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

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

Identification of Dihydromyricetin and Metabolites in Serum and Brain Associated with Acute Anti-Ethanol Intoxicating Effects in Mice.

Dihydromyricetin is a natural bioactive flavonoid with unique GABAA 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 α5ß3γ2 GABAA 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.

Systemic exposure to Ginkgo biloba extract in male F344/NCrl rats: Relevance to humans.

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 Cmax 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.

Flavanol Bioavailability in Two Cocoa Products with Different Phenolic Content. A Comparative Study in Humans.

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.

Cocoa flavanol effects on markers of oxidative stress and recovery after muscle damage protocol in elite rugby players.

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.

Rapid determination of 3', 4'-dimethoxy flavonol-3-ß-d-glucopyranoside in rat plasma by LC-MS/MS method followed by protein precipitation.

A reliable liquid chromatography/tandem mass (LC-MS/MS) method was developed for quantitation of 3', 4'-dimethoxy flavonol-3-ß-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.

Simultaneous determination and pharmacokinetic study of three flavonoid glycosides in rat plasma by LC-MS/MS after oral administration of Rubus chingii Hu extract.

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 C18 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 (r2 > 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.

Simultaneous determination of kaempferol, quercetin, mangiferin, gallic acid, p-hydroxybenzoic acid and chlorpheniramine maleate in rat plasma after oral administration of Mang-Guo-Zhi-Ke tablets by UHPLC-MS/MS and its application to pharmacokinetics.

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 C18 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.

Determination and pharmacokinetics of engeletin in rat plasma by ultra-high performance liquid chromatography with tandem mass spectrometry.

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 (r2=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 (Tmax 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.

Determination of dihydromyricetin in rat plasma by LC-MS/MS and its application to a pharmacokinetic study.

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 t1/2 was 2.05 ± 0.52 h. However, DHM was not readily absorbed and reached Cmax 21.63 ± 3.62 ng/mL at approximately 2.67 h following the oral administration of DHM, and t1/2 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.

Influence of Intestinal Microbiota on the Catabolism of Flavonoids in Mice.

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.

Bioavailability of tomato polyphenols is enhanced by processing and fat addition: Evidence from a randomized feeding trial.

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.

Dietary flavonoid intake and weight maintenance: three prospective cohorts of 124,086 US men and women followed for up to 24 years.

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.

Quercetin Feeding in Newborn Dairy Calves Cannot Compensate Colostrum Deprivation: Study on Metabolic, Antioxidative and Inflammatory Traits.

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-1ß, 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.

Quantification of complanatoside A in rat plasma using LC-MS/MS and its application to a pharmacokinetic study.

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.

The Effects of Oral Quercetin Supplementation on Splanchnic Glucose Metabolism in 1-Week-Old Calves Depend on Diet after Birth.

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.

Determination of dihydromyricetin in rat plasma by LC-MS/MS and its application to a pharmacokinetic study.

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.

Modulation of cellular glucose metabolism in human HepG2 cells by combinations of structurally related flavonoids.

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.

Simultaneous Determination of Typhaneoside and Isorhamnetin-3-O-Neohesperidoside in Rats After Oral Administration of Pollen Typhae Extract by UPLC-MS/MS.

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.

Simultaneous determination by UPLC-MS/MS of seven bioactive compounds in rat plasma after oral administration of Ginkgo biloba tablets: application to a pharmacokinetic study.

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.