Alan Crozier

Dietary phenolics: chemistry, bioavailability and effects on health

There is much epidemiological evidence that diets rich in fruit and vegetables can reduce the incidence of non-communicable diseases such as cardiovascular diseases, diabetes, cancer and stroke. These protective effects are attributed, in part, to phenolic secondary metabolites. This review summarizes the chemistry, biosynthesis and occurrence of the compounds involved, namely the C6-C3-C6 flavonoids-anthocyanins, dihydrochalcones, flavan-3-ols, flavanones, flavones, flavonols and isoflavones. It also includes tannins, phenolic acids, hydroxycinnamates and stilbenes and the transformation of plant phenols associated with food processing (for example, production of black tea, roasted coffee and matured wines), these latter often being the major dietary sources. Events that occur following ingestion are discussed, in particular, the deglycosylation, glucuronidation, sulfation and methylation steps that occur at various points during passage through the wall of the small intestine into the circulatory system and subsequent transport to the liver in the portal vein.We also summarise the fate of compounds that are not absorbed in the small intestine, but which pass into the large intestine where they are degraded by the colonic microflora to phenolic acids, which can be absorbed into the circulatory system and subjected to phase II metabolism prior to excretion. Initially, the protective effect of dietary phenolics was thought to be due to their antioxidant properties which resulted in a lowering of the levels of free radicals within the body.However, there is now emerging evidence that themetabolites of dietary phenolics,which appear in the circulatory systemin nmol/L to low mmol/L concentrations, exertmodulatory effects in cells through selective actions on different components of the intracellular signalling cascades vital for cellular functions such as growth, proliferation and apoptosis. In addition, the intracellular concentrations required to affect cell signalling pathways are considerably lower than those required to impact on antioxidant capacity. The mechanisms underlying these processes are discussed.

Dietary (Poly)phenolics in Human Health: Structures, Bioavailability, and Evidence of Protective Effects Against Chronic Diseases

Human intervention trials have provided evidence for protective effects of various (poly)phenol-rich foods against chronic disease, including cardiovascular disease, neurodegeneration, and cancer. While there are considerable data suggesting benefits of (poly)phenol intake, conclusions regarding their preventive potential remain unresolved due to several limitations in existing studies. Bioactivity investigations using cell lines have made an extensive use of both (poly)phenolic aglycones and sugar conjugates, these being the typical forms that exist in planta, at concentrations in the low-μM-to-mM range. However, after ingestion, dietary (poly)phenolics appear in the circulatory system not as the parent compounds, but as phase II metabolites, and their presence in plasma after dietary intake rarely exceeds nM concentrations. Substantial quantities of both the parent compounds and their metabolites pass to the colon where they are degraded by the action of the local microbiota, giving rise principally to small phenolic acid and aromatic catabolites that are absorbed into the circulatory system. This comprehensive review describes the different groups of compounds that have been reported to be involved in human nutrition, their fate in the body as they pass through the gastrointestinal tract and are absorbed into the circulatory system, the evidence of their impact on human chronic diseases, and the possible mechanisms of action through which (poly)phenol metabolites and catabolites may exert these protective actions. It is concluded that better performed in vivo intervention and in vitro mechanistic studies are needed to fully understand how these molecules interact with human physiological and pathological processes.

Bioavailability of dietary flavonoids and phenolic compounds.

This paper reviews recent human studies on the bioavailability of dietary flavonoids and related compounds, including chlorogenic acids and ellagitannins, in which the identification of metabolites, catabolites and parent compounds in plasma, urine and ileal fluid was based on mass spectrometric methodology. Compounds absorbed in the small intestine appear in the circulatory system predominantly as glucuronide, sulfate and methylated metabolites which seemingly are treated by the body as xenobiotics as they are rapidly removed from the bloodstream. As a consequence, while analysis of plasma provides valuable information on the identity and pharmacokinetic profiles of circulating metabolites after acute supplementation, it does not provide accurate quantitative assessments of uptake from the gastrointestinal tract. Urinary excretion, of which there are great variations with different classes of flavonoids, provides a more realistic figure but, as this does not include the possibility of metabolites being sequestered in body tissues, this too is an under estimate of absorption, but to what degree remains to be determined. Even when absorption occurs in the small intestine, feeding studies with ileostomists reveal that substantial amounts of the parent compounds and some of their metabolites appear in ileal fluid indicating that in volunteers with a functioning colon these compounds will pass to the large intestine where they are subjected to the action of the colonic microflora. A diversity of colonic-derived catabolites is absorbed into the bloodstream and passes through the body prior to excretion in urine. There is growing evidence that these compounds, which were little investigated until recently, are produced in quantity in the colon and form a key part of the bioavailability equation of dietary flavonoids and related phenolic compounds.

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 μmol 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 (Cmax), 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 Cmax values. The short time to reach Cmax (Tmax) 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 Cmax values (145–385 nM) with Tmax 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 μmol) after coffee intake. Feruloylglycine, which is not detected in plasma, was also a major urinary component (20.7 μmol 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 μmol 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.

Polyphenols and health: What compounds are involved?

On the basis of prospective, cross-sectional and intervention studies linking polyphenols to human health, several experimental papers in the literature have tried to evaluate the molecular mechanisms involved in their bioactivity. Polyphenols are reported to in vitro inhibit cancer cell proliferation, reduce vascularisation, protect neurons, stimulate vasodilation and improve insulin secretion, but are often studied as aglycones or as sugar conjugates and at non-physiological concentration. However, it is now well established that polyphenols undergo substantial metabolism after being ingested by humans in dietary relevant amount and that concentrations of plasma metabolites after a normal dietary intake rarely exceed nmol/L. This viewpoint intends to highlight that uncritical judgements made on the basis of the published literature, particularly about toxicity and bioactivity, may sometimes have been misled and misleading and to conclude that i) bioavailability values reported in the literature for phenolic compounds should be strongly reconsidered in the light of the large number of newly identified circulating and excreted metabolites, with particular attention to colonic ring-fission products which are obviously contributing much more than expected to the percentage of their absorption; ii) it is phenolic metabolites, formed in the small intestine and hepatic cells, and low molecular weight catabolic products of the colonic microflora to travel around the human body in the circulatory system or reach body tissues to elicit bioactive effects. Understanding these compounds certainly carries interest for drug-discovery but also for dietary prevention of disease.

Berry flavonoids and phenolics: bioavailability and evidence of protective effects

Berries contain vitamin C and are also a rich source of phytochemicals, especially anthocyanins which occur along with other classes of phenolic compounds, including ellagitannins, flavan-3-ols, procyanidins, flavonols and hydroxybenzoate derivatives. This review examines studies with both human subjects and animals on the absorption of these compounds, and their glucuronide, sulphate and methylated metabolites, into the circulatory system from the gastrointestinal tract and the evidence for their localisation within the body in organs such as the brain and eyes. The involvement of the colonic microflora in catabolising dietary flavonoids that pass from the small to the large intestine is discussed along with the potential fate and role of the resultant phenolic acids that can be produced in substantial quantities. The in vitro and in vivo bioactivities of these polyphenol metabolites and catabolites are assessed, and the current evidence for their involvement in the protective effects of dietary polyphenols, within the gastrointestinal tract and other parts of the body to which they are transported by the circulatory system, is reviewed.

The absorption, metabolism and excretion of flavan-3-ols and procyanidins following the ingestion of a grape seed extract by rats

Rats were fed a grape seed extract (GSE) containing (+)-catechin, (-)-epicatechin and dimers, trimers, tetramers and polymeric procyanidins. Liver, kidney, brain and gastrointestinal (GI) tract together with plasma, urine and faeces were collected over a 24 h period and their flavan-3-ol content was analysed by HPLC with tandem mass spectrometry and diode array detection. Small amounts of the GSE flavan-3-ols moved out of the stomach and into the duodenum/jejunum, and to a greater extent the ileum 1 h after ingestion, and into the caecum after 2 h with relatively small amounts being detected in the colon after 3 h. The GI tract contained the parent GSE flavan-3-ols and procyanidins with only trace amounts of metabolites and there were no indications that proanthocyanidins were depolymerised in the GI tract releasing monomeric flavan-3-ols. Plasma contained exclusively catechin glucuronides and methylated glucuronide metabolites which were also detected in the liver and kidneys. These metabolites were also present in urine together with sulphated metabolites and low amounts of the procyanidin dimers B1, B2, B3 and B4 as well as the trimer C2 and an unknown GSE trimer. The amounts of (+)-catechin and (-)-epicatechin metabolites excreted in urine relative to the quantity of the monomers ingested were 27 and 36 %, respectively, after 24 h. This is similar to the levels of urinary excretion reported to occur by other investigators after feeding (-)-epicatechin to rats and provides further, albeit indirect, evidence that the procyanidin oligomers in the GSE were not depolymerised to monomers to any extent after ingestion. No convincing analytical data were obtained for the presence of flavan-3-ol metabolites in the brain.

Identification of flavonoid and phenolic antioxidants in black currants, blueberries, raspberries, red currants, and cranberries.

The antioxidant capacity (AOC) of black currant, blueberry, raspberry, red currant, and cranberry extracts was determined using the FRAP assay. In addition, the vitamin C content of the berries was determined and phenolic and polyphenolic compounds in the extracts were analyze by reversed-phase HPLC-PDA-MS(3) and by reversed-phase HPLC-PDA with an online antioxidant detection system. A complex spectrum of anthocyanins was the major contributor to the AOC of black currants and blueberries, whereas the lower AOC of red currants and cranberries was due mainly to a reduced anthocyanin content. Raspberries also had a lower anthocyanin content than black currants and blueberries, but there was only a slight decline in the AOC because of the presence of the ellagitannins sanguin H-6 and lambertianin C, which were responsible for 58% of the HPLC-AOC of the berries. Vitamin C was responsible for 18-23% of the HPLC-AOC of black currants, red currants, and cranberries and for 11% of that of raspberries but did not contribute to the AOC of the blueberry extract that was examined. Seven percent of the HPLC-AOC of the cranberry extract was attributable to procyanidin dimers. However, the contribution of polymeric proanthocyanidins to the AOC of the five berries was not determined as when analyzed by reversed-phase HPLC these high molecular weight flavan-3-ols are either retained by the column or eluted as a broad unresolved band.

Analysis of ellagitannins and conjugates of ellagic acid and quercetin in raspberry fruits by LC-MSn.

The use of gradient reversed phase HPLC with diode array and MS(n) detection for the analysis of ellagitannins, ellagic acid conjugates and quercetin conjugates in raspberries (Rubus idaeus L.) is described. MS(n) is a particularly powerful tool for the analysis of trace levels of natural products in impure extracts as interpretation of fragmentation patterns, coupled in some instances with knowledge of HPLC retention properties, can facilitate the partial identification of components when reference compounds are unavailable.

Caffeine: a well known but little mentioned compound in plant science

Caffeine, a purine alkaloid, is a key component of many popular drinks, most notably tea and coffee, yet most plant scientists know little about its biochemistry and molecular biology. A gene from tea leaves encoding caffeine synthase, an N-methyltransferase that catalyses the last two steps of caffeine biosynthesis, has been cloned and the recombinant enzyme produced in E. coli. Similar genes have been isolated from coffee leaves but the recombinant protein has a different substrate specificity to the tea enzyme. The cloning of caffeine biosynthesis genes opens up the possibility of using genetic engineering to produce naturally decaffeinated tea and coffee.