Natural (dihydro)phenanthrene plant compounds are direct activators of AMPK through its allosteric drug and metabolite-binding site.

AMP-activated protein kinase (AMPK) is a central energy sensor that coordinates the response to energy challenges to maintain cellular ATP levels. AMPK is a potential therapeutic target for treating metabolic disorders, and several direct synthetic activators of AMPK have been developed that show promise in preclinical models of type 2 diabetes. These compounds have been shown to regulate AMPK through binding to a novel allosteric drug and metabolite (ADaM)-binding site on AMPK, and it is possible that other molecules might similarly bind this site. Here, we performed a high-throughput screen with natural plant compounds to identify such direct allosteric activators of AMPK. We identified a natural plant dihydrophenathrene, Lusianthridin, which allosterically activates and protects AMPK from dephosphorylation by binding to the ADaM site. Similar to other ADaM site activators, Lusianthridin showed preferential activation of AMPKß1-containing complexes in intact cells and was unable to activate an AMPKß1 S108A mutant. Lusianthridin dose-dependently increased phosphorylation of acetyl-CoA carboxylase in mouse primary hepatocytes, which led to a corresponding decrease in de novo lipogenesis. This ability of Lusianthridin to inhibit lipogenesis was impaired in hepatocytes from ß1 S108A knock-in mice and mice bearing a mutation at the AMPK phosphorylation site of acetyl-CoA carboxylase 1/2. Finally, we show that activation of AMPK by natural compounds extends to several analogs of Lusianthridin and the related chemical series, phenanthrenes. The emergence of natural plant compounds that regulate AMPK through the ADaM site raises the distinct possibility that other natural compounds share a common mechanism of regulation.

A novel ATP-synthase-independent mechanism coupling mitochondrial activation to exocytosis in insulin-secreting cells.

Pancreatic ß-cells sense glucose, promoting insulin secretion. Glucose sensing requires the sequential stimulation of glycolysis, mitochondrial metabolism and Ca2+ entry. To elucidate how mitochondrial activation in ß-cells contributes to insulin secretion, we compared the effects of glucose and the mitochondrial substrate methylsuccinate in the INS-1E insulin-secreting cell line at the respective concentrations at which they maximally activate mitochondrial respiration. Both substrates induced insulin secretion with distinct respiratory profiles, mitochondrial hyperpolarization, NADH production and ATP-to-ADP ratios. In contrast to glucose, methylsuccinate failed to induce large [Ca2+] rises and exocytosis proceeded largely independently of mitochondrial ATP synthesis. Both glucose- and methylsuccinate-induced secretion was blocked by diazoxide, indicating that Ca2+ is required for exocytosis. Dynamic assessment of the redox state of mitochondrial thiols revealed a less marked reduction in response to methylsuccinate than with glucose. Our results demonstrate that insulin exocytosis can be promoted by two distinct mechanisms one of which is dependent on mitochondrial ATP synthesis and large Ca2+ transients, and one of which is independent of mitochondrial ATP synthesis and relies on small Ca2+ signals. We propose that the combined effects of Ca2+ and redox reactions can trigger insulin secretion by these two mechanisms.

Standardized LC×LC-ELSD Fractionation Procedure for the Identification of Minor Bioactives via the Enzymatic Screening of Natural Extracts.

To identify natural bioactive compounds from complex mixtures such as plant extracts, efficient fractionation for biological screening is mandatory. In this context, a fully automated workflow based on two-dimensional liquid chromatography (2D-LC × LC) was developed, allowing for the production of hundreds of semipure fractions per extract. Moreover, the ELSD response was used for online sample weight estimation and automated concentration normalization for subsequent bioassays. To evaluate the efficiency of this protocol, an enzymatic assay was developed using AMP-activated protein kinase (AMPK). The activation of AMPK by nonactive extracts spiked with biochanin A, a known AMPK activator, was enhanced greatly when the fractionation workflow was applied compared to screening crude spiked extracts. The performance of the workflow was further evaluated on a red clover (Trifolium pratense) extract, which is a natural source of biochanin A. In this case, while the crude extract or 1D chromatography fractions failed to activate AMPK, semipure fractions containing biochanin A were readily localized when produced by the 2D-LC×LC-ELSD workflow. The automated fractionation methodology presented demonstrated high efficiency for the detection of bioactive compounds at low abundance in plant extracts for high-throughput screening. This procedure can be used routinely to populate natural product libraries for biological screening.

Unambiguous Characterization of Commercial Natural (Dihydro)phenanthrene Compounds Is Vital in the Discovery of AMPK Activators.

These days, easy access to commercially available (poly)phenolic compounds has expanded the scope of potential research beyond the field of chemistry, particularly in the area of their bioactivity. However, the quality of these compounds is often overlooked or not even considered. This issue is illustrated in this study through the example of (dihydro)phenanthrenes, a group of natural products present in yams, as AMP-activated protein kinase (AMPK) activators. A study conducted in our group on a series of compounds, fully characterized using a combination of chemical synthesis, NMR and MS techniques, provided evidence that the conclusions of a previous study were erroneous, likely due to the use of a misidentified commercial compound by its supplier. Furthermore, we demonstrated that additional representatives of the (dihydro)phenanthrene phytochemical classes were able to directly activate AMPK, avoiding the risk of misinterpretation of results based on analysis of a single compound alone.

Interaction of hesperetin glucuronide conjugates with human BCRP, MRP2 and MRP3 as detected in membrane vesicles of overexpressing baculovirus-infected Sf9 cells.

The citrus flavonoid hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone) is the aglycone of hesperidin, the major flavonoid present in sweet oranges. Hesperetin 7-O-glucuronide (H7G) and hesperetin 3'-O-glucuronide (H3'G) are the two most abundant metabolites of hesperetin in vivo. In this study, their interaction with specific ABC transporters, believed to play a role in the disposition and bioavailability of hesperetin, was studied using Sf9 membranes from cells overexpressing human BCRP (ABCG2), MRP2 (ABCC2) and MRP3 (ABCC3). Both H7G and H3'G were tested for their potential to activate and inhibit ATPase activity, and to inhibit vesicular transport by these transporters. Both H7G and H3'G demonstrated interaction with all tested ABC transporters, especially with BCRP and MRP3. An interesting difference between H7G and H3'G was seen with respect to the interaction with BCRP: H7G stimulated the ATPase activity of BCRP up to 76% of the maximal effect generated by the reference activator sulfasalazine, with an EC(50) of 0.45 µM, suggesting that H7G is a high affinity substrate of BCRP, whereas H3'G did not stimulate BCRP ATPase activity. Only moderate inhibition of BCRP ATPase activity at high H3'G concentrations was observed. This study provides information on the potential of hesperetin glucuronide conjugates to act as specific ABC transporter substrates or inhibitors and indicates that regio-specific glucuronidation could affect the disposition of hesperetin.

Network medicine framework shows that proximity of polyphenol targets and disease proteins predicts therapeutic effects of polyphenols.

Polyphenols, natural products present in plant-based foods, play a protective role against several complex diseases through their antioxidant activity and by diverse molecular mechanisms. Here we develop a network medicine framework to uncover mechanisms for the effects of polyphenols on health by considering the molecular interactions between polyphenol protein targets and proteins associated with diseases. We find that the protein targets of polyphenols cluster in specific neighbourhoods of the human interactome, whose network proximity to disease proteins is predictive of the molecule's known therapeutic effects. The methodology recovers known associations, such as the effect of epigallocatechin-3-O-gallate on type 2 diabetes, and predicts that rosmarinic acid has a direct impact on platelet function, representing a novel mechanism through which it could affect cardiovascular health. We experimentally confirm that rosmarinic acid inhibits platelet aggregation and α-granule secretion through inhibition of protein tyrosine phosphorylation, offering direct support for the predicted molecular mechanism. Our framework represents a starting point for mechanistic interpretation of the health effects underlying food-related compounds, allowing us to integrate into a predictive framework knowledge on food metabolism, bioavailability and drug interaction.

In vivo bioavailability, absorption, excretion, and pharmacokinetics of [14C]procyanidin B2 in male rats.

Procyanidins are important biologically active compounds, but the pathway and extent of absorption and metabolism are controversial. We conducted a mass balance study to evaluate the total radioactivity excreted in urine and feces after oral administration of [(14)C]procyanidin B2 to male rats (n = 5). Urine and feces were collected daily from 0 to 96 h. Absolute bioavailability of (14)C from [(14)C]procyanidin B2 was calculated as approximately 82% using the values for total urinary (14)C. A pharmacokinetic study measured total radioactivity in the blood (n = 9). Blood samples were collected at designated time intervals (0.5-24 h) after administration. Three treatments were used: 1) intravenous, 2) oral higher dose (21 mg/kg b.wt.), and 3) oral lower dose (10.5 mg/kg). Blood concentration of total (14)C reached a maximum at approximately 6 h after ingestion of [(14)C]procyanidin B2 (groups II and III), and area under the curve (AUC) was dependent on oral dose. After intravenous or oral administration the terminal half-lives were similar, whereas 8-fold larger values were obtained after oral dosing for total clearance and the apparent volumes of distribution. These pharmacokinetic differences explain the apparently lower (14)C bioavailability (8-11%) for [(14)C]procyanidin calculated from blood [AUC((0-24))] values. After oral administration of [(14)C]procyanidin B2, 63% was excreted via urine within 4 days. The data suggest that much of the parent compound administered orally is degraded by the gut microflora before absorption and that these microbial metabolites have a different distribution from the compounds circulating after the intravenous dose.

Phase II metabolism of hesperetin by individual UDP-glucuronosyltransferases and sulfotransferases and rat and human tissue samples.

Phase II metabolism by UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) is the predominant metabolic pathway during the first-pass metabolism of hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone). In the present study, we have determined the kinetics for glucuronidation and sulfonation of hesperetin by 12 individual UGT and 12 individual SULT enzymes as well as by human or rat small intestinal, colonic, and hepatic microsomal and cytosolic fractions. Results demonstrate that hesperetin is conjugated at positions 7 and 3' and that major enzyme-specific differences in kinetics and regioselectivity for the UGT and SULT catalyzed conjugations exist. UGT1A9, UGT1A1, UGT1A7, UGT1A8, and UGT1A3 are the major enzymes catalyzing hesperetin glucuronidation, the latter only producing 7-O-glucuronide, whereas UGT1A7 produced mainly 3'-O-glucuronide. Furthermore, UGT1A6 and UGT2B4 only produce hesperetin 7-O-glucuronide, whereas UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15 conjugate both positions. SULT1A2 and SULT1A1 catalyze preferably and most efficiently the formation of hesperetin 3'-O-sulfate, and SULT1C4 catalyzes preferably and most efficiently the formation of hesperetin 7-O-sulfate. Based on expression levels SULT1A3 and SULT1B1 also will probably play a role in the sulfo-conjugation of hesperetin in vivo. The results help to explain discrepancies in metabolite patterns determined in tissues or systems with different expression of UGTs and SULTs, e.g., hepatic and intestinal fractions or Caco-2 cells. The incubations with rat and human tissue samples support an important role for intestinal cells during first-pass metabolism in the formation of hesperetin 3'-O-glucuronide and 7-O-glucuronide, which appear to be the major hesperetin metabolites found in vivo.

Procyanidin B2 catabolism by human fecal microflora: partial characterization of 'dimeric' intermediates.

The catabolism by human fecal microflora of pure procyanidin B2 ((-)-epicatechin-4beta-->8-(-)-epicatechin) has been investigated using a static in vitro culture model. For the first time, 24 catabolites were detected by LC-MS(n) with M(r) greater than 290 indicating that they could not have formed from just one of the epicatechin units in the procyanidin structure. Structures have been assigned on the basis of the fragmentation in the ion trap mass spectrometer and with regard to catabolic pathways known to occur in fecal microorganisms. Twenty of these 'dimeric' catabolites produced fragment ions characteristic of flavanols and/or proanthocyanidins. One catabolite was identified tentatively as either 6- or 8-hydroxy-procyanidin B2. Thirteen were characterized as having been microbially reduced in at least one of the epicatechin units. Five contained an apparently unmodified epicatechin unit but in at least one case this was shown to consist of the B-ring of the "upper" epicatechin unit and the A-ring of the "lower". These 'dimeric' catabolites were detected up to 9h after the start of the incubation and collectively accounted for approximately 20% of the substrate.

Nondairy creamer, but not milk, delays the appearance of coffee phenolic acid equivalents in human plasma.

Chlorogenic acids (CGA) are antioxidants found in coffee. They are becoming of interest for their health-promoting effects, but bioavailability in humans is not well understood. We hypothesized that adding whole milk or sugar and nondairy creamer to instant coffee might modulate the bioavailability of coffee phenolics. Nine healthy participants were asked to randomly drink, in a crossover design, instant coffee (Coffee); instant coffee and 10% whole milk (Milk); or instant coffee, sugar, and nondairy creamer already premixed (Sugar/NDC). All 3 treatments provided the same amount of total CGA (332 mg). Blood was collected for 12 h after ingestion and plasma samples treated using a liquid-liquid extraction method that included a full enzymatic cleavage to hydrolyze all CGA and conjugates into phenolic acid equivalents. Hence, we focused our liquid chromatography-Electrospray ionization-tandem MS detection and quantification on caffeic acid (CA), ferulic acid (FA), and isoferulic acid (iFA) equivalents. Compared with a regular black instant coffee, the addition of milk did not significantly alter the area under the curve (AUC), maximum plasma concentration (C(max)), or the time needed to reach C(max) (T(max)). The C(max) of CA and iFA were significantly lower and the T(max) of FA and iFA significantly longer for the Sugar/NDC group than for the Coffee group. However, the AUC did not significantly differ. As a conclusion, adding whole milk did not alter the overall bioavailability of coffee phenolic acids, whereas sugar and nondairy creamer affected the T(max) and C(max) but not the appearance of coffee phenolics in plasma.

Absorption, conjugation and excretion of the flavanones, naringenin and hesperetin from alpha-rhamnosidase-treated orange juice in human subjects.

We have determined the absorption, conjugation and excretion of naringenin-7-O-rutinoside (narirutin) compared to the corresponding glucoside in an orange juice matrix in human subjects. Healthy volunteers (eight men and eight women), in a double blind, randomised, crossover study, consumed orange juice with (1) natural content of naringenin-7-O-rutinoside; (2) alpha-rhamnosidase-treated to yield naringenin-7-O-glucoside. Blood was sampled at twelve time points and three fractions of urine were collected over 24 h. The area under the plasma-time curve of naringenin from (2) alpha-rhamnosidase-treated orange juice was increased about 4-fold (P < 0.0001), peak plasma concentration (Cmax) was 5.4-fold higher (P < 0.0001) and Tmax was decreased from 311 to 92 min (P = 0.002) compared to untreated orange juice (1), indicating a change in absorption site from the colon to the small intestine. Furthermore, the amount in urine was increased from 7 to 47 % (P < 0.0001) of the dose after consumption of the alpha-rhamnosidase-treated orange juice (2). All urine samples contained both naringenin-7- and -4'-O-glucuronides. In addition, to examine the effect of dose and alpha-rhamnosidase treatment on hesperetin conjugate profiles, a further treatment where (3) orange juice fortified with three times the original content of hesperetin-7-O-rutinoside was used. Five hesperetin metabolites (3'-O-glucuronide; 7-O-glucuronide; 5,7-O-diglucuronide; 3',7-O-diglucuronide; 3'-O-sulphate) were present after all treatments (1-3), with the same profile of the conjugates. The present data show that bioavailability of naringenin is increased by conversion from rutinoside to glucoside, but the profile of the conjugates of flavanones formed and excreted in urine is neither affected by the absorption site nor by a 3-fold change in dose.

Plasma appearance and correlation between coffee and green tea metabolites in human subjects.

Coffee and green tea are two of the most widely consumed hot beverages in the world. Their respective bioavailability has been studied separately, but absorption of their respective bioactive phenolics has not been compared. In a randomised cross-over design, nine healthy subjects drank instant coffee and green tea. Blood samples were collected over 12 h and at 24 h to assess return to baseline. After green tea consumption, (-)-epigallocatechin (EGC) was the major catechin, appearing rapidly in the plasma; (-)-EGC gallate (EGCg) and (-)-epicatechin (EC) were also present, but (-)-EC gallate and C were not detected. Dihydroferulic acid and dihydrocaffeic acid were the major metabolites that appeared after coffee consumption with a long time needed to reach maximum plasma concentration, suggesting metabolism and absorption in the colon. Other phenolic acid equivalents (caffeic acid (CA), ferulic acid (FA) and isoferulic acid (iFA)) were detected earlier, and they peaked at lower concentrations. Summations of the plasma area under the curves (AUC) for the measured metabolites showed 1.7-fold more coffee-derived phenolic acids than green tea-derived catechins (P = 0.0014). Furthermore, we found a significant correlation between coffee metabolites based on AUC. Inter-individual differences were observed, but individuals with a high level of CA also showed a correspondingly high level of FA. However, no such correlation was observed between the tea catechins and coffee phenolic acids. Correlation between AUC and maximum plasma concentration was also significant for CA, FA and iFA and for EGCg. This implies that the mechanisms of absorption for these two classes of compounds are different, and that a high absorber of phenolic acids is not necessarily a high absorber of catechins.

First synthesis, characterization, and evidence for the presence of hydroxycinnamic acid sulfate and glucuronide conjugates in human biological fluids as a result of coffee consumption.

A systematic investigation of the human metabolism of hydroxycinnamic acid conjugates was carried out. A set of 24 potential human metabolites of coffee polyphenols has been chemically prepared, and used as analytical standards for unequivocal identifications. These included glucuronide conjugates and sulfate esters of caffeic, ferulic, isoferulic, m-coumaric and p-coumaric acids as well as their dihydro derivatives. A particular focus has been made on caffeic and 3,4-dihydroxyphenylpropionic acid derivatives, especially the sulfate conjugates, for which regioselective preparation was particularly challenging, and have so far never been identified as human metabolites. Ten out of the 24 synthesized conjugates have been identified in human plasma and/or urine after coffee consumption. A number of these conjugates were synthesized, characterized and detected as hydroxycinnamic acid metabolites for the first time. This was the case of dihydroisoferulic acid 3'-O-glucuronide, caffeic acid 3'-sulfate, as well as the sulfate and glucuronide derivatives of 3,4-dihydroxyphenylpropionic acid.

Journey in the Polyphenol Research World with Ragai Ibrahim.

Dr. Ragai K. Ibrahim, Professor Emeritus at Concordia University, Montréal, Canada, passed away on the November 19, 2017 at the age of 88 years. Dr. Ibrahim dedicated his entire professional life to polyphenols and spent most of his academic career (1967-1997) at the Department of Biology of Concordia University in Montréal. He has been an active member of the Groupe Polyphénols since the beginning. This paper is a tribute to Dr. Ibrahim from some of his former students. An overview of the evolution of polyphenol research since the late 1950s and the outstanding contribution that Dr. Ibrahim had to this topic is given. The input of Dr. Ibrahim's research to the enzymology and genetics of polyphenol biosynthesis is discussed. Furthermore, the links between Dr. Ibrahim's work and some aspects of modern studies on the health benefits of polyphenols are presented.

Metabolite profiling of hydroxycinnamate derivatives in plasma and urine after the ingestion of coffee by humans: identification of biomarkers of coffee consumption.

Human subjects drank coffee containing 412 mumol of chlorogenic acids, and plasma and urine were collected 0 to 24 h after ingestion and were analyzed by high-performance liquid chromatography-mass spectrometry. Within 1 h, some of the components in the coffee reached nanomole peak plasma concentrations (C(max)), whereas chlorogenic acid metabolites, including caffeic acid-3-O-sulfate and ferulic acid-4-O-sulfate and sulfates of 3- and 4-caffeoylquinic acid lactones, had higher C(max) values. The short time to reach C(max) (T(max)) indicates absorption of these compounds in the small intestine. In contrast, dihydroferulic acid, its 4-O-sulfate, and dihydrocaffeic acid-3-O-sulfate exhibited much higher C(max) values (145-385 nM) with T(max) values in excess of 4 h, indicating absorption in the large intestine and the probable involvement of catabolism by colonic bacteria. These three compounds, along with ferulic acid-4-O-sulfate and dihydroferulic acid-4-O-glucuronide, were also major components to be excreted in urine (8.4-37.1 mumol) after coffee intake. Feruloylglycine, which is not detected in plasma, was also a major urinary component (20.7 mumol excreted). Other compounds, not accumulating in plasma but excreted in smaller quantities, included the 3-O-sulfate and 3-O-glucuronide of isoferulic acid, dihydro(iso)ferulic acid-3-O-glucuronide, and dihydrocaffeic acid-3-O-glucuronide. Overall, the 119.9 mumol excretion of the chlorogenic acid metabolites corresponded to 29.1% of intake, indicating that as well as being subject to extensive metabolism, chlorogenic acids in coffee are well absorbed. Pathways for the formation of the various metabolites within the body are proposed. Urinary dihydrocaffeic acid-3-O-sulfate and feruloylglycine are potentially very sensitive biomarkers for the consumption of relatively small amounts of coffee.

Increased bioavailability of hesperetin-7-glucoside compared with hesperidin results in more efficient prevention of bone loss in adult ovariectomised rats.

Hesperidin (Hp), a citrus flavonoid predominantly found in oranges, shows bone-sparing effects in ovariectomised (OVX) animals. In human subjects, the bioavailability of Hp can be improved by the removal of the rhamnose group to yield hesperetin-7-glucoside (H-7-glc). The aim of the present work was to test whether H-7-glc was more bioavailable and therefore more effective than Hp in the prevention of bone loss in the OVX rat. Adult 6-month-old female Wistar rats were sham operated or OVX, then pair fed for 90 d a casein-based diet supplemented or not with freeze-dried orange juice enriched with Hp or H-7-glc at two dose equivalents of the hesperetin aglycone (0.25 and 0.5 %). In the rats fed 0.5 %, a reduction in OVX-induced bone loss was observed regarding total bone mineral density (BMD):+7.0 % in OVX rats treated with Hp (HpOVX) and +6.6 % in OVX rats treated with H-7-glc (H-7-glcOVX) v. OVX controls (P < 0.05). In the rats fed 0.25 % hesperetin equivalents, the H-7-glcOVX group showed a 6.6 % improvement in total femoral BMD v. the OVX controls (P < 0.05), whereas the Hp diet had no effect at this dose. The BMD of rats fed 0.25 % H-7-glc was equal to that of those given 0.5 % Hp, but was not further increased at 0.5 % H-7-glc. Plasma hesperetin levels and relative urinary excretion were significantly enhanced in the H-7-glc v. Hp groups, and the metabolite profile showed the absence of eriodictyol metabolites and increased levels of hesperetin sulphates. Taken together, improved bioavailability of H-7-glc may explain the more efficient bone protection of this compound.

The plant product quinic acid activates Ca2+ -dependent mitochondrial function and promotes insulin secretion from pancreatic beta cells.

BACKGROUND AND PURPOSE: Quinic acid (QA) is an abundant natural compound from plant sources which may improve metabolic health. However, little attention has been paid to its effects on pancreatic beta-cell functions, which contribute to the control of metabolic health by lowering blood glucose. Strategies targeting beta-cell signal transduction are a new approach for diabetes treatment. This study investigated the efficacy of QA to stimulate beta-cell function by targeting the basic molecular machinery of metabolism-secretion coupling. EXPERIMENTAL APPROACH: We measured bioenergetic parameters and insulin exocytosis in a model of insulin-secreting beta-cells (INS-1E), together with Ca2+ homeostasis, using genetically encoded sensors, targeted to different subcellular compartments. Islets from mice chronically infused with QA were also assessed. KEY RESULTS: QA triggered transient cytosolic Ca2+ increases in insulin-secreting cells by mobilizing Ca2+ from intracellular stores, such as endoplasmic reticulum. Following glucose stimulation, QA increased glucose-induced mitochondrial Ca2+ transients. We also observed a QA-induced rise of the NAD(P)H/NAD(P)+ ratio, augmented ATP synthase-dependent respiration, and enhanced glucose-stimulated insulin secretion. QA promoted beta-cell function in vivo as islets from mice infused with QA displayed improved glucose-induced insulin secretion. A diet containing QA improved glucose tolerance in mice. CONCLUSIONS AND IMPLICATIONS: QA modulated intracellular Ca2+ homeostasis, enhancing glucose-stimulated insulin secretion in both INS-1E cells and mouse islets. By increasing mitochondrial Ca2+ , QA activated the coordinated stimulation of oxidative metabolism, mitochondrial ATP synthase-dependent respiration, and therefore insulin secretion. Bioactive agents raising mitochondrial Ca2+ in pancreatic beta-cells could be used to treat diabetes.

Metabolism and transport of the citrus flavonoid hesperetin in Caco-2 cell monolayers.

Metabolism and transport from intestinal cells back into the lumen by ATP-binding cassette (ABC) transporters is believed to limit the bioavailability of flavonoids. We studied metabolism and transport of the citrus flavonoid hesperetin, the aglycone of hesperidin, using a two-compartment transwell Caco-2 cell monolayer system, simulating the intestinal barrier. The role of apically located ABC transporters P-glycoprotein (MDR1/ABCB1), multidrug resistance protein 2 (ABCC2), and breast cancer resistance protein (BCRP/ ABCG2) in the efflux of hesperetin and its metabolites was studied by coadministration of compounds known to inhibit several classes of ABC transporters, including cyclosporin A, GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide], Ko143 [3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester], MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), and PSC-833 (Valspodar). Apically applied hesperetin (10 microM) was metabolized into hesperetin 7-O-glucuronide and hesperetin 7-O-sulfate, identified using high-performance liquid chromatographydiode array detector (DAD), ultraperformance liquid chromatography-DAD-tandem mass spectrometry, and authentic standards, which were transported predominantly to the apical side of the Caco-2 cell monolayer (1.12 cm(2)), at average (S.D.) rates of 14.3 (3.7) and 2.1 (0.8) pmol/min/monolayer, respectively. Hesperetin aglycone also permeated to the basolateral side, and this process was unaffected by the inhibitors used, possibly implying a passive diffusion process. Inhibition studies, however, showed that efflux of hesperetin conjugates to the apical side involved active transport, which from the pattern of inhibition appeared to involve mainly BCRP. Upon inhibition by the BCRP inhibitor Ko143 (5 micro M), the apical efflux of hesperetin conjugates was 1.9-fold reduced (p

Natural compounds analysis using liquid and supercritical fluid chromatography hyphenated to mass spectrometry: Evaluation of a new design of atmospheric pressure ionization source.

The MS hyphenation performance of UniSpray®, a new design of atmospheric pressure ionization source was evaluated in both SFC and RPLC modes. Sensitivity, stability, versatility and matrix effects offered by the UniSpray were assessed in positive and negative ionization modes and systematically compared to an electrospray source (ESI) using 120 natural compounds covering an extended chemical space. In a first instance, a multivariate approach was used to screen and optimize the UniSpray source settings to maximize detection sensitivity. The position of the source capillary against the fixed charged rod was highlighted as the major parameter affecting the detection sensitivity. The sensitivity improvement in Unispray vs. ESI strongly depends on the compounds chemical class and the chromatographic mode. For a few compounds (i.e. anabasine, theanine, caproic acid, fumaric acid and protopanaxatriol), up to a 10-fold increase in sensitivity was noticed with UniSpray. The signal stability over multiple injections was also found to be equivalent between both sources with RSD values on peak intensity lower than 14% on >100 injections, in both chromatographic modes. On complex plant extract, the matrix effects occurring from the secondary metabolites were also found to be comparable between ESI and UniSpray, at least in the positive ionization mode. However, a systematic decrease of MS signal intensity was observed in SFC mode when compounds were ionized using UniSpray in the negative ion mode.

In Vitro Gut Metabolism of [U-13 C]-Quinic Acid, The Other Hydrolysis Product of Chlorogenic Acid.

SCOPE: Quinic acid in its free form is broadly abundant in plants, and can accumulate in copious amounts in coffee, tea, and certain fruits. However, it has been mostly studied as chlorogenic acid, an ester of caffeic and quinic acids. When chlorogenic acid reaches the colon, it is hydrolyzed by microbial esterases releasing caffeic and quinic acids. While biotransformation of chlorogenic and caffeic acids have been elucidated by in vitro and in vivo studies, the gut metabolism of quinic acid has been so far overlooked. METHODS AND RESULTS: [U-13 C]-Quinic acid is submitted to a colonic model using human fecal microbiota for assessing its metabolic fate. The metabolite profiles formed along microbial biotransformation are monitored by a combined metabolomics approach, using both 2D GC- and ultra-HPLC-MS. Six metabolic intermediates are identified by incorporation of isotopic label. CONCLUSION: Two parallel degradation pathways could be proposed: (1) an oxidative route, leading to aromatization and accumulation of protocatechuic acid, and a (2) reductive route, including dehydroxylation to cyclohexane carboxylic acid. Elucidating the biotransformation of food bioactives by the gut microbiota is of relevance for understanding nutrition, interindividual variability and potential effects on human metabolism.