Natural Science 14: 143-1149 (February, 1899)

The Red and Blue Colouring Matters of Flowers.

IN my previous paper (see Natural Science, 1898, xii. pp. 194-199) it was maintained that the tannic chromogen of the blue colouring matter of flowers is not the same as that of the red pigment. The old investigators of this attractive subject were not all of one mind in the matter. The great majority unequivocally held that the blue was the original colour, and that the red was merely a derivative thereof, i.e. merely an unessential modification, or a product of further deoxidation or dehydration. The French chemist Filhol was for a time evidently in a state of considerable doubt and hesitancy. In his last contribution on the subject, dated 25th June 1860, he states that in examining the colouring matter of red, rose, or blue flowers he had been struck with the differences which it presents according as it is taken from such or such a flower; he noticed that a great number of flowers become blue in contact with alkalis, while others become green, and, moreover, that the pigment of the former enjoys a greater stability than that of the others. "At first," he says, "he thought he could conclude that in deep red flowers there exist two distinct colouring matters, one more stable than the other, but he soon saw that it was not so when he operated on solutions of pure cyanine; there are not two kinds of cyanine." It is all the more remarkable, therefore, that the illustrious Berzelius, who had chemically examined the red pigments of cherry and gooseberry fruits and leaves, should conclude that their colouring matter is not, as had been thought, a combination of a blue pigment with an acid. "What had given occasion for this error," he says, "is that by treating the juice of the berries with acetate of lead blue precipitates are obtained, but this blue colour is due to the impurity of the juice in consequence of the presence of citric and malic acids." It is hardly necessary to observe that for a very long time it was well known to chemists that the blue colours of flowers were reddened by acids, and even that the tint of flowers naturally red was made more vivid and brilliant by the addition of a trace of acid. But this fact, remarkable as it is in its way, seemed comparatively trifling so long as the various and divers pigments of plants were regarded as merely modifications of only one fundamental substance. Tainted with German alchemical mysticism, Schubler and Frank, A. P. De Candolle, and Marquart successively sought for and insisted on the close connection and genetic relationship of the reds, blues, and yellows: they went further, they proclaimed that the colours of flowers were all derived from and indissolubly bound to chlorophyll.

Now, it is obvious that no thorough and complete scientific understanding of this subject could possibly be attained so long as this sort of poetically "evolutional" imagination held the field. How eminently, then, does it stand to the credit of those acute and able chemists, Frèmy and Cloez, Martens and Filhol, that they were not moved or deterred in the slightest degree by the priori postulates of their German predecessors, that, in fact, they entirely ignored the connection of these brilliant colours with chlorophyll. No doubt Filhol declared that "there is the most direct analogy between xanthine and chlorophyll," but he never sought to derive the one from the other. He and his collaborateurs examined the floral pigments in and by themselves, they were never troubled with evolutionary imaginations and hypotheses, and hence they led the way to discoveries which fitly served as bases and props for further research and enlightenment. Berzelius had little doubt that pure chlorophyll may be the origin of the yellow and red colouring matters of autumn leaves, but he admits that he was never able to reproduce chlorophyll by means of xanthophyll, nor transform chlorophyll into xanthophyll. Mohl, in 1837, would not admit that chlorophyll has any relation with the red colour of autumn leaves, but he does not deny its intervention, though only indirectly, in the red colour of fruits; there is nothing to show, however, that he considered that there was any chemical connection between these bodies. In 1850 Morot, in criticising Marquart's views, observes that "chlorophyll is not found in the most superficial layers of cells where the blue, violet, and red colouring principles are chiefly found; in the more deeply situated cells of the mesophyll there exists much chlorophyll, and at a certain epoch the red substance is seen to take birth there, but at the same time the chlorophyll persists, and this red substance seems to proceed from cell sap, which is colourless beforehand; this sap by the prolonged action of a weak acid becomes red without passing by blue." This passage seem to me to be the very first distinct and definite declaration on chemical grounds, partly at least, that the green matters of leaves are not connected with the red and blue matters, although, as Morot admits, it may be affirmed that chlorophyll, anthocyan, and the red matter may be modifications of one and the same substance; but it is hardly necessary to rejoin that this is totally different from the affirmation that the blue pigment is a direct derivative by dehydration of chlorophyll itself, as Marquart maintained.

In 1858 Morren published his famous "Dissertation sur les feuilles vertes et colorées," in which he, with great acuteness, insisted that the red and blue colouring matters of plants are not formed from chlorophyll; and the blue colouring matter (anthocyan) is probably, like litmus, the alkali salt of an acid which in the free state constitutes the soluble red pigment (erythrophyll) of autumn leaves. It will be noticed also that Morot had clearly enounced that a red colour could possibly be formed independently in the leaf, in fact it is seen to be produced without having previously passed through a blue tint; in other words, it was not derived from the blue. Here, then, there was a further break up of the olden entanglement, for not only were the blue and red separated from the green, but the red was threatened with divorce from the blue. Now, it was just precisely at this critical point in the scientific enlightenment of the mystery that two papers were published which may rank among the more interesting efforts of an epoch fertile in important researches and discoveries.

In the Botanische Zeitung, dated April 18, 1862, there appeared an article entitled "Some Propositions anent the Physiological Meaning of Tannin and of the Pigments of Plants" by A. Wigand, in which the author, after giving various illustrations, and leaving chlorophyll, anthoxanthin, indigo, etc., out of account, avers that "in general it results from the foregoing as to nearly all blue and red pigments that these proceed from tannin through an only unessential modification." … "A colourless substance dissolved in the cell sap furnishes the basis for anthocyan, and this chromogen is tannin, or rather some modification of tannin (cyaneogen): the transformation of cyaneogen into anthocyan depends on an oxidation." Again, early in 1863 Professor W. Stein suggested that the red colouring matter of flowers was paracarthamin, a red substance which he had obtained by the action of sodium amalgam on the plant-yellow (rutin) that had been discovered by Weiss in 1842.

Notwithstanding the extreme suggestiveness of the declarations just now set forth, appealing as they did most forcibly to the scientific intelligence, it is extremely discreditable that, so far as concerns Germany, they were practically ignored for nearly twenty years. No doubt Wiesner, alluding to Wigand's paper, had published some observations about eight months afterwards, and again in 1872; and Kraus, in 1872, had connected the red coloration of winter leaves with the presence of tannin. But it was not till 1881 that Detmer confirmed the views of Kraus, and H. Pick, in 1883, by an elaborate histological and micro-chemical investigation of young shoots, older stems, petioles, fruits and their peduncles, and autumn leaves, fully and amply extended and ratified the propositions enunciated with such brilliant genius by A. Wigand. "We now," he states, "on the basis of our researches, assert with Wigand, as a fact, that the red pigment is to be found only in plants containing tannin, and that the tannin, at first colourless, can change into the red pigment, which likewise reacts like tannin."

Meanwhile, however, the remarkable differences which are observable in red and blue flowers and their colouring principles towards certain reagents, especially dilute ammonia, or ammonia vapours with or without the presence of added acids, still continued to excite comment and acute controversy. So far back as the year 1824 Macaire of Geneva, having noticed that a red infusion of Viola odorata, when mixed with a vegetable alkaloid, such as quinine or strychnine, gradually retakes the natural blue tint of this flower, suspected that its colour is owing to a combination of its chromule with an alkali. In 1825 Schubler and Frank testified that the infusion of Funkia ovata treated with an acid and then with an alkali may present in the same vessel all the colours of the spectrum. De Candolle was perfectly aware, in 1832, that the infusion of certain red flowers which have been acidified assume a blue colour when treated with alkalis, and, on the other hand, that the acidified infusions of blue flowers do not reassume their original colour when similarly treated. Frèmy and Cloez asserted that anthocyan was reddened by acids and greened by alkalis. Wigand stated that the colouring matter dissolved in acid cell saps became by alkalis first blue and then green. Wiesner, on the other hand, stoutly maintained that anthocyan as such or in itself was always blued by alkalis and never greened: it is only when it is present along with a substance which is coloured yellow by alkalis that it passes by the latter body into green, which thus arises as a mixed coloration. In 1867 Naegeli and Schwendener again asserted that anthocyan is blued by alkalis and then passes into green; and Sacchse, in 1887, expressed concurrence with this view. All this diversity of opinion concerning a matter which, one might imagine, is capable of being settled once and for all by the application of definite tests, would undoubtedly never have come to pass if there had not been "something in it," as they say. The remainder of this paper will be devoted to an attempt to explain precisely what this mysterious "something" is.

In the first place, it will be advisable to enumerate at least some of those flowers whose colouring matters are known to be blued by ammonia vapour and by a dilute solution of ammonia. The following list comprises some of the most notable examples:—Fuchsia, Pelargonium sp., Plumbago, blue sp., Lycium barbarum, Phaseolus multiflorus, Erythrina crista-galli, Echinacea serotina, Impatiens balsamina, Salvia splendens, Polygonum orientale, Camellia, Paeony, and deep-red garden Rose (acidified alcoholic extracts). There are doubtless many more, but the study of these may suffice to throw some light on the subject. Assuming what has been abundantly proved, viz., that tannin constitutes the chromogen of the red and blue floral pigments, and that tannin is not a homogeneous chemical compound, but embraces many varieties, we must ascertain if there exists any kind of tannin in the aforesaid flowers different from that which occurs in the ordinary red or blue flowers which are coloured green by dilute alkalis. Now, it would appear that the majority of flowers contain either rutin or an iron-greening tannin, but a select few, including Fuchsia, Pelargonium, Paeonia, and Camellia, undoubtedly contain an iron-blueing tannin which is no other than gallo-tannin mixed probably in some older specimens with a small quantity of gallic or ellagic acid. The ordinary pink rose contains only a highly phloroglucin-bearing tannin common to the whole order to which the queen of flowers belongs; but in certain garden varieties, wherein the petals have immensely developed with full expansion of parts and profound depth of coloration, a small proportion of gallic acid has doubtless managed to find entrance into the cells of the corolla. In fact, Filhol, Rochieder, and others found gallic acid as a constituent in the well-preserved and intensely purplish crimson cones of the officinal flores Rosae rubrae; and similar remarks will apply to the case of Polygonum. orientale.

The residue of the flowers in the foregoing list belong, it will be observed, to the orders Solanaceae, Plumbaginaceae, Labiatae, Compositae, and Leguminosae, all of which are distinguished by the absence of free phloroglucin, a fact which affords an insurmountable presumption that the tannic chromogen is of a character distinct from that with which we have just dealt. All who have had some experience in the chemistry of herbaceous plants are fully aware that they are brimful of quercetin in the form of rutin, etc., which, however, of itself fails as a basis for brilliant colorific effects, but may, nevertheless, by further de-assimilation develop into tannins which, in the special instances now under review, belong to the distinctively iron-greening class. The question which is now started and remains to be discussed is, whether this iron-greening tannin is adequate to discharge the function of a chromogen competent to evolve a pure blue flower?

All the genera mentioned are, with the exception of Erythrina, capable of producing a blue or purple flower in some of their specific forms. Fuchsia, Plumbago, Lycium, and Salvia produce one or more pure blue efflorescences; while Pelargonium, Phaseolus, Echinacea, Impatiens, Polygonum, Camellia, Paeonia, and Rosa produce purples more or less deep and frequently approaching deep blue. Of all these, Fuchsia, Plumbago, Pelargonium, Lycium, Polygonum, Camellia, Paeonia, and Rosa contain either an iron-blueing tannin or gallic acid in small quantity, and it is the colouring matter of just these flowers which is most distinctively blued by ammonia vapour or solution. In point of fact, I think it must needs be concluded that in all these instances it is the gallic acid resulting from the oxidation of gallo-tannin or of some nearly allied benzene derivative, which is solely responsible for the blue more or less pure and clear which they so beautifully display. Rosa and Polygonum are exceptional, inasmuch as they are genera which contain a highly phlobaphenic tannin, i.e. a chromogen which on advanced oxidation evolves brown-red or muddy anhydrides more than sufficient to neutralise and overcome any tendency to blue coloration incident to the presence of gallic acid.

Nevertheless, it is evident that gallic acid is not the only substance that may officiate as chromogen in the outcoming of vivid and brilliant blues. The genus Linum presents a remarkable phenomenon in this connection. Of some fifty species of this genus about seventeen are pure blue, and the rest are either yellow or purple, lilac, crimson, rose, pink, or pure white. The most astonishing species is L. grandiflorum (coccineum) which is the one crimson amidst a genus predominantly blue: it contains no gallotannin or gallic acid, but there is some rutin and iron-greening tannin, and there is a small amount of free phloroglucin in the plant itself. Now, if ever there was a red flower which is or has been naturally and originally blue it is this one, as the following reactions will show. The alcoholic extract of the petals is of a magenta-red colour, and the filtered aqueous extract of its evaporated residue yields with acetate of lead a blue colour, and then when acetic acid is added in large excess the blue colour still remains; with subacetate of lead it gives a pure blue colour; with bicarbonate of soda a grayish-blue turning grayish-green, and with oxalic acid added a bright scarlet red is obtained; with acetate of magnesium a splendid green, and when acid is added the original red is restored. Thus we see that in the presence of heavy bases the blue coloration persistently remains even in the presence of a considerable excess of free organic acid; with light and feeble bases, on the other hand, the addition of a small quantity of acid determines a distinctly red reaction. And all these facts seems to me to demonstrate what I consider to be quite exceptional, viz., that in Linum coccineum the red coloration of the flowers is produced by the accidental presence of free acid affecting a naturally blue pigment. It is hardly necessary to subjoin, that in the vast majority of cases, whether the flower be either red or blue, the particular tint is not influenced or created by the neutral or acid condition of the cell sap.

By what circumstances, then, is the particular tint created or influenced? In order to answer the question satisfactorily two facts must be minutely considered:—1. Chemical, the presence of quercetin in the form of rutin, etc., in the corolla. 2. Physiological, the possession by the corolla of energetic respiratory and transpiratory functions, with the result that the substances contained in its cells undergo an oxidation more or less vigorous and complete. In my previous paper I showed that the red pigments of flowers were specifically incidental to tannic chromogens which contain phloroglucin in their molecules and yield phlobaphenes, i.e. a series of anhydrides, the lower of which are crimson and the higher are red-brown; while the blue pigments were incidental to acid tannins or to tannins which yield acids on oxidation. Now, only a slight chemical experience is requisite for the understanding that in the latter case the process of oxidation has been more energetic and more complete than in the former case. Even where the blue is apparently the result of a combination of a tannic acid with a base, the effect cannot be produced save under circumstances especially favourable for oxidation. The flowers comprised within the great division Corolliflorae having a gamopetalous corolla, enjoy, by reason of the great expansion of cellular surface, a respiratory and transpiratory activity which the Polypetalae cannot exhibit. We see that all or most of our decidedly blue flowers are gamopetalous, e.g. gentians, bell flowers, Jacob's ladder, convolvulus, speedwells, various labiates, borage, etc. Linum perenne is an example of a bright cobalt blue-coloured polypetalous flower; but it may be readily observed that the petals are very broad and generally large in comparison with the thin and wiry stem and the small strap-shaped leaves; and I know of no valid reason why, under such propitious circumstances, the quercetin of the cell sap may not in the absence as here of any phlobaphenic tannin, develop to the full, and sowise evolve a high oxybenzoic acid which, being immediately transformed into pigment, may not be readily detectable in the free state.

In view of the researches already published, and of my own experiments, I think it may be concluded, that—1. A blue flower is unproducible in species which contain or are capable of forming phlobaphenic tannin, no matter what the development of the inflorescence may amount to. 2. A blue flower is more likely to be produced in a species having a gamopetalous corolla or perianth, and therefore liable to evolve by higher oxidation a certain quantity of a high oxybenzoic acid. 3. In species wherein the tannin natural to the organism is iron-greening and non-phlobaphenic, a blue flower may possibly be producible in a polypetalous corolla, provided always that the petals or perianth be large relatively to the height of the plant and to the size and robustness of its stem and leaves: in this case it is uncertain whether gallic acid is necessary for the production of the effect, but anyway an alkaline compound of an oxybenzoic acid would seem to be indispensable. I hope in a concluding paper to specifically examine these three propositions, and to supply full illustration of their scope and tenor.