Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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0030 The indicator used must have a color at the pH of

the test solution intermediate between the acidic and

basic forms.

0031 The approximate pH of the solution is first dete-

mined using a ‘universal,’ or full-range, indicator,

when an appropriate indicator for the measurement

can be chosen. A quantity of the test solution is

measured into a tube and a measured amount of

indicator added. Tubes of buffer solutions covering

the pH range of the indicator are treated in the same

way. The colors of the tubes are viewed through the

length of the tube against a white background. The

accuracy of the method will depend on the differences

of pH of the buffer solutions. These are typically 0.2,

and the pH of the unknown solution can then be

measured to within +0.1.

0032 The Lovibond Comparator The need to use buffer

solutions is eliminated by using a Lovibond 2000

comparator, in which the color of the test solution is

compared with colored glasses. Standard colored

glass rings are fitted into a disk that can be rotated

and the colored glasses compared with the test solu-

tion. For accurate results, the sample solution and

discs must be viewed against north daylight or in

white light. Indicator discs for 14 different indicators

are available. Special disks are available for the deter-

mination of the pH of blood.

0033 With the Nesslerizer attachment, the solution is

viewed through the depth of the liquid instead of

through its thickness. The proportion of indicator

therefore can be considerably reduced, resulting in

a significant gain in accuracy, particularly when

unbuffered or slightly buffered solutions are being

tested.

0034 Sources of error There are a number of possible

sources of error when making colorimetric

measurements.

0035Slightly buffered solutions. As indicators are

weak acids or bases they will change the pH of

the solution if it is unbuffered or only slightly

buffered. Samples of relatively pure water, and

solutions of a salt of a strong acid and strong

base are the most frequently encountered solutions

of this type. It is recommended that an appropriate

pH meter is used for such solutions.

0036Salt effect. As discussed earlier, equilibrium con-

stants depend on the activities of the components

rather than concentrations, and it is only in very

dilute solutions that activity equals concentration.

Buffer solutions normally used have ionic

strengths of up to 0.2 M. If the solution under

test has a lower ionic strength than the buffer, the

determined pH will be too small. The salt correc-

tion that must be applied is also affected by the

types of ion present and the indicator used.

0037Effect of colloids. Colloid particles in the solution

may preferentially absorb either the acid or the

alkaline form of the indicator, giving totally

erroneous results.

0038Effect of proteins. pH values obtained in the

presence of proteins are unreliable. The effect of

a protein on the determination depends on both

the type of protein and the indicator used. Usually,

the effect is greater when the protein is on the acid

side of its isoelectric point.

0039Effect of finely divided particles. Chemical reac-

tions may occur between the indicator and

particles in the solution, giving erroneous results.

0040Colored solutions. Color comparison is not

possible if the test solution is colored.

0041Nonaqueous solvents. Literature data on indica-

tors apply to aqueous solutions; the indicator equi-

libria, hence the color, will be different in other

solvents. The color of an indicator in, say, a water–

alcohol mixture should be taken as only a rough

guide to the true pH.

tbl0002 Table 2 Some pH indicators and their color changes and ranges

Indicator Color in acid solution Color inalkaline solution pHrange pK

Thymol blue (acid) Red Yellow 1.2–2.8 1.7

Bromophenol blue Yellow Blue 2.8–4.6 4.0

Methyl orange Red Yellow 3.1–4.4 3.7

Methyl red Red Yellow 4.2–6.3 5.1

Litmus Red Blue 5.0–8.0

Bromothymol blue Yellow Blue 6.0–7.6 7.0

Phenol red Yellow Red 6.8–8.4 7.9

Thymol blue (base) Yellow Blue 8.0–9.6 8.9

Phenolphthalein Colorless Red 8.3–10.0 9.6

Universal

Red, orange, yellow Green, blue, violet 3.0–11.0

Full-range

Red, orange, yellow Green, blue, violet 1.0–14.0

These are mixtures of indicators; they give a continuous color change over a wide pH range.

PH – PRINCIPLES AND MEASUREMENT 4505

Electrochemical Method

0042 In order to measure pH by the electrochemical

method, the emf of a reference cell is determined

(see earlier). In this cell, the potential of one electrode

(the hydrogen electrode) changes with pH, and the

potential of the second, reference, electrode does not.

Historically, the hydrogen gas electrode was used as

the pH electrode, but the discovery by Cremer in

1906 that the potential difference across a glass mem-

brane depends on the H

ion concentration on either

side of it led to the development of the glass electrode.

This is the electrode that is now always used for

measuring pH.

0043 Glass electrode In its simplest form, the glass

electrode consists of a thin glass bulb containing a

solution of constant pH, usually hydrochloric acid or

a phosphate buffer with potassium chloride. In this

solution is an electrode, usually silver coated with

silver chloride.

0044 The exchange of ions in the glass membrane for H

ions in the solutions on either side of it to form silanol

(

—

Si-OH) layers is the major factor in determining

the pH response of the electrode, and special sodium

and lithium glasses are used. As this surface layer is

crucial to the correct functioning of a glass electrode,

the pH-sensitive tip is stored in a pH 7 buffer solu-

tion, preferably containing 0.1 mol dm

3

potassium

chloride. As it is most important that the tip does not

dry out, commercial electrodes are supplied with a

storage teat containing the buffer plus potassium

chloride solution.

0045 Glass electrodes give a good linear response in the

pH range 2–9, and up to pH 14 if the salt concen-

tration in the test solution is not too high.

0046 Combination electrodes Historically, the separate

glass and reference electrodes were placed side by

side in the test solution. Such electrodes are still avail-

able but are now used for special applications. Almost

all pH measurements are made with combination

electrodes in which the glass and reference electrodes

are combined together. Although there are many

details that vary from one electrode to another, the

basic design is as shown in Figure 1. The glass mem-

brane is at the bottom and is surrounded by the

reference electrode. This is usually a silver/silver

chloride electrode (silver wire coated with silver

chloride in saturated potassium chloride solution),

and the reference–test solution junction is at a porous

glass frit near the bottom of the electrode. The potas-

sium chloride in the reference cell slowly bleeds

through the frit into solutions being tested and must

be topped up, when necessary, through the filling hole

near the top of the electrode.

0047For many purposes, this electrode is perfectly satis-

factory, but under adverse conditions, the positive

flow of reference electrolyte is reversed, and the

sample solution contaminates the reference cell. This

can be avoided by using a double-junction reference

system in which the reference cell is connected

through a glass frit with an intermediate electrolyte,

which is in turn in contact, through a glass frit, with

the test solution. Such an electrode also eliminates

clogging owing to precipitation of silver chloride at

the glass frit. Also, the intermediate electrolyte can be

changed if that being used reacts with the test solution

at the glass frit junction.

0048Special glass electrodes

Amplified electrode The major experimental

difficulty in measuring the emf of a pH cell is the

high electrical resistance of the glass membrane, com-

monly 10–500 MO. This requires a high-impedance

millivoltmeter and short, screened connections to the

electrode. One solution to this problem is a glass

electrode with a built-in amplifier (Hanna Instru-

ments), where the high-impedance circuitry is in an

integrated circuit encapsulated at the top of the elec-

trode together with a mercury battery. This electrode

is ideal for industrial pH measuring and monitoring

Filling

hole cover

Glass (pH)

electrode

element

Glass (pH)

electrode

electrolyte

pH-sensitive glass bulb

Reference

junction

Reference

electrolyte

Reference

electrolyte

filling hole

Reference electrode

element

fig0001Figure 1 Combination pH electrode. Reproduced from pH:

Principles and Measurement, E ncycl opaed i a of Food Science,

Food Technology and N utrition, Macrae R, Robinson RK and Sadler

MJ (eds), 1993, Academic Press.

4506 PH – PRINCIPLES AND MEASUREMENT

as long, unshielded cables can be used from the

electrode to the pH meter.

0049 Other electrodes pH glass and combination elec-

trodes of many types are available; these include

electrodes in plastic bodies (for durability), micro-

electrodes for small volumes of test solution, elec-

trodes resistant to proteins, and flat electrodes for

pH measurement at surfaces.

0050 pH meters A pH meter is a high-impedance milli-

voltmeter that is designed to convert millivolts to pH.

There are many types available at a range of prices.

Temperature compensation is important, and many

meters have a temperature probe facility for this pur-

pose. Many meters contain microprocessors and are

very easy to use.

0051 To make a pH measurement, it is necessary to

calibrate the meter first. This is done by using two

buffer solutions that, ideally, span the pH of the test

solution. After rinsing the electrode, this is placed in

the test solution and the pH read from the meter.

0052 Accurate pH measurements can be made without

difficulty on test solutions containing dissolved acids,

bases or salts. If, however, the sample is virtually pure

water (e.g., tap, rain, or boiler-feed water) pH meas-

urements are unreliable. This is because (1) the glass

electrode requires a long time to stabilize in a low-

ionic-strength solution; (2) the solution has a

low buffering capacity and is therefore susceptible

to drift as atmospheric carbon dioxide is absorbed;

(3) the low conductivity of the solution allows the

pick-up of electromagnetic noise; and (4) there is

electrical noise created at the reference junction.

Special low-conductivity electrode buffer kits have

been developed (Russell pH Ltd) for such measure-

ments. In these kits, the glass electrode is of extremely

low resistance, and the calibration errors are reduced

by using a buffer of low ionic strength.

0053Very small, portable pH meters are now available. A

good example is the Piccolo (Hanna Instruments).

This meter weighs only 100 g and uses an interchange-

able amplified electrode in a plastic container. It has a

built-in temperature sensor that compensates for tem-

peratures from 0 to 70



C, and it measures pH in the

range 1–13 with an accuracy and resolution of +0.01.

See also: Canning: Principles; Colloids and Emulsions;

Spectroscopy: Visible Spectroscopy and Colorimetry;

Spoilage: Chemical and Enzymatic Spoilage; Molds in

Spoilage; Yeasts in Spoilage

Further Reading

Anonymous (1987) pH Values, 8th edn. Poole, UK: BDH.

Bates RG (1973) Determination of pH. Theory and

Practice, 2nd edn. New York: John Wiley.

Covington A (1989) pH and its Measurement. London:

Royal Society of Chemistry (a chemistry cassette and

workbook).

Galster H (1991) pH Measurement (Fundamentals,

Methods, Applications, Instrumentation) New York:

Wiley-VCH.

PHENOLIC COMPOUNDS

M Murkovic, Graz University of Technology, Graz,

Austria

Background

0001 Polyphenols play an important role in plants as well

as in foods. The main uses of polyphenols in foods are

as colorants and antioxidants. A large amount of

research on polyphenols has focused on their anti-

oxidant properties, since they are thought to have

positive effects on chronic degenerative diseases

(cataracts, age-related macular degeneration, central

neurodegenerative diseases, and diabetes mellitus),

cardiovascular disease, and cancer. Polyphenols are

also used as antioxidants in the food industry to

increase shelf-life, which can be limited due to deteri-

oration because of oxidation reactions, especially fat

oxidation. Not only is oxidized food a problem

because of the resultant rancid aroma. It can also

have implications on human health, since increased

exposure to free radicals can also increase the risk of

these degenerative diseases.

0002The several thousand polyphenols that have been

described in plants can be grouped in several classes.

Distinction between these classes is drawn first on

the basis of the number of constitutive carbon atoms

and then in the light of the structure of the basic

skeleton. In addition, besides simple soluble forms

mainly found in vacuoles, there are also polymerized

forms of varying solubility like the tannins or the

completely insoluble lignins. The groups of polyphe-

nols dealt with in this chapter comprise anthocyanins,

PHENOLIC COMPOUNDS 4507

flavan-3-ols, isoflavones, flavones and flavonols,

hydroxybenzoic acids, and hydroxycinnamic acids.

Anthocyanins

0003 Anthocyanins are the most important group of water-

soluble plant pigments visible to the human eye.

With a few exceptions, e.g., betalains, they are

universal plant colorants and largely responsible for

the cyanic colors of flower petals and fruits. They

may also occur in roots, stems, leaves, and bracts,

accumulating in the vacuoles of epidermal or subepi-

dermal cells.

0004 The main positions of hydroxylation are 3, 5, and 7

in the A ring and 3

and 5

in the B ring (see Figure 1).

Methoxyl groups can be attached to 3

and 5

.In

some cases, the 3-hydroxyl group is removed, and a

few structures are known that have a hydroxyl group

attached to C-6 of the A ring. The anthocyanidins

that are commonly found in fruits are pelargonidin,

cyanidin, delphinidin, peonidin, petunidin, and mal-

vidin. These are all hydroxylated at the positions 3, 5,

and 7. Normally, anthocyanins occur as 3-monosides,

3-biosides, and 3-triosides as well as 3,5-diglycosides

and, in some cases, 3,7-diglycosides. The carbo-

hydrates that are attached to the positions 3, 5, and

7 comprise glucose, galactose, rhamnose, arabinose,

and xylose.

0005 An example of a glycoside is cyanidin 3-rutinosid

(6-O-a-l-rhamnosyl-d-glucose), which is very com-

mon in fruits, most commonly associated with

cyanidin 3-glucoside. Other glycosides are cyanidin

3-sambubioside (2-O-b-d-xylosyl-d-glucose), which

is present in elderberry, redcurrant, and black

raspberry, and cyanidin 3-sophoroside (3-O-b-d-

glucosyl-d-glucose), the main pigment in raspberries,

loganberries, boysenberries, and mangosteen. The

latter also occurs in redcurrants and sour cherries,

but as a minor component (see Table 1).

0006 The color-active form of the anthocyanins shows

an absorption maximum at about 520 nm. This is due

to the flavylium cation structure, which occurs only in

an acidic environment. An increase in pH above 4

leads to a substantial fading of color. Other color

changes occur during oxidation of the anthocyanins,

leading to the browning of foods.

Flavan-3-ols

0007Flavan-3-ols are components in the structure of

proanthocyanidins (condensed tannins) as their

monomers. Flavan-3-ols have been obtained from

several tree species (Quercus, Castandea, Acacia,

Eucalyptus, etc.). Four main flavan-3-ols are found

in fruits. Two are orthodiphenols hydroxylated in the

and 4

positions in the B-ring ([þ]-catechin and

[]-epicatechin), and two are trihydroxylated in

the 3

-, 4

-, and 5

-positions ([þ]-gallocatechin and

[]-epigallocatechin). C-2 and C-3 represent two

centers of asymmetry in the molecule in such a way

that the four mentioned flavan-3-ols in fruits are

grouped in two pairs of diastereomers whose absolute

configurations are as follows: 2R in all cases and

either 3S for (þ)-catechin and (þ)-gallocatechin or

3R for ()-epicatechin and ()-epigallocatechin (see

Figure 2).

0008Unlike in other classes of flavonoids, monomeric

flavan-3-ols are generally found in free rather than

glycosylated or esterified form in fruits (see Table 2).

One exception is the case of the ()-epicatechin-3-O-

gallate identified in grapes. This ester has been found

in several red grape cultivars.

O Glucose

5

3

fig0001 Figure 1 Structure and possible positions of hydroxylation and

methoxylation of anthocyanidins (cyanidin-3-O-glucoside).

tbl0001Table 1 Anthocyanidin content of selected vegetables and

fruits (mg per 100 g fresh weight)

Food Totalanthocyanidin content

Fruits

Blueberry 25–500

Elderberry 300–500

Strawberry 30–50

Cherry, sweet 350–450

Grapes, red 30–750

Vegetables

Cabbage, red 20–40

Onion, red 10–20

5

3

fig0002Figure 2 Structure and possible positions of hydroxylation and

methoxylation of flavans; (()-epicatechin 3-gallate).

4508 PHENOLIC COMPOUNDS

Isoflavones

0009 The isoflavones are a distinctive but large subgroup

of flavonoids. These compounds possess a 3-phenyl-

chroman skeleton that is biogenetically derived by

rearrangement of the flavonoid 2-phenylchroman

system (see Figure 3).

0010 Despite the restricted distribution of the isoflavo-

noids in the plant kingdom, the structural variation is

surprisingly large. This arises not only from the

number and complexity of substituents on the basic

3-phenylchroman system but also from the different

oxidation levels in this skeleton and the presence of

extra heterocyclic rings. Within the flavonoids, the

isoflavones are the largest group of naturally occur-

ring structures.

0011 The enzyme that is responsible for the transform-

ation of the flavones to the isoflavones is named

isoflavone synthase. This enzyme catalyzes the

isomerization of the two flavones (2S)-naringenin

or (2S)-liquiritigenin to the isoflavones genistein and

daidzein, respectively.

0012 Soy foods have received considerable attention for

their role in disease prevention, especially in relation

to heart disease, osteoporosis, and cancer. The re-

search interest in soy focuses on establishing the

physiological effects of isoflavones. The occurrence

of the isoflavonoids is restricted to only a few plants

within the family of the leguminosae and some non-

legume dicotyledons. Soybeans and soyfoods are

practically the only nutritionally relevant dietary

sources of isoflavones. Isoflavones are weak estrogens

in that they bind to estrogen receptors, but they also

have important nonhormonal properties as well. Ini-

tial speculation that soyfoods, and in particular iso-

flavones, might promote bone health was based on

the estrogenic properties of isoflavones. In ovariecto-

mized rodents, isoflavones retard bone loss almost as

effectively as estrogen. Most soyfoods are rich in iso-

flavones and favorably affect bone turnover and

spinal bone mineral density in perimenopausal and

postmenopausal women (see Table 3).

Flavones and Flavonols

0013Flavones are flavonoids characterized by a nonsatu-

rated 3-C chain and have a double bond between C-2

and C-3, like flavonols, with which they differ by the

absence of hydroxyl in the 3-position. It appears that

this simple difference in structure between flavones

and flavonols has very important consequences in the

biogenesis, physiological, and pharmacological roles,

and the phylogenetic and chemotaxonomic significa-

tion of these compounds. Flavones are widely distrib-

uted among the higher plants in the form of aglycones

or glycosides (see Figure 4).

0014The edible portion of some foodstuffs has an un-

usually high concentration of quercetin. The concen-

trations of quercetin analyzed in hydrolyzed samples

from fruits and vegetables are 284–486 mg kg

1

onions, 110 mg kg

1

in kale, 32–45 mg kg

1

in French beans, 30 mg kg

1

in broccoli, 14 mg kg

1

in lettuce, and 8 mg kg

1

in tomatoes. The average

concentration of quercetin in fruits is 15 mg kg

1

with apples having the highest concentration (21–

72 mg kg

1

) (see Table 4). Parsley and thyme are

also major food sources of flavones.

tbl0003Table 3 Content of isoflavones in soy products (mg per 100 g

(ml) fresh weight)

Food Totalisoflavone content

Soya bean 60–400

Tofu 8–70

Soy flour 80–180

Soya milk 3–180

Soy sauce 1–8

tbl0002 Table 2 Content of flavanol monomers in selected vegetables

and fruits (mg per 100 g fresh weight)

Food Total flavan-3-olcontent

Apricot 25

Apple 8

Strawberry 4

Cherry, sweet 22

Grapes (skin) 19

Barley 3–4

Tea 18–35

Flavanols in g per 100 g of dry leaves.

4

5

2

3

fig0003 Figure 3 Structure and possible positions of substitutions of

isoflavone (genistein).

Rutinose

3

fig0004Figure 4 Structure and possible positions of substitutions of

flavones (R ¼OH in flavonols).

PHENOLIC COMPOUNDS 4509

Hydroxybenzoic Acids

0015 Hydroxybenzoic acids have a general structure of the

C6–C1 type derived directly from benzoic acid. Vari-

ations in structure lie in the hydroxylations and meth-

oxylations of the aromatic cycle. Four acids dominate

the family of these compounds: p-hydroxybenzoic

acid, vanillic, syringic, and protocatechuic acids.

The first three are constituents of lignin, from which

they are released by alkaline hydrolysis. Gentisic acid

has a particular pattern of hydroxylation, which can

be related to that of salicylic acid, but it is much less

common. Gallic acid is another well-known plant

acid which participates in the formation of hydrolyz-

able gallotannins. Its dimeric condensation product

(hexahydrodiphenic acid) and the related dilactone,

ellagic acid, are also common plant constituents.

0016 Hydroxybenzoic acids are commonly present in

bound form (see Figure 5). They are the component

of a complex structure like lignins and hydrolyzable

tannins. The content of hydroxybenzoic acids in

foods of plant origin is generally low (see Table 5),

but in some berries and vegetables such as onions and

horse-radish, the content of hydroxybenzoic acids

may be very high.

Hydroxycinnamic Acids

0017Among the fruit phenolics, hydroxycinnamic acid

derivatives play an important role because of both

their abundance and diversity. They all derive from

cinnamic acid and are essentially present as combined

forms of the four basic molecules p-coumaric, caffeic,

ferulic (see Figure 6), and sinapic acids. The free

forms of these acids are very rare in fruits. Two

main types of soluble derivatives have been identified:

first, those involving an ester bond between the carb-

oxylic function of phenolic acid and one of the alco-

holic groups of an organic compound, for example

chlorogenic acid, which has been identified in numer-

ous fruits; second, those involving a glycosidic bond

with one of the phenolic groups of the molecule (e.g.,

p-coumaric acid O-glucoside). The diversity of the

hydroxycinnamic acids encountered in plants and

particularly in fruits thus results from the nature of

the bonds and that of the molecules involved.

0018Hydroxycinnamic acids occur most commonly in

foods of plant origin. Of these, caffeic acid is the

predominant hydroxycinnamic acid in many fruits,

constituting over 75% of total hydroxycinnamic

acids found in plums, apples, apricots, blueberries,

and tomatoes. However, p-coumaric acid is the dom-

inant hydroxycinnamic acid of citrus fruits and pine-

apple. Hydroxycinnamic acids are widely present in

the bound form and are rarely found in free form.

Processing of fruits and vegetables (freezing, steriliza-

tion, and fermentation) contributes to the formation

of free hydroxycinnamic acids in such products (see

Table 6). Chlorogenic acid is found in many foods,

including apples, apricots, berries, peaches, pears,

plums, avocados, and carrots. Chlorogenic acid is

the key substrate for enzymatic browning, particu-

larly in apples and pears. Its content is reduced by

70% during browning of the pear skins.

Analysis of polyphenols

Sample Preparation

0019Prior to extraction, solid food samples have to be

homogenized or crushed using a mortar and pestle.

Soluble phenolic compounds are generally extracted

by means of alcohol–water mixtures. Extraction is

performed using a mixture of ethanol (80%) and

COOH

fig0005 Figure 5 Structure and possible positions of hydroxylation of

hydroxybenzoic acids (gallic acid).

COOH

fig0006Figure 6 Structure and possible positions of hydroxylation of

hydroxybenzoic acids (ferulic acid).

tbl0004 Table 4 Content of flavonols and flavones in selected

vegetables and fruits (mg per 100 g fresh weight)

Food Total flavonol content

Apple 2–4

Apricot 3

Broccoli 9–11

Kale 32–60

Onion 35

Celery, leaf 95

Sweet pepper, red 1

tbl0005 Table 5 Content of hydroxybenzoic acids in selected fruits (mg

per 100 g fresh weight)

Food Totalhydroxybenzoic acid content

Blackberry 8–27

Blackcurrant 4–12

Raspberry 6–10

Strawberry 2–8

4510 PHENOLIC COMPOUNDS

water (20%) of the freeze-dried powder of the food to

be analyzed. Methanol can be used instead of ethanol,

and extraction with methanol gives good results, es-

pecially in the extraction of soluble phenolic com-

pounds from fruits. Oxidation is avoided by

working at low temperatures and, if necessary, by

the addition of antioxidants such as ascorbic acid or

butylated hydroxytoluene. To avoid enzymatic oxida-

tion from polyphenol oxidases, samples may need to

be heated to a temperature of more than 90



C for a

few minutes. These enzymes catalyze the oxidation of

phenols to quinones with subsequent nonenzymatic

rapid polymerization. These oxidases can also be in-

hibited by lowering the pH to below 4.0. Extraction

of anthocyanins is normally carried out under cold

conditions with methanol containing 1% of hydro-

chloric acid to obtain the flavylium cation, which is

stable under acidic conditions.

0020 For analysis of liquid samples like fruit juices, only

dilution, filtration, and, in some cases, clarification

are necessary. Carotenoids that can disturb the analy-

sis should be extracted using nonpolar solvents. To

remove the sugar moieties from the polyphenols,

the glycosides have to be hydrolyzed. The cleaving

off be carried out in acidic, basic, or enzymatic con-

ditions. The hydrolysis of anthocyanins to anthocya-

nidins is often necessary owing to the difficulties of

obtaining anthocyanin standards. This can be done

by refluxing the dry anthocyanins in methanol acid-

ified with 2 M HCl. Alkaline hydrolysis cleaves the

acylated portions of anthocyanins using 2 M NaOH.

During hydrolysis, care has to be taken that no poly-

phenols are destructed or rearrangement takes place

so that artifacts are analyzed.

0021 The water–alcohol extract obtained is a raw ex-

tract containing numerous nonphenolic substances

(carbohydrates, organic acids, proteins, pigments,

etc.). Before these crude extracts can be analyzed by

high-performance liquid chromatography (HPLC),

they should be purified using solid-phase extraction

methods. Chlorophylls and carotenoids can be re-

moved by applying conventional depigmentation

techniques with petroleum ether.

HPLC Analysis

0022The columns used for HPLC analysis in the literature

are almost exclusively reversed phase. Elution

systems are usually binary, with an aqueous acidified

polar solvent such as aqueous acetic acid, phosphoric

acid, or formic acid and a less polar organic solvent

such as methanol or acetonitril. Isocratic elution is

mainly used for routine analysis if only a few poly-

phenols are to be determined.

0023For detection and quantification after separation,

the absorption of ultraviolet or visible light is nor-

mally used. For flavonoids, two absorption bands are

characteristic: the first in the range of 240–285 nm,

which is believed to arise from the A-ring, and the

second with a maximum in the range of 300–550 nm,

which arises from the absorption of the B-ring (see

Table 7). Since the absorption spectrum depends on

the chemical substitution and the conjugated system,

the different groups of flavonoids (flavanons/iso-

flavanons, catechins, flavons/flavonols, etc.) have

distinct absorption bands. The anthocyanins show

absorption above 500 nm, whereas the catechins

have only a small maximum at 280 and no absorption

at higher wavelengths.

0024The combination of liquid chromatography with

mass spectroscopy for the detection of polyphenols

has been increasing in recent years. Mass-selective

detection is especially useful in identifying new struc-

tures and verifying known substances. With a quad-

rupole mainly the unfragmented molecule can be

detected. Using tandem mass spectrometry, the mol-

ecules can be fragmented in a selective way. Thus,

information on the chemical structure of the poly-

phenols can be obtained.

Intake and Bioavailability of Polyphenols

0025In the Western diet, the phenolic acids, catechins,

proanthocyanidins, and anthocyanins dominate

uptake. Especially for epidemiologic studies, it is

necessary to estimate accurately the amount of

tbl0007Table 7 Detections wavelengths of flavonoids

Structure Wavelengthused

foranalysis (nm)

Flavons, flavonols, flavonol glycosides 270

Anthocyanins 500–525

Isoflavones 236, 260, 280

Flavanones and glycosides 280, 290

Catechins 210, 280

tbl0006 Table 6 Content of hydroxycinnamic acid derivatives in

selected fruits (mg per 100 g (ml) fresh weight)

Food Chlorogenic acidisomers

Apple juice 6–20

Beer 0.2–2

Coffee beans, green 6–9 g per 100 g

Coffee beans, roasted 0.2–3 g per 100 g

Hydroxycinnamic acids

Strawberries 1–3

Blackcurrants 5–7

Gooseberries 2–7

Raspberries 2–3

Redcurrants 1–2

PHENOLIC COMPOUNDS 4511

polyphenols consumed. In general, fruits are richer in

polyphenols compared to vegetables. Beverages con-

tribute significantly to the uptake of polyphenols. Red

wine, coffee, and black tea contain high amounts of

polyphenols. Green tea, which also contains large

amounts of these substances, is consumed in East Asia

in high amounts. The uptake of polyphenols is esti-

mated to be 50–200 mg per meal. The phenolic acids

are constituents of coffee, fruits, vegetables, and

cereals. Flavanols, which occur predominantly in

onions,apples,andotherfruitsandvegetables,contrib-

ute about 20 mg per day. Catechins are provided by tea

and various fruits and proanthocyanidines by various

fruits, legume seeds, chocolate, and red wine. Flava-

nones are found in citrus fruits. The total uptake of

flavonoids in the Western diet is estimated to be about

1000 mg per day. Examples of the distribution and

quantity of flavonoids consumed daily include: 44 mg

from cereals; 79 mg from potatoes, bulbs, and roots;

45 mg from peanuts and nuts; and 162 mg from vege-

tables and herbs. The largest portion of flavonoid

intake comes from cocoa, cola, coffee, tea, beer and

wine (420 mg per day), with an additional 290 mg per

day from fruits and juices.

0026 The uptake of 100 mg of polyphenols results in a

concentration of about 300 mM in the gut. Normally,

the plasma levels will not exceed 1 mM. Urinary ex-

cretion varies from less than 1% to about 25%

depending on their gut absorption, enterohepatic

cycling, and metabolism. In particular, the type of

polyphenol and the glycosylation influence bioavail-

ability. The uptake of proanthocyanidines is limited

to monomers and dimers. A higher degree of poly-

merization results in a sharp decrease in bioavailabil-

ity. The metabolism of polyphenols in the small

intestine is mainly via glucuronidation, sulfatation,

and methylation. The colon microflora contributes to

the metabolism of the polyphenols. This has been

investigated in detail for several types of polyphenols,

and especially for quercetin. Most flavonoids enter the

diet as glycosides, with quercetin and rutin being the

most common flavonoid glycosides consumed. The

hydrophilic nature and their relatively high molecular

weight preclude absorption in the small intestine. Fur-

thermore, flavonoid b-glycosides resist intestinal

hydrolases, and consequently, flavonoid glycosides

can pass unaltered into the large intestine. The resident

microflora of the bowel produces glycosidases capable

of releasing the aglycone from its sugar. In addition,

the resident microflora can cleave the pyrone ring (ring

C), producing phenyl acetic and phenyl propionic

acids and other derivatives. But since glycosidase ac-

tivity proceeds at a faster rate than ring cleavage, the

intact flavonoid aglycone can persist in the large intes-

tine with a clear potential for absorption.

Polyphenols as Antioxidants

0027It is well accepted that free-radical-mediated pro-

cesses can lead to chronic degenerating diseases. The

oxidative damage can be found on molecular level as

oxidized lipids, proteins, or DNA. These can be

detected, especially in patients with atherosclerosis,

certain cancers, neurodegenerative diseases, and lung

disorders. Many types of reactive oxygen species

(ROS) and reactive nitrogen species (RNS) have

been shown to induce a certain type of damage that

is associated with disease development. These include

the superoxide radical, hydrogen peroxide, hydroxyl

radical, lipid alkoxyl and peroxyl radicals. Protection

against damage induced by ROS/RNS can be

achieved in several different ways:

0028suppression of free-radical formation by antioxi-

dants;

0029scavenging of radicals by antioxidants to inhibit

initiation of chain reactions and slow down chain

propagation;

0030repair mechanisms;

0031transition metal chelation.

The polyphenols have structural features that charac-

terize their antioxidative potential. This is the presence

of hydrogen-donating substituents and the ability to

delocalize the resulting free electron. Owing to the

delocalization of the electron, the resulting radical

does not have sufficient energy for further radical reac-

tions. The antioxidative properties of flavonoids and

other phenolics from natural sources are assessed by

determining their activity as scavengers of radicals that

arisefrom lipidoxidation or otherbiological processes.

For evaluation of the antioxidant properties of flavo-

noids, the chemical structure can be related to the

activity. Three structural groups are important deter-

minants for radical scavenging: (1) the catechol struc-

ture (3

-dihydroxy) in the B ring, which is the

obvious radical target site; (2) the 2, 3 double bond in

conjugation to the 4-keto function, which is respon-

sible for electron delocalization; and (3) the presence of

both hydroxyl groups (the 3-OH and the 5-OH) for

maximal radical-scavenging potentials. If all of these

structural features are present, a maximum radical-

scavenging potential would be expected.

0032Depending on the method of measuring the anti-

oxidative potential, relative reactivities of structurally

related polyphenols change. This can be seen espe-

cially in low-density-lipoprotein (LDL) oxidation ex-

periments, in which quercetin and luteolin are strong

antioxidants, that protect LDL with high efficiency,

whereas kaempferol does not. In particular, if chela-

tion of metal ions is the protecting mechanism, quer-

cetin is a very strong antioxidant.

4512 PHENOLIC COMPOUNDS

0033 A simple chemical assay broadly applicable in de-

termining the hierarchy of antioxidant activities of

polyphenols measures their ability to reduce the

ABTS-radical cation (2,2

-azinobis (3-ethylben-

zothiazoline-6-sulfonate)). With this method, the

relative ability of antioxidant or radical-scavenging

substances to scavenge the ABTS cation radical in the

aqueous phase is compared to a standard amount of

the synthetic antioxidant Trolox, the vitamin E

analog. Incubation of ABTS

oþ

with peroxidase (met-

myoglobin) and hydrogen peroxide results in the pro-

duction of the radical cation (ABTS

oþ

). This species is

blue–green in color and can be detected at 734 nm.

Antioxidants or radical scavengers in the added

sample suppress this color production to a degree

that is proportional to their concentration. In this

method, the activity of tested substances is expressed

as an equivalent of the millimolar concentration of a

Trolox solution. Table 8 shows the antioxidant activ-

ities of a range of flavonoids in comparison with the

reduction potentials of the flavonoid radicals. It can

be seen that the measured reducing properties of the

phenolics, in terms of their antioxidant activities in

this particular assay system, are consistent with the

reduction potentials of their radicals.

0034 The range of reduction potentials of selected flavo-

noid radicals, relative to those of ascorbic acid and

a-tocopherol, is shown in Table 8, from the more

oxidizable quercetin to kaempferol. These values re-

flect the ability of hydrogen-donating antioxidants to

scavenge the ABTS

oþ

radical cation, absorbing in the

near-infrared region (650–820 nm) compared with

that of ascorbic acid (E

) and Trolox equivalent

antioxidant activity (TEAC). Antioxidants reduce

the absorbance to an extent dependent on the anti-

oxidant activity. Spectral studies have revealed that

the radical site is on the B ring and that the A ring has

no influence on the properties of the radicals from the

B ring in flavonoids with a saturated C ring, such as

catechin. In flavone radicals in which the B ring with

-dihydroxycatechol structure is conjugated

through the C ring 2, 3-double bond, evidence sug-

gests that the radical is on the catechol B ring and that

spectra resemble those of 3,4-dihydroxycinnamate

radicals. The antioxidant activity of flavonoids

depends critically on the part of the molecule with

more efficient electron-donating properties. In most

flavonoids, this is the B ring. Thus, the antioxidant

activity of flavonoids as electron or hydrogen donors

relates to the reduction potentials and reactivities of

the substituent hydroxyl groups. A number of chem-

ical studies on the reactivity of flavonoids with a

range of radicals and the stability of the resultant

antioxidant radicals have also emphasized specific-

ally the role of the catechol structure in the B ring

and unsaturation in the C ring.

Conclusion

0035Substantial epidemiological evidence links high anti-

oxidant status in human populations with a low risk

of degenerative disease. The consumption of large

amounts of fruit and vegetables has been shown un-

equivocally in a considerable number of studies to be

associated with a lowered risk of several kinds of

cancer in a number of different body sites. It remains

to be proven with certainty whether this effect is due

to the content in the fruits and vegetables of antioxi-

dant substances, which are the protective agents, but

substantial circumstantial evidence points to the like-

lihood that antioxidant nutrient and nonnutrient sub-

stances in fruit and vegetables are the most important

anticarcinogenic factors in these foods. Besides

the classical antioxidants vitamin C, vitamin E, and

b-carotene, many phenolic substances are also pre-

sent in foods, including the flavonoids, which may

also contribute to the total antioxidant potential of

the diet and thus may lower the risk of cancer. Similar

considerations apply to arteriosclerosis, with its car-

diovascular and cerebrovascular risk connotations; it

has been clearly shown that antioxidant nutrients can

lower the oxidation of low-density lipoproteins in the

vascular wall, which is thought to be a major early

factor in the development of arteriosclerosis. It is also

possible that antioxidants can play an important role

in delaying the thrombotic events associated with the

onset of heart attack or stroke, and there is thus a

possibility that antioxidants have a dual effect in

lowering the risk of vascular accidents. If it is proven

that antioxidants can lower the risk of these degen-

erative diseases, it is vital that any enhancement of the

diet with antioxidants is safe and free from the risk of

any undesirable side-effects.

See also: Antioxidants: Natural Antioxidants; Role of

Antioxidant Nutrients in Defense Systems;

Chromatography: High-performance Liquid

Chromatography; Soy (Soya) Beans: Properties and

Analysis; Soy (Soya) Cheeses; Soy (Soya) Milk; Soy

(Soya) Bean Oil

tbl0008 Table 8 Reduction potentials (E

) of flavonoid radicals and the

TEAC

Quercetin 0.33 4.7

Epigallocatechin gallate 0.43 4.8

Epigallocatechin 0.42 3.8

Taxifolin 0.5 1.9

Catechin 0.57 2.4

Rutin 0.6 2.4

Kaempferol 0.75 1.3

PHENOLIC COMPOUNDS 4513