Plant Polyphenols: Structure, Occurrence and Bioactivity

Plant Polyphenols: Structure, Occurrence and Bioactivity

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 28 © 2003 Elsevier Science B.V. All rights reserved. 257 PLANT POLYPHENOLS: STRUCTU...

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.

257

PLANT POLYPHENOLS: STRUCTURE, OCCURRENCE AND BIOACTIVITY PffiRGIORGIO PIETTA, MARKUS MINOGGIO, LORENZO BRAMATI ITB-CNR, Via FJli Cervi, 93- 20090 (MI), Italy ABSTRACT: Main dietary plant polyphenols are grouped into structural types and their occurrence in most common foods and beverages is briefly described. The major groups of plant polyphenols which are examined include flavonols, flavones, flavanones, isoflavones, anthocyanins, proanthocyanidins and flavanols. The current evidence on the absorption and the metabolism of each group is discussed. The biochemical and pharmacological activities of plant polyphenols are summarized, including antioxidant and anti-radical activity, chelation of metal ions, modulation of some enzymes activity, anticarcinogenic, antiatherosclerotic, anti-inflammatory, spasmolytic, hepatoprotective, antiviral, antimicrobial and oestrogenic activity, and inhibition of histamine release. An overview on the epidemiological evidence linking the intake of plant polyphenols and diminished risk of chronic diseases is also included.

INTRODUCTION Polyphenols constitute one of the most and widely distributed groups of substances in the plant kingdom, with more than 8000 phenoUc structures currently knovra. They can be divided into at least 10 different classes based upon their chemical structure, ranging from simple molecules, such as phenolic acids, to highly polymerized compounds, such as taimins. Flavonoids constitute the most important group with a common structure of diphenylpropanes (C6-C3-C6), consisting of two aromatic rings linked through three carbons that usually form an oxygenated heterocycle. Based upon the variations in the heterocyclic ring , flavonoids can be subdivided into eight major subclasses, including flavonols, flavones, flavanones, isoflavones, flavanols, anthocyanins, proanthocyanidins and tannins. Plant polyphenols have been of interest for long time owing to their role in plant pigmentation, reproduction and protection against predators and pathogens. Recently, interest in the biological effects of plant polyphenols (particularly flavonoids) has increased, because of the the potential health benefits associated with some dietary polyphenols. This

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contribution provides a brief description of the chemistry of plant polyphenols, their occurrence, bioavailability and bioactivity. CLASSES OF PLANT POLYPHENOLS I. Hydroxy benzoic acids derivatives The hydroxybenzoic acid derivatives (HBAs) are phenolic compounds with a general structure Ce-Ci. The related C6-C2 acids (phenylacetic acids) occur occasionally as minor components of foods. Variations in the basic structure of HBAs include hydroxylation and methoxylation of the aromatic ring, Fig. (1), Table 1.

Fig. (1). Basic structure of hydroxybenzoic acids

Although HBAs can be detected as free acids in some fruits (e.g., gallic acid in persimmons) or after being released during fruit and vegetable processing, they occur mainly as conjugates. For example, gallic acid and its dimer ellagic acid may be esterified with a sugar, usually glucose to produce the so-called hydrolysable tannins. In addition, gallic acid may esterify condensed tannins (i.e., derivatives of flavanols like those present in tea) or quinic acid [1,2]. Four HBAs, namely 4hydroxybenzoic acid (4-HBA), vanillic acid (3-methoxy-4-hydroxy), syringic acid (3,5-dimethoxy-4-hydroxy) and protocatechuic acid (3,4dihydroxy), are constituents of lignin [3]. These acids occur also as esters of glucose.

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Table 1.

Structure of the most common hydroxybenzoic acids

Trivialname

MW (Da)

Position of OH groups

Benzoic acid

122

/

Salicylic acid (2-Hydroxybenzoic acid)

138

2

4-Hydroxybenzoic acid

138

3

Protocatecliuic acid (3,4-Dihydroxybenzoic)

154

3,4

Gallic acid (3,4,5-Trihydroxy benzoic)

170

3,4,5

Vanillic acid (4-Hydroxy-3-methoxybenzoic

168

4

3

Isovanillic acid (3-Hydroxy-4-methoxybenzoic)

168

3

4

Syringic acid (4-Hydroxy-3,5-dimethoxybenzoic)

198

4

3,5

Position of OCH3 groups

Dietary occurrence Some herbs and spices are comparatively rich in various HBAs. After hydrolysis, protocatechuic acid is the dominant HBA in cinnamon bark (23-27 mg/kg), accompanied by saUcylic acid (7 mg/kg) and syringic acid (8 mg/kg). Gallic acid dominates in clove buds (175 mg/kg) and is accompanied by protocatechuic acid (10 mg/kg), genistic acid/4-HBA (7 mg/kg) and syringic acid (8 mg/kg) [4]. The fruit of anise {Pimpinella anisum) contains 730-1080 mg/kg of the glucoside of 4-HBA [3]. The skin of potato tubers contains protocatechuic acid (100-400 mg/kg FW) and vanillic acid (20-200 mg/kg) along with up to 30 mg/kg of gallic, syringic and salicylic acids [5]. Cereals contain also different HBAs. Canadian wheat flours were found to contain vanillic acid (up to 16 mg/kg) and syringic acid (up to 7 mg/kg). Oats contain vanillic acid, 4-HBA and salicylic acid, particularly in the hulls [6]. Alcoholic beverages (wine and beer) have a different content of HBAs. The gaUic acid content of French wines and spirits can reach 31-38 mg/1

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[7]. White wines contain less HBAs than red wines, namely 16-46 and 65-126 mg/1 for white and red Califomian wines [8]. Barley contains vanillic acid (6-17 mg/kg) and syringic acid (1-22 mg/kg), and both are found in malt (12 mg/kg each) and hops (59 and 30 mg/kg). These two acids are foimd in stout, ale and lager beers in the range from 0-2 mg/1) accompanied by gaUic, protocatechuic and 4-HBA (0.1-1.8 mg/1 each) [9]. Table 2 gives an overview of the occurrence of some HBAs in foods [10,11] and Table 3 shows the content of the major HBAs in selected foods [12]. Table 2.

Occurrence of some HBAs in different dietary sources (10,11]

HBAs Benzoic acid Salicylic acid 4-Hydroxy benzoic acid Vanillic acid Syringic acid Protocatechuic acid Gallic acid Table 3.

Dietary source universal component of angiosperms, csp. of berries anise, dill, white mustard, allspice,rosemary,thyme, majoram raspbcny, gooseberry, pecans, anise, fennel vanilla, garden cress, paprika rosemary, basil, thyme, garden cress tarragon, clove, anise, cinnamon, blackberry, blueberry tea, nuts, olive oil

Content of some HBAs in fruits (mg/kg) [12] 4-Hydroxy benzoic acid Food Blackberry Blackcurrant Raspberry Redcurrant Strawberry White currant

6-16

0-6 15-27 10-23 10-36 5-19

Protocatechuic acid 68-189 10-52 25-37

Gallic acid 8-67 30-62 19-38

3-8

-

-

11-44 3-38

2. Hydroxycinnamic acid derivatives Cinnamic acids are ^ra«5'-phenyl-3-propenoic acids differing in their ring substitution, Fig. (2). COOH

I %^ Fig. (2). Basic structure of cinnamic acids

II

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The most common cimiamic acids are caffeic (3,4-dihydroxycimiamic acid), ferulic (3-methoxy-4-hydroxy), sinapic (3,5-dimethoxy-4-hydroxy) and p-coimiaric (4-hydroxy) acid, Table 4 [13]. These compoimds are widely distributed as conjugates, mainly as esters of quinic acid (chlorogenic acids, CGA). Table 4.

Structure of the most common cinnamic acids

Trivialname

MW (Da)

Position of OH groups

Cinnamic acid

148

/

p-Coumaric acid (4-Hydroxycinnamic)

164

4

Cafleic acid (3,4-Dihydroxycinnamic)

180

3,4

Ferulic acid (4-Hydroxy-3-metlioxycinnamic)

194

4

3

Sinapic acid (3,5-Dimetlioxy-4-iiydroxycinnamic)

224

4

3,5

Position of OCH3 groups

Depending on the identity, number and position of the acyl residues, these acids may be divided into the following groups: mono-esters of caffeic, p-coumaric and ferulic acid; di-, tri- and tetra-esters of caffeic acid [14,15]; mixed di-esters of caffeic and ferulic acid or caffeic and sinapic acid [16]; mixed esters of caffeic acid with dibasic aliphatic acids (e.g., oxalic, succinic) [17]. Cinnamic acids may condense with molecules other than quinic acid, including rosmarinic, malic and tartaric acid, aromatic amino acids, choline, mono- and polysaccharides, glycerol, myo-inositol, and different glycosides (anthocyanins, flavonols and diterpenes) [13]. Dietary occurrence There is no doubt that caffeic acid is the cinnamate that occurs most extensively, and the various caffeoylquinic acids (CQA) and dicaffeoylqumic acids (diCQA) are the most ubiquitous conjugates. Usually the 5-isomers dominates, but in some fruit and brassicas the 3isomer is prevalent. Because of the quantity commonly consumed, coffee

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beverage must top the list, with 200 ml instant brew (2% w/v) supplying 50-150 mg CQA, mg. Blueberries, aubergines, apples, cider and green mate are good sources in some populations [13]. However, in view of quantities consumed by other populations, wines could make a significant contribution for tartaric acid conjugates and grapes and grape juice for caftaric acid, respectively. Lettuce is the major source of chicoric (dicaffeoyltartaric) and caffeoylmalic acid (up to 3 mg/100 g), but endive may have twice the concentration. Spinach is ahnost certainly therichestsource of conjugated p-coumaric acid at some 30-35 mg/100 g [18]. Broccoli florets and leafy cruciferous vegetables will be the major source of sugar esters and of conjugated sinapic acid (10 mg/100 g). Tomato and tomato products are likely to be the major source of glucosides at up to 13 mg/100 g in total, and possibly the second richest source of conjugate/7-coumaric acid (3 mg/100 g). Cereal bran and bran-enriched products are the most important source of wall-bound cinnamates with up to 30 and 7 mg ferulate/10 g in maize and wheat bran, respectively. This would make these products the richest dietary source of ferulic acid. However, coffee brew could supply up to 10 mg ferulate (as feruloylquinic acid, FQA) per 200 ml cup [13], and it is the first for conjugated ferulic acid , followed by Citrus juices. Table 5.

Dietary sources of individual cinnamates and each major class of conjugate [13,18]

Name Dietary source Cinnamates CafTeic acid Coffee beverage, blueberries, apples, ciders p-Coumaric acid Spinach, sugar beet fibre, cereals brans Ferulic acid Coffee, citrus juices, sugar beet fibre, cereal brans Broccoli, kale, other leafy brassicas, citrus juices Sinapic acid Conjugates Coffee beverages, blueberries, apples, ciders Caffeoylquinic acids Sweet cherries p-Coumaroylquinic acids Coffee Tartaric conjugates Spinach, lettuce, grapes, wines Malic conjugates 1 Culinary herbs, mixed herbs, possibly stuffing Rosmarinic acid Spinach, sugar beet fibre, cereal brans Cell wall conjugates

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3. 4-oxo-flavonoids: flavonols, flavones, isoflavones, flavanones, chalcones, dihydrochalcones The term 4-oxo-flavonoids includes a group of complex polyphenols that share a common structure of diphenylpropanes (C6-C3-C6) characterized by two aromatic rings and an oxygenated heterocycle, Fig. (3). The three rings are referred to as the A, B, and C (or pyrane) rings. J'

.<^\

11 B 1 ^?\,/^\e^^

1 A % / ^

c II II 0

Fig. (3). Basic structure of 4-oxo-flavonoids

Their biosynthesis derives from the condensation of three acetyl units and of a derivative of hydroxycinnamic acid leading to the formation of a conmion intermediate, tetrahydroxychalcone. This chalcone is precursor of several compounds, the most important being the 4-oxo-flavonoids [19]. The 4-oxo-flavonoids can be distinguished based upon the modifications of the nucleus, including the pyronic cycle saturation, the number and the position of hydroxyl groups and the degree of methylation and glycosylation. In plants,flavonoidsoccasionally occur as aglycones, the most commonly forms being 0-glycoside derivatives. Flavones may also occur as C-glycosides. The bond between the aglycone and sugar moieties is generally located at 7- (flavone, flavanone), 3(flavonols), or 4'-position. The sugars are mono-, di-, tri- and even tetrasaccharides, being D-glucose, L-rhamnose, glucorhamnose, galactose, and arabinose the most frequent [19,20]. Depending on their aglycone structure, at least five different groups of 4-oxo-flavonoids are distinguishable: flavonols, flavones, flavanones, isoflavones and chalcones.

264

Dietary occurrence The concentration of 4-oxo-flavonoids depends on the plant, environmental conditions, the part of the plant consumed, the degree of ripeness as well as on the food processing. Flavonoids are preferentially located in the epidermis: solubilized in the vacuolar sap (especially flavones and flavonols glycosides) or in the epicuticular zone [20]. More detailed information about the occurrence of the different compounds is given in each subchapter. 3.1

Flavonols

Flavonols (about 380 aglycones) are characterized by the presence of a hydroxyl group at position 3, Fig. (4). About 90% of the flavonols have additional hydroxyl at positions 5 and 7.

r^'^4/^

II B 1

l < ^ \

/^^/^'^^K

1 M1 ^ 1 % / ^ S " " ^OH 1 0II Fig. (4). Basic structure of Flavonols

Flavonols occur mainly as O-glycosides and the diversity of the glycoside moiety in this group is noteworthy with about 200 different quercetin and kaempferol glycosides described to date [19]. Table 6 reports the most common flavonols.

265

Table 6.

Structure of the most connnon flavonols Trivialname Aglycones Fisetin Galangin Kaempferol Morin Myricetin Quercetin Rhamnetin Glycosides Quercltrin Rutin

MW (Da)

Position of OH groups

286.24 270.24 286.24 302.24 318.24 302.24 316.27 448.38 610.53

Substituents

Position of tiie substituents

3,7,3',4' 3,5,7 3,5,7,4' 3,5,7,2\4' 3,5,7,3\4\5' 3,5,7,3',4' 3,5,3',4'

OCHj

7

5,7,3\4' 5,7,3',4'

0-Rh 0-Ru

3 3

Rh = rhamnose = 6-dcoxy-L-mannose (C6H12O5); Ru = nitinose == 6-0-D-glucose

Dietary occurrence Flavonols are present in plant foods mainly in the leaves and in the outer parts of plants with quercetin and kaempferol the most common ones. Quercetin and its glycoside are ubiquitous in fruits and vegetables. Conversely, kaempferol and myricetin are less distributed (Table 7) [2123]. Specific quercetin glycosides have been detected in onions (quercetin4'-glucoside and quercetin-3,4'-diglucoside), broccoli (quercetin-3-0sophoroside, kaempferol-3-(9-sophoroside), green beans (quercetin-3-0glucuronide) and tomatoes (rutin = quercetin-3-O-rhamnosyl-glucoside) [24,25], red wine (rutin) and tea (rutin, quercetin-3-(9-glucoside and quercetin-3-O-galactoside) [26]. Preparation of fruits and vegetables for consumption (for example peeling, skinning and cooking) can decrease quercetin and kaempferol content significantly. For example, boiling, microwave cooking and frying of onions or tomatoes involves a decrease in the content of flavonols by 30 to 80% [2].

266

Table 7.

Content of flavonois (expressed in mg/kg or mg/1) in foods determined by HPLC metliods after liydrolysis of tlieir glycosides [21-23]

Food Apple Apricot Bean, French French processed Green Broccoli Currrant, black

Red

Quercetin 20-36

25 39 17 16 30-37

Myricetin

-

-

<12 <3.8

60-72

37

1

8-13 <1.3 15-37 2-12

-

Endive Grape, black White Grape juice 4.4 4.4 Grape fruit juice (fresh) 110-120 Kale Leek 14-79 Lettuce 340-347 Onion 5.7 Orange juice 6-8.6 Strawberry 14-17 Tea, black 2-14 Tomato 63 Cherry tomato i 8.3 Wine, red

3.2

Kaempferol

46

210-470

30

5-12 14-16

-

4.5 4.5 6.2

3.0

7.9

Flavones

Flavones differ from flavonois since the hyciroxyl group at position 3 of the C-ring is missing, Fig. (5).

Fig. (5). Basic structure of flavones

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About 300 different aglycones have been identified, and the most frequently are luteolin, apigenin (especially in parsley) and diosmetin (in Citrusfruits).Among glycosides, the 7-0- and C-forais are very conunon, and are characterized by a carbon-carbon bond between the anomeric carbon of a sugar molecule and the Ce or Cg carbon of the flavone nucleus. Table 8 describes the most common flavones [19]. Table 8.

Structure of the most common flavones

Trivialname Aglycones Apigenin Chrysin Diosmetin Luteolin Pinocembrin Glycosides Isoorientin Isovitexin Orientin Vitexin

MW (Da)

Position of OH groups

270.24 254.24 300.27 286.24 256.24

5,7,4'

448.36 432.36 448.36 432.36

Substituents

Position of the substituents

OCH3

4'

Glue Glue Glue Glue

6 6 8 8

5,7 5,7,3* 5,7,3\4'

5,7 5,7,3',4' 5,7,4' 5,7,3\4' 5.7,4'

Glue = glueosc

Flavones contribute to plant tissue color provided that they occur in high concentrations or are complexed with metal ions. Some flavones participate in taste; for example, the highly methoxylated aglycones nobiletin, sinensetin and tangeretin are responsible for the bitter taste of citrus peel. On the other hand, some glycosylated flavones (for instance neodiosmin and rhoifolin) reduce the bittemess of some substances (limonin, naringin, caffeine, quinine) [2]. Dietary occurrence Flavones are found mainly in grains and herbs and not frequently in fruits. Apigenin and chrysoeriol have been detected in parsley, while cereal grains and herbs contain apigenin and its glycosides as well as luteolin [21,27].

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Table 9.

Content offlavones(expressed in mg/kg or mg/1) in foods determined by HPLC metliods after hydrolysis of tiieir glycosides [21,28] Food Celery leaf Stalk Sweet peper, red

3.3

Luteolin 200 5-20 5-11

Apigenin 750 16-61

-

Isoflavones

Isoflavones are a distinct class of flavonoids which stracturally differ from the commonflavonoidsin B-ring orientation, Fig.(6).

Fig. (6). Basic structure of isoflavones

The isoflavones have a chemical stracture similar to that of mammalian oestrogen 17P-estradiol [28]: the phenoUc ring and a pair of hydroxyl groups separated by a distance comparable to that occurring in mammalian oestrogens are key structural elements of most compounds that bind to oestrogen receptors [29,30]. Thus, isoflavones have recently become best known for their oestrogenic activity, hence the name "phytoestrogens". However, small differences in structures of the individual phytoestrogens can dramatically alter their activity. For instance, daidzein and genistein share identical structures except for an additional hydroxyl group on the A-ring of genistein, but this favours up to five- or six-fold oestrogenic activity of genistein in some assay systems [31].

269

Table 10. Structure of the most common isoflavones Trivialname Aglycones Daidzein Genistein Glycosides Acetyldaidzein Acetylgeiiistin

MW (Da)

Position of OH groups

Substituents

Position of the substituents

254.23 270.23

7,4' 5,7,4'

/ /

/ /

430.23 446.23

7,4' 5,7,4'

Acetyl-gluc Acetyl-gluc

6" 6"

Acetyl-gluc = Acetyl-glucoside

Dietary occurrence Isoflavones are present in plant foods either as aglycones (genistein or daidzein) or - predominantly - as different highly polar and water-soluble glycosides, including acetyl and malonyl glucosides and p-glucosides of daidzein and genistein [32]. Legumes are the main source of isoflavones. Soybeans are particularly rich in daidzein and genistein. Table 11 shows the wide range in total isoflavone concentrations in soya products and other legumes [32,33]. The concentration of isoflavones in soy foods is in the range of 0.1-3.0 mg/g. However, theseflavonoidshave been detected also in black beans, green split peas and clover [34]. Otherflavonoidsof this group, including biochanin A and formononetin, have been found in chick peas, green beans, and sunflower seeds. Table 11.

Content of isoflavones (expressed in ^g/g) in foods [3233]

Food Soya bean Tofu Soy flour Textured soya protein Soya milic Miso Soy clieese Tofu yoghurt Soy sauce Green split peas

3.4

Isoflavones (total) (fig/g)

|xg per average portion size (g)

579-3812 79-674 833-1778 701-1184 34-175 256-890 34-47 151 13-75 73

34740-228720 (60) 10270-87620(130) 16660-35560(20) 28040-47360 (40) 3400-17500(100) 4608-16020(18) 1360-1880(40) 18120(120) 65-375 (5) 2920 (40)

Flavanones, chalcones, dihydrochalcones

270

Flavanones arise from flavones after reduction of the double bond in the heterocycle (position C2/C3), Fig. (7).

Fig. (7). Basic structure of flavanones

Among aglycones, the best known are naringenin and hesperidin. Their glycosylated forms occur commonly as O- or C-glycosides, usually as rutinosides (6-0-a-L-rhamnosyl-D-glucosides) and neohesperidosides (20-a-L-rhamnosyl-D-glucosides) attached at position 7. Flavanones contribute to the flavour of citrus [19]. Table 12 reports the structures of some common flavanones. Table 12.

Structure of some common flavanones

Trivialname Aglycones Eriodictyol Hesperetin Naringenin

MW (Da)

Position of OH groups

288.26 302.28 272.26

Glycosides Hesperidin Naringin

610.57 580.54

Substituents

Position of the substituents

5,7,3*,4' 5,7,3' 5,7,4'

OCH3

4'

5,3' 5,4'

Rli-Gluc; OCH3 O-Rh-Gluc

7;4'

7

Rh = rhamnose = 6-dcoxy-L-mannosc (CeHnOs); Glue = glucose

Flavanones can be easily converted to isomeric chalcones in alkali (or vice versa in acidic media) provided that there is a hydroxyl substituent at position 2' (or 6') of the chalcone. Chalcones are unsaturated and, along with dihydrochalcones, contain an open pyronic cycle and a carbon skeleton numbered in a way different from other flavonoids, Fig. (8, 9). Native chalcone glycosides tend to transform into flavanone glycosides during extraction procedures. Chalcones per se are therefore of restricted occurence in foods [35].

271

OH

rf^^^y

, ^ ^ ^ 4 ^

OH

^==^, OH Fig. (8). Isosaiipiupurin (Chalcone)

OH Fig, (9). Phloretin (Dihydrochalconc)

Dietary occurrence Flavanones are found in a small number of foods. Chick peas (with the flavanone garbanzol), cimiin, pepperaiint (both with hesperidin), hawthom berry, licorice, rowanberry and citrus fruits are among those fews containing molecules of this group. Naringenin and narirutin glycosides are present in hawthomberry and rowanberry; liquoritigenin in licorice roots. Flavanones neohesperidose (such as naringin) are found in grapefruit and are usually bitter; the tasteless flavanone rutinosides (such as hesperidin) are present in oranges [35,36]. Flavanone glucosides are comparatively rare in species but are found for instance in different herbs [37]. Hesperidin and the aglycones naringenin, eriodictyol and hesperitin have been reported in the herbal tea (Honeybush tea) prepared from the legume Cyclopia intermedia [38]. Naringenin and eriodictyol have been reported in potato [5]. Citrusfiruitsand associated products (fiuit juices, peeled freshfruit)are a major dietary source of flavanones (Table 13) [35]. However, the distribution is quite scattered, and much higher concentrations are found in the solid tissues compared to the juice. For example, an individual drinking orange juice (250 ml) will have a daily flavone intake (as aglycones) in the range of 25-60 mg; eating the flesh of a whole orange (200 g) will provide about 125-375 mg. Chalcones are comparatively rare in foods. Naringenin chalcone is present in tomato skin and may be present in juice, paste and ketchup. Acid hydrolysis, commonly applied prior to HPLC, converts the chalcone to the corresponding flavanone (naringenin), which is naturally present only in trace amounts (2-15 mg/kg) in the tomato [23]. Dihydrochalcones (DHCs) are characteristic of apples and derived products (apple juice, cider, pomace etc.), and their content depends on

272

the cultivar. Phloretin 2'-glucoside (phloridzin), phloretin 2'(2"-xylosylglucoside) and 3-hydroxyphloridzin have been identified unequivocally and some investigators have reported phloretin 2' (2"-xylosylgalactoside). They are present in the skin, pulp and especially in tiie seeds, where they account for up to 60% of the total phenols (compared with less than 3% in the epidermis and parenchyma zones). When eating an apple, the seeds and core of the fhiit are usually discarded and thus some of the apple dihydrochalcones are not mgested. Whole apple fruits are processed industrially to produce juices and ciders, and therefore the contribution of these processed products to the intake of DHCs can be higher than that of fresh apples (250 ml of apple juice or cider supply about 1-5 mg phloretin. By contrast, a dessert apple of about 100 g supplies less than 1 mg [39,40]. The dihydrochalcones aspalathin and nothofagin have been identified as the main flavonoids in the South-Afiican Rooibos tea {Aspalathus linearis) [41]. Table 13.

Flavanones characteristic of common citrus fruits

Flavanones Eriocitrin Narirutin Hesperidin Naringin Neohesperidin

Sweet orange (Citrus sinensis) + +++ -

Sour orange (Citrus aurantium) -H^

++

Lemon (Citrus limon) ++ +++ -

Grapefruit (Citrus paradisi) •H-

Trace 4-H-

Trace

Mandarin Lime (Citrus (Citrus aurantifolia) reticulata) ++ +++ +++ -

4. Flavanols (FIavan-3-ols) Flavanols have a C-ring structure similar to that of 4-oxo flavonoids, but they are characterized by the lack of the double bond at the 2-3 position and of the 4-oxo-group, Fig. (10) [19].

Fig. (10). Basic structure of flavanols

273

The flavan-3-ols most occurring in nature are (-f-)-catechin and (-)epicatechin (EC), although gallocatechin and epigallocatechin have also been identified [42], Proanthocyanidins (or condensed tannins) include oligo- and polymeric forms of the monomeric flavanols and will be examined later. Polymerization of monomeric flavanols can occur as a result of auto-oxidation, but more often it is catalyzed by polyphenoloxidase (PPO), an enzyme that is present in most plant tissues [43].

4.1

Catechins - Dietary occurence

Catechins are widely distributed in plants; however, they are rich only in tea leaves, where catechins may constitute up to 25% of dry leaf weight. Catechins of green tea include the flavanols epicatechin, epigallocatechin, and their gallate esters (Table 14). Table 14.

Structure of the most common catechins

MW

Position of OH groups

(+).Catechin (C)

290.3

3(^-0H)

(-)-Epicatechin (EC) (cis form)

290.3

3 (

Epigallocatechin (EGC)

306.4

Epicatechin-gallate (ECg)

442.4

Trivialname

Substituents

Position of the substituents

^»M^»NOH\

3 ( VWSAAVOH)^ 5 »

3 OH

X Epigallocatechin-gallate (EGCg)

458.4

5'

3 OH

The most abundant monomeric flavanols of black tea are (-)epicatechin gallate (ECg) and (-)-epigallocatechin (EGC) and (-)epigallocatechin gallate (EGCg) [44]. Indeed, quantitative analyses of black tea reports levels of 31-79 mg/1 for EC, 5-91 mg/1 for EGC, 18-229 mg/1 for EGCg and 8-110 mg/1 for ECg; for green tea levels from 10-94

274

mg/1 for EC, 20-287 mg/1 for EGC and 60-408 mg/1 for EGCg are found [45]. During fermentation in the preparation of black tea, oxidative polymerization of flavanols occurs with the formation of theaflavin, theaflavingallates, thearubigins, and epitheaflavic acid [44]. Flavanols have been determined in apples, apricots, pears, cherries, peaches and plums [47,48]. The contents of (+)-catechin and (-)-epicatechin in red wine are relatively high (up to 208 mg/1 for catechin and 90 mg/1 for EC) [49]. Low levels of (+)-catechin (-5 mg/1) and (-)-epicatechin (-1 mg/1) have been reported for lager beers [50]. Data on grapes are limited; qualitative studies show the presence of (+)-catechin, (-)-epicatechin and ECg in black and white grape seeds and skins [51]. Recently, chocolate and cocoa have gained interest because of their contents of catechins and related polymers (procyanidin oligomers) [52]. Fruit juices processing may seriously affect flavanol content. For example, the preparation of commercial apple juice decreased the flavanol content in a stepwise manner. In particular, crushing and pressing, storage of the concentrated juice at room temperature and decolorization by treatment with activated carbon destroy theflavanolsalmost entirely [46]. 5. Anthocyanidins, anthocyanins, proanthocyanidins, tannins 5.1

Anthocyanidins and anthocyanins

The fundamental nucleus in anthocyanidins (aglycones) is flavylium chloride. Most of the anthocyanidins are derivatives of 3,5,7trihydroxyflavylium chloride. Thus, the hydroxylation pattems in the natural anthocyanidins fall into the three basic groups of pelargonidin, cyanidin and delphinidin. Anthocyanidins are rarely found in fresh plant material because of their instability [19]. On the other hand, anthocyanins, i.e. the glycosylated anthocyanidins, are an important group of water-soluble pigments occuring in 27 families of food plants (mainly red fruits and vegetables). Fig. (11) [53]. Table 15 shows the most common anthocyanidins and anthocyanins.

275

Fig. (11) Basic structure of anthocyanidins and anthocyanins

Table 15.

Structure of the most common anthocyanidins and anthocyanins MW

Trivialname Anthocyanidins Pelargonidin Cyanidin Delphinidin Malvidin Anthocyanins Pelargonidin-3-glucoside Cyanidin-3-glucoside DeIphinidin-3-glucoside Malvidin-3-glucoside

JM

Position of OH

Substituents

Position of the substituents

271.70 287.70 303.70 331.75

3,5,7,4' 3,5,7,3*,4' 3,5,7,3\4',5' 3,5,7,4*

OCH3

3*,5'

432.70 448.70 499.70 492.70

5,7,4' 5,7,3\4' 5,7,3',4',5' 5,7,4'

OGluc OGluc OGluc OGluc; OCH3,

3 3 3 3;3',5'

The natural anthocyanins vary in: • the basic anthocyanidin skeleton, i.e. the number and position of hydroxyl and methoxyl substituents; • the identity, number and positions(s) at which sugars are attached to the skeleton; the most common sugars are glucose, galactose, rhamnose and arabinose ( as 3-glycosides or 3,5diglycosides). • the extent of sugar acylation and the identity of the acylating agent(s); the most common acylating agents include ciimamic acids (caffeic, p-coumaric, ferulic and sinapic), which may themselves be glycosylated, and a range of aliphatic (for example acetic, malic, malonic, oxalic and succinic acid) and aromatic acids. The anthocyanins occur in the vacuole as an equilibrium of four molecular species: the coloured basic flavylium cation and three

276

secondary structures (the quinoidal base, the carinol pseuodobase and the chalcone pseudobase) [54]. The pH changes the colour intensity of the anthocyanins (for example, by the addition of vinegar or other acids while cooking or processing). Commonly, anthocyanins are red in acid, violet in neutral, and blue in alkaline solution. In fact, when cooking a food that is red, such as red cabbage, it may be helpful to add an acidic substance such as vinegar (or tomato juice or lemon juice) to prevent the food from tuming purple. Anthocyanins contribute significantly to the red purple, and blue color of flowers, many fruits of higher plants, vegetables and associated products, beverages and preserves. Anthocyanins and polymeric pigments derived from anthocyanins by condensation with other flavonoids, are responsible for the color of red wine. It has been recognized that anthocyanin-rich extracts might be used as food additives. Many factors influence the stability of anthocyanins. Heat and light can destroy sensitive anthocyanins during processing of fruits and vegetables. In particular, anthocyanins are rapidly destroyed in the presence of a high sugar concentration; thus processed foods containing large amounts of sugar or syrup would not have the same amount of anthocyanins as their improcessed counterparts [55], Dietary occurrence Anthocyanins are widespread in food plants, with an estimated worldwide consumption of 10000 tonnes from black grapes alone [53]. The anthocyanin content of many fruits and vegetables has been estimated by various methods (Table 16) [56-58]. The main sources of these plant pigments are fresh fruits such as cherries, plums, strawberries, raspberries, blackberries, grapes, red currants and black currants.

277

Table 16.

Content of anthocyanins in foods (expressed in mg/l or mg/lcg) [56-58]

Food Blackberry Blueberry Cherry Cliolceberry Cranberry Currant (blacic) Grape (red) Orange, Blood O'uice) Raspberry, black Raspberry, red Strawberry Cabbage, red Onion Wines, red Wines, Porto + Marsala

5.2

Anthocyanins 1150 825-4200 20-4500 5060-10000 600-2000 1300-4000 300-7500 2000 1700-4200 100-600 150-350

250 up to 250 240-350 140-1100

Proanthocyanidins

Proanthocyanidins (PAs, syn condensed tannins) are polymeric flavan-3ols whose elementary units are linked by C-C and occasionally C-O-C bonds (polymerization degree between 3 and 11), Fig. (12) [19]. Oxidative condensation occurs usually between carbon C4 of the heterocycle and carbons Ce or Cg [59]. A characteristic of PAs is that they yield anthocyanins upon heating in acidic media, hence their name, they yield anthocyanidins (hence their name). Two main types of PAs can be distinguished according to the substitution pattern of their B-ring: • Procyanidins: main constituents are catechin and epicatechin, and are characterized by the presence of two hydroxyl groups (3\ 4') in the B-ring. • Prodelphinidins: main constituten is epigallocatechin, which has three hydroxyl groups (3', A\ 5') in the B-ring.

278

r

^<^°" YY^^K'^^^^OH ./'x OH

HO^

^«^

.0^

^1*

^^5-^

" \ ^ - \ / ° ^ ^ v ^ V , OH

OH

.1

»°\,^'-v^?\^'v^' %

4^

^OH

Dimers: Procyanidin B3: R3' = H

Trimer.

Prodelphinidin B3: RS' = OH

Fig. (12). Basic structure of proanthocyanidins

Dietary occurence Common sources of PAs are fruits, such as apple, strawberry, pear and grape, beverages such as red wme and tea, and chocolate (Table 17) [59]. PAs complex protems, and are responsible for the astringency of foods and beverages (e.g. grape skin and seeds, cider, wine) [19].

279

Table 17.

Content of proanthocyanidins in foods (expressed in mg/1 or mg/lOOg) [59]

Food Apple Apple juice Barley Beer Blackberry Cacao bean Cherry Grape Grape juice Lentil Pear Pear juice Raspberry, red Strawberry Wines, red

5.3

Proanthocyanidins 17-50 nd-298 64-126 3.5-19,5 9-11 260-1200 10-23 1-160 3.546 316-1040 0.7-12 11-74 2-48 2-50 nd-500

Tannins

Tannins are compounds of intermediate to high molecular weight that distinguish them from the groups of low molecular weight plant phenolics. Tannins with a molecular weight up to 30000 DA have been found in certain Leguminosae. One of their main characteristics of tannins is the formation of insoluble complexes with proteins leading to the astringency of taimin-rich foods (for instance tea) because of the precipitation of salivary proteins [59]. Plant tannins are subdivided into two major groups (Table 18) [60]: 1. Hydrolyzable tannins: they consist of a central glucose molecule linked to molecules of gallic acid (gallotannins) or hexahydroxydiphenic acid (ellagitannins), Fig (13). They are readily hydrolyzed, hence their name. The most common hydrolyzable tannin is tannic acid, Fig. (14), which is a gallotannin formed by a pentagalloyl glucose molecule esterified by five gallic acid units.

280

OH HO.

OH

O

COOH

OH

Fig. (13). Structures of gallic (a) and ellagic (b) acid

OH

HO.

X.

/

.OH

CO

OH2C

OH

oc—o

HO"

^

I oc

"OH

OH Fig. (14). Structure of tannic acid

Condensed tannins (= proanthocyanidins): unlike hydrolysable tannins, condensed tannins are polymeric flavans that are not readily hydrolysable. They often consist of molecules of catechin and epicatechin joined by carbon-carbon bonds. Hence catechin and epicatechin are referred to as monomers; oligomers containing 2-4 (epi)catechin units are referred to as oligomeric procyanidins (OPC).

281

Table 18.

Classification of tannins Type of tannin Hydrolyzable tannins: • Gallotannins • EUagitannins and metabolites • Ellagitannin oligomers 2. Condensed tannins: • Proanthocyanidlns: Procyanidlns Prodelphinidins • Galloylated proanthocyanidlns

Example

1.

Pentagalloylglucose Geranin, Corilagin Agrimoniin Epicatechin oligomers Epigallocatechin oligomers

ABSORPTION AND METABOLISM OF SELECTED PLANT POLYPHENOLS IN HUMANS Gut absorption The absorption and the metabolism of dietary polyphenols arte determined primarily by their chemical structure, with glycosylation playing an important role. In fact, glycosylation influences the bioavailability of the polyphenols. It is generally stated that flavonoid glycosides are hydrolyzed before being absorbed [61]. Therefore, the first step of metabolism should involve the removal of the sugar moiety by enzymes-(glycosidases). Glycosidases activity can occur in the food itself (endogenous or added during process) or in the cells of the gastrointestinal mucosa or can be secreted by the colon microflora. Nonenzymatic deglycosylation in the hiraian body, such as in the acid conditions of the stomach, does not occur [62]. The absorption of polyphenols should therefore be controlled by enzyme specificity and distribution. Polyphenols with attached glucose are potential substrates for endogenous human enzymes, while attached rhamnose is not a substrate for human p-glucosidases and so is only cleaved by colon microflora arhamnosidases [63]. Deconjugation and reconjugation reactions in metabolism After the hydrolysis of a polyphenol glycoside to the free aglycone, polyphenols are conjugated by methylation, sulfation, glucuronidation or a combination. However, there are exceptions to this sequence, as

282

supported by different studies [64-68] reporting the absorption of intact polyphenols glycosides. This is a critical point, since the formation of conjugates dramatically alters the biological properties of the circulating metabolites. Furthemiore, it should be reminded that significant differences between the administration of drugs (usually in himdreds of milligrams in one concentrated dose) and the consumption of dietary polyphenols (usually <100 mg in a diluted dose) exist. These differences imply that drugs can readily saturate the metabolic pathways that rely on the supply of cofactors such as UDP-glucuronic acid. Hence, unconjugated drugs are often found in the blood. On the other hand, polyphenols found in food are not expected to saturate the metabolic pathways, therefore being in circulation in the conjugated forms [63]. Metabolism by the gut flora Polyphenols that are not absorbed in the stomach or small bowel will be carried to the colon. Polyphenols that are absorbed, metabolized in the liver and secreted with bile back to the small intestine will also reach the colon. Here, microorganisms degrade both unabsorbed and absorbed flavonoids. Indeed, colonic bacteria produce glycosidases, glucuronidases, sulfatases that can strip flavonoid conjugates of their sugar moieties, glucuronic acids and sulfates [61]. Human intestinal bacteria are able to hydrolyse 6>-glycosides [69] as well as C-glycosides [70]. In addition, the degradation involves the splitting of the heterocyclic oxygen-containing C-ring. Degradation products can be absorbed [71], and subsequently metabolized by enzymes present mainly in the liver, where 3'-0-methylation by catechol-0-methyltransferase, dehydroxylation, p-oxidation, and conjugation with glucuronic acid, sulfate, and glycine occurs [72]. These metabolites are considered to contribute to the biological effects of dietary flavonoids (antioxidants). In general, the metabolism of dietary flavonoids may be sununarized as shown in Fig. (15).

283

INTESTINE

Flavoaoid (agUcones and glycosilated fonns) and products of the microbic metabolism

FECES

Fig. (15). Metabolism of dietary flavonoids. GlcA = glucuronic acid; UGT = uridine 5'diphospoglucuronosyl transferase; Met = methyl; Sulf = sulfate; COMT = catechol-O-methyl transferase; PST = phenol sulfo transferase

1. Flavonols Absorption of flavonols from the diet was long considered to be negligible, because flavonols are present in plants bound to sugars as >8glycosides. Only aglycones were supposed to be absorbable, whereas glycosides were thought to be non- or only marginally absorbable [61]. One of the main flavonols studied is quercetin. Indirect evidence for the presence of quercetin conjugates in humans was obtained by Manach et al [73], who found the presence of quercetin in plasma after the consumption of a complex meal rich in plant products only after pglucuronidase and sulfatase treatment. Furthermore, the authors reported

284

the presence of a methylated derivative of quercetin, isorhamnetin, in three out of the ten subjects. These results are in good accordance with data obtained by Conquer et al [74], where the concentration of quercetin in fasting plasma of a quercetin-supplemented group was 23-fold higher than that of the placebo-group. Urinary excretion of quercetin increased significantly with dose and time after the consimiption of fruit juice (blackcurrant and apple juice in a 1:1 mixture) in humans [75]. Ranges from 0.29-0.47% of ingested quercetin were found in the urine. The presence of quercetin in urine shows that it was absorbed by the gut, but the urinary content does not necessarily reflect absolute absorptive efficiency because absorbed quercetin may be metabolized (conjugated), stored and excreted through other routes such as the biliary tract. However, since quercetin is present in a variety of fruit and vegetables, plasma concentrations or urinary excretion of quercetin may potentially be useful as biomarkers of habitual intake of these foods. The absorption of intact quercetin glycosides has been demonstrated by some authors [64,65,76]. Holhnann demonstrated in ileostomy subjects (who lack colon with the bacterial flora, thus circumventing the problem of microbial degradation), that the quercetin glycosides from regular foods (onions, tea) were far better absorbed than pure aglycone (52%vs24%). LC-ESI-MS analyses allowed the detection of intact flavonol glucosides (rutin) in plasma of healthy volunteers after the consumption of tomato extract [65]. Glycosides of flavonols from onions, such as quercetin-4'-0-glucoside and quercetin-3'-0-methyl-4'-0-glucoside, have been found in the plasma of volunteers with a peak of absorption of 0.5 - 4 h [64]. On the other hand, a diet-controlled cross-over-study [77] with supplementation of quercetin and its glycoside rutin showed that rutin occurred only as conjugated form (with glucuronic acid and/or sulfate groups) in plasma suggesting that this glycoside was not absorbed in its original form. Conversely, quercetin was detected in conjugated as well as unconjugated (aglycone) forms. Interindividual differences in the pharmacokinetics of both compounds were considerable. Sesnik et al [78] found also no intact quercetin glucosides and only traces of aglycone in human plasma after the administration of quercetin-3-glucoside or quercetin-4'-glucoside as an oral solution, while quercetin glucuronides were the major metabolites in plasma.

285

Interestingly, different studies demonstrated that the sugar moiety of quercetin glycosides is an important detemiinant of their absorption and bioavailibility [79-82]. Quercetin-3-rutinoside and quercetin-4'-glucoside are important forms of quercetin in foods. The first one accounts for about 40% of quercetin in black tea [83] and the second one for about 45% of quercetin in onions. [84]. Although the intake of quercetin-3-rutinoside is twice that of quercetin-4'-glucoside, the absorption of quercetin-3rutinoside is only 17% of ingested dose, whereas the absorption of quercetin-4'-glucoside is 52% of ingested dose [76]. Furthermore the bioavailibilty of quercetin-3-rutinoside is only 20% of that of quercetin4'-glucoside [81]. Olthof et al [82] have tested the bioavailibilty of quercetin-3-glucoside in comparison to that of quercetin-4'-glucoside and concluded that both quercetin glucosides are rapidly absorbed in humans, irrespective of the position of the glucose moiety. In addition, this study provided information on the metabohsm of quercetin into isorhamnetin (3'methoxyquercetin). Of the ingested quercetin glucosides, --50% is absorbed in the small intestine and subsequently converted into isorhanmetin, in the liver and in other organs. The 50% of ingested quercetin that is not absorbed in the small intestine is metabolized by the colonic microflora into quercetin aglycone and phenolic acids, which might be absorbed from the colon [73]. The bioavailibility of quercetin-glycosides from onions, containing mainly quercetin-p-glucosides, was superior to that of various quercetin glycosides from apples (containing a mixture of quercetin-p-galactosides and p-xylosides) and of pure quercetin-3-rutinoside (major species in tea). The possible matrix effect of the foods remains unclear. It seems that overall percentage of absorption, determined by measuring plasma levels of flavonols after enzymatic hydrolysis, does not exceed 2-3% of the ingested dose. It is also likely that, as with other micronutrients, the existence of a steady-state concentration of these compounds could result in diminished absorption. Thus, it is conceivable that the major parts of these flavonoids are either degraded to phenolic acids in the large intestine or excreted in the faeces [72]. 2. Flavones Data on the absorption of flavones (namely luteolin) are limited. Shimoi

286

et al [85] investigated the intestinal absorption of iuteolin and iuteolin-70-P-glucoside in rats and humans. The absorption analysis using the rateverted small intestine demonstrated that Iuteolin was converted to glucuronides during passing through the intestinal mucosa and that luteolin-7-O-P-glucoside was absorbed after hydrolysis to Iuteolin. In plasma, either free Iuteolin as well as its monoglucuronide and methylated conjugates were present, while luteolin-7-O-p-glucoside was not detected. This indicates that glucosides may be first hydrolyzed to Iuteolin by the microbacteria. The same authors reported that in humans free Iuteolin and its monoglucuronide have been detected in plasma after oral administration of Iuteolin. 3. Isoflavones Intestinal microflora plays a key role in the metabolism and bioavailibility of isoflavones [86]. After ingestion, soybean isoflavones are hydrolyzed by intestinal glucosidases, which release the aglycones, daidzein and genistein, Fig. (16).

intestinal glucosidases

malonylglucosides acetylglucosides p-glucosides

demethylation dehydroxylation reduction ring cleavage equol dihydrodaidzein 0-desmethylangolensin p-ethylphenol

daidzein genistein

absorption

hepatic conjugation-enterohepatic cycling urinary excretion

Fig. (16). Metabolic fate of soybean isoflavones in humans (Setchell 1999)

287

These aglycones may be absorbed or further metabolized to different metaboUtes, including equol, 0-
288

4. Flavanones, chalcones, dihydrochalcones The flavanones have received less attention in comparison to flavonols and isoflavones, although their intake from the diet can be high and they exhibit promising biological activity. Little infomiation is available about the absorption or the kinetic behavior of the flavanones naringenm, hesperetin and their glycosylated fomis naringin, hesperidin, and narirutin. Studies conceming urinary excretion of these compounds have confimied their bioavailibility from fruits and that they are excreted, at least to some extent, into the urine. The renal excretion of naringin, naringenin and its glucuronides after the consumption of grapefruit juice (20 ml/kg body weight) was investigated by Fuhr et al [96]. Only naringenin and its glucuronides appeared in urine after an average lagtime of 2 h. Neither naringin nor its glucuronides were found. The data suggest that cleavage of the sugar moiety, presumably by intestinal bacteria, is the first step of naringin metabolism. These data were confirmed by Lee et al [97] who detected naringenin glucuronide in urine samples after the administration of grapefruit juice (containing about 214 mg naringin). However, a recent study reported that the glycoside naringin was recovered (0.02% of the administered dose) in urine as unchanged molecule, hence confirming those glycosides are absorbable [66]. The influence of glycosylation on the metabolism of naringenin-7glucoside and its aglycone in the conscious rat model was examined by Choudhury et al [98]. It resulted that via oral route the glycoside group is cleaved by an intestinal enzyme and then the aglycone is glucuronated within the epithelium. By contrast, after intravenous dosing the majority was detected as native glucoside in the urine. Bioavailibility and kinetics of naringenin and hesperetin from orange and grapefiiiit juices was also investigated by Erlimd et al [99]. Both flavonoids were absorbed from the juices with great interindividual variations. The authors hypothesized that these variations were caused by differences in gastrointestinal microflora. Peak plasma concentrations of naringenin and hesperetin were reached between 4.8 and 5.5 h, indicating that an absorption takes place in the distal parts of the small intestine or the colon (where enzymes capable of cleaving theflavonoidglycosides in question are present).

289

5. Flavanols - Catechins Early studies (in the 1970s) on the pharmacokinetics of (+)-catechin revealed that tiiis flavanol is absorbed from the gastromtestinal tract following administration to healthy volunteers (4.2 g in the form of gelatin capsules) [100]. (•f)-Catechin was excreted in the urine together with several imidentified metabolites, and the amount excreted within 24 h was about 7.5% of the administered dose. Other authors suggested that catechins are converted to glucuronyl derivatives in the intestinal mucosa and are further metabolized by methylation, sulfation and conjugation with glucuronic acid, sulfate and glycine [101]. Indeed, Lee et al [102] determined flavanol conjugates in human plasma after the ingestion of green tea. EGCg was mainly present as a sulfate conjugate (65%), followed by the free form (20%) and the glucuronide (15%). EGC on the other hand was mainly present as glucuronide (60%), followed by sulfate (30%). About 10% was detected as imconjugated EGC aglycone. More recently. Da Silva et al [103] detected the presence of glucuronides, O-methylglucuronide sulfate, and glucuronide sulfate of epicatechin (EC) in plasma of rats fed epicatechin. Absorption of (-)-epicatechin from chocolate has been studied by different authors [104-106]. Baba et al [104] found maximum levels of total EC metabolites in plasma after 2 h of chocolate or cocoa intake. Sulfate, glucuronide and sulfoglucuronide conjugates of non-methylated EC were the main metabolites present rather than methylated forms. In urine samples, excretion of total EC metabolites within 24 h was about 30% of total EC intake after chocolate and 25 % after cocoa consumption. A 12-fold increase in plasma epicatechin concentration from 22 to 257 nmol/L was reported by Rein et al [105] after consmnption of 80 g semisweet (procyanidin rich) chocolate within 2 h after ingestion. The total antioxidant capacity of plasma increases of 31% within the same time, and plasma 2-thiobarbituric acid reactive substances decreased up to 40%. These data support that consimiption of chocolate increases plasma epicatechin concentrations and decreases plasma baseline oxidation products. These results have been confirmed in another study by Wang et al [106]. The bioavailability and metabolism of catechins was studied in humans after consumption of black tea containing 15.48 mg of EGC, 36.54 mg of

290

EC, 16.74 mg of EGCg and 31.1 mg of ECg [107]. Plasma concentrations of EGC, EC and EGCg increased significantly reaching peak plateau between 5 and 8 h (peak levels of 145,174 and 20.1 nmol/L respectively). ECg on the other hand increased linearly over the 24h-period, peaking at 50.6 nmol/L. Urinary excretion of EGC and EC paralleled the rise in plasma levels. EGC, EC and ECg peaked at 5 h whereas EGCg at Ih. Fecal catechin excretion varied widely from subject to subject, but it was significantly different from baseline for all catechins. Furthermore the authors calculated the percentage of ingested catechins. Only 1.68% of the total catechins consumed (400 mg) was found in the plasma, urine and feces, providing evidence that catechins undergo considerable metabolism and/or degradation either in the gastrointestinal tract or in the body after absorption. After ingestion of green tea infiision (400 mg of total catechins), epigallocatechin gallate (EGCg) and epicatechin gallate (ECg) were detected in human plasma with an significant increase of these two free catechins after enzymatic hydrolysis with glucuronidase/sulfatase, indicating their presence in plasma mainly in the conjugated form [108]. At the same time, detectable amounts of final 60 mg catechin metabolites were found in plasma and urine, including 4-hydroxybenzoic acid, 3,4dihydroxybenzoic acid, 3-methoxy-4-hydroxy-hippuric acid and 3methoxy-4-hydroxybenzoic acid. LC/ESI-MS analyses were applied to determine urinary glucuronidated and sulfated tea catechins after the administration of green tea to humans, mouse and rats [109]. The major conjugates were identified as monoglucuronides and monosulfates of EGC and EC. Besides these metabolites, also 0-methyl-EGC-O-glucuronides, 0-sulfates and Omethyl-EC-0-sulfates in human urine were detected. Furthermore, the ring-fission metabolites of EGC and (-)-epicatechin, 5-(3',4',5'trihydroxyphenyl)-x-valerolactone and 5-(3 ',4'-dihydroxyphenyl)-yvalerolactone respectively, have been detected in the monoglucuronide and monosulfate forms. It is not known whether tea catechin conjugates possess any biological activities. In a study by Manach et al [110], the glucuronic/sulfate conjugates were shown to have the same electrochemical behaviour as the parent drug. Considering that the oxidation potential of chemicals may represent their antioxidant capacity, the electrochemical behaviour of the conjugates suggests that they are effective antioxidants. Nevertheless, because the glucuronic acid/sulfate conjugates are generally more

291

hydrophilic than the parent compound, the tissue distributions of these metabolites are likely to be more limited than those of the parent catechins [111]. 6. Hydroxycinnamates, hydroxybenzoic acids Non-ruminants possess several intestinal Na'^^-dependent saturable transport systems. These include the well-known sodium-glucose cotransporter (SGLTl), responsible for the active uptake of glucose, and it appears to be specific for cinnamic and ferulic acid and possibly for other hydroxy-cinammic acids [112]. Healthy volunteers have shown to excrete caffeic, p-coumaric and ferulic acid in the urine after the consumption of various fruits [113]. The excretion of free ferulic acid in urine peaked after 7 h at a concentration of-'T jiM after the consumption of tomatoes (36-73 g containing 21-44 mg ferulic acid). The concentration of free ferulic acid plus glucuronide (and possibly sulfate) conjugates exceeded 20 |aM and accounted for some 11-25% of the dose [114]. Simonetti et al [115] studied the plasma levels of caffeic acid after consumption of 100,200, 300 ml of red wine (caffeic acid content of 9.01 mg/L) that provided about 0.9, 1.8, and 2.7 mg of caffeic acid, respectively. The highest plasma levels of caffeic acid was reached 60 min after ingestion and decreased to basal values within 180 min (for 100 and 200 ml) and within 240 min (for 300 ml). Both, the absence of caffeic acid in plasma before the trial and its significant, dose-dependent increment after red wine ingestion suggest that it may be a possible marker of consimiption of beverages containing this acid. Furthermore, 200 and 300 ml red wine intake produced a significant increase in plasma total antioxidant capacity (TRAP). Conceming HBAs, their metabolism involves conjugation with sulfate, glucuronate and glycine. Methylation may also occur, as may demethylation, dehydroxylation and decarboxylation (this only if there is a 4-hydroxyl) [3].

292

7. Anthocyanins, proanthocyanidins a.

Anthocyanins

Dietary anthocyanins have gained much attention based on the recognition of the "French paradox'* which led to the suggestion that some components of red wine (in particular anthocyanins) may protect against coronary heart disease. Limited evidence on the absorption of intact anthocyanins exist until today. There are reports, based upon spectral properties from DAD-HPLC of anthocyanin-like substances in plasma [116] and urine after acidification [117]. However, the ^max observed for the components in urine was at 430 nm. Anthocyanins should not have an absorption peak around this wavelength. Thus, the detected compounds appeared to be anthocyanin metabolites [68]. These spectroscopic data do not provide conclusive evidence for the absorption of intact anthocyanins. Conversely, anthocyanins from elderberry were detected in human plasma using a more selective approach based on HPLC coupled to UV detection (512 nm) [67]. These findings have been confirmed by a recent study on the detection of anthocyanins in their unchanged glycosylated form in urine and plasma after ingestion of 720 mg anthocyanins [68]. b.

Proanthocyanidins (PA)

The complexity and the lack of commercial pure standards of PA make their analysis difficult. Their absorption depends on their degree of polymerization. In some in vitro experiments, only PA dimers and trimers, but not polymers with an average polymerization degree of 7, were absorbed through an intestinal epithelium cell monolayer [118]. Experiments in chicken and sheep showed that polymeric PAs were not absorbed through gut barrier [119,120]. Evidence occurred that polymeric proanthocyanidins could be degraded by the colonic microflora into low-molecular-weight compounds, which would be subsequently absorbed. The group of Deprez [118] investigated their metabolism by human colonic microflora incubated in vitro in anoxic conditions using non-labeled and ^"^C-labeled

293

purified proanthocyanidin polymers. Degradation occurred almost totally after 48 h of incubation; metabolites identified were low-molecularweight phenolic acids: phenylacetic, phenylpropionic and phenylvaleric acids, monohydroxylated mainly in meta or para-position. It is supposed that, once fermentation products have crossed the intestinal barrier, they reach the liver through the portal vein, where they are further metabolized by dehydroxylation, methylation or conjugation to sulfate esters or glucuronides as it has been shown for other flavonoids [59]. POLYPHENOLS: BIOCHEMICAL AND PHARMACOLOGICAL PROPERTIES Polyphenols are endowed with different biological activities, including: 1. 2. 3. 4. 5. 6. 7. 8. 9.

Antioxidant/anti-radical activity and chelation of metal ions Modulation of some enzymes activity Anticarcinogenic activity Antiatherosclerotic activity Anti-inflammatory activity Inhibition of histamine release and spasmolytic activity Hepatoprotective activity Antiviral and antimicrobial activity Oestrogenic activity

1. Antioxidant and anti-radical activity, chelation of metal ion Polyphenols can act as antioxidants by a number of potential pathways. The most important is likely to be by free radical scavenging, in which the polyphenol can break the radical chain reaction. Polyphenols are effective antioxidants in a wide range of chemical oxidation systems, being capable of scavenging peroxyl radicals, alkyl peroxyl radicals, superoxide, hydroxyl radicals, nitric oxide and peroxynitrate in aqueous and organic environments [121]. This activity is due to the ability of donating an H atom from an aromatic hydroxyl group to a free radical, and the major ability of an aromatic structure to support an unpaired electron by delocalization around the 7i-electron system. Phenolic acids

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and flavonoids may be good antioxidants, particularly those possessing the catechol-type structure [122-125]. Phenolic acids and flavonoids (PP-H) can also act as free radicalchain (ROO*) reaction terminator, as follow: ROO* + PP-H -> ROO-H -f PP* The PP* radical is relatively stable and could react in another reaction as terminator, as follow: ROOVPP*->ROO-PP The interaction between flavonoids and phenolic acids with other physiologic antioxidants, such as ascorbate or tocopherol, is another possible antioxidant pathway for these compounds [72,126,127]. Nevertheless, like must other antioxidants, flavonoids may also act as prooxidant in particular circumstances [128,129]. Phenolic acids and flavonoids can also act as chelating agents, complexing transition metals that are responsible of the initiation of peroxidative processes (Fenton and Haber-Weiss reactions). This property is much stronger in phenolics having a catechol, pyrogallol, or 3-hydroxy4-carbonyl group [130]. 2. Modulation of some enzymes activity Interaction of low molecular weight molecules, such as phenolic acids andflavonoids,with macromolecules can modify their chemical-physical properties. Different studies confirm the ability of phenolics in modulating some enzymes, such as hydrolases, transferases, kinases, oxidases, hydroxylases, glutathione S-transferase, nitric-oxide synthase, cytochrome P450 systems, ATPases, lipases, phospholipases, adenylate cyclase, RNA and DNA polymerase, human DNA ligase I, ribonuclease, reverse transcriptase, topoisomerase, aromatase, hyaluronidase, elastase, HIV-1 proteinase and integrase, aldose reductase [131,132]. Specifically, enzymes such as xantine-oxidase, nitric oxide synthase, phospholipase, cyclooxygenase and lipoxygenase, involved in the production of free radicals in biologic systems, are also inhibited, in vitro, from different polyphenols [132,133].

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Overall, these activities may explain the correlation between polyphenols and their anticancer, antithrombotic, anti-atherogenetic and anti-inflammatory effects. However, more research is needed to determine which of these activities can realistically be translated into clinical effects. 3. Anticarcinogenic activity The antioxidant effect, the modulation of some enzymes and induction of apoptosis are at the root of the anticancer mechanism. Carcinogenesis is a multi stage process of genetic change that may be initiated by increased and persistent damage to DNA causing permanent alterations in the genetic message when the cell replicates its DNA and divides. ROS (Reactive Oxygen Species) are potential carcinogens because they can induce structural damage to DNA by oxidation, methylation, depurination and deamination reactions. The ability of some polyphenols to reduce or inhibit the oxidative damage to DNA is well documented. For example chlorogenic acid inhibits DNA damage in vitro caused by peroxynitrite [134], coumaric acids are good free radical scavengers [135], caffeic and dihydroxybenzoic acids are able to inhibit iron induced DNA damage [135,136]. Chlorogenic acid can inhibit DNA damage caused by monochloramine [137]. Many kind of flavonoids are able to scavenge free radicals protecting DNA from oxidation, including the isoflavone genistein [138,139], theflavonesluteolin and apigenin [140], the flavanol epigallocatechin gallate [141], the flavonols myricetin and kaempferol [140], quercetin [140-142], and its glycosylated derivatives, quercetin-3glycoside, quercitrin and rutin [140]. DNA damage may be also reduced by metal binding properties of flavonoids, as demonstrated for rutin and quercetin [143]. Another mechanism in reducing carcinogenesis by polyphenols is the modulation of the enzymatic system that is in charge of the metabolization of carcinogenic molecules. The cytochrome P450 family of enzymes metabolizes a large number of procarcinogens to reactive intermediates, which bind covalently to DNA and can induce mutation. Severalflavonoidsare able to inhibit the activity of this family enzymes, as reported by different authors [144-149]. The inhibition of this activity byflavonoidsis directly correlated to their antimutagenic properties. The glutathione transferases (GST), together with the tripeptide glutathione (GSH), conjugate the highly reactive and potentially

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carcinogenic substances, making such molecules more polar, thereby facilitating their excretion. Flavanones, flavones, flavonols increase hepatic GST levels and activity in rats [148,150]. Furthermore, by modification of gene expression, some polyphenols may prevent or reverse carcinogenesis, inducing apoptosis or inhibiting neoplastic transformation. Gallic acid induces selective cell death in cancer cell [151], and hamster fed caffeic acid with the diet were shown to be less susceptible to the effect of methylazoxymethanol, an initiator of colon carcinogenesis [152]. Caffeic acid phenethyl ester from propolis, given to mice bearing a germline mutation in the Ape gene and spontaneously developing numerous intestinal adenomas by 15 weeks of age, decreased tumor formation by 63% at a dietary level of 0.15% and the examination of intestinal tissue from treated animals showed that tumor prevention was associated with increased enterocytes apoptosis [153]. Protocatechuic acid from Hibiscus sahdariffa induced apoptosis in human leukemia cells [154]. Green tea polyphenols and epigallocatechin gallate increase fragmentation in several human and rodent carcinoma cells, but not in normal epidermal keratinocytes [155]. Epigallocatechin gallate also induces apoptosis in transformed fibroblast [156] and in human histiolytic lymphoma U937 cells [157]; theasinensin D, theaflavin, theaflavin digallate induced apoptosis in the same lymphoma cells [157]. Quercetin, rutin, morin, gallic acid and tannic acid inhibited the growth of human prostate cancer cell (LNCaP) at different concentrations, and induced apoptosis [158]. 4. Antiatherosclerotic activity Cardiovascular heart diseases (CHD) are considered as the clinical expression of advanced atherosclerosis. One of the initial steps in atherogenesis is the oxidative modification of LDL and the uptake of the modified lipoprotein particles by macrophages, which in tum become lipid laden cholesterol-rich cells, so-called foam cells [159]. An accumulation of foam cells in the arterial wall is the fu-st visible sign of atherosclerosis and is termed fatty streak, the precursor to the development of the occlusive plaque [160]. It is well known that oxidation of LDL can be initiated in vitro by incubating isolated LDL particles with cells (macrophages, lymphocytes, smooth muscle cells, or endothelial cells), metal ions (copper or iron), enzymes, oxygen radicals, or UV-light. However less is known about the mechanisms by which

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LDL becomes oxidized in vivo. There is evidence that LDL is protected against oxidation in plasma by water-soluble antioxidative substances, such as ascorbic acid, uric acid, or bilirubin. Thus, it is likely that the majority of oxidative modification of LDL occurs in the artery wall, where LDL is largely isolated from the plasmatic antioxidants. Recent evidence suggests that metal ions (copper or iron) and the enzymes myeloperoxidase and lipoxygenase play major parts in the modification of LDL [161]. In in vitro studies the oxidation of LDL by endotheUal cells, macrophage and Cu"*^ can be inhibited by a wide range of polyphenols and polyphenol-rich extracts [162-164]. Such effects may be due to polyphenols by direct scavenging of the oxidizing species, by regeneration of a-tocopherol in LDL [165], by their ability in binding metal ion and LDL protein [166]. In addition to in vitro studies, several animal models and trials with human subjects indicate that ingestion of polyphenols increases the resistance of LDL oxidation ex vivo [167,168]. Some polyphenols inhibit platelet aggregation reducing the risk of thrombosis [171-173]. This effect may be due to a series of interaction of flavonoids in different biochemical pathways, such as by inhibition of cyclooxygenase and lipoxygenase, that are involved in the arachidonic acid metabolism in the platelets, or by inhibition of the formation of tromboxane and of the receptor function of the same [173-176]. Regular consumption of wine, tea and chocolate has been associated to the reduction of platelet aggregation, cardio-vascular diseases and thrombosis [171,177-179]. 5. Anti-inflammatory activity Inflammation is a highly complex biochemical protective response to cellular injury. It is important in the maintenance of homeostasis when the organism is challenged by noxious agents or by tissue mechanical injury. Inflammation is associated with a drastic rise in the number of polymorphonuclear leukocytes and monocytes in the affected tissue and with the release of inflanunatory mediators such as prostaglandins and cytokines. Under normal conditions, inflammation results in the complete recovery of the integrity of the affected tissue, but if the response to the triggering stimulus is not subjected to tight regulation.

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cellular and extracellular components of the organism adjacent to the inflammation site can be injured, inducing a condition known as chronic inflammatory disease. A chronic inflammatory like environment characterizes the pathogenesis of various diseases such as atherosclerosis, arthritis, and Crohn's disease, and it is thought to be among the causative factors of more than 30% of human cancers. For these reasons it is very important to localize and reduce the inflammatory response. In this scenario, polyphenols are involved as immunomodulatory and anti-inflammatory agents, scavenging ROS and modulating the activity of key enzymes of the inflammatory response [172,180482]. 6. Inhibition of histamine release and spasmolytic activity The flavonoids quercetin, hyperoside and isoquercetin, present in the ethanolic extract of Drosera madagascariensis, are inducers of spasmolytic and anti-inflammatory effects in guinea-pig ileum by affecting cholinergic M3 and histamine HI receptors [183]. In an in vitro study by Yamada et al [184], triphenols, such as pyrogallol and gallic acid, and among flavonols, myricetin, inhibited histamine release from rat peritonei cells. Pyrogallol and gallic acid, and also o- and /?- diphenols, such as catechol and hydroquinone and all flavonols tested, strongly suppressed leukotriene B4 release in the same cells. Another in vitro study on rat basophilc leukemia cells demonstrated that, among tea polyphenols, (-)-epigallocatechin gallate (EGCg), (-)-epigallocatechin (EGC) and (-)-epicatechin gallate (ECg) have different inhibitory effects on histamine release induced by a calcium ionophore, with the following magnitude: EGCg>ECg>EGC [185]. In the same study was also demonstrated that pyrogallol and gallic acid exert inhibitory activity and a mixture of these two compounds inhibited histamine release as strongly as EGCg. 7. Hepatoprotective activity Polyphenols are also endowed to have hepatoprotective effects. For example, quercetin reduces liver oxidative damage, ductural proliferation and fibrosis in biliary-ostructed rats, suggesting that it may

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be a useful liver protective agent in patients with biliary obstruction [186]. 8. Antiviral and antimicrobial activity Polyphenols may act as antimicrobial and antiviral agents as demonstrated by several studies in vitro. Polyphenol-rich extracts from various plants, such as Betula pubescens^ Epilobium angustifolium, Perillafrutescens, Pinus sylvestris, Rubus chamaemorus, Rubus idaeus. Solarium tuberosum, propolis and pure compounds, were tested to evaluate their antimicrobial activity against different bacteria and yeasts species, such as Bacillus subtilis, Escherichia coli, Mycobacterium tuberculosis H37Rv, Pseudomonas aeruginosa. Salmonella spp, Staphylococcus aureus. Streptococcus piogenes, Aspergillus niger, Candida albicans, Saccharomyces cerevisiae and showed growth inhibitory and bactericidal effect at different concentrations [187-192]. Naturally occurring flavonoids with antiviral activity have been recognized since the 1940s [193]. Quercetin, morin, rutin, taxifolin, dihydrofisetin, leucocyanidin, pelargonidin chloride, apigenin, catechin, hesperidin, and naringin have been reported to possess antiviral activity against some of 11 types of viruses [193]. (-)-Epigallocatechin gallate and theaflavin digallate inhibited the infectivity of both influenza A virus and influenza B virus in Madin-Darby canine kidney cells in vitro [194]. 9. Oestrogenic activity Plant-derived oestrogens may exert both oestrogenic and antioestrogenic effects, depending on several factors, including their concentration, the concentrations of endogenous oestrogens, and individual characteristics, such as gender and menopausal status [195,196]. The anti-oestrogenic activity of phytoestrogens may be partially explained by their competition with endogenous 17p-estradiol for oestrogen receptors [197]. Many of the potential health benefits of phytoestrogens may be attributable to features that do not involve oestrogen receptors, such as their influence on enzymes, protein synthesis, cell proliferation, angiogenesis, calcium transport, Na"^/K"*" adenosine triphosphatase, growth factor action, vascular smooth muscle cells, lipid oxidation, and cell differentiation. Phytoestrogens may have

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favorable effects on the risk of cardiovascular disease and are thought to be hypocholesterolemic, anticarcinogenic, antiproliferative, antiosteoporotic, and hormone altering [195,196,198,199]. Finally, flavonoids can bind to structural proteins and this feature could explain their ability to enhance the integrity of connective tissue. EPIDEMIOLOGIC EVIDENCE HEALTH BENEFITS

OF PLANT POLYPHENOL

1. Risk of CHD diseases Several epidemiological studies have reported inverse relation between intakes of flavonols and flavones and cardiovascular heart diseases (CHD). In a prospective study of 3454 men and women (age 55 years and older), a significant inverse association between the intake of catechinrich tea and radiographically quantified aortic atherosclerosis was found [200]. Similarly, inverse association between the consumption of red wine and CHD mortality (French paradox) have been suggested [201]. This beneficial effect of red wine may be due to the antioxidant ability of the wine phenolics to inhibit the oxidation of LDL to an atherogenic form [202], In the Zupthen Elderly Study [203] flavonol and flavone intake at baseline in 1985 of approximately 800 men (aged 65-85 years) was determined using the cross-check dietary history method. Men were divided into tertiles of flavonol and flavone intake. After five years of follow-up 43 men died from heart disease in this period. Flavonol and flavone intake, expressed as tertiles, was inversely associated with mortality from coronary heart disease and to a lesser extent with the incidence of first myocardial infarction. Furthermore, the association between long-term flavonol and flavone intake and risk of stroke in a cohort of 552 middle-aged Dutch men free fi"om history of stroke at baseline was also investigated within this study. Men were divided into quartiles of flavonol and flavone intake, and followed for 15 years. During this period 42 men had a first stroke event. Flavonol and flavone intake was strongly inversely associated with stroke risk. In both studies, the men in the highest category of flavonol and flavone intake (>30mg/day) had about one-third the risk of getting the disease compared

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with men in the lowest category. The major sources of dietary quercetin and other flavonols were revealed as tea and onions (fruits and vegetables had minor importance). The same authors [204] confirmed these results in the Seven Country Study. The contribution of flavonols and flavones in explaining the variance in coronary heart disease mortaUty rates across 16 cohorts from seven countries was studied. Flavonol and flavone intake was inversely correlated with mortality from coronary heart disease. Thesefindingare in line with the results of a cohort study in Finnland [205], where a significant inverse gradient was observed between dietary intake of flavonoids and total and coronary mortality. A modest but not significant inverse correlation between the intake of flavonols and flavones and subsequent mortality rates was found in a prospective cohort study of US Health Professionals by Rimm et al [206]. The authors do not exclude thatflavonoidshave a protective effect in men with established coronary heart disease although strong evidence was missing. Also other studies failed to demonstrate a significant statistical association between the intake of polyphenols and CHD. In Great Britain for instance coronary and total mortality even rose with the intake of the majorflavonolsource, tea [207]. The most likely explanation for the latter observation is that in this study tea consumption merely acted as a marker for a lifestyle that favours the development of cardiovascular disease. Indeed, men with the highest intake of tea and flavonols tended to be manual workers, and they smoked more and ate more fat [208]. 2. Risk of cancer The epidemiological evidence for a beneficial support of polyphenols in cancer disease is contradictory and less clear than its role in CHD. The Zupthen Elderly Study found a weak inverse association between flavonoid intake from fruit and vegetables sources and cancer of the alimentary and respiratory tracts combined [209]. The same authors observed no independent association with mortality from other causes between flavonoid intake and cancer mortality in the Seven Country Study [204]. ICnekt et al [210] studied the relation between the intake of flavonoids and subsequent cancer among 9959 finnish men and women during a follow-up in 1967-1991, An inverse association was observed between the intake offlavonoidsand incidence of all sites of cancer combined. Of

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the major flavonoid sources, the consumption of apples showed an inverse association with lung cancer incidence. The cancer protective effects of black and green tea consiraiption, important sources of flavonol in specific countries, have been investigated mainly in case-control studies. Kohlmeier et al [211] evaluated the epidemiologic literature about tea and cancer prevention, concluding that cohort studies do not suggest a protective role for tea drinking in the total risk of cancer. Site-specific studies give a more complex picture. For example, a protective effect of green tea on the development of colon cancer is suggested. On the other hand, evidence for black tea is less clear, with some indication of a risk of colon or rectal cancer associated with regular use of black tea. In another cohort study of a Japanese population, researcher surveyed more than 8000 individuals over 40 years of age on their living habits, including daily consumption of green tea. Results found a negative association between green tea consumption and cancer incidence, especially among females drinking more than 10 cups per day [212]. 3, Vasoprotective effects (Hypertension) Experimental studies have shown that the administration of green teaenriched water to laboratory animals is associated with a reduction in blood pressure [213]. Different epidemiologic studies have suggested that drinking either green or black tea may lower cholesterol concentration and blood pressure [214,215]. In a epidemiological study of Japanese women, a history of stroke was less common among those who drank more green tea. There was no statistically significant reduction in blood pressure alone among those women who drank more tea [206]. 4. Oestrogenic effects Phytoestrogens represent a family of plant compounds that have been shown to have both oestrogenic and anti-oestrogenic properties. Accumulating evidence from molecular and cellular biology experiments, animal studies and, to a limited extent, human clinical trials suggests that phytoestrogens may potentially confer health benefits related to

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cardiovascular diseases, cancer, osteoporosis, and menopausal symptoms. These potential health benefits are consistent with the epidemiological evidence that the risk of heart disease, various cancers, osteoporotic fractures, and menopausal symptoms is lower among populations that consume plant-based diets, particularly among cultures with diets that are traditionally high in soy products. One study over 9 months noted a significant reduction in total cholesterol in premenopausal women when they consumed soy products with 45 mg conjugated isoflavones/day in comparison to levels during a control period when they were fed isoflavone-free soy products. The treatment group difference was significant despite the small sample size and the selection of healthy, normocholesterolemic women who had limited room for detectable improvements [216]. The pattem of soy intake and its association with blood lipid concentrations in the Hong Kong Chinese population was studied in a total of 500 men and 510 women with an age range of 24-74 years by Ho et al [217]. In men, soy intake and total plasma cholesterol were negatively correlated (r = 20.09, P = 0.04), as were soy intake and LDL cholesterol fr = 20.11, P = 0.02). The respective values in women <50 y old were r = 20.11, P = 0.04 and r = 20.11,P = 0.05. In the Framingham Offspring Study a group of 939 postmenopausal women was studied to correlate the association between dietary phytoestrogen intake and metabolic cardiovascular risk factors. Mean blood pressure, waist-hip ratio (WHR) and lipoprotein levels were determined in quartile categories of dietary phytoestrogen (isoflavones and lignans) intake. In the highest quartile of intake of isoflavones, plasma triglyceride levels were 0.16 mmol/L lower (95% CI, -0.30 to 0.02) compared with the lowest quartile of isoflavones and the mean cardiovascularriskfactor metabolic score was 0.43 points lower (95% CI, -0.70 to -0.16) than the lowest quartile [218]. Hormone-related cancers of the breast, ovary, endometrium, and prostate have been reported to vary by as much as 5 to 20-fold between populations. Migrant studies indicate that the difference is largely attributable to environmental factors rather than genetics [219,220]. The highest rates of these cancers are typically observed in populations with Westem lifestyles that include relatively high fat, meat-based, low fiber diets, whereas the lowest rates are typically observed in Asian populations with Eastem lifestyles that include plant-based diets with a high content of phytoestrogens [219,221].

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In a case-control study Ingram et al [222] reported a significant reduction in breast cancer risk among both premenopausal and postmenopausal women who consumed phytoestrogens. In a study of Asian-Americans of Chinese, Japanese, and Filipino heritage, it was reported that tofii consumption was significantly and inversely associated with breast cancer [223]. Similar findings were reported from a case-control study of women in Singapore in which soy intake was inversely, and animal products intake was positively, associated with breast cancer, although these findings were significant only among premenopausal, not postmenopausal women [224]. Soy and fiber consumptions were both associated with a decreased risk of endometrial cancer among the multiethnic population of Hawaii, a finding that was limited to women who had never used oestrogens and had never been pregnant [225]. In a study conducted in Boston and Helsinki, it was demonstrated that the lowest excretion of enterolactone and equol was found in a group of postmenopausal breast cancer patients compared to healthy omnivorous and vegetarian women [226]. The continual loss of bone mass in the elderly is a natural process of aging. Women have a higher incidence of osteoporotic fractures than men due to their lower peak bone mass, but in addition, the abrupt decrease in oestrogen secretion in postmenopausal women accelerates bone loss. Currently, osteoporosis-related fractures are lower in Asia than in most Westem communities, possibly due to the phytoestrogen-rich soybeans and vegetables consumed in large quantities in the Asian diet [227]. Investigation about rates of hip fracture in Hong Kong and the U.S. reported that for men and women 85 yr of age or more, the rates in Hong Kong were roughly one third the rates in the U.S. [228]. REFERENCES: [1] [2] [3] [4] [5] [6] [7]

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