VOL. 5, No. 12
CHEMISTRY ou RED AND BLUEP I G ~ N T SI1.
1615
THE CHEMISTRY OF THE RED AND BLUE PIGMENTS OF FLOWERS AND FRUITS. PART I1 ERNESTH. H U N ~ S MASSACWSBTTS S, INSTITUrS OF TECANOLOOY, C~RIDOE. MASSACWSBTTS
The Occurrence and Isolation of the Anthocyanins Having thus described1 the very simple relationships existing between pelargonidine, cyanidine, and delphinidine, and digressed briefly to illustrate the methods by which their supposed structures have been so abundantly confirmed by synthesis, it will now be appropriate to consider the natural occurrence of the corresponding anthocyanins in the flowers and fruits. Twenty-one glucosidic compounds of these anthocyanidines have been isolated by the work of Willstatter and Karrer. They have usually been designated by names in which a root indicating the plant source is terminated by the suffix "in" to emphasize the glucosidic character and to distinguish the substance from the corresponding anthocyanidine obtained on hydrolysis. In many cases these root names are derived from the Latin botanical nomenclature. In the accompanying Table I the names of these twenty-one anthocyanins are so tabulated as to indicate not only the source of the material but also the particular anthocyanidine and the number and kind of carbohydrate molecules with which it is associated. The parenthetical numbers refer to the bibliography at the end of this article. Mixtures of anthocyanins usually occur in any given source but this table lists only those individuals which actually have been isolated. The entries under the heading of indefinite combinations represent cases in which the indicated anthocyanidine was detected but where the exact mode of combination was not fully examined. The presence of the designated organic acids was detected from salvianin, monardein, gentianin, and delphmin: nothing is yet known with regard to the mode of combination of these acids in the original anthocyanin molecule. The proportion of anthocyanin in the various flowers varies through a wide range. In the first case studied by Willstatter, that of the blue cornflower, the cyanin content is only about 0.75y0 of the weight of the dried flowers. Since subsequent experience showed that this is the smallest percentage of coloring matter in any of the flowers studied, the glory of the achievement is all the greater. In the more deeply colored blossoms, however, the pigment contents are generally higher. In deep blue-violet cornflowers the cyanin content has risen to 3.6% of the dry weight: in the deep bordeaux shades it may be 13-1470. The cyanin content of the deep red dahlia constitutes 20% of its dry weight and represents the best source of this particular glucoside. 'See THISJOURNAL, 5, 1392-8 (Nov., 1928).
Pelargonidine
Cyanidine
Delphinidine
MONOGLUCOSIDES Callistephinsummer aster Asterin-summer aster (14) Gentianin-ommon blue Chrysanthemin-winter gentian (18). Hydrolysis (14) Pelarganenin-from sal- aster (13) gives also 9-hydroxy vinin of salvia (12) cinnamic acid MONOGALACIDSIDES Idaein-anberry (8) DIGL~COSIDES Pelargonin-scarlet pelar- Cyanin-blue corniiower(6) Delphiuin-larkspur (10) Cysnin4eep purple corn- Hydrolysis gives also @gonium (9) hydrovyhenzoic add flower (11) Pelarganin-orange-red Cyanin-rose (7) dahlia (11) Pelargonin-red cornflower Cyanin-deepreddahlia(11) Mekocyanin - double (11) purple-scarlet poppy (17) Salvianint* Salviu -redsalvia (12) Salvinin 'Hydrolysis gives also malonic acid Monardein-bee-balm (18) Hydrolysis gives Monardin (possibly identical with pelargonin) and p-hydroxycinnamic acid Punicin-pomegranate (18)
1
RH~~OGLUCOSIDBS Keracyanin-sweet cherry Violanin-blue-black pansy (16) (15) Vicin-dark red vetch (18) Prunicyanin-sloe (15) Sambucin-elderberries (18)
I ~ E F I N I COMBINATTONS TE Anthocyanidines from rad- Anthocyanidines from zinishes (12), scarlet red nia, gaillardia, gladiolus, sneeze-weed, tulip, nasgladialas (13) turtium, raspberry, red currant, fruit of mountain ash (13, 15)
T h e percentage:pigment obtained f r o m t h e dried flowers is listed in Table 11.
petals of some other
TABLEI1 Anthocyaoin
mowers
% pigment
Delphinin Peonin Salvianin Malvin Pelargonin Chrysanthemin Callistephin Asterin Althaein Petunin Violanin
Wild purple delphinium Deep violet-red peony Red salvia Wild mallow Searlet pelargonium Deep red chrysanthemum
1.75 33.5 8 6.4 6.67.1 7
Asters Hollyhack Petunia Dark blue-black pansy
The proportion of violanin in the pansy represents the highest anthocyanin content found during Willstatter's investigations. The processes for the isolation of the pure anthocyanins from flowers and fruits represent in some respects the most outstanding feature of Willstatter's remarkable work. Quite apart from the obvious difficulties inherent with work'mg with the necessarily large quantities of raw material and solvent and the troublesome solubility relations already mentioned, many other difficult chemical problems had to be solved. For example, it was necessary to devise not only some means of separating the anthocyanins (themselves glucosides) from the accompanying starches, gums, pentosans, and other water-soluble or colloidal material, but also methods of minimizing a tendency on the part of certain of the pigments to change over during treatment to a colorless form. Although these studies of the isolation of anthocyanins are marvels of ingenuity and patience and will be an inspiration to any chemist who will refer to the original papers it is impossible to speak of details here. For raw materials there could be used either the fresh flowers or commercially dried petals. The objections to the use of fresh flowers arose on one hand from the consequent necessity of carrying out their treatment at fixed times and places and involving the simultaneous collection of a very large number of blossoms, and on the other from the possible complications due to the presence in the fresh flowers of plant enzymes in active form. Substitution of dried for fresh petals obviated both these difficulties but hazarded the loss of anthocyanin through decomposition during the dryiig. This difficulty proved to be negligible, however, and dried petals were mainly employed. In certain cases, like that of the red poppy, it proved necessary to use the fresh blossoms: Willstatter (Ref. 17) mentions the interesting f a d that although the poppy field at the experiment station was picked clean each day, the next morning found a fresh brilliant red surface of new blossoms.
The solvent chosen for the extraction of the pigment from the flower petals or berry skins varied according to the nature of the particular anthocyanin. In the case of the cornflower, water alone sufficed: for the rose, hollyhock, mallow, peony, and huckleberry, a dilute (273 solution of hydrochloric acid in methyl alcohol was employed: for the larkspur and scarlet pelargonium, dilute alcohol proved suitable: glacial acetic acid was used for the cranberry, grape, salvia, winter and summer aster, petunia, and pansy. Following the prelimmary extraction the anthocyanin was precipitated in very impure form by addition of another liquid, usually ether, and the crnde precipitate thus obtained subjected to an exhaustive and laborious series of fractional solutions and reprecipitations. These operations ultimately raised the purity of the material to a degree which warranted further treatment by conversion to and crystallization of the picrate or chloride or both. Throughout the procedures Willstatter followed the progress of the purification by colorimetric comparison of the samples with known standards prepared from preliminary experiments. Since the colors both of red and blue flowers appear to be traceable to the same anthocyanin or at least to a few similarly constituted individuals, the question arises as to how the great variation in effects of shade and tint as well as color itself may be accounted for. The answer appears to be two-fold in character, including on one hand the results attributable to mixtures of anthocyanins one with another or with pigments of other types, and on the other the variation of a given anthocyanin produced by its environment. In pursuing the first of these causes it may be noted that in a number of instances several diierent anthocyanins were found in the same source. Thus from the dark purple-red cornflower both cyanin and pelargonin were isolated: in one violet-red variety of Pelargoniu~zzonale more cyanin was found than the expected pelargonin. Furthermore, it appears that the characteristic anthocyanin may vary from one variety to another of the same species: for example, although certain deep brownish red dahlias contained as much as 19.4Y0 cyanin and formed the most favorable source of this material, yet other scarlet red varieties contained 4.0-5.670 pelargonin (Ref. 12). Since each anthocyanin possesses a characteristic color under given conditions it is plain that considerable color variation may arise from pigment intermixture. Furthermore, the admixture of yellow pigments with the anthocyanins strongly influences the resultant color. There are three types of these: first, the inert carotinoids, principally carotin, a hydrocarbon (C40Hs6), which is responsible for the color of carrots, and xanthophyll (CIoHssOp),one of its oxidation products: second, the glucosidic flavone and flavonol colors; and third, the little known dissolved pigments designated as anthochlors. As another important source of color variation for cases where pigment
VOL. 5, NO. 12
CHEMISTRY OF REDAND BLUE PIGMENTS I1
1619
intermixture may not be involved there should be considered the variation in concentration of the particular anthocyanin which is present. Certain of these are able to isomerize to a colorless form and this cause alone may serve to produce color variations in different parts of the same flower. Even apart from this phenomenon, light and deep colored specimens of the same flower often showed pronounced differences in pigment content. To the instances already cited in discussing pigment content may he added another: the bluish red blossoms of one pelargonium contained 0.97% pelargonin on the dried weight, another deep violet-red specimen gave 14.1%. The same diierence in concentration of colored pigments may even be exhibited in a single flower: in one large purple dahlia the cyanin content of the inner petals was 23.7'%,that of the outer petals 15.1%. Even more interesting than these predictable concentration effects is the influence of the acid or basic character of the cell fluids, or in other words their pH. It will he recalled that the anthocyanins are amphoteric substances which form salts both with acids and bases. It was concluded by Willstatter that the red forms of anthocyanin represented combinations with acids, the blue forms salt formation with alkalies, and their purple forms an intermediate neutral condition. In the scarlet pelargonium the anthocyanin was found combined with tartaric acid; in the cranberry, citric, benzoic, and gallic acid o c m abundantly; the blue cornflower was found to contain its cyanin largely in the form of potassium salt; the violet blossoms of larkspur contained delphinin in neutral condition. Karrer has reasoned that if this he true then blue flowers should yield a higher percentage of ash than red and, noting the meager data, has reported (Ref. 19) the results of analysis of some flower ash. These are reproduced in Table 111.
MS -
Red blossoms
Pomegranate Peony Pink Blue blossoms t
1.37
Traces ?
Violet
....
Wmd mallow
3.5
ComEower "Black malve"
Traces Traces
* Data for individual metals and radicals is given in per cent of total ash. It is plain that these data supPo& this view.
1620
JOURNAL o* CBEMICAG ED~CAT~ON
DECEMBER, 1928
Bibliography (6) Willstitter and Everest, "I. The Pigment of the Corn Flower," Ann., 401,189-232 (1913). (7) Willstitter and Nolan, "11. The Pigment of the Rose," Ann., 408, 1-14 (1914). (8) Willstitter and Mallism, "111. The Pigment of the Cranberry," Ann., 408, 15-41 (1914). (9) WiVstitter and Bolton, "IV. The Pigment of the Scarlet Pelarganium," Ann., 408, -1 (1914). (10) WiNstitter and Hieg, "V. An Anthocyan of the Larkspur," Ann., 408, 61-82 (1914). (11) Willstitter and Mallison, "X. Variation in the Color of Flowen." Ann.. 408, 14742 (1914). (12) Willstitter and Bolton. "XI. The Anthocyan of the Red Salvias," Ann.,, 412, 113-36 (1916). (13) Willstitter and Bolton, "XII. The Anthocyan of the Winter Aster (Chrysanthemum)," Ann., 412, 13748 (1916). (14) Willstitter and Burdick, "XIII. Two Anthocyans of the Summer Aster," Ann., 412, 149-64 (1916). (15) Willstitter and Zollinger, "XIV. The Pigments of the Cherry and Sloe," Ann., 412, 164-78 (1916). (16) Willstitter and Weil, "XV. The Anthocyan of the Violet Pansy," Ann., 412, 178-94 (1916). (17) Willstitter and Weil, "XVIII. The Color of the Poppy. I," Ann., 412, 231-51 (1916). (18) Karrer and Widmer, "Investigation of the Plant Coloring Matten. I. The Constitution of Certain Anthocyanidines," HA. Ckim. Acla, 10, -3 (1927). "11. Further investigations," Ibid., 10, 67-86 (1927). (19) Karrer, Widmer, Helfenstein, Hiirliman, Nievergelt, and Monsarrat-Thorns, "IV. The Anthocyans and Anthoeyanidines," HA. Ckim. Ada, 10, 729-57 (1927).
