Flavonoids and phenolic compounds in foods have attracted great interest since the 1990s owing to growing evidence of their
beneficial effects on human health. The interest was stimulated mainly by epidemiological studies indicating an inverse associa-tion between intake of foods rich in these compounds and the incidence of non-communicable diseases such as cardiovascular diseases, diabetes mellitus, and cancer.202For example, studies carried out by various investigators suggested a protective effect of flavonol (2) and flavone (3) intake on (i) the risk of fatal or non-fatal coronary artery diseases,203 (ii) the risk of lung cancer,204,205(iii) the incidence of asthma204and (iv) the impair-ment of pulmonary functions.206
Since the phenolics in fruits and vegetables, cocoa, chocolate, red wine, green tea and other dietary sources exhibit potent free radical-scavenging properties in vitro, their main role in vivo was thought to be as antioxidants involved in protection against lipid peroxidation. However, in the last decade, the fate and mode of action of these compounds has turned out to be more complex than originally expected.144,207,208Generalisations are invidious, and there are always exceptions. Nevertheless, one can say that only a small percentage of the phenols and polyphenols consumed ever reaches the tissues, and very little of this absorbed material retains the structure found in the plant.
The percentage of intake that is absorbed varies with structure (for example being significantly modulated by which sugars are attached to a flavonoid aglycone), and the food matrix. Any single phenol or polyphenol generates several metabolites, perhaps as many as 20 in the case of quercetin glycosides although two or three usually dominate.147The exact yields and proportions of metabolites from any substrate will vary not only with the individual’s genetic profile but also with the composition and competence of that individual’s intestinal microflora. Any biological effects produced by these metabolites will be a func-tion of the concentrafunc-tion achieved at the relevant site and the susceptibility of the organelle (receptor, enzyme, transporter, etc.) that again might vary with the individual’s genetic profile. It is not possible to quantify the magnitude of the variation produced by these factors, but it would not be unreasonable to assume an order of magnitude overall.
As discussed in Section 6, most dietary polyphenolics are modified during absorption from the small intestine, with the formation of glucuronide, methyl and sulfate metabolites, while in the large intestine breakdown to phenolic acid and non-phenolic catabolites occurs. Consequently, the compounds that reach cells and tissues are chemically, biologically and (in many instances) functionally distinct from the dietary form, and such features underlay their bioactivity. This, in addition to the fact that very low levels of dietary flavonoids and related compounds are actually absorbed and appear in the bloodstream (generally the maximum is <10 mmol/L in total, and such maxima are transient), implies that their mechanism of action goes beyond the modulation of oxidative stress.209The capacity of flavonoids and their metabolites to bind to proteins is another factor that must be considered when determining the overall bioactivity.
Manach et al.210 have reported the existence of intermolecular bonds between serum albumin and quercetin metabolites, which supports its slow elimination from the body. Similarly, ()-epi-gallocatechin-3-O-gallate (27) possesses a high affinity for blood proteins211 which, potentially, could extend its half-life in the circulatory system. However, volunteer studies indicate that in normal dietary circumstances the half-life is shorter than the ª The Royal Society of Chemistry 2009
interval between repeat consumption, indicating that there is little opportunity for metabolite concentrations to increase during the day, and any slight daytime accumulation is likely to be eliminated overnight. Many of the commodities so far examined in volunteer studies might only be consumed once a day, but the significance of this rapid elimination is well illus-trated by the data in for green tea (Fig. 8 and Table 10) that would often be consumed several times a day.
There is now emerging evidence that some phytochemicals, at concentrations that might be achieved under normal dietary circumstances, can exert modulatory effects in cells through selective actions on multiple intracellular signalling cascades, which are vital for cellular functions such as growth, prolifera-tion and death (apoptosis).207For example, trans-resveratrol (71) has been shown to target many components of intracellular signaling pathways, including pro-inflammatory mediators, regulators of cell survival and apoptosis, and tumor angiogenic and metastatic switches, by modulating a distinct set of upstream kinases, transcription factors and their regulators.212The iden-tification of these molecular targets is an important first step that must be attained before the molecular mode(s) of action can be elucidated and understood, and this understanding is essential in order to formulate dietary strategies that might manage or even prevent certain non-communicable diseases. Emphasis on the mode of action in subsequent sections will cover investigations that either deal with clinical and preclinical studies or in vitro investigations that use either phenolic compounds and/or their main in vivo metabolites at concentrations that, at least, approach what might be achieved through diet.
7.1 Significance of phenolic metabolites for human health There are a large number of reports on in vitro studies into the effect of individual phenolics or plant extracts on various aspects of human health. However, many of these investigations lack any physiological significance because of the high doses used, typi-cally of parent compounds, such as quercetin, rather than their conjugated mammalian metabolites or microbial degradation products (see Section 6). In order for any metabolite of any dietary phenolic to exert in vivo a biologically significant effect, then it must be sufficiently potent to exert that effect at 50% of its transient plasma Cmax. So far as we are aware, such potency has yet to be demonstrated, and as a consequence only a handful of in vitro studies have used concentrations sufficiently low to have any relevance to potential bioactivities in vivo.
Enterolactone (238), a phytoestrogenic lignan metabolite formed by the action of colonic microflora, has been ascribed various health-benefiting properties. A high concentration of plasma enterolactone is associated with a lower risk of acute coronary events,213 breast cancer,214,215 colorectal adenomas216 and prostate cancer.217 More recently, enterolactone has been shown to have direct inhibitory effects associated with cancer cell growth and is able to modulate the cell-signaling pathway.218 Enterolactone (238) competes with E2 for the type II estrogen receptor, induces sex-hormone-binding globulin219 and influ-ences steroid-metabolising enzymes and synthesis, thus poten-tially reducing proliferation of hormone-dependant cancer.215
Platelet activation and subsequent aggregation play a major role in the pathogenesis of myocardial infarction and ischaemic heart disease. Hence, promoting an optimal platelet function via the reduction of platelet hyper-reactivity using dietary solutions is considered a feasible approach for the maintenance of cardiovascular health. An investigation on this topic has assayed the anti-platelet activity of physiologically relevant concentra-tions of the anthocyanins delphinidin-3-O-rutinoside (239), cyanidin-3-O-glucoside (158), cyanidin-3-O-rutinoside (159), and malvidin-3-O-glucoside (41), and their putative colonic metabolites, dihydroferulic acid (209), 3-(3-hydrox-yphenyl)propionic acid (212), 3-hydroxyphenylacetic acid (202) and 3-methoxy-4-hydroxyphenylacetic acid (203), both sepa-rately and in combination. Anti-thrombotic properties were exhibited by 10 mmol/L dihydroferulic acid, and 3-(3-hydrox-yphenyl)propionic acid, 1 mmol/L delphinidin-3-rutinoside and a mixture of all the test compounds.220
The isoflavones daidzein (49) and genistein (50) are known to have estrogenic properties because of their structural similarity to estradiol (52), and both compounds elicit or selectively modulate estrogenic responses by binding estrogen receptors ERa and ERb, with greater affinity for ERa.221However, equol (221), a gut bacterial metabolite of daidzein (49), exhibits greater binding affinity to ERa and posses higher antioxidant capacity than its parent molecule.222 Furthermore, equol at mmol/L concentrations is more active than soy isoflavones in competing for binding to thromboxane A2 receptor in human platelets, thus eliciting its anti-platelet activity. The order of the relative affinity in competing for binding was equol (221) > genistein (50) >
daidzein (49) > glycitein (240) > genistein-7-O-glucoside (195) >
daidzein-7-O-glucoside (222) > glycitein-7-O-glucoside (241).223 However, only 20–30% of the human population are equol producers.224Thus, the use of dietary sources of isoflavones as a treatment for those at risk of thrombus formation and atherosclerosis will require evaluation of the patient’s ability to convert daidzein (49) to equol (221).223
7.2 Tissue or organ targets of phenolics
To understand the mechanism of action of dietary phenolics and their derivatives, it is necessary to identify their target sites.
However, data on tissue distribution are very scarce even in experimental animals. Microautoradiography of mice tissues ª The Royal Society of Chemistry 2009
after administration of either radiolabeled ()-epigallocatechin-3-O-gallate (27) or resveratrol (71) indicated that radioactivity is unequally incorporated into the cells of organs.225Phenolics have also been detected in the brain,226heart, kidney, spleen, pancreas and reproductive organs of mice and rats.227 After acute inges-tion of a nutriinges-tionally appropriate dose of the flavonol [2-14C]quercetin-40-glucoside (172) by rats, a number of glucu-ronide and methylated quercetin metabolites formed in the small intestine and subsequently small amounts, ca. 4% of intake, were excreted in urine. Once the flavonols reached the caecum and colon they were rapidly degraded to phenolic acids, principally 3-hydroxyphenylacetic acid (202) and benzoic acid (242), most of which (over a 72 h period) were rapidly excreted in urine without any noticeable build-up in the circulatory system. In this instance there is, therefore, no evidence of significant quantities of quer-cetin-derived compounds binding to albumin and elimination from the body being slowed, as proposed by Manach and colleagues.210 There was also no marked accumulation of radioactivity in any of the body tissues, including the brain.228
Following the ingestion of isoflavones by humans, the pres-ence of daidzein (49) and genistein (50) and their metabolites equol (221), enterolactone (238) and enterodiol (243) have been detected in prostate tissue.229In another study, after ingesting ca.
300 mmol of daidzein (49), concentrations up to 6 mmol/L of equol (221) accumulated in the breast tissue.230 When 20 men scheduled for surgery consumed 1.42 L of green tea and black tea on a daily basis for 5 days before radical prostatectomy, tea flavan-3-ols, specifically ()-epigallocatechin (25) and ()-epi-gallocatechin-3-O-gallate (27), were found to accumulate in prostate samples.231
Recently, immunochemistry has been used successfully to demonstrate the target sites of a flavan-3-ol in humans. A monoclonal antibody specific for ()-epicatechin-3-O-gallate (27) was developed, and this antibody detected immunoreactive materials in human atherosclerotic lesions, specifically localized in the macrophage-derived foam cells, but not in the normal aorta.232This is especially interesting as it is known that unme-tabolised ()-epicatechin-3-O-gallate (27) appears in the human circulatory system after the ingestion of green tea,154but why the concentration is higher in damaged tissue than in healthy tissue is not known. Similar methodology was also used to reveal that quercetin-3-O-glucuronide (180), a known plasma flavonol metabolite,147accumulates in macrophage-derived foam cells in human atherosclerotic lesions where it is converted to the agly-cone quercetin (15) and is associated with a subsequent reduction in lesion size.233This suggests a role for human b-glucuronidase which is released at sites of inflammation, but the exact specificity of this enzyme towards the flavonoids and non-flavonoid conjugates is not known, and certain glucuronide conjugates may have greater potential than others. In this regard it is interesting to note that quercetin-3-O-glucuronide (180), one of
the major human metabolites of some quercetin glycosides, inhibits angiogenesis in vitro, whereas quercetin-30-O-sulfate (197), another major metabolite, promotes angiogenesis.234 These results may provide insights into a mechanism for the anti-atherosclerotic actions of flavan-3-ols and flavonols.
7.3 Potential mode of action of phenolic compounds and their metabolites
Many phenolic compounds are potent effectors of biologic processes and have the capacity to influence disease risk via several complementary and overlapping mechanisms. In this section, current knowledge on mechanisms by which dietary phenolic compounds play a role in preventing degenerative pathologies will be summarized. In particular, the complex interactions between these dietary molecules and their molecular targets including the cell signaling pathways and response will be discussed.
7.3.1 NF-kB signaling pathway. NF-kB (nuclear factor kappa B) is a redox-sensitive transcription factor that regulates numerous physiological functions and is involved in the patho-genesis of various diseases. NF-kB regulates the expression of cytokines, inducible nitric oxide synthase (iNOS), cyclo-oxge-nase 2 (COX-2), growth factors and inhibitors of apoptosis.
Pathological dysregulation of NF-kB is associated with inflam-matory disease such as asthma,235Crohn’s disease and ulcerative colitis,236 and is also involved in the pathophysiology of auto-immune disorders, such as rheumatoid arthritis, as well as neurodegenerative diseases and cancer. Potent inhibitors of NF-kB include curcumin (188),237resveratrol (71),238ellagic acid (56), and ()-epigallocatechin-3-O-gallate (27).239Curcumin at doses of 10 to 30 mmol/L inhibits NF-kB activation of human prostate cancer cells.240
7.3.2 Activator protein-1 (AP-1). AP-1 is another class of redox-sensitive transcriptional factor with important roles in normal development and the response to stress. AP-1 activation is linked to growth regulation, cell transformation, inflamma-tion, and innate immune response. AP-1 has been implicated in regulation of genes involved in apoptosis and proliferation, and may promote cell proliferation by activating the cyclin D1 gene, and repressing tumor-suppressor genes, such as p53, p21cip1/
waf1 and p16. Several phenolic compounds, such as green tea flavan-3-ols, quercetin (15), trans-resveratrol (71) and curcumin (188), have been shown to suppress the AP-1 activation process.241
7.3.3 Phase II enzyme activation and Nrf2. Anthocyanins were shown to induce phase II antioxidant and detoxifying enzymes in cultured cells.242Treatment of rat liver clone 9 cells and non-cancerous breast cells with anthocyanins, albeit at high concentrations (20–50 mM), enhanced antioxidant capacity through the activation of both NADPH:quinone reductase and three glutathione-related enzymes, glutathione reductase, gluta-thione peroxidase, and glutagluta-thione S-transferase.243 Other phenolic compounds have also been implicated in the induction of phase II enzymes and they can be considered as potential candidates for preventing tumour development.244 5-O-ª The Royal Society of Chemistry 2009
Caffeoylquinic acid (64) increases the activity of the phase II detoxifying enzymes GST and NADPH quinone oxidoreductase in mouse epidermal cells.245 Similar results were also obtained with drinks rich in phenolic compounds such as tea and mate.246 The mechanism by which phenolic compounds exhibited these effects is through activation of the antioxidant response element upstream of genes that code for these enzymes. Recently, nuclear transcription factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2) has been shown to be a critical transcription factor that binds to the antioxidant response element in the promoter region of a number of genes encoding for antioxidant enzymes in several types of cells and tissues.247Phenolic compounds such as gallic acid (54), p-coumaric acid (58) and ferulic acid (60), albeit at a high dosage of 100 mg/kg body weight, significantly increase levels of Nrf2 and thus up-regulate the gene expression of cardiac antioxidant enzymes in the heart of male Sprague-Dawley rats.248 Similarly, curcumin (188), trans-resveratrol (71), and the synthetic analogues caffeic acid phenethyl ester (244) and 40 -bromoflavone (245), exhibit chemopreventive properties through stimulating Nrf2.249
7.3.4 Mitogen-activated protein kinase (MAPK) signaling pathway. MAPKs are a family of highly conserved groups of signalling proteins; they are divided into three main groups, (i) the extracellular signal-regulated protein kinase (Erk), (ii) c-Jun N-terminal kinase/stress-activated protein kinases (JNK) and (iii) p38MAPK. Typically, JNK and p38MAPKcascades are activated by environmental stresses and pro-inflammatory cytokines such as tumour necrosis factor (TNF), interleukin-1 (IL-1), IL-2 and IL-17, and they are largely associated with the promotion of inflammation, pain and programmed cell death.250MAPKs have also been implicated in the regulation of Phase II enzyme gene expression and induction of apoptosis.251Green tea flavan-3-ols, specifically 25 mmol/L ()-epigallocatechin-3-O-gallate (27), which appears in the bloodstream (albeit at ca. 100 nM concentrations154) suppress tumorigenesis through induction of the antioxidant-response element and MAPK (ERK, JNK and p38) in several chemical-induced animal carcinogenesis models in a dose- and time-dependent manner.251
(+)-Catechin (22) and quercetin (15) exhibit cardiovascular protection through suppressing PAI-1 expression in human coronary artery endothelial cells in vitro through activating the ERK, JNK and p38 signalling pathways.252 In a more recent study, quercetin (15) at mM concentrations exerted anti-adipo-genesis activity in a dose-dependent manner by inducing apoptosis of mature adipocytes through the modulation of the ERK and JNK pathways, which play pivotal roles during apoptosis.253 5-O-Caffeoylquinic acid (64) exhibits chemo-preventive effects on A549 human cancer cells in vitro in a dose-dependent manner at mmol/L concentrations, mainly through its up-regulation of the MAPK signaling pathway. In addition, it also suppressed the ROS-mediated NF-kB and AP-1 signalling
pathways, thus providing protection against environmental carcinogen-induced carcinogenesis.245