Influence of diabetes on plasma pharmacokinetics and brain bioavailability of grape polyphenols and their phase II metabolites in the Zucker diabetic fatty rat.

SCOPE: The effect of diabetes on the pharmacokinetics, bioavailability and brain distribution of grape polyphenols and select metabolites was studied in the Zucker diabetic fatty (ZDF) rat model. METHODS AND RESULTS: (ZDF) rats and their lean controls (LN) were dosed with a Standardized Grape Polyphenol (SGP) Mixture consisting of grape seed extract, Concord grape juice and resveratrol (RES) by oral gavage for 10 days. An 8-h pharmacokinetic study was performed. After 24 h, a second dose of SGP was administered and 1 h later animals were sacrificed and brain tissue was harvested. Plasma, urine, and brain tissue were analyzed for grape polyphenols. ZDF rats exhibited significantly diminished Cmax for all catechin, epicatechin, quercetin and resveratrol conjugated metabolites. Bioavailability was significantly lower in ZDF rats for methylated flavan-3-ol, RES, and quercetin metabolites. Significantly lower levels of metabolites of RES, quercetin, and flavan-3-ols were found in brains of ZDF rats. There was no significant difference between ZDF and LN in anthocyanins in plasma and no anthocyanins were detectable in brain extracts. ZDF rats showed significantly higher urinary excretion for all polyphenols. CONCLUSION: Diabetes may alter the overall bioavailability of some polyphenols in plasma and brain in part due to higher urinary clearance.

Thyroid status, insulin sensitivity and glucose tolerance in overweight and obese adults before and after 36 weeks of whey protein supplementation and exercise training.

UNLABELLED: Research suggests that subclinical hypothyroidism (SHT) influences insulin sensitivity and glucose tolerance. Reductions in thyroid stimulating hormone (TSH) concentrations are associated with exercise training (ExTr), which improves insulin sensitivity and glucose uptake. PURPOSE: A secondary analysis of previously published data was conducted to examine the relationship between SHT, TSH and glucose homeostatic control at baseline and to assess the impact of ExTr on thyroid status and how SHT affects changes in insulin sensitivity after ExTr. MATERIALS AND METHODS: Data were obtained from a 36-week ExTr and whey protein supplementation intervention trial. Subjects (n = 304, 48 ± 7 years, females = 186) were randomized to a specific whey protein group (0, 20, 40, or 60 g per day) and all subjects participated in a resistance (2 d/wk) and aerobic (1 d/wk) training program. Testing was conducted at baseline and post-intervention. RESULTS: At baseline, 36% (n = 110) and 12% (n = 35) of subjects were classified with SHT based on the TSH ≥ 3 µIU/L or TSH ≥ 4.5 µIU/L cut-offs, respectively. No association was found between baseline TSH and baseline measures of glucose homeostatic control. Whey protein supplementation did not influence intervention outcomes. Post-intervention (n = 164), no change was observed in TSH. SHT did not affect changes in insulin sensitivity following ExTr. CONCLUSION: These results support that the health benefits of ExTr for the management of insulin resistance (IR) are not blunted by SHT.

Plasma bioavailability and regional brain distribution of polyphenols from apple/grape seed and bilberry extracts in a young swine model.

SCOPE: The pharmacokinetics, bioavailability, and regional brain distribution of polyphenols from apple-grape seed extract (AGSE) mixture and bilberry extract were studied after 3 weeks of dosing in weanling pigs. MATERIALS AND METHODS: Weanling piglets were treated for 3 weeks with extracts of (AGSE) or bilberry extracts, using a physiological (27.5 mg/kg) or supplement (82.5 mg/kg) dose. A 24-h pharmacokinetic study was conducted and brain tissue was harvested. Major flavan-3-ol and flavonol metabolites including catechin-O-ß-glucuronide, epicatechin-O-ß-glucuronide, 3'O-methyl-catechin-O-ß-glucuronide, 3'O-methyl-epicatechin-O-ß-glucuronide, quercetin-O-ß-glucuronide, and O-methyl-quercetin-O-ß-glucuronide were analyzed in plasma, urine, and regional brain extracts from AGSE groups. Anthocyanidin-O-galactosides and O-glucosides of delphinidin (Del), cyanidin (Cyn), petunidin (Pet), peonidin (Peo), and malvidin (Mal) were analyzed in plasma, urine, and brain extracts from bilberry groups. CONCLUSION: Significant plasma dose-dependence was observed in flavan-3-ol metabolites of the AGSE group and in Mal, Del and Cyn galactosides and Pet, Peo, and Cyn glucosides of the bilberry groups. In the brain, a significant dose dependence was found in the cerebellum and frontal cortex in all major flavan-3-ol metabolites. All anthocyanidin glycosides, except for delphinidin, showed a dose-dependent increase in the cerebellum.

Green Tea Increases the Concentration of Total Mercury in the Blood of Rats following an Oral Fish Tissue Bolus.

Fish has many health benefits but is also the most common source of methylmercury. The bioavailability of methylmercury in fish may be affected by other meal components. In this study, the effect of green tea on the bioavailability of methylmercury from an oral bolus of fish muscle tissue was studied in rats and compared to a water treated control group and a group treated with meso-2,3-dimercaptosuccinic acid (DMSA), a compound used medically to chelate mercury. Rats were given a single oral dose of fish tissue via gavage and one of the treatments. Rats were given access to food for 3 h at 12 h intervals. They were dosed with each of the treatments with each meal. Blood samples were collected for 95 hours. Green tea significantly increased the concentration of total mercury in blood relative to the control, whereas DMSA significantly decreased it. In addition, feeding caused a slight increase in blood mercury for several meals following the initial dose.

Role of intestinal microbiota in the generation of polyphenol-derived phenolic acid mediated attenuation of Alzheimer's disease ß-amyloid oligomerization.

SCOPE: Grape seed polyphenol extract (GSPE) is receiving increasing attention for its potential preventative and therapeutic roles in Alzheimer's disease (AD) and other age-related neurodegenerative disorders. The intestinal microbiota is known to actively convert many dietary polyphenols, including GSPE, to phenolic acids. There is limited information on the bioavailability and bioactivity of GSPE-derived phenolic acid in the brain. METHODS AND RESULTS: We orally administered GSPE to rats and investigated the bioavailability of 12 phenolic acids known to be generated by microbiota metabolism of anthocyanidins. GSPE treatment significantly increased the content of two of the phenolic acids in the brain: 3-hydroxybenzoic acid and 3-(3´-hydroxyphenyl)propionic acid, resulting in the brain accumulations of the two phenolic acids at micromolar concentrations. We also provided evidence that 3-hydroxybenzoic acid and 3-(3´-hydroxyphenyl)propionic acid potently interfere with the assembly of ß-amyloid peptides into neurotoxic ß-amyloid aggregates that play key roles in AD pathogenesis. CONCLUSION: Our observation suggests important contribution of the intestinal microbiota to the protective activities of GSPE (as well as other polyphenol preparations) in AD. Outcomes from our studies support future preclinical and clinical investigations exploring the potential contributions of the intestinal microbiota in protecting against the onset/progression of AD and other neurodegenerative conditions.

Targeting multiple pathogenic mechanisms with polyphenols for the treatment of Alzheimer's disease-experimental approach and therapeutic implications.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease of aging and currently has no cure. Its onset and progression are influenced by multiple factors. There is growing consensus that successful treatment will rely on simultaneously targeting multiple pathological features of AD. Polyphenol compounds have many proven health benefits. In this study, we tested the hypothesis that combining three polyphenolic preparations (grape seed extract, resveratrol, and Concord grape juice extract), with different polyphenolic compositions and partially redundant bioactivities, may simultaneously and synergistically mitigate amyloid-ß (Aß) mediated neuropathology and cognitive impairments in a mouse model of AD. We found that administration of the polyphenols in combination did not alter the profile of bioactive polyphenol metabolites in the brain. We also found that combination treatment resulted in better protection against cognitive impairments compared to individual treatments, in J20 AD mice. Electrophysiological examination showed that acute treatment with select brain penetrating polyphenol metabolites, derived from these polyphenols, improved oligomeric Aß (oAß)-induced long term potentiation (LTP) deficits in hippocampal slices. Moreover, we found greatly reduced total amyloid content in the brain following combination treatment. Our studies provided experimental evidence that application of polyphenols targeting multiple disease-mechanisms may yield a greater likelihood of therapeutic efficacy.

Role of standardized grape polyphenol preparation as a novel treatment to improve synaptic plasticity through attenuation of features of metabolic syndrome in a mouse model.

SCOPE: Metabolic syndrome has become an epidemic and poses tremendous burden on the health system. People with metabolic syndrome are more likely to experience cognitive decline. As obesity and sedentary lifestyles become more common, the development of early prevention strategies is critical. In this study, we explore the potential beneficial effects of a combinatory polyphenol preparation composed of grape seed extract, Concord purple grape juice extract, and resveratrol, referred to as standardized grape polyphenol preparation (SGP), on peripheral as well as brain dysfunction induced by metabolic syndrome. METHODS AND RESULTS: We found dietary fat content had minimal effect on absorption of metabolites of major polyphenols derived from SGP. Using a diet-induced animal model of metabolic syndrome (DIM), we found that brain functional connectivity and synaptic plasticity are compromised in the DIM mice. Treatment with SGP not only prevented peripheral metabolic abnormality but also improved brain synaptic plasticity. CONCLUSION: Our study demonstrated that SGP, comprised of multiple bioavailable and bioactive components targeting a wide range of metabolic syndrome related pathological features, provides greater global protection against peripheral and central nervous system dysfunctions and can be potentially developed as a novel prevention/treatment for improving brain connectivity and synaptic plasticity important for learning and memory.

Identification of brain-targeted bioactive dietary quercetin-3-O-glucuronide as a novel intervention for Alzheimer's disease.

Epidemiological and preclinical studies indicate that polyphenol intake from moderate consumption of red wines may lower the relative risk for developing Alzheimer's disease (AD) dementia. There is limited information regarding the specific biological activities and cellular and molecular mechanisms by which wine polyphenolic components might modulate AD. We assessed accumulations of polyphenols in the rat brain following oral dosage with a Cabernet Sauvignon red wine and tested brain-targeted polyphenols for potential beneficial AD disease-modifying activities. We identified accumulations of select polyphenolic metabolites in the brain. We demonstrated that, in comparison to vehicle-control treatment, one of the brain-targeted polyphenol metabolites, quercetin-3-O-glucuronide, significantly reduced the generation of ß-amyloid (Aß) peptides by primary neuron cultures generated from the Tg2576 AD mouse model. Another brain-targeted metabolite, malvidin-3-O-glucoside, had no detectable effect on Aß generation. Moreover, in an in vitro analysis using the photo-induced cross-linking of unmodified proteins (PICUP) technique, we found that quercetin-3-O-glucuronide is also capable of interfering with the initial protein-protein interaction of Aß(1-40) and Aß(1-42) that is necessary for the formation of neurotoxic oligomeric Aß species. Lastly, we found that quercetin-3-O-glucuronide treatment, compared to vehicle-control treatment, significantly improved AD-type deficits in hippocampal formation basal synaptic transmission and long-term potentiation, possibly through mechanisms involving the activation of the c-Jun N-terminal kinases and the mitogen-activated protein kinase signaling pathways. Brain-targeted quercetin-3-O-glucuronide may simultaneously modulate multiple independent AD disease-modifying mechanisms and, as such, may contribute to the benefits of dietary supplementation with red wines as an effective intervention for AD.

Whey protein supplementation does not affect exercise training-induced changes in body composition and indices of metabolic syndrome in middle-aged overweight and obese adults.

Little is known about the effects of different quantities of whey protein on exercise training-induced changes in body composition and indices of metabolic syndrome in middle-aged overweight and obese adults. Therefore, we examined the effects of consuming 0.8-MJ supplements with 0 (n = 126), 10 (n = 112), 20 (n = 44), or 30 (n = 45) g whey protein twice daily in conjunction with resistance (2 d/wk) and aerobic (1 d/wk) exercise training in a double-blind, randomized, placebo-controlled, community-based 9-mo study in men (n = 117) and women (n = 210); (age: 48 ± 7.9 y; BMI: 30.0 ± 2.8 kg/m(2)). Whey protein supplementation did not influence any of the following outcomes, some of which were affected by training. Among all participants, strength increased by 15 ± 12% (P < 0.001) and maximal oxygen uptake capacity (VO(2)max) increased by 9 ± 15% (P < 0.001). Body weight was unchanged (0.1 ± 3.7 kg, P = 0.80), lean body mass increased by 1.9 ± 2.8% (0.95 ± 1.3 kg, P < 0.001), and fat mass decreased by 2.6 ± 9.4% (-0.86 ± 3.1 kg, P = 0.001). Oral-glucose-tolerance testing showed that plasma glucose AUC was unchanged (-18.0 ± 170 mmol/L· 3 h, P = 0.16), insulin AUC decreased by 2.6 ± 32% (-7.5 ± 29 nmol/L· 3 h, P = 0.01), and HOMA-IR (0.2 ± 2.0, P = 0.81) and the insulin sensitivity index (0.3 ± 3.0, P = 0.63) were unchanged. Plasma concentrations of TG; total, LDL, and HDL cholesterol; C-reactive protein; plasminogen activator inhibitor-1; blood pressure; and waist circumference were unchanged. Whey protein supplementation did not affect exercise training-induced responses in body composition and indices of metabolic syndrome in middle-aged overweight and obese adults who maintained body weight.

Determining the effects of antioxidants on oxidative stress induced carbonylation of proteins.

There is potential that the pathological effects of oxidative stress (OS) associated diseases such as diabetes could be ameliorated with antioxidants, but this will require a clearer understanding of the pathway(s) by which proteins are damaged by OS. This study reports the development and use of methods that assess the efficacy of dietary antioxidant supplementation at a mechanistic level. Data reported here evaluate the impact of green tea supplementation on oxidative stress induced post-translational modifications (OSi-PTMs) in plasma proteins of Zucker diabetic fatty (ZDF) rats. The mechanism of antioxidant protection was examined through both the type and amount of OSi-PTMs using mass spectrometry based identification and quantification. Carbonylated proteins in freshly drawn blood samples were derivatized with biotin hydrazide. Proteins thus biotinylated were selected from plasma samples of green tea fed diabetic rats and control animals by avidin affinity chromatography, further fractionated by reversed phase chromatography (RPC); fractions from the RPC column were tryptic digested, and the tryptic digest was fractionated by RPC before being identified by tandem mass spectrometry (MS/MS). Relative quantification of peptides bearing carbonylation sites was achieved for the first time by RPC-MS/MS using selective reaction monitoring (SRM). Seventeen carbonylated peptides were detected and quantified in both control and treated plasma. The relative concentration of eight was dramatically different between control and green tea treated animals. Seven of the OSi-PTM bearing peptides had dropped dramatically in concentration with treatment while one increased, indicating differential regulation of carbonylation by antioxidants. Green tea antioxidants were found to reduce carbonylation of proteins by lipid peroxidation end products most, followed by advanced glycation end products to a slightly lower extent. Direct oxidation of proteins by reactive oxygen species (ROS) was protected the least by green tea.

Tracking deposition of a 14C-radiolabeled kudzu hairy root-derived isoflavone-rich fraction into bone.

Hairy roots were induced in four genotypes from three kudzu species (Pueraria montana var. lobata, P. lobata and P. phaseoloides) in vitro using Agrobacterium rhizogenes to stimulate rapid secondary metabolite synthesis. Hairy roots from P. montana var. lobata (United States Department of Agriculture no. PI 434246) yielded the highest puerarin and total isoflavone content and the greatest new biomass per growth cycle among the genotypes evaluated. Hairy roots from this genotype were selected for radiolabeling using (14)C-sucrose as a carbon source. Isoflavones from radiolabeled kudzu hairy root cultures were extracted with 80% methanol, partitioned by solvent extraction, and then subfractionated by Sephadex LH-20 gel filtration. Radiolabeled isoflavones were isolated in a highly enriched fraction, which contained predominantly puerarin, daidzin and malonyl-daidzin and had an average radioactivity of 8.614 MBq/g (232.8 µCi/g) dry fraction. The (14)C-radiolabeled, isoflavone-rich fraction was orally administered at a dose of 60 mg/kg body weight to male Sprague-Dawley rats implanted with a jugular catheter, a subcutaneous ultrafiltrate probe and a brain microdialysate probe. Serum, interstitial fluid, brain microdialysate, urine and feces were collected using a Culex(®) Automated Blood Collection System for 24 h. At the end of this period, rats were sacrificed and major tissues were collected. Analysis by a scintillation counter confirmed that a bolus dose of (14)C-radiolabeled, isoflavone-rich kudzu fraction reached bone tissues, which accumulated 0.011%, 0.09% and 0.003% of the administered dose in femur, tibia and vertebrae, respectively. Femurs extracted with 80% methanol were analyzed by high-performance liquid chromatography with electrospray ionization-mass spectrometry and were found to contain trace quantities of puerarin, daidzein and puerarin glucuronide. This study demonstrates that kudzu isoflavones and metabolites are capable of reaching bone tissues, where they may contribute to the prevention of osteoporosis and the promotion of bone health.

Pharmacokinetics and tissue distribution of 14C-labeled grape polyphenols in the periphery and the central nervous system following oral administration.

Grape polyphenols confer potential health benefits, including prevention of neurodegenerative diseases. To determine the absorption and tissue distribution of the complex grape polyphenol mixture, (14)C-labeled polyphenols were biosynthesized by grape cell suspension cultures, during co-incubation with radioisotopically labeled sucrose, and fractionated into polyphenolic subfractions. The pharmacokinetics and distribution of grape polyphenols into blood, brain, and peripheral interstitial fluid were determined by tracking the (14)C label. The blood peak (14)C concentration of the fractions ranged from 15 minutes to 4 hours. Absorption and tissue distribution varied greatly between fractions. Concentrations in interstitial fluid were lower than in blood. The amount of residual label in the brain at 24 hours ranged from 0.1% to 1.7% of the dose, depending on the fraction. (14)C label found in the brain tissue and brain microdialysate indicated that grape polyphenols or their metabolites are able to cross the blood-brain barrier. Using (14)C-labeled plant polyphenols it is possible to track the compounds or their metabolic products into any tissue and determine distribution patterns in spite of low concentrations. A central question regarding the potential role of dietary polyphenolics in neurodegenerative research is whether they are bioavailable in the brain. Our observations indicate that some grape-derived polyphenolics do reach the brain, which suggests their potential value for applications in neurodegenerative disorders.

Chocolate matrix factors modulate the pharmacokinetic behavior of cocoa flavan-3-ol phase II metabolites following oral consumption by Sprague-Dawley rats.

The impact of carbohydrates and milk on the bioavailability of catechin (C) and epicatechin (EC) from chocolate has been previously studied. However, little data exist regarding potential modulation of the phase II metabolism by these chocolate matrix factors. The objectives of this study were to assess the impact of matrix composition on qualitative and quantitative profiles of circulating catechins and their metabolites following administration of commercially relevant chocolate confections. Sprague-Dawley rats were administered 1.5 g of a confection (reference dark, high sucrose, or milk chocolate) by intragastric gavage, and plasma samples were collected over 8 h. High-performance liquid chromatography-mass spectrometry analysis was performed to quantify C, EC, and their metabolites. The predominant metabolites were O-glucuronides (two metabolites) and O-Me-O-glucuronides (three metabolites). Plasma concentrations of metabolites were generally the highest for high sucrose treatment and lowest for milk treatment, while the reference dark treatment generally resulted in intermediate concentrations. The O-Me-(+/-)-C/EC-O-beta-glucuronide (peak 4) was significantly higher for the high sucrose treatment (2325 nM h) versus the milk treatment (1300 nM h). Additionally, C(MAX) values for (+/-)-C/EC-O-beta-glucuronide (peak 3) and two O-Me-(+/-)-C/EC-O-beta-glucuronides (peaks 4 and 6) were significantly higher for the high sucrose treatment (4012, 518, and 2518 nM, respectively) versus the milk treatment (2590, 240, and 1670 nM, respectively). Milk and sucrose appear to modulate both metabolism and plasma pharmacokinetics and, to a lesser extent, the overall bioavailability of catechins from chocolate confections.

Influence of chocolate matrix composition on cocoa flavan-3-ol bioaccessibility in vitro and bioavailability in humans.

Conflicting data exist regarding the influence of chocolate matrices on the bioavailability of epicatechin (EC) from cocoa. The objective of this study was to assess the bioavailability of EC from matrices varying in macronutrient composition and physical form. EC bioavailability was assessed from chocolate confections [reference dark chocolate (CDK), high sucrose (CHS), high milk protein (CMP)] and cocoa beverages [sucrose milk protein (BSMP), non-nutritive sweetener milk protein (BNMP)], in humans and in vitro. Six subjects consumed each product in a randomized crossover design, with serum EC concentrations monitored over 6 h post consumption. Areas under the serum concentration-time curve (AUC) were similar among chocolate matrices. However, AUCs were significantly increased for BSMP and BNMP (132 and 143 nM h) versus CMP (101 nM h). Peak serum concentrations (C(MAX)) were also increased for BSMP and BNMP (43 and 42 nM) compared to CDK and CMP (32 and 25 nM). Mean T(MAX) values were lower, although not statistically different, for beverages (0.9-1.1 h) versus confections (1.8-2.3 h), reflecting distinct shapes of the pharmacokinetic curves for beverages and confections. In vitro bioaccessibility and Caco-2 accumulation did not differ between treatments. These data suggest that bioavailability of cocoa flavan-3-ols is likely similar from typical commercial cocoa based foods and beverages, but that the physical form and sucrose content may influence T(MAX) and C(MAX).

Bioavailability of gallic acid and catechins from grape seed polyphenol extract is improved by repeated dosing in rats: implications for treatment in Alzheimer's disease.

The present study explored the bioavailability and brain deposition of a grape seed polyphenolic extract (GSPE) previously found to attenuate cognitive deterioration in a mouse model of Alzheimer's disease (AD). Plasma pharmacokinetic response of major GSPE phenolic components was measured following intragastric gavage of 50, 100, and 150 mg GSPE per kg body weight. Liquid chromatography-mass spectrometry (LC-MS) analysis identified gallic acid (GA), catechin (C), and epicatechin (EC) in plasma of rats gavaged acutely with GSPE. Additionally, 4-methylgallic acid (4-OMeGA), 3'-methylcatechin (3'-OMeC), and 3'-methylepicatechin (3'-OMeEC) were identified as circulating metabolites of GSPE phenolic constituents. Cmax for individual GSPE constituents and their metabolites increased in a dose-dependent fashion (with increasing GSPE oral dose). Repeated daily exposure to GSPE was found to significantly increase bioavailability (defined as plasma AUC0-8h) of GA, C, and EC by 198, 253, and 282% relative to animals receiving only a single acute GSPE dose. EC and C were not detectable in brain tissues of rats receiving a single GSPE dose but reached levels of 290.7 +/-45.9 and 576.7 +/- 227.7 pg/g in brain tissues from rats administered GSPE for 10 days. This study suggests that brain deposition of GA, C, and EC is affected by repeated dosing of GSPE.

Pharmacokinetics of green tea catechins in extract and sustained-release preparations.

Catechins are a major constituent of green tea. For green tea to have cancer therapeutic benefit, catechin concentrations in the range of 100 nM are required continuously until apoptosis (programmed cell death) is induced. To prolong elevated plasma and interstitial concentrations of catechins, a sustained-release formulation of green tea extract was tested and compared to a commercial green tea extract (Tegreen97®). Sustained-release formulations are usually developed in the pharmaceutical industry to slowly deliver the compound over a period of time and increase the dosing interval. Plasma and interstitial fluid (ISF) pharmacokinetics of catechins were determined following an oral dose in the rat. The sustained-release formulation profile included multiple smaller peaks of total catechins in both plasma and ISF. Interstitial fluid profiles of green tea extract indicate that higher catechins concentration and longer duration in tissue than in blood may make a sustained-release form unnecessary.

Effect of long-term oral administration of green tea extract on weight gain and glucose tolerance in Zucker diabetic (ZDF) rats.

There have been some claims that green tea reduces weight and lowers blood glucose in diabetes. Intraperitoneal injections of green tea catechins in diabetic rats have shown beneficial effects. To determine if oral administration of green tea would prevent development of diabetes, young Zucker diabetic rats were dosed with green tea extract containing 50-125 mg/kg of Epigallocatechin gallate (EGCG) starting at 7 weeks of age, before the appearance of excessive weight gain and glucose elevation. While there was a trend toward lower weight gain and average daily glucose, there was no statistically significant difference.

Tea catechin synergies in inhibition of cancer cell proliferation and of a cancer specific cell surface oxidase (ECTO-NOX).

The anticancer properties of tea catechins are most frequently attributed to the principal catechin (-)-epigallocatechin-3-gallate (EGCg). Efficacy was evaluated using growth of cultured HeLa cells and inhibition of the enzymatic activity of a putative cell surface tea target enzyme, a cancer-associated cell surface-located NADH oxidase (ECTO-NOX) designated tNOX. The amounts of EGCg required to inhibit by both criteria was reduced 10 times by combination with inactive catechins such as (-)-epicatechin (EC), (-)-epigallocatechin (EGC) or (-)-epicatechin-3-gallate (ECG). Various synthetic mixtures based on purified catechins and decaffeinated tea extracts treated enzymatically to reduce the ester bond-containing catechins varying in EGCg content from 0.065 to 40% were of comparable efficacy to decaffeinated green tea extracts as long as EGCg was present and the ratio of total catechins to EGCg + EGC was about 1.5. Such mixtures appear to offer potential cancer protection and therapeutic advantages over those of EGCg alone through lowered toxicity of the mixture to normal cells and for more efficient blood delivery of orally-administered catechins to a tumour site.