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Flavonoid Compounds in Foods

BY E. C. BATE-SMITH

Low Temperature Research Station, University of Cambridge, and Department of Scientific and Industrial Research, Cambridge, England

CONTENTS Page

. . . . . . . . . . . . . . . . . . . . . . 267

1. Properties Depending upon Their General Phenolic Character.. . . . . . . 268 a. Destruction of Ascorbic Acid.. . . . . . . . . . . . . . . . . . . b. Reactions with Metals.. . . . . . . . . . . . . . . . . . . . . . . . . . . c. Antioxidant Action. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

a. Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

c. Pharmacological Action. . . . . . . . . . . . . . . . . . . . . . . 275

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 111. The Genetic Situation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

1. Genetic Situation in Fruits and Vegetables.. . . . . . . . . . . . . . . . . . . . . . . 280 a. Apples. . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Peaches.. . . . . . . . . . . . . . . . . . . . . . . . . . c. Grapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. Potatoes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 e. Onions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

IV. Systematic Distributiori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 1. Anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

a. Zsoflavones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 2. Anthoxanthins . . . . . . . . . . . . . . . . . . . . . . . . .

3. Catechins and Leuco-anthocyanins. . . . . . . . . . . . .

2. Tea. . . . . . .

c. Fermentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

. . . . . . . . . . . . . . . . . . . . . . . . . .

261

262 E. C. BATE-SMITH

I . INTRODUCTION

The task of reviewing the subject of flavonoid compounds in foods has been made very much easier by the appearance recently of several reviews dealing with particular aspects of the subject. One of these, “Theories of the Biogenesis of Flavonoid Compounds” (Geissman and Hinreiner, 1952), is especially valuable in providing a complete survey of these compounds, in tabular form, and a discussion of their chemical and biological relationships. The definition of flavonoid compounds employed by these authors is as follows: “The flavonoid compounds are characterised by their possession of a Cs-C8-Ce carbon skeleton con- sisting of two aromatic rings linked by an aliphatic three-carbon chain. Chiefly on the basis of the oxidation state of this aliphatic fragment the very large number of compounds included in the flavonoid classification is subdivided into such well-known types as anthocyanins, flavones, chalcones, etc.”

Flavone, the type substance of the whole class, which occurs as a dust on the leaves and stems of certain species of Primula, has the structure (I). The other naturally occurring compounds of this class have a number of phenolic hydroxyl or methoxyl groups substituted in each of the two aromatic rings, usually in certain characteristic patterns (Table I) e.g., apigenin (11). Flavones substituted with hydroxyl specifically in the

6 Co

I

111 IV

0

V VI

FLAVONOID COMPOUNDS IN FOODS 263

3 position are known as flavonols, e.g., quercetin (111). In the flavanones, e.g., naringenin (IV), and flavanonols, e.g., taxifolin (V), the double bond between carbon atoms 2 and 3 is reduced; together with the isoflavones (VI), these four types are sometimes collectively known as anthoxanthins.

The catechins, and probably the leuco-anthocyanins (whose structure is not completely known) are derived from a reduced flavone, i.e., a flavan molecule; catechins, e.g., catechin (VII), being flavan-3-01s. Anthocyanins, in the colored anionic form in which they usually occur, are flavylium salts (e.g., cyanidin, VIII). The anthocyanins may, however, occur in a colorless form, known as a pseudo base, possibly (IX),

+

VII VIII

and the leuco-anthocyanins probably have some structure such as (X), related both to this pseudo-base form of the anthocyanins and to the catechins.

IX X

Closely related to the flavones and anthocyanins are the coumarins (XI) and chlorogenic acid (XII), which may be regarded as derivatives of cinnamic acid. O;:,; Ho(j CHOH

CH-COOCk ‘CHOH I I ‘c< CH, CH2

\ / \ CH

\Ci/OH)COOH

XI XI1

These, although not, properly speaking, flavonoid in structure, have hydroxylation patterns similar to that of the B ring in flavones (Table I) and must therefore be kept in view in any survey of flavonoid compounds.

Cinnamic acids

Coumarm

Anthocyanidin:

Catechins

Flavonols

CH=CH. COOH p-Coumaric

TABLE I The Principal Naturally Occurring Flavonoid, and Some Related, Compounds

H O G

CH=CH.COOH Caffeic

Pelargonidin Cyanidin

I Catechins

Kaempfeml R = H Fisetin I R- OH Quercetin

C H , O G

CH=CH .COOH Ferulic

Scopoletin

Peonidin

Isorhamnetin

CH= CH . COOH Sinapic

R = H Fraxetin R = CH, Isofraxidin

+ OR

HO ooH \ &*OH OR'

OH CH R = R' = H Delphinidin R'-H, R-CHI Petunidin R' = R = CHI h5alvidin

Gallocatechins

K = H Robinetin R - OH hlvricetin

Flavones

Flavanonols

Flavanones

Chalcones

Isoflavones

Apigenin

H O P O o H ,CHOH

OH co Katsurenin

(Aromadendrin)

R - H Liquiretigenin R= OH Xaringenin

HO' ' ? H a o H U c 6 C H

Dahlia chalcone

R = R' - H Daidzin R - OH, R' - H Genistein R = H , R'-CHa Formononetin R - OH, R E CHI Biochanin-A

Luteolin I Chrysoeriol I Tricin

I Taxifolin I Ampelopsin

I R = H Butin Homoeriodictyol R = OH Ericdictyol I

Butein I

Orobin


The only other types of flavonoid substances which have to be con. sidered are benzalcoumaranones (aurones, cf., Bate-Smith and Geissman, 1951), e.g., sulphuretin (XIII, R = H) and aureusidin (XIII, R = OH), chalcones, e.g., butein (XIV), and dihydrochalcones, e.g., phloretin (XV). The relationships between these types are admirably illustrated by Geissman and Hinreiner (1952). Table I1 is a somewhat modified

TABLE^ I1 Equivalent Level of Oxidation of 3-Carbon Fragments

Catechins -CHz.CHOH.CHOH- Leuco-antho- Structure uncertain

Anthocyanins -CHz.CO.CO cyanins

or -CHOH.CHOH.CO-

Dihydrochalcones -CO.CHn.CH2 Chalcones Flavtmones } --CO.CHz.CHOH . Flavones -CO.CHz.CO-

Benaalcoumaranones -C0.C0.CH2- Flavanonols -CO.CHOH.CHOH- Flavonols -CO.CO.CHOH-

version of their chart. It shows in a purely formal way the level of oxidation of each of the three carbon atoms of the central ring or chain. It is interesting to note that in the jlavanone series no higher state of oxidation of the Ca fragment than that of the flavonols is possible; and similarly no higher state of oxidation in the JEavan series is possible than that of the anthocyanins.

XIII XIV

From the examples given in Table I it will be noted that variation within a type is due to hydroxylation and methoxylation of the two benzene nuclei. As pointed out above, an important point to note is that the pattern of hydroxylation and methoxylation is common to a great many types. In some families of plants, and especially in the Rutaceae and Malvaceae, still more highly hydroxylated and methoxylated flavonoids occur. Some of these are dealt with in connection with citrus fruits (p. 289).


1. Glycosidation

With the exception of the catechins and possibly the leuco-anthocyanins, the flavonoid compounds occur in the plant as glycosides in which certain of the phenolic hydroxyl groups are combined with sugar residues. The sugar-free molecules shown in Table I are termed aglycones. If an extract of plant material is chromatographed on paper, numerous spots will be found which give the typical reactions for flavonoid compounds. Usually these are clearly visible by their fluorescence in ultraviolet light, and many change both their visible and fluorescent colours on fuming with ammonia. If the extract is hydrolyzed with mineral acid, a chromatogram of the extract now shows only a few spots-often only one-reacting as described. The numerous spots on the original chromatogram are, in fact, numerous glycosyl forms of just a few parent substances. The variety of glycosyl forms arises both from the number and variety of sugars that can combine with any one phenolic hydroxyl group and from the numerous hydroxyl groups capable of glycosylation present on the flavonoid molecule. Sugars which commonly occur in glycosyl combination with flavonoid substances include galactose, arabinose, xylose, and, especially, glucose and rhamnose. These can, and often do, occur not only attached as single sugar residues to particular hydroxyl groups, but as di- or tri- saccharides, and several positions on the same molecule may be so glycosylated. Thus it is quite possible for three or four different glycosides of each parent phenolic compound to be clearly visible on a chromatogram. I n such circumstances the particularization of any one of these as a constituent of the foodstuff in question (unless it far outweighs all other flavonoid constituents in quantity or is known to have some property of especial importance in regard to the behavior of the food) would be more misleading than instructive. Furthermore, the practice of giving specific glycosides-or even specific aglycones-names derived, as has usually been the case, from the botanical species from which they were first isolated, cannot be extended to the hosts of new compounds which, we can anticipate, will be isolated and characterized with the help of chromatographic methods. Finally, it remains to be seen how much of the work on the characterization or identification of flavonoid compounds recorded in the literature is reliable.

So far as is known, the catechins are never glycosylated, and the leuco-anthocyanins seem also to occur, as a rule, uncombined with sugars. Johnson et al. (1951) report, however, that the main component of peach tannin (which from the description they give resembles in every respect a leuco-anthocyanin) is associated with carbohydrate, since glucose appears on hydrolysis with HC1.


The catechins in tea occur mainly in the form of 3-galloyl esters such as catechin-3-gallate (XVI).

XVI

Many anthocyanins are “ acylated ” with aliphatic or aromatic hydroxy- acids, attached as a rule to the glycosyl sugar residues.

11. PROPERTIES OF FLAVONOID COMPOUNDS SIGNIFICANT IN FOODS 1 . Properties Depending upon Their General Phenolic Character

I n spite of their frequently brilliant color, i t is not so much this property which is important in foods as their tendency to undergo discoloration. This is due to their general phenolic character which allows them to serve as effective substrates for oxidase action. They are, in fact, of all classes of phenolic substances, those most universally present in the plant kingdom. Seldom does the analysis of a plant extract fail to reveal one or more substances of flavonoid character, and if these are absent, chlorogenic acid or one of the closely related coumarins is almost certain to be present. Thus the flavonoid compounds and coumarins are the most commonly available substrates, actual or potential, for polyphenoloxidase or peroxidase activity.

Little work has been done with the specific aim of showing that flavonoid substances are competent to act as substrates for phenolase, but what has been done allows no room for doubt that they are. The most extensive work in this connection, that of Roberts and Wood (1951a, b, 1953) with tea oxidases and polyphenols, will be discussed in detail later on (p. 292). Having established that the mixed polyphenolic substances were acted upon by the purified phenolase from tea, they proceeded to show that individual catechins, flavonols, chalcones, etc., were similarly attacked. They showed, moreover, that certain glycosides of flavonols were highly resistant to attack by the enzyme, although the corresponding aglycones were themselves readily acted upon. In the author’s own laboratory (Baruah and Swain, 1952) it has, similarly, been shown that the glycosides quercitrin and rutin are not acted upon by potato polyphenolase, whereas the corresponding aglycone, quercetin, is rapidly oxidized.

It is evidently of great importance whether the phenolic compounds are present in the free state, or as glycosides, in the tissues of the plant.

FLAVONOID COMPOUNDS I N FOODS 269

Where both active polyphenoloxidases and competent phenolic substrates are present, the result of the action of the one upon the other is to produce a discoloration of the tissues, usually brown and therefore known as “enzymic browning.” A review of this subject has appeared so recently (Joslyn and Ponting, 1951) that there is no need to embark on any detailed description of i t here. Browning ensues when tissues are damaged by cutting or bruising, by physiological injury such as storing fruits and vegetables in inappropriate atmospheres, and by freezing and thawing. It is, in fact, an indication of post-mortem change, and is, in almost all circumstances connected with food, undesirable. What interests us here is whether, by attention to the particular nature of the flavonoid substrates, any means of control of enzymic browning suggests itself.

Occasionally, but only very occasionally, a proper development of browning is a desired step in the preparation of a food product. This is so, for instance, in the manufacture of tea and cider. It is necessary in these few instances t o ensure that the right phenolic substrates are present in the raw material and that the conditions are right for the enzymes to act upon them.

a. Destruction of Ascorbic Acid. An indirect outcome of the un- restrained action of polyphenoloxidase in plant tissues is the total and rapid destruction of the ascorbic acid in the tissue. This might, in certain circumstances, be a more serious disadvantage from the viewpoint of the use of the material as food (for instance in citrus products, and vitamin concen- trates) than the discoloration itself. Enzymic browning does not, in fact, begin until all, or almost all, of the ascorbic acid has been destroyed (Reid (1952) in apple juice; Miller and Heilmann (1952) in pineapple).

The reason for this is that the polyphenol, in the course of oxidation, can reversibly transfer oxygen to ascorbic acid, being itself reduced to its original state. The sequence in such a cycle of changes might be indicated as follows:

0 OH It 0 OH

where R is a substituent group such as

OH CO acid, and DHA dehydroascorbic acid (cf. Bate-Smith and Morris, 1952).


b. Reactions with Metals. In common with other phenols, the flavonoid compounds give color reactions with a number of heavy metals. In many instances insoluble complexes are formed, and this enables those compounds which are usefully colored to be employed as dyes. It was, in fact, on account of their value as dyes that the chemistry of these substances was first studied, and especially so by A. G. Perkin. The colors produced by treatment with ferric salts are often characteristic of particular hydroxyl groupings ; thus the catechol grouping gives a greenish reaction, the pyrogallol grouping a blue or black reaction, and the resorcinol grouping a reddish reaction. With the flavones and related compounds having a ketonic group a t carbon atom-4 and a Bhydroxyl group, ferric chloride gives a deep brown color. Usually, with their multiplicity of hydroxyl groups on two benzene nuclei, the flavonoid compounds give rather undifferentiated reactions with ferric chloride, in which green or greenish-brown shades are commonest because of the greatest frequency of occurrence of the catechol grouping.

An interesting reaction is that of the flavones with aluminum ions. The very faintly yellow-colored flavones can be made to dye tissues a bright yellow. An instance occurred within the author’s experience, when a tripe-dresser complained that when boiled with onions in an aluminum kettle his tripe became bright yellow in color. This was due to the quercetin glycosides (rutin, etc.) present in the yellow-skinned onions he had happened to use. Had he chosen purple-skinned or white-skinned varieties this difficulty would probably not have been encountered, since these contain much smaller quantities of flavone (cf. p. 283).

The chelation of metal ions has been discussed by Clark and Geissman (1949). They suggest that several sites might be concerned in chelation. In quercetin, for instance, sites A and B can both be considered as competent to form chelate complexes (XVII). Site C is perhaps even more likely to be involved.

w- C H

XVII

Reference to Table I will show what a large number of flavonoid compounds have either or both of these active groupings. “That chelation

FLAVONOID COMPOUNDS IN FOODS 27 1

with copper actually takes place with these substances can be shown qualitatively by the formation of coloured substances (in solution or as insoluble precipitates) when copper sulphate is added to their alcoholic solution ” (Clark and Geissman, 1949).

This ability to chelate heavy metal ions has been brought forward to account for some pharmacological activities shown by flavonoid compounds. Clark and Geissman showed, however, that the potentiation of effects of epinephrine by flavonoid compounds, tested upon an isolated smooth muscle preparation and attributable to the metal-chelating properties of the compounds, did not correspond with their vitamin-P activities as reported in the literature (cf. p. 275).

c. Ant ioxidant Action. In common with other phenolic compounds, flavonoid compounds, especially the more highly hydroxylated members, might be expected to have antioxidant properties. If we consider, for instance, the constitution of such well-known and effective antioxidants as the alkyl gallates (XVIII) and nordihydroguaiaretic acid (XIX), it is easy to understand how the catechins, their gallate esters, and perhaps

XVIII XIX

even such commonly occurring representatives as cyanin and quercetin might be quite powerful antioxidants. Lard and beef fat can be considerably stabilized by stirring the melted fat for a few minutes with commercial tannic acid or other crude tannin, followed by filtration (Spannuth et al., 1946). Quercitrin has been tested as an antioxidant for walnuts (Cruess and Armstrong, 1947). Although it afforded some protection, i t was not so effective as nordihydroguaiaretic acid. Some unpublished results (Banks, 1943) show that extracts of flower petals can exert a strong protective action against the haem-catalyzed oxidation of unsaturated fatty acids. Especially effective were extracts of wallflowers, peonies, azaleas, and roses, whereas those of pale-colored lupins, white clover, and calendula were least effective. The antioxidant action seems to have been shared by, but was not confined to, the anthocyanin pigment. It seems highly probable that it was due to the phenolic constituents in general, of which the flavonoid compounds form the major fraction.

2. Properties of Certain Classes of Flavonoid Compounds

a. Color. Several classes of these compounds possess brilliant coloring properties. Pre-eminent among these are the anthocyanins, both by the


depth and quality of their color, and by the frequency of their occurrence. Although there is a general tendency for the color to become bluer in hue from left to right of Table I ( i e . , with increasing hydroxylation of the flavylium radical), circumstances within the cell may profoundly modify the color produced by the same anthocyanin. Thus the blue cornflower and the red rose are both pigmented with cyanin (cyanidin-3,5-diglucoside), but in the former petal the cyanin is “co-pigmented” with apigenin, itself colorless, which has the, a t present unexplained, property of bluing the color of cyanin. Acid conditions will cause a reddening of the hue of anthocyanins, alkaline conditions, a bluing, so that the pH of the cell-sap, or, in the case of processed foods, of the aqueous phase, will be highly important in determining the color of a food containing anthocyanin pigment.

The chalcones are yellow in color, tending towards orange as hydroxylation increases. Although they do not appear to have been reported in foods, they may well be present, since butein occurs in flowers of the Papilionaceae (Butea frondosa) and several of the tribe Heliantheae of the Compositae, of which the artichoke (Helianthus tuberosus) is a member. They may be met with as added coloring matters; for instance safflower contains a chalcone, carthamin. They may, moreover, be produced from flavanones during the processing of foods containing these flavonoids, since the flavanone ring readily opens on heating, the isomeric forms tending towards an equilibrium :-

The Zeuco-anthocyanins are themselves colorless, but give rise to red- brown colored products when heated in aqueous solution, especially if the solution is strongly acid. The color of stewed pears may well be due to leuco-anthocyanins present in the fresh tissues, but this has, apparently, never been investigated. Several classes of flavonoid compounds, themselves colorless, are unstable to alkaline or oxidizing conditions, producing brown discolorations (quite independently of enzymic browning). The calechins and Jlavanonols are examples of such types of substances. The latter, especially, can be detected by the formation of an orange-brown stain when fumed for some time with ammonia vapor.

The Jlavones themselves are almost colorless. Yellow colors in plant tissues are usually due to carotenoid pigments, especially the xantho- phylls and their epoxides, a group of pigments beyond the scope of the


present review. Even in flowers, only in rare instances such as Butea frondosa, a few Scrophulariaceae, Papaveraceae, and Heliantheae have yellow pigments been shown to be other than carotenoid. The absolute limit of coloration to be expected of flavones is probably indicated by the pale yellow color of cotton flowers, which contain glycosides of quercetin (111) and gossypetin (XX).

OH

XX

b. Taste. Except for naringin, the 7-rhamnoglucoside of naringenin, a flavanone, the flavonoid compounds are not conspicuously endowed with taste. Naringin is extremely bitter, and i t is to this substance that the bitterness of grapefruit (cf. Kesterson and Hendrickson, 1953) and sour oranges' is due. If, however, we distinguish (as we must) astringency from bitterness, the flavonoid compounds are by no means of negligible importance; on the contrary, some of them, for instance the catechins, are among the most important astringent substances in nature. Astrin- gency is, strictly, concerned with the sense of touch, and is due to the coagulation of the proteins of the saliva and mucous epithelium by combination with the astringent body (cf. E. G. McDonough, 1935). The action is akin to that which occurs during tanning, and these substances are, therefore, to be regarded as tannins. Furthermore, we have reason to suppose (Bate-Smith and Swain, 1953a) that the leuco-anthocyanins also belong to this class of materials, and they are even more widespread than the catechins. Such of them as have been tested are, in fact, strongly astringent. This property is, in general, undesirable in foods. Where a food is apt t o contain astringent substances, varieties are bred and selected for cultivation in which astringency is reduced to a tolerable level. Examples of this are apples and peaches. To proceed in the process of elimination of astringency beyond a certain point often, however, results in flatness and insipidity, so that the possession of factors for astringency is, in many food plants, from the genetic point of view a desirable character. Ciders, for instance, are blended to a degree of astringency adjudged optimal for the consumer market concerned. Wines, especially red wines, are also required to have a proper degree of

* Bitterness in navel orange juice (cf. Marsh, 1953) is due to the presence of limonin, which is not a flavonoid compound.


astringency. An important part of the art of chocolate manufacture seems to lie in the selection of varieties which, after an intricate series of processes, will yield a finished product of exactly the desired degree of astringency. In each of the instances mentioned the astringent constitutents are flavonoid in nature. Incidentally the bitterness of chocolate is quite unconnected with its astringency, and is largely due to the presence of the purines theobromine and caffeine. These questions have been fully discussed in a recent article (Bate-Smith, 1954a).

The realization that the leuco-anthocyanins are probably the commonest representatives in Nature of the substances known generally, but indefinitely, as tannins enables a great deal of existing information to be brought within the framework of this review. “Tannin” is a heading commonly included in tables of the composition of foods, but it is well recognized that the analytical figures recorded under this heading are no more than an indication of the approximate amounts of materials which react with the reagent employed (usually permanganate in the cold) under standard conditions. As Charley and Harrison (1939) remark, with regard to the application of a modification of the permanganate method to the analysis of fruit juices: “This estimation is particularly useful when bitter fruit is available and when it is desirable to blend the astringency equally throughout other bulks of juice . . . This method does not give a true tannin figure, but the data obtained do bear a definite positive relationship to the astringency of the juice.” It might be asked what is a “true tannin figure,” short of a detailed analysis of the concen- tration of every specific substance possessing tanning properties in the system!

Potassium permanganate reacts in the cold, in weakly acid solution, with many other substances than tannins. An indication of the kinds of substances contributing to the permanganate titer of a plant extract is given by the data in Table 111, representing results (unpublished) obtained a t Long Ashton Research Station,2 Bristol, England. (C j . also Williams, 1953 and Kaiser, Pollard and Williams, 1953.)

Clearly, from these data, any ortho- or paradihydroxybenzene derivative will react with permanganate, with the absorption of 4-5 equivalents of oxygen, but neither monohydroxy nor metadihydroxy derivatives are reactive unless the molecule is otherwise open to attack (as in the side chain of cinnamic acid and its derivatives). The same workers have also shown that the Folin-Denis reagent is even more general in its reactivity -monohydroxy as well as dihydroxyphenols are included in the range of substances with which it reacts. Clearly, a good deal more discrimina- tion is needed in the methods employed before chemical data recorded 2 By Miss M. E. Kieser, Dr. A. Pollard, and Mr. A. H. Williams.


under the heading “tannins” can be related to any particular class of compound.

As it happens, the catechins and leuco-anthocyanins can be diff eren- tiated from other known tannin-like substances in that they give a red color-reaction with vanillin and concentrated hydrochloric acid ( c f . Bate-Smith and Swain, 1953a). Although at present employed only as a qualitative test for their detection, this reaction might well serve as a basis for their quantitative estimation, since it depends on the 5,7-dihydroxy- substitution of the A ring, which is common to all the known

TABLE I11 Titer of Various Compounds Reacted with Cold, Weakly Acid Permanganate

Atoms oxy- Atoms oxygen equiva- gen equiva-

Substance wt. KMn04 titer Substance wt. KMnOl titer Mol. lent to Mol. lent to

Catechol 110 4 . 6 Tannic acid 322 7 . 9 Resorcinol 110 - Cinnamic acid 148 3 . 9 Hydroquinone 110 5 . 2 Caffeic acid 180 6 . 7 p-Hydroxybenzoic acid 138 - Chlorogenic acid 354 6 . 7 Protocstechuic acid 154 4 . 7 Quinic acid 192 2 : 4 Dihydroxybenzoic Tyrosine 181

- -

acid 154 - Dihydroxy-

acid 154 5 . 1 Catechin 290 4 . 3 2: 5 Dihydroxybenzoic phenylalanine 197 5 . 5

- Gallic acid 170 5.8 Phlorizin 436

catechins and leuco-anthocyanins. (A carbonyl group a t position 4 interferes with the reaction so that flavonoid compounds containing this group, such as flavones and flavanones, do not react.)

Catechins and leuco-anthocyanins readily form condensation products, and it seems to be more particularly the condensation products of intermediate size which possess tanning properties, i . e . , the property of cross-linking and precipitating proteins. These products, either pre- existing or formed in the expressed juices on standing, contribute to the turbidity, “body,” and other properties, desirable or undesirable, of beverages such as wine, beer, and tea.

Finally, it has been observed that in the presence of “tannins” the fading of anthocyanins is considerably retarded. This may be not the least important, in food technology, of the properties of this very important group of flavonoid substances.

c . Pharmacological Action. In recent years the flavonoid compounds have come into prominenceit might almost be said have achieved notoriety-in connection with their so-called vitamin-P activity. The concept was first employed by Armentano el al. (1936) in explanation of

276 E. C . BATE-SMITH

the improvement in capillary resistance after administration of the juicc of Hungarian red peppers or of lemons. The substance responsible was not ascorbic acid. An active preparation from lemon juice (2 g. from 200 kg. lemons) believed to be a mixture of flavones, was called “citrin.” The main component, hesperidin, was tested by Scarborough and his collaborators (1938, 1940, 1945) and found to produce certain of the effects described by the earlier workers. Lavollay et al. (1943), of a number of flavonoid compounds studied, found d-epicatechin (prepared by epimerization of d-catechin) to be the most active. Activity is not, however, restricted to one or two specific substances. It seems to be shared in greater or lesser degree by most flavonoid compounds and is, if only on these grounds, considered to depart from proper vitamin character. The whole question has been reviewed quite recently by Scarborough and Bacharach (1949).

Hesperidin derivatives have been shown to act as inhibitors of hyaluronidase (Beiler and Martin 1947, 1948) and, as a rather remarkable application of this property, hesperidin solubilized by phosphorylation has been shown to suppress fertility (Sieve, 1952). It is suggested that the phosphorylated hesperidin, which can be administered orally, pro- duces its effect by inhibiting the liquefaction of a hyaluronic acid gel which is necessary before the spermatozoon can penetrate the ovum. Since the results vary with different preparations, Martin (1953) suggests that the effect is due to one particular component of a complex mixture of phosphorylated derivatives. Insofar as both the aglycone hesperetin and its 7-rhamnoglucoside hesperidin, unless phosphorylated, are extremely insoluble, it is unlikely that any such action can be expected of the flavanones themselves when present in foodstuffs.

The isoflavone genistein (cf. Table I), which is present in subterranean clover (Trifolium subterraneum) and probably in other Trifolieae, pos- sesses oestrogenic properties (Bradbury and White, 1951, 1953). It seems likely that genistein is the causal agent in the sheep infertility problem first encountered in Western Australia in 1941, since this has been shown to be due to excessive ingestion by the sheep of oestrogenic substance present in subterranean clover (Bennetts and Underwood, 1951) .a

A direct action of flavones on smooth muscle (distinct from the potentiation of epinephrine action mentioned earlier) has been reported. Most conspicuous is the action on rabbit intestine reported by Ferguson (1948) and Ferguson et a2. (1949, 1950) and offered as a possible explanation of bloat in ruminants following overconsumption of herbage plants

8 Information kindly supplied by Dr. G . S. Pope, National Institute for Research in Dairying, Shinfield, Reading, England.


such as lucerne (alfalfa). Lucerne was shown by these authors, a t certain stages of growth, to have an exceptionally high content of tricin (see Table I), but it could not be demonstrated that the oral administration of flavones produced the condition. This does not rule out the possibility that paresis of the rumen is a contributory cause of bloat, since the etiology of the condition is extremely complicated.

The pharmacological action of coumarins and related unsaturated lactones has been reviewed recently by Haynes (1948).

d. Fate in the Body. The little that is known about the flavonoids after ingestion or injection (cf. Scarborough and Bacharach, 1949) suggests that they are excreted more or less unchanged in the urine. In the case of the bright purple-red pigment betanin of the beetroot, which is a nitrogenous anthocyanin (c f . p. 285), certain individuals possess the idiosyncracy of excreting the pigment in the urine. Czimmer (1937) made the interesting observation that after administration of pelargonin the urine is a t first colored red, but later is colorless, becoming red when acidified. This suggests that the anthocyanin is excreted as the pseudo- base. Czimmer (1936) also reported that, after administration of flavonol (probably quercetin) glucoside extracted from Forsythia sp., only the urine of carnivora contained the pigment, that of herbivora being free from flavonol. The likeliest explanation would seem to be that the pigment is destroyed in the rumen or caecum of the herbivorous animal.

An effect of administration of the drug uva ursae is to cause darkening of the urine. Although leuco-anthocyanins are present in the plant, the effect is probably due to the glucoside of hydroquinone, arbutin, that the plant, in common with many Ericaceae, also contains.

There is perhaps one misconception which might be corrected here, and that is that the catechins are intestinal astringents. In actual fact, in the case of the tea catechins at least, the reverse is the case; they have a cathartic effect.

e. Toxicity. All workers are agreed that, phlorizin excepted, flavonoid compounds, administered orally, are virtually nontoxic. If, in fact, this were not the case, the very considerable amounts which are consumed in one form or another in fruits and vegetables would long ago have made their presence unpleasantly felt. Some invertebrates accumulate large quantities of flavone in their organs, conspicuously moths of the family Sphingidae, whose wing color is due to flavone pigmentation (Thompson, 1926). Flavones are also accumulated in the epidermal tissues of the snail Heliz pomatia (Kubista, 1950). There is, however, evidence that flavones and chalcones are toxic to certain bacteria (Schrauffstiitter, 1948; Schrauffstatter and Bernt, 1949).


111. THE GENETIC SITUATION

The kinds of foods that are grown are determined by their desirability for direct consumption or by their suitability for consumption after manufacturing operations. Each particular kind of food exists in many varieties, some of superior, some of inferior, quality, and new, superior varieties are continually being produced by the breeder. It is an advantage to the breeder to have as much information as possible about the separate variable properties which are concerned in the over-all “quality ” of a product. In the previous section we have discussed some of these properties, and the various ways in which flavonoid compounds may affect them.

Some of the genetic variations in which flavonoid compounds are concerned can be discerned by mere visual inspection: such variations, for instance, as the presence or absence of red or yellow color in the skin or flesh of fruits; others, such as astringency, can be discerned by simple organololeptic test. But when, as is frequently the case, the genetic situation is complicated, it may be important to the breeder to know, for instance, exactly what pigments are present in a particular strain, or how to detect variants possessing undeveloped characters which may be elicited in further crossings.

The need for deeper exploration of such genetic situations, and the importance, in certain manufacturing processes, attaching to the presence or absence of particular s~bstances,~ make it likely that from these direc- tions of plant breeding and food technology detailed knowledge of the nature and distribution of the flavonoid compounds in foods is most likely to come. So far as the genetic situation is concerned, more information is, however, at present being obtained from studies not immediately connected with the use of plants as foods. From these studies it is already apparent that in the plant different classes of flavonoid compounds are biosynthetically related. (By way of contrast, it is equally apparent that there is no biosynthetic connection between the flavonoids and the carotenoids.) This means, for instance, that where a red anthocyanin pigment is suppressed, a yellow chalcone or a virtually colorless flavone may appear in its place, or that colored varieties may appear from the crossing of two colorless ones. Chemical studies related to investigations of pigment inheritance have also shown the impossibility of inferring, from appearance alone, the precise genetic makeup of a particular phenotype. A realization of the complexity of the genetic situation is

4 In this connection, a paper by Marsh (1953), although it concerns the occurrence of a substance, limonin, which is not a flavonoid compound, in navel orange juice, is especially pertinent.


especially desirable when we come to consider the records in the literature of the systematic distribution of this or that flavonoid compound in edible plants.

The most detailed investigations to date in this field have been carried out with flower petal pigments. The cases of Dahlia variabilis and Antir- rhinum majus are particularly instructive. In the dahlia, the genetic situation has been worked out by Lawrence (1931), and chemical studies by Lawrence and Scott-Moncrieff (1935), Price (1939), Bate-Smith and Swain (1935b), and Nordstrom and Swain (1953a) have shown that the following flavonoid compounds in various combinations, and numerous glycosyl forms, may be present:

Genes

A , B ilnthocyanidins: Pelargonidin, cyanidin Flavones: Apigenin, luteolin Flavanones: Naringenin (eriodictyol?)

Their occurrence is determined by the genes A , B, I , and Y , as indicated. A and B differ in the depth of anthocyanin coloration they determine. In an “abnormal white’’ form, still another “pigment,” which gives a deep mahogany color when fumed with ammonia and which is possibly a flavanonol, is present. The genetic status of this form has not yet been ascertained, but it may perhaps represent the recessive condition, a b y i.

A particularly important aspect of Lawrence and Scott-Moncrieff’s work is that they showed the following interactions of genes:

1. Y suppresses I . 2. Y in presence of A or B suppresses cyanidin formation. 3. I in presence of A or B suppresses pelargonidin formation. The position in D. variabilis is complicated by the fact that it is a

polyploid, and each gene can therefore be multiplex. Some of the genes are additive in their effects, others fully effective in the simplex condition. Similar or even greater complexity can be expected in large numbers of cultivated plants, so many of which are hybrid and polyploid in their genetic makeup.

In still finer detail, an even greater complexity in the representation of specific glycosides is revealed, Thus in a blue-mauve variety of dahlia (constitution probably A1--4b12--4y), Nordstrom and Swain (1953a) found the following specific glycosides: cyanidin (3.5?) arabino glucoside, and traces of a pelargonidin diglucoside ; apigenin-7- and 4’-glucoside and 7-rhamnoglucoside (rhoifolin) ; luteolin-5-glucoside and 7-diglucoside as well as a naringenin monoglucoside and an unknown -diglucoside.

E l b Chalcones: 2’,4,4’Trihydroxychalcone, butein

6 Nordstrom and Swain (1953b), more recently still, have shown the aurone 8 ~ 1 -

phuretin (cf. p. 266, S I I I ) to be present in some yellow dahlias.


Similarly in parsley, which in the literature is quoted as containing the glycoside apiin 7-(apiosyl-glucosyl)-apigenin, Nordstrom et al. (1953) have found, in addition to apiin, 7-(apiosyl-glucosyl)-luteolin, an apigenin glucoside, and a naringenin derivative.

In garden forms of Antirrhinum majus yellow flowers contain the aurone glucoside aureusin (XXI, R = glucose), luteolin and apigenin gly-

OH

XXI

cosides, and a naringenin glycoside. Here the formation of luteolin is sepa- rately controlled by a specific gene, and its presence or absence cannot be detected by the phenotypic appearance of the flowers, which are equally yellow whether it be present or not (Seikel and Geissman, 1950).

The moral to be drawn from these and other studies with similar outcome is that the reporting of the occurrence, in a particular species, of a particular member of this class of substances does not mean that it is present in every variety of that species, nor that other flavonoid compounds may not also be present. Only occasionally, however, will it be of practical significance whether any particular glycoside of a particular flavonoid compound, is present; i t will, as a rule only be important to know whether members of a particular group, such as flavones, flavonols, catechins, leuco-anthocyanins, etc., are present, since it is the properties of these substances as a group which will usually matter.

One reservation must, however, be made, that is, as to whether the particular member is competent to act as a substrate for the polyphenoloxidases present. We have already (p. 268) seen that the 3-glycosides of quercetin and myricetin are not attacked by the tea polyphenolase system, whereas the aglycones themselves are readily attacked. It may prove to be important, in any tissue, whether the specific glycosides present are attacked by the enzyme present, or introduced from other sources, as in products of mixed origin. Such considerations as these un- doubtedly operate in the manufacture of such products as cider and Perry.

1. Genetic Situation in Fruits and Vegetables

a. Apples. Owing to the degree of polyploidy of cultivated forms (Darlington and Moffett, 1930), variation in characters is almost con- tinuous. The inheritance and distribution of anthocyanins are controlled


by a number of genes, and red color (due to cyanidin glycosides) appears to be dominant over absence of red (Wellington, 1924). White-fleshed varieties are of two kinds, one of which (heterozygous for yellow) carries a suppressed factor for yellow flesh color. Although yellow-skinned apples contain glycosides of quercetin (Sando, 1937), it is not clear to what extent yellow skin color or yellow flesh color is due to these compounds or to carotenoids6-a case in point where more precise information would assist in the interpretation of breeding results. The apple certainly contains catechins, leuco-anthocyanins, and many other flavonoid compounds, as well as chlorogenic acid (Bradfield et al., 1952).

b . Peaches. An excellent example of the linking together of breeding experiments and critical examination for edible quality is provided by the work, summarized by Bailey and French (1949), that has been done on peaches. It is, in the first place, appreciated that “edible quality” is rather an elusive and complex thing, and has itself to be analyzed so that its components can be separated for purposes of scoring in the families raised. Even so, many of the component characteristics have to be evaluated subjectively, so that assessment is a matter of personal taste. “Hence in this particular area of fruit genetics it is a question of whether genetic analysis measures the taste of the peach or the taste of the taster.” The characteristics recorded were (1) flavor characteristics : acids, sugars, tannins, and essential oils; (2) feel in the mouth: firmness, mealiness, stringiness. Characteristics in the fruit other than those concerned with edibility were skin color, color around the pit, and flesh color. In the determination of “catechol tannin” we have at least some sort of an objective measure of astringency, that is, the component in edible quality with which flavonoid compounds are associated.

Both red skin color and tannin are transmitted from parent to off- spring, high color and high tannin giving correspondingly high scores in the seedlings produced, and vice versa (Blake, 1939-40 ; Weinberger, 1944). A white-fleshed variety, “Sunbeam,” which does not become brown on wounding, was found by Kertesz (1933) to contain no flavonoid compounds other than tannins and anthocyanins-a suggestion supported by the work of Johnson et al. (1951). Their description of the main component of the peach tannin preparation corresponds exactly with that of a leuco-anthocyanin (cf. p. 267). d-Catechin and chlorogenic acid are also present.

Yellow flesh color is due to carotenoid pigment (McKinney, 1937), so that the genetic situation as between white and yellow flesh does not concern us here.

6 The yellow color of the flesh of the variety Tydeman’s Late Orange (Cox’s Orange x Laxton’s Superb) is due to carotenoid pigment (Bate-Smith, unpublished).


c. Grapes. The anthocyanin pigment of the skins of European black grapes (Vitis vinifera) was shown by Willstatter and Zollinger (1915, 1917) to be the 3-glucoside of malvidin (oenidin). American black grapes are crosses from V . labrusca, V . riparia, and V . aestivalis with V . vinifera. Anderson and his collaborators (1923, 1926, 1928) and Shiner and Anderson (1929) found that the pigment from these grapes seldom gave the correct theoretical methoxyl content, and also gave an intense reaction with ferric chloride, which malvidin does not give. From the variety “ Ives,” regarded by Hedrick as “practically pure V . labrusca,” both malvidin and monomethylated delphinidin (petunidin) were isolated. Crosses known to contain V . vinifera blood always contained malvidin, and i t was concluded that the factor for malvidin formation carried by V . vinifera was dominant in these crosses.

In crosses of colored with white grapes, white skin color is always recessive to colored (Hedrick and Anthony, 1915).

White grapes contain quercetin and its 3-glucoside, isoquercitrin (Williams and Wender, 1952a). Here the flavone and the anthocyanin have quite different hydroxyl substitution patterns. Vitis vinifera and V . heterophylla humifolia were reported by Robinson and Robinson (1933, 1934) to contain leuco-anthocyanin yielding cyanidin on digestion with hydrochloric acid, and Durmishidze (1951) and Durmishidze and Bukin (1951) report 1-gallocatechin amounting to 45-54% of the total tannin present. This fruit provides therefore an interesting example of the varied states of oxidation and methylation in which the different classes of flavonoid compounds exist in identical or nearly related species.

d . Potatoes. As an example of similar genetic work on vegetables, that on the potato is outstanding. The skin color of the tubers may vary from “white” to (a) red or (b) blue or purple, and in intensity from very faint to deep. Various authors differ as to the genetic interpretation of the results of crossing, but most postulate the presence of a basic gene, acting together with a red-producing ( R ) or a purple-producing gene ( P ) . As in the garden dahlia and in apples, polyploidy is likely to complicate the genetic interpretation. As regards flesh color, purple flesh in the variety Congo Black was dominant to white (Krantz, 1922). (It seems, in fact, to be generally true that presence of anthocyanin is dominant to absence.) The pigment in yellow-fleshed varieties is mainly carotenoid, but other phenolic compounds may be present in the flesh including chlorogenic acid, coumarins (scopoletin?), and the substance of at present unknown constitution responsible for the blackening of some varieties during cooking. The amount of this substance, if not in fact its presence or absence, is almost certainly an inheritable varietal factor.

Work currently in progress a t the Agricultural Research Council’s


Potato Genetics Station, Cambridge, England,’ on Solanum rybinii, suggests a somewhat different genetic interpretation for this diploid cultigen. The shoots of all seedlings are colored with anthocyanin, so that all plants have the faculty for pigment production. The tubers may have blue, red, or colorless skins; for colorless skins the presence in a homo- zygous condition of a recessive inhibitory factor seems to be necessary and this is epistatic to color-producing factors. Red pigmentation is recessive to blue. If P is the factor for blue pigment, p will represent the red-skinned condition; if i is the factorial condition for suppression of color, I will represent the necessary condition for color development. Other genes (some of which are linked with those for tuber skin-color) control flower color, flecked patterning of the flower, and “spectacle” patterning around the eyes of the tuber. The blue pigment appears to be due to a cyanidin glycoside, and the red to a pelargonidin glycoside, but this is probably too simple a view because in some genotypes the two pigments may occur together.

e. Onions. In this vegetable, genetically controlled factors can be manipulated to the advantage both of the cultivator and the food proc- essor. Forms may occur having red, yellow, or white bulbs, the first containing in the outer scales a glycoside of cyanidin, the second glycosides of quercetin, and the last neither pigment. When pigment is present it is accompanied by protocatechuic acid, which effectively protects the bulb against invasion of the pathogenic fungus Colletotrichum circinans. The presence of quercetin can be undesirable in some food- processing operations; an instance has already been described (p. 270). According to Rieman (1931) allelomorphic genes, W , Wv, and w govern red pigment, yellow pigment, and white condition, respectively, W being dominant to Wu and Wu to w. Clarke et al. (1944) postulate a basic gene, c, for any pigment, a gene R for red, the recessive T producing yellow. Either of these interpretations would imply that the formation of cyanidin is alternative to that of quercetin, and involves an additional step in the synthetic process. Such a relationship is understandable when the anthocyanin and flavone are as similar in structure as these two substances are.

IV. SYSTEMATIC DISTRIBUTION

1 . Anthocyanins

Most of what we know about the systematic distribution of the anthocyanins, and in fact a good deal of their chemistry also, is due to Sir Robert Robinson and his collaborators. The results of their studies up to 1939 were summarized and discussed in a paper in the Philosophical

7 By K. S. Dodds and D. H. Long.


Transactions of the Royal Society of London (Lawrence et al., 1939). In this paper the occurrence of pelargonidin, cyanidin, peonidin, malvidin, petunidin, and delphinidin in the flowers of more than 400 species of plants was recorded, with their glycosidic condition. The arrangement of the data in systematic order of families allowed any regularity in the distribution, if present, to be discerned, but no striking regularity, relatable to systematic affinities, was apparent. Overall, however, the data clearly showed the predominance of cyanidin (including peonidin) over pelargonidin and delphinidin (including malvidin and petunidin) in flowering plants. It was further observed that the occurrence of cyanidin was also correlated in some degree with a woody habit in the plant. Particularly relevant from our present point of view are the data for the distribution of anthocyanins in fruits, also recorded in this paper. The data for those of the fruits which are edible are produced in Table IV.

TABLE IV Anthocyanins Present in Edible Fruits

Common name

Banana, red Bilberry Cowberry Cranberry, American

Eggplant Eggplant, common

Elderberry

Grape Fig Gean Mulberry Plum Pomegranate Raspberry Sloe Strawberry, alpine Strawberry, Virginian

Botanical name

Musa coccinea Vaccinium myrtillus Vaccinium vitis-idaea Vaccinium (Ozycoccus)

Solanum melongena Solanum melongena

var. esculentum Sambucus nigra

Vitis vinifera Ficus can'ca Prunus avium var. Morus nigra Prunus communis var. Punica granatum Rubus idaeus Prunus spinosa

macrocarpus

Anthocyanin

Pelargonidin-3-monoside Malvidin-Bmonoside Cyanidin-3-monoside (galactose) Peonidin-3-monoside (glucose)

Delphinidin-3-pentoseglucoside Delphinidin-3-bioside

Cyanidin-3-pentoseglucoside and

Malvidin-3-monoside Cyanidin-3-monoside Cyanidin-3-monoside Cyanidin-3-monoside Cyanidin-3-monoside Delphinidin diglycoside Cyanidin-3-bioside Cyanidin-3-pentoseglucoside

monoside

Pelargonidin-3-monoside Fragaria vesca Fragaria virginiana

Apart from these, very few identifications of anthocyanins in foods seem to have been made, though some of those listed have since been confirmed. Thus Sondheimer and Kertesz (1948) have confirmed that the pigment of strawberries is pelargonidin monoglucoside. In Jonathan and Stayman Winesap apples the pigment has been identified as cyanidin-3- galactoside (Sando, 1937).

In vegetables, betanin, the pigment of red beets, calls for special note.


This is a nitrogenous anthocyanin, and its constitution has not yet been established8 (cf. Pucher et al., 1938; Chmielewska, 1938). Apart from the Chenopodiales in which they are repeatedly found the nitrogenous anthocyanins are limited in their distribution to four other orders, but only a few of the species mentioned are used as food: in Cactales, Opuntia spp., and in Caryophyllales, Mesembryanthemum spp. Chmielewska el al. (1936, 1938) have found that the anthocyanin of red cabbage is a bioside of cyanidin acylated with sinapic acid, and that of a purple- skinned variety of potato (Chmielewska, 1935) a bioside of malvidin acylated with p-coumaric acid; otherwise no identifications of the anthocyanins in vegetables appear to have been attempted.

'

2. Anthozanthins

No survey of the systematic distribution of flavones, flavonols, and flavanones has been made comparable with that carried out on the anthocyanins. This is due in the first place to the absence, until recent years, of any tests for their identification comparable in simplicity with those available for the anthocyanins; and in the second place, to the wider range of variety of these compounds and their glycosides found in nature.

The valuable summary in Klein's Handbuch der Pjlanzenanalyse, Vol. I11 (2), 1933, enables a general view to be made of the identifications recorded up to that date. The warning previously given must however be repeated here, that conclusions from such identifications must be drawn with caution because of the variability of these compounds within a species from one genotype to another, and also because the reported presence of one of these compounds in a plant does not necessarily mean that others are absent. Indeed, chromatography shows a t once how many and varied are the flavonoid compounds present in most plant extracts (see for instance Fig. 1, p. 293). Usually, however, when hydrolyzates of the extracts are examined, these are found to derive from no more than one or two aglycones. It is therefore more important as a rule to know what aglycones are present than to try to determine in detail and with great labor the constitution of a few of the glycosidic combinations in which they happen to occur in the particular specimens examined.

Bearing these considerations in mind, there seems nevertheless to be no doubt that the analog of cyanidin, quercetin, is by far the most widely distributed of the flavonols. In Klein's Handbuch it is reported as occurring in 87 species, representing 47 genera, the next most frequent, kaempferol, and myricetin, being reported in 14 and 17 species, respectively. Of the flavones, those derived from catechol, i.e., luteolin and its 8 Although Chemical Abstracts, Index, 1949 gives the structure for betanin chloride a8

4'-amino-5, 7-dihydroxyflavylium chloride, no authority for this can be traced.


methyl ethers, are again the commonest. In the flavanones, hesperitin, the 4’-methylated analog of luteolin, has been most frequently reported. There is, however, some confusion in nomenclature regarding this substance and its glycoside, hesperidin, and the records of its occurrence cannot be completely trusted (cf. Klein’s Handbuch, Zoc. cit., p. 849).

There are fairly strong indications, from these recorded data, that the flavones (and flavanones) occur for the most part in herbaceous plants, whereas the flavonols occur more particularly in woody ones. The flavones are reported as occurring in 28 families of the dicotyledons, 20 of which are placed by Hutchinson (1946) in Herbaceae, and only 8 in Lignosae; whereas the flavonols are reported in 47 families of Lignosae and in only 15 of Herbaceae. The same tendency has been noted by the author in chromatographic studies of the anthoxanthins in flower petals. These studies have also revealed the universality of these compounds in flowering plants. It is exceptional to find a plant in which flavones or flavonols are not present, and frequently two, three, or four aglycones occur together. Only rarely is it possible to identify these by chromatographic evidence alone, and it must be long before a really adequate picture can be formed of their systematic distribution.

a. Isojiavones. These appear to be very restricted in their distribution, having been reported so far only in Leguminosae, in Iris, and in one species of Prunus. The isoflavones in Soja have received most attention. It is now fairly certain that the structures of isogenistein and methyl isogenistein reported by Okano and Beppu (1939) are incorrect (Baker et al., 1952) and that the Soja isoflavones are daidzein and genistein on1 (Baker, personal communication). The yellow pigments of Osage orange (Maclura pomifera, Moraceae) are complex isoflavones, having a second fused pyran ring in the 7, 8 position. They bear some resemblance in structure to the hop resins, humulone and lupolone, so important in beer manufacture. Two other flavonoid compounds, also occurring only in Moraceae, should be mentioned here, viz., morin (XXII) and cyano- maclurin (XXIII). The latter is probably ring-closed between the 2’( = 6’) and 3 positions. It occurs in the jackfruit, Artocarpus integrifolia.

XXII XXIII

b. Aurones. Besides aureusidin (XXI, R = H), isolated from Antirrhi- num majus, and leptosidin (XXIV), isolated from Coreopsis and Leptosyne


species by Geissman and co-workers, Shimokoriyama and Hattori (1953) have recently isolated sulphuretin (XXV) from Cosmos sulphureus. These substances are, therefore, of more frequent occurrence than, up to recently, has been recognized, and if present in foods would, because of their brilliant golden-yellow color, be of considerable importance as pigments.

S X I V 0

s s v c . Chalcones. These occur only infrequently and have not in fact been

reported to occur in any common food-plant ; but since they may readily be formed by opening of the pyrone ring from flavanones, they may be found as secondary products from foodstuffs containing flavanones. This seems to be especially likely in the case of hesperidin, and i t has been suggested that the resulting chalcone imparts a bitter taste t o the fruit (Kotodi, 1950). They are much more deeply colored, yellow to orange, than the corresponding flavanone, and any such formation of chalcone may therefore have a marked effect on color.

Phlorizin is a dihydrochalcone. Apart from its common occurrence in rosaceous trees, and especially in the root bark, it is otherwise reported as occurring only in Micromelum teprocarpum, a relative of Citrus. Although present in the seeds, shoots, and leaves of apples, it appears to be entirely absent from the flesh-a fortunate fact in view of its poisonous character.

3. Catechins and Leuco-anthocyanins

As a first approximation, the systematic distribution of the catechin- leuco-anthocyanin group of substances can be taken to coincide with that of “tannins,” as recorded in the botanical literature, since the condensed, or catechin, tannins, which this group comprises, outnumber by far the hydrolyzable (tannic acid) group (cf. Rottsieper, 1946; Russell, 1935). The tanniniferous families of the dicotyledons are listed by Metcalfe and Chalk (1950). These include almost all the woody plants


from which edible products are obtained, but herbaceous families are notable for their scanty representation. The expectation is, therefore, that catechins and leuco-anthocyanins will generally be absent from the tissues of herbaceous plants, and this expectation is fully realized, at least in so far as the dicotyledons are concerned (Bate-Smith and Lerner, 1954).

In Klein’s Handbuch (Zoc. cit., p. 410), very few identifications of catechins are recorded. Apart from tea and cacao, recent work has shown them to be present in: (1) apples (epicatechin, commonly, catechin in some cider varieties (Bradfield, 1952, Williams, 1952)); (2) pears (epicatechin only in perry varieties in small amounts (Williams, 1952)) ; (3) peaches (d-catechin? (Johnson et al., 1951)).

Leuco-anthocyanins were reported by Robinson and Robinson (1933, 1934) to occur in almonds, grapes, apples, walnuts, chestnuts, and brazil nuts: and in the seed coats of groundnuts, runner beans, and loquats. An analysis of the anthocyanidins produced from all the specimens examined by these authors (most of them heartwoods) showed a great preponderance of cyanidin (84%), followed by delphinidin. Bate-Smith (1954b) reports a similar preponderance of cyanidin-producing sources, the remainder producing delphinidin. (It is interesting to note that the natural catechins, derived from catechin and gallocatechin, are restricted to the same pattern of hydroxyl substitution as, it seems, are the leuco-anthocyanins.)

A wider systematic survey of the occurrence of leuco-anthocyanins in leaves (Bate-Smith and Lerner, 1954) has shown that in dicotyledons these substances are, with few exceptions, confined to woody families (those included by Hutchinson in his division Lignosae). Outside this division, so far the only species giving a positive reaction are in the families Polygonaceae, Plumbaginaceae (which contain many woody species), Primulaceae Crassulaceae, Ficoidaceae (Aizoaceae) , and Oxali- daceae. All of these, except Plumbaginaceae and Primulaceae, provide edible products, mainly leafy vegetables or salad plants.

I n the monocotyledons, the presence of leuco-anthocyanins, in some part of the plant at least, appears to be the rule, even among the herbaceous members. Iris pseudacorus, for instance, recognized as a tanniniferous plant, gives an intensely strong leuco-anthocyanin reaction in leaves and rhizome. Sugar cane, although negative in the aerial growth, gives a positive reaction in the rootstock. Cereals such as sorghum and barley contain tannins; the amount varies widely in different varieties of the former (Menaul, 1923). The tannin in the latter is of importance in relation to the control of turbidity in beer. Luers and Stauber (1931) state that barley tannin forms a red phlobaphene compound on heating,


and appears to belong to the same group as quebracho tannin (Le., the condensed tannins). In asparagus, the tannin which is sometimes present may give rise to complaints of excessive astringency (Culpepper and Moon, 1935). All too little information is given, however, in these and similar cases, for a decision to be made as to the precise nature of the tannins present.

4 . General Tendencies in Distribution

Following pp indications that can be most clearly discerned in the case of the leuco-anthocyanins, there is a tendency for the organs of woody plants to be distinguished from those of herbaceous plants by the types of flavonoid compounds that are found in them. The data relating to anthocyanins can be reviewed in this light, and what was there re- marked about the preponderance of cyanidin in woody plants will be found to hold for the Lignosae and Herbaceae of Hutchinson’s classification. If the argument were taken to its extreme limit, the situation would be represented as follows:

Lignosae Herbaceae

Cyanidin types predominate.

Flavonols predominate. Leuco-anthocyanins and/or catechins uau-

Pelargonidin and delphinidin types pre-

Flavones and flavanones predominate. Leuco-anthocyanins and catechins usually

dominate.

ally present. absent.

Generally speaking, the expectation will be for the lignose situation to be found in fruits and nuts, and the herbaceous in leafy vegetables. These expectations are being realized in the most recent work of Williams, Ice and Wender (1952) and Williams and Wender (1952a, b, 1953) on the flavonols of fruits. Studies such as these, employing chromatographic methods for the isolation and unambiguous identification of flavonoid compounds, are most valuable, and their extension to leafy vegetables and seeds would add immensely to our knowledge of the occurrence and significance of these substances in foods as a whole.

V. SOME SPECIAL CASES

1 . Citrus Fruits0

In these fruits occur the following highly methoxylated flavones and flavanones which have not been reported as occurring in any other natural source :

8 Cf. also footnotes on pp. 273 and 278.


Auranetin Tangeretin

Nobiletin Ponkanetin

as well as naringenin, isosakuranetin, eriodictyol, and hesperitin (cf. Table I).

As is so often the case with cultivated plants, the systematic botany of citrus fruits is exceedingly confused, especially since the recognized species seem to be almost completely interfertile. Swingle, in Webber and Batchelor’s The Citrus Industry (1938), recommends the terminology given in the second column of Table V. Since other authors, whose work will be quoted, have employed other names, synonyms are given in the third column.

TABLE V

Common name (Swingle) Synonyms Botanical name

Citron Citrus medica Lemon Citrus limon Lime Citrus aurantifolia Sweet orange Citrus sinensis Citrus aurantium vars. Sour (bitter, or Seville) orange Citrus aurantium Grapefruit Citrus paradisi }Citrus decumana Pummel0 (shaddock) Citrus grandis Tangerine (mandarin orange) Citrus reticulata Citrus nobilis

Ponkan tangerine

Trifoliate orange

Citrus deliciosa Citrus nobilis var. deliciosa

Citrus reticulata Citrua poonensis

Poncirus trifoliata Pseudaegle trifoliata Citrus trifoliata, etc.

var. austera

Swingle (loc. cit., p. 392) gives the following distributions of flavones

Hesperidin in C. limon, C . aurantium, C . sinensis, C. medica. Naringin in C. aurantium, C . paradisi, C. grandis. Eriodictyol in C . limon, C. sinensis.

and flavanones:


Tangeretin in C . reticulata. “ Aurantamarin,” presumably naringin, is reported (according to

C. Tanret, 1886) as responsible for most of the bitter taste of the sour orange (C. aurantium).

“ Ponciridin,” presumably isosakuranetin, in Poncirus trifoliata. Tseng (1938) found nobiletin in the drug chen-pi prepared from the

Patnayak et al. (1942) found the following distributions: Hesperidin in C . aurantium vars. Batavias and Kamatas; C . medica

Naringin in C . decumana vars. Shaddock and Mathrepala. Auraneting in C . aurantium var. Kamatas. The bitterness of C . decumana vars. was attributed by these authors

to naringin. Ichikawa and Yamashita (1941) isolated ponkanetin from the Ponkat

tangerine, C. poonensis. Yamamoto and Oshima (1931) reported the isolation from the skins

of C . l imon f . ponderosa a new glycoside, citronetin, to which they ascribed the constitution of a 7-rhamnoglucoside of 5,7-hydroxy-2‘-methoxyflavanone. In the same year, Shinoda and Sato, referring to the synthesis of citronetin, ascribe the constitution 5,7-hydroxy-3’methoxyflavanone. Neither of these constitutions would appear reasonable by analogy with the other co-occurring flavonoid compounds in citrus fruit. It should be considered whether citronetin is not, in fact, identical with isosakuranetin (5,7-hydroxy-4’-methoxyflavanone), which bears the same relation to naringenin as does hesperitin to eriodictyol.

It would be expected (cf. Section 111) that the particular flavonoid compounds found in hybrids would depend, in the first place, on whether their formation was a dominant or recessive character. In the Sampson tangelo (C. paradisi X C . reticulata) Nelson (1934) found no nobiletin. Krewson and Couch (1948) report the occurrence of rutin (quercetin-3- rhamnoglucoside) in the Satsumelo ( C . paradisi x C . reticulata var. of Satsuma group), which presumably indicates its presence also in a t least one of the parent forms.

The chances of using the specific distribution of flavonoid compounds either for the identification of the origin of a citrus product, or for clari- fication of taxonomy (as Swingle suggests), will depend on the development of easy methods of identification. With such completely methoxylated compounds as nobiletin and tangeretin this is not an easy matter, because of the absence of any phenolic hydroxyl function. For

9 This name substituted by V. V. S. Murti, S. Rangaswami, and T. R.. Seshadri, 1948

skins of C. nobilis.

vars. Naranja and Dabba.

Proc. Ind. Acad. Sci. 28A, 19, for earlicr aurantin.


this reason also, these substances cannot, of course, be glycosylated, and they occur not in the cell sap but in the oil glands of the fruits. They do not even make deeper-colored salts with bases, because of the absence of hydroxyl groups, and are therefore difficult to locate on chromatograms. The best hope would seem to lie in demethylation and identification of the resulting nor-compounds.

In addition to these flavones and flavanones, many varieties of citrus fruits contain anthocyanin pigment. This does not appear to have been identified, but that in blood oranges has been studied by Matlack (1931), Carrante (1941), and Patan& (1948). The color of pink grapefruit and Indian pummel0 is due to lycopene, a carotenoid (Matlack, 1934, 1935).

Citrus fruits also contain, in greater or lesser degree, " phlobaphene tannin." Hardy and Warneford (1925) reported that the coloring matter of lime juice (variety not specified) was a sap-soluble phlobatannin. The author has been unable to confirm this observation in limes of West Indian origin; nor has Mrs. N. H. Lerner (personal communication) in West African limes. Faint phlobaphene reactions were, however, obtained with the albedo of commercial sweet oranges and tangerines, varieties unknown. These also gave fairly strong, but rather anomalous, vanillin reactions.

Technical uses of naringin as a by-product of the citrus industry are discussed by Kesterson and Hendrickson (1953).

2. Tea

The polyphenols of tea comprise catechins, flavones, and simpler molecules such as gallic acid, the whole system presenting a picture of the greatest complexity even in the green leaf before fermentation (cf. Fig. 1). The changes during fermentation increase the complexity still more.

a. Catechins. Roberts and Wood (1951a, b, 1953), from their own work and that of Tsujimura and Bradfield and his co-workers (for references see Roberts, 1952) conclude that the naturally occurring catechins are Z-epicatechin, d-catechin, 1-epigallocatechin, d-gallocatechin, and the 3-galloyl esters of Z-epicatechin and Z-epigallocatechin, the last pre- dominating markedly over all other catechins. During the manufacture of green tea, the steaming or immersion in boiling water partially epimer- izes the catechins, so that in green tea, as Roberts and Wood (1951b) showed, the unnatural optical isomers may be found. These are separated from the natural ones when extracts are chromatographed on paper with water as the mobile phase.

b. Flawones. The large number of individual anthoxanthins revealed on a chromatogram of an extract of unfermented tea (Roberts and Wood, 1951b) are probably glycosyl forms of a much smaller number of agly-


cones. Osima and Ka (1936) reported the presence of kaempferol and Tsujimura (1941) that of quercetin and kaempferol in green tea. I n a chromatogram of the aglycones from fresh leaves, the author has observed myricetin, quercetin, and kaempferol, the first in greatest amount. It is likely that it is from glycosides of these three flavonols that all the spots giving flavone reactions derive. Other “ anthoxanthin” spots are due to chlorogenic acid and similar substances (Roberts and Wood, 1953).

c. Fermentation. In this complicated process, oxidation and condensation of the polyphenolic substances play a major part. The rolling

R F

0 0.2 0.4 0.6 0. I I I r I +

I

FIG. 1. Map showing location of the chief anthoxanthins found in tea-leaf juice. Crosses indicate positions of identified polyphenols (I - 10). Color reagent, NHs vapor. Solvents left to right, phenol; downwards n-butanol-acetic acid (Roberts and Wood, 1951a).

process is, in effect, a deliberate bruising treatment which allows rapid enzymic oxidation, as described earlier (p. 268), to take place. As might be expected, the catechol and pyrogallol residues of the catechins are strongly attacked by the polyphenoloxidase system, with the formation of dark-colored oxidation products. The anthoxanthins, possibly because they are present almost wholly in glycosidic combination, show little change during fermentation.

In fermenting juice, the gallocatechins are oxidized before the catechins and epicatechins are attacked. In the earlier stages of fermentation gallic acid shows a marked increase, presumably by liberation from ester combination. Similar changes are observed in fermenting tea leaf, in which, as the catechins disappear, more complex substances are formed which are probably condensation products (Harrison and Roberts, 1939).

d. Bearing on Quality in Tea. The term “quality11 in tea has a rather specialized significance, about which there is not at present any general agreement. Various factors contribute to over-all quality, of which


“quality,” in this specialized sense, is one. A particular taster might, for example, recognize “strength,” “briskness,” body, flavor, etc., and also, in addition, “quality.” These factors, it will be observed, are not clear-cut in their attribution to specific physical properties, and it is therefore unlikely that they can be accounted for in terms of specific chemical constituents. “Strength,’] relating especially to taste, will be referable in part to the bitterness imparted by caffeine, in part to the astringency and taste (if any) which the “tannins” and other polyphenols may contribute. “Briskness ” is probably related to astringency specifically, but acidity may also play a part. “Body” and L‘smoothnessJ’ are likely to be concerned with the colloidal constituents, that is, to the degree of condensation of the polyphenolic constituents. Flavor is concerned mainly with the volatile constituents, i.e., the essential oils and esters. “Quality,’] it has been suggested, may be defined as an especial attractiveness that a particular tea may possess, in respect of any of these factors or a combination of them, but especially, perhaps, in relation to flavor. Finally, the color of the infusion is a factor to be taken account of in quality. This, above all, is related to the flavonoid constituents of the leaf and the changes they undergo during fermentation. It is towards an understanding of the processes which take place during fermentation, and how they might be more closely controlled to the advantage of the finished product, that research in tea chemistry is now being most actively pursued.

3. Cacao

Traditionally, there are two main types of cacao, Forastero (“for- eign”) in which the beans are purple in section, and Criollo (“native”) in which they are cream-colored. The purple pigment of the former was shown by Robinson (cf. Knapp, 1937) to be a cyanidin-3-glycoside. The latter were shown by Knapp and Hearne (1939) to contain a leuco- anthocyanin and Forastero beans were found by Hallaslo (1949) to contain a leuco-anthocyanin in addition to anthocyanin. Both types contain Z-epicatechin (Freudenberg el al., 1932).

With the aid of chromatography, Forsyth (1948, 1949, 1952a, b) has, during the last few years, immeasurably advanced our knowledge of the polyphenolic substances in cacao and their changes during fermentation. There are three anthocyanins in Forastero beans, the two main pigments being a cyanidin monoglycoside, probably of glucose, and an acylated cyanidin bioside, probably of arabinose and glucose; and the third, in minor amounts, a diglycoside yielding glucose and arabinose in the pro- portion about 3 : 1.

10 Unpublished work of the British Food Manufacturing Industries Research Asso- ciation.


The leuco-anthocyanins consist of two main fractions one, sugar-free, RE about 0.4 in butanol-acetic acid, and one remaining on the origin when chromatographed and yielding sugar on hydrolysis. Both yield cyanidin when digested with hydrochloric acid.

There appear to be at least six catechin-like constituents, of which I-epicatechin is the principal one, with d-catechin, I-epigallocatechin, and d-gallocatechin also present.

For isolation of these various constituents in bulk, fractionation with ethyl acetate and chromatography on cellulose pulp were employed.

a. Fermentation of Cacao. In practice, the fermentation of cacao is carried out in sweat boxes, in which the pods are heaped. The pulp sur- rounding the beans ferments, producing heat and acetic acid, and killing the beans. Forsyth has shown that the fermentation is anaerobic, and that the browning of the beans, which was previously thought to be due to the action of polyphenoloxidase, is nonoxidative. It is mainly accompanied by a destruction of the anthocyanins, and their condensation to products resembling the nonmotile leuco-anthocyanin fraction. Oxidation only takes place during the subsequent sun-drying of the beans. The changes during fermentation are vitally important in determining the flavor and aroma of the final product.

This review was prepared as part of the program of work of the Food Investigation Organization of the Department of Scientific and Industrial Research. The author wishes to acknowledge the help he has received from Professor T. Wallace and the staff of the Long Ashton Horticultural Research Station, and Dr. E. A. H. Roberts, Dr. E. B. Hughes, Dr. K. S. Dodds, and Dr. T. Swain.

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