Christian G. Krueger

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.

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.

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.

Vascular Response to Zotarolimus-Coated Balloons in Injured Superficial Femoral Arteries of the Familial Hypercholesterolemic Swine

Background— Drug-coated balloons are rapidly emerging as a therapeutic alternative for the interventional treatment of peripheral vascular disease. The purpose of this study was to test the hypothesis that an angioplasty balloon coated with the mTOR inhibitor zotarolimus (ZCB) would inhibit neointimal hyperplasia in a novel injury-based superficial femoral artery model in the familial hypercholesterolemic swine. Methods and Results— A total of 44 familial hypercholesterolemic swine were included (12 designated to study tissue pharmacokinetics and 32 to study safety and efficacy). Fogarty balloon denudation was performed in all superficial femoral artery segments, followed by balloon angioplasty. In the pharmacokinetic study, a total of 24 ZCBs (300 &mgr;g/cm2) were used. Zotarolimus was detected in arterial tissue at 5 minutes (162 ng/mg of tissue), 24 hours (5.9 ng/mg of tissue), and 28 days (0.007 ng/mg of tissue) after ZCB inflation. In the safety and efficacy study, superficial femoral artery segments were randomized to either high-dose (600 &mgr;g/cm2, n=16), low-dose (300 &mgr;g/cm2, n=16), or paired uncoated balloons (high-dose ZCB control, n=16; low-dose ZCB control, n=16). At 28 days, the percentage of angiographic stenosis was similar among all tested groups. Histological analysis demonstrated a reduction in neointimal formation in both ZCB groups compared with controls (high-dose ZCB 44% reduction, P=0.007; low-dose ZCB 22% reduction, P=0.08). There was no evidence of delayed arterial healing or vascular toxicity in any of the ZCB groups. Conclusions— The single delivery of zotarolimus via coated balloon is feasible, and therapeutic levels are maintained up to 28 days. The ZCB technology appears to be effective in the reduction of neointimal proliferation in the superficial femoral artery of the familial hypercholesterolemic swine.

Quantifying and characterizing proanthocyanidins in cranberries in relation to urinary tract health

The “A-type” proanthocyanidins in cranberry fruit (Vaccinium macrocarpon Ait.) are bioactive components associated with prevention of urinary tract infections (UTI). Cranberry juice, fruit (fresh and dried), functional foods, and cranberry dietary supplements are promoted for prevention of UTI and for maintenance of urinary tract health (UTH), on the basis of their content of cranberry proanthocyanidins (c-PAC) with “A-type” interflavan bonds. With increasing consumer use of cranberries for maintenance of UTH and an expanding number of commercial cranberry products of different types, the availability of unified methods for measuring levels of c-PAC is important. This review discusses quantitative and qualitative analysis of c-PAC with “A-type” interflavan bonds in relation to their biological activity for UTI prevention. The integrity (including authenticity, standardization, efficacy, and safety) of cranberry fruit, juices, and dietary supplements may now be measured by using recent advances in mass spectrometry, liquid chromatography, production of c-PAC standards, and improved simple quantitative techniques.

Comparison of isolated cranberry (Vaccinium macrocarpon Ait.) proanthocyanidins to catechin and procyanidins A2 and B2 for use as standards in the 4-(dimethylamino)cinnamaldehyde assay.

The 4-(dimethylamino)cinnamaldehyde (DMAC) assay is currently used to quantify proanthocyanidin (PAC) content in cranberry products. However, this method suffers from issues of accuracy and precision in the analysis and comparison of PAC levels across a broad range of cranberry products. Current use of procyanidin A2 as a standard leads to an underestimation of PACs content in certain cranberry products, especially those containing higher molecular weight PACs. To begin to address the issue of accuracy, a method for the production of a cranberry PAC standard, derived from an extraction of cranberry (c-PAC) press cake, was developed and evaluated. Use of the c-PAC standard to quantify PAC content in cranberry samples resulted in values that were 2.2 times higher than those determined by procyanidin A2. Increased accuracy is critical for estimating PAC content in relationship to research on authenticity, efficacy, and bioactivity, especially in designing clinical trials for determination of putative health benefits.

Deconvolution of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry isotope patterns to determine ratios of A-type to B-type interflavan bonds in cranberry proanthocyanidins

A method to deconvolute overlapping isotope patterns in positive mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was developed to determine ratios of A- to B-type interflavan bonds in proanthocyanidins that were isolated from cranberry (Vaccinium macrocarpon, Ait.) press cake (c-PAC). Precision and accuracy was validated for binary mixtures of procyanidins A2 and B2. Deconvolution of c-PAC spectra indicated that oligomers with one or more A-type interflavan bonds occur in a higher proportion than oligomers with all B-type interflavan bonds. c-PAC with at least one A-type bond accounted for more than 91% of the oligomers between trimers and undecamers. The c-PAC isotope patterns are highly repeatable, suggesting that the method can be applied to authentication, standardization and efficacy of cranberry products in relationship to urinary tract health. This is the first time MALDI-TOF MS has been used for estimating ratios of A- to B-type bonds in PAC.

Cranberry Proanthocyanidins Improve the Gut Mucous Layer Morphology and Function in Mice Receiving Elemental Enteral Nutrition

BACKGROUND Lamina propria Th2 cytokines, interleukin (IL)-4 and IL-13, stimulate goblet cell (GC) proliferation and MUC2 production, which protect the intestinal mucosa. Elemental enteral nutrition (EEN) reduces tissue IL-4 and impairs barrier function. Proanthocyanidins (PACs) stimulate oral mucin levels. We hypothesized that adding PAC to EEN would maintain Th2-without stimulating Th1-cytokines and preserve luminal MUC2 vs EEN alone. MATERIALS AND METHODS Seventy mice were randomized to 5 diet groups-standard chow, intragastric EEN, or EEN with lowPAC, midPAC (50 mg), or highPAC (100 mg PAC/kg BW)-for 5 days, starting 2 days after gastric cannulation. Ileal tissue was analyzed for histomorphology and the cytokines IL-4, IL-13, IL-1β, IL-6, and TNF-α by enzyme-linked immunosorbent assay. MUC2 was measured in intestinal washes. RESULTS EEN lowered IL-13 (P < .05) compared with standard chow, whereas IL-4 was not significant (P < .07). LowPAC and midPAC increased IL-13 (P < .05), whereas highPAC increased both IL-4 and IL-13 (P < .05) compared with EEN. All EEN diets reduced (P < .05) crypt depth compared with the chow group. Compared with standard chow, GC numbers and luminal MUC2 were reduced with EEN (P < .05). These effects were attenuated (P < .05) with midPAC and highPAC. No changes were observed in tissue Th1 cytokines. CONCLUSIONS Adding PACs to EEN reverses impaired intestinal barrier function following EEN by improving the gut mucous layer and function through increased GC size and number as well as levels of MUC2 and ileal IL-4 and IL-13.

Food-compatible method for the efficient extraction and stabilization of cranberry pomace polyphenols.

Cranberry pomace is a byproduct of cranberry processing and is comprised of seeds, skins and stems of the cranberry fruit. While cranberry pomace contains beneficial polyphenols, including proanthocyanidins and anthocyanins, it is not a palatable source of these compounds and is typically discarded. In this study, we have developed and optimized a method to extract polyphenols from cranberry pomace using aqueous ethanol, a food grade solvent. Biochemical characterization of the pomace extract showed the presence of a broad range of polyphenols also present in cranberry juice concentrate. By co-drying cranberry pomace extract with a protein-rich food matrix, such as soy protein isolate (SPI), we have developed a method to produce a cranberry polyphenol-SPI complex (CBP-SPI) containing 10% cranberry polyphenols. Unlike dried cranberry pomace extract alone, proanthocyanidins, anthocyanins and total polyphenols were found to be highly stable at 37 °C in the CBP-SPI powder. The extraction and stabilization of cranberry pomace polyphenols using SPI provides an innovative approach for utilizing pomace in the development of novel food ingredients.