Lilian B.M. Tijburg

The antioxidant properties of theaflavins and their gallate esters — radical scavengers or metal chelators?

The antioxidant properties of theaflavins and their gallate esters were studied by investigating their abilities to scavenge free radicals in the aqueous and lipophilic phases. The total relative antioxidant activities in the aqueous phase were assessed by measuring their direct ABTS·+ radical scavenging abilities, and by their efficacies in inhibiting the degradation of deoxyribose induced by iron. The propensities for enhancing the resistance of LDL to oxidation mediated by Cu2+ were also measured. The results show that the hierarchy of reactivity of these compounds as antioxidants is: theaflavin digallate > 3′‐monogallate=3‐monogallate > theaflavin. Spectroscopic studies show that all the compounds chelate iron and copper; enhanced absorbance in the visible region is observed in the case of the iron‐digallate complex, but not with copper.

Bioavailability of catechins from tea : the effect of milk

Objectives: To assess the blood concentration of catechins following green or black tea ingestion and the effect of addition of milk to black tea.Design: Twelve volunteers received a single dose of green tea, black tea and black tea with milk in a randomized cross-over design with one-week intervals. Blood samples were drawn before and up to eight hours after tea consumption.Setting: The study was performed at the Unilever Research Vlaardingen in The Netherlands.Subjects: Twelve healthy adult volunteers (7 females, 5 males) participated in the study. They were recruited among employees of Unilever Research Vlaardingen.Interventions: Green tea, black tea and black tea with semi-skimmed milk (3 g tea solids each).Results: Consumption of green tea (0.9 g total catechins) or black tea (0.3 g total catechins) resulted in a rapid increase of catechin levels in blood with an average maximum change from baseline (CVM) of 0.46 μmol/l (13%) after ingestion of green tea and 0.10 μmol/l (13%) in case of black tea. These maximum changes were reached after (mean (s.e.m.)) t=2.3 h (0.2) and t=; 2.2 h (0.2) for green and black tea respectively. Blood levels rapidly declined with an elimination rate (mean (CVM)) of t½=4.8 h (5%) for green tea and t½=6.9 h (8%) for black tea. Addition of milk to black tea (100 ml in 600 ml) did not significantly affect the blood catechin levels (areas under the curves (mean (CVM) of 0.53 h. μmol/l (11%) vs 0.60 h. μmol/l (9%) for black tea and black tea with milk respectively.Conclusion: Catechins from green tea and black tea are rapidly absorbed and milk does not impair the bioavailability of tea catechins.

Dietary non-tocopherol antioxidants present in extra virgin olive oil increase the resistance of low density lipoproteins to oxidation in rabbits

Consumption of a range of dietary antioxidants may be beneficial in protecting low density lipoprotein (LDL) against oxidative modification, as studies have demonstrated that antioxidants other than vitamin E may also function against oxidation of LDL in vitro. In the present study, the effect of polyphenol antioxidants on the susceptibility of LDL to copper-mediated oxidation was investigated after feeding semi-purified diets to 3 groups of New Zealand white (NZW) rabbits. All diets comprised 40% energy as fat with 17% energy as oleic acid. Dietary fatty acid compositions were identical. Oils with different polyphenol contents were used to provide the dietary source of oleic acid-refined olive oil, extra virgin olive oil and Trisun high oleic sunflower seed oil. Polyphenolic compounds (hydroxytyrosol and p-tyrosol) could only be detected in the extra virgin olive oil. Vitamin E was equalised in all diets. LDL oxidizability in vitro was determined by continuously monitoring the copper-induced formation of conjugated dienes after 6 weeks of experimental diet feeding. The lag phase before demonstrable oxidation occurred was significantly increased in the high polyphenol, extra virgin olive oil group (P < 0.05) when compared with combined results from the low polyphenol group (refined olive oil and Trisun), even though the LDL vitamin E concentration in the high polyphenol group was significantly lower. The rate of conjugated diene formation was not influenced by the presence of dietary polyphenols. Results demonstrate that antioxidants, possibly phenolic compounds which are present only in extra virgin olive oil, may contribute to the endogenous antioxidant capacity of LDL, resulting in an increased resistance to oxidation as determined in vitro.

Effects of green tea, black tea and dietary lipophilic antioxidants on LDL oxidizability and atherosclerosis in hypercholesterolaemic rabbits

The hypothesis that tea or dietary lipid-soluble antioxidants reduce atherogenesis by lowering the oxidizability of low-density lipoprotein (LDL) was investigated. Five groups of 20 female New Zealand white rabbits were fed a restricted amount of a high-fat (30 en%) semipurified diet supplemented with cholesterol (0.15%, w/w) for 21 weeks. The vitamin E content of the control diet was 40 mg/kg diet. The animals received either green tea or black tea in their drinking water or vitamin E (200 mg/kg diet) or beta-carotene (20 mg/kg). The serum cholesterol concentrations (in the order of 18-23 mmol/l) were not significantly different between the groups. Vitamin E was substantially increased as compared to controls in vitamin E supplemented animals (3-fold within 8 weeks in plasma and LDL; P < 0.01) and weakly (1.2-fold) by green and black tea (P < 0.05). Green tea consumption tended to reduce aortic lesion formation by 31% (24 +/- 3.2% versus 35 +/- 5.7% for control animals P = 0.11), while black tea, vitamin E and beta-carotene had no effect. This was in contrast to the resistance of isolated LDL to oxidation induced at high copper concentration. Green and black tea induced a 13% and 15% (P < 0.05) prolongation of the lag phase, respectively, with a correspondingly lower oxidation rate, while vitamin E increased the lag phase by 63% (P < 0.01) with a concomitant diminution of the oxidation rate and beta-carotene had no effect. Regression analysis showed that there was no relationship between the extent of atherosclerosis and LDL oxidizability or plasma malondialdehyde as marker of in vivo lipid peroxidation. The results of the present study raise the question whether LDL oxidizability (at least when tested at high induction rate ex vivo) is a primary causal mechanism in atherosclerosis in the cholesterol-fed rabbit. The suitability of the cholesterol-fed rabbit with extreme hypercholesterolaemia as a model to study antiatherosclerotic properties of dietary antioxidants, such as the tested polyphenols, is discussed.

Plasma concentrations of individual tea catechins after a single oral dose in humans

1. Ten healthy volunteers ingested 1.5 mmole epicatechin gallate (ECg), epigallocatechin (EGC) or epigallocatechin gallate (EGCg) in a randomized crossover design. After deconjugation, catechins in plasma and 24-h urine samples were determined by high-performance liquid chromatography (HPLC). Antioxidant activity was measured in plasma by determining ferric reducing activity (FRAP). 2. The catechin levels in plasma after ingestion were significantly different: EGC rose quickly with a short elimination half-life (t 1/2 elim = 1.7 h), ECg was intermediate in rise but slowest in decline (t 1/2 elim = 6.9h), EGCg was slowest in rise but intermediate in decline (t 1/2 elim = 3.9 h). At 24 h, EGC and EGCg had returned to base levels, but ECg was still elevated. Peak maximum varied between 1.3 (EGCg) and 5.0 µmol l -1 (EGC). 3. Very limited interconversion (ECg → epicatechin, EGCg → EGC) occurred indicating that degallation is not required for uptake. 4. Up to 13.6% of the ingested EGC (partly methylated) was excreted in the urine, but ECg or EGCg were not detected. 5. EGC and ECg produced an increase in antioxidant activity in plasma, but with EGCg, no statistically significant effect was found. 6. The pattern of uric acid in plasma showed a clear resemblance with that of FRAP and linear regression analysis indicated a very significant relationship (R 2 = 0.88, p < 0.0001). 7. It is concluded that tea catechins differ significantly in their pharmacokinetic behaviour.

Addition of milk does not affect the absorption of flavonols from tea in man.

Tea is a major source of flavonols, a subclass of antioxidant flavonoids present in plant foods which potentially are beneficial to human health. Milk added to tea, a frequent habit in the United Kingdom, could inhibit absorption of tea flavonoids, because proteins can bind flavonoids effectively. Eighteen healthy volunteers each consumed two out of four supplements during three days: black tea, black tea with milk, green tea and water. A cup of the supplement was consumed every 2 hours each day for a total of 8 cups a day. The supplements provided about 100 μmol quercetin glycosides and about 60 – 70 μmol kaempferol glycosides. Addition of milk to black tea (15 ml milk to 135 ml tea) did not change the area under the curve of the plasma concentration-time curve of quercetin or kaempferol. Plasma concentrations reached were about 50 nM quercetin and 30 – 45 nM kaempferol. We conclude that flavonols are absorbed from tea and that their bioavailability is not affected by addition of milk.

Influence of feeding different vegetables on plasma levels of carotenoids, folate and vitamin C. Effect of disruption of the vegetable matrix

Carotenoids, folate and vitamin C may contribute to the observed beneficial effects of increased vegetable intake. Currently, knowledge on the bioavailability of these compounds from vegetables is limited. We compared the efficacy of different vegetables, at the same level of intake (i.e. 300 g/d), in increasing plasma levels of carotenoids, folate and vitamin C and we investigated if disruption of the vegetable matrix would enhance the bioavailability of these micronutrients. In an incomplete block design, sixty-nine volunteers consumed a control meal without vegetables and three out of four vegetable meals (i.e. broccoli, green peas, whole leaf spinach, chopped spinach; containing between 1.7 and 24.6 mg beta-carotene, 3.8 and 26 mg lutein, 0.22 and 0.60 mg folate and 26 and 93 mg vitamin C) or a meal supplemented with synthetic beta-carotene (33.3 mg). Meals were consumed for 4 d and fasting blood samples were taken at the end of each period. Consumption of the spinach-supplemented meal did not affect plasma levels of beta-carotene, although the beta-carotene content was 10-fold those of broccoli and green peas, which induced significant increases in plasma beta-carotene levels (28 (95% CI 6.4, 55)% and 26 (95% CI 2.6, 54)% respectively). The beta-carotene-supplemented meal increased plasma concentrations of beta-carotene effectively (517 (95% CI 409, 648)%). All vegetable meals increased the plasma concentrations of lutein and vitamin C significantly. Broccoli and green peas were, when expressed per mg carotenoid consumed, also more effective sources of lutein than spinach. A significant increase in plasma folate concentration was found only after consumption of the spinach-supplemented meal, which provided the highest level of folate. Disruption of the spinach matrix increased the plasma responses to both lutein (14 (95% CI 3.7, 25)%) and folate (10 (95% CI 2.2, 18)%), whereas it did not affect the response to beta-carotene. We conclude that the bioavailabilities of beta-carotene and lutein vary substantially among different vegetables and that the bioavailabilities of lutein and folate from spinach can be improved by disruption of the vegetable matrix.

Dialysis of isolated low density lipoprotein induces a loss of lipophilic antioxidants and increases the susceptibility to oxidation in vitro

We determined the effects of different dialysis conditions on the antioxidant content, duration of the lag phase and oxidation rate of LDL. Dialysis for 22 h resulted in a 56%--66% reduction in the concentrations of beta-carotene, lycopene and alpha-tocopherol. The lag phase of copper-induced oxidation of freshly isolated LDL was considerably longer than that of LDL dialysed for 22 or 44 h. Our data show that dialysis may result in LDL preparations with antioxidant compositions that are not truly representative of freshly isolated lipoproteins.

Comparison of the effects of dietary vitamin E on in vivo and in vitro parameters of lipid peroxidation in the rabbit

This study has investigated the effect of dietary vitamin E on markers of antioxidant status. Four groups of rabbits received diets containing 30 energy percent (en%) total fat (7.8 en% contributed by linoleic acid) for 12 weeks. D,1-alpha tocopheryl acetate was added to the diets to obtain a range of vitamin E concentrations (49, 114, 179, or 775 tocopherol equivalents per kg diet). Increased vitamin E concentrations were demonstrated in plasma lipoproteins and erythrocyte membranes following supplementation, and dietary effects on lipid peroxidation were investigated by (i) monitoring a fluorescent parinaric acid probe incorporated into erythrocyte membranes in vivo, (ii) determination of malondialdehyde and oxysterols in plasma, and (iii) investigation of the susceptibility of low density lipoprotein (LDL) to copper-induced conjugated diene formation in vitro. No effects of vitamin E were observed on parinaric acid oxidation in vivo or on the accumulation of lipid peroxidation products in plasma, but the resistance of LDL to oxidation in vitro increased significantly as vitamin E was supplemented to the diets. Our results demonstrate that under these dietary conditions (7.8 en% linoleic acid) increasing the vitamin E content of plasma and erythrocytes approximately two-fold does not reduce the level of lipid peroxidation in vivo, indicating sufficient antioxidant capacity on the lowest vitamin E diet. In contrast, LDL became more resistant to an extreme oxidative stress applied in vitro. The relevance of these assays to currently proposed mechanisms of atherosclerosis is discussed.

Total antioxidant activity of low density lipoproteins and the relationship with α-tocopherol status

A rapid method is described for measuring the antioxidant activity of low density lipoproteins. Studies were undertaken on individuals attending a hyperlipidaemia clinic, an unsupplemented group and a group after supplementation with 300 mg dl‐α‐tocopherol acetate for nine weeks. The results show a positive correlation between the antioxidant activity and α‐tocopherol content of LDL in the supplemented group.