Modern Methods of Plant Analysis: 3: 463-467 (1955)
Ed. K. Paech & M. V. Tracey

Anthocyanins, Chalcones, Aurones and Flavones.

C. Identification by Color Reactions.
I. Color Tests on Fresh Plant Tissues and Crude Extracts.

The presence of flavonoid constituents in plant tissues can often be demonstrated by the testing of simple aqueous or alcoholic extracts or samples of the tissues themselves. Tests on fresh tissues are most successful with flower petals, in which interference with color reactions by chlorophyll and non-flavonoid phenolic substances is minimal. Extracts of plant samples can be subjected to a rough fractionation for the purpose of testing for the presence of specific classes of compounds and as a guide in later larger-scale isolation studies.

Anthocyanins are immediately recognizable. Red to blue pigmentation is in most cases due to the presence of anthocyanins, and the colors of red-orange, brown or "black" flower petals are usually due to the presence of mixtures of anthocyanins and "background" pigments of flavonoid or carotenoid types. Exposure of anthocyanin-containing tissues to the fumes of concentrated aqueous ammonia causes color changes to violet or blue. When the anthocyanin is accompanied by flavones, ammonia vapor causes the appearance of green-blue to green colorations.

II. Color Reactions of Flavonoid Compounds.

1. Anthocyanins and Anthocyanidins.

The scheme developed by ROBINSON and ROBINSON (1931) for the rapid analysis of anthocyanins in plant extracts depends primarily upon observations of color changes, coupled with estimations of the distribution of the pigments between organic and aqueous solvent phases. The following is a description of the procedure developed by these workers for the examination of flower petal extracts:

A cold, aqueous 1% hydrochloric acid of the fresh petals is prepared. Frequently some purification is necessary before satisfactory color reactions can be obtained.

1. Solutions of diglycosides can be repeatedly extracted with amyl alcohol. Occasionally this is sufficient.

2. The pigment is taken up in a mixture of amyl alcohol (2 parts) and acetophenone (1 part) containing picric acid. The organic layer is separated, filtered, diluted with ether and shaken with 1% hydrochloric acid. The aqueous solution is completely freed of picric acid by repeated extractions with ether.

3. Monglycosides can be purified as in 2. by extraction with cyclohexanone-picric acid or ethyl acetate-picric acid, followed by dilution of the organic phase with light petroleum ether and extraction of the pigment with 1% hydrochloric acid. The aqueous solution is then extracted with benzene, cyclohexanone and again with benzene. Occasionally the process has to be repeated.

Examination of the Anthocyanin. (1) If the color of the solution is orange-red, dilute a sample with alcohol. Peonidin derivatives become bluer than pelargonidin derivatives.

(2) Test for complex (acylated) diglycosides: A sample of the original solution is shaken with amyl alcohol to roughly determine the distribution number. A second extraction is made in the case of anthocyanins of high distribution number. In the latter case a test must be made for complex diglycosides. The solution is boiled in a test tube while hot 20% aqueous sodium hydroxide is added drop by drop until the color changes through green to yellow or brownish-yellow. After boiling for a few seconds, concentrated hydrochloric acid is added dropwise until the appearance of the color of the reconstituted anthocyanin indicates that the solution is acidic. One more drop is added, and after 15 seconds the distribution is again observed: monoglycosides: no change; complex diglycosides: distribution falls almost to zero.

(3) On addition of sodium acetate to the original solution, the following colors are observed:

Callistephin: dull brownish violet red.
Pelargonin: bright bluish red.
Peonidin-3-glycosides: similar to callistephin, but brighter.
Peonin: red-violet.
Cyanin: violet.
Mecocyanin, chrysanthemin: violet-red.
Malvin: bright violet.
Oenin: dull violet.
Delphinidin glycosides: blue-violet to blue.

This reaction is subject to interference from iron salts and tannins.

Examination of the Anthocyanidin. The original solution is mixed with somewhat less than an equal volume of concentrated (12 N) hydrochloric acid and boiled for about 30 seconds. The solution is cooled and extracted with amyl alcohol, and the organic layer washed with water and with 1% hydrochloric acid. A large excess of benzene is added and the pigment extracted with a small portion of 1% hydrochloric acid. The amount of benzene needed to decolorize the upper layer gives some information about the anthocyanidin, the more highly hydroxylated pigments being driven into the aqueous layer with smaller amounts of benzene.

Anthocyanidin. Amount of benzene required.
Delphinidin, 3-4 times the volume of amyl alcohol.
Petunidin: 4-5 times the volume of amyl alcohol.
Cyanidin: 5-6 times the volume of amyl alcohol.
Malvidin: 5-6 times the volume of amyl alcohol.
Peonidin: 8-9 times the volume of amyl alcohol.
Pelargonidin: 10-11 times the volume of amyl alcohol.

The filtered anthocyanin solution is again extracted with amyl alcohol and the process repeated Finally the aqueous solution is washed thoroughly with benzene to remove traces of the alcohol. The following tests are performed:

1. A portion of the solution is extracted with amyl alcohol and sodium acetate is added; after observation, a drop of ferric chloride solution is added and the tube gently shaken.

2. A portion of the solution is shaken with an equal volume of the "cyanidin reagent" (a mixture of 1 part cyclohexanol and 5 parts toluene), and the upper layer transferred to a narrow tube for observation.

3. A portion is shaken with air and half it volume of 10% aqueous sodium hydroxide added. This is immediately followed by concentrated hydrochloric acid and amyl alcohol. Recovery of the anthocyanin in this "oxidation test" is noted.

4. A portion is shaken with 5% solution of picric acid in a mixture of methyl amyl ether (1 part) and anisole (4 parts): the "delphinidin reagent".

The various anthocyanidins exhibit the following behavior in the above tests:

Pelargonidin (3,4',5,7-tetrahydroxy). AmOH—NaOAc: violet-red; no change when FeCl3 is added. Largely extracted by the cyanidin reagent, completely by the delphinidin reagent. Not destroyed in the oxidation test.

Peonidin (3,4',5,7-tetrahydroxy-3'-methoxy). Differs from pelargonidin in the color reactions of the anthocyanins derived from it.

Cyanidin (3,3',4',5,7-pentahydroxy). Reddish-violet when NaOAc is added to the amyl alcohol extract over water; FeCl3 changes the violet to a bright, clear blue (which may be confused with malvidin containing a trace of a ferric-reacting anthocyanidin). Imparts a rose-red color to the cyanidin reagent (malvidin a very weak mauve). Fairly stable to the oxidation test. Extraction by the delphinidin reagent is not complete from dilute solution.

Malvidin (3,4',5,7-tetrahydroxy-3',5'.dimethoxy). Gives a slightly bluer color in the AmOH—NaOAc test than cyanidin, and FeCl3 does not change it. The oxidation test leaves malvidin largely unchanged. Not extracted by the cyanidin reagent; completely extracted by the delphinidin reagent. Pure malvidin is of rare occurrence.

Delphinidin (3,3',4',5,7-hexahydroxy). Gives a blue solution in AmOH on the addition of NaOAc. Destroyed in the oxidation test. Not extracted by the cyanidin reagent or the delphinidin reagent.

Color Reactions of Anthocyanins.

Pelargonin (3, 5-diglycoside). Violet coloration with aqueous Na2CO3; this becomes greenish-blue on addition of acetone. Decisive confirmation is obtained by adding one-quarter the volume of concentrated HCl to the solution and boiling for about a minute. On extraction with AmOH a green fluorescence due to pelargonenin. will be observed.

Pelargonidin-3-glycosides (e. g., callistephin). Red-violet color with Na2CO3 that is rather stable towards NaOH. No other anthocyanin type behaves similarly.

Peonin (3,5-diglycoside). Blue coloration with Na2CO3.

Cyanin (3,5-diglycoside). Rich, pure blue with Na2CO3, unstable to NaOH.

Malvin (3,5-diglycoside). Bright greenish-blue with Na2CO3.

Oenin (malvidin-3-glucoside). Blue-violet with Na2CO3, unchanged by NaOH.

When new anthocyanins are encountered the tests described above are useful in allowing an estimate to be made of the extent and pattern of hydroxylation, but are incapable of establishing the details of new structures. In such cases the isolation of the crystalline pigment in amounts sufficient for degradation experiments (KARRER et al., 1927, 1929, 1932), is necessary. The color tests are likewise useless in elucidating the nature of the sugars present in the glycosides. The subtle differences in the behavior in such tests between glucosides and galactosides, for example, would not permit their positive identification by this means.

Distinctions between glycosides containing different sugar types — hexosides, pentosides, methylpentosides, dihexosides or pentosehexosides — can be made with reasonable certainty by careful measurements of the distribution of anthocyanins between amyl alcohol and dilute hydrochloric acid. This procedure, extensively used by ROBINSON and his co-workers, ía described in detail by ROBINSON and TODD (1932).

2. Flavones, Chalcones and Aurones.

Flavones and Flavonols are readily detected in white or pale yellow tissues (e. g., white flowers) by the ammonia tat, white tissues turning yellow, and yellow tissues usually deepening in shade. While this test is not specific for any single class of flavonoid substances, it is sensitive to those polyphenolic carbonyl compounds represented by the flavones, flavanones, chalcones and xanthones. Xanthones are, however, of rare occurrence in plants. The addition of alkali to a crude or partially purified plant extract can serve a substitute for the ammonia test, the presence of flavones, etc. being recognized by the appearance of a yellow coloration.

The presence of flavanones and flavonols and their glycosides can be confirmed by testing an alcoholic extract of the plant material, freed of anthocyanins, waxes and chlorophyll, with magnesium and concentrated hydrochloric acid. The appearance of a pink to magenta color indicates the presence of flavonols (and their 3-ethers and glycosides), flavanones or flavanolones. Experiments in the writer's laboratory have shown that the sensitivity of the magnesium hydrochloric acid teat is such that about 50 micrograms of quercetin can be detected with ease when this amount is present in approximately 0.5 ml. of ethanol. The use of more dilute solutions and the presence of colored impurities will reduce the sensitivity of the test, but it is safe to say that amounts of flavonols considerably less than one milligram can be detected with ease. Flavanones and flavanonols give reduction products similar in color to those formed from flavonols possessing corresponding hydroxilation patterns, and can be detected in comparable concentrations (see p. 471).

Flavones which lack the 3-hydroxyl group do not usually respond to the magnesium-hydrochloric acid reduction test. Application of this test to samples of pure flavones such as apigenin and luteolin results in the appearance of colored reduction products, but the colors produced are yellow-orange to orange-red; consequently, small amounts of flavones in plant extracts are not detectable with certainty by this test, the presence of colored impurities of whatever king usually being sufficient to mask them.

Chalcones and Aurones impart bright yellow to orange-yellow colors to flower petals and yellow-green colors to chlorophyll-containing tissues; but since most yellow flowers are pigmented by carotenoids, the chalcone and aurone pigments cannot be recognized with certainty simply by the visual appearance of the living tissues. They can be readily detected, however, by the color change from yellow to red or red-orange in the ammonia test. Chalcones and aurones behave so nearly alike in this respect that the test does not distinguish between them.

Flavanones dissolve in cold, dilute alkali to give nearly colorless to yellow solutions. On heating, isomerization (ring opening) to the corresponding chalcones occurs, with the formation of deep yellow to red colors.

III. Color Reactions of Individual Classes of Flavonoid Compounds.

Ferric Chloride. The production of colors with ferric chloride is a general property of all classes of polyhydroxy flavonoid compounds. This property is of limited use in the examination of crude plant extracts except to show the presence of ferric-reacting substances because of the non-specific nature of the reaction, but is often of value in support of experiments in which partially methylated compounds or degradation products are formed.

When two or more hydroxyl groups are present in a flavone or structurally related compound, the color given with ferric chloride is seldom unequivocally diagnostic. Ortho-dihydroxyl groups often, though not invariably, give green colors (catechin, eriodictyol, 7-0-methyl eriodictyol, protocatechuic acid); but the utility of this generalization is limited by the fact that green colors are also given by many compounds which do not contain the ortho-dihydroxyl grouping. BRIGGS and LOCKER (1951b) have discussed the use of the ferric reaction, with examples drawn from their work and the extensive studies of SESHADRI and his co-workers.