Elke Richling

The Biological Relevance of Direct Antioxidant Effects of Polyphenols for Cardiovascular Health in Humans Is Not Established

Human studies provide evidence for beneficial effects of polyphenol-rich foods on cardiovascular health. The antioxidant activity of polyphenols potentially explains these effects, but is the antioxidant activity a reliable predictor for these effects? An International Life Sciences Institute Europe working group addressed this question and explored the potential of antioxidant claims for polyphenols in relation to cardiovascular health by using the so-called Process for the Assessment of Scientific Support for Claims on Foods project criteria. In this process, analytical aspects of polyphenols, their occurrence in foods, dietary intake, and bioavailability were reviewed. Human studies on polyphenols and cardiovascular health were reviewed together with methods for biomarkers of oxidative damage and total antioxidant capacity (TAC). In retrospective studies, F2-isoprostanes and oxidized LDL, the most reliable biomarkers of lipid peroxidation, and measures for TAC showed the expected differences between cardiovascular disease patients and healthy controls, but prospective studies are lacking, and a causal relationship between these biomarkers and cardiovascular health could not be established. Therefore, the physiological relevance of a potential change in these biomarkers is unclear. We found limited evidence that some types of polyphenol-rich products modify these biomarkers in humans. A direct antioxidant effect of polyphenols in vivo is questionable, however, because concentrations in blood are low compared with other antioxidants and extensive metabolism following ingestion lowers their antioxidant activity. Therefore, the biological relevance of direct antioxidant effects of polyphenols for cardiovascular health could not be established. Overall, although some polyphenol-rich foods exert beneficial effects on some biomarkers of cardiovascular health, there is no evidence that this is caused by improvements in antioxidant function biomarkers (oxidative damage or antioxidant capacity).

Influence of apple polyphenols on inflammatory gene expression

Apples (Malus spp., Rosaceae) and products thereof contain high amounts of polyphenols which show diverse biological activities and may contribute to beneficial health effects, like protecting the intestine against inflammation initiated by chronic inflammatory bowel diseases (IBD). IBD are characterized by an excessive release of several proinflammatory cytokines and chemokines by different cell types which results consequently in an increased inflammatory response. In the present study we investigated the preventive effectiveness of polyphenolic juice extracts and single major constituents on inflammatory gene expression in immunorelevant human cell lines (DLD-1, T84, MonoMac6, Jurkat) induced with specific stimuli. Besides the influence on proinflammatory gene expression, the effect on NF-kappaB-, IP-10-, IL-8-promoter-, STAT1-dependent signal transduction, and the relative protein levels of multiple released cytokines and chemokines were studied. DNA microarray analysis of several genes known to be strongly regulated during gastrointestinal inflammation, combined with quantitative real-time PCR (qRT-PCR) revealed that the apple juice extract AE04 (100-200 microg/mL) significantly inhibited the expression of NF-kappaB regulated proinflammatory genes (TNF-alpha, IL-1beta, CXCL9, CXCL10), inflammatory relevant enzymes (COX-2, CYP3A4), and transcription factors (STAT1, IRF1) in LPS/IFN-gamma stimulated MonoMac6 cells without significant effects on the expression of house-keeping genes. A screening of some major compounds of AE04 revealed that the flavan-3-ol dimer procyanidin B(2 )is mainly responsible for the anti-inflammatory activity of AE04. Furthermore, the dihydrochalcone aglycone phloretin and the dimeric flavan-3-ol procyanidin B(1 )significantly inhibited proinflammatory gene expression and repressed NF-kappaB-, IP-10-, IL-8-promoter-, and STAT1-dependent signal transduction in a dose-dependent manner. The influence on proinflammatory gene expression by the applied polyphenols thereby strongly correlated with the increased protein levels investigated by human cytokine array studies. In summary, we evaluated selected compounds responsible for the anti-inflammatory activity of AE04. In particular, procyanidin B(1), procyanidin B(2), and phloretin revealed anti-inflammatory activities in vitro and therefore may serve as transcription-based inhibitors of proinflammatory gene expression.

Analysis of heterocyclic aromatic amines in wine by high-performance liquid chromatography-electrospray tandem mass spectrometry.

High-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) was used for the analysis of heterocyclic aromatic amines (HAAs) in 25 wines from various regions. In 24 wines under study HAAs were detected in the low ng/l range. 2-Amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C) were found to be the most widely distributed HAAs in the wines under study. There was a difference in the presence of HAAs in white and red wines. In the latter, higher concentrations were found, with MeA alpha C in amount ranging from < 7.5 to 107 ng/l. Compared to the quantities of HAAs evaluated in meat and meat products, the amounts found in wines are 100-1000-fold lower.

Preparation and Comparative Release Characteristics of Three Anthocyanin Encapsulation Systems

Bilberries (Vaccinium myrtillus L.) and their major polyphenolic constituents, anthocyanins, have preventive activities inter alia against colon cancer and inflammatory bowel diseases. However, anthocyanins are sensitive to environmental conditions; thus their bioavailability in the gastrointestinal tract is an important determinant of their in vivo activity. In the study reported here, the potential benefits of encapsulating an anthocyanin rich bilberry extract (BE) on anthocyanin stability were investigated. Nonencapsulated BE and three different BE loaded microcapsule systems were incubated in simulated gastric fluid (SGF) and fed state simulated intestinal fluid (FeSSIF). After exposure to these media, released anthocyanins were identified and quantified by HPLC with UV/Vis detection. Although a rapid release of anthocyanins was observed within the first 20 min, encapsulation of anthocyanins doubled the amount of available anthocyanins after 150 min of incubation. These results illustrate the ability of encapsulation to inhibit early degradation of anthocyanins in the intestinal system.

High performance liquid chromatography analysis of anthocyanins in bilberries (Vaccinium myrtillus L.), blueberries (Vaccinium corymbosum L.), and corresponding juices.

UNLABELLED In the present study the anthocyanin content of commercially available bilberry juices and fresh fruits were quantified by using 15 authentic anthocyanin standards via high performance liquid chromatography with an ultra-violet detector (HPLC-UV/VIS). Delphinidin-3-O-glucopyranoside, delphinidin-3-O-galactopyranoside, and cyanidin-3-O-arabinopyranoside were the major anthocyanins found in juices, nectar, and fresh bilberries. In contrast, fresh blueberries had higher concentrations of malvidin-3-O-arabinopyranoside and petunidin-3-O-galactopyranoside. Up to 438 mg anthocyanins per 100 g fresh weight (2762 mg/100 g dry weight (DW)) were detected in blueberries from various sources, whereas bilberries contained a maximum of 1017 mg anthocyanins per 100 g fresh weight (7465 mg/100 g DW). Commercially available bilberry and blueberry juices (n= 9) as well as nectars (n= 4) were also analyzed. Anthocyanin concentrations of juices (1610 mg/L to 5963 mg/L) and nectar from bilberries (656 mg/L to 1529 mg/L) were higher than those of blueberry juices (417 mg/L) and nectar (258 mg/L to 386 mg/L). We conclude that using several authentic anthocyanin references to quantify anthocyanin contents indicated them to be up to 53% and 64% higher in fresh bilberries and blueberries, respectively, than previously reported using cyanidin-3-O-glucoside. This study has also demonstrated that commercially available juices produced from bilberries contain much higher anthocyanin concentrations than those from blueberries. PRACTICAL APPLICATION We have investigated the contents of a special class of antioxidants, namely anthocyanins in blueberry and billberry fruits and juices commercially available in Germany. To achieve reliable data we have used authentic standards for the first time. We think that our results are important in the field of nutritional intake of this important class of polyphenols and fruit juice companies get a closer insight in the occurrence of these antioxidants in market samples to be used in food composition databases and for nutritional survey.

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.

High performance liquid chromatography —Electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) for the analysis of heterocyclic aromatic amines (HAA)

SummarySensitive and selective detection of the sixteen most abundant heterocyclic aromatic amines (HAA) has been achieved by application of high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) in combination with selected reaction monitoring (SRM). Detection limits between 0.1 and 50 ng mL−1 were established by use of HAA model solutions.

Dose‐dependent absorption of chlorogenic acids in the small intestine assessed by coffee consumption in ileostomists

SCOPE Until now, the question of how the ingested doses of chlorogenic acids (CGA) from coffee influence their absorption and metabolism remains unresolved. To assess absorption in the small intestine, we performed a dose-response study with a randomized, double-blinded, crossover design with ileostomist subjects. METHODS AND RESULTS After a polyphenol-free diet, the volunteers consumed, on three separate occasions, coffee with different total CGA contents (high 4525 μmol; medium 2219 μmol; low 1053 μmol). CGA concentrations in plasma, ileal effluent, and urine were subsequently determined by HPLC-DAD-ESI-MS and -ESI-MS/MS. The results show that the consumption of higher CGA concentrations leads to a faster ileal excretion. This corresponds to a renal excretion of 8.0 ± 4.9% (high), 12.1 ± 6.7% (medium), and 14.6 ± 6.8% (low) of total CGA and metabolites. Glucuronidation of CGA became slightly greater with increasing dose. After enzyme treatment, the area under the curve (AUC)(0-8h) for CGA metabolites in plasma was 4412 ± 751 nM × h(0-8) (-1) (high), 2394 ± 637 nM × h(0-8) (-1) (medium), 1782 ± 731 nM × h(0-8) (-1) (low), respectively. Additionally, we were able to identify new metabolites of CGA in urine and ileal fluid. CONCLUSION We conclude that the consumption of high CGA concentrations via coffee might influence the gastrointestinal transit time and consequently affect CGA absorption and metabolism.

Application of atmospheric pressure ionization HPLC-MS-MS for the analysis of natural products

SummaryThe development of techniques utilizing atmospheric pressure ionization, namely atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), has pioneered the coupling of liquid chromatography (HPLC) with mass spectrometry in recent years. Both ESI and APCI generate ions from polar and labile biomaterials with remarkable ease and efficiency. In particular, the use of HPLC with tandem mass spectrometry (MS-MS) opens further dimensions in the field of bioorganic analysis. Thus, HPLC-MS-MS provides the tools for direct elucidation of the structure and variety of polar natural compounds in complex matrices. In order to develop efficient and straightforward strategies for the analysis of polar natural products, the potential and the limitations of these hyphenated analytical techniques are discussed using heterocyclic aromatic amines, fumonisins, acylated glycoconjugates and regioisomeric fatty acid hydroperoxides as examples.

Anthocyanin-rich Purple Wheat Prolongs the Life Span of Caenorhabditis elegans Probably by Activating the DAF-16/FOXO Transcription Factor

Colored cereals attract public attention due to their potential antioxidant properties and corresponding health benefits. Purple wheat, rich in anthocyanins, is one of the newly developed cereals on the market. Cyanidin-3-O-glucoside (42.6%) is the predominant anthocyanin in purple wheat, followed by peonidin-3-O-glucoside (39.9%) and malvidin-3-O-galactoside (17.4%). To investigate the potential antiaging and antioxidant properties of purple wheat, the nematode Caenorhabditis elegans was chosen as an experimental model organism. It was found that an anthocyanin-rich methanolic extract of purple wheat extended the mean life span of wild type worms and of mev-1(hn1) mutants, which are sensitive to oxidative stress, by 10.5 and 9.2%, respectively. Life span extension depends on the transcription factor DAF-16; no significant increase of longevity was seen in daf-16 (mgDf50) mutant worms. Translocation of DAF-16/FOXO to the nucleus implies that the transcription factor DAF-16/FOXO was activated under purple wheat treatment by inhibition of the insulin/IGF-1-like signaling pathway which includes the insulin receptor DAF-2. Moreover, purple wheat increased stress response in C. elegans as well as reduced oxidative stress. Anthocyanins of purple wheat apparently exhibit beneficial effects in C. elegans. They may exert similar properties in humans, which is an issue to be explored in future studies.