Maria Margalef

Serum Metabolites Of Proanthocyanidin-Administered Rats Decrease Lipid Synthesis In Hepg2 Cells

The regular consumption of flavonoids has been associated with reduced mortality and a decreased risk of cardiovascular diseases. The proanthocyanidins found in plasma are very different from the original flavonoids in food sources. The use of physiologically appropriate conjugates of proanthocyanidins is essential for the in vitro analysis of flavonoid bioactivity. In this study, the effect of different proanthocyanidin-rich extracts, which were obtained from cocoa (CCX), French maritime pine bark (Pycnogenol extract, PYC) and grape seed (GSPE), on lipid homeostasis was evaluated. Hepatic human cells (HepG2 cells) were treated with 25 mg/L of CCX, PYC or GSPE. We also performed in vitro experiments to assess the effect on lipid synthesis that is induced by the bioactive GSPE proanthocyanidins using the physiological metabolites that are present in the serum of GSPE-administered rats. For this, Wistar rats were administered 1 g/kg of GSPE, and serum was collected after 2 h. The semipurified serum of GSPE-administered rats was fully characterized by liquid chromatography tandem triple quadrupole mass spectrometry (LC-QqQ/MS(2)). The lipids studied in the analyses were free cholesterol (FC), cholesterol ester (CE) and triglycerides (TG). All three proanthocyanidin-rich extracts induced a remarkable decrease in the de novo lipid synthesis in HepG2 cells. Moreover, GSPE rat serum metabolites reduced the total percentage of CE, FC and particularly TG; this reduction was significantly higher than that observed in the cells directly treated with GSPE. In conclusion, the bioactivity of the physiological metabolites that are present in the serum of rats after their ingestion of a proanthocyanidin-rich extract was demonstrated in Hep G2 cells.

A rapid method to determine colonic microbial metabolites derived from grape flavanols in rat plasma by liquid chromatography-tandem mass spectrometry.

This study describes the development and validation of a liquid chromatography-mass spectrometry method for determination of a large number of flavanol colonic derivatives in biological samples. The method was validated with rat plasma after the intake of grape seed flavanols. The minimum plasma volume necessary to maintain good recovery values within the range of 83-110% for all of the standards was determined by micro solid-phase extraction (μ-SPE). In total, 16 commercial standards were used to measure 30 different phenolic compounds present at low concentration levels (micromolar). The chromatographic method enabled reliable quantification of plasma colonic flavanol derivatives with low limits of detection and quantification, achieving values of 0.03 nM and 0.10 nM, respectively. The developed method can be readily applied to determine all of the flavanol metabolites that are most likely responsible for the majority of biological effects of poorly absorbed flavanols.

A dose–response study of the bioavailability of grape seed proanthocyanidin in rat and lipid-lowering effects of generated metabolites in HepG2 cells

Hyperlipidemia is one of the principal causes of cardiovascular disease and proanthocyanidins (PAs) regulate lipid homeostasis. This study aims to evaluate the concentration of PAs in rat serum after the administration of different doses of PAs and to determine the capacity of these metabolites to reduce de novolipid synthesis in HepG2 cells. Two hours after oral administration of different doses of a grape seed proanthocyanidin extract (GSPE) (1000, 375, 250 and 125mg/kg), serum was semi-purified and characterised by HPLC-ESI-MS/MS before analysing the synthesis and secretion of lipids in HepG2 cells. Results showed a dose-dependent appearance of metabolised PAs in serum at doses up to 375mg/kg and saturation at 1000mg/kg of GSPE. A reduction in cholesterol esters (CE), free cholesterol (FC) and triglycerides (TG) synthesis was observed without dose-dependence when the cells were treated with PAs metabolites. Moreover, a low dose of metabolites (125mg/kg) was sufficient to reduce FC and TG synthesis. In conclusion, the study demonstrated that PAs metabolise in a dose-dependent manner up to 370mg/kg but not dose-dependent effect was shown in reducing the de novosynthesis of lipids.

Regulation of vascular endothelial genes by dietary flavonoids: structure-expression relationship studies and the role of the transcription factor KLF-2

Physiological concentrations (1 μM) of 15 flavonoids were evaluated in human umbilical vein endothelial cells in the presence of hydrogen peroxide (H₂O₂) for their ability to affect endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) expression in order to establish the structural basis of their bioactivity. Flavonoid effects on eNOS transcription factor Krüpple like factor-2 (KLF-2) expression were also evaluated. All studied flavonoids appeared to be effective compounds for counteracting the oxidative stress-induced effects on vascular gene expression, indicating that flavonoids are an excellent source of functional endothelial regulator products. Notably, the more effective flavonoids for KLF-2 up-regulation resulted in the highest values for eNOS expression, showing that the increment of eNOS expression would take place through KLF-2 induction. Structure-activity relationship studies showed that the combinations of substructures on flavonoid skeleton that regulate eNOS expression are made up of the following elements: glycosylation and hydroxylation of C-ring, double bond C2=C3 at C-ring, methoxylation and hydroxylation of B-ring, ketone group in C4 at C-ring and glycosylation in C7 of A-ring, while flavonoid features involved in the reduction of vasoconstrictor ET-1 expression are as follows: double bond C2=C3 at C-ring glycosylation in C7 of A-ring and ketone group in C4 of C-ring.

Gender-related similarities and differences in the body distribution of grape seed flavanols in rats

SCOPE Dietary flavanols produce beneficial health effects, and once absorbed, they are recognized as xenobiotics and undergo phase-II enzymatic detoxification. Flavanols health-promoting properties are mainly attributed to their metabolic products. This work aimed to elucidate whether rats of the opposite sex exhibited differences in the metabolism and distribution of ingested flavanols. METHODS AND RESULTS Acute doses of grape seed polyphenols were administered to male and female rats. After 1, 2 and 4 h, plasma, liver, mesenteric white adipose tissue (MWAT), brain and hypothalamus flavanol metabolites were quantified by HPLC-MS/MS. Results indicated important sex-related quantitative differences in plasma and brain. Moreover, remarkable sex-related differences in the distributions and types of flavanol metabolites were also observed between liver and brain. CONCLUSIONS This study demonstrated that sex differentially influences the metabolism and distribution of flavanols throughout the bodies of rats, which may affect the physiological bioactivities of flavanols between males and females.

The blood pressure effect and related plasma levels of flavan-3-ols in spontaneously hypertensive rats

We studied the short-term antihypertensive effect of flavan-3-ols (-)-epicatechin, (+)-catechin and (-)-catechin, in spontaneously hypertensive rats (SHR). Plasma metabolites and the corresponding plasma antioxidant capacity were determined. All the assayed flavan-3-ols decreased systolic blood pressure (SBP) in SHR. Their antihypertensive effects were less pronounced than that of Captopril (50 mg kg(-1)) and were not shown in normotensive Wistar-Kyoto rats. 6 mg kg(-1) (-)-epicatechin caused the maximum decrease in SBP. The maximum effects of the catechin monomers were observed post-administration of 0.5 mg kg(-1) of flavan-3-ols, (-)-catechin being the least effective among the three assayed compounds. Glucuronide and methyl glucuronide metabolites were obtained in the flavan-3-ol treated SHR, but it was not possible to relate the antihypertensive effect of the assayed flavan-3-ols with a concrete plasma metabolite or with their antioxidant effect. In conclusion, the studied flavan-3-ols could be responsible for the antihypertensive effect of cocoa products.

Lack of tissue accumulation of grape seed flavanols after daily long-term administration in healthy and cafeteria-diet obese rats.

After ingestion flavanols are metabolized by phase-II enzymes and the microbiota and are distributed throughout the body depending on several factors. Herein we aim to evaluate whether flavanols are tissue-accumulated after the long-term administration of a grape seed polyphenol extract (GSPE) in rats and to study if compounds present in tissues differ in a cafeteria-diet obesity state. For that, plasma, liver, mesenteric white adipose tissue (MWAT), brain, and aorta flavanol metabolites from standard chow-diet-fed (ST) and cafeteria-diet-fed (CAF) rats were analyzed by high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) 21 h after the last 12-week-daily GSPE (100 mg/kg) dosage. Results showed that long-term GSPE intake did not trigger a flavanol tissue accumulation, indicating a clearance of products at each daily dosage. Therefore, results suggest that polyphenol benefits in a disease state would be due to a daily pulsatile effect. Moreover, obesity induced by diet also influences the metabolism and bioavailability of flavanols in rats.

Flavanol plasma bioavailability is affected by metabolic syndrome in rats

Flavanols, which exert several health benefits, are metabolized after ingestion. Factors such as the host physiological condition could affect the metabolism and bioavailability of flavanols, influencing their bioactivities. This study aimed to qualitatively evaluate whether a pathological state influenced flavanol plasma bioavailability. Standard and cafeteria (CAF) diet fed rats, a robust model of metabolic syndrome (MeS), were administered 1000mg/kg of flavanol enriched grape seed polyphenol extract (GSPE). Flavanols and their metabolites were quantified by HPLC-MS/MS in plasma before and at 2, 4, 7, 24, and 48h after GSPE ingestion. Results showed that in CAF administered rats the maximum time of plasma flavanol concentration was delayed and these animals presented higher levels of plasma phase-II metabolites as well as altered microbial metabolites. In conclusion, this study demonstrated that MeS pathological state modified flavanol bioavailability, supporting the hypothesis that flavanol metabolism, and therefore flavanol functionality, depend on the organisms state of health.

Chronic administration of grape-seed polyphenols attenuates the development of hypertension and improves other cardiometabolic risk factors associated with the metabolic syndrome in cafeteria diet-fed rats

The effects of grape-seed polyphenols against the development of hypertension and other cardiometabolic conditions associated with the metabolic syndrome (MetS) were studied in rats fed a high-fat, high-carbohydrate diet, known as the cafeteria (CAF) diet. Two groups of Wistar rats were fed standard (STD) or CAF diets for 12 weeks. The CAF diet-fed rats were administered different doses of a low-molecular-weight grape-seed polyphenol extract (LM-GSPE) (25, 100 and 200 mg/kg per d) or vehicle daily, and the STD diet-fed rats were administered LM-GSPE (100 mg/kg per d) or vehicle using ten animals per group. Body weight (BW), waist perimeter (WP) and systolic and diastolic blood pressures (BP) by the tail-cuff method were recorded weekly. The animals were housed in metabolic chambers every 2 weeks to estimate daily food and liquid intakes and to collect faeces and urine samples. The plasma lipid profile was analysed at time 0 and on the 4th, 7th, 10th and 12th weeks of the experiment. Moreover, plasma leptin was measured at the end of the experiment. Results demonstrated that LM-GSPE, when administered with the CAF diet, attenuated the increase in BP, BW, WP and improved lipid metabolism in these animals. However, although the 25- and 100-mg/kg per d doses were sufficient to produce beneficial effects on BP and lipid metabolism, a 200-mg/kg per d dose was necessary to have an effect on BW and WP. The present findings suggest that LM-GSPE is a good candidate for a BP-lowering agent that can also ameliorate other conditions associated with the MetS.