Dose-Related Antihypertensive Properties and the Corresponding Mechanisms of a Chicken Foot Hydrolysate in Hypertensive Rats.

The antihypertensive properties of different doses of a chicken foot hydrolysate, Hpp11 and the mechanisms involved in this effect were investigated. Spontaneously hypertensive rats (SHR) were administered water, Captopril (50 mg/kg) or Hpp11 at different doses (25, 55 and 85 mg/kg), and the systolic blood pressure (SBP) was recorded. The SBP of normotensive Wistar-Kyoto (WKY) rats administered water or Hpp11 was also recorded. Additionally, plasmatic angiotensin-converting enzyme (ACE) activity was determined in the SHR administered Hpp11. Moreover, the relaxation caused by Hpp11 in isolated aortic rings from Sprague-Dawley rats was evaluated. Hpp11 exhibited antihypertensive activity at doses of 55 and 85 mg/kg, with maximum activity 6 h post-administration. At this time, no differences were found between these doses and Captopril. Initial SBP values of 55 and 85 mg/kg were recovered 24 or 8 h post-administration, respectively, 55 mg/kg being the most effective dose. At this dose, a reduction in the plasmatic ACE activity in the SHR was found. However, Hpp11 did not relax the aortic ring preparations. Therefore, ACE inhibition could be the mechanism underlying Hpp11 antihypertensive effect. Remarkably, Hpp11 did not modify SBP in WKY rats, showing that the decreased SBP effect is specific to the hypertensive state.

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 organism's 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.

Rat health status affects bioavailability, target tissue levels, and bioactivity of grape seed flavanols.

SCOPE: Studying the flavanol metabolism is essential to identify bioactive compounds, as beneficial effects of flavanols have been attributed to their metabolic products. However, host-related factors, including pathological conditions, may affect flavanol metabolism and, thus, their bioactivity. This study aims to elucidate whether hypertension affects grape seed flavanol metabolism, influencing their bioactivity in relation to hypertension. METHODS AND RESULTS: Grape seed flavanols' effect on blood pressure (BP) was studied in spontaneously hypertensive rats (SHR) and healthy Wistar rats 6 h after grape seed extract administration (375 mg/kg). Animals were then sacrificed, and plasma bioavailability and aorta distribution of flavanol metabolites were studied by HPLC-MS/MS in both the groups. Grape seed flavanols were only able to decrease BP in SHR. Plasma total flavanol metabolites showed similar levels, being the difference noticed in specific metabolites' concentrations. Specifically, microbial metabolites showed quantitative and qualitative differences between both health states. Moreover, aorta total concentrations were found decreased in SHR. Interestingly, flavanol microbial metabolites were specifically increased SHR aortas, showing qualitative differences in small phenolic forms. CONCLUSION: This study demonstrates important differences in bioactivity and target tissue metabolite levels between healthy and diseased rats, indicating potential metabolites responsible of the anti-hypertensive effect.

Age related differences in the plasma kinetics of flavanols in rats.

Dietary flavanols produce beneficial health effects; once absorbed, they are recognized as xenobiotics and undergo Phase-II enzymatic detoxification. However, flavanols with a degree of polymerization greater than 2 reach the colon, where they are subjected to microbial metabolism and can be further absorbed and undergo Phase-II reactions. In this sense, flavanols' health-promoting properties are mainly attributed to their metabolic products. Several age-related physiological changes have been evidenced, and it is known that flavanols' bioavailability is affected by internal factors. Therefore, this study aimed to elucidate whether animals of different ages, specifically young and adult rats, exhibit differences in their flavanol metabolism and plasma bioavailability. To accomplish this, an acute dose of a grape seed polyphenol extract was administered to male rats; after 2, 4, 7, 24 and 48 h, flavanols and their Phase-II and microbial metabolites were quantified by HPLC-ESI-MS/MS in plasma. The results indicated important age-related quantitative differences in plasma flavanol metabolites. Interestingly, adult rats presented a remarkable reduction in flavanol absorption and Phase-II flavanol metabolism. Consequently, microbial-derived flavanol metabolism is triggered by higher flavanol affluence in the colonic tract. Furthermore, young rats presented a faster metabolic profile than adult rats. Hence, our results indicate that the physiological bioactivities of flavanols may depend on age.

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.

Acute administration of single oral dose of grape seed polyphenols restores blood pressure in a rat model of metabolic syndrome: role of nitric oxide and prostacyclin.

PURPOSE: The aims of this study were to evaluate the antihypertensive effectiveness of different doses of grape seed polyphenols in cafeteria diet-fed hypertensive rats (CHRs) and to establish the mechanism involved in the blood pressure (BP) lowering effect of these compounds in this experimental model of metabolic syndrome (MS). METHODS: Male 8-week-old Wistar rats were fed cafeteria or standard (ST) diet for 10 weeks. After this, the antihypertensive effect of a single oral administration of a polyphenol grape seed extract (GSPE) was tested at different doses (250, 375 and 500 mg/kg) in CHRs. BP was recorded before and 2, 4, 6, 8, 24 and 48 h post-administration. The hypotensive effect of GSPE was also proved in ST diet-fed rats. Additionally, in other experiment, CHRs were orally administered 375 mg/kg GSPE. Four hours post-administration, the rats were intraperitoneally administrated 30 mg/kg NG-nitro-L-arginine methyl ester (L-NAME) or 5 mg/kg indomethacin [inhibitors of nitric oxide (NO) and prostacyclin synthesis, respectively]. BP was recorded initially and 6 h post-administration. RESULTS: GSPE produced a decrease in SBP and DBP, the most effective dose (375 mg/kg) showing an antihypertensive effect in CHRs similar to the drug captopril, and did not affect BP of ST diet-fed rats. The antihypertensive effect was completely abolished by L-NAME and partially inhibited by indomethacin. CONCLUSIONS: GSPE acts as an antihypertensive agent in a rat model of hypertension associated with MS. The change in endothelium-derived NO availability is one of the mechanisms involved in the antihypertensive effect of GSPE in CHRs. Additionally, endothelial prostacyclin contributes to the effect of GSPE on arterial BP.

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.

Tissue distribution of rat flavanol metabolites at different doses.

Flavanols are metabolized in the small intestine and the liver to produce their glucuronidated, sulfated or methylated conjugates that can be body distributed or excreted in the urine. However, the intake of large amounts of flavanols is not directly related to their bioavailability. This study aims to investigate the administered dose dependence of flavanols' conjugation and body distribution. In this study, different doses of a grape seed proanthocyanidin extract (GSPE; 125, 250, 375 and 1000 mg/kg) were orally administered to male Wistar rats. Tissues were collected 2h after GSPE administration. Flavanols were quantified by HPLC-MS/MS. Results show that the majority of GSPE metabolites are located in the kidney, followed by the liver. Lower concentrations were found in mesenteric white adipose tissue (MWAT) and the brain. Moreover, flavanol metabolites followed a tissue-specific distribution pattern independent of dosage. In the kidney, glucuronidated metabolites were the most abundant; however, in the liver, it was mainly methyl-glucuronidated metabolites. In MWAT, free flavanols were dominant, and methylated metabolites were dominant in the brain. Concentration within a tissue was dependent on the administered dose. In conclusion, flavanol metabolites follow a tissue-specific distribution pattern and only the tissue concentration of flavanol metabolites is dependent on the administered dose.

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.

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.

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.