclerotic disease. This clinical review focuses on contrast-enhanced ultrasound (CEUS) as a noninvasive application for identifying and quantifying carotid and coronary artery VV and intraplaque neovascularization. These novel approaches could potentially impact the clinicians ability to identify individuals with premature cardiovascular disease who are at high risk. Once clinically validated, the uses of CEUS may provide a method to noninvasively monitor therapeutic interventions. In the future, the therapeutic use of CEUS may include ultrasound-directed, site-specific therapies using microbubbles as vehicles for drug and gene delivery systems. The combined applications for diagnosis and therapy provide unique opportunities for clinicians to image and direct therapy for individuals with vulnerable lesions.

High Molecular Weight Persimmon (Diospyros kaki L.) Proanthocyanidin: A Highly Galloylated, A-Linked Tannin with an Unusual Flavonol Terminal Unit, Myricetin

MALDI-TOF MS suggested that the high molecular weight proanthocyanidin (condensed tannin) from persimmon (Diospyros kaki L.) pulp comprised a heteropolyflavanol series with flavan-3-O-galloylated extenders, flavan-3-ol and flavonol terminal units, and A-type interflavan linkages. Thiolysis-HPLC-ESI-MS with DAD, electrochemical, and ESI-MS detection confirmed a previously unreported terminal unit, the flavonol myricetin, in addition to the typical flavan-3-ols catechin and epigallocatechin gallate. The extender units were epicatechin, epigallocatechin, (epi)gallocatechin-3-O-gallate, and (epi)catechin-3-O-gallate. The crude tannin had a high prodelphinidin content (65%) and a high degree of 3-O-galloylation (72%). The material was fractionated on Toyopearl TSK-HW-50-F to yield fractions distinguished by degree of polymerization (DP). Thiolysis suggested that the persimmon tannin was composed of polymers ranging from 7 to 20 kDa (DP 19-47), but sizes estimated by GPC were 50-70% smaller. The crude material was chemically degraded with acid to yield products that were amenable to NMR and ESI-MS analysis, which were used to establish for the first time that persimmon tannin has a mixture of B-type and A-type linkages.

Characterisation of tannins and in vitro protein digestibility of several Lotus corniculatus varieties

Seven birdsfoot trefoil (BFT) varieties (Lotus corniculatus) grown in Sweden, were harvested at the 50% flowering stage and analysed for tannins by the radial diffusion and HCl–butanol methods. The flavan-3-ol composition of different BFT tannins was determined by HPLC. Tannins were isolated and examined for their molecular weight distributions by HPLC gel permeation chromatography (GPC) and MALDI-TOF mass spectrometry. Ruminal protein degradability was determined in vitro and related to tannin chemistry. Tannin concentrations of the BFT varieties were generally low and ranged between 0.3–1.0% (radial diffusion assay) and 0.2–1.7% (HCl–butanol assay) on a DM basis. The delphinidin:cyanidin ratios showed considerable variation ranging from 16:84 to 33:67 amongst the seven varieties. GPC analysis revealed small differences between the varieties with most of the variation occurring in the relative proportions of the higher molecular weight tannins. MALDI-TOF mass spectrometry of tannins from two varieties gave well-resolved spectra of tetramers, pentamers and hexamers. Oligomers up to the decamers were also detectable. Each of these oligomers had a subset of structures incorporating catechin/epicatechin (CE) and gallocatechin/epigallocatechin (GE) units. Some homopolymers containing CE units only (i.e. procyanidins), but none with GE units only (i.e. prodelphinidins), were detected. Most mixed CE/GE oligomers of all sizes contained one or two GE units. There were significant differences (P≤0.05) in vitro N-degradability between four varieties. The data suggest that degradability of the soluble proteins in birdsfoot trefoil were negatively correlated to tannin concentrations (R2=0.93) despite the fact that their overall concentrations were very low.

A Method for Isolating Condensed Tannins from Crude Plant Extracts with Trivalent Ytterbium

A method to precipitate condensed tannin from crude plant extracts using trivalent ytterbium has been developed. The new method requires less time and resources than the condensed isolation procedure using Sephadex LH-20 recommended by Hagerman (1991, Tannin Analysis, Miami University, Oxford, OH, USA). The absorbance of the preparations obtained by precipitation with trivalent ytterbium was similar to the preparations obtained with the original isolation procedure, when the acid butanol method (Porter et al 1986, Phytochemistry1 223–230) was used to measure condensed tannins. Condensed tannins were isolated from crude plant extracts of three plant species, Desmodium ovalifolium, Gliricidia sepium and Manihot esculenta, and the condensed tannin content of the lyophilised leaf tissue was determined. For each plant species, the amounts of the soluble, insoluble and fibre-bound condensed tannins were estimated using five different standards. These standards included two tannin preparations obtained either by (1) isolation with Sephadex LH-20, or (2) by the precipitation with trivalent ytterbium and three external standards: (3) cyanidin, (4) delphinidin and (5) purified quebracho (Schinopsis balansae). When external standards were used (cyanidin, delphinidin, purified quebracho), it was likely that the condensed tannin content of the plant tissue would be under- or overestimated. When an internal standard based on the isolated tannin from the respective plant species was used, accurate estimates were obtained.

Purple Carrot (Daucus carota L.) Polyacetylenes Decrease Lipopolysaccharide-Induced Expression of Inflammatory Proteins in Macrophage and Endothelial Cells

Carrots ( Daucus carota L.) contain phytochemicals including carotenoids, phenolics, polyacetylenes, isocoumarins, and sesquiterpenes. Purple carrots also contain anthocyanins. The anti-inflammatory activity of extracts and phytochemicals from purple carrots was investigated by determining attenuation of the response to lipopolysaccharide (LPS). A bioactive chromatographic fraction (Sephadex LH-20) reduced LPS inflammatory response. There was a dose-dependent reduction in nitric oxide production and mRNA of pro-inflammatory cytokines (IL-6, IL-1beta, TNF-alpha) and iNOS in macrophage cells. Protein secretions of IL-6 and TNF-alpha were reduced 77 and 66% in porcine aortic endothelial cells treated with 6.6 and 13.3 microg/mL of the LH-20 fraction, respectively. Preparative liquid chromatography resulted in a bioactive subfraction enriched in the polyacetylene compounds falcarindiol, falcarindiol 3-acetate, and falcarinol. The polyacetylenes were isolated and reduced nitric oxide production in macrophage cells by as much as 65% without cytotoxicity. These results suggest that polyacetylenes, not anthocyanins, in purple carrots are responsible for anti-inflammatory bioactivity.

Proanthocyanidins and other phenolics in Acacia leaves of Southern Africa

Four provenances each of Acacia karroo, Acacia nilotica, Acacia tortilis, Acacia Senegal, Acacia erioloba and Faidherbia albida were sampled at Cyrene, Lucydale and Mahiye in the Matopos Research Station. Provenances were not replicated within site. The leaves and leaf stalks sampled were dried at 55°C and then analysed for total phenolics and proanthocyanidins (PAs). Variations in content of PAs and total phenolics of the Acacias were studied. Three assay methods for phenolics were evaluated. Total phenolics were estimated by precipitation with trivalent ytterbium after extraction with acetone:water (7:3 v/v) or colorimetrically by the Folin-Ciocalteu method. PAs were estimated colorimetrically by the butanol-HCI method. The results from the colorimetric methods are reported as absorbance units (au). A. Senegal contained the lowest level of ytterbium precipitated phenolics (Ybppt) (mean = 47 g/kg DM), while A. nilotica contained the highest (mean = 298 g/kg DM). Amounts of Ybppt for A. karroo, A. erioloba and F. albida were not significantly different (P > 0.05) from one another. The amount of Ybppt for A. tortilis was significantly different (P 0.05). A. senegal was again the lowest in total phenolics detected by the Folin-Ciocalteu method (A 675 = 0.370 au/g DM), while A. nilotica was the highest (A 675 = 5.261 au/g DM). A. karroo, F. albida and A. tortilis were not significantly different (P > 0.05) from one another. PAs were lowest for A. senegal (A 550 = 0.040 au/g DM) and highest for A. karroo (A 550 = 2.011 au/g DM). Values for A. tortilis, F. albida and A. erioloba were not significantly different (P > 0.05) from one another. For all the three assay methods, there were no significant differences (P > 0.05) between Cyrene and Mahiye sites. The regression analysis of PAs on Ybppt showed the relationship to be highly significant (P < 0.05) for A. karroo, F. albida and A. erioloba (R 2 =0.904, 0.892, 0.884, respectively, and significant for A. tortilis (R 2 = 0.680). It was poor for A. Senegal and A. nilotica (R 2 = 0.347 and 0.460, respectively). The relationship between total phenolics and Ybppt was similarly good for F. albida, A. erioloba, A. nilotica and A. karroo (R 2 = 0.957, 0.808, 0.792, and 0.725, respectively) but poor for A. senegal, and A. tortilis (R 2 = 0.413 and 0.004, respectively).