ANTHOCYANINS

Y R R=H, resorcinol

2.5 ANTHOCYANINS

2.5.1 Chemistry and distribution

The anthocyanins are the most important and widespread group of col- ouring matters in plants. These intensely coloured water-soluble pig- ments are responsible for nearly all the pink, scarlet, red, mauve, violet and blue colours in the petals, leaves and fruits of higher plants. The anthocyanins are all based chemically on a single aromatic structure, that of cyanidin, and all are derived from this pigment by addition or sub- traction of hydroxyl groups or by methylation or by glycosylation (see Fig. 2.9).

Anthocyanins

OR

67

H

HO HO

Pelargonidin

OR

HO

Delphinidin, R=R'=H Petunidin, R=Me, R'=H Malvidin, R=R'=Me

OH

OR' HO

OH

HO

Cyanidin, R=H Peonidin, R=Me

OH OH

RO

Cyanidin 3-glucoside, R=H Cyanidin 3,5-diglucoside, R=glucose

Betanidin, R=H Betanin, R=glucose

Fig. 2.9 Structures of anthocyanins and betacyanins.

There are six common anthocyanidins (anthocyanin aglycones formed when anthocyanins are hydrolysed with acid), the magenta-coloured cyanidin being by far the most common. Orange-red colours are due to ,pelargonidin with oneless hydroxyl group than cyanidin, while mauve, purple and blue colours are generally due to delphinidin, which has one morehydroxyl group than cyanidin. Three anthocyanidin methyl ethers

68 Phenolic compounds

..

are also quite common: peonidin derived from cyanidin; and petunidin and malvidin, based on delphinidin. Each of these six anthocyanidins occurs with various sugars attached as a range of glycosides (Le. as anthocyanins). The main variation is in the nature of the sugar (often glucose, but may also be galactose, rhamnose, xylose or arabinose), the number of sugar units (mono, di- or tri-glycosides) and the position of attachment of sugar (usually to the 3-hydroxyl, or to the 3- and 5- hydroxyIs, see Fig. 2.9).

A significant number of anthocyanins occur acylated with either an organic acid such as malonic or with an aromatic acid such as p-coumaric acid. Acylation is commonly through the sugar unit of the 3-position and both types of acylation may be present in the same molecule.

Delphinidin, cyanidin and, to a much less extent, pelargonidin may also be formed in acid-hydrolysed plant tissue from colourless polymeric tannins present, from compounds originally known as leucoanthocy- anidins and now as proanthocyanidins or flavolans. Production of anthocyanidin in this way constitutes the main method for detecting these colourless substances in plants. They are regularly present in wood and leaf of woody plants, but very often appear in the flowers of these plants.

In such cases, ifthe tissue is already coloured, it is not always clear in preliminary analysis whether the anthocyanidin detected is derived from anthocyanidins or from anthocyanin pigment already present.

While anthocyanins are nearly universal in vascular plants (they have been detected in a few mosses, in young fern fronds as well as in gymno- sperms and angiosperms), they are replaced by a superficially similar group of pigments, the betacyanins, in one order of higher plants, the Centrospermae. The best known betacyanin is betanin (aglycone:

betanidin), the major pigment of beetroot, Beta vulgaris. Betacyanins are chemically distinct from anthocyanins (see Fig. 2.9), being derived biosyn- thetically from the aromatic amino acid, 3,4-dihydroxyphenylalanine. The restriction of betacyanins to the Centrospermae (except one family, the Caryophyllaceae) is of considerable taxonomic interest and methods for distinguishing between anthocyanins and betacyanins are discussed in a later section.

A more detailed account of the plant anthocyanins may be found in Harborne (1967a).

2.5.2 Recommended techniques (a) General precaution

Anthocyanins are unstable in neutral or alkaline solution and, even in acid solution, the colour may fade slowly due to exposure to light.

Anthocyanins must therefore be extracted from plants with solvents con-

Anthocyanins

69

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4'

.: I,

,/

^""

taining acetic or hydrochloric acids (e.g. methanol containing 1% cone.

HCl) and solutions should be stored in the dark and preferably refriger- ated. Ifmalonylated anthocyanins are present, it is important to avoid contact with HCI during extraction and subsequent chromatography.

(b) Anthocyanidins

Fresh petal (or other cyanic tissue) is heated in 2MHCI in a test tube for 40min at 100°C. The coloured extract is cooled and decanted from the plant tissue. The cooled extract is twice washed with ethyl acetate to remove flavones, the ethyl acetate layers being discarded and the remain- ing aqueous layer being heated at 80°C for 3 min to remove the last traces of ethyl acetate. The pigment is then extracted into a small volume of amyl alcohol, which can be pipetted off and concentrated to dryness by heating on a watchglass on a boiling waterbath. (N.B. overheating destroys the pigment). The anthocyanidin in the residue is dissolved in 2-4 drops of methanolic HCI and chromatographed one-dimensionally on paper in Forestal and formic acid solvents. The six common anthocyanidins can be distinguished on the basis of differences inRFand colour (Table 2.10 and see Fig. 2.7). The two pigments cyanidin and petunidin run fairly close together and, if a mixture of these two pigments is suspected, a longer time should be allowed for chromatographic separation (24-30 h, instead of the usual 16-18h).

Table 2.10 Properties of the common anthocyanidins

Visible

R_F(xl00) in max. (nm) Colour

Visible in MeOH- shift with

Pigment* Forestal Formic RAW colour HCI AICl₃t

Pelargonidin 68 33 80 red 520

Cyanidin 49 22 68 } 535 +

Peonidin 63 30 71 magenta

532

Delphinidin 32 13 _{42 }} 546 +

Petunidin 46 20 52 purple 543 +

Malvidin 60 27 58 542

Solvent key: Forestal=conc.HCl-HOAc-H₂0 (3: 30: 10): Formic=conc.HCl-HC0₂H-H₂0 (2:5:3); BAW= n-BuOH-HOAc-H₂0 (4: 1:5).

"There are about six other known anthocyanidins, but these are all rare in occurrence. For the properties, see Harbome (l967a).

tThis bathochromic shift to blue colours with AICl) may be observed in the visible spectrum by measuring the spectrum in methanol-Q.01% HCl and then adding 5% ale.AICl~it may also be detected by spraying or dipping chromatograms (which have been thoroughly dried to remove acid vapour) in 5% aIc. AICl).

70 Phenolic compounds

Anthocyanidins can be further identified by measuring their visible spectra and by noting whether the spectrum undergoes a bathochromic shift in the presence of aluminium chloride, a specific test for the catechol nucleus (Table 2.10). Reductive cleavage or alkaline degradation can also be carried out, to yield different phenolic acids according to the nature of the hydroxylation pattern in the original pigment. Identification proce- dures for simple phenols and phenolic acids are given in Section 2.2.

(c) Anthocyanins

Extraction of small amounts of fresh coloured petals by crushing in a narrow specimen tube with a minimum amount of methanol containing 1%conc. HCI provides (within 10-15 min) an extract sufficiently concen- trated for direct PC. Alternatively, larger amounts of tissue may be mac- erated for 5 min in methanol-HCI, the macerate filtered or centrifuged and the extract concentrated in vacuo at 35-40°C, until the volume is

Table 2.11 R_rvalues and sources of some common anthocyanins*

R_r(X100) in

Anthocyanin BAW BuHCI 1% HCI Petal source

Mon oglycosides

Pelargonidin 3-glucoside 44 38 14 Chinese Aster

Cyanidin 3-glucoside 38 25 07 Chrysanthemum

Malvidin 3-glucoside 38 15 06 Primula polyanthus

Diglycosides

Pelargonidin 3,5-diglucoside 31 14 23 Pelargonium

Cyanidin 3-rhamnosylglucoside 37 25 19 Snapdragon

Peonidin 3,5-diglucoside 31 10 17 Peony

Delphinidin 3,5-diglucoside 15 03 08 Verbena

Triglycosides

Cyanidin 3-rhamnosylglucoside-

5-glucoside 25 08 36 Cape Primrose

Cyanidin 3-(2^G-glucosylrhamno-

sylglucoside) 26 11 61 Begonia coccineus

Acylated Diglucoside

Pelargonidin 3-(p-coumaryl-

glucoside)-5-glucoside 40 46 19 Solanum tuberosum

Solvent key: BAW =n-BuOH-HOAc-HzO (4: 1:5); BuHCl =n-BuOH-2M HCl(1 :1, top layer); 1% HCl =conc.HCl-HzO (3: 97).

.. Visible colour depends on nature of the aglycone: pelargonidin glycosides are orange-red, cyanidin glycosides deep red, etc. The 3,5-diglycosides of pelargonidin, peonidin and malvidin can be further distinguished by the fluorescent colours they give inUVlight, other glycosides give dull to bright colours.

Anthocyanins

71

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reduced to about one-fifth of the original extract. The anthocyanins are chromatographed one dimensionally on paper in BAW, BuHCI and 1% HCI, against one or more marker solutions. Colours, R~

and sources of some of the common anthocyanins are shown in Table 2.11. One of the main factors determining RF is the number of sugars attached to the anthocyanin, and increasing glycosylation reduces the RFin BAWand BuHCI but leads to a higher R_F in 1% HCI (compare the RFs of the cyanidin mono-, di- and triglycosides in Table 2.11).

Glycosides of the different anthocyanidins can be recognized by their visual colours on chromatograms, since pelargonidin glycosides are orange, cyanidin and peonidin glycosides magenta and delphinidin glycosides mauve.

Some anthocyanins occur in more complex combined form, acylated with a simple organic acid or with a cinnamic acid. They can be identified by the effect that acylation has onRFvalue (Table 2.11) and by the fact that, on re-chromatography, they give rise to two spots due to the lability of the acyl group. A test for acylation in anthocyanins is alkaline hydrolysis at room temperature (under nitrogen), which removes the acyl group and causes a change inRF•

(d) Malonylated anthocyanins

Some 30% of higher plants have anthocyanins with malonic (or similar) acid attached to them. Such pigments are zwitterionic and are readily distinguished from other anthocyanins by their mobility during paper electrophoresis in acetate buffer at pH 4.4 at 40 V/ cm and 1rnA/cm for 1.Sh. The malonyl residue is labile and pigments have to be handled in weak acid, e.g. methanol-acetic acid-water (10: 1 :9).

Malonylated pigments have higher R_Fvalues in BAW and lesser mobi- lities on HPLC than the corresponding unacylated anthocyanins. The presence of malonic acid can be confirmed after deacylation and by H202oxidation to give the corresponding malonylglucose (Takedaet al., 1986).

(e) Authentic markers

Anthocyanidins are not readily available commercially, but marker solu- tions can be obtained from readily available plant sources as follows:

pelargonidin from pelargonium flowers or red radish; cyanidin from cop- per beech leaf or blackberry; peonidin from peony flower; delphinidin from blue delphinium flower or skin of eggplant (aubergine); petunidin from black grapes or red wine; malvidin from mallow flowers. Sources of some anthocyanins are given in Table 2.11. For further details of pigment sources, see Harborne (1967a).

72

Phenolic compounds 2.5.3 Alternative techniques

(a) Thin layer chromatography

Anthocyanidins will separate on silica gel in ethyl acetate-formic acid-2M

HCI (85: 6: 9), but the colour of the spots fades fairly rapidly after separa- tion. It is important to use standard-grade silica gel, since the traces of metal present aid the separation of peonidin and malvidin from cyanidin and delphinidin. Two-dimensional TLC of anthocyanidins on microcrystalline cellulose has been carried out in formic acid-eonc. HCI- water 00: 1:3), and amyl alcohol-acetic acid-water (2: 1:1) (Nybom, 1964).

Anthocyanins can be separated by TLC on cellulose or on mixtures of cellulose and silica gel (Asen, 1965) using similar solvents to those on paper.

(b) High peiformance liquid chromatography

This method using reverse-phase columns such as ~Bondapak CI8 or LiChrosorb RP-18 is ideal for the quantitative analysis of anthocyanins in floral tissues (Stracket al., 1980).Ithas also been applied successfully to the sepal extracts of Poinsettia, where cultivars can be distinguished by quantitative differences in the five pigments present (Stewartet al., 1980).

Acidic solvents are necessary, such as water-acetic acid-methanol (71 : 10: 19) or varying proportions of 1.5% H3P04,20% acetic acid and 25%

acetonitrile in water.

2.5.4 Practical experiment

(a) Distinction between anthocyanins and betacyanins

As mentioned above, while anthocyanins are almost universal in their occurrence in plants, they are replaced by a different type of water-soluble purple pigment in the Centrospermae. Plants in all but one of the families of this order have nitrogen-containing betacyanins. The most common betacyanin is betanin, the pigment of the beetroot. Other members of the Centrospermae from which betacyanins have been isolated are various cacti, bougainvillea, pokeweed,Phytolacca and Amaranthus.

Betacyanins differ from anthocyanins in being much more unstable to acid hydrolysis, in their colours at different pHs and in their chromato- graphic and electrophoretic properties, so that it is easy to distinguish them by simple colour tests. Since the two classes of pigment are mutually exclusive in their occurrence (i.e. they never occur together in the

I

Anthocyanins 73

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same plant), these tests can be carried out on crude pigment extracts.

Betacyanins also differ from anthocyanins in that, when ingested in food (e.g. as beetroot) they are not metabolized by some 14% of the human population and their colour is excreted unchanged in the urine; hence the condition known as 'beeturia'.

(b) Procedure

(1) Students can prepare methanolic HCI extracts from anyone of the many anthocyanin sources (e.g. elderberry, raspberry, blackberry, copper beech leaves, almost any red to purple flowering planO, solu- tion (A) and from beetroot, solution (B) as being the most accessible betacyanin source. After maceration, the extracts should be filtered (with celite) and concentratedin vacuoto about one fifth of the original volume. Students may then be provided with four 'unknown' purple- red pigment extracts, solutions (C-F) and told to identify the nature of the pigment present.

(2) Tests, as indicated in Table 2.12, are carried out on all six solutions (A- F). The results obtained with A and B are then used to decide what pigments are present in (C-F). The first four tests shown in Table 2.12 are sufficient for most purposes; the latter two tests can be added if the necessary time and equipment are available.

(c) Additional experiments

(1) Anthocyanins in coloured petals in any series of ornamental plants available may be separated by PC in BAWand 1%HCI of methanolic HCI extracts of fresh tissues. Marker solutions of pelargonidin 3,5- diglucoside (from pelargonium flowers), cyanidin 3,5-diglucoside (most red roses) and delphinidin 3,5-diglucoside (Verbena, Del- phinium) should be included on all chromatograms. The nature of the aglycone and the approximate number of sugar residues can be determined from RFs and visible colours for each of the flower anthocyanins which separate.

(2) Anthocyanins in different colour types of the same plant may be compared by PC in BAW and the biochemical effects of genes control- ling anthocyanin synthesis be noted. Plants with a wide range of colour types suitable for such study include snapdragon, sweet pea, Streptocarpus and anemone. In the sweet pea, Lathyrus odoratus, for example it will be found that purple, violet and lilac varieties contain glycosides of malvidin, petunidin and delphinidin, carmine varieties glycosides of cyanidin and peonidin and scarlet shades glycosides of pelargonidin.

74

Phenolic compounds

Table 2.12 Tests for distinguishing anthocyanin and betacyanin·

Test 1.

Heat with2M HCI for 5 min at 100°C 2.

Add2MNaOH dropwise 3.

Chromatography in 1%aqueous HCI 4.

Chromatography in BAW

5.

Visible spectrum in Methanol-HCI 6.

Paper electrophoresis at pH 2-4

Anthocyanin response

colour stable (can be extracted into amyl alcohol)

changes to blue-green and slowly fades low to intermediateRF

moderateRF00-40)

maximum in the range 505-535nm

moves towards cathode

Betacyanin response

colour vanishes

changes to yellow

highRF

very low^RF(00-10)

maximum in the range 532-554nm

moves towards anode

• These colour tests can be carried out on crude pigment extracts; for anthocyanin, any deep red-coloured flowers may be extracted or certain fruits (e.g. blackberry, raspberry, straw- berry); for betacyanin, beetroot extract is convenient. The responses are those shown for such crude extracts; purified pigments may behave slightly differently.

2.6 FLAVONOLS AND FLAVONES