Filter aid selection allows modulating the vanadium concentration in beverages.

This study reports a clear-cut relationship of the technological use of specific filter aids with highly variable vanadium levels in beer, wine, and fruit juices. First, the previously reported broad range of vanadium levels was confirmed in 68 commercial beverages by ICP-MS. Since cloudy apple juices exhibited significantly lower vanadium amounts than clear apple juices, filter aids used for clarification were analyzed and found to contain substantial and highly variable amounts of vanadium, particularly in all analyzed diatomite (38-368 mg vanadium per kg filter aid), but not in perlite products (<4 mg/kg). Subsequent pilot-scale precoat filtration experiments (170 L/batch) proved vanadium to be released from diatomite (Kieselguhr), increasing its levels from ca. 2.1-2.6 µg/kg unfiltered to 27-201 µg/kg filtered juice, depending on the use of diatomites high or low in vanadium. Thus, filter aid selection was shown to modulate the vanadium concentrations in clarified beverages.

Anthocyanins from fruit juices improve the antioxidant status of healthy young female volunteers without affecting anti-inflammatory parameters: results from the randomised, double-blind, placebo-controlled, cross-over ANTHONIA (ANTHOcyanins in Nutrition Investigation Alliance) study.

Anthocyanins (ACN) can exert beneficial health effects not only through their antioxidative potential but also through modulation of inflammatory parameters that play a major role in CVD. A randomised cross-over study was carried out to investigate the effects of ACN-rich beverage ingestion on oxidation- and inflammation-related parameters in thirty healthy female volunteers. The participants consumed 330 ml of beverages (placebo, juice and smoothie with 8·9 (SD 0·3), 983·7 (SD 37) and 840·9 (SD 10) mg/l ACN, respectively) over 14 d. Before and after each intervention, blood and 24 h urine samples were collected. Plasma superoxide dismutase (SOD) and catalase activities increased significantly after ACN-rich beverage ingestion (P<0·001), whereas after placebo juice ingestion no increase could be observed. Plasma glutathione peroxidase and erythrocyte SOD activities were not affected. An increase in Trolox equivalent antioxidant capacity could also be observed after juice (P<0·001) and smoothie (P<0·01) ingestion. The plasma and urinary concentrations of malondialdehyde decreased after ACN-rich beverage ingestion (P<0·001), whereas those of 8-OH-2-deoxyguanosine as well as inflammation-related parameters (IL-2, -6, -8 and -10, C-reactive peptide, soluble cluster of differentiation 40 ligand, TNF-α, monocyte chemoattractant protein-1 and soluble cell adhesion molecules) were not affected. Thus, ingestion of ACN-rich beverages improves antioxidant enzyme activities and plasma antioxidant capacity, thus protecting the body against oxidative stress, a hallmark of ongoing atherosclerosis.

Apple procyanidins affect several members of the ErbB receptor tyrosine kinase family in vitro.

Complex polyphenol-rich extracts from apples are known to inhibit the activity of the epidermal growth factor receptor (EGFR) in vitro. The aim of the present study was to identify the bioactive constituents of the apple juice extract which contribute substantially to this potentially chemopreventive effect and to address the question whether the effect is specific to the EGFR or whether other members of the ErbB-receptor family might also be affected. Apple-derived dihydrochalcones and their respective glycosides were found to decrease EGFR activity under cell-free conditions with IC50-values ranging from 0.4 ± 0.1 to 267.0 ± 50.0 µM but showed no activity on human cancer cells. The concentration of quercetin or its glycosides in the extract was too low to contribute substantially to the EGFR-inhibitory properties. In contrast, fractions derived from the apple juice extract comprising ≥86% oligomeric procyanidins (OPCs) suppressed the activity of the EGFR in cell culture with an IC50 ∼ 100 µg mL(-1). In addition, the activity of further members of the ErbB-receptor family was potently inhibited, with ErbB3 receptor activity being most potently decreased (IC50 ∼ 10 µg mL(-1)). From the apple polyphenols identified so far OPCs were found to add the highest contribution to the inhibitory effects towards members of the ErbB-receptor family. Considering the crucial role of the ErbB-receptors in carcinogenesis, these results support the hypothesis that apple-derived OPCs as well as OPC-rich apple preparations might be of interest with respect to chemoprevention.

Solar UVB response of bioactives in strawberry (Fragaria × ananassa Duch. L.): a comparison of protected and open-field cultivation.

Strawberries (Fragaria × ananassa Duch. cvs. Everest, Elsanta) were grown in a tunnel covered with two films, which were distinguished in their ultraviolet transparency, as well as under open-field conditions. One applied film was not transparent for UVB radiation, and the second film transmitted 70% of UVB radiation. During the present study, the nutritional value and quality parameters of the fruits were evaluated. Strawberries were UV-unresponsive in view of the content of ascorbic acid and sum parameters like total anthocyanins and antioxidant capacity measured with TEAC (trolox equivalent antioxidant capacity), ORAC (oxygen radical absorbance capacity) and total phenols. These parameters were mainly affected by sampling date and cultivar. However, HPLC analysis showed that individual phenolics were affected in the absence of UV radiation. The content of the anthocyanin cyanidin 3-glucoside and the flavonols quercetin 3-glucuronide and kaempferol 3-glucoside was decreased in the fruits grown under UV blocking film compared to open-field grown strawberries. By means of the UV transparent film the content of the mentioned flavonoids could be enhanced up to similar amounts like in open-field grown strawberries. All other phenolics were not consistently affected by UV radiation. This result was independent of cultivar.

Polyphenolic apple extracts: effects of raw material and production method on antioxidant effectiveness and reduction of DNA damage in Caco-2 cells.

A diet rich in fruits and vegetables is commonly perceived to be associated with reduced cancer risk, attributed to its high content of polyphenols. As apples represent a major polyphenol source in Western countries, we studied differentially produced extracts (1-100 microg/mL): two from different apple juices (AEs), one from pomace (APE), and one peel extract (PE) on their potential to reduce DNA oxidation damage and induce antioxidant defense in Caco-2 cells. Additionally, we measured direct antioxidant capacity (TEAC/ORAC) of the extracts. Quercetin-rich PE and APE most effectively diminished DNA damage and ROS level after 24 h incubation (PE > APE), whereas the AEs were only moderately effective. GPx activity was diminished for all extracts, with AEs > APE > PE. Direct antioxidant activity decreased in the order AEs > PE > APE, displaying no significant correlation with cellular markers. In conclusion, apple phenolics at low, nutritionally relevant concentrations may protect intestinal cells from ROS-induced DNA damage, mediated by cellular defense mechanisms rather than by antioxidant activity.

Antioxidant effectiveness of phenolic apple juice extracts and their gut fermentation products in the human colon carcinoma cell line caco-2.

Apples represent a major dietary source of antioxidative polyphenols. Their metabolic conversion by the gut microflora might generate products that protect the intestine against oxidative damage. We studied the antioxidant effectiveness of supernatants of fermented apple juice extracts (F-AEs, 6 and 24 h fermentation) and of selected phenolic degradation products, identified by HPLC-DAD-ESI-MS. Cell free antioxidant capacity of unfermented apple juice extracts (AEs) was decreased after fermentation by 30-50%. In the human colon carcinoma cell line Caco-2, F-AEs (containing <0.5% of original AE-phenolics) decreased the reactive oxygen species (ROS) level more efficiently than the F-blank (fermented without AE) but were less effective than the respective AEs. Similarly, antioxidant effectiveness of individual degradation products was lower compared to respective AE constituents. Glutathione level was slightly increased and oxidative DNA damage slightly decreased by fermented AE03, rich in quercetin glycosides. In conclusion, F-AEs/degradation products exhibit antioxidant activity in colon cells but to a lesser extent than the respective unfermented AEs/constituents.

Histone-deacetylase inhibition and butyrate formation: Fecal slurry incubations with apple pectin and apple juice extracts.

OBJECTIVE: Butyrate plays a major role among the short-chained fatty acids formed by the microbial flora of the colon. It is considered to be an important nutrient of the colon mucosa and has been shown to trigger differentiation and apoptosis of colon-derived cells in culture. Inhibition of histone deacetylase (HDAC) seems to play a central role in these effects. Butyrate was thus suggested to act as a chemopreventive metabolite that can prevent the occurrence of colorectal cancer, one of the most abundant types of cancer in Western industrialized countries. Some polymeric carbohydrates such as pectin, resistant to digestion in the small intestine, have been shown to serve as substrates for butyrate formation by the microflora of the colon. METHODS: In this study we investigated fermentation supernatants (FSs) from incubations of human fecal slurry with apple pectin and with polyphenol-rich apple juice extracts (AJEs). RESULTS: We found that FSs from fermentations with pectin were rich in butyrate and very active in HDAC inhibition in nuclear extracts prepared from the colon tumor cell lines HT-29 and Caco-2 and in intact HeLa Mad 38 cells bearing a reporter gene driven by HDAC inhibition. The butyrate levels explained most of the HDAC-inhibitory potency in FSs from pectin-rich fermentations. FSs from fermentations with AJEs showed lower butyrate yields but comparable HDAC inhibition. Combined incubations of pectin with AJEs led to effects similar to those with FSs from incubations with pectin as the only substrate added. These effects could not be explained by a direct HDAC-inhibitory potency of AJEs. Furthermore, the FSs were not cytotoxic at the HDAC-inhibitory concentrations. CONCLUSION: These findings suggest that butyrate is the most relevant HDAC inhibitor formed in fermentations of human fecal slurry with apple pectin, whereas addition of AJEs leads to the formation of butyrate and other, yet unknown, HDAC inhibitors.

Apple polyphenols and products formed in the gut differently inhibit survival of human cell lines derived from colon adenoma (LT97) and carcinoma (HT29).

Colorectal tumor risks could be reduced by polyphenol-rich diets that inhibit cell growth. Here, apple polyphenols were studied for effects on the survival of colon adenoma (LT97) and carcinoma-derived (HT29) cell lines. Three apple extracts (AEs) from harvest years 2002-2004 were isolated (AE02, AE03, and AE04) and fermented in vitro with human fecal flora. Extracts and fermentation products were analyzed for polyphenols with HPLC. The cells were treated with AEs (0-850 microg/mL) or fermented AEs (F-AEs, 0-9%), and survival was measured by DNA staining. All AEs contained high amounts of polyphenols (311-534 mg/g) and reduced cell survival (in LT97 > HT29). AE03 was most potent, possibly because it contained more quercetin compounds. Fermentation of AEs resulted in an increase of short chain fatty acids, and polyphenols were degraded. The F-AEs were approximately 3-fold less bioactive than the corresponding AEs, pointing to a loss of chemoprotective properties through fermentation.

Cytochrome P450 1A1 expression and activity in Caco-2 cells: modulation by apple juice extract and certain apple polyphenols.

Cytochrome P450 (CYP) 1A1 plays a role in drug metabolism of intestinal cells (e.g., by activating certain chemical carcinogens such as polycyclic aromatic hydrocarbons into carcinogenic metabolites). In the human colon carcinoma cell line Caco-2, we investigated the effects of a defined polyphenolic apple juice extract (AJE), the major principle flavonoid/dihydrochalkone constituents quercetin and phloretin, and the corresponding prototype glycosides rutin and phlorizin on CYP1A1 expression and activity. Incubations were carried out with or without the potent aryl hydrocarbon receptor agonist/CYP1A1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AJE and quercetin acted as weak inducers of CYP1A1 mRNA and protein, and AJE, quercetin, and phlorizin led to a slight induction of CYP1A1-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity. However, AJE, quercetin, and phloretin were highly effective in suppressing CYP1A1 induction in co-incubations of the cells with 1 nM TCDD. The antagonistic effects were seen on the levels of mRNA, enzyme protein, and catalytic activity. In contrast, the related glycosides rutin and phlorizin were inactive as inducers or inhibitors. Inhibition of CYP1A1 induction was not related to general cytotoxicity, which could be completely abolished by the addition of ascorbic acid/alpha-tocopherol. AJE, quercetin, and phloretin also antagonized the TCDD-mediated induction of a reporter gene driven by a regulatory sequence of the human CYP1A1 gene promoter. Our findings suggest that apple juice extract can antagonize TCDD-mediated CYP1A1 induction by interfering with AhR-dependent gene transcription and by inhibiting the catalytic activity of CYP1A1. These effects may result in reduced metabolic activation of certain chemical carcinogens, in particular, under conditions of sustained AhR activation.

Physiological effects of extraction juices from apple, grape, and red beet pomaces in rats.

In comparison to classical fruit juice processing, polyphenols and dietary fiber can be extracted from pomace by means of pectinases and cellulases. In the present study, rats were fed with such produced extraction juices from apples, grapes, and red beets as drinking fluids instead of water for 4 weeks to evaluate their physiological effects. In all test groups, the intake of extraction juices was greater as compared to control (water intake), resulting in a higher urine excretion. In the apple and grape group, pH values in feces was lower than control. Administration of extraction juices from apples increased fecal counts of Lactobacillus and Bifidobacterium. More acetate and total short-chain fatty acids appeared in intestinal contents of the apple and red beet group. Furthermore, the intestinal contents of test groups contained higher concentrations of primary bile acids, cholesterol, and cholesterol metabolites but lower concentrations of secondary bile acids. The total amount of steroids excreted by these groups was also greater than control. Quercetin and isorhamnetin appeared in urine of rats fed extraction juices from apples and grapes; in urine of the former group, phloretin was found also. Administration of the extraction juices, enriched in secondary plant metabolites and dietary fiber, resulted in beneficial nutritional effects in rats.

An anthocyanin/polyphenolic-rich fruit juice reduces oxidative DNA damage and increases glutathione level in healthy probands.

Oxidative cell damage is involved in the pathogenesis of atherosclerosis, cancer, diabetes and other diseases. Uptake of fruit juice with especially high content of antioxidant flavonoids/polyphenols, might reduce oxidative cell damage. Therefore, an intervention study was performed with a red mixed berry juice [trolox equivalent antioxidative capacity (TEAC): 19.1 mmol/L trolox] and a corresponding polyphenol-depleted juice (polyphenols largely removed, TEAC 2.4 mmol/L trolox), serving as control. After a 3-week run-in period, 18 male probands daily consumed 700 mL juice, and 9 consumed control juice, in a 4-week intervention, followed by a 3-week wash-out. Samples were collected weekly to analyze DNA damage (comet assay), lipid peroxidation (plasma malondialdehyde: HPLC/fluorescence; urinary isoprostanes: GC-MS), blood glutathione (photometrically), DNA-binding activity of nuclear factor-kappaB (ELISA) and plasma carotenoid/alpha-tocopherol levels (HPLC-DAD). During intervention with the fruit juice, a decrease of oxidative DNA damage (p<5x10(-4)) and an increase of reduced glutathione (p<5x10(-4)) and of glutathione status (p<0.05) were observed, which returned to the run-in levels in the subsequent wash-out phase. The other biomarkers were not significantly modulated by the juice supplement. Intervention with the control juice did not result in reduction of oxidative damage. In conclusion, the fruit juice clearly reduces oxidative cell damage in healthy probands.

Apple flavonoids inhibit growth of HT29 human colon cancer cells and modulate expression of genes involved in the biotransformation of xenobiotics.

Flavonoids from fruits and vegetables probably reduce risks of diseases associated with oxidative stress, including cancer. Apples contain significant amounts of flavonoids with antioxidative potential. The objectives of this study were to investigate such compounds for properties associated with reduction of cancer risks. We report herein that apple flavonoids from an apple extract (AE) inhibit colon cancer cell growth and significantly modulate expression of genes related to xenobiotic metabolism. HT29 cells were treated with AE at concentrations delivering 5-50 microM of one of the major ingredients, phloridzin ("phloridzin-equivalents," Ph.E), to the cell culture medium, with a synthetic flavonoid mixture mimicking the composition of the AE or with 5-100 microM individual flavonoids. HT29 cell growth was inhibited by the complex extract and by the mixture. HT29 cells were treated with nontoxic doses of the AE (30 microM, Ph.E) and after 24 h total RNA was isolated to elucidate patterns of gene expression using a human cDNA-microarray (SuperArray) spotted with 96 genes of drug metabolism. Treatment with AE resulted in an upregulation of several genes (GSTP1, GSSTT2, MGST2, CYCP4F3, CHST5, CHST6, and CHST7) and downregulation of EPHX1, in comparison to the medium controls. The enhanced transcriptional activity of GSTP1 and GSTT2 genes was confirmed with real-time qRT-PCR. On the basis of the pattern of differential gene expression found here, we conclude that apple flavonoids modulate toxicological defense against colon cancer risk factors. In addition to the inhibition of tumor cell proliferation, this could be a mechanism of cancer risk reduction.

Inhibitors of the epidermal growth factor receptor in apple juice extract.

The polyphenol-rich extract of a consumer-relevant apple juice blend was found to potently inhibit the growth of the human colon cancer cell line HT29 in vitro. The epidermal growth factor receptor (EGFR) and its subsequent signaling cascade play an important role in the regulation of cell proliferation in HT29 cells. The protein tyrosine kinase activity of an EGFR preparation was effectively inhibited by the polyphenol-rich apple juice extract. Treatment of intact cells with this extract resulted in the suppression of the subsequent mitogen-activated protein kinase cascade. Amongst the so far identified apple juice constituents, the proanthocyanidins B1 and B2 as well as quercetin-3-glc (isoquercitrin) and quercetin-3-gal (hyperoside) were found to possess substantial EGFR-inhibitory properties. However, as to be expected from the final concentration of these potential EGFR inhibitors in the original polyphenol-rich extract, a synthetic mixture of the apple juice constituents identified and available so far, including both proanthocyanidins and the quercetin glycosides, showed only marginal inhibitory effects on the EGFR. These results permit the assumption that yet unknown constituents contribute substantially to the potent EGFR-inhibitory properties of polyphenol-rich apple juice extract. In summary, the polyphenol composition of apple juice possesses promising growth-inhibitory properties, affecting proliferation-associated signaling cascades in colon tumor cells.

Dietary fiber-rich colloids from apple pomace extraction juices do not affect food intake and blood serum lipid levels, but enhance fecal excretion of steroids in rats.

The aim of this study was to investigate the effects of colloids isolated from apple pomace extraction juices (so-called B-juices) produced by enzymatic liquefaction on food intake, levels of blood serum lipids, and fecal excretion of bile acids (BA) and neutral sterols (NS) in vivo. Ten male Wistar rats per group were fed diets containing either no apple dietary fiber (DF) (control), a 5% supplementation with juice colloids, or an alcohol-insoluble substance (AIS) from apples for 6 weeks. Apple DF in diets led to lower weight gain in rats fed with B-juice colloids (P< 0.05). For these rats, food intake was not affected but was highest with feeding AIS (10% more than control) to cover energy requirements. The supplementation of diet with apple DF from extraction juices or AIS had minor effects on blood serum lipids. In rats fed either juice colloids or AIS, up to 30% (5.31 micromol/g dry weight) and 88% (7.69 micromol/g dry weight) more primary BA were excreted in feces, respectively, as compared to that in the control group (4.10 micromol/g dry weight) (P < 0.05). In cecal contents, a 15% (juice colloids) to 37% (AIS) increase in primary BA was found. In contrast, concentrations of secondary BA were lower in feces of test groups (P < 0.05). Excretion of total BA and NS was higher in rats fed apple DF (P < 0.05). Our study is the first to prove that there are beneficial physiologic effects of apple DF isolated from pomace extraction juices produced by enzymatic liquefaction. These results may help to develop such innovative juice products that are rich in DF of fruit origin for diminishing the lack of DF intake.

Effects of dietary fibre-rich juice colloids from apple pomace extraction juices on intestinal fermentation products and microbiota in rats.

Effects of colloids isolated from apple pomace extraction juices (so-called B-juices) produced by enzymic liquefaction on food intake, body and faecal weights, short-chain fatty acid (SCFA) profile and selected intestinal microbiota were investigated in rats. Ten male Wistar rats per group were fed diets without any apple dietary fibre (DF) (control) or supplement with 5 % B-juice colloids or an alcohol-insoluble substance (AIS) from apples for 6 weeks. Rats fed with apple DF (5 % B-juice colloids or AIS) gained less weight than control rats (P<0.05). B-juice colloids did not affect food intake, whereas feeding AIS resulted in a 10 % higher food consumption than in control rats. Both juice colloids and AIS increased the weight of caecal contents in rats and lowered luminal pH values (P<0.05). In addition, SCFA concentrations and total yields were also raised (P<0.05) in caecum of these rats indicating good fermentability of apple substrates by gut microflora. Distinctly higher concentrations of acetate and propionate were found in intestinal contents of juice colloid-fed rats (P<0.05), whereas AIS also increased butyrate yield. Changes in microbiota due to apple DF in diets were restricted in the caecum to the Eubacterium rectale cluster (AIS; P<0.05) and in faeces to the Bacteroidaceae (juice colloids and AIS; P<0.05). The present study shows the physiological effects of apple DF isolated from pomace extraction juices produced by enzymic liquefaction on intestinal fermentation. Results may be helpful for the development of such innovative juice products that are rich in DF of fruit origin.