Recommending flavanols and procyanidins for cardiovascular health: current knowledge and future needs

Data on the potential health benefits of dietary flavanols and procyanidins, especially in the context of cardiovascular health, are considerable and continue to accumulate. Significant progress has been made in flavanol analytics and the creation of phytonutrient-content food databases, and novel data emanated from epidemiological investigations as well as dietary intervention studies. However, a comprehensive understanding of the pharmacological properties of flavanols and procyanidins, including their precise mechanisms of action in vivo, and a conclusive, consensus-based accreditation of a causal relationship between intake and health benefits in the context of primary and secondary cardiovascular disease prevention is still outstanding. Thus, the objective of this review is to identify and discuss key questions and gaps that will need to be addressed in order to conclusively demonstrate whether or not dietary flavanols and procyanidins have a role in preventing, delaying the onset of, or treating cardiovascular diseases, and thus improving human life expectancy and quality of life.

Phenolic compounds in raw and cooked rice (Oryza sativa L.) and their inhibitory effect on the activity of angiotensin I-converting enzyme

Whole rice has been widely studied due to the abundance of bioactive compounds in its pericarp. Some of the beneficial effects of these compounds on human health have been attributed to their antioxidant and other biological activities, such as enzyme inhibition. In this work, we evaluated the contents of total, soluble and insoluble phenolic compounds of 6 red and 10 non-pigmented genotypes of whole rice as well as their inhibitory effect on the activity of angiotensin I-converting enzyme (ACE). The effects of cooking on phenolics and their inhibitory activities were also investigated. Red genotypes showed high content of phenolics, mainly soluble compounds, at an average of 409.7 mg ferulic acid eq./100 g, whereas overall lower average levels (99.4 mg ferulic acid eq./100 g) at an approximate soluble/insoluble compound ratio of 1:1 were observed in non-pigmented rice. Pigmented rice displayed a greater inhibitory effect on ACE than non-pigmented rice. In fact, a significant correlation between the content of soluble phenolics and ACE inhibition was observed (r = 0.8985, p < 0.05). In addition to significantly reducing the levels of total phenolics and ACE inhibition, cooking altered the soluble/insoluble compound ratio, especially among red rice genotypes. (C) 2011 Elsevier Ltd. All rights reserved.

Composition of phenolic acids content in apple (Malus sp) pomace

The present work aimed the study of phenolic acids composition in apple pomace of Gala and Fuji cultivars. Phenolic acids were fractionated in phenolic acids, esterified and insoluble and analyzed by gas chromatography-mass spectrometry (GC-MS). Sixteen phenolic acids were identified in apple pomace samples. Total phenolic acids in apple pomace from Gala and Fuji cultivars were, in dry weight, 93.94 mg/g and 68.38 mg/g, respectively. Content of free phenolic acids in apple pomace from Gala cultivar was 29.11 mg/g and the following acids were identified: salicylic, protocatequinic, quinic, p-coumaric, gallic, propylgallate and synapic. Content of free phenolic acids in apple pomace from Fuji cultivar was 16.03 mg/g and the following acids were identified: salicylic, protocatequinic, gallic, ferulic and sinapic. Salicylic was the predominant free phenolic acids found in both cultivars, consisting of 91.67% and 63.57% of the free phenolic acids in Gala and Fuji cultivars, respectively. Chlorogenic acid (1.147 mg/g) was found only in apple pomace from Fuji cultivar. Content of esterified phenolic acids in apple pomace from Gala and Fuji cultivars were 53.75 mg/g and 48.29 mg/g, respectively. It was verified that the predominant esterified phenolic acid in pomace from apple Gala is derived from salicylic acid (52.76 mg/g). Acids derived from gallic acid (0.175 mg/g), propylgallate acid (0.198 mg/g), ferulic acid (0.159 mg/g) and sinapic acid (0.140 mg/g) were also found in Gala cultivar. Regarding to pomace from cultivar Fuji, the main esterified phenolic acid found is also derived from salicylic acid (47.42 mg/g) followed by gallic acid (0.270 mg/g), benzoic acid (0.194 mg/g) and sinapic acid (0.115 mg/g). Content of insoluble phenolic acids in apple pomace from Gala and Fugi cultivars were, in dry weight, 11.08 mg/g and 4.05 mg/g, respectively Insoluble phenolic acids derived from salicylic acid were found in higher concentrations in apple pomace from both cultivars.

Effect of Toasting Intensity at Cooperage on Phenolic Compounds in Acacia (Robinia pseudoacacia) Heartwood

The phenolic composition of heartwood from Robinia pseudoacacia, commonly known as false acacia, before and after toasting in cooperage was studied by HPLC-DAD and HPLC-DAD/ESI-MS/MS. A total of 41 flavonoid and nonflavonoid compounds were identified, some tentatively, and quantified. Seasoned acacia wood showed high concentrations of flavonoid and low levels of nonflavonoid compounds, the main compounds being the dihydroflavonols dihydrorobinetin, fustin, tetrahydroxy, and trihydroxymethoxy dihydroflavonol, the flavonol robinetin, the flavanones robtin and butin, and a leucorobinetinidin, none of which are found in oak wood. The low molecular weight (LMW) phenolic compounds present also differed from those found in oak, since compounds with a beta-resorcylic structure, gallic related compounds, protocatechuic aldehyde, and some hydroxycinnamic compounds are included, but only a little gallic and ellagic acid. Toasting changed the chromatographic profiles of extracts spectacularly. Thus, the toasted acacia wood contributed flavonoids and condensed tannins (prorobinetin type) in inverse proportion to toasting intensity, while LMW phenolic compounds were directly proportional to toasting intensity, except for gallic and ellagic acid and related compounds. Even though toasting reduced differences between oak and acacia, particular characteristics of this wood must be taken into account when considering its use in cooperage: the presence of flavonoids and compounds with beta-resorcylic structure and the absence of hydrolyzable tannins.

Long-term stability of dentin matrix following treatment with various natural collagen cross-linkers

Objectives: Collagen disorganization is one of the main degradation patterns found in unsuccessful adhesive restorations. The hypothesis of this study was that pretreatment using natural collagen cross-linking agents rich in proanthocyanidin (PA) would improve mechanical properties and stability over time of the dentin collagen and, thus, confer a more resistant and lasting substrate for adhesive restorations. Methods: PA-based extracts, from grape seed (GSE), cocoa seed (CSE), cranberry (CRE), cinnamon (CNE) and acai berry (ACE) were applied over the demineralized dentin. The apparent elastic modulus (E) of the treated dentin collagen was analyzed over a 12 month period. Specimens were immersed in the respective solution and E values were obtained by a micro-flexural test at baseline, 10, 30, 60, 120 and 240 min. Samples were stored in artificial saliva and re-tested after 3, 6 and 12 months. Data was analyzed using ANOVA and Tukey test. Results: GSE and CSE extracts showed a time-dependent effect and were able to improve [240 min (MPa): GSE = 108.96 +/- 56.08: CSE = 59.21 +/- 24.87] and stabilize the E of the organic matrix [12 months (MPa): GSE = 40.91 +/- 19.69; CSE = 42.11 +/- 13.46]. CRE and CNE extracts were able to maintain the E of collagen matrices constant over 12 months [CRE = 11.17 +/- 7.22; CNE = 9.96 +/- 6.11; MPa]. ACE (2.64 +/- 1.22 MPa) and control groups immersed in neat distilled water (1.37 +/- 0.69 MPa) and ethanol-water (0.95 +/- 0.33 MPa) showed no effect over dentin organic matrix and enable their degradation and reduction of mechanical properties. Significance: Some PA-based extracts were capable of improving and stabilizing collagen matrices through exogenous cross-links induction. (C) 2011 Elsevier Ltd. All rights reserved.

Mechanical characterization of proanthocyanidindentin matrix interaction

Objectives To characterize the properties of dentin matrix treated with two proanthocyanidin rich cross-linking agents and their effect on dentin bonded interfaces. Methods Sound human molars were cut into 0.5mm thick dentin slabs, demineralized and either treated with one of two cross-linking agents (grape seedGSE and cocoa seedCOE extracts) or left untreated. The modulus of elasticity of demineralized dentin was assessed after 10 or 60min and the swelling ratio after 60min treatment. Bacterial collagenase was also used to assess resistance to enzymatic degradation of samples subjected to ultimate tensile strength. The effect of GSE or COE on the resindentin bond strength was evaluated after 10 or 60min of exposure time. Data were statistically analyzed at a 95% confidence interval. Results Both cross-linkers increased the elastic modulus of demineralized dentin as exposure time increased. Swelling ratio was lower for treated samples when compared to control groups. No statistically significant changes to the UTS indicate that collagenase had no effect on dentin matrix treated with either GSE or COE. Resindentin bonds significantly increased following treatment with GSE regardless of the application time or adhesive system used. Significance Increased mechanical properties and stability of dentin matrix can be achieved by the use of PA-rich collagen cross-linkers most likely due to the formation of a PAcollagen complex. The short term resindentin bonds can be improved after 10min dentin treatment.(C) 2010 Academy of Denta lMaterials. Published by Elsevier Ltd. All rights reserved.

Effect of cocoa powder in the prevention of cardiovascular disease: biological, consumption and inflammatory biomarkers. A metabolomic approach

Numerous health benefits have been attributed to cocoa and its derived products in the last decade including antioxidant, anti-platelet and positive effects on lipid metabolism and vascular function. Inflammation plays a key role in the initiation and progression of atherosclerosis. However, cocoa feeding trials focused on inflammation are still rare and the results yielded are controversial. Health effects derived from cocoa consumption have been partly attributed to its polyphenol content, in particular of flavanols. Bioavailability is a key issue for cocoa polyphenols in order to be able to exert their biological activities. In the case of flavanols, bioavailability is strongly influenced by several factors, such as their degree of polymerization and the food matrix in which the polyphenols are delivered. Furthermore, gut has become an active site for the metabolism of procyanidins (oligomeric and polymeric flavanols). Estimation of polyphenol consumption or exposure is also a very challenging task in Food and Nutrition Science in order to correlate the intake of phytochemicals with in vivo health effects. In the area of nutrition, modern analytical techniques based on mass spectrometry are leading to considerable advances in targeted metabolite analysis and particularly in Metabolomics or global metabolite analysis. In this chapter we have summarized the most relevant results of our recent research on the bioavailability of cocoa polyphenols in humans and the effect of the matrix in which cocoa polyphenols are delivered considering both targeted analysis and a metabolomic approach. Furthermore, we have also summarized the effect of long-term consumption of cocoa powder in patients at high risk of cardiovascular disease (CVD) on the inflammatory biomarkers of atherosclerosis.

Effect of cocoa powder in the prevention of cardiovascular disease: biological, consumption and inflammatory biomarkers. A metabolomic approach

Numerous health benefits have been attributed to cocoa and its derived products in the last decade including antioxidant, anti-platelet and positive effects on lipid metabolism and vascular function. Inflammation plays a key role in the initiation and progression of atherosclerosis. However, cocoa feeding trials focused on inflammation are still rare and the results yielded are controversial. Health effects derived from cocoa consumption have been partly attributed to its polyphenol content, in particular of flavanols. Bioavailability is a key issue for cocoa polyphenols in order to be able to exert their biological activities. In the case of flavanols, bioavailability is strongly influenced by several factors, such as their degree of polymerization and the food matrix in which the polyphenols are delivered. Furthermore, gut has become an active site for the metabolism of procyanidins (oligomeric and polymeric flavanols). Estimation of polyphenol consumption or exposure is also a very challenging task in Food and Nutrition Science in order to correlate the intake of phytochemicals with in vivo health effects. In the area of nutrition, modern analytical techniques based on mass spectrometry are leading to considerable advances in targeted metabolite analysis and particularly in Metabolomics or global metabolite analysis. In this chapter we have summarized the most relevant results of our recent research on the bioavailability of cocoa polyphenols in humans and the effect of the matrix in which cocoa polyphenols are delivered considering both targeted analysis and a metabolomic approach. Furthermore, we have also summarized the effect of long-term consumption of cocoa powder in patients at high risk of cardiovascular disease (CVD) on the inflammatory biomarkers of atherosclerosis.

Enriquecimento de compostos fenólicos de folhas de Inga edulis por extração em fase sólida: quantificação de seus compostos majoritários e avaliação da capacidade antioxidante

A phenolic fraction was obtained from of the acetone-water-acetic acid extract of Inga edulis leaves, by liquid-liquid partition and SPE-C18 cartridges. This method provided an increase of 108, 66, 51, 50 and 36% of flavonols, proanthocyanidins, total polyphenols, gallotannins and flavanols, respectively. The major phenolics in purified fraction were procyanidin B2, catechin and myricetin-3-O-α-L-rhamnopyranoside, which achieved increases of 111, 47 and 45%, respectively, after SPE. Acid hydrolysis confirmed the presence of procyanidins, prodelphinidins and glycosylated flavonoids.

Composition of Hawthorn (Crataegus spp.) Fruits and Leaves and emblic Leafflower (Phyllanthus emblica) Fruits

Hawthorn (Crataegus sp.) is widely distributed in the northern hemisphere (Asia, Europe and North America). It has been used as a medicinal material and food for hundreds of years both in Europe and in China. Clinical investigations and other research suggest that extracts of hawthorn fruits and leaves have multiple health effects including hypolipidaemic, anti-atherosclerotic, hypotensive, cardioprotective and blood vessel relaxing activities. Hawthorn fruit extracts have also displayed antioxidant and radical scavenging activities. Emblic leafflower fruit (Phyllanthus emblica) is widely used in Chinese and Indian traditional medicine. It has been found to have anti-cancer, hypoglycaemic and hypolipidaemic activities as well as cardioprotective effects and antioxidant activity. The fruit is currently used as a functional food targeted at obese people in China. Phenolic compounds, procyanidins (PCs), flavonols and C-glycosyl flavones in hawthorn and hydrolysable tannins in emblic leafflower fruits are considered among the major bioactive compounds in these berries. Moreover, hawthorn and emblic leafflower fruits are rich in vitamin C, triterpenoids, fruit acids, sugar alcohols and some other components with beneficial effects on the health of human beings. The aim of the thesis work was to characterise the major phenolic compounds in hawthorn fruits and leaves and emblic leafflower fruits as well as other components contributing to the nutritional profile and sensory properties of hawthorn fruits. Differences in the content and compositional profile of the major phenolic compounds, sugars, acids and sugar alcohols within various origins and species of hawthorn were also investigated. Acids, sugars and sugar alcohols in the fruits of different origins/cultivars belonging to three species (C. pinnatifida, C. brettschneideri and C. scabrifolia) of hawthorn were analysed by gas chromatography (GC-FID) and mass spectrometry (Publication I). Citric acid, quinic acid, malic acid, fructose, glucose, sorbitol and myo-inositol were found in all the subspecies. Sucrose was present only in C. scabrifolia and three cultivars of C. pinnatifida var. major. Forty-two phenolic compounds were identified/tentatively identified in fruits of C. pinnatifida var. major by polyamide column chromatography combined with high-performance liquid chromatograph-electrospray ionisation mass spectrometry (HPLC-ESI-MS) (Publication II). Ideain, chlorogenic acid, procyanidin (PC) B2, (-)-epicatechin, hyperoside and isoquercitrin were the major phenolic components identified. In addition, 35 phenolic compounds were tentatively identified based on UV and mass spectra. Eleven major phenolic compounds (hyperoside, isoquercitrin, chlorogenic acid, ideain, (-)-epicatechin, two PC dimers, three PC trimers and a PC dimer-hexoside) were quantified in the fruits of 22 cultivars/origins of three species of Chinese hawthorn by HPLC-ESI-MS with single ion recording function (SIR) (Publication III). The fruits of the hawthorn cultivars/origins investigated fell into two groups, one rich in sugars and flavonols, the other rich in acids and procyanidins. Based on the compositional features, different biological activities and sensory properties may be expected between cultivars/origins of the two groups. The results suggest that the contents of phenolic compounds, acids, sugars and sugar alcohols may be used as chemotaxonomic information distinguishing the hawthorn species from each other. Phenolic compounds in fruits and leaves of C. grayana and their changes during fruit ripening/harvesting were investigated using HPLC-UV-ESI-MS (Publication IV). (-)-Epicatechin, PC B2 and C1, hyperoside and a quercetin-pentoside were the major phenolic compounds in both fruits and leaves. Three C-glycosyl flavones (a luteolin-C-hexoside, a methyl luteolin-C-hexoside and an apigenin-C-hexoside) were present in leaves in abundance, but only at trace levels in fruits. Ideain and 5-O-caffeoylquinic acid were found in fruits only. Additionally, eleven phenolic compounds were identified/tentatively identified in both leaves and fruits (three B-type PC trimers, two B-type PC tetramers, a quercetin-rhamnosylhexoside, a quercetin-pentoside, a methoxykaempferol-methylpentosylhexoside, a quercetin-hexoside acetate, a methoxykaempferol-pentoside, chlorogenic acid and an unknown hydroxycinnamic acid derivative). The total content of phenolic compounds reached the highest level by the end of August in fruits and by the end of September in leaves. The compositional profiles of phenolic compounds in fruits and leaves of C. grayana were different from those of C. pinnatifida, C. brettschneideri, C. scabrifolia, C. pinnatifida. var. major, C. monogyna, C. laevigata and C. pentagyna. Phenolic compounds in emblic leafflower fruits were characterised by Sephadex LH-20 column chromatography combined with HPLC-ESI-MS (Publication V). A mucic acid gallate, three isomers of mucic acid lactone gallate, a galloylglucose, gallic acid, a digalloylglucose, putranjivain A, a galloyl-HHDP-glucose, elaeocarpusin and chebulagic acid represented the major phenolic compounds in fruits of emblic leafflower. In conclusion, results of this study significantly increase the current knowledge on the key bioactive and nutritional components of hawthorn and emblic leafflower fruits. These results provide important information for research on the mechanism responsible for the health benefits of these fruits.

Interactions of tea tannins and condensed tannins with proteins

Binding parameters for the interactions of four types of tannins: tea catechins, grape seed proanthocyanidins, mimosa 5-deoxy proanthocyanidins,and sorghum procyanidins (mDP=17), with gelatin and bovine serum albumin (BSA) have been determined from isothermal titration calorimetry data. Equilibrium binding constants determined for the interaction with gelatin were in the range 10(4) to 10(6) M-1 and in the order: sorghum procyanidins > grape seed proanthocyanidins > mimosa 5-deoxy proanthocyanidins > tea catechins. Interaction with BSA was generally weaker, with equilibrium binding constants of <= 10(3) M-1 for grape seed proanthocyanidins, mimosa 5-deoxy proanthocyanidins and tea catechins, and 10(4) M-1 for the sorghum procyanidins. In all cases the interactions with proteins were exothermic and involved multiple binding sites on the protein. The data are discussed in relation to the structures and the known nutritional effects of the condensed tannins.

Flavonoid supplement improves leg health and reduces fluid retention in pre-menopausal women in a double-blind, placebo-controlled study

Flavonoid extracts derived from plant foods have been shown to benefit certain types of fluid retention. However, no studies have investigated these compounds for use in premenstrual fluid retention, a complaint common among women with otherwise normal menstrual cycles. Therefore, we conducted a double-blind, placebo-controlled, pilot study into the effect of a daily flavonoid extract (Colladeen(R), 320 mg oligomeric procyanidins) on premenstrual fluid retention. Fluid retention was assessed at baseline and throughout 4 menstrual cycles of the intervention using validated questionnaires. Leg girth was also measured at baseline and at the end of the study. Thirty subjects completed the study (n = 18 active treatment; n = 12 placebo). Although no significant changes in leg girth measurements were noted, there was a significant improvement in subjective "leg health" scores after flavonoid treatment compared to placebo (p = 0.013). Furthermore, this was accompanied by an improvement in reported premenstrual fluid retention nearing significance (p = 0.066). We conclude that flavonoids supplements may provide a new therapeutic direction to counter premenstrual fluid retention and improve leg health. A larger study is now warranted.

Flavonoid supplement improves leg health and reduces fluid retention in pre-menopausal women in a double-blind, placebo-controlled study

Flavonoid extracts derived from plant foods have been shown to benefit certain types of fluid retention. However, no studies have investigated these compounds for use in premenstrual fluid retention, a complaint common among women with otherwise normal menstrual cycles. Therefore, we conducted a double-blind, placebo-controlled, pilot study into the effect of a daily flavonoid extract (Colladeen(R), 320 mg oligomeric procyanidins) on premenstrual fluid retention. Fluid retention was assessed at baseline and throughout 4 menstrual cycles of the intervention using validated questionnaires. Leg girth was also measured at baseline and at the end of the study. Thirty subjects completed the study (n = 18 active treatment; n = 12 placebo). Although no significant changes in leg girth measurements were noted, there was a significant improvement in subjective "leg health" scores after flavonoid treatment compared to placebo (p = 0.013). Furthermore, this was accompanied by an improvement in reported premenstrual fluid retention nearing significance (p = 0.066). We conclude that flavonoids supplements may provide a new therapeutic direction to counter premenstrual fluid retention and improve leg health. A larger study is now warranted.

Deciphering the complexity of sainfoin (Onobrychis viciifolia) proanthocyanidins by MALDI-TOF mass spectrometry with a judicious choice of isotope patterns and matrixes

Use of superdihydroxybenzoic acid as the matrix enabled the analysis of highly complex mixtures of proanthocyanidins from sainfoin (Onobrychis viciifolia) by MALDI-TOF mass spectrometry. Proanthocyanidins contained predominantly B-type homopolymers and heteropolymers up to 12- mers (3400 Da). Use of another matrix, 2,6-dihydroxyacetophenone, revealed the presence of A-type glycosylated dimers. In addition, we report here how a comparison of the isotopic adduct patterns, which resulted from Li and Na salts as MALDI matrix additives, could be used to confirm the presence of A-type linkages in complex proanthocyanidin mixtures. Preliminary evidence suggested the presence of A-type dimers in glycosylated prodelphinidins and in tetrameric procyanidins and prodelphinidins.

Structurally-related (−)-epicatechin metabolites in humans: assessment using de novo chemically synthesized authentic standards

Accumulating data suggest that diets rich in flavanols and procyanidins are beneficial for human health. In this context, there has been a great interest in elucidating the systemic levels and metabolic profiles at which these compounds occur in humans. While recent progress has been made, there still exist considerable differences and various disagreements with regard to the mammalian metabolites of these compounds, which in turn is largely a consequence of the lack of availability of authentic standards that would allow for the directed development and validation of expedient analytical methodologies. In the present study, we developed a method for the analysis of structurally-related flavanol metabolites using a wide range of authentic standards. Applying this method in the context of a human dietary intervention study using comprehensively characterized and standardized flavanol- and procyanidin-containing cocoa, we were able to identify the structurally-related (−)-epicatechin metabolites (SREM) postprandially extant in the systemic circulation of humans. Our results demonstrate that (−)-epicatechin-3′-β-D-glucuronide, (−)-epicatechin-3′-sulfate, and a 3′-O-methyl(−)-epicatechin-5/7-sulfate are the predominant SREM in humans, and further confirm the relevance of the stereochemical configuration in the context of flavanol metabolism. In addition, we also identified plausible causes for the previously reported discrepancies regarding flavanol metabolism, consisting to a significant extent of inter-laboratory differences in sample preparation (enzymatic treatment and sample conditioning for HPLC analysis) and detection systems. Thus, these findings may also aid in the establishment of consensus on this topic.