R. Bitsch

Assessment of antioxidant activity by using different in vitro methods

In this study, six common tests for measuring antioxidant activity were evaluated by comparing four antioxidants and applying them to beverages (tea and juices): Trolox equivalent antioxidant capacity assay (TEAC I-III assay), Total radical-trapping antioxidant parameter assay (TRAP assay), 2,2-diphenyl- l -picrylhydrazyl assay (DPPH assay), N , N -dimethyl- p -phenylendiamine assay (DMPD assay), Photochemiluminescence assay (PCL assay) and Ferric reducing ability of plasma assay (FRAP assay). The antioxidants included gallic acid representing the group of polyphenols, uric acid as the main antioxidant in human plasma, ascorbic acid as a vitamin widely spread in fruits and Trolox ® as water soluble vitamin E analogue. The six methods presented can be divided into two groups depending on the oxidising reagent. Five methods use organic radical producers (TEAC I-III, TRAP, DPPH, DMPD, PCL) and one method works with metal ions for oxidation (FRAP). Another difference between these tests is the reaction procedure. Three assays use the delay in oxidation and determine the lag phase as parameter for the antioxidant activity (TEAC I, TRAP, PCL). They determine the delay of radical generation as well as the ability to scavenge the radical. In contrast, the assays TEAC II and III, DPPH, DMPD and FRAP analyse the ability to reduce the radical cation (TEAC II and III, DPPH, DMPD) or the ferric ion (FRAP). The three tests acting by radical reduction use preformed radicals and determine the decrease in absorbance while the FRAP assay measures the formed ferrous ions by increased absorbance. Gallic acid was the strongest antioxidant in all tests with exception of the DMPD assay. In contrast, uric acid and ascorbic acid showed low activity in some assays. Most of the assays determine the antioxidant activity in the micromolar range needing minutes to hours. Only one assay (PCL) is able to analyse the antioxidant activity in the nanomolar range. Black currant juice showed highest antioxidant activity in all tests compared to tea, apple juice and tomato juice. Despite these differences, results of these in vitro assays give an idea of the protective efficacy of secondary plant products. It is strongly recommended to use at least two methods due to the differences between the test systems investigated.

Bioavailability and Biokinetics of Anthocyanins From Red Grape Juice and Red Wine

In a comparative study, 9 healthy volunteers ingested a single oral dose of 400 mL red grape juice or red wine with dose-adjusted anthocyanin content (283.5 mg or 279.6 mg, resp) in crossover. The content of anthocyanin glucosides was detected in plasma and urinary excretion. Additionally, the plasmatic antioxidant activity was assessed after intake. Based on the plasma content, biokinetic criteria of the single anthocyanins were calculated, such as AUC, cmax, tmax, and the elimination rate t1/2. The urinary excretion of total anthocyanins differed significantly and amounted to 0.18% (red wine) and 0.23% (red grape juice) of the administered dose. Additionally, the plasmatic antioxidant activity increased to higher levels after juice ingestion compared to wine. The intestinal absorption of the anthocyanins of red grape juice seemed to be improved compared to red wine, suggesting a possible synergistic effect of the glucose content of the juice. The improved absorption resulted in an enhanced plasmatic bioactivity.

Effect of grape processing on selected antioxidant phenolics in red wine

Wine, particularly red wine, is an important source of polyphenols and several studies have shown that moderate wine consumption is associated with a reduced risk of coronary heart disease. It has been hypothesized that these antioxidant compounds may be responsible for the potential beneficial effects of wine. The influence of different vinification techniques (fermentation on skin [A], mash heating [B], and the combination of both [C]) on the antioxidant capacity and the phenolic composition of red wines (Spatburgunder [Pinot Noir], Lemberger, and Cabernet Franc) were tested in the present study. The highest concentrations of anthocyanins, flavan-3-ols, flavonols, stilbenes, and antioxidant capacity were found in the red wines which were produced under the conditions of C, followed by B and A.

In vivo antioxidative capacity of a composite berry juice

In order to test the health protective potential of a special antioxidant-rich juice (containing 30% white grape-, 25% black-currant-, 15% elderberry-, 10% sour cherry-, 10% blackberry- and 10% aronia-juice), the bioavailability of its most important bioactive compounds (anthocyanins and ascorbic acid) and the influence of juice consumption on plasma antioxidant capacity and plasma malondialdehyde (MDA) was assessed by six healthy volunteers. The juice ingestion (400 ml) resulted in a significantly increased plasmatic antioxidant capacity after 2 h (30%) and significantly decreased plasma MDA after 4 h (18%). The cumulative urinary excretion of ascorbic acid and anthocyanins was 79 and 0.06% of the ingested amount. From the present findings it can be concluded that various juice antioxidants are variably absorbed and are active as antioxidants in vivo.

Rosehip -- a "new" source of lycopene?

Lycopene, an efficient antioxidant and singlet oxygen quencher, is mainly delivered within the human diet out of tomatoes and tomato products. Processing liberates this carotenoid from complexes with proteins and thus makes it more bioavailable. Rosehip, a wild fruit which is used more often recently to produce mark, jams and juices, showed remarkable contents of lycopene (12.9-35.2 mg/100 g) with an unexpected isomer pattern.

Effects of ingestion of tomatoes, tomato juice and tomato puree on contents of lycopene isomers, tocopherols and ascorbic acid in human plasma as well as on lycopene isomer pattern

Tomatoes are an important part of the diet. Lycopene, the predominant carotenoid in tomatoes, is hypothesised to mainly mediate the health benefits of tomato products. Anticancer activity of tomato products and lycopene has been suggested by numerous studies. The aim of the present study was to investigate the effect of ingestion of three different tomato-based foodstuffs on plasma contents of lycopene, tocopherols and ascorbic acid. Because isomers of lycopene may have different biological activities, a special interest was to look how the lycopene isomer pattern is changed depending on the matrix of tomato products. Following a 2-week depletion phase volunteers ingested 12.5 mg lycopene/d for 4 weeks comprising tomatoes, tomato juice or tomato purée. The basal levels of lycopene in plasma were comparable for all groups and decreased significantly during the 2 weeks of depletion to approximately half of the basal values. Following intervention, plasma lycopene concentration increased significantly. Conversely, supplementation did not significantly affect levels of tocopherols and ascorbic acid in plasma. Regarding isomers of lycopene, the (Z)-lycopene:(all-E)-lycopene plasma isomer ratio was significantly changed during the study for all groups. A remarkable enrichment of the relative contents of (5Z)-lycopene was observed during the depletion period, which supports the hypothesis that lycopene (Z)-isomers are formed within the human body after ingestion of (all-E)-lycopene. After dietary intervention with lycopene-rich products the isomer ratios returned to those observed at the start of the study. Further investigations will clarify the process of isomerisation in more detail.

Urinary Excretion of Cyanidin Glucosides and Glucuronides in Healthy Humans After Elderberry Juice Ingestion

In a pilot study with 6 females and 1 male, the metabolism of various cyanidin glucosides after oral administration of elderberry juice was investigated. The anthocyanin metabolites were detected in urinary excretion. After ingestion of a bolus quantity of 3.57 g total anthocyanins in a 150 mL elderberry juice concentrate, 0.053% of the administered dose was excreted in urine as glucosidically bound cyanidins within the first 5 hours. Only 0.003% of the ingested anthocyanin glucosides was excreted as cyanidin glucuronide, suggesting that this conversion step might be of minor importance in urinary excretion.

Assessment of thiamin status in chronic renal failure patients, transplant recipients and hemodialysis patients receiving a multivitamin supplementation.

The thiamin status of patients with chronic renal failure (CRF, n = 14), dialysis patients (DP, n = 24) and patients after renal transplantation (RT, n = 19) was assessed. Thiamin intake was calculated at mean levels of 1.26 mg/d (CRF), 0.83 mg/d (DP) and 1.42 mg/d (RT). Corresponding mean plasma concentrations were 64.2 nmol/l (CRF), 78.3 nmol/l (DP) and 55.1 nmol/l (RT). Thiamin supplements of 1.5 mg or 8.0 mg orally given to patients of the DP-group after each dialysis session showed slightly higher thiamin concentrations in plasma. Transketolase activity coefficients (ETK-AC) were in the same range (1.11...1.19) except for RT-patients who had a slightly but not significantly higher ETK-AC of 1.22. During dialysis treatment (DT), thiamin plasma concentrations dropped to 75 and/or 82% in patients supplemented with 1.5 and/or 8.0 mg. They both reached initial levels again 44 hours later. Despite large inter-individual differences, thiamin concentrations increased in the non-supplemented DP-group. ETK-AC did not change after a 14-day interruption of supplementation and did not deteriorate after a single dialysis session, both in supplemented and non-supplemented patients. A daily thiamin supplementation which complies with the RDA for healthy subjects is indicated in DP and is sufficient to keep thiamin status within the normal range.

High thiamine diphosphate concentrations in erythrocytes can be achieved in dialysis patients by oral administration of benfotiamine

AbstractObjective: The influence of either orally administered S-benzoylthiamine-O-monophosphate (benfotiamine) or thiamine nitrate on the thiamine status was tested in a randomised, two-group comparison study in 20 end-stage renal disease (ESRD) patients. Main outcome measures were the pharmacokinetics of thiamine diphosphate (TDP) in blood, the in vitro erythrocyte transketolase activity, its activation coefficient (α-ETK) and the TDP concentration in erythrocytes. Methods: After ingestion of a single dose of either 100 mg thiamine nitrate (corresponding to 305 μmol thiamine) or 100 mg benfotiamine (corresponding to 214 μmol thiamine), the blood levels of thiamine phosphate esters were analysed by means of high-performance liquid chromatography for a 24-h period. The TDP concentration in erythrocytes was calculated using the haematocrit and TDP concentration in blood. Erythrocyte transketolase activity and α-ETK were measured before and 10 h after administration. The pharmacokinetics of TDP in blood were compared with healthy subjects of other studies retrieved from database query. Results: Regarding the blood concentrations of TDP, the patients with ESRD had a 4.3 times higher area under the concentration–time curve after benfotiamine administration than after thiamine nitrate. After benfotiamine administration, the peak plasma concentration of TDP exceeded that in healthy subjects by 51%. In the ESRD patients, after 24 h, the mean TDP concentration in erythrocytes increased from 158.7 ± 30.9 ng/ml initially to 325.8 ± 50.9 ng/ml after administration of benfotiamine and from 166.2 ± 51.9 ng/ml to 200.5 ± 50.0 ng/ml after thiamine nitrate administration. The ratio between the maximum erythrocyte TDP concentration and basal concentration was 2.66 ± 0.6 in the benfotiamine group and 1.44 ± 0.2 in the group receiving thiamine nitrate (P < 0.001). After 24 h, it was 2.11 ± 0.4 and 1.23 ± 0.2, respectively. The transketolase activity increased from 3.54 ± 0.7 μkat/l initially to 3.84 ± 0.6 μkat/l after benfotiamine intake (P=0.02) and from 3.71 ± 0.8 μkat/l to 4.02 ± 0.7 μkat/l after thiamine nitrate intake (P=0.08). Likewise, α-ETK decreased from initially 1.10 ± 0.07 to 1.04 ± 0.04 (P=0.04) and from 1.12 ± 0.05 to 1.08 ± 0.06 (P=0.09). After 24 h, the phosphorylation ratio in whole blood decreased from 12.9 ± 6.9 initially to 5.6 ± 3.2 after benfotiamine administration (P=0.02) and from 13.5 ± 7.3 to 9.0 ± 4.8 (P=0.03) after administration of thiamine nitrate. No correlation between erythrocyte TDP concentration and transketolase activity and/or α-ETK was observed in ESRD patients, either before or 10 h after administration. Conclusion: Compared with thiamine nitrate, the oral administration of benfotiamine leads to higher TDP concentrations in erythrocytes accompanied with a significant improvement of the erythrocyte transketolase activity in ESRD patients.

Bioavailability of antioxidative compounds from Brettacher apple juice in humans

The human bioavailability of ascorbic acid and polyphenolics such as chlorogenic acid and caffeic acid resp. from apple juice produced from the polyphenolic rich variety Brettacher was tested. After intake of 700 ml juice the antioxidant capacity in the plasma of human volunteers assessed by the TRAP-test increased significantly by 52% during the following 2 h. The cumulative urinary excretion of ascorbic acid within 7 h after intake was measured to 104% the ingested dose of the juice. Caffeic acid as the intestinal cleavage product of chlorogenic acid was excreted to only 0.56% of the dose in the juice. The results demonstrate in view of the favourable absorptive attributes and the influence on the plasmatic antioxidative capacity of these juice components that Brettacher apple juice may be suitable as a functional food.