Simultaneous determination of five constituents of areca nut extract in rat plasma using UPLC-MS/MS and its application in a pharmacokinetic study.

Areca nuts have been used as a traditional Chinese medicine (TCM) for thousands of years. Recent studies have shown that it exhibits good pharmacological activity and toxicity. In this study, the pharmacokinetics of five major components of areca nut extract in rats were investigated using a highly sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method. Arecoline, arecaidine, guvacoline, guvacine, and catechin were separated and quantified accurately using gradient elution with mobile phases of (A) water containing 0.1 % formic acid-10 mM ammonium formate, and (B) methanol. The constituents were detected under a timing switch between the positive and negative ion modes using multiple reaction monitoring (MRM). Each calibration curve had a high R2 value of >0.99. The method accuracies ranged -7.09-11.05 % and precision values were less than 14.36 %. The recovery, matrix effect, selectivity, stability, and carry-over of the method were in accordance with the relevant requirements. It was successfully applied for the investigation of the pharmacokinetics of these five constituents after oral administration of areca nut extract. Pharmacokinetic results indirectly indicated a metabolic relationship between the four areca nut alkaloids in rats. For further clarification of its pharmacodynamic basis, this study provided a theoretical reference.

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.

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.

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.

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.

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.

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.

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.

Effects of Supplemental Epigallocatechin Gallate in the Diet of Broilers Exposed to Fluoride Intoxication.

We evaluated the effects of dietary epigallocatechin gallate (EGCG) on the performance, biochemical parameters, and liver histopathology of fluoride-intoxicated broiler chickens. In total, 160 1-day-old male broiler chicks (Ross PM3 strain) were collected and assigned to four groups (40 animals each), with four replicates. The control group received a basal diet; the F group received 800 mg/kg fluoride; the EGCG group received 400 mg/kg EGCG; and the EGCG+F group received 400 mg/kg EGCG and 800 mg/kg fluoride. The live weight (LW) of F-treated chicks was significantly lower than that of the controls. In the F-treated groups, feed intake (FI) and LW values were lower, but the feed conversion ratio (FCR) was higher than those of the controls. The ratio of heart weight to LW was found to be the highest in the F-treated groups. Alkaline phosphatase (ALP), aspartate aminotransferase (AST), and total oxidant status (TOS) levels in the F-treated groups were significantly higher, whereas the increase in total cholesterol levels was insignificant than those in the control group. In the EGCG+F group, AST, total cholesterol, and TOS levels decreased to a level comparable to those in the control group. Histopathological evaluation revealed that there were mild changes in the portal region in the EGCG+F group; additionally, there was an improvement in liver morphology in the EGCG+F group compared to that in the F group. Thus, EGCG has potent antioxidant and regenerative effects that can ameliorate the detrimental effects of fluoride toxicity on blood parameters and the liver.

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.

Lack of pharmacokinetic interaction between fluvastatin and green tea in healthy volunteers.

PURPOSE: The objective of this study is to assess the effects of green tea and its major catechin component, (-)-epigallocatechin gallate (EGCG), on CYP2C9-mediated substrate metabolism in vitro, and the pharmacokinetics of fluvastatin in healthy volunteers. METHODS: The metabolism of diclofenac and fluvastatin in human recombinant CYP2C9 was investigated in the presence of EGCG. In a randomized three-phase crossover study, 11 healthy volunteers ingested a single 20-mg dose of fluvastatin with green tea extract (GTE), containing 150 mg of EGCG, along with water (300 mL), brewed green tea (300 mL), or water (300 mL) after overnight fasting. Plasma concentrations of fluvastatin and EGCG were measured by ultra-performance liquid chromatography with fluorescence detection and a single mass spectrometer. RESULTS: EGCG inhibited diclofenac 4'-hydroxylation and fluvastatin degradation with IC50 of 2.23 and 48.04 µM, respectively. Brewed green tea used in the clinical study also dose-dependently inhibited the metabolism of diclofenac and fluvastatin in vitro. However, no significant effects of GTE and brewed green tea were observed in plasma concentrations of fluvastatin. The geometric mean ratios with 90% CI for area under the plasma concentration-time curve (AUC0-∞) of fluvastatin were 0.993 (0.963-1.024, vs. brewed green tea) and 0.977 (0.935-1.020, vs. GTE). CONCLUSIONS: Although in vitro studies indicated that EGCG and brewed green tea produce significant inhibitory effects on CYP2C9 activity, the concomitant administration of green tea and fluvastatin in healthy volunteers did not influence the pharmacokinetics of fluvastatin.

Quercetin and fisetin enhanced the small intestine cellular uptake and plasma levels of epi-catechins in in vitro and in vivo models.

Quercetin and fisetin, known as catechol-containing flavonoids, could positively affect the absorption of catechins due to their strong affinity for catechol-O-methyl transferase (COMT), which can methylate and cause the excretion of catechins. The current study examined the effect of quercetin and fisetin on the absorption of epi-catechins (ECs) by using a Caco-2 cell line and an in vivo model. The intestinal transport of total catechins by Caco-2 cells was enhanced from 1.3- to 1.6-fold and 1.4- to 1.7-fold by adding quercetin and fisetin, respectively, compared to the control. It was even higher in the treatment with a mixture of quercetin and fisetin. While EC had the highest value of intestinal transport (169% of the control) in 10% quercetin treatment, EGC (235%), EGCG (244%), and ECG (242%) were significantly transported in the treatment with a 5% mixture of quercetin and fisetin (p < 0.05). In an in vivo pharmacokinetic study, the values of the area under the plasma concentration-time curve (AUC, ng h mL-1) were also higher in rats orally administered EGCG with 10% quercetin (365.5 ± 25.5) or 10% fisetin (825.3 ± 46.7) than in those administered EGCG only (111.3 ± 13.1). Methylated quercetin and methylated fisetin were determined to be m/z 317.24 and m/z 301.25 [M + H]+ with their own product ions, respectively. The results indicate that quercetin or fisetin is superior to ECs for methylation by COMT.

Global testing of shifts in metabolic phenotype.

INTRODUCTION: Current metabolomics approaches to unravel impact of diet- or lifestyle induced phenotype variation and shifts predominantly deploy univariate or multivariate approaches, with a posteriori interpretation at pathway level. This however often provides only a fragmented view on the involved metabolic pathways. OBJECTIVES: To demonstrate the feasibility of using Goeman's global test (GGT) for assessment of variation and shifts in metabolic phenotype at the level of a priori defined pathways. METHODS: Two intervention studies with identified phenotype variations and shifts were examined. In a weight loss (WL) intervention study obese subjects received a mixed meal challenge before and after WL. In a polyphenol (PP) intervention study obese subjects received a high fat mixed meal challenge (61E% fat) before and after a PP intervention. Plasma samples were obtained at fasting and during the postprandial response. Besides WL- and PP-induced phenotype shifts, also correlation of plasma metabolome with phenotype descriptors was assessed at pathway level. The plasma metabolome covered organic acids, amino acids, biogenic amines, acylcarnitines and oxylipins. RESULTS: For the population of the WL study, GGT revealed that HOMA correlated with the fasting levels of the TCA cycle, BCAA catabolism, the lactate, arginine-proline and phenylalanine-tyrosine pathways. For the population of the PP study, HOMA correlated with fasting metabolite levels of TCA cycle, fatty acid oxidation and phenylalanine-tyrosine pathways. These correlations were more pronounced for metabolic pathways in the fasting state, than during the postprandial response. The effect of the WL and PP intervention on a priori defined metabolic pathways, and correlation of pathways with insulin sensitivity as described by HOMA was in line with previous studies. CONCLUSION: GGT confirmed earlier biological findings in a hypothesis led approach. A main advantage of GGT is that it provides a direct view on involvement of a priori defined pathways in phenotype shifts.