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Volume 26 | Number 8 | 2009 NPR Pages 965–1096

Natural Product Reports

www.rsc.org/npr

Current developments in natural products chemistry

Volume 26 | Number 8 | August 2009 | Pages 965–1096

ISSN 0265-0568

REVIEW

Alan Crozier, Indu B. Jaganath and Michael N. Clifford Dietary phenolics: chemistry,

bioavailability and effects on health 0265-0568(200908)26:8;1-U

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Dietary phenolics: chemistry, bioavailability and effects on health

Alan Crozier,*

^a

Indu B. Jaganath

^b

and Michael N. Clifford

^c

Received 12th March 2009

First published as an Advance Article on the web 13th May 2009 DOI: 10.1039/b802662a

Covering: up to the end of 2008

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–C₆flavonoids – 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 the metabolites of dietary phenolics, which appear in the circulatory system in nmol/L to low mmol/L concentrations, exert modulatory 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.

1 Introduction

2 Classification of phenolic compounds 2.1 Flavonoids

2.2 Non-flavonoids

3 Significant dietary sources of phenolics and polyphenolics 3.1 Beverages

3.1.1 Tea 3.1.2 Coffee 3.1.3 Cocoa 3.1.4 Wines 3.1.5 Beer 3.1.6 Cider 3.2 Fruits 3.3 Vegetables

3.4 Miscellaneous minor commodities

4 Biosynthesis of phenolics and polyphenolics 5 Potential for genetically-modified produce

6 Bioavailability of phenolics and polyphenolics 6.1 Flavonols

6.1.1 Onion quercetin-O-glucosides

6.1.2 Tomato juice quercetin-3-O-rutinoside 6.2 Orange juice flavanones

6.3 Dihydrochalcones 6.4 Flavan-3-ols

6.4.1 Green tea and flavan-3-ol monomers 6.4.2 Procyanidins

6.5 Anthocyanins 6.6 Isoflavones 6.7 Ellagitannins

7 Paradigm shift on the possible mode of action of phenolics

7.1 Significance of phenolic metabolites for human health 7.2 Tissue or organ targets of phenolics

7.3 Potential mode of action of phenolic compounds and their metabolites

7.3.1 NF-kB signaling pathway 7.3.2 Activator protein-1 (AP-1)

7.3.3 Phase II enzyme activation and Nrf2

7.3.4 Mitogen-activated protein kinase (MAPK) signaling pathway

8 Concluding comments 9 References

aGraham Kerr Building, Division of Ecology and Evolutionary Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK. E-mail: a.crozier@bio.gla.ac.uk

bMalaysian Agricultural, Research and Development Institute, Kuala Lumpur, Malaysia. E-mail: indu@mardi.gov.my

cFood Safety Research Group, Centre for Nutrition and Food Safety, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK. E-mail: M.Clifford@Surrey.ac.uk

ª The Royal Society of Chemistry 2009

REVIEW www.rsc.org/npr | Natural Product Reports

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1 Introduction

Current dietary advice is that for optimum health people should consume on a daily basis five portions of fruit and vegetables each comprising at least 80 grams.¹The epidemiological evidence for the benefit of consuming a diet that is high in fruit and vegetables is quite compelling. The evidence for specific vegetables, and indeed specific phytochemicals, is less convincing and the best simple advice that can be given is to recommend as much variety as possible. Phytochemicals are plant secondary metabolites, i.e. substances that in planta have little or no role in photosynthesis, respiration or growth and development, but which may accumulate in surprisingly high concentrations.²

Unlike the traditional vitamins, phytochemicals as dietary components are not essential for short-term well-being, and

whereas the body has specific mechanisms for the accumulation and retention of vitamins, in contrast, phytochemicals are treated as non-nutrient xenobiotics and metabolised so as to eliminate them efficiently. The flavonoids and allied phenolic and polyphenolic compounds, including tannins and derived polyphenols, form one major group of phytochemicals. We here review data relating to those dietary commodities that make a particular contribution to the intake of phenols and polyphenols, either because the commodity is unusually rich, or consumed in large quantities, or is otherwise of note. However, it cannot be overstressed that published analytical data may not be truly representative of the individual component in a particular diet.

The lack of comprehensive and reliable data for the phyto- chemical content of raw foods severely limits the insights that can be obtained from epidemiological studies. This is compounded by a lack of information relating to changes in content and character, i.e. the production of derived polyphenols caused by food processing, and the physiological consequences of the gut microbial and mammalian metabolism of both native and derived phytochemicals once consumed.

2 Classification of phenolic compounds

Phenolics are characterized by having at least one aromatic ring with one or more hydroxyl groups attached. In excess of 8000 phenolic structures have been reported and they are widely dispersed throughout the plant kingdom³– many occur in food.

Phenolics range from simple, low molecular weight, single- aromatic-ring compounds to the large and complex tannins and derived polyphenols. They can be classified by the number and arrangement of their carbon atoms (Table 1) and are commonly found conjugated to sugars and organic acids. Phenolics occurring naturally in healthy plant tissue can be classified into two groups, the flavonoids and the non-flavonoids: traditionally processed foods and beverages, such as black tea, matured red wine, coffee and cocoa, may contain phenolic transformation

Indu Jaganath

Indu Jaganath is a senior research officer at The Biotech- nology Research Centre at the Malaysian Agricultural Research and Development Institute. She obtained a Master of Science from the University of California, Riverside, and a PhD at the University of Glasgow. Her earlier research areas include pollination biology and ethnobotany. Currently her research is focused on the discovery of chemically and biologically active natural products in tropical plants. Her research group uses metabolomics and genomics approaches to understand and delineate the metabolic pathways involved in the production of these biologically active compounds.

Mike Clifford

Mike Clifford is a Food Science graduate of the University of Reading. He obtained a PhD at the University of Strathclyde.

He taught at Grimsby College of Technology for eight years and joined the University of Surrey in 1979, where he founded and led the Food Safety Research Group. He is now Emeritus Professor of Food Safety. He has published extensively on the analysis and characterisation of phenols, especially those in coffee (chlorogenic acids) and black tea (thearubigins), and their metabolism by the gut microflora and their effects on the consumer.

Alan Crozier

Alan Crozier is a Botany graduate of the University of Durham. He obtained a PhD at Bedford College, University of London, carried out post- doctoral research at the Univer- sity of Calgary and lectured at the University of Canterbury in New Zealand before joining the University of Glasgow where he is currently Professor of Plant Biochemistry and Human Nutrition. He has published extensively on plant hormones and purine alkaloids in coffee and teas. The activities of his research group are currently focussed on dietary flavonoids and phenolic compounds in fruits, vegetables and beverages, including teas and fruit juices, and their fate within the body following ingestion in relation to their potentially beneficial effects on health.

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products that are best described as ‘derived polyphenols’.

Tannins are the active ingredients of traditional plant extracts used to convert hides to leather and occur widely in foods and beverages but at concentrations too low to tan hides.

2.1 Flavonoids

Flavonoids are polyphenolic compounds comprising 15 carbons, with two aromatic rings connected by a three carbon bridge, hence C₆–C₃–C₆ (1). They are the most numerous of the phenolics and are found throughout the plant kingdom.⁴ They are present particularly in the epidermis of leaves and the skin of fruits.

The main sub-classes of dietary flavonoids are flavonols (2), flavones (3), flavan-3-ols (4), anthocyanidins (5), flavanones (6) and isoflavones (7), while those that are comparatively minor components of the diet are dihydroflavonols (8), flavan-3,4-diols (9), coumarins (10), chalcones (11), dihydrochalcones (12) and aurones (13). The basic flavonoid skeleton can have numerous substituents. Hydroxyl groups are usually present at the 4⁰-, 5- and 7-positions. Sugars are very common, with the majority of flavonoids existing naturally as glycosides. Whereas both sugars and hydroxyl groups increase the water solubility of flavonoids, other substituents, such as methyl groups and isopentyl units, make flavonoids lipophilic.

Flavonols (2) are the most widespread of the flavonoids, being dispersed throughout the plant kingdom with the exception of algae. They are also not found in fungi. The distribution and structural variations of flavonols are extensive and have been well documented.⁵The main dietary flavonols, kaempferol (14), quercetin (15), isorhamnetin (16) and myricetin (17), are most commonly found as O-glycosides. Conjugation occurs most Table 1 Basic structural skeletons of phenolic and polyphenolic

compounds.

Skeleton Classification Basic structure

C6–C1 Phenolic acids

C6–C2 Acetophenones

C6–C2 phenylacetic acid

C6–C3 Hydroxycinnamic acids

C6–C3 Coumarins

C6–C4 Naphthoquinones

C6–C1–C6 Xanthones

C6–C2–C6 Stilbenes

C6–C3–C6 Flavonoids

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frequently at the 3-position of the C-ring but substitutions can also occur at the 5-, 7-, 4⁰-, 3⁰- and 5⁰-carbons. Although the number of aglycones is limited there are numerous flavonol conjugates, with more than 200 different sugar conjugates of kaempferol alone. The levels of flavonols found in commonly consumed fruits, vegetables and beverages are well documented.⁶ However, sizable differences are found in the amounts present in seemingly similar produce, possibly due to seasonal changes and varietal differences;⁷ effects of processing will also have an impact.

Flavones (3), such as apigenin (18) and luteolin (19), lack oxygenation at C3 but otherwise may have a wide range of substitutions including hydroxylation, methylation, O- and C- alkylation and glycosylation. Most flavones occur as 7-O- glycosides. Flavones are not distributed widely, with significant occurrences being reported in only celery, parsley and some herbs. Polymethoxylated flavones, such as tangeretin (20) and nobiletin (21), have been found in citrus species.

Flavan-3-ols (4) are non-planar by virtue of their saturated C3 element and are the most structurally complex subclass of flavonoids, ranging from the simple monomers (+)-catechin (22) and its isomer ()-epicatechin (23), which can be hydroxylated to form gallocatechins (24, 25) and also undergo esterification with gallic acid (26, 27), through to complex structures including the oligomeric and polymeric proanthocyanidins, which are also known as condensed tannins. The two chiral centres at C2 and C3 of the flavan-3-ols produce four isomers for each level of B- ring hydroxylation, two of which, (+)-catechin and ()-epicatechin, are widespread in nature whereas ()-catechin (28) and

(+)-epicatechin (29) are comparatively rare.⁸The oligomeric and polymeric proanthocyanidins have an additional chiral centre at C4 of each additional flavan-3-ol unit. Pairs of enantiomers are not resolved on the commonly used reverse-phase HPLC columns, and so are easily overlooked. Although difficult to visualise, these differences in chirality have a significant effect on the 3-D structure of the molecules, as illustrated in Fig. 1 for the (epi)gallocatechin-3-O-gallates. Although this has little, if any, effect on their redox properties or ability to scavenge small unhindered radicals,⁹ it can be expected to have a more pronounced effect on their binding properties and hence any phenomenon to which the ‘lock and key’ concept is fundamental, e.g. enzyme–substrate, enzyme–inhibitor or receptor–ligand interactions. Humans fed ()-epicatechin (23) excrete some (+)-epicatechin (29), indicating ring opening and racemisation, possibly in the gastrointestinal tract.¹⁰Transformation can also occur during food processing.¹¹

Type B proanthocyanidins are formed from (+)-catechin (22) and ()-epicatechin (23) with oxidative coupling occurring between the C4 of the heterocycle and the C6 (30) or C8 positions (31) of the adjacent unit to create oligomers or polymers. Type A proanthocyanidins have an additional ether bond between C2 and C7 (32). Proanthocyanidins can occur as polymers of up to 50 units. Proanthocyanidins that consist exclusively of (epi)catechin units are called procyanidins, and are the most abundant type of proanthocyanidins in plants. The less common proanthocyanidins containing ()-epiafzelechin (33) and (+)-afzelechin (34) or (epi)gallocatechin (24, 25) sub-units are called propelargonidins and prodelphinidins, respectively. Many condensed tannins

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contain more than one monomer. Flavan-3-ol monomers are extensively transformed during the traditional processing of wines, cocoa and black tea, in the latter case yielding theaflavins, theacitrins and thearubigins (see Section 3.1.1)

Anthocyanidins (5) are widely dispersed throughout the plant kingdom, being particularly evident in fruit and flower tissue where they are responsible for red, blue and purple colours. They are also found in leaves, stems, seeds and root tissue. The most common anthocyanidins are pelargonidin (35), cyanidin (36), delphinidin (37), peonidin (38), petunidin (39) and malvidin (40).

In plant tissues these compounds are invariably found as sugar conjugates that are known as anthocyanins, which may also be conjugated to hydroxycinnamates and organic acids such as acetic acid (41–44). Although glycosylation can take place on carbons 3, 5, 7, 3⁰and 5⁰, it occurs most often on C3. In certain products, such as matured red wines and ports, chemical and enzymic transformations occur, and an increasing number of ‘anthocyanin- derived polyphenols’ are being found (see Section 3.1.4).

The flavanones (6) are non-planar and have a chiral center at C2. In the majority of naturally occurring flavanones, ring C is attached to the B-ring at C2 in the a-configuration. Flavanones are present in especially high concentrations in citrus fruits. The most common flavanone glycoside is hesperetin-7-O-rutinoside Fig. 1 Computer-generated stereochemical projections for flavan-3-ol

diastereoisomers. epigallocatechin-3-O-gallate (EGCG) and gallocatechin-3-O-gallate (GCG). Three-dimensional structures computed by Mr J. Warren Dryman, Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK.

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(hesperidin) (45), which along with narigenin-7-O-rutinoside (narirutin) (46) is found in citrus peel. Flavanone rutinosides are tasteless. In contrast, flavanone neohesperidoside conjugates such as hesperetin-7-O-neohesperidoside (neohesperidin) (47) from bitter orange (Citrus aurantium) and naringenin-7-O-neohesperidoside (naringin) (48) from grapefruit peel (Citrus para- disi) are intensely bitter.

Isoflavones (7) have the B-ring attached at C3 rather than C2.

They are found almost exclusively in leguminous plants, with the highest concentrations occurring in soy bean (Glycine max).¹² The isoflavones, daidzein (49) and genistein (50), and the coumestan, coumestrol (51) from lucerne and clovers (Trifolium spp.), have sufficient oestrogenic activity to seriously affect the reproduction of grazing animals such as cows and sheep, and are termed phyto-oestrogens. These isoflavonoids appear to mimic the steroidal hormone oestradiol (52) which blocks ovulation. The consumption of legume fodder by animals must, therefore, be restricted, or low-isoflavonoid producing varieties selected. This is clearly an area where it would be beneficial to produce genetically modified isoflavonoid-deficient legumes.

Dietary consumption of genistein and daidzein from soy products is thought to reduce the incidence of prostate and breast cancers in humans. However, the mechanisms involved are different. Growth of prostate cancer cells is induced by and dependent upon the androgen testosterone (53), the production of which is suppressed by oestradiol. When natural oestradiol is insufficient, the isoflavones can lower androgen levels and, as a consequence, inhibit tumour growth. Breast cancers are

dependent upon a supply of oestrogens for growth, especially during the early stages. Isoflavones compete with natural oestrogens, restricting their availability and thereby suppressing the growth of the cancerous cells. There was concern that neonates and infants could be adversely affected by excessive intakes of isoflavones in soy-protein-based human milk-replacers, and the levels have been voluntarily reduced by industry as a precaution.¹³

2.2 Non-flavonoids

The main non-flavonoids of dietary significance are the C₆–C₁ phenolic acids, most notably gallic acid (54), which is the biosynthetic precursor of hydrolysable tannins, the C₆–C₃ hydroxycinammates and their conjugated derivatives, and the polyphenolic C6–C2–C6stilbenes (Table 1).

Gallic acid is the commonest phenolic acid, and occurs widely as complex sugar esters in gallotannins such as 2-O-digalloyl- tetra-O-galloyl-glucose (55) but these are found only to a limited extent in dietary components. Non-sugar galloyl esters in grapes, wine, mangoes, green tea and black tea are the major source of gallic acid in the human diet. The related ellagic acid (56) and ellagitannins, such as sanguiin H-10 (57), which is found in raspberries (Rubus idaeus) and strawberries (Fragaria ana- nassa), are also present in a number of fruits including pomegranate (Punica granatum), blackberries (Rubus spp.), persimmon (Diospyros kaki) as well as walnuts (Juglans regia), hazelnuts (Corylus avellana) and oak-aged wines.

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The most common hydroxycinnamates are p-coumaric acid (58), caffeic acid (59), ferulic acid (60) and sinapic acid (61), with caffeic acid dominating. These occur as conjugates, for example with tartaric acid or quinic acid, collectively referred to as chlorogenic acids. Chlorogenic acids, principally 3-O-, 4-O- and 5-O-caffeoylquinic acids (62–64), form ca. 10%

of green robusta coffee beans (processed seeds of Coffea canephora). Regular consumers of coffee may have a daily intake in excess of 1 g, and these for many people will be the major dietary phenols.

Derivatives of phenylvaleric acid (65), phenyl-lactic acid (66), phenylpropionic acid (67), phenylmandelic acid (68) and phe- nylhydracrylic acid (69) rarely occur preformed in food but are colonic microflora metabolites of many dietary phenols and polyphenols that are readily absorbed, and may in part be responsible for some biological effects associated with diets rich in polyphenols (see Section 7.0).

Stilbenes have a C₆–C₂–C₆structure (Table 1) and are phy- toallexins produced by plants in response to disease, injury and stress.¹⁴ The main dietary source of stilbenes is resveratrol (3,5,4⁰-trihdroxystilbene) from red wine and peanuts (Arachis ª The Royal Society of Chemistry 2009

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hypogaea)¹⁵with lesser amounts found in berries, red cabbage (Brassica oleracea), spinach and certain herbs. Resveratrol occurs as cis and trans isomers (70, 71), and trans-resveratrol and trans-resveratrol-3-O-glucoside (trans-piceid) (72) have been detected in pistachio nuts (Pistacia vera).¹⁶The woody root of the noxious weed Polygonum cuspidatum (Japanese knotweed or Mexican bamboo) has been shown to contain very high levels of trans-resveratrol and its glucoside, with concentrations of up to 377 mg per 100 g dry weight.¹⁷As well as resveratrol, red wines can also contain trans-piceatannol (3,3⁰,4,5⁰-tetrahydrox- ystilbene) (73) and trans-astringin, its 3-O-glucoside (74).¹⁸trans- Resveratrol is transformed by Botrytis cinerea, a fungal grapevine pathogen, to pallidol (75) and resveratrol trans-dehydrodimer (76), and both these compounds have been detected in grape cell cultures along with the 11-O- and 11⁰-O-glucosides of resveratrol trans-dehydrodimer (77, 78).¹⁹Viniferins are another family of oxidised resveratrol dimers, and trans-d-viniferin (79) and smaller amounts of its isomer trans-3-viniferin (80) have been detected in Vitis vinifera leaves infected with Plasmopara viticola (downy mildew).²⁰

trans-Resveratrol (71) has gained significant worldwide attention because of its ability to inhibit or retard a wide variety of animal diseases²¹ that include cardiovascular disease²² and cancer.²³It has also been reported to increase stress resistance and enhance longevity.²⁴ The protective effects of red wine consumption are regularly attributed to resveratrol.²⁵However, this is highly unlikely as the levels of resveratrol in red wines are low, and for humans to ingest the quantity of resveratrol that

affords protective effects in animals they would have to drink in excess of 100 L of red wine per day.²⁶

3 Significant dietary sources of phenolics and polyphenolics

It is not possible to rank commodities in terms of their production of phenols and polyphenols per annum. However, on a global scale, the most important commodities are those that are rich in polyphenols and widely consumed in large quantities such as green tea, black tea, red wine, coffee and cocoa/chocolate.

Generally, fruits, and especially vegetables, are a poor second because of their much lower contents. No staples are rich in these phytochemicals, but along with herbs and spices, nuts, algae and olive oil, they are potentially significant for supplying certain phenols and polyphenols of restricted botanical occurrence.² Because of constraints on space, our emphasis will be on those commodities making the greatest quantitative contribution, but this is not to suggest that the minor dietary components are unimportant.

3.1 Beverages

3.1.1 Tea. Tea prepared from the leaves of Camellia spp. is one of the most widely consumed beverages in the world.

Approximately 3.2 million metric tons of dried leaf are produced annually, of which 20% is green tea and 2% is oolong, the remainder being black tea. In all cases the raw material is young leaves, the tea flush, which are preferred as they have a higher flavan-3-ol content and elevated levels of active enzymes. The highest quality teas utilise ‘two leaves and a bud’, with progressively lower quality taking four or even five leaves.²⁷Although ª The Royal Society of Chemistry 2009

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produced from similar plant material, these teas differ markedly in the nature of phenols and polyphenols that they contribute to the diet because of differences in their manufacture.

There are basically two types of green tea.²⁸The Japanese type utilises a shade-grown hybrid leaf with comparatively low flavan- 3-ol levels and high amino acid content, including theanine. After harvesting the leaf is steamed rapidly to inhibit polyphenol oxidase and other enzymes. Chinese green tea traditionally uses selected forms of Camellia sinensis var. sinensis and dry heat (firing) rather than steaming, giving a less efficient inhibition of the polyphenol oxidase activity and allowing some transformation of the flavan-3-ols.

In the production of black tea there are two major processes – the ‘orthodox’ and the ‘cut–tear–curl’ processes.^29,30In both the objective is to achieve efficient disruption of cellular compart- mentation bringing phenolic compounds into contact with polyphenol oxidases and activating many other enzymes. A detailed account of the processes is beyond the scope of this article (see ref. 30) but oxidation for 60–120 min at about 40C before drying is representative.

When harvested, the fresh tea leaf is unusually rich in polyphenols (ca. 30% dry weight) and this changes with processing even during the manufacture of commercial green tea, and progressively through semi-fermented teas to black teas and those with a microbial processing stage. Flavan-3-ols are the dominant polyphenols of fresh leaf. Usually

()-epigallocatechin-3-O-gallate (27) dominates, occasionally taking second place to ()-epicatechin-3-O-gallate (26), together with smaller but still substantial amounts of (+)-catechin (22), ()-epicatechin (23), (+)-gallocatechin (24), ()-epigallocatechin (25) and ()-epiafzelchin (33). The minor flavan-3-ols also occur as gallates, and ()-epigallocatechin (25) may occur as a digallate, esterified with p-coumaric acid or caffeic acid, and with various levels of methylation.³¹ There are at least 15 flavonol glycosides, comprising mono-, di- and tri-glycosides based upon kaempferol (14), quercetin (15) and myricetin (17), and various permutations of glucose, galactose, rhamnose, arabinose and rutinose.^32–34Three C-glycosides of apigenin (18),³⁵several caffeoyl- and p-coumaroylquinic acids (chlorogenic acids) and galloylquinic acids and at least 27 proanthocyanidins, including some with ()-epiafzelchin units, also occur.³⁶In addition, some forms have a significant content of hydrolysable tannins, such as strictinin (81),³⁷perhaps indicating an affinity with C. japonica, C. sasanqua and C. oleifera,³⁸whereas others contain chalcan–

flavan dimers known as assamaicins (82).³⁹

In green teas, especially those of Japanese production, most of these various polyphenols survive and can be found in the mar- keted product. In Chinese green teas and the semi-fermented teas such as oolong, some transformations occur, for example leading to the production of theasinensins (83) (flavan-3-ol dimers linked 2/2⁰), oolong homo-bis-flavans linked either 8/8⁰(84) or 8/

6⁰, oolongtheanin (85) and 8C-ascorbyl-epigallocatechin-3-O-

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gallate (86).⁴⁰In black tea production the transformations are much more extensive, with some 90% destruction of the flavan-3- ols in orthodox processing and even greater transformation in cut–tear–curl processing. Some losses of 5-O-galloylquinic acid (theogallin) (87), quercetin glycosides and especially myricetin glycosides have been noted, and recent studies on thearubigins (88) suggest that theasinensins (83) and possibly proanthocyanidins may also be transformed. Pu’er tea is produced by a microbial fermentation of black tea. Some novel compounds have been isolated and it is suggested that they form during the fermentation.⁴¹These include two new 8-C-substituted flavan-3- ols, puerins A and B (89, 90), two known cinchonain-type phenols, epicatechin-[7,8-bc]-4-(4-hydroxyphenyl)-dihydro- 2(3H)-pyranone (91) and cinchonain Ib (92), and 2,2⁰,6,6⁰-tet- rahydroxydiphenyl (93). However, various cinchonains have previously been reported in unfermented plant material.⁴²

It is generally considered that polyphenol oxidase, which has at least three isoforms, is the key enzyme in the fermentation processes that produce black teas, but there is also evidence for an important contribution from peroxidases, with the essential hydrogen peroxide being generated by polyphenol oxidase.⁴³The

primary substrates for polyphenol oxidase are the flavan-3-ols which are converted to quinones. These quinones react further, and may be reduced back to phenols by oxidising other phenols, such as gallic acid (54), flavonol glycosides and theaflavins (94), that are not direct substrates for polyphenol oxidase.⁴⁴

Many of the transformation products are still uncharacterised.

The best known are the various theaflavins and theaflavin gallates (94), characterised by their bicyclic undecane benztropolone nucleus, reddish colour and solubility in ethyl acetate.

These form through the Michael addition of a B-ring trihydroxy (epi)gallocatechin quinone to a B-ring dihydroxy (epi)catechin quinone prior to carbonyl addition across the ring and subsequent decarboxylation.⁴⁵However, it is now accepted that the theasinensins (83) form more rapidly and may actually be theaflavin (94) precursors.^46,47 Theaflavonins (95) and theogallinin (96) (2/2⁰-linked theasinensin analogues formed from ()-epigallocatechin (23)/()-epigallocatechin-3-O-gallate (27) and iso- myricetin-3-glucoside (97) or 5-O-galloylquinic acid (87), respectively) have also been found in black tea.⁴⁶

Coupled oxidation of free gallic acid or ester gallate produces quinones that can replace (epi)gallocatechin quinone leading to

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(epi)theaflavic acids (98) and various theaflavates (99).⁴⁸Inter- action between two quinones derived from trihydroxy precursors can produce benztropolone-containing theaflagallins (100)⁴⁹or yellowish theacitrins (101) that have a tricyclic dodecane nucleus.⁵⁰Mono- or di-gallated analogues are similarly formed from the appropriate gallated precursors, and in the case of theaflavins coupled oxidation of benztropolone gallates can lead to theadibenztropolones (102) (and higher homologues at least in model systems). Oxidative degallation of ()-epigallocatechin-3- O-gallate (27) produces a pinkish-red desoxyanthocyanidin, tri- cetanidin (103).⁵¹

The brownish water-soluble thearubigins (88) are the major phenolic fraction of black tea, and these have been only partially characterised. Masses certainly extend to ca. 2000 daltons. Early reports that these were polymeric proanthocyanidins⁵²probably arose through detection of proanthocyanidins that had passed through from the fresh leaf unchanged. The few structures that have been identified include dibenztropolones (102) where the

‘chain extension’ has involved coupled oxidation of ester gallate,⁵³ theanaphthoquinones (104) formed when a bicyclo-undecane benztropolone nucleus collapses back to a bicyclo-decane nucleus⁵⁴and dehydrotheasinensins (105).⁵⁵Production of higher- mass thearubigins could involve coupled oxidation of gallate esters yielding tribenztropolones, etc., coupled oxidation of large mass precursors such as proanthocyanidin gallates or theasinensin gallates (83) rather than flavan-3-ol gallates,⁵⁶or interaction of

quinones with peptides and proteins. Though long anticipated, 8⁰- ethylpyrrolidinonyl-theasinensin A (106), the first such product containing an N-ethyl-2-pyrrolidinone moiety, was only isolated from black tea in 2005.⁵⁷It is probably formed from a theasinensin (83) and the quinone-driven Strecker aldehyde produced by decarboxylation of theanine (107). Much remains to be done in this area, and it is interesting to note that for consumers of black tea, consumption of these uncharacterised derived polyphenols at ca. 100 mg per cup greatly exceeds their consumption of chemically-defined polyphenols such as flavonoids.⁵⁸

Green and semi-fermented teas retain substantial amounts of the flavan-3-ols, but they decline progressively with increased fermentation and are lowest in cut–tear–curl black teas. Beverages from green, semi-fermented and black teas also have significant contents of flavonol glycosides and smaller amounts of chlorogenic acids, flavone-C-glycosides (including luteolin-8-C-glucoside (108)) and 5-O-galloylquinic acid (87), which are less affected by processing but may vary more markedly with the origin of the fresh leaf.^32,34.59The black tea beverage uniquely contains theaflavins (94) and to a greater extent the high molecular weight thearubigins (88), which are responsible for the astringent taste of black tea and the characteristic red-brown colour. Thearubigins are difficult to analyse, since they either do not elute from or are not resolved on reverse-phase HPLC columns. Indirect estimates indicate that they comprise around 80% of the phenolic components in black tea infusions.⁶⁰Details of how some of the phenolic ª The Royal Society of Chemistry 2009

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compounds in green tea are modified by fermentation to produce black tea are presented in Table 2.⁶¹Further changes may occur during the domestic brewing process and production of instant tea beverages. The flavan-3-ols may epimerise, producing for example ()-catechin (28) and (+)-epicatechin (29).⁶²

3.1.2 Coffee. In economic terms, coffee is the most valuable agricultural product exported by third-world and developing countries, amounting to ca. six million metric tonnes per annum.²⁷The green coffee bean is the processed, generally non- viable, seed of the coffee cherry. Commercial production exploits Table 2 Concentration of the major phenolics in infusions of green and black tea manufactured from the same batch of Camellia sinensis leaves.^61,^a

Compound Green tea Black tea

Black tea content as a percentage of green tea content

Gallic acid (54) 6.0 0.1 125 7.5 2083

5-O-Galloylquinic acid (87) 122 1.4 148 0.8 121

Total gallic acid derivatives 128 273 213

(+)-Gallocatechin B (24) 383 3.1 n.d. 0

()-Epigallocatechin (25) 1565 18 33 0.8 2.1

(+)-Catechin (22) 270 9.5 12 0.1 4.4

()-Epicatechin (23) 738 17 11 0.2 1.5

()-Epigallocatechin-3-O-gallate (27)

1255 63 19 0.0 1.5

()-Epicatechin-3-O-gallate (26) 361 12 26 0.1 7.2

Total flavan-3-ols 4572 101 2.2

3-O-Caffeoylquinic acid (62) 60 0.2 10 0.2 17

5-O-Caffeoylquinic acid (64) 231 1.0 62 0.2 27

4-O-p-Coumaroylquinic acid (149) 160 3.4 143 0.2 89

Total hydroxycinammate quinic esters

451 215 48

Quercetin-O-rhamnosylgalactoside 15 0.6 12 0.2 80

Quercetin-3-O-rutinoside (153) 131 1.9 98 1.4 75

Quercetin-3-O-galactoside (150) 119 0.9 75 1.1 63

Quercetin-O-rhamnose-hexose- rhamnose

30 0.4 25 0.1 83

Quercetin-3-O-glucoside (117) 185 1.6 119 0.1 64

Kaempferol-rhamnose-hexose- rhamnose

32 0.2 30 0.3 94

Kaempferol-galactoside 42 0.6 29 0.1 69

Kaempferol-rutinoside 69 1.4 60 0.4 87

Kaempferol-O-glucoside 102 0.4 69 0.9 68

Kaempferol-arabinoside 4.4 0.3 n.d. 0

Unknown quercetin conjugate 4 0.1 4.3 0.5 108

Unknown quercetin conjugate 33 0.1 24 0.9 73

Unknown kaempferol conjugate 9.5 0.2 n.d. 0

Unknown kaempferol conjugate 1.9 0.0 1.4 0.0 74

Total flavonols 778 570 73

Theaflavin (94) n.d. 64 0.2 N

Theaflavin-3-gallate (94) n.d. 63 0.6 N

Theaflavin-3⁰-gallate (94) n.d. 35 0.8 N

Theaflavin-3,3⁰-digallate (94) n.d. 62 0.1 N

Total theaflavins n.d. 224 N

aData expressed as mg/L standard error (n ¼ 3). n.d. – not detected. Green and black teas prepared by infusing 3 g of leaves with 300 mL of boiling water for 3 min.

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the seeds of Coffea arabica (so-called arabica coffees) accounting for ca. 70% of the world market and C. canephora (so-called robusta coffees) accounting for ca. 30%. Although the method of processing the coffee cherry and the extracted beans has subtle effects on the sensory properties of the beverage obtained, the effects on the delivery of polyphenols are comparatively slight,^27,63and will not be described here.

Green coffee beans are one of the richest dietary sources of chlorogenic acids comprising 6–10% on a dry-weight basis. 5-O- Caffeoylquinic acid (62) is by far the dominant chlorogenic acid, accounting for some 50% of the total. This is accompanied by significant amounts of 3-O- and 4-O-caffeoylquinic acid (63, 64), the three analogous feruloylquinic acids and 3,4-O-, 3,5-O- and 4,5-O-dicaffeoylquinic acids (109–111).⁶⁴Recently, many minor mono-acyl and diacyl chlorogenic acids involving also p-coumaric acid (58) and 3,4-dimethoxycinnamic acid (112) have been characterised in green coffee beans⁶⁵along with a series of amino acid conjugates.⁶⁶Robustas, with the possible exception of those from Angola, have a significantly greater content of chlorogenic acids than arabicas.⁶⁷

The commercial beans are roasted at air temperatures as high as 230C for a few minutes, or at 180C for up to ca. 20 min.

During roasting there is a progressive destruction and transformation of chlorogenic acids with some 8–10% being lost for every 1% loss of dry matter, but substantial amounts survive to be extracted into domestic brews and commercial soluble coffee powders. For many consumers coffee beverage must be the major dietary source of chlorogenic acids.⁶³ Regular coffee drinkers will almost certainly have a greater intake of chlorogenic acids than flavonoids.⁵⁸While a portion of the green bean chlorogenic acids is completely destroyed, some is transformed during roasting. Early in roasting when there is still adequate water content, isomerisation (acyl migration) occurs accompanied by some hydrolysis, releasing the cinnamic acids and quinic acid. Later in roasting the free quinic acid epimerises and lacto- nises, and several chlorogenic lactones including 3-O- and 4-O- caffeoyl-1,5-quinide (113, 114)† also form.⁶⁸The cinnamic acids may be decarboxylated and transformed to a number of simple phenols and a range of phenylindans, probably via

decarboxylation and cyclisation of the vinylcatechol interme- diate.⁶⁹Two of these rather unstable compounds, 1,3-trans- and 1,3-cis-tetrahydroxyphenylindan (115, 116), have been found in roasted and instant coffee at 10–15 mg/kg.

3.1.3 Cocoa. Cocoa (Theobroma cacao) is a tree which originated in the tropical regions of South America. There are two forms sufficiently distinct as to be considered subspecies. Criollo developed north of the Panama isthmus and Forastero in the Amazon basin, the latter accounting for 90% of world production. Extraction of the seeds, fermentation and conversion to chocolate and cocoa are described elsewhere.^27,70These processes will cause some transformation of the native polyphenols, but although there is a role for polyphenol oxidase and a roasting at ca. 150C, these transformations are even less well characterised than those of tea or coffee processing. Cocoa and chocolate as consumed have been characterised only with regard to the surviving untransformed flavan-3-ols and proanthocyanidins.

The major polyphenols in fresh beans are (+)-catechin (22), ()-epicatechin (23) and oligomeric procyanidins ranging from dimers to decamers. Trace quantities of quercetin-3-O-glucoside (117) and quercetin-3-O-arabinoside (118) also occur.⁷¹ Indi- vidual procyanidins that have been identified include the B5 and B2 dimers (30, 31) and the trimer C1 (119).⁷²N-Caffeoyl-3-O- hydroxytyrosine (clovamide) (120) and N-p-coumaroyl-tyrosine

† To avoid confusion, non-IUPAC numbering is used for the quinide moiety in 113 and 114.

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(deoxyclovamide) (121) are also present.⁷³ These compounds along with the proanthocyanidins contribute to the astringent taste of unfermented cocoa beans and roasted cocoa nibs, but not to the same degree as other amides, in particular cinnamoyl-L- aspartic acid (122) and caffeoyl-L-glutamic acid (123).⁷⁴During fermentation and processing, the conversion of many of the phenolic components to insoluble brown polymeric compounds takes place and the level of soluble polyphenols can fall by ca.

90%. As a consequence, there are large variations in the flavan-3- ol monomer and procyanidin content of commercial cocoas, and many brands of milk chocolate are largely depleted of flavan-3- ols.⁷⁵As a further complication it has recently been reported that chocolate has a significant content of ()-catechin (28), which is absorbed less readily than its (+)-isomer (22).⁷⁶

3.1.4 Wines. Wine is basically the fermented juice of Vitis vinifera grapes with a minimum alcohol level of 8.5% by volume.

The wild grapevine originated in the Far East (Mesopotamia) and Egypt, and evidence for wine production dates from Neolithic times. Today, wines are produced from numerous varieties of grapes, including Cabernet Sauvignon, Merlot, Pinot Noir, Syrah, Cinsault, Rondinella, Sangiovese, Nebiolo, Grenache, Tempra- nillo, Tannat and Carignan. The main commercial producers are located in France, Italy, Australia, New Zealand, Spain, Chile, Argentina, California and South Africa, as well as Bulgaria, Romania, southern Brazil, and (more recently) China and India.

A wide variety of processes are used in the making of red wine.

Typically, however, black grapes are pressed and the juice (‘must’), together with the crushed grapes, undergo alcoholic fermentation for 5–10 days at ca. 25–28 C. The solids are removed and the young wine subjected to a secondary or malo- lactic fermentation during which malic acid is converted to lactic acid and carbon dioxide. This softens the acidity of the wine and adds to its complexity and stability. The red wine is then matured in stainless steel vats or (in the case of higher quality vintages) in oak barrels for varying periods, before being filtered and bottled.

White wines are produced from both black and, more traditionally, white varieties of grapes. The berries are crushed gently rather than pressed to prevent breaking of stems and seeds. Solid material is removed and the clarified juice fermented typically between 16C and 20C for 5 days. The resultant must then undergoes malo-lactic fermentation, before maturation, filtra- tion and bottling.

Wines are produced from an assortment of grape cultivars grown under climatic conditions that can vary substantially not only in different geographical regions but also locally on a year- to-year basis. To complicate matters further, grapes at different stages of maturity are used, and vinification and ageing proce- dures are far from uniform. It is hardly surprising, therefore, that wines are extremely heterogeneous in terms of their colour, flavour, appearance, taste and chemical composition.^72,77 In general, however, red wines, and to a much lesser extent white wines, are an extremely rich source of a variety of phenolic and polyphenolic compounds.

In the making of red wine, with prolonged extraction, the fermented must can contain up to 40–60% of the phenolics originally present in the grapes. Subtle changes in these grape- derived phenolic components occur during the ageing of the wines, especially when carried out in oak barrels or, as in recent years, during exposure to chips of oak wood. Consequently, there is a wide range in the level of phenolics between different red wines, the concentration of flavonols, for instance, varying by more than 10-fold and the overall level of phenolics by almost 5- fold (Table 3).⁷⁸ Information on variations in the levels of a number of phenolic compounds in comprehensive range of French red wines have been published.^79,80

The phenolics in red wines are the hydroxycinnamate–tartaric acid conjugates, coutaric acid (124), caftaric acid (125) and fer- taric acid (126), malvidin-3-O-glucoside (41) and other anthocyanins with lower levels of gallic acid (54), stilbenes and flavonols. From the data presented in Table 3 it is evident that the levels of the flavan-3-ol monomers (+)-catechin (22) and ()-epicatechin (23) are not high and that there is a large discrepancy between the levels of phenolics measured by HPLC and the total phenolics determined by the Folin-Ciocalteau assay. Among the ‘missing ingredients’ that were not measured ª The Royal Society of Chemistry 2009

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by HPLC are proanthocyanidin B1–4dimers (127, 31, 128, 30), the C1and C2trimers (129, 130)⁸¹and oligomeric and polymeric forms with respective mean degrees of polymerisation of 4.8 and 22.1.⁸² The equivalent mean degrees of polymerisation of proanthocyanidins in grapes were 9.8 and 31.5, indicating that substantial changes in flavan-3-ol composition occur during fermentation and aging of the wines. Among the processes involved is the formation of compounds corresponding to malvidin-3-O-glucoside (41) linked through a vinyl bond to either (+)-catechin, ()-epicatechin or the procyanidin dimer B3(131–

133).⁸³Similar blue-coloured compounds with the flavan-3-ols linked to malvidin-3-O-(6⁰⁰-O-p-coumaroyl)glucoside (44) have also been detected in red wines (134–136).⁸⁴The production of pyruvate and acetaldehyde by yeast during fermentation of Tempranillo grapes has been associated with the formation of

malvidin-3-O-glucoside-pyruvic acid (vitisin A) (137) and malvidin-3-O-glucoside-4-vinyl (vitisin B) (138).⁸⁵

The production of white wine results in either low levels or an absence of skin- and seed-derived phenolics, so the overall level of phenolics can be much lower than that found in many red wines.⁸⁶ This observation is reflected in a more detailed comparison of the constituents of French red wines, dry white wines and sweet white wines summarised in Table 4.⁸⁰

3.1.5 Beer. Beer is an alcoholic beverage made from malted grains, usually barley (Hordeum vulgare) or wheat (Triticum vulgare), hops (Humulus lupulus), yeast (Saccharomyces spp.) and water. It contains a range of phenolic and polyphenolic compounds, derived partly from the barley (70%) and partly from the hops (30%). Flavan-3-ols are found in both hops and malt. These include monomers such as (+)-catechin (22) and ()-epicatechin (23), and the dimers procyanidin B3 (128) and prodelphinidin B₃ (139). Trimers also occur, although a more recent study, albeit with one unnamed American beer, reported an absence of high molecular weight polymeric proanthocyanidins and an average degree of polymerisation of only 2.1.⁸⁷The malt contributes most of the simple phenolics such as 3,4-dihy- droxybenzoic acid (protocatechuic acid) (140), caffeic acid (59) and ferulic acid (60), with small amounts of these compounds also being found in hops.

Table 3 Range of concentrations of phenolic compounds in 15 red wines of different geographical origin.^77,^a

Phenolic Range (mg/L)

Total flavonols 5–55

trans-Resveratrol (71) and trans- resveratrol-3-O-glucoside (72)

1–18

Gallic acid (54) 8–71

Total hydroxycinnmates 66–124

(+)-Catechin (22) and ()-epicatechin (23)

8–60

Free and polymeric anthocyanins 41–150

Total phenols 824–4059

aTotal phenols measured by colorimetric Folin-Ciocalteau assay; other estimates based on HPLC analyses that did not detect proanthocyanidins.

Table 4 Average concentration of selected phenolic compounds in 34 red, 11 dry white and 7 sweet white French wines.^79,^a

Red wine Dry white wine Sweet white wine Flavan-3-ols

(+)-Catechin (22) 41 6 15 8 4.2 0.7 ()-Epicatechin (23) 29 3 12 9 1.4 0.3 Procyanidin B1(127) 15 2 5.1 2.3 3.4 0.4 Procyanidin B2(31) 27 5 8.9 4.9 3.0 0.5 Procyanidin B3(128) 59 7 13 5 10 2 Procyanidin B4(30) 5.2 1.0 4.0 2.5 2.0 1.1 Total flavan-3-ols 177 22 59 31 24 1 Gallic acid (54) 30 2 4.0 2.1 5.8 1.1 Caffeic acid (59) 11 1 3.4 0.5 1.6 0.3 Caftaric acid (125) 51 4 33 6 14 3

Anthocyanins 22 19 n.d. n.d.

aData expressed as in mg/L as mean values standard error. n.d. – not detected.

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Hops contain quercetin conjugates and the prenylflavonoid xanthohumol (141), which during the brewing process undergoes substantial conversion to the flavanone isoxanthohumol (142), which predominates in most beers. Other prenylflavonoids found in beers include desmethylxanthohumol (143), 6- and 8-pre- nylnaringenin (144, 145) and 6-geranylnaringenin (146).⁸⁸ The quantity of phenolics in beer has not been widely studied, but in

general low and sub-mg quantities per litre are present. De Pascual-Teresa et al.⁸⁹determined the flavan-3-ol content of a red wine and a beer and found 17.8 and 7.3 mg/L, respectively.

Considering serving size, the potential flavan-3-ol intake from these two sources is similar.

3.1.6 Cider. Cider is an alcoholic beverage produced by yeast fermentation of apples (Malus domestica), typically specific cider varieties rather than dessert apples, the varieties often but not always blended. A survey of English ciders found that the overall level of phenolics ranged from 44 to 1559 mg/L.

The principal components were 5-O-caffeoylquinic acid (62), and procyanidins. Also present were (+)-catechin (22), ()-epicatechin (23), the dihydrochalcones phloretin-2⁰-O- glucoside (phloridzin) (147) and phloretin-2⁰-O-(2⁰⁰-O-xylosyl)- glucoside (148), hydroxycinnamates and trace amounts of quercetin glycosides.^90,91

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Chlorogenic acids and flavan-3-ols are the two classes that are important in the cider industry due to their physiochemical properties. 5-O-Caffeoylquinic acid (64) is a key substrate for endogenous polyphenol oxidases, with further reactions from the products formed giving cider its yellow-brown colouring.⁹² Phenolics of apples are implicated in cider quality, being involved in astringent and bitter tastes. The degree of polymerisation of procyanidins is directly involved in the balance of bitterness and astringency. Bitterness is due to oligomeric procyanidins with a degree of polymerisation of 2–5, whereas procyanidins with a degree of polymerisation of 6–10 are more involved in astringency.⁹³

The method of production has been shown to affect the phenolic content, with modern techniques of pneumatically pressed cider fermented in stainless steel vats decreasing levels more slowly than the more traditional methods of pressing and fermenting in wooden barrels.⁹⁴Oxidation which occurs during the juice extraction has also been linked with a reduction in the level of polymeric procyanidins.⁹⁵Fining to clarify the cider has been shown to decrease the procyanidin content.⁹⁰

3.2 Fruits

Apples (Malus domestica) and pears (Pyrus communis) are among the main sources of proanthocyanidins in the diet.⁹⁶ Apples and apple products are extensively consumed. They are a good source of flavonoids and phenolic compounds, containing 2310–4880 mg/kg.⁹⁷The principal ingredients include 5-O-caffeoylquinic acid (64) which occurs together with small quantities of phloretin-2⁰-O-glucoside (146), phloretin-2⁰-O-(2⁰⁰-O-

xylosyl)glucoside (148) and 4-O-p-coumaroylquinic acid (149).

Apples are an important source of flavonols, containing quercetin-3-O-glucoside (117), quercetin-3-O-galactoside (150), quercetin-3-O-rhamnoside (151), quercetin-3-O-xyloside (152), quercetin-3-O-rutinoside (153), quercetin-3-O-arabinopyroside (154) and quercetin-3-O-arabinofuranoside (155). They also contain flavan-3-ols, including ()-epicatechin (23) and its procyanidin dimers (B₁(127) and B₂(31)) and oligomers, these latter especially in cider apples.⁹⁸The procyanidins have been shown to have an average degree of polymerisation of between 3.1 and 8.5.⁹⁹An anthocyanin, cyanidin-3-O-galactoside (156), is found in the skin of red apple varieties.

The total phenolic content of some cultivars of pears is between 1235 and 2500 mg/kg in the peel and 28–81 mg/kg in the flesh.¹⁰⁰The phenolic composition of pears is very similar to that of apples, containing 5-O-caffeoylquinic acid (64), 4-O-p- ª The Royal Society of Chemistry 2009

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coumaroylquinic acid (149), procyanidins and quercetin glycosides. The main difference in the phenolic content of apples and pears is the presence of 1-hydroxyphenyl-4-O-glucoside (arbutin) (157) in pears and the dihydrochalcones in apples.¹⁰¹The average degree of polymerisation of procyanidins in some varieties of pears has been shown to be as high as 44.¹⁰²

There is increasing usage of nectarines (Prunus persica var.

nectarina) a smooth-skinned variety of peach. Peaches and nectarines contain cyanidin-3-O-glucoside (158), cyanidin-3-O- rutinoside (159), quercetin-3-O-glucoside (117) and quercetin-3- O-rutinoside (153), and other stone fruits (cherries, plums, prunes) are not greatly different. Stone fruits are characterised by a greater content of 3-O-caffeoylquinic acid (62) than 5-O-caffeoylquinic acid (64).¹⁰³Canning and storage of nectarines causes a reduction in the contents of (+)-catechin (22), ()-epicatechin (23) and proanthocyanidins including procyanidin B1(127).¹⁰⁴

Citrus fruits are significant sources of flavonoids, principally flavanones, which are present in both the juice and the tissues that are ingested when fruit segments are consumed. These tissues are a particularly rich source but are only consumed as an accidental adjunct to the consumption of the pulp. It is difficult to estimate dietary intake in such cases because it is so heavily dependent on the amount of tissue surrounding the segments after peeling, and on the method of analysis.¹⁰⁵Citrus peel, and to a lesser extent the segments, also contain the conjugated flavanone naringenin-7-O- rutinoside (46) as well as hesperetin-7-O-rutinoside (45), which is included in dietary supplements and is reputed to prevent capil- lary bleeding. Naringenin-7-O-neohesperidoside (48) from grapefruit peel and hesperetin-7-O-neohesperidoside (47) from bitter orange are intensely bitter flavanone glycosides. Orange juice contains polymethoxylated flavones such as tangeretin (20), nobiletin (21), scutellarein (160) and sinensetin (161), which are found exclusively in citrus species. The relative levels of these compounds can be used to detect the illegal adulteration of orange juice with juice of tangelo fruit (Citrus reticulata).

The red colour of ripe mango (Mangifera indica) peel is due to cyanidin-3-O-galactoside (156). The peels also contains several

quercetin and kaempferol glycosides, the principal flavonols being quercetin-3-O-glucoside (117) and quercetin-3-O-galactoside (150), a xanthone C-glucoside, mangiferin (162), and smaller amounts of its isomer isomangiferin (163), an array of gallotannins, and C-glucosides and galloyl derivatives of the benzo- phenones maclurin (164) and iriflopheone (165).¹⁰⁶Mango latex also contains the contact allergen 5-(12-heptadecenyl)resorcinol (166),¹⁰⁷which may contaminate the peel but not normally the fruit itself. Mango extracts are used widely in traditional medi- cines for treating a number of conditions including diarrhoea, diabetes and skin infections,¹⁰⁸ and mangiferin is reported to inhibit bowel carcinogenesis in rats.¹⁰⁹

Commercial pomegranate juice is being consumed in increasing quantities. Some, but not all, of the products have a high antioxidant content attributable to gallagic acid (167), an analogue of ellagic acid (56) containing four gallic acid (54) residues, and punicalin (168), the principal monomeric ellagitannin in which gallagic acid is bound to glucose. Punicalagin (169) is a further ellagitannin in which ellagic acid, as well as gallagic acid, is linked to the glucose moiety. Pomegranates contain 3-O-glucosides and 3,5-O-diglucosides of cyanidin (36) and delphinidin (37) and several ellagic acid derivatives.¹¹⁰There is evidence that consumption of pomegranate juice can have a favourable impact on cardiovascular disease¹¹¹ and have protective effects against colon and prostate cancer.¹¹²

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3.3 Vegetables

Carrots contain a range of chlorogenic acids including 3-O- and 5- O-caffeoylquinic acids (62, 64), 3-O-p-coumaroylquinic acid (170), 5-O-feruloylquinic acid (171) and 3,5-O-dicaffeoylquinic acid (110). The chlorogenic acids are found in orange, purple, yellow and white carrots, the level of 5-O-caffeoylquinic acid in purple carrots, at 540 mg/kg, being almost 10-fold higher than the amounts present in the other varieties.¹¹³ Chlorogenic acids are considered a carrot root fly attractant, and varieties resistant to this pest have been bred to have low caffeoylquinic acid contents.¹¹⁴

Onions provide a range of flavonols that are of comparatively restricted occurrence, principally quercetin-4⁰-O-glucoside (172) and quercetin-3,4⁰-O-diglucoside (173) with smaller amounts of isorhamnetin-4⁰-O-glucoside (174) and other quercetin conjugates.¹¹⁵Yellow onions form one of the main sources of flavonols

in the Northern European diet, the edible flesh containing between 280 and 490 mg/kg.⁷ Even higher concentrations are found in the dry outer scales.¹¹⁶ By contrast, leeks have been found to contain only 10–60 mg/kg of kaempferol and no quercetin. White onions are all but devoid of flavonols. Red onions like their yellow counterparts are rich in flavonols and also contain up to 250 mg/kg of anthocyanins,¹¹⁷ among the major components being cyanidin-3-O-(6⁰⁰-malonyl)glucoside (175) and cyanidin-3-O-(6⁰⁰-malonyl)laminaribioside (176).¹¹⁸ Flavonols are of chemotaxonomic interest, and many commodities, e.g. broccoli¹¹⁹and spinach,¹²⁰contain distinctive forms, but in most cases intake is low. Flavonols are generally concentrated in the leaf or peel, and cherry tomatoes, because of their high skin:volume ratio, are an especially rich source of quercetin-3-O-rutinoside (153).¹²¹ The extent to which this is transferred to processed products is largely unknown.

Although widely consumed in fresh and processed forms, there is relatively little information on the phytochemicals present in most legumes of dietary significance. The notable exception is soybean (Glycine max) which contains the isoflavones diadzein- 7-O-(6⁰⁰-O-malonyl)glucoside (177) and genistein-7-O-(6⁰⁰-O- malonyl)glucoside (178), and their aglycones (49, 50).¹²² The levels of isoflavones in soybeans have been reported to range from 560–3810 mg/kg, which is two orders of magnitude higher than the amounts detected in other legumes. Fermented soya products can be comparatively rich in the aglycones as hydrolysis of the glycosides can occur.¹²³ Products whose manufacture involves heating at 100C, such as soy milk and tofu, contain reduced quantities of isoflavones, the principal components being daidzein and genistein glucosides, which form as a result of degradation of the malonyl- and acetylglucosides.¹²⁴

As far as other legumes are concerned, peanuts (Arachis hypogaea) contain 5,7-dimethoxyisoflavone (179), broad beans (Vicia faba) are a relatively rich source of flavan-3-ols (containing more than 150 mg/kg⁸⁹), while French beans (Phaseolus vul- garis) can contain substantial quantities of quercetin-3-O- glucuronide (180).¹²⁵Pinto beans and red kidney beans contain in excess of 5 g/kg of proanthocyanidins, principally as prodelphinidins and propelargonidins, most with a degree of polymerisation >4.¹²⁶