Journal of the Science of Food and Agriculture J Sci Food Agric 80:1094±1117 (2000)

ReviewProanthocyanidins and tannin-like compounds– nature, occurrence, dietary intake and effectson nutrition and healthCelestino Santos-Buelga1 and Augustin Scalbert2*1Unidad de Nutricion y Bromatologıa, Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno, E-37007Salamanca, Spain2Laboratoire des Maladies Metaboliques et Micronutriments, INRA, F-63122 Saint-Genes-Champanelle, France

(Rec

* CoCham

# 2

Abstract: Proanthocyanidins (syn condensed tannins) are complex ¯avonoid polymers naturally

present in cereals, legume seeds and particularly abundant in some fruits and fruit juices. They share

some common structural featuresÐphenolic nature and high molecular weightÐwith phenolic

polymers found in black tea and red wine (called here tannin-like compounds). The polymeric nature

of proanthocyanidins makes their analysis and estimation in food dif®cult. For this reason, little is

known about their consumption, although they likely contribute a large part of the daily polyphenol

intake. They also share common physicochemical properties: they form stable complexes with metal

ions and with proteins and are, like other polyphenols, good reducing agents. Many of their biological

effects of nutritional interest derive from these properties. As metal ion chelators, they in¯uence the

bioavailability of several minerals. The nutritional signi®cance of the non-speci®c complexation of

proteins is less clear. As reducing agents, they may participate in the prevention of cancers, both of the

digestive tract and inner organs. They may also protect LDLs against oxidation and inhibit platelet

aggregation and therefore prevent cardiovascular diseases. In vitro, animal and human studies on the

prevention of these chronic diseases are reviewed with particular attention to wine and tea poly-

phenols. The lack of data on their bioavailability and the paucity of human studies are emphasised.

# 2000 Society of Chemical Industry

Keywords: absorption; antioxidants; bioavailability; black tea; burden; cancer; cardiovascular diseases;condensed tannins; fruits; fruit juices; metabolism; metal ion bioavailability; polyphenols; proanthocyanidins;red wine review; tannin-like compounds; tea; wine

INTRODUCTIONThe term `tannin' has been employed classically to

designate the substances of vegetable origin capable of

transforming fresh hide into leather. Tannins are

widespread in plants and in food of plant origin, in

particular in fruits, legume seeds, cereal grains and

different beverages (wine, tea, cocoa, cider). They

have been de®ned by Bate-Smith and Swain as `water-

soluble phenolic compounds having molecular weights

between 500 and 3000 and, besides giving the usual

phenolic reactions, have special properties such as the

ability to precipitate alkaloids, gelatin and other

proteins'.1 It is precisely this capacity to precipitate

proteins, in particular the salivary proteins in the oral

cavity, which is believed to give them an astringent

character easily recognised in tannin-rich food. This

property is essential to explain their role in plant

protection against pathogens2 or to deter herbivores

from feeding on tannin-rich plants.3

eived 2 November 1999; accepted 10 November 1999)

rrespondence to: Augustin Scalbert, Laboratoire des Maladiespanelle, France

000 Society of Chemical Industry. J Sci Food Agric 0022±5142/2

Tannins are classically divided into two groups.

Hydrolysable tannins are esters of phenolic acids and a

polyol, usually glucose. The phenolic acids are either

gallic acid in gallotannins or other phenolic acids

derived from the oxidation of galloyl residues in

ellagitannins.4 Proanthocyanidins (PAs), forming the

second group of tannins, are far more common in our

diet. They are polymers made of elementary ¯avan-3-

ol units. A key feature of PAs is that they yield

anthocyanidins upon heating in acidic media, hence

their name.

Structurally, tannins possess 12±16 phenolic groups

and 5±7 aromatic rings per 1000 units of relative

molecular mass.5 This feature, together with their high

molecular weight, clearly makes the tannins and

similar phenolic polymers found in processed products

such as red wine or black tea different both in structure

and properties from the low-molecular-weight pheno-

lic acids and monomeric ¯avonoids. The phenolic

Metaboliques et Micronutriments, INRA, F-63122 Saint-Genes-

000/$17.50 1094

Proanthocyanidins and tannin-like compounds

polymers, formed by enzymatic and/or chemical

transformation of simple ¯avanols, PAs and other

phenolic compounds, are called here tannin-like

compounds in analogy to lignin and lignin-like

compounds.6 In common with lignin-like compounds,

their structures are ill-de®ned and their structural

characterisation suffers from a lack of adequate

methods. We adopt here the word tannin-like com-pounds as, although not belonging to the two well-

characterised chemical classes of tannins, they share all

features included in their de®nition given above and

also likely share some of their biological properties.

The purpose of this paper is to describe the

biological properties of PAs and tannin-like com-

pounds. Their main chemical propertiesÐinteraction

with proteins, complexation of metal ions and redu-

cing capacityÐare reviewed with regard to their

biological properties. Much has been written in the

last 30 years on the antinutritive effects of tannins in

animals which ingest larger quantities of tannins than

humans. The signi®cance of these results in human

nutrition is discussed. However, emphasis is given to

the putative protective effects of PAs and tannin-like

compounds against cancers and cardiovascular dis-

eases.

CHEMICAL STRUCTURE OF PROANTHOCYANIDINSPAs are polymeric ¯avan-3-ols whose elementary units

are linked by C±C and occasionally C±O±C bonds.

The ¯avan-3-ol units have the typical C6±C3±C6

¯avonoid skeleton. The three rings are distinguished

by the letters A, B and C (Fig 1). They differ

structurally according to the number of hydroxyl

groups on both aromatic rings and the stereochemistry

of the asymmetric carbons of the heterocycle. The

most common PAs in food are procyanidins (PCs)

with a 3',4'-dihydroxy substitution on the B-ring and

prodelphinidins (PDs) with a 3',4',5'-trihydroxy sub-

stitution. PCs or mixed PC/PDs are most common in

food. Propelargonidins with 4'-hydroxy B-rings are

relatively rare in food sources. So far a unique

propelargonidin dimer has been isolated as a minor

compound in oolong tea7 and propelargonidins were

Figure 1. Basic structure of proanthocyanidins: R1, R2=H,propelargonidins; R1=H, R2=OH, procyanidins; R1, R2=OH,prodelphinidins.

J Sci Food Agric 80:1094±1117 (2000)

found by acid depolymerisation to account for 3% of

the total PAs in tea.8,9 The three carbons C2, C3 and

C4 of the ¯avanol heterocycle are asymmetric and may

occur in different con®gurations. With some very rare

exceptions,10,11 the con®guration of C2 is R. Flavan-

3-ol units with the 2S con®guration are distinguished

by the pre®x enantio (ent-). The stereochemistry of the

C2±C3 linkage may be either trans (2R, 3S) or cis (2R,

3R) as in (�)-(gallo)catechin and (ÿ)-epi(gallo)cate-

chin polymers respectively. The inter¯avan bond at

C4 is always trans with respect to the hydroxy group at

C3.5

The most usual inter¯avanol linkages are C±C

bonds established between the C4 of one ¯avanol unit

(`extension or upper unit') and the C8 or C6 of

another (`lower unit'). Such PAs belong to the so-

called B-type (dimeric) and C-type (trimeric) PAs.

Compounds with doubly linked units (one C±C and

one C±O; `A-type' linkage) have also been reported in

some food sources such as tea leaf, cocoa and

cranberry fruits.12 In these A-type PAs an additional

ether linkage between the C2 of the upper unit and the

oxygen-bearing C7 or C5 of the lower one is formed in

addition to the usual C4±C8 or C4±C6 bond.

Initially, oligomeric PAs were named by an alpha-

numeric system, with a letter A, B or C to describe the

type of inter¯avanol linkage; a number was added to

the letter as they were detected.13 A new nomenclature

was later introduced to name an increasing number of

new structures. It is based on that utilised for the

polysaccharides.14,15 In this nomenclature the ele-

mentary units of the oligomers are designated with the

name of the corresponding ¯avan-3-ol monomers.

The inter¯avanol linkage and its direction are indi-

cated in parentheses with an arrow (4 →) and its

con®guration at C4 is described as a or b. In type-A

doubly linked PAs, both linkages are indicated within

the parentheses. It is unnecessary to indicate the

oxygen in the additional ether bond since it is obvious

from the substitution pattern of catechin lower units.16

For instance, according to this nomenclature, pro-

cyanidin dimer B1 becomes epicatechin-(4b→8)-

catechin and dimer A2 becomes epicatechin-(2b→7,

4b→8)-epicatechin (Fig 2).

Flavanol units can bear various acyl or glycosyl

substituents. The most common acyl substituent is

gallic acid which forms an ester with the hydroxyl in

the C3 position, as in tea17 and wine.18 Several glyco-

sylated PA oligomers have also been characterised.

The sugar is generally linked to the hydroxyl group at

the C3 position,19±23 but also at the C5 position.24

Although PA heterosides are less frequently reported

than other ¯avonoid glycosides, their occurrence may

be underestimated, as sugars are frequently associated

with puri®ed PA polymers.25±27

Since the elucidation in the 1960s of the PA basic

structure,28,29 more than 200 PA oligomers with

polymerisation degree as high as 530±32 have been

identi®ed and fully characterised.12 However, the bulk

of PA polymers in plants usually have a higher degree

1095

Figure 2. Structures of some proanthocyanidin dimers and trimers of the A,B and C types.

C Santos-Buelga, A Scalbert

of polymerisation (DP). Such polymers are then

characterised by chemical degradation in the presence

of a nucleophile, usually phloroglucinol33,34 or

benzylmercaptan,18,26,35,36 and NMR spectro-

scopy.34,35,37,38 Such methodologies allow charac-

terisation of the nature of terminal and inner ¯avanol

units and the average length of the chain. More

recently, the introduction of electrospray mass spec-

trometry techniques coupled to liquid chromatogra-

phy led to a more detailed characterisation of PA

polymers39,40 and to the non-ambiguous identi®cation

of a PA molecule with a DP of 17 in an extract of cider

apple.41

The polymerisation degree was found to vary widely

with the species, tissues or methods of extraction. For

example, the average DP of extracts prepared from

barks originating from different tree species varied

between 3 and 8.42 In cider apple extracts it varied

between 4 and 11 according to the tissue zones in the

fruit.43 In these same apple samples, PAs with the

highest DPs were better extracted by aqueous acetone

than aqueous methanol.43 Some PAs resist extraction,

either because of a poor solubility or because of

secondary chemical reactions with the insoluble

matrix.42 High-molecular-weight PAs are usually

more strongly absorbed on a polar matrix such as

silica than PAs of lower molecular weight.18

Figure 3. Basic structure of theasinensins (from Ref 17): R1, R2=H orgalloyl.

TANNIN-LIKE PHENOLIC POLYMERSDuring post-harvest storage and processing of fruits

and vegetables some oxidative transformations of PAs,

related ¯avonoid monomers and other polyphenols

may occur, giving rise to the formation of new

1096

compounds that share common structural features

with tannins: high molecular weight and similar

number of phenolic rings per mass unit. They also

share common properties, the most evident being

astringency. They are formed by irreversible oxidative

reactions catalysed by enzymes such as polyphenol-

oxidases or by metal ions.5 The ®rst step is the

conversion of o-dihydroxy- (eg catechins) and tri-

hydroxy- (eg gallocatechins) phenyl groups to highly

reactive o-quinones which further react with various

nucleophiles such as other phenols, thiols or amines to

form a wide range of products. The formation of such

products usually results from the crushing of plant

tissues as in the preparation of fruit pureÂes and the

fermentation of tea leaves or grapes; plant compart-

ments are destroyed and polyphenols in the vacuole

and oxidases in the cytoplasm, initially kept apart, are

mixed. The most widely consumed tannin-like poly-

mers are found in tea and wine.

Tannin-like compounds in teaThe outstanding characteristic of the green tea ¯ush is

its very high concentration of polyphenol metabolites,

principally ¯avan-3-ols. Among these, (gallo)catechins

and their gallate esters highly predominate. PAs and

other ¯avan-3-ol dimers in which the B-rings of the

two monomers are linked by a biphenyl C±C bond are

also present (Fig 3).17 These last dimers are similar to

those that can be formed in vitro from catechin in the

presence of polyphenoloxidases (Fig 4).44

The characteristic colour and sensory properties of

semi-fermented oolong and fermented black teas are

generated during their manufacturing process, during

which the colourless (gallo)catechins present in fresh

tea leaves are oxidised both enzymatically and non-

enzymatically to give two major groups of pigments:

thea¯avins and thearubigins. Eventually, in black teas,

some 75% of the substrate ¯avan-3-ol may be con-

verted to thearubigins, whereas 10% accounted for the

formation of thea¯avins and approximately 15% of

¯avanols would remain unchanged.5 Thearubigins

may account for 15±20% of the dry weight in black

tea leaves and for 25±35% of the soluble solids in

infusions.45

Thea¯avins are yellow/orange pigments resulting

from the oxidative coupling of the dihydroxy- and

trihydroxybenzyl B-rings of the (gallo)catechins (Fig

J Sci Food Agric 80:1094±1117 (2000)

Figure 4. Dimers formed by enzymatic oxidation of (�)-catechin (from Ref44).

Figure 6. Structure of theacitrin A (from Ref 45).

Proanthocyanidins and tannin-like compounds

5). The major thea¯avins have been early identi®ed46

and novel compounds of this group further character-

ised.45,47 Thea¯avins are important tea components as

they contribute to the coppery colour of the tea brew as

well as to its brightness and briskness.48 Thearubigins

constitute a heterogeneous group of complex, poorly

de®ned red/brown polymers whose structures are

largely unknown and which would be formed from

thea¯avins and ¯avanol substrates by random poly-

merisation and other oxidative reactions. Some yellow

pigments have been recently isolated from the thea-

rubigin fractions of an Assam black tea and one of

them has been identi®ed as theacitrin A (Fig 6).45 The

molecular weight of thearubigins was originally sug-

gested to vary between 700 and 40000,49 but more

recent studies of thearubigin fractions using size

exclusion HPLC and calibration against neutral

condensed tannins has suggested masses up to

approximately 2500.9,50

Tannin-like compounds in wineDuring storage and aging of red wines a change in

colour from red to tawny occurs which is attributed to

the progressive loss of anthocyanins and the formation

of more complex pigments. The formation of these

pigments is not completely understood. The conden-

sation of anthocyanins and ¯avanols may be direct or

may involve the participation of acetaldehyde or

glyoxylic acid cross-links, as suggested by studies with

model solutions.

The ¯avanols in acidic media may undergo electro-

philic substitution by acetaldehyde on the electro-

negative positions C6 or C8 of their phloroglucinol

A-ring, giving rise to the formation of condensation

products which are colourless when derived from only

¯avanols51±53 or reddish/violet when anthocyanins are

also present.54±58 Acetaldehyde forms ethyl bridges

between ¯avonoid units (Fig 7). The condensed

products can further polymerise through their un-

Figure 5. Structure of theaflavin A (from Ref 46).

J Sci Food Agric 80:1094±1117 (2000)

occupied reactive positions to form ever greater

complexes which ®nally precipitate.52,53,56 The acet-

aldehyde is produced during fermentation either

enzymatically or by coupled oxidation with auto-

oxidised polyphenols such as catechins or PAs.59,60

Similar condensations were also recently shown to

be mediated by glyoxylic acid which results from the

oxidation of grape tartaric acid. Flavanols form

colourless products in which the units are linked

through a carboxy-methine bridge,61 which rearrange

into yellowish/orange pigments showing an absorption

maximum at around 440±460nm.61,62 Both lac-

tone63,64 and xanthylium structures have been pro-

posed (Fig 8).65

The formation of xanthylium derivatives was also

observed after direct condensation of catechins and

synthetic ¯avylium ions.55,66±68 Similar condensation

was suggested to explain the formation of yellowish

pigments which appeared in solutions containing

grape anthocyanins and ¯avanols.69,70 However, it

was recently shown that such pigments were derived

from the glyoxylate-mediated condensation of ¯ava-

nols with no involvement of anthocyanins.63,71 Ac-

cording to Somers,72 no xanthylium pigments but

¯avylium derivatives would be formed after the direct

condensation of anthocyanins and ¯avanols. The

recent ®nding of a red pigment in wine, whose mass

spectrum perfectly ®ts with a dimer formed from the

condensation between malvidin 3-monoglucoside and

Figure 7. Acetaldehyde-mediated condensation of flavanols.

1097

Figure 8. Products proposed for thecondensation of flavanols mediated byglyoxylic acid, according to Ref 64 (lactonestructure) and Ref 65 (xanthyliumstructure).

C Santos-Buelga, A Scalbert

(epi)catechin (Fig 9), supports the proposal of

Somers.73

Other red anthocyanin-derived pigments have also

been found in wines and named vitisins by Bakker and

co-workers,74,75 whose structure contains two meso-

meric ¯avylium forms (Fig 10). These compounds

derive from the cycloaddition on C4 and the hydroxy

group at position C5 in the anthocyanins of substances

such as 4-vinylphenol,76 pyruvic acid, acetaldehyde77

or ethyl-¯avanol adducts.62

Such condensation reactions involving catechins,

PAs and anthocyanins result in the formation of

relatively stable polymeric pigments of high molecular

weight which may partially precipitate during wine

storage. Molecular weights of 2000 and 4000 were

estimated after 5 and 10years of aging respectively.

They are associated with a change in the wine quality

such as a modi®cation of the hue and a decrease in

astringency. Somers72 has suggested that these com-

pounds account for up to 80% of the pigmentation of

red wines after 10years of aging. The involvement of

PAs in such condensation reactions may explain the

Figure 9. Proposed structure for a red pigment isolated from wine (fromRef 73).

Figure 10. Basic structure and mesomeric forms of vitisin pigments.

1098

loss of astringency as has been established for

persimmon.78

ANALYSIS AND ESTIMATION OFPROANTHOCYANIDINSMost polyphenols can be detected and estimated by

chromatographic techniques as they give a well-

de®ned peak or spot. Reverse phase HPLC with UV

detection was initially applied to the analysis of PA

dimers and trimers by Lea and co-workers79,80 and has

become the technique of choice for the analysis of

individual PA dimers and trimers. However, PA

oligomers are often present as complex mixtures in

food extracts, which makes their separation dif®cult.

This problem was partially solved by the development

of new detection methods such as post-column

reaction with N-dimethylaminocinnamaldehyde

(DMACA) and electrospray mass spectrometry. The

®rst method allows the speci®c detection of ¯avanols

in complex mixtures and overcomes the problems of

interference with other UV-absorbing phenolic com-

pounds.81,82 This procedure has been successfully

applied to the analysis of catechins and PAs in plant

extracts11,83 and in foodstuffs and beverages.84±86

However, the identi®cation of the PA peaks often

remains dif®cult owing to the lack of reference

substances, and quantitative data are usually expressed

in catechin equivalents. The same can be said for the

increasingly used HPLC±ESI-MS techniques which

offer an easy way to recognise PA oligomers by their

molecular weight.40

As previously stated, the bulk of PAs in a plant

extract are polymers of higher molecular weight. They

form a continuous set of molecules of increasing

molecular weight and cannot be resolved by chroma-

tographic techniques. The methods used for their

estimation differ in their basic principles and speci®-

city, and none of them can be considered totally

satisfactory.87 Methods used for the global determina-

tion of PAs in foods and plant materials can be divided

into ®ve groups.

1. Methods based on characteristic phenol reactions.

PAs are rich in phenolic groups reducing the Folin

J Sci Food Agric 80:1094±1117 (2000)

Proanthocyanidins and tannin-like compounds

reagent88 or iron(III) in the Prussian blue reac-

tion.89,90 Obviously, these are not speci®c proce-

dures; other phenolic compounds and easily

reducing compounds such as ascorbic acid are

simultaneously determined.

2. Assays based on the ability of tannin to precipitate

proteins.91±93 These assays also lack speci®city,

since other substances capable of precipitating

proteins may also occur in the same materials (eg

hydrolysable tannins), and differences in af®nity

may exist depending on the nature of the tannin

compounds and on the protein used in the assay.

This kind of assay may be more appropriate for

estimating astringency rather than quantifying the

tannin content.94

3. Methods which take advantage of the capacity of

the ¯avanols to react with aromatic aldehydes in

strongly acidic media and form a coloured

adduct. The reagents usually employed are

vanillin95±97 and 4-dimethylaminocinnamaldehyde

(DMACA);82,98 the reaction takes place with both

catechins and PAs. The reactivity of the com-

pounds varies depending on their structure and

degree of polymerisation, which makes it dif®cult to

compare the results obtained with samples of

different ¯avanol compositions.

4. Methods based on the depolymerisation of PAs by

heating in acidic medium and on the colorimetric

determination of the anthocyanidins released.99,100

These assays are speci®c for PAs, since the

monomeric ¯avan-3-ols and other phenolics do

not give these pigments. The yield of the reaction is

low owing to the formation of polymeric side-

products (phlobaphenes); it depends on the condi-

tions employed and on the structure of the

procyanidins present in the sample.

5. Thiolysis based on the depolymerisation of the

condensed tannins in the presence of benzylmer-

captan.26,43 Addition of this nucleophilic reagent,

forming a thioether with the C4 of each polymer

unit, limits the formation of side-products.

Thioether yields commonly reach 40±70% of the

theoretical yields. The degradation products

derived from either procyanidins or prodelphini-

dins are further separated and quanti®ed by either

GC or HPLC.101 The unambiguous determination

of the products by chromatography makes these

methods very speci®c for PAs and well adapted to

the estimation of insoluble PAs42 or of PAs only

present in minute amounts in plant extracts.

Furthermore, information on the nature of the

constitutive ¯avanol units and degree of polymer-

isation can be simultaneously obtained. It is

expected that such a method, although more

time-consuming than other colorimetric methods,

will develop in the future owing to its high

speci®city.

Results obtained by the different methods are often

not comparable owing to their distinct speci®city or to

J Sci Food Agric 80:1094±1117 (2000)

the lack of a suitable standard. They are usually

expressed in gallic acid (Folin assay), tannic acid

(protein complexation), catechin (vanillin assay) or

cyanidin (acid depolymerisation in anthocyanidins)

equivalents. However, good correlations between

results obtained by different assays, eg vanillin assay

and thiolysis, are usually found.26

Reliable determination of PAs in plant materials also

depends on their extraction, which varies with the

solvent system used.87 Aqueous acetone usually give

the best yields, but a variable proportion of PAs still

resist extraction, particularly in aged or oxidised

tissues.42,102

OCCURRENCE IN FOOD AND DIETARY BURDEN OFPROANTHOCYANIDINSAlthough relevant progress has recently been made in

de®ning the distribution and content of other ¯avo-

noids in foods, including monomeric catechins, little

information is yet available on PAs. Table 1 sum-

marises available data on PA content in foods and

beverages. Large discrepancies according to authors

are observed for a given product or commodity. They

can be explained by differences in the assays used for

PA estimation or in the nature of the sample analysed:

variety, stage of ripeness, part of the fruit considered,

processing into foodstuffs. It is even more dif®cult to

estimate tannin-like compounds such as those of tea or

wine, which may account for the major part of the

polyphenols present.

This lack of reliable values for PA content in food

makes it impossible to accurately evaluate their dietary

intake. Table 2 shows the average consumption of

products of plant origin which contribute most to the

Spanish diet. Data from Tables 1 and 2 can be

combined to roughly estimate the dietary intake of PAs

in Spain. It may thus range from several tens to several

hundreds of milligrams of PAs per day, the main

sources being apples, pears, grapes and red wine.

This estimate may vary from country to country and

with dietary habits, which may or may not include rich

sources of tannins such as tea, wine and berries. An

estimate of 460mgdayÿ1 was made by KuÈhnau134 for

the average intake of bi¯avans by Americans. No detail

was given on what was meant by `bi¯avan', which we

suppose to be equivalent to oligomeric PAs. A notice-

able fact is that vegetables hardly contribute to the PA

intake even though they are good sources of other

¯avonoids.84

It is also impossible to estimate the relative intake of

PAs of low polymerisation degree (dimers and trimers)

likely absorbed through the gut barrier (see below) and

that of PAs of high molecular weight, more abundant

in our diets, that may not be absorbed before being

metabolised by the colon micro¯ora. Analysis of both

total PAs by thiolysis and dimers by HPLC showed

that in cider apple, oligomers are at least three times

more abundant than dimers (Guyot S, pers commun).

Although no detailed study has been published on

1099

Table 1. Proanthocyanidin content in different foodstuffs and beverages

Content a Analytical procedure

Proanthocyanidin

type b References

Cereals and pulses (mg per 100g)

Lentil 316±1040 (dry weight) Vanillin assay PC, PD 103, 104

tr±2.4 HPLC 105

Faba bean nd±740 Vanillin assay PC 106

Sorghum nd±3900 Vanillin assay PC 107

Barley 64±126 HPLC PC, PD 108

Fruits and berries (mg per 100g)

Apple 17±50 (dry weight) Vanillin assay PC 109

tr±15 HPLC 110, 111

Pear 0.7±12 HPLC PC 112

Grape 1±160 HPLC PC, PD, gall 113, 114

Sweet cherry 10±23 HPLC PC, PD 115

Blueberry 1±7 HPLC PC, PD 115

Red raspberry 2±48 HPLC PC, PD 115

Strawberry 2±50 HPLC PC, PD, gall 115

Blackberry 9±11 HPLC PC 115

Juices and drinks (mglÿ1)

Apple juice nd±298 HPLC PC 110, 111, 116, 117

Apple pureÂe 16±43 HPLC PC 118

Peach pureÂe 9.5±24 HPLC PC 118

Pear juice 11±74 HPLC PC 119

Grape juice 3.5±46 HPLC PC 117, 120

Cider 2290±3710 Photometry at 280nm PC 121, 122

Red wine nd±500 HPLC PC, PD 85, 114, 123±127

White wine tr±7 HPLC PC 85, 127±129

Rose wine tr±43 HPLC PC 85, 127

Sherry wine 45±54 HPLC PC 130

Wine vinegar 4±414 Vanillin assay PC 95

Beer 3.5±19.5 DMACA assay PC, PD 131, 132

Others (mg per 100g)

Cacao bean 260±1200 Not given PC 133

a Expressed as catechin equivalent. HPLC values only include oligomers (DP=2±3).b PC, procyanidins; PD, prodelphinidins; gall, galloylated derivatives.

C Santos-Buelga, A Scalbert

dimers in particular, it is likely that dimers B1 and B2

are the most extensively consumed as they often are

the main dimers in major dietary PA sources (Table

3). Although the four dimers B1±B4 are commonly

found in mixtures in fruits, B1 predominates in

wine114 and B2 in apple,83 pear119 and cacao.133

PHYSICOCHEMICAL PROPERTIES OFPROANTHOCYANDINS AND BIOLOGICAL EFFECTSMost studies on ¯avanol biological activity, either invitro or in animals, have been carried out with

monomeric catechins or with plant extracts containing

both catechins and PAs. Very few authors have used

pure condensed tannins or catechin-free extracts with

de®ned PA composition. The effects more usually

checked are those related to their af®nity for proteins

and their antioxidant and radical scavenging activities.

Studies also exist that evaluate tannin±metal com-

plexation and antimicrobial and antimutagenic/anti-

carcinogenic properties. The number of phenolic

functional groups in one PA molecule is at the origin

of three essential properties: formation of complexes

1100

with proteins, formation of chelates with metal ions

and reducing capacity. Their biological effects depend

on these essential properties.

Interactions with proteinsProtein±tannin interactions have been extensively

studied and reviewed by Haslam and co-

workers.5,136,137 These interactions are essentially a

dynamic surface phenomenon, generally reversible,

which basically involves proteins that possess an open,

random coil type conformation, and whose principal

leading forces are hydrophobic effects, reinforced by

the establishment of hydrogen bonds. The hydrogen

bridges are basically established between phenolic

groups, as proton donors, and the carbonyl groups of

the peptide bonds, as proton acceptors.138 The

strength of the interactions depends both on the

nature of the protein and on the nature of the PA

molecule.

A competitive binding assay was developed to

compare the af®nity of a given PA preparation to

various proteins, and af®nities were shown to vary by

three orders of magnitude. Proline-rich proteins

J Sci Food Agric 80:1094±1117 (2000)

Table 2. Products of plant origin included among those which contribute to95% of the caloric intake in the Spanish diet

Group Product Daily average intake (g)

Fruits Orange 83

Apple 42

Melon 34

Banana 26

Pear 23

Peach 20

Watermelon 20

Fruit juices 16

Grape 15

Tangerine 15

Cereal products Bread 194

Rice 22

Wheat ¯our 16

Cookies 14

Pasta 10

Vegetables Potato 145

Tomato 40

Lettuce 23

Onion 17

French bean 13

Pepper 11

Carrot 10

Pulses Chickpea 8

Alcoholic drinks Red wine 69

Beer 38

Source: Instituto Nacional de Estadistica, 1991.

Proanthocyanidins and tannin-like compounds

(PRPs) such as collagen, gelatin, salivary proteins and,

to a lesser extent, casein showed the highest af®-

nity.139,140 Glycosylation of proteins may also affect

their binding af®nity to PAs. Carbohydrate residues in

salivary glycoproteins may enhance the af®nity and the

speci®city of the interaction,141 whereas in enzymes

such as a-amylase142 or invertase143 they were

Table 3. Contents of proanthocyanidin oligomers in some foodstuffs (mg per 100g

Proanthocyanidins

B1 B2 B3 B4 B7 C1

Grape 9±26 4±19 3±18 3±15 tr 3±15

Red wine 1±218 tr±73 tr±66 tr±36 nd±18 tr±24

Rose wine tr±24 nd±4 nd±4

White wine nd±2 nd±2 nd±2

Sherry wine 12±21 7±13 10±15 3±9

Apple tr±1 tr±5 tr±4

Apple juice nd±28 nd±139 nd±3 nd±2 nd±12

Apple pureÂe 1.6±7.6 14±35.5

Proanthocyanidins

B3 GC-C C2 GC-GC-C GC-C-C C-GC

Barley 14±28 27±36 3±11 14±24 11±25 6±14

Lager beer 0.7±2.6 1.1±2.5 0.2±0.3 0.4±0.7 0.1

E, epicatechin;C, catechin; GC, gallocatechin; B1, E-(4,8)-C; B2, E-(4,8)-E; B3, C-

C-(4,8)-C-(4,8)-C; EEC, E-(4,8)-E-(4,8)-C; GC-C, GC(4,8)C; GC-GC-C, GC(4,8)GC(

J Sci Food Agric 80:1094±1117 (2000)

suggested to decrease their af®nity for tannins and

their resistance to tannin inhibition.

The strength of the interactions also depends on the

structure of PAs and in particular on their molecular

weight, degree of galloylation and hydroxylation

pattern. PA trimers were shown to precipitate various

proteins126 or to inhibit a b-glucosidase144 more

ef®ciently than the related dimers. When a mixture

of PAs was allowed to interact with salivary proteins,

the PAs forming a precipitate with the proteins were

again the larger PA molecules, with an average poly-

merisation degree of 7, and those left in the super-

natant were mainly dimers and trimers.145 Similar

conclusions were reached by comparing procyanidin

dimer, trimer and a non-galloylated mixture of

procyanidins with an average polymerisation degree

of 5.94 The presence of galloyl groups also results in a

major increase in af®nity for proteins.94,126,146 On the

other hand, the hydroxylation pattern of the B-ring in

synthetic PAs was shown to have no in¯uence on the

af®nity for bovine serum albumin.147

Some of the effects of PAs or PA-rich food sources

on human physiology, both bene®cial and detrimental,

can be explained by their ability to interact with

proteins (enzymes, toxins, hormones, etc). It has been

known for a long time that monogastric animals such

as rats fed a regime rich in tannins may suffer from a

reduction of weight gain.148,149 Similarly, consump-

tion of PA-rich faba beans by Egyptian boys reduces

the net protein utilisation, which can be restored by

dehulling.150

Such effects have been attributed to a direct inter-

action with digestive enzymes or with dietary protein

substrates in the gut. However, experiments with

labelled dietary proteins showed that the low apparent

nitrogen digestibility was not explained by an inhibi-

tion of the digestion of dietary protein but rather by an

increase in endogenous protein secretion and in

particular of salivary PRPs.151 Other mechanisms

) and beverages (mglÿ1)

EEC Others References

5±18 <7 (galloyled dimers) 113, 114

tr±49 <10 (galloyled dimers) 85, 113, 114, 123±125, 127

85, 127

85, 127±129

130

<1 (B5) 110, 111

0 14±36 (unknown) 111, 116, 117

118

-C

108

131, 132, 135

(4,8)-C; B4, C-(4,8)-E; B5, E-(4,6)-E; B7, E-(4,6)-C; C1, E-(4,8)-E-(4,8)-E; C2,

4,8)C; GC-C-C, GC(4,8)C(4,8)C; C-GC-C, C(4,8)GC(4,8)C.

1101

C Santos-Buelga, A Scalbert

must thus still be found to explain the general effects of

tannins on food digestion by monogastric animals or

humans. PAs also have other physiological effects in

the gut that may counteract their antinutritional

properties: levels of some digestive proteases and

lipases152±155 and of biliary acids156 are increased

upon feeding on tannin-rich diets. The pancreatic

biliary juice was shown to neutralise the inhibitory

effects of grape seed tannins on brush border hydrolase

activities.157

The secretion of salivary PRPs may also counteract

tannin biological action. Salivary PRPs form com-

plexes with PAs which may be stable throughout the

digestion process, and this would allow the absorption

and retention of nutritionally more useful food

proteins.158 The level of salivary PRPs secreted by

herbivores,159 and possibly also by humans,136 is

enhanced by the dietary exposure to tannins. An

isoprenaline/tannin-dependent region has been found

that plays a role in the regulation of PRP expression.160

Feeding rats with high-tannin diets causes dramatic

changes in gene expression of parotid glands,161

induces a hypertrophy of the parotid salivary glands

and increases the ¯ow of saliva and the secretion of

PRPs.162,163 The secretion of salivary PRPs is thus

seen as a defence mechanism against dietary tannins.

The detrimental effects of PAs on digestion are

dose-dependent. In rats they are effective at doses of

1% of the diet or higher. Lower doses such as 0.2%

have no effect.149 This last dose is still higher than that

found in a regular human diet, which does not usually

exceed 0.1%. It was calculated that such a PA intake

corresponds to a maximal concentration in the gut

lumen of 1.2glÿ1 (or 4mM catechin unit equiva-

lent).164 This concentration is lower than that needed

for PAs to inhibit 50% of the activity of soluble

proteases in model solutions (about 2.5g lÿ1),165 but

suf®cient to signi®cantly inhibit brush border enzyme

activities in Caco-2 gut epithelial cells grown invitro.166 Tea thea¯avins and thearubigins were also

shown to inhibit rat intestinal sucrase and a-glucosi-

dase at 0.25glÿ1.167 However, it is doubtful that PAs

still inhibit such enzymic activity in vivo in the

presence of a large excess of other dietary or

endogenous proteins that may also bind tannins.

PAs may also show preventive or therapeutic

properties against gastrointestinal diseases. Tannin-

containing plants are commonly used in folk medicine

to treat diarrhoea. The quantities administered are

then much higher than those regularly consumed with

our diet, up to 0.6g kgÿ1 body weight in infants.168

The antidiarrhoeal effect of tannins is commonly

attributed to the unspeci®c complexation of mucosal

proteins in the gut with formation of a protective layer.

Radiolabelled PAs were also shown to be adsorbed on

the apical surface of intestinal epithelium Caco-2

cells.164 Such an adsorption of PAs on the gut mucosa

is probably at the origin of the use of tannic acid in

barium enema for colon examination.169 Other

mechanisms that may explain the antidiarrhoeal

1102

effects of tannins are the complexation of secretagogue

compounds such as cholera toxin170 and rhein171 or

the inhibition of intestinal motility.172

PAs may prevent dental caries173 by inhibiting the

activity of glycosyl transferases which catalyse the

formation of water-insoluble glucan from glu-

cose174,175 or by inhibiting the growth of cariogenic

streptococci.176,177

Similar non-speci®c interactions between tannins

and proteins in the gut lumen may be biologically

signi®cant but are less likely to occur in the inner

organs owing to the poor absorption of polymeric PAs

(see below). The less astringent PA dimers and trimers

or the low-molecular-weight PA metabolites formed

by the gut micro¯ora, once absorbed through the gut

barrier, may well form complexes with plasma proteins

or with enzymes and membrane receptors in the inner

organs.178 However, the nutritional signi®cance of

these interactions will be very different from that of

interactions taking place in the gut lumen.

Complexation of metal ionsPAs with their o-dihydroxyphenyl groups are excellent

chelators of iron(III). At neutral pH and in the

presence of PC dimer B2179 or of a galloyl ester of

glucose,180 all iron was found in the form of mono-

nuclear ferric complex with the catecholate groups of

two ligands. They also form complexes with Al(III)

and Cu(II).179,181 PA±metal ion complexes easily

precipitate at neutral pH as long as the concentration

of the ligand is not too high relative to the metal ion

concentration.182

Complexation of such metal ions by PAs has some

well-established consequences in biology.183 The

consumption of polyphenol-rich beverages such as

wine184 or tea185 inhibits the absorption of non-haem

dietary iron through the gut barrier. Such an inhibition

is explained by the formation of stable polyphenol/

iron(III) complexes in the gut.186 The ligand must be

consumed together with iron(III) to inhibit its

absorption.187 This inhibition of iron absorption by

polyphenols depends on the presence of other

nutrients. Red wine polyphenols inhibit iron absorp-

tion from a simple bread roll meal184 but had a limited

effect on that ingested with a more complex composite

meal.188 The absorption of the radiolabelled iron

complexed in haemoglobin haem is not affected by tea

polyphenols when the protein is cooked as in gravy.189

This means that haem iron is not exchanged with

polyphenol ligands. Other nutrients such as ascorbic

acid will remove the inhibition of iron absorption by

polyphenols,190 probably by reducing the complexed

iron(III) into the poorly co-ordinated iron(II).

The large majority of polyphenols regularly con-

sumed with our diet, and not only PAs, have o-

dihydroxyphenyl groups in their structures (exceptions

are citrus ¯avanones, soy iso¯avones and ferulic acid

esteri®ed to dietary ®bres). Both low- and high-

molecular-weight polyphenols are all likely to inhibit

non-haem iron bioavailability,191 although little is

J Sci Food Agric 80:1094±1117 (2000)

Proanthocyanidins and tannin-like compounds

known on the effect of their chemical structure on iron

absorption. In vitro experiments with Caco-2 cells

showed that procyanidin polymers (polymerisation

degree of 7) were more inhibitory at identical mass

concentration than the related procyanidin dimer B1

or (�)-catechin (Mila I, Huneau J-F and Scalbert A,

unpublished). However, these results still need to be

con®rmed by in vivo experiments.

In terms of human health, it is advisable for

population groups most susceptible to developing iron

de®ciency (infants, children, pregnant women) to

avoid an excessive consumption of PA- and poly-

phenol-rich beverages and foods or to avoid their

consumption together with meals. On the other hand,

the consumption of black tea with meals has been

suggested as a strategy for reducing iron absorption in

patients with iron overload disorders.192

The in¯uence of PAs and other polyphenols on the

gut absorption of metal ions other than Fe(III) is less

documented but may also be signi®cant. The con-

sumption of tea was shown to inhibit the absorption of

aluminium in both humans193 and rats.194 Paradoxi-

cally, tea is one of the main sources of aluminium for

regular tea drinkers.195 However, it is largely present

as complexes with tea catechins196 which limit its

bioavailability. Similarly, dietary PAs and other poly-

phenols may inhibit the absorption of aluminium from

other sources as suggested by epidemiology studies.

Aluminium in drinking water has been repeatedly

associated with an increase in Alzheimer disease

risk,197 whereas the moderate consumption of wine

(3±4 glasses per day) has been associated with a

reduction of the risk of senile dementia.198 These

protective effects of wine might be explained on the

basis that PAs and other polyphenols in wine form

complexes with aluminium in drinking water, and

these complexes are not absorbed.

The in¯uence of tea on copper(II) absorption has

been studied in rats.199±201 Interestingly, tea con-

sumption caused an increase in copper absorption, in

the plasma level of ceruloplasmin and in the copper

retention in tissues, particularly in the liver. In the

absence of data on individual phenolic compounds,

one may only speculate on the nature of the com-

pounds responsible for these effects. Polyphenols form

the bulk of the dry matter in tea decoctions. Another

bioavailability experiment was carried out with (�)-

catechin, but it failed to show an increase in copper

retention in rats.200 More experiments should be

carried out with other compounds and in volunteers.

Zinc has a low af®nity for polyphenols, particularly

at acidic and neutral pH. This is consistent with the

lack of a signi®cant effect of tea, wine and beer on its

bioavailability in humans.202,203 In a study with rats

fed tea, zinc retention was increased in the tibia, but

this was not correlated with the apparent absorption of

zinc.200 Zinc retention was not increased in soft tissues

such as liver.

Altogether, these data show that PAs and tannin-

like polyphenols may inhibit iron(III) and aluminium

J Sci Food Agric 80:1094±1117 (2000)

and possibly increase copper(II) metal ion bioavail-

ability. The explanation of these differences is likely to

be found in their relative af®nities for PAs. Once

absorbed, PAs are largely metabolised. One of the

main consequences is that they lose their o-dihydroxy-

phenyl functionality to form simple phenols (see

below). The nutritional signi®cance of metal ion

complexation by PAs may then be largely limited to

the gut as is that of protein complexation. However,

once absorbed and metabolised, they may retain part

of their reducing capacity.

Antioxidant and radical scavenging activitiesA number of highly reactive oxygen species such as

singlet oxygen 1O2 and O2

.ÿ, OH., NO

.and alkyl

peroxyl free radicals are regularly produced in our

body. They cause damage to lipids, proteins and DNA

and participate in pathogenesis and aging.204 Humans

possess a wide array of antioxidant physiological

defences which scavenge radicals, chelate metals

involved in their formation and repair damage. The

consumption with the diet of polyphenols together

with other natural antioxidants such as vitamins C and

E and carotenoids also contributes to these

defences.205

All polyphenols are able to scavenge singlet oxygen

and O2

.ÿ, OH., NO

.and alkyl peroxyl radicals through

electron-donating properties, generating a relatively

stable phenoxyl radical. For ¯avonoids with an o-

dihydroxyphenyl group as B-ring and a fully saturated

C-ring, such as in (gallo)catechins and most PAs, the

radical site is at the B-ring and the substitution of the

A-ring has only a limited in¯uence on the reduction

potentials of the semiquinone radical formed.206

These semiquinone radicals are quite stable.207

Gallocatechins with a 3',4',5'-trihydroxyphenyl B-ring

are more ef®cient scavengers than catechins.206,208

Galloylation, adding a similar 3,4,5-trihydroxyphenyl

group to the molecule, also reinforces their scavenging

properties.208

PAs, owing to the lack of easily available substrates,

have received much less attention than their mono-

meric counterparts. They were shown to scavenge

O2

.ÿ, OH.209,210 and the synthetic radical cation

ABTS.�211 in aqueous solutions, often as ef®ciently

as quercetin or butylated hydroxytoluene.211 Galloyla-

tion increased the scavenging capacity of both

PAs210,211 and thea¯avins.212 The doubly linked

A-type dimers were less effective than their B-type

counterparts.209,211 The in¯uence of the polymerisa-

tion degree was not as clear. In some experiments, no

difference was observed between monomers, dimers

and trimers.210 In other experiments, for a given per

weight concentration, the scavenging capacity in-

creased up to the trimers then decreased for higher

molecular weights,213 or was the same for catechin

monomers and dimers and then decreased for larger

polymerisation degree.211 Such discrepancies are

likely attributed to the differences in the antioxidant

assay used, to the structure of the PAs tested or to the

1103

C Santos-Buelga, A Scalbert

presence of some residual impurities in the PA

fractions. Both the nature of the monomer units and

the position of the intermonomeric linkage had no

signi®cant in¯uence on the scavenging activity.211

PAs were also shown to inhibit the radical peroxida-

tion of lipids: in these experiments, peroxidation of a

methyl linoleate dispersion or of phosphatidylcholine

liposomes was induced by the OH.

radical211 or by

AAPH or AIBN azo-initiators.214,215 In these systems,

galloylation had this time a negative effect on their

antioxidant capacity (AOC).211 Two PA fractions with

an average polymerisation degree of 8 and 10

respectively showed an AOC (in weight units)

comparable with that of (ÿ)-epigallocatechin gallate,

whereas in two other studies the AOC decreased as the

polymerisation degree increased from monomers to

hexamers.211,216

Rice-Evans et al suggested that differences in the

polyphenol structure±activity relationship for experi-

ments carried out in the homogenous aqueous phase

and in lipid emulsion were explained by variations in

the partition between the lipid and the aqueous

phase.208 However, Plumb et al measured the partition

coef®cient in octanol of a few PA molecules and found

that it did not explain the different behaviour of PAs in

water or in lipid emulsion.211 Results on inhibition of

lipid peroxidation are more dif®cult to interpret than

those obtained in water solution, as protection by

polyphenols can be explained by several mechanisms,

either by scavenging the radical used to initiate lipid

peroxidation or the lipid peroxyl radicals themselves,

or by inhibiting the formation of the initiating radical

through chelation of the transition metal (Fe(III) or

Cu(II)) added together with a reducing agent to

generate OH.

radicals. In some experiments, addition

of Cu(II) together with polyphenols (which are

themselves reducing agents) accelerates the peroxida-

tion of methyl linoleate.

The data described above show that PAs, at least in

some well-de®ned conditions, behave as radical

scavengers or antioxidants, in a way similar to other

phenolic compounds having an o-dihydroxyphenyl

group. In other experimental conditions, eg in the

presence of transition metals, they may behave as pro-

oxidants. As stressed by Aust,217 model systems

involving unknown mechanisms which cannot be

readily associated with or related to the oxidation of

some biomolecules should be discouraged. In order to

ascertain the role of PAs in the protection against

disease, it will be necessary to de®ne:

. the right target involved in the disease pathogenesis

(lipid, protein, DNA, etc);

. the physicochemical environment of the target (pH,

concentrations, presence of transition metals, redox

state, homogenous or heterogeneous reaction,

etc)Ðthis will help to select or develop the most

appropriate assay reproducing the in vivo condi-

tions.

Low-density lipoproteins (LDLs) which transport

1104

lipids in our blood are one such target. Their oxidative

modi®cation plays a pivotal role in atherosclerosis,218

and much work has been published showing a

protective role of polyphenols when added to LDLs

in vitro in the presence of Cu(II) or Cu(I) ions or

AAPH azo-initiator. All wines, particularly red

wines,219±222 as well as puri®ed PAs223,224 protected

LDLs against oxidation. The oxidation inhibition by

wines varied in proportion to their phenolic con-

tent.225 The limit of such studies is that the poly-

phenols tested are different from those present in our

plasma. Often, nearly nothing of the phenolic com-

pound ingested survives in the body, as it is largely

metabolised by tissues or by the colon micro¯ora (see

below).

It is thus not only necessary to know the target and

its physicochemical environment but also to know if

the antioxidant effectively reaches the target. In the

absence of such data it will be dif®cult to extrapolate

the results obtained in vitro to the in vivo situation, and

in particular to establish the nature of the dietary

phenolic antioxidants providing the most ef®cient

protection against chronic diseases.

BIOAVAILABILITY OF PROANTHOCYANIDINSIn comparison to (�)-catechin, very few studies have

concerned the bioavailability of PAs in animals and

none in humans. This is due to the complexity of PA

structures, which makes their analysis dif®cult, and to

the lack of commercial pure standards. Feeding rats

and mice with a radiolabelled mixture of PAs led to the

identi®cation in urines and faeces of some ill-de®ned

PAs (54% of the total radioactivity in urines) and of

low-molecular-weight products such as hippuric acid,

ethylcatechol and various phenolic acids.226 The

radioactivity reached all the tissues of the animal

within a few hours following ingestion.227 The PA

mixture used in these studies was obtained from a

whole grape by ethyl acetate extraction and is made of

oligomeric PAs (largely dimers), (�)-catechin and

(ÿ)-epicatechin.228 A doubt thus still remains on the

origin of these low-molecular-weight metabolites, as

they could also be formed from the catechin mono-

mers present in the extract.

Other authors used chromatography on Sephadex

LH20 to purify radiolabelled PA polymers.229,230

These fractions were likely free of sugars, proteins

and monomeric catechins, as the column was washed

with water and methanol before elution by aqueous

acetone.27 The whole radioactivity was recovered in

the faeces and in the bowel for both chickens229 and

sheep,230 showing that polymeric PAs were not

absorbed through the gut barrier.

More recently, we compared the in vitro absorption

of radiolabelled procyanidin dimer, trimer and poly-

mers (average polymerisation degree of 7) and the

related (�)-catechin through a cell monolayer derived

from the human intestinal cell line Caco-2.164 The

monomer, dimer and trimer were all absorbed to a

J Sci Food Agric 80:1094±1117 (2000)

Proanthocyanidins and tannin-like compounds

similar extent through the cell layer, whereas the

polymers were not absorbed and partially adhered to

the cell surface. The procyanidin polymer was also

shown to be degraded by a human colonic micro¯ora

grown in vitro and anaerobically into low-molecular-

weight phenolic acids which might well be absorbed invivo through the colon.164 A similar degradation of the

procyanidin dimer B3 by a rat micro¯ora was reported

earlier.231 This contradicts the common view of a

passive transit of PAs through the bowel. This will

have to be con®rmed by in vivo studies.

Once PAs or their fermentation products have

crossed the intestinal barrier, they reach the liver

through the portal vein, where they are further

metabolised. Three monomethylated and two di-

methylated derivatives were formed when the dimer

B3 was allowed to react in the presence of a methyl

donor (SAM) and a fresh liver homogenate obtained

from a human biopsy.164 No in vivo study has yet been

reported, but PAs should be extensively metabolised,

dehydroxylated, methylated or conjugated to sulphate

esters or glucuronides as has been shown for other

¯avonoids.232,233 These reactions both limit the

potential formation of toxic quinones234 and facilitate

excretion of PAs in the form of anionic derivatives. It is

likely that no more than traces of PAs with intact o-

dihydroxyphenyl groups survive in the tissues. This is

why it is essential to study not so much the biological

effects of intact PAs as they are ingested but rather

those of the metabolites as they reach the target

tissues. A pioneer study was recently published in

which quercetin conjugates were shown to retain part

of the antioxidant activity of quercetin when added invitro to LDLs.235

PROANTHOCYANIDINS, TANNIN-LIKECOMPOUNDS AND CANCER PREVENTIONProanthocyanidins and cancer preventionMany experimental studies have suggested a protec-

tive role of tannins against cancers in humans. Most of

these works were carried out with tannic acid as it is

easily available. However, tannic acid is a mixture of

gallotannins virtually absent from the human diet and,

for this reason, it is clearly not the most appropriate

tannin preparation for such studies. PAs, the most

abundant tannins in our diet, have received much less

attention. Pure PA dimers and oligomers have been

tested both in in vitro assays and animals. The 3,3'-di-

O-gallate derivative of B2 procyanidin dimer showed

cytotoxic activity against the human leukaemic cell line

HL-60.236 Its activity (EC50=119mM) was close to

that of epigallocatechingallate (EGCG). The same

derivative of B2 dimer also inhibited a melanoma cell

line but was inactive against several other tumour cell

lines.237 Several galloylated dimers were also shown to

inhibit the growth of human lung and colon carcinoma

cell lines (IC50=30±60mM). The non-galloylated

dimers, A-type dimers and trimers and a galloylated

pentamer were less active.238

J Sci Food Agric 80:1094±1117 (2000)

Protein kinase C (PKC) involved in signal transduc-

tion and tumour promotion was inhibited by various

PAs when tested in vitro.239 PAs also inhibited tumour

promotion in the mouse epidermis test when induced

by the 12-O-tetradecanoylphorbol 13-acetate (TPA;

acting on the phorboid receptor PKC).240,241 The

same structure±activity relationship was observed as

for protein complexation in general: activity increases

with the polymerisation and galloylation degrees and is

reduced by the presence of an additional intermono-

meric linkage (A-type PAs).

In all these experiments, PAs were in direct contact

with the malignant cells. No experiments have so far

been carried out on either animals or humans with PAs

given orally aiming at showing an association with

cancers. Miyamoto et al have injected galloylated

dimers and trimers (5±10mgkgÿ1 body weight)

intraperitoneally to mice 4 days before intraperitoneal

inoculation with sarcoma-180 cells but failed to

demonstrate any protective effects.242

Tea polyphenols and cancer preventionBlack tea, rich in phenolic polymers, has received more

attention than PAs per se as a putative protective

beverage against various cancers. In most studies, tea

extracts were given orally to rats or mice simulta-

neously treated with a carcinogenic agent. Both black

and green teas showed protective effects.243 Black tea

is usually as active as green tea, but sometimes less244

or more so.245 The nature of the active molecules in

tea is not precisely known. EGCG, the main phenolic

compound of green tea, also present but at a much

lower concentration in black tea, has been suggested as

the active chemopreventer. When male C3H mice

treated with diethylnitrosamine were given varying

amounts of green and black teas in their drinking

water, an inverse correlation was observed between the

intake of EGCG and the number of lung tumours.

However, no correlation was observed with the

number of liver tumours.246 Therefore EGCG is likely

not the only protecting agent present in tea.

Both non-phenolic and phenolic compounds other

than EGCG may also play a role in cancer prevention

by tea. A caffeine solution was found to be as active as

a black tea containing the same caffeine concentration

against lung tumorigenesis induced by 4-(methyl-

nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a

nicotine-derived carcinogen.247 Decaffeinated green

and black teas were also less inhibitory than regular

teas against UVB-induced carcinogenesis in SKH-1

mice. Their activity was restored by addition of

caffeine.244 However, caffeine is certainly not the only

protecting agent, as decaffeinated green and black teas

still retain signi®cant anticarcinogenic246 and anti-

mutagenic activities.248

In comparison to green tea ¯avanol monomers such

as EGCG, few studies have been carried out with the

black tea phenolic polymers. In the Ames test, black

tea phenolic polymers were found to inhibit the

mutagenicity of various mutagenic agents.249

1105

C Santos-Buelga, A Scalbert

Thea¯avins of black tea inhibited the mutagenicity

of 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyri-

dine (PhIP) and 2-amino-3-methylimidazo[4,5-f]qui-

noline (IQ).250,251 They also inhibited the trans-

formation of a mouse epidermal cell line252 and the

proliferation of a human epidermoid carcinoma cell

line induced by TPA or the epidermal growth factor253

and promoted apoptosis of human lymphoma254 and

stomach cancer255 cell lines. In animals, thea¯avins

inhibited the N-nitrosomethylbenzylamine-induced

oesophageal tumorigenesis when given orally to rats256

and the NNK-induced lung tumorigenesis when fed to

A/J mice.257

Despite such well-established evidence of anti-

carcinogenic activity of tea in animals, and in

particular of tea polyphenols, no clear evidence could

be drawn from epidemiological studies on the protec-

tive effects of tea in humans, possibly because of an

insuf®cient control of confounding factors and of the

qualitative and quantitative aspects of tea consump-

tion.258±261

Wine polyphenols and cancersWine, the other major source of tannin-like phenolic

polymers, is generally considered as pro-carcinogenic,

particularly for the upper digestive tract, owing to its

ethanol content.262±264 A recent report con®rmed that

beer and spirit consumption increases the risk of

oropharyngeal and oesophageal cancer, but it also

showed that this risk decreases with the proportion of

wine in the total alcohol intake.265 The relative risk

even decreased to 0.5 for moderate drinkers (7±21

drinks per week) who include over 30% wine in their

alcohol intake, as compared with non-drinkers. These

differences between alcoholic beverages were tenta-

tively explained by the differences in nitrosamine

content, particularly low in wine and high in beer.266

Alternatively, it could be explained by the presence in

urine of some protective constituents counteracting

the toxic effects of alcohol. Red wine solids, ie

dealcoholised red wine largely made of polyphenols

and polysaccharides, delayed tumour onset in trans-

genic mice which spontaneously develop tumours.267

Several authors have suggested that resveratrol, a

potent anticarcinogenic phenolic compound identi®ed

in a medicinal plant268 and also present in wine,225

could be responsible for these effects. However, its

content does not exceed 2mglÿ1 in red wine and is still

lower in white wine.225 We rather think that other

polyphenols are responsible for these effects, most

probably PAs with a minimum concentration of

350mglÿ1,114 or tannin-like phenolic polymers which

may account for the bulk of total polyphenols

(2500mglÿ1) in red wine.225 With their high af®nity

for proteins, they may directly interact with the

epithelium of the upper digestive tract and exert their

protective effects without being absorbed through the

intestinal barrier. A contribution of wine polyphenols

to the prevention of cancer of inner organs is also not

excluded. Wine was found to decrease the risk of lung

1106

cancer, whereas other alcoholic beverages such as beer

or spirits increased the risk.269,270

Are proanthocyanidins carcinogenic?If the large majority of the studies currently published

today tend to demonstrate anticarcinogenic properties

of polyphenols, others have also underlined their

potential carcinogenic effects.271 The consumption

of PA-rich betel nuts has received much attention as it

is associated with oesophageal cancer.272 When

applied to the cheek pouch of hamster, betel nut

induced precancerous lesions or carcinoma in the

mouth and forestomach.273 PA-rich wine and grape

juice,274 but not tea and tannic acid,275 betel fruit

extracts276 or pure PAs,277 were shown to be muta-

genic in the Ames test. Various reports also showed

carcinogenic properties of tannic acid or PA-rich

extracts when administered as subcutaneous injections

to rats,278±280 but failed to show any effect when given

in drinking water.278,281 Similar observations have

been made with quercetin, which is highly mutagenic

in the Ames test but not carcinogenic in animals. This

has been tentatively explained by dose±response

effects282 and by metabolic inactivation and in

particular by O-methylation of its redox-cycling o-

dihydroxyphenyl group.283 Although no direct evi-

dence of a similar metabolism of PAs in animals or

humans has so far been provided, the formation of

mono- and dimethylated derivatives of the B3 pro-

cyanidin dimer by a cell-free liver extract prepared

from a human biopsy in the presence of a methyl donor

has been reported164 and would contribute to limit the

toxicity of PAs.

The association of betel nut consumption and

oesophageal cancer could then be explained, either

by the exceedingly high amount of PA intake with

regular chewing of betel nut, by the existence of

confounding factors (social class, smoking, etc) or by

the presence of other non-identi®ed carcinogenic

compounds. It is noteworthy that betel nuts also

contain large amounts of alkaloids284 that might also

be carcinogenic. Sorghum rich in PAs has also been

claimed to explain cases of oesophageal cancer.285

However, this is contradictory to ecological studies

showing a low risk of oesophageal cancer in regions

where sorghum is a main staple.286,287 Several epi-

demiologists reported a positive association between

the consumption of tea and oesophageal cancer. It was

later found that this is not explained by the intake of

tea polyphenols or other tea compounds but by the

high temperature of hot tea.258,260 Taken together,

these data show that PAs per se, as well as tea or wine

phenolic polymers, are probably not carcinogens but

more likely anticarcinogens in humans.

PROANTHOCYANIDINS, TANNIN-LIKECOMPOUNDS AND PREVENTION OFCARDIOVASCULAR DISEASESAn association between PA consumption and cardio-

J Sci Food Agric 80:1094±1117 (2000)

Proanthocyanidins and tannin-like compounds

vascular diseases (CVDs) has never been examined in

humans, but several epidemiologists have studied the

effects of the consumption of wine and black tea rich in

tannin-like phenolic polymers on CVDs.

Wine and tea polyphenols and prevention ofcardiovascular diseasesThe few existing data on black tea were reviewed by

Tijburg et al. 288 Three of the ®ve studies showed a

protective effect against CVDs, whereas the remaining

two showed either no association or a detrimental

effect. Such discrepancies were believed to be due to

the existence of residual confounding factors. The

protective effect of the moderate consumption of wine

against CVDs has received more attention. A moder-

ate consumption of alcohol has consistently been

associated with a lower risk of coronary heart disease

(CHD).289±291 In all ecological292±295 and in some

prospective296,297 and case control298,299 studies, wine

was found to be more protective than beer and spirits.

These data have been critically discussed by Rimm etal,295 who underlined the potential bene®ts of beer

and spirit (and not only wine) consumption and the

limitations of ecological studies; the strong inverse

association observed with wine consumption by some

authors might also be explained by differences in

drinking patterns or lifestyle. These last epidemio-

logical data thus suggest that alcohol consumption

limits CVD risk but do not exclude an additional role

of the polyphenol fraction.

Consumption of PAs or polyphenol-rich wine and

tea has been associated with several CVD risk factors

such as blood pressure and plasma cholesterol level.

Regular tea consumers (over 5 cups per day) in a

Norwegian cohort had a lower systolic blood pressure

and serum cholesterol concentration (6±9mgdlÿ1

reduction) than those consuming less than a cup per

day.300 An even greater reduction of serum cholesterol

in tea drinkers (18±29mgdlÿ1) was observed in a

cohort study carried out in Israel.301 In mice and rats

fed high-cholesterol diets, tea, (�)-catechin and grape

seed PAs reduced the serum cholesterol level.302,303

However, all randomised trials failed to show any

effects of black or green tea intake on blood pressure

and serum cholesterol.304±308 Effects of polyphenol

intake could be masked by the simultaneous presence

of hypercholesterolaemic caffeine309,310 in tea or by

differences in genotypes and in particular in apolipo-

protein E genotypes.307

A protective role of PAs and wine and tea

polyphenols against CVDs has also been suggested

by various experimental studies. The two most largely

studied mechanisms that may prevent atherosclerosis

are the inhibition of LDL oxidation218 and the

inhibition of platelet aggregation.311 PAs and tannin-

like phenolic polymers may contribute to both mech-

anisms.

Protectants against LDL oxidationAs shown above, PAs and tannin-like compounds

J Sci Food Agric 80:1094±1117 (2000)

inhibit LDL oxidation in vitro, but such results cannot

be extrapolated to humans as most PAs and tannin-

like compounds in their native form do not reach inner

tissues. However, intake of either red wine,312±314

dealcoholised red wine,315 red wine polyphenols316 or

tea317 by volunteers increases either the AOC of

plasma in the hours following ingestion or the basal

AOC after regular ingestion for up to 4 weeks. Plasma

AOC values re¯ect the capacity of plasma to scavenge

an exogenous radical or endogenous radicals such as

lipid peroxyl radicals produced upon addition of a

radical chain reaction initiator.318 An increase in

plasma AOC thus shows that some of the ingested

polyphenols have been absorbed and that their

reducing capacity is at least partially preserved.

Expectedly, such an increase in plasma AOC was not

observed when white wine314,315 or vodka319 relatively

poor in polyphenols was ingested in place of red wine.

LDL oxidability has been measured ex vivo after

ingestion of wine or tea or of polyphenols isolated from

these beverages. Only one such study in which

volunteers consumed a glass of red wine with lunch

and supper meals showed an increase in the lag phase

of copper(II)-promoted LDL oxidation; no delay in

LDL oxidation was observed after white wine con-

sumption.320 The authors suggested that some of the

red wine polyphenols had been integrated in LDLs

and contributed to inhibit their oxidation. These

results have been critically discussed by de Rijke etal, who could not observe any protection of LDLs even

after consuming 5 glasses of red wine per day for 4

weeks.321 Several other similar studies with red

wine,322 red wine polyphenols316 or tea poly-

phenols306 led to the same conclusions. This absence

of effects was explained by an insuf®cient absorption

of red wine or tea polyphenols.321 A more likely

explanation is that polyphenol metabolites, largely

conjugated, are too polar to be incorporated into

LDLs and are thus lost when LDLs are isolated for exvivo oxidation measurements. Polar polyphenol

metabolites present in the plasma could protect LDLs

as ef®ciently as ascorbic acid323 without being

incorporated in the lipoprotein.

Other authors have measured the oxidative state of

plasma (rather than the ex vivo oxidability of LDL)

sampled after volunteers had drunk tea and wine. This

procedure gives an integrated view of the physiological

status of the plasma as well as some indirect evidence

of polyphenol bioavailability. With one exception in

which the a-tocopherol level in LDLs was higher after

consumption of red wine polyphenols,316 all other

volunteer studies with wine and tea failed to show any

clear effect on either sparing of endogenous anti-

oxidants315,321 or any reduction in the levels of

oxidised lipids.306 This failure might be explained by

the short duration of the volunteer experiments, as the

concentration of oxidised lipids in plasma, liver and

kidney of rats was signi®cantly reduced when the rats

were fed with tea for 19 months.324 Altogether these

results give support to protective effects of wine and

1107

C Santos-Buelga, A Scalbert

tea polyphenols against tissue lipid oxidation. More

data on the bioavailability of tea and wine polyphenols

are still needed to evaluate the relative contribution of

low-molecular-weight phenolic compounds and phe-

nolic polymers.

Inhibition of platelet aggregationThe second mechanism by which PAs and tannin-like

polymers may prevent CVDs is inhibition of platelet

aggregation. In vitro data in the literature are contra-

dictory. Cotton bract PAs induced the release of

platelet serotonin and stimulated platelet aggregation

in the absence of any other inducers, a mechanism

which could contribute to the pulmonary symptoms

associated with byssinosis in cotton workers.325,326

However, these agonist effects may not be con®rmed

in vivo for the following reasons: the doses were much

higher (260mM catechin equivalent) than those reason-

ably expected in the plasma; two PA dimers, the most

bioavailable PAs, were ineffective;327 when platelet-

rich plasma was used in lieu of washed platelets,

platelet activation was decreased over 50-fold.328 On

the contrary, the structurally related EGCG at a

similar concentration (660mM) did not activate plate-

lets but inhibited their aggregation when induced by

collagen, thrombin or the platelet activating factor.329

Experiments were carried out on animals or

volunteers given either red wine, white wine or grape

juice in order to determine the respective contribu-

tions of alcohol and wine polyphenols to the inhibition

of thrombosis. In rats, red wine and alcohol-free red

wine increased the bleeding time and reduced the

thrombus weight as well as the platelet adhesion to

collagen.330 No effect was observed with either white

wine or ethyl alcohol. Administration of red wine or

grape juice intragastrically also eliminated the cyclic

¯ow reductions in the stenosed coronary arteries of

dogs and also inhibited the aggregation of platelets

measured ex vivo after induction with collagen.331

Here again, white wine showed no effect. Red wine,

but not white wine or ethyl alcohol, inhibited the

rebound effect of thrombin-induced platelet aggrega-

tion in rats 18h after withdrawing the alcoholic

beverage from the diet.332 Such a platelet rebound

effect is known to provoke stroke in the hours

following episodes of drunkenness. Grape juice, but

not orange or grapefruit juice, when fed to monkeys for

7 days, also inhibited the collagen-induced ex vivoplatelet aggregation.333 A crude preparation of grape

polyphenols (as well as ethyl alcohol) fed to rats

inhibited the thrombin-induced platelet aggrega-

tion.334 Black tea exerted a similar inhibition on

platelet aggregation when administered intragastrically

to dogs.288 All these data on animals show that red

wine and tea polyphenols inhibit platelet aggregation

when taken orally.

However, all studies on humans failed to show any

differences in collagen- or thrombin-induced ex vivoplatelet aggregation after red wine or white wine

intake.335,336 Furthermore, ingestion of red wine and

1108

an equivalent quantity of alcohol added to fruit juice

similarly inhibited the collagen-induced platelet aggre-

gation, whereas dealcoholised red wine had no

effect.337 The reasons for these discrepancies between

animal and human studies still need to be clari®ed.

CONCLUSIONSBiological effects of PAs and tannin-like phenolic

polymers commonly consumed by humans are ex-

pressed at two different sites in our body: either in the

lumen of the digestive tract or in the inner tissues. In

the gut they trigger adaptative responses (secretion of

various endogenous proteins and biliary acid) which

limit their adverse effects. They in¯uence the bioavail-

ability of some nutrients. The only one for which there

is clear evidence of their effects is iron. PAs are one of

the main compounds in our diet affecting iron

bioavailability; an excessive PA consumption may

cause anaemia in people at high risk of iron de®ciency,

especially as therapeutic iron supplements usually

consist of inorganic iron. On the other hand, they

may be effective to treat iron overload. Their in¯uence

on aluminium absorption is not as well documented

and deserves further attention. The effects on protein

digestibility, clear in animals consuming larger

amounts of PAs, has no established nutritional

signi®cance in humans.

PAs and tannin-like phenolic polymers may con-

tribute to prevent the cancers of the digestive tract.

The recent results of Gronbaek et al on cancers of the

upper digestive tract265 showing protective effects of

red wine that cannot be attributed to alcohol are very

encouraging. They show that phenolic polymers

directly in contact with the digestive mucosa may well

have an anticarcinogenic action.

Anticarcinogenic effects of PAs and tannin-like

compounds may not be limited to the digestive tract.

Both in vitro and animal studies showed that PAs and

wine and tea polyphenols prevent different cancers of

inner organs. However, epidemiological studies have

so far failed to demonstrate any clear association

between tea and wine consumption and the prevention

of such cancers. This is possibly explained by the

existence of confounding factors (mode of consump-

tion, lifestyle, presence of caffeine in tea, etc).

Tannin-like compounds in tea and red wine may

also well prevent cardiovascular diseases. They in-

crease the plasma antioxidant capacity and probably

inhibit oxidation of LDLs (the apparent contradictions

in the results obtained by different authors are likely

explained by the methodology used). They reduce the

systolic blood pressure and the level of plasma

cholesterol, at least in animal and cohort studies.

However, this has not been con®rmed in randomised

trials. They inhibit platelet aggregation both in vitroand in animal experiments and may thereby prevent

thrombosis. However, this last observation could not

be con®rmed in human studies.

PAs and tannin-like compounds might thus prevent

J Sci Food Agric 80:1094±1117 (2000)

Proanthocyanidins and tannin-like compounds

both cancers and cardiovascular diseases. The respec-

tive properties of polyphenols and non-phenolic

compounds present in foods and beverages (eg

caffeine in tea or alcohol in wine) are beginning to be

clari®ed. It remains dif®cult to determine the precise

nature of the polyphenols responsible for these effects.

They could also be attributed to the presence of low-

molecular-weight polyphenols or to only a fraction of

PAs and tannin-like compounds of lower molecular

weight and more easily absorbed through the gut

barrier. We believe that PAs and tannin-like com-

pounds account for a major part of the polyphenols

consumed daily. However, both their intake and

bioavailability in humans remain largely unknown

owing to analytical limits imposed by the complexity of

their chemical structures. It is essential when con-

sidering a complex product such as a wine, tea or fruit

juice to determine the nature of the polyphenols

absorbed and biologically active.

There is also a paucity of human studies. More are

needed to determine the origin of the contradictions

observed between such studies and animal or in vitrostudies. Hopefully we will discover that such contra-

dictions are explained by the limits of the experimental

protocols applicable to volunteers. It is under these

conditions that we will obtain de®nitive proofs of their

role in disease prevention which will form a sound base

needed to design new food products with real health

bene®ts.

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