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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.

Dihydromyricetin is a natural bioactive flavonoid with unique GABA A receptor activity with a putative mechanism of action to reduce the intoxication effects of ethanol. Although dihydromyricetin's poor oral bioavailability limits clinical utility, the promise of this mechanism for the treatment of alcohol use disorder warrants further investigation into its specificity and druggable potential. These experiments investigated the bioavailability of dihydromyricetin in the brain and serum associated with acute anti-intoxicating effects in C57BL/6J mice. Dihydromyricetin (50 mg/kg IP) administered 0 or 15-min prior to ethanol (PO 5 g/kg) significantly reduced ethanol-induced loss of righting reflex. Total serum exposures (AUC0 → 24) of dihydromyricetin (PO 50 mg/kg) via oral (PO) administration were determined to be 2.5 µM × h (male) and 0.7 µM × h (female), while intraperitoneal (IP) administration led to 23.8-fold and 7.2- increases in AUC0 → 24 in male and female mice, respectively. Electrophysiology studies in α5β3γ2 GABA A receptors expressed in Xenopus oocytes suggest dihydromyricetin (10 µM) potentiates GABAergic activity (+43.2%), and the metabolite 4-O-methyl-dihydromyricetin (10 µM) negatively modulates GABAergic activity (-12.6%). Our results indicate that administration route and sex significantly impact DHM bioavailability in mice, which is limited by poor absorption and rapid clearance. This correlates with the observed short duration of DHM's anti-intoxicating properties and highlights the need for further investigation into mechanism of DHM's potential anti-intoxicating properties.

Ginkgo biloba extract (GBE) is a popular botanical dietary supplement used worldwide and the safety of use is a public health concern. While GBE is a complex mixture, the terpene trilactones and flavonol glycosides are believed to elicit the pharmacological and/or toxicological effects of GBE. In a National Toxicology Program (NTP) 2-year rodent bioassay with GBE, hepatotoxicity was observed in rodents (≥100 mg/kg in rats, ≥ 200 mg/kg in mice). Subsequently, questions arose about whether or not the GBE used in NTP studies was representative of other GBE products and how rodent doses are related to human doses. To address these, we generated systemic exposure data for terpene trilactones in male rats following oral administration of 30, 100, and 300 mg/kg GBE test article from the 2-year bioassay. Dose-normalized C max and AUC ∞ for terpene trilactones from the current study were within 5-fold of published rodent studies using a standardized GBE preparation. Comparison of our rat systemic exposure data at 100 mg/kg GBE to published human data following ingestion of 240 mg GBE-containing product showed that the rat/human exposure multiple was 3-22, for terpene trilactones. These data demonstrate the relevance of NTP rodent toxicity data to humans., (Published by Elsevier Ltd.)

Cocoa has beneficial health effects partly due to its high flavanol content. This study was aimed at assessing the absorption and metabolism of polyphenols in two soluble cocoa products: a conventional (CC) and a flavanol-rich product (CC-PP). A crossover, randomized, blind study was performed in 13 healthy men and women. On two different days, after an overnight fast, volunteers consumed one serving of CC (15 g) or CC-PP (25 g) in 200 mL of semi-skimmed milk containing 19.80 mg and 68.25 mg of flavanols, respectively. Blood and urine samples were taken, before and after CC and CC-PP consumption, and analyzed by high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QToF-MS). Up to 10 and 30 metabolites were identified in plasma and urine, respectively. Phase II derivatives of epicatechin were identified with kinetics compatible with small intestine absorption, although the most abundant groups of metabolites were phase II derivatives of phenyl-γ-valerolactone and phenylvaleric acid, formed at colonic level. 5-(4'-Hydroxyphenyl)-γ-valerolactone-sulfate could be a sensitive biomarker of cocoa flavanol intake. CC and CC-PP flavanols showed a dose-dependent absorption with a recovery of 35%. In conclusion, cocoa flavanols are moderately bioavailable and extensively metabolized, mainly by the colonic microbiota.

Objectives: Strenuous exercise can impair athletic performance due to muscular inflammation and oxidative stress. Antioxidants such as cocoa flavanols have been used as a supplement to prevent oxidative stress; however, the benefits of dietary antioxidants for athletic performance after muscle soreness (MS) is unclear. The purpose of this study was to examine the effects of cocoa flavanols after a MS inducing protocol., Methods: In a randomized, double-blinded design, 13 male collegiate rugby players consumed either chocolate milk (CHOC) or chocolate milk with additional cocoa flavanols (CocoaCHOC) during a 7-d loading phase. MS was induced by a drop jump protocol on day 5 of the intervention. Athlete performance was assessed with vertical-jump and yo-yo tests and subjective measures of soreness 5 d before and 2 d post-MS protocol. Urinary markers of oxidative stress (isoprostanes) were assessed before and 48 h post-MS., Results: No changes were observed between the groups over time for isometric torque (P = .63), vertical jump performance (P = .39), and yo-yo testing (P = .57) between the trials. No interaction was found in isoprostanes levels between the trials (CocoaCHOC baseline: 88 ± 0.38 pg/mL and 48 h post-MS: 81 ± 0.53 pg/mL; P = .82; and CHOC baseline: 98 ± 0.96 pg/mL and 48 h post-MS: 96 ± 0.38 pg/mL; P = .59). No main effect (treatment × time; P = .58) was observed for isoprostanes. Although not significant, the CocoaCHOC group ran 97 meters further than the CHOC group in the yo-yo test., Conclusions: Cocoa flavanols added to a post-exercise recovery beverage for 7 d has no oxidative stress or athletic performance benefits., (Copyright © 2018. Published by Elsevier Inc.)

A reliable liquid chromatography/tandem mass (LC-MS/MS) method was developed for quantitation of 3', 4'-dimethoxy flavonol-3-β-d-glucopyranoside (DF3G) in rat plasma using pantoprazole as the internal standard. An Agilent C18 column (100 mm × 3 mm, 3.5 μm) was performed for chromatographic separation with the mobile phase composed of methanol-water (containing formic acid) at a flow rate of 0.4 mL/min. A triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source in positive ion mode was applied to quantitative analysis by multiple reacting monitoring (MRM). The MRM precursor-product ion transition of DF3G and Pantoprazole (IS) were m/z 461.1 → 299.2 and 383.9 → 200.2, respectively. Calibration curves were recovered in a concentration range of 1-500 ng/mL for plasma with a limit of lower quantification (LLOQ) of 1 ng/mL. The intra-day and inter-day precision were not >10%. The accuracy of DF3G ranged from -10% to 0.53% in quality control (QC) samples at three concentrations. DF3G was not degraded during the analysis and the storage period. All the data were validated in accordance with the FDA bioanalytical method validation guideline. The LC-MS/MS method was successfully employed in the pharmacokinetic study of the DF3G after oral administration and intravenous injection in rats., (Copyright © 2018. Published by Elsevier B.V.)

Mang-Guo-Zhi-Ke tablets (MGZKTs) is an effective Chinese patent medicine. It contains mango leaf extract as the main raw material and the antihistamine drug, chlorpheniramine maleate is included in the formulation. However, its pharmacokinetic effect is rarely reported. A highly sensitive, reliable and rapid high-throughput method using ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) was used to simultaneously determine kaempferol, quercetin, mangiferin, p-hydroxybenzoic acid, gallic acid and chlorpheniramine maleate in rat plasma after oral administration of MGZKTs. The method was successfully developed and fully validated to investigate the pharmacokinetics of MGZKTs. Chloramphenicol and clarithromycin were used as internal standards (IS). A practicable protein precipitation procedure with methanol was adopted for sample preparation. The samples were separated on an Acquity UHPLC Syncronis C 18 column (100 × 2.1 mm, 1.7 μm) using 0.1% formic acid-acetonitrile as the mobile phase. The flow rate was set at 0.4 mL/min. The obtained calibration curves were linear in the concentration range of ~1-1000 ng/mL for plasma (r > 0.99). Method validation results met the criteria reported in the US Food and Drug Administration guidelines. Quercetin, p-hydroxybenzoic acid and kaempferol were absorbed rapidly and reached the peak concentration between 0.16 and 0.25 h. This validated that the UHPLC-MS/MS method was successfully applied to study the pharmacokinetic parameters of the six compounds in rat plasma after oral administration of MGZKTs. This evidence will be useful for the clinical rational use of Mang-Guo-Zhi-Ke tablets., (Copyright © 2017 John Wiley & Sons, Ltd.)

A simple and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of isoquercitrin, kaempferol-3-O-rutinoside and tiliroside in rat plasma. Plasma samples were deproteinized with methanol and separated on a Hypersil Gold C 18 column (2.1 × 50 mm, i.d., 3.0 μm) using gradient elution with the mobile phase of water and methanol at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed with negative ion electrospray ionization in selected reaction monitoring mode. All analytes showed good linearity over their investigated concentration ranges (r 2  > 0.99). The lower limit of quantification was 1.0 ng/mL for isoquercitrin and 2.0 ng/mL for kaempferol-3-O-rutinoside and tiliroside, respectively. Intra- and inter-day precisions were <8.2% and accuracy ranged from -11.5 to 9.7%. The mean extraction recoveries of analytes and IS from rat plasma were >80.4%. The assay was successfully applied to investigate the pharmacokinetic study of the three ingredients after oral administration of Rubus chingii Hu to rats., (Copyright © 2017 John Wiley & Sons, Ltd.)

Context: The pharmacokinetics properties of dihydromyricetin (DHM) are still unknown., Objective: This study investigates the pharmacokinetic characteristics of DHM using a sensitive and reliable LC-MS/MS method., Materials and Methods: A rapid and sensitive LC-MS/MS method was developed for the determination of DHM in male Sprague-Dawley rat plasma. Twelve rats were equally randomized into two groups, including the intravenous group (2 mg/kg) and the oral group (20 mg/kg). Blood samples (250 μL) were collected at designated time points and analyzed using this method. The pharmacokinetic parameters were calculated using DAS 3.0 pharmacokinetic software., Results: The calibration curve was linear within the range of 0.5-200 ng/mL (r > 0.998) with the lower limit of quantification at 0.5 ng/mL. After the intravenous injection, DHM reached a maximum concentration of 165.67 ± 16.35 ng/mL, and t 1/2 was 2.05 ± 0.52 h. However, DHM was not readily absorbed and reached C max 21.63 ± 3.62 ng/mL at approximately 2.67 h following the oral administration of DHM, and t 1/2 was 3.70 ± 0.99 h. The MRT for the intravenous group and the oral group were 2.62 ± 0.36 and 5.98 ± 0.58 h, respectively. The AUC (0-t) for the intravenous group and the oral group were 410.73 ± 78.12 and 164.97 ± 41.76 ng·L/mL, respectively, so the absolute bioavailability of DHM was 4.02% which was poor., Discussion and Conclusion: The results indicated that the bioavailability was poor. Further work needs to be conducted to investigate the reason for poor bioavailability and improve this situation.

Engeletin, a bioactive flavonoid, has attracted much attention recently by virtue of its multiple biological (anti-diabetic and anti-inflammatory) activities. Despite signifying many therapeutic applications researches indicating quantification or pharmacokinetics of engeletin in biological matrix are still lacking. Here, a simple, sensitive, accurate and reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) approach for the quantification of engeletin in rat plasma was developed and fully validated for the first time. Plasma samples were processed with acetonitrile by a single step protein precipitation and the separation was achieved on a ZORBAX Eclipse Plus C18 Rapid Resolution High Definition column with a gradient acetonitrile-water mobile phase. Quantification of engeletin was carried out by electrospray ionization tandem mass spectrometry in multiple reaction monitoring (MRM) mode with negative ionization. Results revealed that the approach was linearity from 5 to 5000ng/mL (r 2 =0.9937) and proved to be precise (better than 12.3%) and accurate (-3.3%-5.2%). The developed approach was successfully employed to pharmacokinetic study of engeletin following peroral and intravenous administration to rats. The results of pharmacokinetics demonstrated rapid engeletin absorption (T max of 15min) after oral administration, extensive distribution after three different dosages and an absolute bioavailability of ∼1.53%. The developed method and pharmacokinetic data can provide a meaningful basis for further studies on engeletin., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Although in vitro studies have shown that flavonoids are metabolized into phenolic acids by the gut microbiota, the biotransformation of flavonoids by intestinal microbiota is seldom studied in vivo. In this study, we investigated the impact of the gut microbiota on the biotransformation of 3 subclasses of flavonoids (flavonols, flavones, and flavanones). The ability of intestinal microbiota to convert flavonoids was confirmed with an in vitro fermentation model using mouse gut microflora. Simultaneously, purified flavonoids were administered to control and antibiotic-treated mice by gavage, and the metabolism of these flavonoids was evaluated. p-Hydroxyphenylacetic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, hydrocaffeic acid, coumaric acid, and 3-(4-hydroxyphenyl)propionic acid were detected in the serum samples from the control mice after flavonoid consumption. The serum flavonoid concentrations were similar in both groups, whereas the phenolic metabolite concentrations were lower in the antibiotic-treated mice than in the control mice. We detected markedly higher flavonoids excretion in the feces and urine of the antibiotic-treated mice compared to the controls. Moreover, phenolic metabolites were upregulated in the control mice. These results suggest that the intestinal microbiota are not necessary for the absorption of flavonoids, but are required for their transformation., (© 2016 Institute of Food Technologists®.)

Scope: Tomato contains a variety of phenolics associated with health-promoting properties. However, the effects of processing and the addition of oil during tomato sauce preparation on microbial metabolism of phenolics in the small intestine are still unclear., Methods and Results: An open, controlled, randomized, and crossover feeding trial with 40 healthy volunteers was carried out to analyze the metabolites in plasma and urine after the consumption of tomato and tomato sauces, with tomato sauce enriched with refined olive oil (ROOE) and without refined olive oil (oil-free: OF). Ten phenolics in plasma and 93 metabolites in urine were quantified. Processing tomatoes into sauce enhanced the bioavailability of flavanones, flavanols, and some hydroxycinnamic acids, as reflected by the increase in the area under the plasma concentration versus time curve. An increase in their plasma half-life was also observed, particularly after ingestion of ROOE, possibly favored by enterohepatic circulation. A wide variety of gut microbial metabolites was also detected, namely flavanones, hydroxycinnamic acids, flavonols, hydroxyphenylpropanoic acids, hydroxyphenylacetic acids, and hydroxybenzoic acids., Conclusions: Flavanones and flavonols in ROOE presented higher bioavailability, suggesting that the processing undergone by the raw tomato improved their absorption., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Complanatoside A is a flavonol glycoside isolated from Astragalus complanatus, and currently it is used as a quality control index for A. complanatus in the 2010 edition of the Chinese Pharmacopoeia. For the first time, a simple and sensitive LC-MS/MS method was developed for the determination of complanatoside A in rat plasma over the range of 2.3-575 ng/mL. Complanatoside A was extracted from plasma by a protein precipitation procedure, separated by LC and detected by MS/MS in positive electrospray ionization mode. The method was validated for selectivity, carryover, sensitivity, linearity, extraction recovery, matrix effect, accuracy, precision and stability studies. The lower limit of quantification was established at 2.3 ng/mL. Intra- and inter-day precisions (LLOQ, low-QC, med-QC and high-QC) were <7.9%, and accuracies were between 94.0 and 105.1%. Matrix effect was acceptable (97.9-103.0%) and extraction recovery was reproducible (88.5-94.4%). Complanatoside A was stable in the investigated conditions. The method was applied to the pharmacokinetics of complanatoside A in rats. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Objective: To examine whether dietary intake of specific flavonoid subclasses (including flavonols, flavones, flavanones, flavan-3-ols, anthocyanins, and flavonoid polymers) is associated with weight change over time., Design: Three prospective cohort studies., Setting: Health professionals in the United States., Participants: 124,086 men and women participating in the Health Professionals Follow-up Study (HPFS), Nurses' Health Study (NHS), and Nurses' Health Study II (NHS II)., Main Outcome Measure: Self reported change in weight over multiple four year time intervals between 1986 and 2011., Results: Increased consumption of most flavonoid subclasses, including flavonols, flavan-3-ols, anthocyanins, and flavonoid polymers, was inversely associated with weight change over four year time intervals, after adjustment for simultaneous changes in other lifestyle factors including other aspects of diet, smoking status, and physical activity. In the pooled results, the greatest magnitude of association was observed for anthocyanins (-0.23 (95% confidence interval -0.30 to -0.15) lbs per additional standard deviation/day, 10 mg), flavonoid polymers (-0.18 (-0.28 to -0.08) lbs per additional SD/day, 138 mg), and flavonols (-0.16 (-0.26 to -0.06) lbs per additional SD/day, 7 mg). After additional adjustment for fiber intake, associations remained significant for anthocyanins, proanthocyanidins, and total flavonoid polymers but were attenuated and no longer statistically significant for other subclasses., Conclusions: Higher intake of foods rich in flavonols, flavan-3-ols, anthocyanins, and flavonoid polymers may contribute to weight maintenance in adulthood and may help to refine dietary recommendations for the prevention of obesity and its potential consequences., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Immaturity of the neonatal immune system is causative for high morbidity in calves and colostrum intake is crucial for acquiring passive immunity. Pathogenesis is promoted by reactive oxygen species accumulating at birth if counter-regulation is inadequate. The flavonol quercetin exerts antioxidative and anti-inflammatory effects that may enhance neonatal health. The aim of this work was to study effects of quercetin feeding on metabolic, antioxidative and inflammatory parameters in neonatal calves to investigate whether quercetin could compensate for insufficient colostrum supply. Twenty-eight newborn calves were assigned to two dietary groups fed colostrum or milk-based formula on day 1 and 2 and milk replacer thereafter. From day 2 onwards, 7 calves per diet group were additionally fed quercetin aglycone (50 mg/(kg body weight × day)). Blood samples were taken repeatedly to measure plasma concentrations of flavonols, glucose, lactate, total protein, albumin, urea, non-esterified fatty acids, triglycerides, cholesterol, insulin, glucagon, cortisol, immunoglobulins, fibrinogen, haptoglobin and serum amyloid A. Trolox equivalent antioxidative capacity, ferric reducing ability of plasma, thiobarbituric acid reactive species and F2-isoprostanes were analyzed to evaluate plasma antioxidative status. Expression of tumor necrosis factor, interleukin-1α, interleukin-1β, serum amyloid A, haptoglobin, fibrinogen, C-reactive protein, catalase, glutathione peroxidase and superoxide dismutase mRNA were measured in liver tissue on day 8. Plasma flavonol concentrations were detectable only after quercetin-feeding without differences between colostrum and formula feeding. Plasma glucose, lactate, total protein, immunoglobulins, triglycerides, cholesterol, trolox equivalent antioxidative capacity and thiobarbituric acid reactive species were higher after colostrum feeding. Body temperature, fecal fluidity and plasma concentrations of cortisol and haptoglobin were higher in formula- than in colostrum-fed groups. Hepatic mRNA expression of tumor necrosis factor was higher after quercetin feeding and expression of C-reactive protein was higher after formula feeding. Data confirm that colostrum improves neonatal health and indicate that quercetin feeding cannot compensate for insufficient colostrum supply.

Background: Inadequate colostrum supply results in insufficient intake of macronutrients and bioactive factors, thereby impairing gastrointestinal development and the maturation of glucose metabolism in neonatal calves. The flavonoid quercetin has been shown to have health-promoting properties, including effects in diabetic animals. However, quercetin interacts with intestinal glucose absorption and might therefore exert negative effects in neonates., Objective: We evaluated the interaction between neonatal diet and quercetin feeding on splanchnic glucose metabolism in neonatal calves., Methods: Calves (n = 28) were assigned to 4 groups and fed either colostrum or a milk-based formula on days 1 and 2 and supplemented daily with 148 μmol quercetin aglycone/kg body weight [colostrum with quercetin (CQ+)/formula with quercetin (FQ+)] or without this substance [colostrum without quercetin (CQ-)/formula with quercetin (FQ-)] from days 2-8. From day 3 onward, all calves received milk replacer. A xylose absorption test was performed on day 3, and on day 7, blood samples were collected to study glucose first-pass uptake after [(13)C6]-glucose feeding and intravenous [6,6-(2)H2]-glucose bolus injection. Plasma concentrations of metabolites and hormones were measured by taking additional blood samples. A biopsy specimen of the liver was harvested on day 8 to measure the mRNA expression of gluconeogenic enzymes., Results: Higher postprandial plasma concentrations of glucose, lactate, urea, adrenaline, noradrenaline, insulin, and glucagon on day 7 in colostrum-fed calves indicate that metabolic processes were stimulated. Postabsorptive xylose and glucose plasma concentrations each increased by an additional 26%, and splanchnic glucose turnover decreased by 35% in colostrum-fed calves, suggesting improved glucose absorption and lower splanchnic glucose utilization in colostrum-fed calves. Quercetin supplementation resulted in higher noradrenaline concentrations and enhanced peak absorption and oxidation of [(13)C6]-glucose by 10%. Liver mitochondrial phosphoenolpyruvate carboxykinase mRNA abundance was reduced by 34% in colostrum-deprived calves., Conclusions: Feeding colostrum during the first 2 d of life is crucial for maturation of splanchnic glucose metabolism in calves. Supplementing quercetin improves gastrointestinal absorption capacity, particularly in colostrum-deprived calves., (© 2015 American Society for Nutrition.)

Ampelopsis grossedentata (Hand.-Mazz.) W.T. Wang has long been used as a traditional Chinese medicinal herb among the indigenous people in the Yangtze River region of China. Dihydromyricetin (DMY) is the most abundant (approximately 30%) and bioactive constituent in A. grossedentata (Hand.-Mazz.) W.T. Wang, and recent studies have demonstrated its various pharmacological activities. In the present study, a first specific, sensitive, rapid and reliable LC-MS/MS method for the determination of DMY in rat plasma was developed and validated. The plasma samples were prepared with protein precipitation method, and chromatographic separation was performed on a Welch Ultimate XB-C18 column (50 × 2.1 mm, 5 μm) using a gradient elution with water and acetonitrile. The mass spectrometry (MS) analysis was conducted in negative ionization mode with multiple reaction monitoring (MRM) transitions at m/z 319.1→192.8 for DMY and m/z 609.0→301.2 for rutin (IS). The plasma concentration profiles and pharmacokinetic parameters were analyzed after oral administration of dextroisomer and racemate DMY at the dose of 100 mg/kg in rats. The method validation was conducted over the calibration range of 10.0-5000 ng/ml with the intra- and inter-day precision and accuracy within 12.0% (RSD) and 5.6% (RE). The recoveries, matrix effect and stability under different conditions were all proved acceptable. The values of Tmax, AUC(0-∞) and Vd were significantly different between the groups of dextroisomer and racemate DMY (P<0.05), and pharmacokinetic results revealed their poor absorptions into blood, probably high tissue distributions and slow elimination processes. The present study will provide helpful information for the further studies and future clinical applications of DMY., (Copyright © 2015 Elsevier B.V. All rights reserved.)

For the first time, a selective and rapid ultra-performance liquid chromatography method with tandem mass spectrometric (UPLC-MS/MS) detection for simultaneous determination of typhaneoside and isorhamnetin-3-O-neohesperidoside in rat plasma was developed and validated, which was applied to the pharmacokinetic study of Pollen Typhae extract. The separation was carried out on an ACQUITY UPLC(TM) BEH C18 column with gradient elution using mobile phase including acetonitrile and water (containing 0.1% formic acid). The flow rate was 0.4 mL/min. The detection was conducted by means of electrospray ionization mass spectrometry in negative ion mode with multiple reaction monitoring. The assays were linear over the concentration range of 0.5-100 ng/mL, and the lower limit of quantification was 0.5 ng/mL for typhaneoside and isorhamnetin-3-O-neohesperidoside. The method was validated in terms of intra- and interday precision (<9.37%), accuracy (within ±10.91%), linearity, specificity and stability, and has been successfully applied to a pharmacokinetic study of Pollen Typhae extract in rats after oral administration., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Scope: Insulin-regulated glucose metabolism in cells is critical for proper metabolic functioning, and insulin resistance leads to type 2 diabetes. We performed a human study to assess the availability of structurally related dietary flavonols and tested their ability to affect cellular glucose uptake, metabolism, and glucose transporter gene expression in a liver HepG2 cell model., Methods and Results: Eight healthy volunteers consumed a meal containing galangin, kaempferol, quercetin, and myricetin. In plasma, myricetin was absent, but the others were present, mostly as conjugates. In HepG2 cells, a combination of galangin, kaempferol, and quercetin (5 μM each) for 12 h increased the acute uptake of [U-(14)C]-glucose and 2-[U-(14)C]-deoxyglucose by almost 100 and ∼10%, respectively. All of the combinations increased glucose metabolism, but the effect on transport was less pronounced and mixed. A mixture of all flavonols significantly increased mRNA expression of the main glucose transporter Glut1 in HepG2 cells., Conclusion: These results for the first time show the presence of galangin conjugates in human plasma, and allow direct comparison between absorption of flavonols. A combination of flavonols has the potential to modulate sugar metabolism, both uptake into cells as evident from effects on deoxyglucose, and also further cellular glucose metabolism., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A rapid, reliable, and sensitive method was developed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with an electrospray ionization (ESI) source for determination of seven bioactive compounds in rat plasma after oral administration of Ginkgo biloba tablets (GBTs). The method simultaneously detects bilobalide (BB), ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC), quercetin (QCT), kaempferol (KMF), and isorhamnetin (ISR) for pharmacokinetic study. The analytes and internal standard (IS) were extracted from rat plasma by acetidin. An MS/MS detection was conducted using multiple reaction monitoring (MRM) and operating in the negative ionization mode. The calibration curve ranges were 5-500, 5-500, 2.5-250, 1-100, 1-100, 1-100, and 1-100 ng/ml for BB, GA, GB, GC, QCT, KMF, and ISR, respectively. The mean recovery of the analytes ranged from 68.11% to 84.42%. The intra- and inter-day precisions were in the range of 2.33%-9.86% and the accuracies were between 87.67% and 108.37%. The method was used successfully in a pharmacokinetic study of GBTs. The pharmacokinetic parameters of seven compounds were analyzed using a non-compartment model. Plasma concentrations of the seven compounds were determined up to 48 h after administration, and their pharmacokinetic parameters were in agreement with previous studies.

To establish a method for the determination of astilbin, peoniflorin, rasmarinci acid, isofraxidin and liquiritin contained in Shaolin Xiaoyin tablets, in order to lay a foundation for designing late-stage dosage forms and clinical medication schemes. In this paper, efforts were made to establish a method for the determination of the blood concentration of the five components and study the in vivo pharmacokinetics in rats. The blood concentration was determined by HPLC. Phenomenex C18 column (4.6 mm x 250 mm, 5 microm) was adopted and eluted with methanol-acetonitrile-0.05% formic acid, the flow rate was 0.8 mL x min(-1), and the wavelength was 275 nm. The samples were processed by the solid phase extraction method. After oral administration of Shaoling Xiaoyin tablets, the rat bloods were collected at different time points to determine the blood concentrations. The experimental results showed that the baseline separation could be adopted for the five components, and astilbin, peoniflorin, rasmarinci acid, isofraxidin and liquiritin showed good linear relations within ranges of 2.48-248, 0.213 6-21.36, 0.531-53.1, 0.704-70.4, 0.253-25.3 mg x L(-1). All the five components could be absorbed in blood and excreted quickly. The method established in this paper is rapid and accurate, and could be used for in vivo analysis on preparations containing similar components. The main components in Shaoling Xiaoyin tablets could be absorbed and excreted quickly, and thus suitable to be made into sustained release tablets. Common preparations are required to be taken for 4-6 times a day.

Aim: To establish an LC-MS/MS method for determination of isorhamnetin-3-O-neohesperidoside and investigate its application on pharmacokinetic study in rats., Methods: Eight rats were given 5 mg·kg(-1) isorhamnetin-3-O-neohesperidoside after intravenous administration. A highly sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of isorhamnetin-3-O-neohesperidosidein rat plasma using rutin as internal standard. The analytes and rutin (internal standard) were extracted with methanol followed by a rapid isocratic elution with 10 mmol·L(-1) ammonium acetate buffer/methanol (20 : 80, V/V) on a C18 column (150 mm × 2.1 mm, I.D., 5 μm) and subsequent analysis by mass spectrometry in the multi-eaction-monitoring mode., Results: The assays were linear over the concentration range of 0.01-10 μg·mL(-1) for isorhamnetin-3-O-neohesperidosidein rat plasma. The lower limit of quantifications for isorhamnetin-3-O-neohesperidoside was 0.01 μg·mL(-1)., Conclusion: The validated method is successfully applied to determine the plasma concentrations of isorhamnetin-3-O-neohesperidosidein in rats., (Copyright © 2013 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Quercetin has been shown to be a potent antioxidant, acts hepatoprotectively, and affects glucose and lipid metabolism in monogastrics. If this is also true in ruminants, quercetin could be beneficial in periparturient high-yielding dairy cows by ameliorating the negative effects of free radical formation and reducing the severity of liver lipidosis and ketosis. In a first attempt to evaluate effects of a long-term quercetin treatment, we intraduodenally administered twice daily 18 mg of quercetin (Q)/kg of body weight to 5 late-lactation (215d in milk) dairy cows over a period of 28 d. Frequent blood samples were taken before and during administration to determine plasma concentrations of flavonols and metabolites. Before and after 1 and 4 wk of Q administration, glycogen and fat content as well as mRNA expression of selected genes were measured in liver biopsies. Furthermore, euglycemic, hyperinsulinemic, and hyperglycemic clamp studies were conducted before and after 2 wk of Q administration. During the experiment, dry matter intake and most other zootechnical data remained unchanged. Milk protein content was increased in wk 2 and 4 of Q administration compared with basal values, whereas fat and lactose contents of milk remained unchanged. Plasma nonesterified fatty acids, γ-glutamyl transferase, cholesterol, glutamate dehydrogenase, triglyceride, and albumin concentrations, as well as liver fat and glycogen concentrations, were not affected by Q supplementation. Plasma glucose and β-hydroxybutyrate concentrations in plasma decreased and increased, respectively, under the influence of quercetin. During hyperglycemic clamp conditions, the relative increase of plasma insulin was higher after 2 wk of Q administration, and a tendency for an increased rQUICKI (revised quantitative insulin sensitivity check index) was observed. The relative mRNA expression levels of selected genes related to glucose metabolism, fat metabolism, and antioxidative status were not altered after 1 or 4 wk of Q supplementation. In conclusion, the effects on insulin release and sensitivity support the assumption that administration of Q could have positive effects on the metabolic adaption of high-yielding cows to early lactation. The increase of milk protein content in response to Q supplementation needs to be verified., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Sarcandra glaber is a common traditional Chinese medicine used to treat psoriasis and other infectious diseases, isofraxidin and astilbin are the main components of it. In order to study the pharmacokinetics of Sarcandra glabra, an HPLC method for simultaneous determination of isofraxidin and astilbin in rat plasma was established. Plasma samples were prepared using solid phase extraction method. C(18) column with a guard was used, mobile phase was consisted of A (methanol) and B (0.1% aqueous acetic acid) with gradient elution as follows: 0 - 4min, A: 35%, B: 65%; 4 - 10min, A: 35% - 45%, B: 65% - 55%; 10 - 20min, A: 45%, B: 55%. The flow rate was 1.2 mL/min from 0 to 4 min, 1.0 mL/min from 4 to 20 min. The detection wavelength was 300 nm. A linear correlation between drug amount and peak area was established for isofraxidin in the range of 20-320 ng and for astilbin in the range of 19-304 ng. The recovery was over 68% for both compounds, the accuracy was within 8%, and the inter-day and intra-day precisions were all less than 8%. The pharmacokinetics of isofraxidin and astilbin was studied after orally administration the extract of Sarcandra glabra.

Scope: One major health problem in westernized countries is dysregulated fatty acid and cholesterol metabolism that causes pathologies such as metabolic syndrome. Previous studies from our group have shown that proanthocyanidins, which are the most abundant polyphenols in the human diet, regulate lipid metabolism and are potent hypolipidemic agents. The noncoding RNAs, miR-33 and miR-122, regulate genes that are involved in lipid metabolism., Methods and Results: Here, we show that grape seed proanthocyanidins rapidly and transiently repressed the expression of miR-33 and miR-122 in rat hepatocytes in vivo and in vitro. Furthermore, the miR-33 target gene ATP-binding cassette A1 and the miR-122 target gene fatty acid synthase were also modulated by proanthocyanidins. Specifically, ATP-binding cassette A1 mRNA and protein levels were increased, and fatty acid synthase mRNA and protein levels were reduced after the miRNA levels were altered., Conclusion: These results suggest that proanthocyanidin treatment increased hepatic cholesterol efflux to produce new HDL particles by repressing miR-33, and it reduced lipogenesis by repressing miR-122. These results highlight a new mechanism by which grape seed proanthocyanidins produce hypolipidemia through their effects on miRNA modulators of lipid metabolism., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A simple, rapid and sensitive liquid chromatography tandem mass spectrometry method is presented for the simultaneous determination of oleanolic acid, p-coumaric acid, ferulic acid, kaemperol and quercetin in rat plasma. Glycyrrhetinic acid was used as an internal standard, and sample pretreatment consisted of a liquid-liquid extraction. Chromatographic separation was achieved on a Gemini 110A C18 column (50 × 2.0 mm i.d., 5 µm) by gradient elution with a mobile phase consisting of methanol, acetonitrile and 0.01% formic acid in water. Tandem mass spectrometric detection was conducted using multiple reaction monitoring under negative ionization mode. Calibration curves offered linear ranges of two orders of magnitude with r > 0.99. The method was validated in terms of matrix effect, intra-day and inter-day precision, accuracy, linearity, specificity and stability. The relative standard deviation of intra-day and inter-day variations ranged from 2.66 to 14.74% and 1.9 to 14.55%. No substantial endogenous interference from blank plasma was observed. The method has been successfully applied to a pharmacokinetic study of Oldenlandia diffusa extract after oral administration in rats.

Aims: Flavanol-rich chocolate (FRC) is beneficial for vascular and platelet function by increasing nitric oxide bioavailability and decreasing oxidative stress. Congestive heart failure (CHF) is characterized by impaired endothelial and increased platelet reactivity. As statins are ineffective in CHF, alternative therapies are a clinical need. We therefore investigated whether FRC might improve cardiovascular function in patients with CHF., Methods and Results: Twenty patients with CHF were enrolled in a double-blind, randomized placebo-controlled trial, comparing the effect of commercially available FRC with cocoa-liquor-free control chocolate (CC) on endothelial and platelet function in the short term (2 h after ingestion of a chocolate bar) and long term (4 weeks, two chocolate bars/day). Endothelial function was assessed non-invasively by flow-mediated vasodilatation of the brachial artery. Flow-mediated vasodilatation significantly improved from 4.98 ± 1.95 to 5.98 ± 2.32% (P = 0.045 and 0.02 for between-group changes) 2h after intake of FRC to 6.86 ± 1.76% after 4 weeks of daily intake (P = 0.03 and 0.004 for between groups). No effect on endothelial-independent vasodilatation was observed. Platelet adhesion significantly decreased from 3.9 ± 1.3 to 3.0 ± 1.3% (P = 0.03 and 0.05 for between groups) 2 h after FRC, an effect that was not sustained at 2 and 4 weeks. Cocoa-liquor-free CC had no effect, either on endothelial function or on platelet function. Blood pressure and heart rate did not change in either group., Conclusion: Flavanol-rich chocolate acutely improves vascular function in patients with CHF. A sustained effect was seen after daily consumption over a 4-week period, even after 12 h abstinence. These beneficial effects were paralleled by an inhibition of platelet function in the presence of FRC only.

Objective: To establish a SPE-HPLC method for analyzing astilbin in rats serum and explore the effects of Yinxieling (YXL) prescription and Smilacis Glabrae Rhizoma on the pharmacokinetic characteristics of effective components., Method: Male Sprague-dawley rats were administrated YXL and Smilacis Glabrae Rhizoma respectively. At different time points, serum concentration of astilbin was extracted by Solid Phase Extraction (SPE) and determined using HPLC method. Chromatographic separation was achieved on a reversed-phase Phenomenex C18 column with the mobile phase of methanol-acetonitrile-formic acid water solution (0.05% formic acid) and gradient elution, temperature of bar was 24 degrees C, flow rate was 0.8 mL x min(-1)., Result: The method showed excellent linearity over the concentration range 0.266-53.1 mg x L(-1) of astilbin (r = 0.996). The extract recoveries were from 79.0% to 89.1%. Significant diffenerce in pharmacokinetic parameter of astilbin including t1/2, Cmax, AUC(0-t), AUC(0-infinity) and MRT were obtained through non-compartment model after oral administration of YXL prescription comparing with Smilacis Glabrae Rhizoma., Conclusion: The method was applied to a pharmacokinetic study of astilbin. It indicated that the bioavailability of astilbin in YXL enhanced in rats and one of the reasons may be that components of prescription affect the pharmacokinetics of astilbin in vivo.

Objective: To establish a RP-HPLC method for simultaneous determination of total quercetin, kaempferol and isorhamnetin in rat plasma after oral administration of Folium Mori extract (FME)., Methods: After a single dose of FME (110 mg/kg) was taken, rat plasma samples were collected. The samples were hydrolyzed with hydrochloric acid (c=3.0 mol/L), the mixed solution was extracted with ether acetone mixture. The total quercetin, kaempferol and isorhamnetin in plasma samples were determined by HPLC, pharmacokinetic parameters were calculated by DAS 3.0 software., Results: The method was linear over the concentration ranges of 0.0545-8.70, 0.0954-14.7 and 0.0545-8.55 μg/ml for quercetin, kaempferol and isorhamnetin, respectively (r=0.9979, 0.9993, 0.9981). The absolute recoveries were 85.3%-86.1%, 79.4%-86.7% and 62.8%-89.7%, respectively and the assay recoveries were all from 94.7% to 107%. The relative standard deviation (RSD) of intra-and inter-day were less than 9.5% and 9.8%, respectively. The main pharmacokinetic parameters were as follows: T(1/2z) was 92.7, 67.9 and 54.2 h; Tmax was 0.400, 0.400 and 3.87 h; AUC(0-∞) was 68.0, 67.5 and 32.8 mg/h/L; MRT(0-∞) was 128, 85.2 and 72.0 h for quercetin, kaempferol and isorhamnetin, respectively., Conclusion: The method established in this study is accurate, reliable and reproducible, and can be applied for determination of total quercetin, kaempferol and isorhamnetin in rat plasma after oral administration of FME; the pharmacokinetic studies showed that the distribution of drugs is rapid and elimination is very slow.

In the present study the effect of quercetin and its major metabolites quercetin-3-glucuronide (Q3G) and isorhamnetin on inflammatory gene expression was determined in murine RAW264.7 macrophages stimulated with lipopolysaccharide. Quercetin and isorhamnetin but not Q3G significantly decreased mRNA and protein levels of tumor necrosis factor alpha. Furthermore a significant decrease in mRNA levels of interleukin 1β, interleukin 6, macrophage inflammatory protein 1α and inducible nitric oxide synthase was evident in response to the quercetin treatment. However Q3G did not affect inflammatory gene expression. Anti-inflammatory properties of quercetin and isorhamnetin were accompanied by an increase in heme oxygenase 1 protein levels, a downstream target of the transcription factor Nrf2, known to antagonize chronic inflammation. Furthermore, proinflammatory microRNA-155 was down-regulated by quercetin and isorhamnetin but not by Q3G. Finally, anti-inflammatory properties of quercetin were confirmed in vivo in mice fed quercetin-enriched diets (0.1 mg quercetin/g diet) over 6 weeks., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: To observe the effect of serum containing Tengcha total flavonoid and dihydromyricetin on proliferation and apoptosis of HepG2 cells., Method: Serum containing respectively Tengcha total flavonid, dihydromyricetins and CTX and control serum were prepared by serological pharmacology method. MTT assay was used to observe the proliferation inhibition rate of HepG2 cells after incubated with different kinds of serum. Inverted microscope was utilized to observe the morphological changes after HepG2 cells were treated with different serum. AnnexinV/7AAD double label method was used to detect earlier period apoptosis cells., Result: Both serum containing 20% Tengcha total flavonid and serum containing 20% dihydromyricetin could restrain the HepG2 cells proliferation at different levels and the morpholological changes of apoptosis were observed. AnnexinV/7AAD double label method showed that the earlier period apoptosis cells rates were increased by serum containing 20% Tengcha total flavonoid, but serum containing 20% dihydromyricetin did not show influence on the earlier period apoptosis cells., Conclusion: Tengcha total flavonoid can restrain the HepG2 cells proliferation and induce earlier period apoptosis cells.

It is undisputed that terpene lactones and flavonoid glycosides of Ginkgo biloba are responsible for most of the extracts (e.g., EGb 761®) pharmacological actions. This investigation focused on the pharmacokinetic and the ability of the flavonoid constituents to cross the blood-brain barrier in rats, after single (600 mg/kg) or repeated (8 days, 100, or 600 mg/kg) oral administration of EGb 761®, and their distribution in different areas of the brain. For this purpose, we developed an HPLC-fluorescence method for the determination of the Ginkgo flavonoid metabolites (quercetin, kaempferol, and isorhamnetin derivatives) in the brain and plasma. A single dose of 600 mg/kg EGb 761® resulted in maximum plasma concentrations of 176, 341, and 183 ng/mL for quercetin, kaempferol, and isorhamnetin/tamarixetin, respectively and in maximum brain concentrations of 291 ng/g protein for kaempferol and 161 ng/g protein for isorhamnetin/tamarixetin. In comparison, the repeated administration of the same dose for 8 days led to an approximate 4.5-fold increase in the plasma concentration for quercetin, 11.5-fold increase for kaempferol, and 10-fold increase for isorhamnetin/tamarixetin. In the brain, an approximate 2-fold increase was observed for kaempferol and isorhamnetin/tamarixetin. About 90% of the determined flavonoids were distributed in the hippocampus, frontal cortex, striatum, and cerebellum, which together represent only 38% of the whole brain., (© Georg Thieme Verlag KG Stuttgart · New York.)

Larvae of nine species of sawflies (Symphyta) were fed with the foliage of three birch species, after which the larval hemolymph composition was studied by HPLC-DAD and HPLC-ESI-MS. The hemolymph of sawfly larvae contained high concentrations of flavonol oligoglycosides (tri-, tetra-, penta-, and hexaglycosides) that could not be found in the larval foliar diet. In addition, there were significant between-sawfly species differences in both flavonoid composition and concentration (from 0.6 to 12.3 mg/ml) of the hemolymph. This suggested that the studied species have different biosynthetic activities for the synthesis of flavonoid oligoglycosides. Variation in the foliar diets did not cause differences in the hemolymph composition. Our hypothesis is that sawflies use foliar flavonoid monoglycosides rather than flavonoid aglycones to produce these new types of oligoglycosides. These findings open up new possibilities for understanding the more holistic role of flavonoids in insect biochemistry and complex interactions between plants and herbivores.

Epidemiologic studies have shown that populations that consume more fruits and vegetables have lower incidences of some diseases. These health effects have largely been attributed to flavonoid intake and bioavailability. However, no published data on the estimated flavonol and flavone intake of Chinese adults are currently available. Considering reports that food frequency questionnaires (FFQs) have been shown to provide good measurements of energy, macronutrient, and micronutrient intakes, we hypothesized that FFQ may be used to estimate intake of dietary flavonol and flavone. The two 7-day 24-hour dietary recalls (24-HDRs) and plasma levels were used as reference criteria. A total of 128 subjects each completed two 7-day 24-HDR and 2 FFQs, and 92 subjects donated 2 plasma samples. Pearson correlation coefficients and the agreement of quartile categorization between the FFQ and each reference instrument were conducted. Pearson correlation coefficients between 2 FFQs were 0.62 for flavonol and 0.65 for flavone and ranged from 0.48 (quercetin) to 0.63 (luteolin) (all P < .05). Pearson correlation coefficients between FFQ and 24-HDR were 0.62 for flavonol and 0.68 for flavone and ranged from 0.36 (quercetin) to 0.63 (luteolin) (all P < .05). Between the FFQ and plasma samples, Pearson correlation coefficients were 0.52 for flavonol and 0.41 for flavone and ranged from 0.32 (quercetin) to 0.44 (kaempferol) (all P < .05). The complete and partial agreement by quartiles ranged from 70% to 89%. The findings indicate that administering FFQ is a reliable and accurate method of assessing dietary intake of flavonol and flavone., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Green tea containing 634 micromol of flavan-3-ols was ingested by human subjects with an ileostomy. Ileal fluid, plasma, and urine collected 0-24 h after ingestion were analysed by HPLC-MS. The ileal fluid contained 70% of the ingested flavan-3-ols in the form of parent compounds (33%) and 23 metabolites (37%). The main metabolites effluxed back into the lumen of the small intestine were O-linked sulphates and methyl-sulphates of (epi)catechin and (epi)gallocatechin. Thus, in subjects with a functioning colon substantial quantities of flavan-3-ols would pass from the small to the large intestine. Plasma contained 16 metabolites, principally methylated, sulphated, and glucuronidated conjugates of (epi)catechin and (epi)gallocatechin, exhibiting 101-256 nM peak plasma concentration and the time to reach peak plasma concentration ranging from 0.8 to 2.2 h. Plasma pharmacokinetic profiles were similar to those obtained with healthy subjects, indicating that flavan-3-ol absorption occurs in the small intestine. Ileostomists had earlier plasma time to reach peak plasma concentration values than subjects with an intact colon, indicating the absence of an ileal brake. Urine contained 18 metabolites of (epi)catechin and (epi)gallocatechin in amounts corresponding to 6.8+/-0.6% of total flavan-3-ol intake. However, excretion of (epi)catechin metabolites was equivalent to 27% of the ingested (-)-epicatechin and (+)-catechin.

Glucuronidation and excretion of sea buckthorn and lingonberry flavonols were investigated in a postprandial trial by analyzing the intact forms of flavonol glycosides as well as glucuronides in plasma, urine, and feces. Four study subjects consumed sea buckthorn (study day 1) and lingonberry (study day 2) breakfasts, and blood, urine, and fecal samples were collected for 8, 24, and 48 h, respectively. Both glycosides and glucuronides of the flavonol quercetin as well as kaempferol glucuronides were detected in urine and plasma samples after the consumption of lingonberries; 14% of flavonols in urine were glycosides, and 86% were glucuronidated forms (wt %). After the consumption of sea buckthorn, 5% of flavonols excreted in urine were detected intact, and 95% as the glucuronides (wt %). Solely glucuronides of flavonols isorhamnetin and quercetin were found in plasma after the consumption of sea buckthorn berries. Only glycosides were detected in the feces after each berry trial. Flavonol glycosides and glucuronides remained in blood and urine quite long, and the peak concentrations appeared slightly later than previously described. The berries seemed to serve as a good flavonol supply, providing steady flavonol input for the body for a relatively long time.

Epidemiological studies suggest that a diet high in flavonoids protects against chronic diseases such as CVD and cancer. The objective of the present study was to evaluate the relationship between the intake of quercetin, kaempferol, isorhamnetin, apigenin and luteolin and their corresponding plasma concentrations, and further to explore whether these flavonoids can serve as biomarkers of their intake. Flavonoid intake and their plasma concentrations were analysed in ninety-two subjects consuming their habitual diet. Flavonoid intake was estimated with 7-d dietary records using available data on the flavonoid content of food. Plasma flavonoid concentrations were quantified by HPLC. In addition, we undertook a dietary intervention study to investigate plasma apigenin concentration after the consumption of celery leaf. The mean intake estimates of quercetin, kaempferol, isorhamnetin, apigenin and luteolin amounted to 13.58, 14.97, 12.31, 4.23 and 8.08 mg/d, respectively. The corresponding mean plasma concentrations were 80.23, 57.86, 39.94, 10.62 and 99.90 nmol/l. The mean 7 d intake of five flavonoids was positively correlated to their corresponding plasma concentrations, with correlation coefficients ranging from 0.33 to 0.51 (P < 0.05). In the dietary intervention study, the plasma apigenin concentration rose after celery leaf ingestion, and fell within 28 h to below the limit of detection (2.32 nmol/l). The present results suggest that quercetin, kaempferol, isorhamnetin, apigenin and luteolin are bioavailable from the diet. The levels of fasting plasma flavonoids seem to be suitable biomarkers of short-term intake. The combination of plasma flavonoids with their intake may prove useful when the possible health-protective effects of flavonoids are studied.

The influence of the dietary flavonol quercetin on the pharmacokinetics of the HMG-CoA reductase inhibitor simvastatin was investigated in pigs. Simvastatin (0.25mg/kg body weight) was orally administered to six pigs either without or with quercetin (10mg/kg). In addition, simvastatin was administered to three pigs that had received a diet supplemented with the flavonol over a period of 1 week. Daily quercetin intake was 10mg/kg in these animals. Co-ingestion of quercetin with the statin did not alter area under the concentration time curve (AUC(0-->infinity)), time to achieve maximum plasma concentration (t(max)) or half-life (t(1/2)) of simvastatin. However, there was a trend towards a reduction of the maximum plasma concentration (C(max)) of simvastatin when quercetin was administered concomitantly (P=0.06). As compared to controls, AUC(0-->infinity) of simvastatin was significantly decreased after feeding the quercetin-supplemented diet for 1 week. The plasma ratio of simvastatin and its acid metabolite was neither altered by the concomitant quercetin ingestion nor by feeding of the flavonol over a period of 1 week. We conclude that chronic ingestion of high doses of the flavonol quercetin will decrease the bioavailability of simvastatin to a significant extent.

Background: Epidemiological studies indicate beneficial effects of flavonoids on cardiovascular disease (CVD) risk., Aim of the Study: To study the effect of flavonoid-rich sea buckthorn berry (SBB) on circulating lipid markers associated with CVD risk and plasma flavonol concentration. Also investigated was whether changes in the circulating flavonol concentrations correlate with the SBB induced changes in C-reactive protein (CRP) concentration observed previously., Subjects and Methods: In all 229 healthy participants completed the randomized double-blind study and consumed daily 28 g of SBB or placebo for 3 months. Fasting blood samples for the analysis of lipid markers and flavonols were obtained at the beginning and end of the study., Results: Compared to the placebo, the consumption of SBB increased the plasma concentration of the flavonols quercetin and isorhamnetin significantly [treatment differences 3.0 ng/ml (P = 0.03) and 3.9 ng/ml (P < 0.01), respectively]. The increase of kaempferol concentration was not significant [treatment difference 0.7 ng/ml (P = 0.08)]. SBB did not affect the serum total, HDL, LDL cholesterol, or the serum triacylglycerol concentrations. There was no correlation between the changes in flavonol and CRP concentrations of participants., Conclusions: The consumption of SBB significantly increased the fasting plasma concentration of quercetin and isorhamnetin indicating that it is a good dietary source of flavonols. However, this did not convert to affecting the circulating concentrations of lipid markers in healthy, normolipidemic adults having healthy diets.

We herein describe the development of an LC-MS method for simultaneous determination of astilbin and 3'-O-methylastilbin in rat plasma. A simple liquid-liquid extraction procedure was followed by injection of the extracts on to a Shim-pack C(18) column (150 mm x 2.0 mm I.D., 5 microm) with gradient elution and detection in negative ionization mode. Initially, the method was validated regarding linearity, accuracy and precision. The correlation coefficients of all the calibration curves showed good linearity (r> 0.999) within test ranges, and the relative deviation was less than 10% for intra- and inter-day assays. Besides, this method was also validated for its stability, extraction efficiency, matrix effect and so on. Finally, this proposed method was successfully applied to rat pharmacokinetic study and yielded the most comprehensive data on systemic exposure of them to date.

The aim of this study was to determine the effect of dietary quercetin supplementation on blood lipids and TNF-alpha levels according to the apoE genotype in apoE3 and apoE4 targeted gene replacement mice. In a two-factorial design female apoE3 and apoE4 mice were fed semi-synthetic diets without (controls) and with quercetin (2 mg/g diet) for 6 weeks. Feeding the quercetin-supplemented diets significantly increased plasma levels of quercetin and isorhamnetin both in apoE3 and apoE4 mice. There was no significant effect of apoE genotype on plasma quercetin levels. ApoE3 and apoE4 transgenic mice exhibited similar plasma levels of apoE and cholesterol which were not significantly affected by dietary quercetin supplementation. In mice receiving the basal diet without quercetin supplementation, levels of TNF-alpha in whole blood stimulated ex vivo with lipopolysaccharide were higher in apoE3 as compared to apoE4 transgenic mice. Dietary quercetin significantly lowered levels of TNF-alpha by 44 % in apoE3 mice relative to apoE3 mice receiving the unsupplemented diets. In apoE4 mice a moderate (20 %) but not significant decrease in TNF-alpha levels in response to the quercetin supplementation was evident. Following quercetin supplementation TNF-alpha levels were similar between apoE3 and apoE4 transgenic mice. Current findings indicate that apoE3 mice are more responsive to the TNF-alpha lowering properties of dietary quercetin supplementation as compared to apoE4 animals.

Background: Essential hypertension is characterized by reciprocal relations between endothelial dysfunction and insulin resistance. Cocoa flavanols stimulate production of the vasodilator nitric oxide from vascular endothelium., Objective: The objective was to test the hypothesis that consumption of cocoa may simultaneously lower blood pressure, improve endothelial dysfunction, and ameliorate insulin resistance in subjects with essential hypertension., Design: We conducted a randomized, placebo-controlled, double-blind, crossover trial of a flavanol-rich cocoa drink (150 mL twice a day, approximately 900 mg flavanols/d) in individuals with essential hypertension (n = 20). Antihypertensive medications were discontinued before study enrollment. After a 7-d cocoa-free run-in period, cocoa or flavanol-poor placebo (approximately 28 mg flavanols/d) treatment for 2 wk was followed by a 1-wk washout and then crossover to the other treatment arm. Blood pressure was measured thrice weekly. At baseline and after each treatment period, we assessed insulin sensitivity (hyperinsulinemic-isoglycemic glucose clamp) and insulin-stimulated changes in brachial artery diameter and forearm skeletal muscle capillary recruitment (Doppler ultrasound with or without microbubble contrast)., Results: Cocoa treatment for 2 wk increased insulin-stimulated changes in brachial artery diameter when compared with placebo [median percentage increase from baseline (25th-75th percentile): 8.3 (4.2-11.3) compared with 5.9 (-0.3 to 9.6); P < 0.04]. Nevertheless, cocoa treatment did not significantly reduce blood pressure or improve insulin resistance and had no significant effects on skeletal muscle capillary recruitment, circulating plasma concentrations of adipocytokines, or endothelial adhesion molecules., Conclusions: Daily consumption of flavanol-rich cocoa for 2 wk is not sufficient to reduce blood pressure or improve insulin resistance in human subjects with essential hypertension. This trial was registered at clinicaltrials.gov as NCT00099476.

Our aim was to investigate the effects of an oral supplementation of quercetin at 3 different doses on plasma concentrations of quercetin, parameters of oxidant/antioxidant status, inflammation, and metabolism. To this end, 35 healthy volunteers were randomly assigned to take 50, 100, or 150 mg/d (group Q50-Q150) quercetin for 2 wk. Fasting blood samples were collected at the beginning and end of the supplementation period. Compared with baseline, quercetin supplementation significantly increased plasma concentrations of quercetin by 178% (Q50), 359% (Q100), and 570% (Q150; P < 0.01 for all). High interindividual variation was found for plasma quercetin concentrations (36-57%). Quercetin did not affect concentrations of serum uric acid or plasma alpha- and gamma-tocopherols, oxidized LDL, and tumor necrosis factor-alpha, or plasma antioxidative capacity as assessed by the ferric-reducing antioxidant potential and oxygen radical absorbance capacity assays. In addition, serum lipids and lipoproteins, body composition, and resting energy expenditure did not significantly change during quercetin supplementation. Pharmacokinetics of quercetin were investigated in a subgroup of 15 volunteers. The areas under the plasma concentration-time curves ranged from 76.1 mumol.min.L(-1) to 305.8 mumol.min.L(-1) (50- and 150-mg dosages, respectively). Median maximum plasma concentrations of quercetin (431 nmol/L) were observed 360 min after intake of 150 mg quercetin. In conclusion, daily supplementation of healthy humans with graded concentrations of quercetin for 2 wk dose-dependently increased plasma quercetin concentrations but did not affect antioxidant status, oxidized LDL, inflammation, or metabolism.

Although the flavonol quercetin is intensively investigated, our knowledge about its bioavailability and possible target organs is far from being complete. The aim of this study was to check the potential of quercetin to accumulate in various tissues after long-term dietary treatment compared with a single treatment with flavonol. Pigs ingested either a single dose of quercetin aglycone (25 mg/kg body weight; Expt. 1) or received the flavonol twice a day at the same dose mixed into their regular meals (i.e 50 mg.kg(-1).d(-1)) for 4 wk (Expt. 2). In both experiments, we took plasma and tissue samples 90 min after the final meal and analyzed them using HPLC. Additionally, the specific activity of the enzyme beta-glucuronidase was measured in selected tissues. Higher flavonol concentrations than in plasma were found in only the liver (Expt. 1) or the intestinal wall and kidneys (Expt. 2). All tissues except blood plasma contained a variable amount of deconjugated quercetin in the range of 30-100% of total flavonols. However, the specific beta-glucuronidase activity was not correlated with the proportions of deconjugated flavonols in the various tissues. Long-term dietary intake of the flavonol did not lead to a greater accumulation in any tissue compared with the single treatment. Flavonol concentrations only exceeded the plasma concentration within organs involved in its metabolism and excretion, including liver, small intestine, and kidneys.

Objectives: Our goal was to test feasibility and efficacy of a dietary intervention based on daily intake of flavanol-containing cocoa for improving vascular function of medicated diabetic patients., Background: Even in fully medicated diabetic patients, overall prognosis is unfavorable due to deteriorated cardiovascular function. Based on epidemiological data, diets rich in flavanols are associated with a reduced cardiovascular risk., Methods: In a feasibility study with 10 diabetic patients, we assessed vascular function as flow-mediated dilation (FMD) of the brachial artery, plasma levels of flavanol metabolites, and tolerability after an acute, single-dose ingestion of cocoa, containing increasing concentrations of flavanols (75, 371, and 963 mg). In a subsequent efficacy study, changes in vascular function in 41 medicated diabetic patients were assessed after a 30-day, thrice-daily dietary intervention with either flavanol-rich cocoa (321 mg flavanols per dose) or a nutrient-matched control (25 mg flavanols per dose). Both studies were undertaken in a randomized, double-masked fashion. Primary and secondary outcome measures included changes in FMD and plasma flavanol metabolites, respectively., Results: A single ingestion of flavanol-containing cocoa was dose-dependently associated with significant acute increases in circulating flavanols and FMD (at 2 h: from 3.7 +/- 0.2% to 5.5 +/- 0.4%, p < 0.001). A 30-day, thrice-daily consumption of flavanol-containing cocoa increased baseline FMD by 30% (p < 0.0001), while acute increases of FMD upon ingestion of flavanol-containing cocoa continued to be manifest throughout the study. Treatment was well tolerated without evidence of tachyphylaxia. Endothelium-independent responses, blood pressure, heart rate, and glycemic control were unaffected., Conclusions: Diets rich in flavanols reverse vascular dysfunction in diabetes, highlighting therapeutic potentials in cardiovascular disease.

A simple and sensitive high-performance liquid chromatographic method was developed for the simultaneous determination of dihydromyricetin (1) and myricetin (2) in rat plasma after orally administrating the decoction of Ampelopsis grossedentata. Plasma samples were acidified with 0.375% phosphoric acid and extracted with ethyl acetate. Analysis of the extract was performed on reversed-phase C(18) column with a gradient eluent composed of acetonitrile and 0.04% phosphoric acid. The flow rate was kept at 1 ml/min and the detection wavelength was set at 290 and 370 nm for 1 and 2, respectively. The calibration curves were linear in the range of 0.247-4.114 microg/ml and 0.150-2.501 microg/ml for 1 and 2, respectively. The intra-day and inter-day precisions were better than 4.9 and 6.2%, respectively. The limits of detection (LOD) for 1 and 2 in plasma were 21.600 and 52.530 ng/ml, and the limits of quantification (LOQ) were 0.247 and 0.150 microg/ml, respectively. The mean recoveries for 1 and 2 were 92.0 and 93.3%, respectively. The accuracy and precision were well within the acceptable range and R.S.D. of measured rat samples was less than 7.5%. This validated method has been successfully applied in the pharmacokinetics study of dihydromyricetin and myricetin in vivo after orally administrating the decoction of A. grossedentata to rats.

Few reports have considered the effects of dietary fiber on plasma quercetin and the intestinal flora. We investigated the effects of pectin on the plasma and fecal flora of mice fed a diet supplemented with the quercetin glycoside rutin. Male mice were randomly divided into 2 groups, which were fed a pectin-rutin (PR) or cellulose-rutin (CR) diet for 14 d. Plasma quercetin and isorhamnetin metabolites were measured by high-performance liquid chromatography. Feces were immediately processed with bacteriological procedures. The fecal flora was investigated. Plasma quercetin and isorhamnetin concentrations were significantly higher in the PR diet group, as was the plasma isorhamnetin/quercetin ratio. The composition of the intestinal flora differed between the 2 dietary groups. The total number of fecal bacteria was significantly larger in the PR group, in which most types of bacteria were more abundant, with the exceptions of bifidobacteria, fusiform-shaped bacteria, and staphylococci. The lower gut seemed to be the major absorption site for rutin. Pectin might thus enhance the bioavailability of quercetin from rutin by altering the metabolic activity of the intestinal flora and/or gut physiological function.

A SPE-HPLC method was developed and validated for the simultaneous determination of flavonols, isoquercitrin (1), hibifolin (2), myricetin (3), quercetin-3'-O-d-glucoside (4) and quercetin (5) in rat plasma and urine after oral administration of the total flavonoids from Abelmoschus manihot (TFA). The astragalin (6) and kaempferol (7) were used as internal standards (IS). Plasma and urine samples were pretreated by solid-phase extraction using Winchem C(18) reversed-phase cartridges. Analysis of the plasma and urinary extract was performed on YMC-Pack ODS-A C(18) and Thermo ODS-2HYEPRSIL C(18) reversed-phase column, respectively and a mobile phase of acetonitrile-0.1% phosphoric acid was employed. HPLC analysis was conducted with different elution gradients. The flow rate was 1.0 mL/min and the detection wavelength was set at 370 nm. Calibration ranges in plasma for flavonols 2-5 were at 0.011-2.220, 0.014-2.856, 0.022-4.320, and 0.028-5.600 microg/mL, respectively. In urine calibration ranges for flavonols 1, 2, 4 and 5 were at 2.00-16.00, 8.56-102.72, 2.70-21.60, and 3.00-24.00 microg/mL, respectively. The RSD of intra- and inter-day was less than 5.40% and 4.89% in plasma, and less than 3.96% and 6.85% in urine for all the analyses. A preliminary experiment to investigate the plasma concentration and urinary excretion of the flavonols after oral administration of TFA to rats demonstrated that the present method was suitable for determining the flavonols in rat plasma and urine.

Extracts from Ginkgo biloba leaves (family Ginkgoaceae) have antioxidant and free radical scavenging effects, largely attributed to the flavonols, which are a major class of functional components in ginkgo extracts. In order to facilitate analysis of systemic exposure to ginkgo-derived products in animals and/or humans, we developed a liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based method that is capable of routinely monitoring plasma levels of ginkgo flavonols. We used an initial acidic hydrolysis step to convert the plasma ginkgo flavonol conjugates into their aglycone forms [quercetin (QCT), kaempferol (KMF) and isorhamnetin (ISR)] prior to EtOAc-based extraction and subsequent LC/MS/MS-based analysis. Comparative studies showed that the use of a mobile phase containing an extremely low concentration of HCOOH (0.01 per thousand) dramatically improved the electrospray ionization efficiency of the analytes in the negative ion mode; the efficiencies were approximately 4-, approximately 8- and approximately 20-fold higher for QCT, KMF and ISR, respectively, versus the results obtained using an electrolyte-free mobile phase, or approximately 2-, approximately 3- and approximately 4-fold higher, respectively, versus the results obtained using a mobile phase containing the more commonly utilized concentration of HCOOH (1 per thousand). In addition, use of the low concentration of HCOOH also decreased undesired matrix effects. These favorable effects have been referred to as 'LC-electrolyte effects'. Due to structural differences in the B-ring substituent, different types of precursor-to-product ion pairs (m/z 301 --> 151 for QCT, 285 --> 187 for KMF, and 315 --> 300 for ISR) were used for the selected reaction monitoring of the analytes. In addition, the chromatographic conditions were optimized on the basis of an initial scouting of matrix effects on analyte ionization. Despite the absence of an internal standard, the validation results consistently demonstrated that our bioassay is valid, reproducible, and reliable. The newly developed assay provided lower limits of quantification of 1.3, 1.3 and 0.4 pg on-column for QCT, KMF and ISR, respectively, which is more sensitive than any previously reported method for determining ginkgo flavonols. Finally, the assay suitability was demonstrated in a pilot pharmacokinetic measurement of a pharmaceutical ginkgo product in a beagle dog. This newly developed method should prove useful for wide-scale monitoring of ginkgo flavonol plasma concentrations for both pharmaceutical investigations and clinical applications.