Non-enzymatic degradation of flavan-3-ols by ascorbic acid- and sugar-derived aldehydes during storage of apple juices and concentrates produced with the innovative spiral filter press.

Potentially health-promoting concentrations of flavan-3-ols were previously shown to be retained in apple juices produced with the emerging spiral filter press. Due to the novelty of this technology, the factors governing the stability of flavan-3-ol-rich apple juices have only scarcely been studied. Therefore, we produced flavan-3-ol-rich apple juices and concentrates (16, 40, 70 °Brix) supplemented with ascorbic acid (0.0, 0.2, 1.0 g/L) according to common practice. Flavan-3-ols (DP1-7) and twelve flavan-3-ol reaction products were comprehensively characterized and monitored during storage for 16 weeks at 20 and 37 °C, employing RP-UHPLC- and HILIC-DAD-ESI(-)-QTOF-HR-MS/MS. Flavan-3-ol degradation followed a second-order reaction kinetic, being up to 3.5-times faster in concentrates (70 °Brix) than in single strength juices (16 °Brix). Furthermore, they diminished substantially faster compared to other phenolic compounds. For instance, after 16-weeks at 20 °C, the maximum loss of flavan-3-ols (-70 %) was greater than those of hydroxycinnamic acids (-18 %) and dihydrochalcones (-12 %). We observed that flavan-3-ols formed adducts with sugars and other carbonyls, such as 5-(hydroxymethyl)furfural and the ascorbic acid-derived L-xylosone. Increased degradation rates correlated particularly with increased furan aldehyde levels as found in concentrates stored at elevated temperatures. These insights could be used for optimizing production, distribution, and storage of flavan-3-ol-rich apple juices and other foods and beverages.

Anthocyanin-Rich Berry Extracts Affect SN-38-Induced Response: A Comparison of Non-Tumorigenic HCEC-1CT and HCT116 Colon Carcinoma Cells.

Chemotherapy with irinotecan (CPT-11), the pro-drug of the highly cytotoxic SN-38, is among the standard-of-care treatments for colorectal cancer. To counteract undesired toxic side effects on healthy tissue such as the intestinal epithelium, the use of preparations rich in polyphenols with anti-oxidative and anti-inflammatory properties such as anthocyanins has been proposed. In the present study, the question of whether non-tumorigenic human epithelium cells (HCEC-1CT) can be protected against the cytotoxic impact of SN-38 by anthocyanin-rich polyphenol extracts without compromising the desired therapeutic effect against tumor cells (HCT-116) was addressed. Hence, single and combinatory effects of anthocyanin-rich polyphenol extracts of elderberry (EB), bilberry (Bil), blackberry (BB) and black currant (BC) with the chemotherapeutic drug SN-38 were investigated. Out of the extracts, BB showed the most potent concentration-dependent cytotoxicity alone and in combination with SN-38, with even stronger effects in non-tumorigenic HCEC-1CT cells. In cytotoxic concentrations, BB decreased the level of DNA/topoisomerase I covalent complexes in HCEC-1CT cells below base level but without concomitant reduction in SN-38-induced DNA strand breaks. The herein reported data argue towards an interference of anthocyanins with successful treatment of cancer cells and a lack of protective properties in healthy cells.

Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites Attenuate LPS/Nigericin-Induced Inflammasome Activation by Inhibiting ASC Speck Formation in THP-1 Monocytes.

Inflammasomes are multi-protein complexes, which are formed in response to tissue injury, infections, and metabolic stress. However, aberrant inflammasome activation has been linked to several inflammatory diseases. Anthocyanins have been reported to attenuate NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, but the influence of grape/blueberry anthocyanins and especially their gut-derived metabolites on NLRP3 inflammasome activation in human monocytes remains unclear. Therefore, human leukemic monocytes (THP-1 cells, Tohoku Hospital Pediatrics-1 cells) were preincubated with different concentrations of grape/blueberry anthocyanins, homovanillyl alcohol, or 2,4,6-trihydroxybenzaldehyde (THBA) before the NLRP3 inflammasome was activated by lipopolysaccharide and/or nigericin. Apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, as well as ASC and NLRP3 protein expression, were determined using flow cytometry. Caspase-1 activity was measured in cultured cells, and pro-inflammatory cytokine secretion was determined using enzyme-linked immunosorbent assays. Anthocyanins and their metabolites had no effect on ASC or NLRP3 protein expression. However, THBA significantly inhibited ASC speck formation in primed and unprimed THP-1 monocytes, while caspase-1 activity was significantly declined by grape/blueberry anthocyanins. Furthermore, reduced inflammasome activation resulted in lower pro-inflammatory cytokine secretion. In conclusion, our results show for the first time that grape/blueberry anthocyanins and their gut-derived metabolites exert anti-inflammatory effects by attenuating NLRP3 inflammasome activation in THP-1 monocytes.

Analytical characterization of flavan-3-ol-rich apple juices produced with the innovative spiral filter press technology.

The retention of flavan-3-ols and other phenolic compounds during apple juice production at pilot plant scale (200 kg, cv. Boskoop) was investigated. An oxygen-excluding spiral filter press and a conventional decanter were used with and without pectinase mash treatment. Phenolic compounds were comprehensively identified and quantitated by RP-UHPLC and HILIC, both coupled to DAD-FLD and DAD-ESI(-)-QTOF-HR-MS/MS. These techniques combined with using a NIST cocoa flavan-3-ol standard allowed for the first time an individual quantification of flavan-3-ol fractions (DP 1-7) in apple juices. Spiral filter-pressed juices were exposed to less oxidation and exhibited four times higher total phenolic compound levels than decanter-made juices (1016 vs. 262 mg/L). Apple juices derived from pectinase-treated mashes had lower total phenolic compound levels than their non-treated counterparts. However, those made by spiral filter press still retained significantly higher concentrations (780 vs. 104 mg/L). Flavan-3-ols were especially well retained by spiral filter press processing, reaching unprecedentedly high concentrations of up to 713 mg/L. A 280 mL serving of non-treated spiral filter-pressed juice would therefore suffice to provide the daily intake of 200 mg flavan-3-ols, equaling the dose of cocoa flavan-3-ols associated with an authorized European health claim for healthy blood flow.

In Vitro Inhibitory Potential of Different Anthocyanin-Rich Berry Extracts in Murine CT26 Colon Cancer Cells.

Anti-oxidant, -inflammatory, and -carcinogenic activities of bioactive plant constituents, such as anthocyanins, have been widely discussed in literature. However, the potential interaction of anthocyanin-rich extracts with routinely used chemotherapeutics is still not fully elucidated. In the present study, anthocyanin-rich polyphenol extracts of blackberry (BB), bilberry (Bil), black currant (BC), elderberry (EB), and their respective main anthocyanins (cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-sambubioside) were investigated concerning their cytotoxic and DNA-damaging properties in murine CT26 cells either alone or in combination with the chemotherapeutic agent SN-38. BB exerted potent cytotoxic effects, while Bil, BC, and EB only had marginal effects on cell viability. Single anthocyanins comprised of the extracts could not induce comparable effects. Even though the BB extract further pronounced SN-38-induced cytotoxicity and inhibited cell adhesion at 100-200 µg/mL, no effect on DNA damage was observed. In conclusion, anti-carcinogenic properties of the extracts on CT26 cells could be ranked BB >> BC ≥ Bil ≈ EB. Mechanisms underlying the potent cytotoxic effects are still to be elucidated since the induction of DNA damage does not play a role.

Mass Spectrometry-Based Proteomic Profiling of a Silvaner White Wine.

The comprehensive identification of the proteome content from a white wine (cv. Silvaner) is described here for the first time. The wine protein composition isolated from a representative wine sample (250 L) was identified via mass spectrometry (MS)-based proteomics following in-solution and in-gel digestion methods after being submitted to size exclusion chromatographic (SEC) fractionation to gain a comprehensive insight into proteins that survive the vinification processes. In total, we identified 154 characterized (with described functional information) or so far uncharacterized proteins, mainly from Vitis vinifera L. and Saccharomyces cerevisiae. With the complementarity of the two-step purification, the digestion techniques and the high-resolution (HR)-MS analyses provided a high-score identification of proteins from low to high abundance. These proteins can be valuable for future authentication of wines by tracing proteins derived from a specific cultivar or winemaking process. The proteomics approach presented herein may also be generally helpful to understand which proteins are important for the organoleptic properties and stability of wines.

Additive and synergistic antifungal effects of copper and phenolic extracts from grape cane and apples.

BACKGROUND: Organic viticulture seeks sustainable alternatives for eco-toxic copper fungicides to control downy mildew caused by Plasmopara viticola. (Poly)phenol-rich extracts of agricultural byproducts are known to possess antifungal activity, but high production costs often limit their actual implementation. RESULTS: We developed and produced novel ligninsulfonate-based grape cane extract (GCE) formulations and an apple extract on a pilot plant scale, including a detailed (poly)phenol characterization by high-performance liquid chromatography photodiode array mass spectrometry (HPLC-PDA-MS). Our GCE formulations alone reduced downy mildew disease severity in greenhouse trials by 29%-69% in a dose-dependent manner, whereas a standard application of the copper-based agent alone reached ~56%. When applied together, disease severity was diminished by 78%-92%, revealing a synergistic effect that depended on the mixture ratio. Combining GCE formulations with the apple extract, additive effects were found (80% disease severity reduction). CONCLUSION: The studied plant extracts are proposed to both substitute for and synergistically reinforce copper fungicides in grapevine downy mildew control. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Recombinant Thaumatin-Like Protein (rTLP) and Chitinase (rCHI) from Vitis vinifera as Models for Wine Haze Formation.

Cross-linking net aggregates of thermolabile thaumatin-like proteins (TLPs) and chitinases (CHIs) are the primary source of haze in white wines. Although bentonite fining is still routinely used in winemaking, alternative methods to selectively remove haze proteins without affecting wine organoleptic properties are needed. The availability of pure TLPs and CHIs would facilitate the research for the identification of such technological advances. Therefore, we proposed the usage of recombinant TLP (rTLP) and CHI (rCHI), expressed by Komagataella phaffii, as haze-protein models, since they showed similar characteristics (aggregation potential, melting point, functionality, glycosylation levels and bentonite adsorption) to the native-haze proteins from Vitis vinifera. Hence, rTLP and rCHI can be applied to study haze formation mechanisms on a molecular level and to explore alternative fining methods by screening proteolytic enzymes and ideal adsorptive resins.

Influence of Plasma-Isolated Anthocyanins and Their Metabolites on Cancer Cell Migration (HT-29 and Caco-2) In Vitro: Results of the ATTACH Study.

Cancer mortality is mainly due to metastasis. Therefore, searching for new therapeutic agents suppressing cancer cell migration is crucial. Data from human studies regarding effects of anthocyanins on cancer progression, however, are scarce and it is unclear whether physiological concentrations of anthocyanins and their metabolites reduce cancer cell migration in vivo. In addition, interactions with chemotherapeutics like 5-fluorouracil (5-FU) are largely unknown. Thus, we combined a placebo-controlled, double-blinded, cross-over study with in vitro migration studies of colon cancer cell lines to examine the anti-migratory effects of plasma-isolated anthocyanins and their metabolites (PAM). Healthy volunteers (n = 35) daily consumed 0.33 L of an anthocyanin-rich grape/bilberry juice and an anthocyanin-depleted placebo juice for 28 days. PAM were isolated before and after intervention by solid-phase extraction. HT-29 and Caco-2 cells were incubated with PAM in a Boyden chamber. Migration of HT-29 cells was significantly inhibited by PAM from juice but not from placebo. In contrast, Caco-2 migration was not affected. Co-incubation with 5-FU and pooled PAM from volunteers (n = 10), which most effectively inhibited HT-29 migration, further reduced HT-29 migration in comparison to 5-FU alone. Therefore, PAM at physiological concentrations impairs colon cancer cell migration and may support the effectiveness of chemotherapeutics.

Comparative Evaluation of Juices from Red-Fleshed Apples after Production with Different Dejuicing Systems and Subsequent Storage.

In this work, two vintages (2019 and 2020) of red-fleshed 'Weirouge' apples were processed with the innovative spiral filter press technology to investigate juice production in an oxygen-reduced atmosphere. After pressing, a more brilliant red color and appreciably higher amounts of oxidation-sensitive constituents (ascorbic acid, anthocyanins, and colorless (poly)phenols) were seen in spiral filter pressed juices compared to those produced with conventional systems (horizontal filter press and decanter). In a subsequent stability study (24 weeks storage at 4, 20, and 37 °C), the color and phenolic compounds were monitored and differences in the juices produced with the different pressing-systems were widely maintained during the storage period. The analyses of the anthocyanins and colorless (poly)phenols were conducted by UHPLC-DAD-ESI-QTOF-HR-MS/MS and UHPLC-DAD. The spiral filter press emerged as a promising technology for the production of juices with a more attractive color and a better retention of oxidation-sensitive constituents during processing and storage compared to conventional juices.

Variability of Constitutive Stilbenoid Levels and Profiles in Grape Cane (Vitis vinifera L.) Depending upon Variety and Clone, Location in the Vineyard, Pruning Time, and Vintage.

Stilbenoids in grape cane (Vitis vinifera L.) are bioactive compounds relevant for plant defense and the potential valorization of this byproduct. Our screening of grape cane from 102 varieties showed constitutive stilbenoid levels in a wide range (557-7748 mg/kg of dry weight). Analyses of genetically distinct clones of selected cultivars unraveled that intravarietal variability (e.g., cv. Riesling, 3236-6541 mg/kg) was higher than that across samples from a single clone but different vineyard positions (3017-3710 mg/kg). Furthermore, stilbenoid levels in samples obtained in October, December, and February (3 years, 2017-2019) showed pronounced quantitative and qualitative variability and the highest yields upon December pruning. For instance, vitisin B and ε-viniferin in cv. Pinot Noir and Accent were predominant in 2017 and 2019 (both >90% of total stilbenoids) but not in 2018 (both <55%) when temperatures were high and precipitation low. In brief, we report the variability of stilbenoid levels in grape cane depending upon genetic and environmental factors.

Haze Formation and the Challenges for Peptidases in Wine Protein Fining.

To meet consumer expectations, white wines must be clear and stable against haze formation. Temperature variations during transport and storage may induce protein aggregation, mainly caused by thaumatin like-proteins (TLPs) and chitinases (CHIs), which thus need to be fined before bottling of the wine. Currently, bentonite clay is employed to inhibit or minimize haze formation in wines. Alternatively, peptidases have emerged as an option for the removal of these thermolabile proteins, although their efficacy under winemaking conditions has not yet been fully demonstrated. The simultaneous understanding of the chemistry behind the cleavage of haze proteins and the haze formation may orchestrate alternative methods of technological and economic importance in winemaking. Therefore, we provide an overview of wine fining by peptidases, and new perspectives are developed to reopen discussions on the aforementioned challenges.

Exploring Genotype-by-Environment Interactions of Chemical Composition of Raspberry by Using a Metabolomics Approach.

Promoting the consumption of fruits is a key objective of nutrition policy campaigns due to their associated health benefits. Raspberries are well appreciated for their remarkable flavor and nutritional value attributable to their antioxidant properties. Consequently, one of the objectives of present-day raspberry breeding programs is to improve the fruit's sensory and nutritive characteristics. However, developing new genotypes with enhanced quality traits is a complex task due to the intricate impacts genetic and environmental factors have on these attributes, and the difficulty to phenotype them. We used a multi-platform metabolomic approach to compare flavor- and nutritional-related metabolite profiles of four raspberry cultivars ('Glen Ample', 'Schönemann', 'Tulameen' and 'Veten') grown in different European climates. Although the cultivars appear to be better adapted to high latitudes, for their content in soluble solids and acidity, multivariate statistical analyses allowed us to underscore important genotypic differences based on the profiles of important metabolites. 'Schönemann' and 'Veten' were characterized by high levels of anthocyanins and ellagitannins, respectively, 'Tulameen' by its acidity, and 'Glen Ample' for its content of sucrose and ß-ionone, two main flavor contributors. Our results confirmed the value of metabolomic-driven approaches, which may foster the development of cultivars with enhanced health properties and flavor.

Identification of intact peptides by top-down peptidomics reveals cleavage spots in thermolabile wine proteins.

Prevention of haze formation in wines is challenging for winemakers. Thermolabile proteins in wines, notably thaumatin-like proteins (TLPs) and chitinases (CHIs), undergo structural changes under varying physicochemical conditions, resulting in protein aggregation and visible haze in bottled products. Peptidases are an alternative fining method, although an effective proteolysis under typical winemaking conditions (acidic pH and low temperature) is difficult to achieve. In this study, tryptic peptides from TLPs and CHIs were identified by MS-based peptidomics (top-down proteomics) after exposure of scissile bonds on the protein surface. As proposed by the theory of limited proteolysis, protein conformational changes following temperature and pH variations allowed the detection of enzyme-accessible regions. Protein structure visualization and molecular dynamics simulations were used to highlight cleavage spots and provide the scientific basis for haze formation mechanisms. The described method offers a tool to the search for ideal enzymes to prevent wine haze.

Influence of polysaccharides on wine protein aggregation.

Polysaccharides are the major high-molecular weight components of wines. In contrast, proteins occur only in small amounts in wine, but contribute to haze formation. The detailed mechanism of aggregation of these proteins, especially in combination with other wine components, remains unclear. This study demonstrates the different aggregation behavior between a buffer and a model wine system by dynamic light scattering. Arabinogalactan-protein, for example, shows an increased aggregation in the model wine system, while in the buffer system a reducing effect is observed. Thus, we could show the importance to examine the behavior of wine additives under conditions close to reality, instead of simpler buffer systems. Additional experiments on melting points of wine proteins reveal that only some isoforms of thaumatin-like proteins and chitinases are involved in haze formation. We can confirm interactions between polysaccharides and proteins, but none of these polysaccharides is able to prevent haze in wine.

Temperature variations at nano-scale level in phase transformed nanocrystalline NiTi shape memory alloys adjacent to graphene layers.

The detection and control of the temperature variation at the nano-scale level of thermo-mechanical materials during a compression process have been challenging issues. In this paper, an empirical method is proposed to predict the temperature at the nano-scale level during the solid-state phase transition phenomenon in NiTi shape memory alloys. Isothermal data was used as a reference to determine the temperature change at different loading rates. The temperature of the phase transformed zone underneath the tip increased by ∼3 to 40 °C as the loading rate increased. The temperature approached a constant with further increase in indentation depth. A few layers of graphene were used to enhance the cooling process at different loading rates. Due to the presence of graphene layers the temperature beneath the tip decreased by a further ∼3 to 10 °C depending on the loading rate. Compared with highly polished NiTi, deeper indentation depths were also observed during the solid-state phase transition, especially at the rate dependent zones. Larger superelastic deformations confirmed that the latent heat transfer through the deposited graphene layers allowed a larger phase transition volume and, therefore, more stress relaxation and penetration depth.

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.

Moderate effects of apple juice consumption on obesity-related markers in obese men: impact of diet-gene interaction on body fat content.

PURPOSE: The effect of polyphenol-rich cloudy apple juice (CloA) consumption on plasma parameters related to the obesity phenotype and potential effects of interactions between CloA and allelic variants in obesity candidate genes were assessed in obese men. METHODS: In this controlled, randomized, and parallel study, n = 68, non-smoking, non-diabetic men with a BMI ≥27 kg/m(2) received 750 mL/day CloA (802.5 mg polyphenols) or 750 mL/day control beverage (CB, isocaloric equivalent to CloA) for 4 weeks. Further, study participants were genotyped for single-nucleotide polymorphisms in PPARγ (rs1801282), UCP3 (rs1800849), IL-6 (rs1800795), FABP2 (rs1799883), INSIG2 (rs7566605), and PGC1 (rs8192678) genes. At the beginning and at the end of intervention plasma lipids, distinct adipokines and cytokines as well as anthropometric parameters were determined. RESULTS: CloA compared to CB had no significant effect on plasma lipids, plasma adipokine and cytokine levels, BMI, and waist circumference. However, CloA consumption significantly reduced percent body fat compared to CB (∆ % body fat: CloA: -1.0 ± 1.3 vs. CB: -0.2 ± 0.9, p < 0.05). The IL-6-174 G/C polymorphism showed an interaction with body fat reduction induced by CloA. Solely in C/C, but not in G/C or G/G variants, a significant reduction in body fat after 4 weeks of CloA intervention was detectable. CONCLUSION: The observed diet-gene interaction might be a first indication for the impact of individual genetic background on CloA-mediated bioactivity on obesity-associated comorbidities.

Colonic availability of polyphenols and D-(-)-quinic acid after apple smoothie consumption.

SCOPE: The aim of this study was to determine the amounts of polyphenols and D-(-)-quinic acid reaching the ileostomy bags of probands (and thus the colon in healthy humans) after ingestion of apple smoothie, a beverage containing 60% cloudy apple juice and 40% apple puree. METHODS AND RESULTS: Ten healthy ileostomy subjects each ingested 0.7 L of apple smoothie (a bottle). Their ileostomy bags were collected directly before and 1, 2, 4, 6 and 8 h after smoothie consumption, and the polyphenol and D-(-)-quinic acid contents of the ileostomy fluids were examined using HPLC-DAD and HPLC-MS/MS. The total polyphenol and D-(-)-quinic acid content of the apple smoothie was determined to be 1955.6±124.6 mg/0.7 L, which is very high compared to cloudy apple juices. The most abundant substances found in the ileostomy bags were oligomeric procyanidins (705.6±197.9 mg), D-(-)-quinic acid (363.4±235.5 mg) and 5-caffeoylquinic acid (76.7±26.8 mg). Overall recovery of ingested polyphenols and D-(-)-quinic acid in the ileostomy bags was 63.3±16.1%. CONCLUSIONS: The amounts of polyphenol and D-(-)-quinic acids reaching the ileostomy bags are considerably higher after apple smoothie consumption than after the consumption of cloudy apple juice or cider. These results suggest that the food matrix might affect the colonic availability of polyphenols, and apple smoothies could be more effective in the prevention of chronic colon diseases than both cloudy apple juice and apple cider.

Apple juice intervention modulates expression of ARE-dependent genes in rat colon and liver.

BACKGROUND: The risk of cancer and other degenerative diseases is inversely correlated with consumption of fruits and vegetables. This beneficial effect is mainly attributed to secondary plant constituents such as polyphenols, supposed to play a major role in protection against ROS (reactive oxygen species)-associated toxicity. AIM OF THE STUDY: To elucidate the potential of differently manufactured apple juices (clear AJ/cloudy AJ/smoothie, in comparison with a polyphenol-free control juice) to modulate expression of ARE-dependent genes. METHODS: In male Sprague-Dawley rats (n = 8/group; 10d juice intervention, 4d wash-out; 4 treatment cycles), expression of target genes (superoxide dismutase, SOD1/SOD2; glutathione peroxidase, GPX1/GPX2; γ-glutamylcysteine ligase, GCLC/GCLM; glutathione reductase, GSR; catalase, CAT; NAD(P)H:quinone oxidoreductase-1, NQO1 and transcription factor erythroid-derived 2-like-2, Nrf2) was quantified with duplex RT-PCR, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control. RESULTS: In colon and liver of rats consuming polyphenol-free control juice, rather similar basic expressions were observed (relative GAPDH ratios ranging from 2 to 0.7 and 2.5-0.3, respectively). In the distal colon, apple juice intervention slightly but significantly induced most genes (e.g. GPX2, GSR, CAT, Nrf2; p < 0.001), whereas in the liver only GPX1 and NQO1 mRNA were up-regulated; other hepatic target genes were not affected or down-regulated (SOD1, SOD2, GCLC/M, GSR), concomitant with the absence of Nrf2 induction. Induction of antioxidant gene expression differed with juice type (cloudy AJ > clear AJ ~ smoothie). CONCLUSION: Taken together, the results underline the potential of polyphenol-rich apple juice to increase the expression of ARE-dependent antioxidant genes.