Excess serum Na level in rats administered with high doses of (-)-epigallocatechin gallate-casein nanoparticles prepared with sodium caseinate.

(-)-Epigallocatechin gallate (EGCG)-incorporated casein nanoparticles benefit from excellent antioxidant, anti-inflammatory and anti-cancer activities due to their synergistic efficiency, but few studies have evaluated their safety. In this study, the EGCG-casein nanoparticles (EGCG-NPs) formulated using caseinate by ultrasonic treatment were evaluated for their subacute toxicity. The subacute toxicity test of EGCG-NPs through 28-day oral administration in rats did not exhibit adverse effect, with a no-observed-adverse-effect level (NOAEL) of at least 5.0 g per kg body weight (BW) per day, which was equivalent to 500 mg per kg BW EGCG per day. However, the serum Na level in females and males treated with 10.0 g per kg BW EGCG-NPs increased significantly as compared to the control rats (P < 0.05). Similar indications appeared in rats treated with 10.0 g per kg BW pure casein nanoparticles without EGCG, which indicated that high doses of caseinate nanoparticles result in an excess serum Na level. Therefore, we should consider the safety of the nanoparticle formulation of caseinate when it is used as a loading nutrient and a functional substance in foods.

Circulating Structurally Related (-)-Epicatechin Metabolite Species and Levels after Sustained Intake of a Cocoa Powder High in Polyphenols Are Comparable to Those Achieved after a Single Dose.

BACKGROUND: While the bioavailability of cocoa polyphenols, particularly of the monomer (-)-epicatechin, has been investigated after a single-dose intake, the effect of sustained cocoa consumption on the metabolic profile of the structurally related (-)-epicatechin metabolites (SREMs) has not been investigated. METHODS: A randomized, controlled crossover clinical trial in healthy young adults (18-40 year) was conducted to evaluate SREMs after consumption of a single-dose and after daily consumption of 1.3 g of polyphenol-rich cocoa powder for 28 days. The circulating SREMs were measured by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). RESULTS: Twenty subjects (eleven males and nine females) were enrolled. The SREMs concentrations increased to 1741 ± 337 nM after a single-dose and to 1445 ± 270 nM after sustained supplementation. Sulfate conjugates showed higher levels in females (p < 0.05). The epicatechin-3'-glucuronide (E3'G) and epicatechin-3'-sulfate (E3'S) were the most abundant metabolites in all subjects. A high intra-individual correlation (r = 0.72, p < 0.001) between SREMs concentrations after single-dose and sustained supplementation was observed. The antioxidant capacity of plasma did not change in response to the intervention and was not correlated with any of the SREMs. CONCLUSION: The individual SREMs profile and concentrations after a 28-day supplementation are comparable to those after a single dose.

Validation of a high-throughput method for the quantification of flavanol and procyanidin biomarkers and methylxanthines in plasma by UPLC-MS.

Nutritional biomarkers are critical tools to objectively assess intake of nutrients and other compounds from the diet. In this context, it is essential that suitable analytical methods are available for the accurate quantification of biomarkers in large scale studies. Recently, structurally-related (-)-epicatechin metabolites (SREMs) and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone metabolites (gVLMs) were identified as biomarkers of intake of flavanols and procyanidins, a group of polyphenol bioactives. This study aimed at validating a high throughput method for the quantification of SREMs and gVLMs in plasma along with methylxanthines (MXs), dietary compounds known to interact with flavanol and procyanidin effects. To accomplish this, a full set of authentic analytical standards were used to optimize a micro solid phase extraction method for sample preparation coupled to HPLC-MS detection. Isotopically-labelled standards for all analytes were included to correct potential matrix effects on quantification. Average accuracies of 101%, 93% and 103% were obtained, respectively, for SREMs, gVLMs and MXs. Intra- and inter-day repeatability values were <15%. The method showed linear responses for all analytes (>0.993). Most SREMs and gVLMs had limits of quantifications <5 nM while limits of quantification of MXs were 0.2 µM. All analytes were stable under different tested processing conditions. Finally, the method proved to be suitable to assess SREMs, gVLMs and MXs in plasma collected after single acute and daily intake of cocoa-derived test materials. Overall, this method proved to be a valid analytical tool for high throughput quantification of flavanol and procyanidin biomarkers and methylxanthines in plasma.

Low Plasma Appearance of (+)-Catechin and (-)-Catechin Compared with Epicatechin after Consumption of Beverages Prepared from Nonalkalized or Alkalized Cocoa-A Randomized, Double-Blind Trial.

Flavan-3-ols are claimed to be responsible for the cardioprotective effects of cocoa. Alkalized cocoa powder (ALC), commonly used for many non-confectionary products, including beverages, provides less (+)-catechin, (-)-epicatechin, and procyanidins and more (-)-catechin than nonalkalized cocoa powder (NALC). This may affect the plasma appearance of monomeric flavan-3-ol stereoisomers after consumption of NALC vs. ALC. Within a randomized, crossover trial, 12 healthy nonsmokers ingested a milk-based cocoa beverage providing either NALC or ALC. Blood was collected before and within 6 h postconsumption. (+)-Catechin, (-)-catechin, and epicatechin were analyzed in plasma by HPLC as sum of free and glucuronidated metabolites. Pharmacokinetic parameters were obtained by a one-compartment model with nonlinear regression methods. For epicatechin in plasma, total area under the curve within 6 h postconsumption (AUC0-6h) and incremental AUC0-6h were additionally calculated by using the linear trapezoidal method. After consumption of NALC and ALC, (+)-catechin and (-)-catechin were mostly not detectable in plasma, in contrast to epicatechin. For epicatechin, total AUC0-6h was different between both treatments, but not incremental AUC0-6h. Most kinetic parameters were similar for both treatments, but they varied strongly between individuals. Thus, epicatechin is the main monomeric flavan-3-ol in plasma after cocoa consumption. Whether NALC should be preferred against ALC due to its higher (-)-epicatechin content remains unclear with regard to the results on incremental AUC0-6h. Future studies should investigate epicatechin metabolites in plasma for a period up to 24 h in a larger sample size, taking into account genetic polymorphisms in epicatechin metabolism and should consider all metabolites to understand inter-individual differences after cocoa intake.

Physiologically relevant screening of polyphenol-rich commercial preparations for bioactivity in vascular endothelial cells and application to healthy volunteers: A viable workflow and a cautionary tale.

This study describes the screening of 13 commercially-available plant extracts for pharmacological activity modulating vascular function using an endothelial cell model. A French maritime pine bark extract (FMPBE) was found to have the greatest effect upon nitric oxide availability in control (181% ± 36% of untreated cells) and dysfunctional cells (132% ± 8% of untreated control cells). In healthy volunteers, the FMPBE increased plasma nitrite concentrations 8 h post-consumption compared to baseline (baseline corrected median 1.71 ± 0.38 (25% IQR) and 4.76 (75% IQR) µM, p < 0.05). This was followed by a placebo-controlled, healthy volunteer study, which showed no effects on plasma nitrite. It was confirmed that different batches of extract had been used in the healthy volunteer studies, and this second batch lacked bioactivity, assessed using the in vitro model. No difference in plasma catechin levels was seen at 8 h following supplementation between the studies (252 ± 194 nM versus 50 ± 64 nM, p > 0.05), however HPLC-UV fingerprinting showed that the new batch had a 5-15% in major constituents (including procyanidins A2, B1 and B2) compared to the original batch. This research describes a robust mechanism for screening bioactive extracts for vascular effects. It also highlights batch variability as a significant limitation when using complex extracts for pharmacological activity, and suggests the use of in vitro systems as a tool to identify this problem in future studies.

A colorimetric sensor array for detection and discrimination of antioxidants based on Ag nanoshell deposition on gold nanoparticle surfaces.

There is growing interest in developing a high-performance sensor array for detection and discrimination of antioxidants owing to their widespread use and essential role in the human body. The present work unveils a novel colorimetric sensor array for colorimetric discrimination of antioxidants based on the red, green, and blue alteration (ΔRGB) pattern recognition. In this sensor array, three concentrations of AgNO3 were used as sensing elements, and gold nanoparticles (AuNPs) were employed as a colorimetric probe. In the presence of antioxidants, the sensor array produces unique colorimetric response patterns for the discrimination of these antioxidants due to different reactivities between three different concentrations of AgNO3 and each antioxidant, leading to deposition of different quantities of Ag nanoshells on the surface of AuNPs, enabling an excellent discrimination of six antioxidants (catechin, epigallocatechin 3-gallate, epicatechin, epigallocatechin, epicatechin 3-gallate, and gallocatechin) at a 20 nM level, when linear discriminant analysis (LDA), hierarchical cluster analysis (HCA), centroid diagram, spidergram, and color contour profiles were smartly combined. Furthermore, different concentrations of antioxidants and binary antioxidant mixtures, even ternary mixtures, could also be discriminated with this sensor array. Finally, the sensor array was successfully used for the discrimination of antioxidants in serum samples, demonstrating its potential applications in the diagnosis of antioxidant-related diseases.

The profiling and identification of the absorbed constituents and metabolites of Naoshuantong capsule in mice biofluids and brain by ultra- fast liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry.

Naoshuantong capsule (NSTC) is an oral traditional Chinese medicine formula used widely in the clinic for ischemic stroke. The absorbed ingredients and metabolites of NSTC have never been reported before. In this study, a method incorporating rapid resolution liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was used to identify absorbed ingredients and metabolites after oral administration of NSTC. A total of 15 constituents were detected and identified as prototypes of NSTC. 109 metabolites related to catechin, gallic acid, paeoniflorin, chlorogenic acid, protocatechuate, typhaneoside, ß-elemene, calycosin were identified in serum, urine and brain. 19 metabolites of typhaneoside, 3 metabolites of ß-elemene, 12 metabolites of calycosin were reported for the first time. This is the first time to explore the absorption and metabolism of NSTC. The work will provide helpful information for further research of the mechanism and application of NSTC.

Improved oral bioavailability of the anticancer drug catechin using chitosomes: Design, in-vitro appraisal and in-vivo studies.

Catechin hydrate is a phytopharmaceutical with promising anticancer effects but poor bioavailability. This study aimed to elaborate catechin loaded chitosan-tethered liposomes (chitosomes) to enhance catechin oral bioavailability. Nanocarriers were optimized via ethanol injection method followed by physicochemical, ex vivo and biological appraisal in male Wistar albino rats. Results demonstrated that chitosomes possessed excellent nanosize of 137 nm, monodispersity (PDI < 0.2) and high Zeta potential of +36.8 mV. Additionally, chitosomes showed significant improvement in digestive stability against bile salt with enhanced ex-vivo intestinal permeation. Pharmacokinetic studies revealed the significant potential of chitosomes to enhance catechin bioavailability (AUC, Cmax) and sustain its effect (Tmax). In conclusion, elaborated chitosomes are promising nanoplatforms to enhance catechin oral efficacy with lower dose, side effects, administration frequency and higher patient compliance.

Development of a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for simultaneous determination of epigallocatechin-3-gallate, silibinin, and curcumin in plasma and different tissues after oral dosing of Protandim in rats and its application in pharmacokinetic and tissue distribution studies.

Protandim is an over-the-counter herbal dietary supplement. The key components of Protandim, i.e., epigallocatechin-3-gallate (EGCG), silibinin (SIL), and curcumin (CUR) were simultaneously analyzed through a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method in plasma and different tissues after administration of Protandim in rats. The developed and validated method was employed to assess the pharmacokinetic profiles and the accumulation of EGCG, SIL, CUR in rat plasma and tissue homogenates. The plasma and tissue homogenates were subjected to liquid-liquid extraction and separated on a Hypurity C18 column (50 × 4.6 mm) with a gradient elution of water and acetonitrile. Mass spectrometric detection was performed in the multiple reaction monitoring mode (MRM) following the transitions: m/z 457.3/169.3, m/z 481.3/125.0, m/z 367.3/149.3 and m/z 609.4 /300.2 for EGCG, SIL, CUR, and RU (rutin), respectively. The concentrations of all the analytes in the range from 2 to 1000 ng/mL showed linear relationships with respective peak areas in different matrices. For all matrices, the values of inter-day and intra-day precisions and accuracies were less than 10.3% of the nominal concentration. The matrix effect, extraction recovery, dilution integrity, and stability values were all within acceptable levels. This method was successfully applied for determining the pharmacokinetics and tissue distribution of the components in rats after the intragastrical administration of a single-dose (364.5 mg/kg) or multiple-doses (1458 mg/kg) of Protandim. The data showed that EGCG, SIL, and CUR did not accumulate in rats after multiple doses of Protandim, and the three main components were distributed mainly in the small intestine.

A sensitive UPLC-MS/MS method for simultaneous determination of polyphenols and theaflavins in rat plasma: Application to a pharmacokinetic study of Da Hong Pao tea.

A method based on ultra-performance liquid chromatography-tandem mass spectrometry has been developed for the rapid and simultaneous determination of five catechins and four theaflavins in rat plasma using ethyl gallate as internal standard. The pharmacokinetic profiles of these compounds were compared after oral administration of five kinds of Da Hong Pao tea to rats. Biosamples processed with a mixture of ß-glucuronidase and sulfatase were extracted with ethyl acetate-isopropanol. Chromatographic separation was achieved by gradient elution using 10 mm HCOONH4 solution and methanol as the mobile phase. Analytes were detected using negative ion electrospray ionization in multiple reaction monitoring mode. The lower limits of quantification were 1.0, 0.74 and 0.5 ng/mL for theaflavins, two catechins and three catechins, respectively. The validation parameters were well within acceptable limits. The average half-lives (t1/2 ) in blood of the reference solution group was much shorter than those of tea samples. The values of AUC0-t and Cmax of the polyphenols and theaflavins exhibited linear pharmacokinetic characteristics which were related to the dose concentration.

ProDiet: A Phase II Randomized Placebo-controlled Trial of Green Tea Catechins and Lycopene in Men at Increased Risk of Prostate Cancer.

Epidemiologic studies suggest that diet can alter prostate cancer risk. This study aimed to establish the feasibility and acceptability of dietary modification in men at increased risk of prostate cancer. Men were invited with a PSA level of 2.0-2.95 ng/mL or 3.0-19.95 ng/mL with negative prostate biopsies. Randomization (3 × 3 factorial design) to daily green tea and lycopene: green tea drink (3 cups, unblinded) or capsules [blinded, 600 mg flavan-3-ol ()-epigallocatechin-3-gallate (EGCG) or placebo] and lycopene-rich foods (unblinded) or capsules (blinded, 15 mg lycopene or placebo) for 6 months. Primary endpoints were randomization rates and intervention adherence (blinded assessment of metabolites) at 6 months with secondary endpoints of acceptability (from interviews), safety, weight, blood pressure, and PSA. A total of 133 of 469 (28.4%) men approached agreed to be randomized and 132 were followed-up (99.2%). Mean lycopene was 1.28 [95% confidence intervals (CI), 1.09-1.50, P = 0.003] times higher in the lycopene capsule group and 1.42 (95% CI, 1.21-1.66; P < 0.001) times higher in the lycopene-enriched diet group compared with placebo capsules. Median EGCG was 10.7 nmol/L (95% CI, 7.0-32.0) higher in in the active capsule group and 20.0 nmol/L (95% CI, 0.0-19.0) higher in the green tea drink group compared with placebo capsules (both P < 0.001). All interventions were acceptable and well tolerated although men preferred the capsules. Dietary prevention is acceptable to men at risk of prostate cancer. This intervention trial demonstrates that a chemoprevention clinical trial is feasible. Cancer Prev Res; 11(11); 687-96. ©2018 AACR.

Assessing the respective contributions of dietary flavanol monomers and procyanidins in mediating cardiovascular effects in humans: randomized, controlled, double-masked intervention trial.

Background: Flavanols are an important class of food bioactives that can improve vascular function even in healthy subjects. Cocoa flavanols (CFs) are composed principally of the monomer (-)-epicatechin (∼20%), with a degree of polymerisation (DP) of 1 (DP1), and oligomeric procyanidins (∼80%, DP2-10). Objective: Our objective was to investigate the relative contribution of procyanidins and (-)-epicatechin to CF intake-related improvements in vascular function in healthy volunteers. Design: In a randomized, controlled, double-masked, parallel-group dietary intervention trial, 45 healthy men (aged 18-35 y) consumed the following once daily for 1 mo: 1) a DP1-10 cocoa extract containing 130 mg (-)-epicatechin and 560 mg procyanidins, 2) a DP2-10 cocoa extract containing 20 mg (-)-epicatechin and 540 mg procyanidins, or 3) a control capsule, which was flavanol-free but had identical micro- and macronutrient composition. Results: Consumption of DP1-10, but not of either DP2-10 or the control capsule, significantly increased flow-mediated vasodilation (primary endpoint) and the concentration of structurally related (-)-epicatechin metabolites (SREMs) in the circulatory system while decreasing pulse wave velocity and blood pressure. Total cholesterol significantly decreased after daily intake of both DP1-10 and DP2-10 as compared with the control. Conclusions: CF-related improvements in vascular function are predominantly related to the intake of flavanol monomers and circulating SREMs in healthy humans but not to the more abundant procyanidins and gut microbiome-derived CF catabolites. Reduction in total cholesterol was linked to consumption of procyanidins but not necessarily to that of (-)-epicatechin. This trial was registered at clinicaltrials.gov as NCT02728466.

Short-term supplementation with flavanol-rich cocoa improves lipid profile, antioxidant status and positively influences the AA/EPA ratio in healthy subjects.

We evaluated the short-term effects of a flavanol-rich cocoa (FRC) on lipid profile and selected oxidative stress biomarkers such as oxidized low-density lipoprotein (oxLDL), glutathione (GSH), and F2-isoprostane. We also assessed whether FRC modulates plasma levels of polyunsaturated fatty acids (PUFA) in healthy individuals. The subjects (n=48) were randomly assigned to a low-cocoa group (1 g/d; ~55 mg flavanols) (n=16), middle-cocoa group (2 g/d; ~110 mg flavanols) (n=16), or a high-cocoa group (4 g/d; ~220 mg flavanols) (n=16). The samples were collected at baseline, at 1, 2, and 4 h post initial consumption of FRC, and after 4 weeks of FRC supplementation. The peak plasma concentration of (-)-epicatechin metabolites reached a maximum level (578±61 nM; P<.05) at 2 h after ingestion of FRC. After 4 weeks, total cholesterol (-12.37±6.63; P<.0001), triglycerides (-3.81±2.45; P<.0001), plasma LDL (-14.98±6.77; P<.0001), and oxLDL (-95.61±41.69; P<.0001) decreased in the high-cocoa group, compared with baseline. We also found that plasma high-density lipoprotein (HDL) (+3.37±2.06; P<.0001) concentrations increased significantly in the same group. Total GSH significantly increased in all FRC-treated groups (+209.73±146.8; P<.0001), while urinary F2-isoprostane levels decreased in the middle- (-0.73±0.16; P<.0001) and high-cocoa (-1.62±0.61; P<.0001) groups. At the end of the four-week study, a significant reduction of arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio was observed in the low-(-2.62±2.93; P=.003), middle- (-5.24±2.75; P<.0001) and high-cocoa (-7.76±4.96; P<.0001) groups, compared with baseline. Despite the small sample size used in this study, these data extend previous clinical and experimental studies, providing new insights into the health benefits of cocoa flavanols.

Plasma tea catechins and risk of cardiovascular disease in middle-aged Japanese subjects: The JPHC study.

BACKGROUND AND AIMS: Although a potential benefit of drinking green tea has been suggested to reduce the development of cardiovascular disease, no study has investigated the relationship between plasma tea catechin and risk of cardiovascular disease. METHODS: A prospective, nested case-control study was conducted to examine the association between plasma tea catechin and risk of stroke and coronary heart disease (CHD) in a cohort of 29,876 men and women aged 40-69 years without history of heart disease, stroke or cancer. Participants completed a survey and donated blood samples between 1990 and 1994, and were followed-up through 2008. A total of 1132 stroke cases and 209 CHD cases, matched 1:1 to controls (n = 1132) for stroke and 1:2 to controls (n = 418) for CHD, were included in the analysis. RESULTS: We found no significant association between plasma tea catechin and the incidence of stroke or CHD in either men or women. However, we found that high plasma levels of epigallocatechin gallate (EGCG) were associated with reduced risk of stroke in non-smoking men; the adjusted odds ratio (95% CI) for the highest vs. non-detectable levels was 0.53 (0.29-0.98). The respective OR in male smokers was 1.23 (0.75-2.16). A significant interaction by smoking status was found for the highest vs. non-detected plasma EGCG in relation to stroke (p-for-interaction: p = 0.09). CONCLUSIONS: Plasma tea catechin was not associated with reduced risks of either stroke or CHD, while a protective effect of certain tea catechin on stroke risk is suggested for male non-smokers.

Comparative analysis of fecal phenolic content between normal and obese rats after oral administration of tea polyphenols.

Tea polyphenols (TP) have many health benefits, but most are metabolized into low molecular-weight phenolic acids after oral administration. In the present study, the absorption, metabolism, and excretion of catechins in rats fed a normal chow diet and in obese rats fed a high-fat and high-sugar (HFHS) diet were compared. After a ten-day oral administration of TP (500 mg per kg bw), the plasma levels of (-)-epigallocatechin gallate (EGCG) and (-)-gallocatechin gallate (GCG) in obese rats were significantly lower than those in the normal group. In obese rats, the fecal levels of EGCG, (-)-epicatechin gallate (ECG) and GCG were significantly enhanced. Ten phenolic metabolites of TP were quantitatively analyzed, and the results showed that 4-hydroxyphenylacetic acid was the primary metabolite in feces and plasma. The plasma and fecal concentrations of 4-hydroxyphenylacetic acid in the obese group were significantly lower than those in normal rats, but the levels of 4-hydroxyphenylpropionic acid in plasma and feces were increased. The content of other phenolic acids was also dramatically changed. These results suggested that a HFHS diet might influence the excretion of tea catechins, leading to insufficient metabolism of catechins by the gut microflora.

Development and validation of a high-throughput assay for the quantification of multiple green tea-derived catechins in human plasma.

A rapid, accurate and robust method for the determination of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) concentrations in human plasma has been developed. The method utilizes protein precipitation following enzyme hydrolysis, with chromatographic separation and detection using reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). Traditional issues such as lengthy chromatographic runtimes, sample and extract stability, and lack of suitable internal standards have been addressed. The method has been evaluated using a comprehensive validation procedure, confirming linearity over appropriate concentration ranges, and inter/intra-batch precision and accuracies within suitable thresholds (precisions within 13.8% and accuracies within 12.4%). Recoveries of analytes were found to be consistent between different matrix samples, compensated for using suitable internal markers and within the performance of the instrumentation used. Similarly, chromatographic interferences have been corrected using the internal markers selected. Stability of all analytes in matrix is demonstrated over 32 days and throughout extraction conditions. This method is suitable for high-throughput sample analysis studies.

Phenolic metabolites in plasma and tissues of rats fed with a grape pomace extract as assessed by liquid chromatography-tandem mass spectrometry.

Grape pomace extract (GPE) is a rich and relatively low-cost source of phenolic compounds. However, little is known about the main GPE metabolites in mammals, which could help explain the observed health-promoting effects. This study investigated the presence of parent compounds from flavanol, flavonol and stilbene families and their metabolites in rat plasma and tissues after an acute intake of GPE in doses of 300 and 600 mg kg/body weight. The measurement of free compounds and their metabolites was performed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results showed the presence of epicatechin, epicatechin methyl-glucuronide, epicatechin methyl-sulphate, catechin, catechin-glucuronide, quercetin methyl-glucuronide, resveratrol-3-glucuronide, resveratrol-4-glucuronide and resveratrol-3-sulphate in plasma, which was dose dependent. The most abundant measured compound in plasma was epicatechin-glucuronide. The presence of glucuronidated and methyl-glucuronidated forms of catechin were observed in the liver at both doses, while epicatechin-glucuronide and methyl-glucuronide were detected only upon intake of 600 mg GPE/kg body weight. At this dose epicatechin-glucuronide and methyl-glucuronide were also detected in muscle, and catechin methyl-glucuronide in adipose tissue. Results show the main GPE metabolites present in rat tissues after oral consumption, contributing to better understand the health benefits of GPE and its potential utilization as a functional ingredient.

The effect of tea consumption on the steroid profile.

Green tea (GT), along with its flavonol epigallocatechin-3-gallate (EGCG), has shown to inhibit the UGT2B17 isoenzyme, which is highly involved in the glucuronidation of testosterone (T) and its metabolites. Since the steroid profile (SP) is composed of urinary concentrations of T and related metabolites excreted in both the free and the glucuronide fractions, GT consumption could alter the SP, leading to misunderstanding in doping controls. The aim of the present work was to study the effect of GT consumption on the SP. This study was performed with 29 male volunteers, which could be classified in 2 arms depending on their T/E values (0.12 ± 0.02, n = 12; 1.64 ± 0.90, n = 17). The clinical protocol was designed to evaluate the effect of GT administration on the SP biomarkers. Participants were asked to consume GT with a high content of EGCG for 7 days (5 GT beverages along the whole day for days 1-6 and 9 GT beverages on day 7, corresponding to 520 and 936 mg/day of EGCG, respectively). Urine samples were collected before and during GT consumption at different time periods. The SP was measured using gas chromatography-mass spectrometry. The excretion rates of the SP metabolites did not change after GT consumption. Moreover, the individual evaluation of the subject's steroidal biological passport resulted in normal sequences. The results obtained show that GT consumption does not distort the establishment of normal ranges of SP parameters. Therefore, GT consumption does not need to be considered a confounding factor in the SP evaluation.

Quantitative Analysis of Four Catechins from Green Tea Extract in Human Plasma Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry for Pharmacokinetic Studies.

Green tea is consumed as a beverage worldwide and has beneficial effects, such as a lower risk of cardiovascular disease and cancer. A quantitative analysis of the beneficial components in plasma is important for understanding the potential health benefits of green tea. Four catechins­epigallocatechin-3-gallate (EGCG), epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epicatechin (EC)­which account for the majority of the components of green tea, were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In this study, a validated method was optimized to obtain the blood concentrations after the one-time ingestion of 630 mg green tea extract with digoxin and then after the ingestion of 630 mg green tea repeatedly for 15 days. The calibration curve, including the LLOQ, was constructed over 1⁻500 ng/mL for EGCG, ECG, and EGC and 0.1⁻50 ng/mL for EC. The method for inter- and intra-validation was applied, acceptable for both accuracy and precision. We successfully developed an appropriate UPLC-MS/MS method for human plasma with good reproducibility and sensitivity. Thus, this method could be applied for future preclinical and clinical studies on EGCG, ECG, EGC, and EC.

Absorption kinetics of vitamin E nanoemulsion and green tea microstructures by intestinal in situ single perfusion technique in rats.

The absorption kinetics of food ingredients such as nanoemulsified vitamin E and green tea microstructures were evaluated by the intestinal in situ single perfusion technique. Absorption rate, sub-acute oral toxicity and organ morphology in a rat model were examined. The intestinal in situ single perfusion technique and HPLC analysis were applied to investigate the absorption rate of selected materials by examining time-dependent changes in the serum levels of catechin and dl-α-tocopherol. The acute toxicity test and histopathological evaluation were applied to analyze the safety of microsized green tea and nanosized vitamin E in a rat model. Total serum dl-α-tocopherol levels significantly increased with nanosized vitamin E administration (P<0.05). Rats treated to nanosized vitamin E until 90min after administration showed significantly increased absorption rate of serum dl-α-tocopherol levels at each time point (10min interval) (P<0.001). Rats administered 2000mg/kg of nanosized vitamin E and microsized green tea did not show signs of acute toxicity or death after 14days of observation. In addition, macroscopic analysis showed that there were no changes in representative organ sections of rats following the oral administration of food-related nanoscale materials. We successfully demonstrated that using nanosized vitamin E increased absorption rate to a greater extent than normal food-related material, and these results occurs via safety analyses on food-related nanoscale materials for human consumption. These results could be useful for the design and development of novel nanoemulsified vitamin E and microsized green tea formulations that can overcome the problem of their bioavailability and improve their efficacy while still maintaining their essential therapeutic efficacies.