Proanthocyanidins in barley and malt analyzed by pressurized liquid extraction, solid-phase extraction and HPLC

Aim of this work is to develop a convenient method for the determination of proanthocyanidins in barley and malt. In a second step this method is applied to 61 barley and malt samples of different varieties, proveniences and growing years. In the brewing industry proanthocyanidins are of special interest. Main activities of the proanthocyanidins are related to undesired formation of chill haze and to the positively valued augmentation of the antioxidative capacity of beer. The detailed mechanisms are still under discussion. It is clear, that the positive and negative effects of proanthocyanidins depend on their quantity and quality. So determination of the sum of proanthocyanidins does not give sufficient information to discuss their action. Selective analysis of proanthocyanidins is time and labor consuming.  Especially sample preparation requires a lot of manual work. Thus,  this work presents a fully automated and therefore fast and reliable  method for sample preparation of barley and malt followed by HPLC-UV. The here described method bases on extraction using pressurized  liquid extraction (PLE). Essentially it is a static solid/liquid  extraction with high pressure and eventually high temperature in  stainless steel extraction cells. Using the Accelerated Solvent  Extractor (ASE) by Dionex, up to 24 samples in a series can be  extracted automatically. For the first time, commercially available  polyamide cartridges are used for proanthocyanidins. SPE is  accomplished automatically by a liquid handling robot, the Automated  Sample Preparation with Extraction Cartridges-device (ASPEC) by  ABIMED and Gilson. The aspec takes the extracts from the ase and  carries out the complete spe procedure. The resulting solution is  ready to inject into the HPLC, that separates and quantifies six  proanthocyanidins and catechin in one run of 90 min. Sample extraction and extract clean-up are coupled online. This  coupling was developed by abimed and Dionex and is tested and  established under real laboratory conditions for the first time.  Within 24 hours 16 samples can be analyzed, about 6 hours of manual  work is needed. Recovery of the overall method is 70–91%,  reproducibility is 2.3–6.4%. With this method 61 barley and malt samples of the growing years 1998– 2001 from four locations including summer and winter barley varieties  are analyzed. The annual and local variation of absolute contents of  proanthocyanidins appears to interfere varietal differences, so  differentiation between the samples is not possible. The ratio of  several pairs of proanthocyanidins (the relative quantitative  polyphenolic fingerprint) is characteristic for the variety and can  be used to control authenticity. In addition, the here presented method is supposed to be applicable  to samples taken during the brewing process and to other food  samples. Two examples are given: monitoring beer filtration and  analyzing proanthocyanidins in the seeds of the açaí fruit from  northern Brazil. Since proanthocyanidins are discussed to have  positive effects on health, there is a market for functional food  with naturally high or enriched content of proanthocyanidins. Hence  it is necessary to control such products.Proanthocyanidine in Gerste und Malz analysiert mit Hochdruck-Flüssig-Extraktion, Festphasenaufreinigung und HPLC Ziel dieser Arbeit ist, eine leicht durchf ührbare Methode zur  selektiven Bestimmung von Proanthocyanidinen in Gerste und Malz zu  entwickeln. Als zweiter Schritt wird diese Methode auf 61 Gersten-  und Malzproben verschiedener Sorten, Anbauorte und Jahrgänge angewandt. Im Brauereiwesen spielen die Proanthocyanidine eine wichtige Rolle.  Die wichtigsten Eigenschaften der Proanthocyanidine sind die Bildung  von unerwünschten Kältetrübungen und die positiv bewertete Erhöhung  der antioxidativen Kapazität des Bieres. Die einzelnen Reaktionen und  genauen Mechanismen sind noch Gegenstand wissenschaftlicher  Diskussion. Fest steht, dass die positiven und negativen Effekte der  Proanthocyanidine von ihrer Art und Menge abhängen. Daher bringt die  Bestimmung von Summenparametern keine ausreichende Information, um  die Wirkungen zu diskutieren. Die selektive Analyse von Proanthocyanidinen ist sowohl zeit- als  auch arbeitsaufwendig – vor allem die Probenvorbereitung erfordert  viel Handarbeit. Hier wird eine vollautomatische und daher schnelle  und zuverlässige Methode für die Probenvorbereitung von Gerste und  Malz zur anschließenden HPLC-UV-Analyse vorgestellt. Die hier beschriebene Methode basiert auf der Extraktion mittels  beschleunigter Lösemittelextraktion (pressurized liquid extraction  (PLE)). Im wesentlichen ist dies eine statische fest/flüssig- Extraktion unter hohem Druck und ggf. hoher Temperatur in  Edelstahlzellen. Mit dem Accelerated Solvent Extractor (ASE) von  Dionex können bis zu 24 Proben in Serie automatisch extrahiert werden. Der zweite Schritt der Probenvorbereitung ist die Aufreinigung mit  Festphasenextraktion (solid-phase extraction (SPE)). Zum ersten Mal  werden kommerziell erhältliche Polyamid-Kartuschen zur Aufreinigung  von Proanthocyanidinen eingesetzt. Die SPE wird vollautomatisch von  einem Pipettierrobotor, dem Automated Sample Preparation with  Extraction Cartridges-Gerät (ASPEC) von ABIMED und Gilson,  durchgeführt. Der ASPEC übernimmt die Extrakte von der ASE und führt  die komplette SPE durch. Die resultierende Lösung kann direkt in die  HPLC injiziert werden, die sechs Proanthocyanidine und Catechin  innerhalb eines Laufes von 90 min trennt. Die Probenextraktion und -aufreinigung sind online gekoppelt. Diese  Kopplung wurde von ABIMED und Dionex entwickelt und wird zum ersten  Mal unter realen Labor-Bedingungen getestet und eingesetzt. Innerhalb  von 24 Stunden können 16 Proben analysiert werden, dabei sind etwa 6  Stunden Personaleinsatz erforderlich. Die Wiederfindung der gesamten  Methode beträgt 70–91%, die Reproduzierbarkeit 2.3–6.4%. Mit dieser Methode wurden 61 Gersten- und Malzsorten, darunter  Sommer- und Wintergersten der Jahrgänge 1998–2001 von vier Anbauorten  analysiert. Die jährlichen und lokalen Unterschiede überlagern die  Sortenunterschiede, so dass keine Differenzierung zwischen den Proben  möglich ist. Das Verhältnis bestimmter Paare von Proanthocyanidinen  (der relative quantitative polyphenolische Fingerabdruck) ist jedoch  charakteristisch für die Gerstensorte und kann zur  Authentizitätskontrolle genutzt werden. Außerdem kann die hier vorgestellte Methode auch eingesetzt werden,  um Proben aus dem Brauprozess oder andere Lebensmittel zu  untersuchen. Zwei Beispiele sind angeführt: die Überwachung der  Bierfiltration und die Analyse von Samen der nordbrasilianischen Açaí- Frucht. Da Proanthocyanidine als gesundheitsförderlich diskutiert  werden, gibt es einen Markt für Lebensmittel, die natürlicherweise  viele Proanthocyanidine enthalten oder damit angereichert sind.  Daraus ergibt sich die Notwendigkeit, solche Produkte zu überprüfen

Antioxidant Capacity and Polyphenolic Composition as Quality Indicators for Aqueous Infusions of Salvia officinalis L. (sage tea)

Sage (Salvia officinalis L.) is used as an herbal medicinal product, with the most typical form of application as infusion with boiling water (sage tea). The well-established traditional uses include symptomatic treatment of mild dyspeptic complaints, the treatment of inflammations in the mouth and the throat, and relief of excessive sweating and relief of minor skin inflammations. In this study, sage teas prepared from commercially available products were chemically analyzed for polyphenolic content using liquid chromatography, for antioxidant potential using the oxygen radical absorbance capacity method, and for the Folin–Ciocalteu (FC) index. The sage teas showed a high variation for all parameters studied (up to 20-fold differences for rosmarinic acid). Univariate and multivariate analyses showed that the antioxidant potential, which varied between 0.4 and 1.8 mmol trolox equivalents/100 mL, was highly dependent on rosmarinic acid and its derivatives. The FC index also showed a high correlation to these polyphenols, and could therefore be used as a screening parameter for sage tea quality. The considerable differences in polyphenolic composition and antioxidant capacity between the brands lead to a demand for quality standardization, especially if these sage teas are to be used for therapeutic purposes. Further research also appears to be necessary to characterize the dose–benefit relationship, as sage may also contain a constituent (thujone) with potentially adverse effects

Efficacy and Safety of Pomegranate Medicinal Products for Cancer

Preclinical in vitro and in vivo studies demonstrate potent effects of pomegranate preparations in cancer cell lines and animal models with chemically induced cancers. We have carried out one systematic review of the effectiveness of pomegranate products in the treatment of cancer and another on their safety. The PubMed search provided 162 references for pomegranate and cancer and 122 references for pomegranate and safety/toxicity. We identified 4 clinical studies investigating 3 pomegranate products, of which one was inappropriate because of the low polyphenol content. The evidence of clinical effectiveness was poor because the quality of the studies was poor. Although there is no concern over safety with the doses used in the clinical studies, pomegranate preparations may be harmful by inducing synthetic drug metabolism through activation of liver enzymes. We have analysed various pomegranate products for their content of anthocyanins, punicalagin, and ellagic acid in order to compare them with the benchmark doses from published data. If the amount of coactive constituents is not declared, patients risk not benefiting from the putative pomegranate effects. Moreover, pomegranate end products are affected by many determinants. Their declaration should be incorporated into the regulatory guidance and controlled before pomegranate products enter the market

No role for epigallocatechin gallate (EGCG)

Consumption of tea is inversely associated with cardiovascular diseases. However, the active compound(s) responsible for the protective effects of tea are unknown. Although many favorable cardiovascular effects in vitro are mediated by epigallocatechin gallate (EGCG), its contribution to the beneficial effects of tea in vivo remains unresolved. In a randomised crossover study, a single dose of 200 mg EGCG was applied in three different formulas (as green tea beverage, green tea extract (GTE), and isolated EGCG) to 50 healthy men. Flow-mediated dilation (FMD) and endothelial-independent nitro-mediated dilation (NMD) was measured before and two hours after ingestion. Plasma levels of tea compounds were determined after each intervention and correlated with FMD. FMD significantly improved after consumption of green tea containing 200 mg EGCG (p < 0.01). However, GTE and EGCG had no significant effect on FMD. NMD did not significantly differ between interventions. EGCG plasma levels were highest after administration of EGCG and lowest after consumption of green tea. Plasma levels of caffeine increased after green tea consumption. The results show that EGCG is most likely not involved in improvement of flow-mediated dilation by green tea. Instead, other tea compounds, metabolites or combinations thereof may play a role

Quantitative evaluation of the beneficial effects in the mdx mouse of epigallocatechin gallate, an antioxidant polyphenol from green tea

In two separate previous studies, we reported that subcutaneous (sc) or oral administration of (−)-epigallocatechin-3-gallate (EGCG) limited the development of muscle degeneration of mdx mice, a mild phenotype model for Duchenne muscular dystrophy (DMD). However, it was not possible to conclude which was the more efficient route of EGCG administration because different strains of mdx mice, periods of treatment and methods of assessment were used. In this study, we investigated which administration routes and dosages of EGCG are the most effective for limiting the onset of dystrophic lesions in the same strain of mdx mice and applying the same methods of assessment. Three-week-old mdx mice were injected sc for 5 weeks with either saline or a daily average of 3 or 6 mg/kg EGCG. For comparison, age-matched mdx mice were fed for 5 weeks with either a diet containing 0.1% EGCG or a control diet. The effects of EGCG were assessed quantitatively by determining the activities of serum muscle-derived creatine kinase, isometric contractions of triceps surae muscles, integrated spontaneous locomotor activities, and oxidative stress and fibrosis in selected muscles. Oral administration of 180 mg/kg/day EGCG in the diet was found the most effective for significantly improving several parameters associated with muscular dystrophy. However, the improvements were slightly less than those observed previously for sc injection started immediately after birth. The efficacy of EGCG for limiting the development of dystrophic muscle lesions in mice suggests that EGCG may be of benefit for DMD patients

Altered Error Processing following Vascular Thalamic Damage: Evidence from an Antisaccade Task

Event-related potentials (ERP) research has identified a negative deflection within about 100 to 150 ms after an erroneous response – the error-related negativity (ERN) - as a correlate of awareness-independent error processing. The short latency suggests an internal error monitoring system acting rapidly based on central information such as an efference copy signal. Studies on monkeys and humans have identified the thalamus as an important relay station for efference copy signals of ongoing saccades. The present study investigated error processing on an antisaccade task with ERPs in six patients with focal vascular damage to the thalamus and 28 control subjects. ERN amplitudes were significantly reduced in the patients, with the strongest ERN attenuation being observed in two patients with right mediodorsal and ventrolateral and bilateral ventrolateral damage, respectively. Although the number of errors was significantly higher in the thalamic lesion patients, the degree of ERN attenuation did not correlate with the error rate in the patients. The present data underline the role of the thalamus for the online monitoring of saccadic eye movements, albeit not providing unequivocal evidence in favour of an exclusive role of a particular thalamic site being involved in performance monitoring. By relaying saccade-related efference copy signals, the thalamus appears to enable fast error processing. Furthermore early error processing based on internal information may contribute to error awareness which was reduced in the patients

Profiling Trait Anxiety: Transcriptome Analysis Reveals Cathepsin B (Ctsb) as a Novel Candidate Gene for Emotionality in Mice

Behavioral endophenotypes are determined by a multitude of counteracting but precisely balanced molecular and physiological mechanisms. In this study, we aim to identify potential novel molecular targets that contribute to the multigenic trait “anxiety”. We used microarrays to investigate the gene expression profiles of different brain regions within the limbic system of mice which were selectively bred for either high (HAB) or low (LAB) anxiety-related behavior, and also show signs of comorbid depression-like behavior

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Can home-brewed Benifuuki green tea deliver health-relevant amounts of 3''-O-methyl epigallocatechin gallate?

Benifuuki is a cultivar of tea (Camellia sinensis) that contains 3''-O-methylated epigallocatechin gallate (EGCg3''Me). In vitro, animal and human studies have shown positive health effects of Benifuuki tea (or more specifically of EGCg3''Me), such as allergy mitigation or prevention of cardiovascular diseases. In this article, suitability of the ISO 14502-2 method (slightly modified), that has been set up for the analysis of other flavanols in tea, was shown to be suitable for EGCg3''Me and validated. Eight Benifuuki green tea samples from Japan were analyzed and contained 2.3 to 10.5 mg EGCg3''Me per g in the leaves. Additionally, tea infusions were brewed according to the suppliers' recommendations and at 100 °C. The content of EGCg3''Me in the infusions was compared to the effective doses in human trials. Three Benifuuki infusions brewed at recommended temperatures of 60 or 85 °C deliver in less than 2.5 L at least 34 mg of EGCg3''Me, which is the minimal dose showing health effects in human studies. Using water of 100 °C, the EGCg3''Me concentration in the infusions is roughly doubled. Some infusions exceeded the safe level of caffeine of 400 mg within drinkable volumes

Screening of Thymoquinone Content in Commercial <i>Nigella sativa</i> Products to Identify a Promising and Safe Study Medication

(1) Background: Thymoquinone (TQ) is the leading compound accounting for the pharmacological effects of Nigella sativa seed oil, also known as black seed oil. This study aimed to analyze the TQ content of commercial black seed oils and black seed oil-containing capsules to obtain information on the quality of the products and to find a promising and safe study medication for a putative clinical study. (2) Methods: Six black seed oils and five black seed oil-containing capsules were analyzed. TQ was quantified using a validated method consisting of a simple methanolic extraction and a fast HPLC-UV analysis. (3) Results: The TQ content varied from 3.08 to 809.4 mg/100 g (mean). The highest TQ content was found in a bottled oil, which might be considered for a clinical study. A dose of 4 mL of this oil per day contains 30 mg TQ, which is unlikely to be harmful. Based on the literature, a safe daily TQ dosage appears to be <48.6 mg per adult. (4) Conclusions: These findings suggest that black seed products should be regulated regarding TQ content to enable consumers to buy black seed food supplements of known content for the maintenance and improvement of health