The Natural Organic Colouring Matters
By
Arthur George Perkin, F.R.S., F.R.S.E., F.I.C., professor of colour chemistry and dyeing in the University of Leeds
and
Arthur Ernest Everest, D.Sc., Ph.D., F.I.C., of the Wilton Research Laboratories; Late head of the Department of Coal-tar Colour Chemistry; Technical College, Huddersfield
Longmans, Green and Co.
39 Paternoster Row, London
Fourth Avenue & 30th Street, New York
Bombay, Calcutta, and Madras
1918
Kaikki kuvat (kemialliset kaavat) puuttuvat // None of the illustrations (of chemical formulas) included.
The red, blue, and purple pigments of this group being the cause of some of nature's most beautiful and vivid colour effects, it is not at all surprising that at quite an early date chemists began to attempt the investigation of these pigments. Despite this, it is only within the last few years that anything definite concerning the chemistry of this group has become known.
The term Anthocyan appears to have been introduced by Marquart (Die Farben der Blüten, Bonn, 1835) to designate the blue pigments present in flowers. Later there arose the belief that the red and purple flower pigments were all merely different forms of the same blue anthocyan or, as Fremy and Cloëz styled it, cyanin - and that the variation of colour was merely due to the nature of the cell sap; this resulted in the name anthocyan being indiscriminately applied to all of them. The present use of the term anthocyan to designate a large class of naturally occurring plant pigments gradually became general as from time to time evidence accumulated to show that the red, purple, and blue pigments differed considerably among themselves.
As early as 1836, Hope, in a paper read before the Royal Society of Edinburgh (March 21; cf. J. f. prakt. Chem., (10), 269 (1837)), concluded, as the result of experiments on a large number of different kinds of flowers, that the pigments, or chromules, present were formed from faintly coloured chromogens, by a variety of changes. Of these chromogens, according to Hope, there were two types, one called by him Erythrogen, which by the action of acids yielded red pigments, and a second, named by him Xanthogen, which with alkalis gave rise to yellow pigments. He concluded that, in orange, red, purple, and blue flowers both were present, whereas in yellow and white flowers only xanthogen was found. From his examination of leaves, he concluded that chlorophyll was accompanied by xanthogen, but that, excepting in cases where reddening was obvious e.g. autumn leaves no erythrogen was present.
In the following year, Berzelius (Annalen, 1837, 21, 262) published the results of experiments on the pigments present in some berries, as cherry, black-currant, and in autumn leaves, e.g. red-currant, and of his attempts to purify and isolate them. For the red-leaf pigments he suggested the name Erythrophyll (leaf red), but pointed out that there might be objections to this term, as the pigments of flowers and berries appeared to belong to the same class, an indication of the expansion of the term anthocyan to cover other than flower pigments.
In his attempts to prepare the pure pigments, Berzelius made use of the precipitation, by means of lead acetate, of the insoluble lead salts of these pigments, and of their regeneration by means of sulphuretted hydrogen. Berzelius did not obtain any pure pigments, but the above-mentioned method, either as used by him, or with such small modifications as the decomposition of the lead salt by means of hydrochloric acid instead of with sulphuretted hydrogen, has been employed by a large number of later workers, though by this means only Grafe (1906) and Willstätter (1916) have succeeded in obtaining crystalline products. Berzelius was not of the opinion that all these pigments could be considered as the same blue substance changed by variation in the cell sap.
The next work of interest was that of Morot (Annales des Sc. nat., (3), 13, 1 60 (1849-1850)) who attempted to prepare the blue pigment of the cornflower by repeated precipitation of its aqueous solutions by means of alcohol. He did not thus obtain a pure product, but the method is of interest in that, improved by the use of modern apparatus, it constitutes the first step of the process whereby Willstätter and Everest isolated the pure corn-flower pigment. Impurities containing nitrogen were present in Morot's products, and in the presence of nitrogen he saw a possible connection between this pigment and chlorophyll, but it should be noted that he was doubtful whether this nitrogen was really a constituent of the pigment. He described the decolorisation on standing in solution which is characteristic not only of this, but also of nearly all anthocyans. That this decolorisation observed by Morot occurred in other cases was proved by the work of Fremy and Cloëz (Journ. de Ph. et de Chim., (3), 25, 249), who, moreover, by allowing such a decolorised solution to evaporate in the air, whereby the colour returned as the solution became concentrated, showed that the pigment was not destroyed by this change. They, however, looked upon the decolorisation as the result of a reduction of the pigment. These workers used the cornflower, violet, and iris for their experiments on blue pigments, and for those on red ones, the dahlia, rose, and peony. In each instance they attempted to purify the pigment by use of the lead salt, as described by Berzelius, but in no case, however, did they obtain a pure product.
Fremy and Cloëz discussed the general ideas then current regarding the plant pigments, and pointed out the uselessness of assuming, as so many.workers about that time did, that a relationship existed between chlorophyll and the blue and yellow pigments, for, as pure chlorophyll had not then been obtained, and the flower pigments were almost uninvestigated, no reliable conclusions could be drawn. They suggested that all anthocyans were one and the same substance which they called cyanin and that the colour variations were due to the properties of the particular plant sap. They distinguished three flower pigments: (1) Cyanin (red or blue); (2) Xanthins (yellow, insoluble in water); (3) Xantheïns (yellow, soluble in water). Here a clear distinction is made between the carotin derivatives, corresponding to (2), and the flavone and flavonol derivatives, to (3), both of which occur as yellow flower pigments. These authors considered that (1) and (3) were in no way related to each other, for although they almost invariably found (3) occurring in flowers containing (1), they never observed a blue flower turn yellow, nor a yellow flower turn blue.
Filhol (Comptes rend., 39, 194; J. pr. Chem., 1854, 63, 78) investigated qualitatively a large number of flowers and confirmed previous workers' observations that yellow pigments - for which he retained the name Xanthogen - were present, not only in yellow and white, but also in red, purple, and blue flowers. He concluded that, with some few exceptions, all the red, purple, and blue pigments were derived from the same anthocyan. He examined the decolorisation of anthocyans in solution, and finding that the addition of acid caused the reappearance of colour, concluded that the decolorisation could not be the result of a reduction, as suggested by Fremy and Cloëz. In his opinion this was due to the mixing of the pigments with other contents of plant cells, from which they were kept apart in the living plant.
Martens in 1855 (cf. Jahres., 1855, 657) attacked the problem from another point of view, attempting to elucidate the mode of formation of the anthocyans in plants. He further confirmed the presence of the yellow pigments, for which he used the name Xanthein of Fremy and Cloëz in flowers containing anthocyan, and as the result of his work was led to put forward the hypothesis that both yellow and red pigments have their origin in a faintly yellow substance produced in the sap of all plants, which by oxidation, particularly under the influence of alkalis and light, produces the different yellow pigments, from which by further action of light and oxygen, the red pigments are formed. It is interesting to note that the relationship thus suggested by Martens as existing between the yellow pigments and the anthocyans is that which has been revived in more recent years by Wheldale; Keeble, Armstrong and Jones, and others (see below).
In 1859 Morren put forward the suggestion that the blue flower pigments (anthocyans) were the alkali salts of acids which in the free state are red, and for which he used the name Erythrophyll (cf. Berzelius). His conception of the blue pigments has been confirmed by recent work, but not so that regarding the red colouring matters.
A number of workers have, at different times, attempted to prepare pure anthocyan pigments by making use of their lead salts (cf. Berzelius); thus Glénard (Comptes rend., 47, 268; Jahres., 1858, 476), working with red wine, and using ethereal hydrochloric acid for the decomposition of his lead salt, obtained a pigment which he called Oenolin, and for which he put forward the nitrogen-free formula C20H16O10. His preparation was, however, by no means pure. Senier (cf. Jahres., 1878, 970), using Rosa gallica, and decomposing the lead compound, suspended in alcohol, either by sulphuric acid or sulphuretted hydrogen, prepared a pigment, for the lead salt of which he gave the formula C21H29O30Pb2. Heise (cf. Chem. Zentr., 1889, 2, 953) by similar means prepared two pigments (A and B) from red wine, using sulphuretted hydrogen for decomposing the lead salts, and suggested that Glénard's compound was a mixture of these. His examination, however, was not complete. Glan (Dissertation, Erlangen, 1892), examining the pigment of the deep-red hollyhock, also obtained two products, and in 1894, Heise (cf. Chem. Zentr., 1894, 2, 846) further prepared two pigments (A and B) from the bilberry (in this case using ethereal hydrochloric acid to decompose the lead salt), and obtaining them in a fairly pure, but amorphous condition, showed that the one (B) was a glucoside of the other (A). He gave analyses and formulæ, but these have proved to be incorrect, though the relative amount of glucose which he found to be present in his glucoside (B) has proved to be approximately accurate.
The result of this work of Heise and Glan was to produce a general tendency to consider that the anthocyan pigments were present in plants both as glucosides and non-glucosides, the former predominating somewhat. Molisch, in 1905, decided in favour of their being glucosides, but Grafe, who carried out a continuation of Molisch's work on a preparative scale, reverted to the earlier ideas. The results of recent work have definitely proved that in all investigated cases these pigments occur as glucosides possibly accompanied in a few instances (cf. Annalen, 1916, 412, 195) by a small percentage of sugarfree pigment, the non-glucosides isolated by the above workers being almost entirely the result of hydrolysis during their preparation.
In 1895 Weigert published (Jahrber. der k. k. 6nol. and pomol., Lehranstalt inKlosterneuburg, 1894-1895) a classification of the anthocyan pigments, thereby completely dispelling the one pigment idea that had so often been brought forward. As already stated, the views of earlier workers upon this point differed considerably; thus Berzelius was of the opinion that more than one anthocyan pigment existed, whereas Fremy and Cloë'z considered that all red, violet, and blue flowers contained the same blue pigment (cyanin), its colour having been changed by the conditions prevailing in the various cell saps. Filhol, as also Wigand (Bot. Ztg., 1862, 123), likewise asserted that all red and blue flower colours were produced by different forms of one and the same anthocyan, and Hausen (Die Farbstoffe der Bliiten und Früchten, Würzburg, 1884, p. 8) went still further, being of the opinion that not only all red colours in flowers, but also those in fruits, were due to one and the same pigment. Wiesner (Bot. Ztg., 1862, 392), however, cast considerable doubt upon the identity of all these compounds.
Weigert, as a result of the comparison of the behaviour of the anthocyan pigments with various reagents, in particular with regard to the colour of their lead salts, and the colour changes that took place on addition of acid or alkali, distinguished two classes of these compounds, the first Wine-red group such as gave blue or bluegreen lead salts, and whose acidified solution, on addition of alkali became blue or blue-green; and a second the Beetroot-red group which gave red lead salts, and whose acid solution showed no change of colour, or slight change to violet-red, on being made alkaline. The crude anthocyan pigments are, however, so varied in their reactions, that this simple classification of Weigert by no means covers all cases, for even by comparison only of the colour changes on acidification or on being made alkaline, and by formation of the lead compounds, quite a number of sub-groups can be observed.
Overton (Pring. Jahrber. f. wiss. Bot., 1899, 33, 222) also came to the conclusion that a considerable number of different anthocyan pigments existed.
In all the above-mentioned work, either qualitative results only were aimed at, or the preparations were amorphous and lacked the essential characteristics of chemically pure products. From these observations, however, it had become clear that in the anthocyans a large class of new pigments were awaiting chemical investigation, and moreover, in the light of the work of Heise and Glan, it was evident that at least certain of these must be looked upon as belonging to a class of nitrogen-free glucosides. This view was expressed by Molisch in 1905.
In 1903 two papers were published by Griffiths (Ber., 36, 3959; and Chem. News, 88, 249) which, had they been followed up, might have had considerably greater influence on this field of work than has been the case. He describes the preparation for the first time of an anthocyan in a crystalline condition. His work was carried out with geranium and verbena flowers, but the pigment obtained from the latter contained nitrogen and sulphur and was doubtless impure; that from the geranium contained neither of these elements, and was the only one analysed. His method of preparation consisted in extracting the petals with 90 per cent, alcohol, and after filtration evaporating in vacua. The residue thus obtained was extracted with absolute alcohol, filtered, and the filtrate again evaporated in vacuo, when the pigment separated in crystalline form. He did not attempt to decide whether the pigment was a glucoside or not. The description of his experiments is very superficial and imperfect; thus, for example, the fact that a fresh 90 per cent, alcoholic extract of geranium petals has a fine scarlet colour, but passes in a few minutes to a practically colourless solution, which, however, regains its original colour as evaporation takes place, is not even mentioned.
Very different in character from these papers of Griffiths is the beautifully clear and descriptive publication of the botanist Molisch (Bot. Ztg., 1905, 145), and a greater incentive to research upon these pigments than lies in this paper one cannot easily imagine. Molisch, after giving a summary of the literature dealing with the then very doubtful appearance of solid anthocyan pigments in plants, described how, on examination of a number of anthocyan-containing flowers and leaves, he found that these pigments existed not only in solution in the cell sap, but were, in many cases, present also in the solid state, sometimes as small spheres, sometimes as definitely crystalline formations. Of some of the more well-defined cases he gave illustrations. Having described the appearance of anthocyan crystals in the living plant, he followed up these observations with a description of his attempts to obtain crystals of these pigments outside the plant. In this in several cases (pelargonium, rose, Anemone fulgens) he was successful, and gave illustrations of the resultant microscopic crystals. Having thus established the fact that some of these pigments readily crystallise, he pointed out that it should not be difficult to prepare them in sufficiently large quantities for chemical examination. In a very slightly modified form, the method whereby Molisch obtained his crystals is so simple and certain, that it is worth describing. One or two petals of the flower are laid upon a piece of glass, slightly larger than a microscope slide and upon which are one or two drops of 75 per cent, acetic acid, the cell structure is then broken down by rolling a glass rod over them, leaving the petals flattened on the glass with the cell sap beside or upon them. Two or three more drops of 75 per cent, acetic acid are then placed on the petals, and a microscope slide pressed down upon them. The whole is then placed under a clockglass (to ensure slow evaporation), and after some twelve to twentyfour hours crystals begin to appear, either round the edge of the slide, or round or on the petals. Scarlet pelargonium gives the best results and very rarely fails; in some cases, using these flowers, clusters of crystals may be obtained so large that they are readily discernible by means of the naked eye.
As mentioned above, Grafe was moved by the work of Molisch to attempt the chemical investigation of some of these pigments. He published three papers (Sitzber. k. Akad. d. Wiss., Vienna, 1906, 975; 10+0. 1033; and 1911, 75); in the first he described experiments with red cabbage leaves and rose petals, from neither of which could he obtain any crystalline pigment; from the blueblack berries of Ligustrum vulgare he obtained a crystalline product, but was unable to obtain any agreement in his analyses of it, and finally, with the flowers of the hollyhock (Althaea rosea), he obtained two pigments, one crystalline, the other amorphous, of which he gave analyses. In each case he used the lead compound for the preparation of his pigment, and decomposed this by means of sulphuretted hydrogen. After separation of the hollyhock pigment in this manner, he further purified it by means of alcohol and ether.
To the crystalline compound he gave the formula C14H16O6, the amorphous product C20H30O13; he considered the latter to be a glucoside, as from it he obtained glucose by hydrolysis, but apparently did not examine the non-glucoside pigment produced by this reaction. In his second paper he continued his investigation of the hollyhock pigment, and discussed the formation of anthocyans in the plant. The third paper of the series contains an account of his further attempts to prepare the pigment of the red cabbage in a crystalline form. Despite the fact that Molisch had failed to obtain crystals by his method, Grafe attempted to produce them by using the same process on a larger scale. Failing in his attempts, he tried dialysis previously used in other cases by Portheim and Scholl (Ber. deut. Bot. Ges., 1908, 26a, 480) as a means of purification, but again failed to obtain any crystalline product. Grafe then turned his attention to the pigment of the scarlet pelargonium, which had so readily yielded crystals. He was successful in obtaining this pigment in a fine crystalline condition (microphotographs of the crystals were given) by carrying out Molisch's experiment on a large scale, employing the lead salt method, or dialysis. Besides the crystalline pigment, Grafe obtained, as with the hollyhock, an amorphous compound, and in this case also considered the latter to be a glucoside, whereas the former was sugar-free. The crystalline compound he described as very unstable and deliquescent; as this is not true of the pure pigment his crystals must have contained impurities for it he gave the formula C18H25O13. To the amorphous substance he gave the formula C24H44O20, and from it by hydrolysis obtained glucose; here again he does not appear to have examined the resulting non-glucoside pigment. Of the crystalline substance he obtained 10 grams, together with 15 grams of the amorphous compound, from some 28 kilograms of fresh petals, which would point to a slight preponderance only of the glucoside in the petals if no hydrolysis took place during its isolation.
Having thus overcome the experimental difficulties involved, and having for the first time obtained considerable quantities of an anthocyan in crystalline condition, it is to be regretted that Grafe drew such incorrect conclusions from his results. Doubtless he had in mind those of Heise and Glan, that both glucoside and non-glucoside pigments were present in the plants they examined, for, on finding that his amorphous product reduced Fehling's solution, but that his crystalline substance did not, he concluded that the former only was a glucoside, and convinced himself of this by its hydrolysis whereby he obtained glucose. Recent work has proved that Grafe's amorphous product must have been an impure specimen of the glucoside containing reducing sugars, whereas the crystalline substance was the glucoside in a practically pure condition; the possibility that such glucosides when pure do not appreciably reduce Fehling's solution never appears to have occurred to Grafe, and as a result he never seems to have attempted to hydrolyse his crystalline pigments. He concluded that his work, together with that of Heise and Glan, proved the co-existence, in the cases examined, of glucoside and non-glucoside in these plants. This conclusion has, however, been proved erroneous by the work of Willstätter and Everest, who have shown that only the glucoside there exists, accompanied in a few instances by a small percentage of sugar-free pigments.
* This oxidation may, or may not, be accompanied by polymerisation.During the years covered by this series of papers by Grafe, a considerable amount of work had been published by botanists, dealing with the formation of the anthocyans in plants. Miss Wheldale, as the result of much botanical work, came to the conclusion that the anthocyans are derived from colourless or faintly coloured chromogens (probably flavone or xanthone derivatives) by oxidation, most probably as the result of the action of peroxidases. She considered that the chromogens were produced by hydrolysis of glucosides that existed in the plant, this reaction being reversible. An essential feature of her theory is that the oxidation of the chromogen, with production of anthocyan, can only take place after the hydrolysis of the glucoside. To represent these changes she proposed the following scheme:
Glucoside + H2O → Chromogen + sugar,
then,
Oxidation of chromogen* → Anthocyan pigment.
As chemical evidence of the latter part of these changes, Nierenstein and Wheldale (Ber., 1911, 44, 3487) and Nierenstein (Ber., 1912, 45, 499) brought forward products obtained by the oxidation of quercetin and chrysin respectively with chromic acid, which they described as "Anthocyan-like" products. The reactions from which they drew this conclusion were, however, by no means sufficient to show that any relationship exists between these compounds and the anthocyans. In this connection it is necessary to mention the observation of Perkin (Chem. Soc. Trans., 1913, 650), that gossypetin by oxidation in alkaline solution yields a substance (gossypeton) which is coloured deep blue by alkalis and red by acids. He pointed out the bearing of this observation upon the theory of Miss Wheldale. The fact, however, that this pigment is somewhat stable to alkalis is not in agreement with the properties of such anthocyans as have as yet been investigated, and indeed Perkin expresses doubt as to its existence as such in the cotton flower itself.
Keeble, Armstrong, and Jones (Proc. Roy. Soc., B., 1912, 85, 215; B., 1913, 86, 308, and 318; B., 1913, 87, 113; and Keeble and Armstrong, Jour. Genetics, 1913, 2, 277) have published a number of interesting papers upon the formation of anthocyans, and the hypothesis which they support is very similar to that of Miss Wheldale, but they part company with that author in regard to the process necessary subsequent to hydrolysis of the glucosides, for they maintain that the oxidation must be preceded by reduction of the non-glucosidal flavone or flavonol derivative.
In these experiments Keeble, Armstrong, and Jones came very near to the discovery of the true relationship that exists between the anthocyans and the flavones, for, in the light of later work described below, it is obvious that by some misfortune their reductions were carried too far (cf. Everest, Proc. Royal Soc., 1914* B., 87, 449).
In 1913 Willstätter and Everest (Annalen, 401, 189) published an account of investigations upon the anthocyan pigments, and in particular of the pigment of the corn-flower, and in this communication the first of a series by Willstätter and his collaborators some important conclusions were arrived at. It was proved that the blue form of corn-flower pigment was a potassium salt, the free compound being violet in colour, whereas the red form was not this latter, as had always been assumed by previous workers, but an oxonium salt in which the pigment was combined with an equivalent of some mineral or plant acid. The anthocyans were found to be most stable when in the form of these oxonium salts. It was also definitely proved that the decolorisation in solution, so often mentioned by other workers, was not due to reduction.
Having obtained the corn-flower pigment pure and crystalline in the form of its chloride, they proved that it was a disaccharide, since on hydrolysis 2 molecules of glucose were split off from each molecule of pigment; the sugar-free pigment was obtained in a finely crystalline condition. Microphotographs of the crystals were given. The pure glucoside does not reduce Fehling's solution.
By careful oxidation of cyanidin with hydrogen peroxide, a yellow crystalline product was obtained which in its reactions closely resembled a flavonol colouring matter.
To prevent confusion these authors proposed the terms anthocyanins and anthocyanidins for the glucoside and non-glucoside pigments respectively, and in agreement with this assigned to the glucoside present in the corn-flower the name introduced by Fremy and Cloëz cyanin, whereas to the sugar-free pigment obtained by hydrolysis of cyanin the name cyanidin was given.
Willstätter and Everest introduced a method whereby they were able to distinguish between glucoside pigments of the anthocyan series and the corresponding sugar-free pigments. This depends upon the difference that exists between these classes of pigment in their distribution between amyl alcohol and aqueous acid. It has been very carefully studied by Willstätter and his collaborators in the course of their later work, and by but very slight modification it has been possible to utilise it in many cases to determine, with very considerable certainty, whether a given anthocyan pigment is a mono- or disaccharide, or a sugar-free pigment; and indeed in certain instances (e.g. chrysanthemum pigments) to separate mixtures of mono-, di-, and non-glucosides by its means, and show clearly the presence of all three. There are, however, limitations to its use for these purposes, dependent upon circumstances that are dealt with below.
Under suitable conditions of acid concentration Willstätter and Everest used dilute sulphuric acid ca. N-2N; whereas in later work Willstätter and Zollinger use 0.5 per cent, hydrochloric, or sulphuric acid. The disaccharide anthocyans (with few exceptions) are almost quantitatively retained by the aqueous layer in the form of their oxonium salts when a solution of the pigment in aqueous acid is shaken with amyl alcohol; the monosaccharide derivatives, however, pass partially into the amyl alcohol layer the percentage of the total pigment taken up by the alcohol being to a certain extent characteristic of the individual pigment. This may be quantitatively removed again by repeated washing with dilute aqueous acid, whereas, when sugar-free pigments are subjected to this test, the amyl alcohol layer takes the pigments quantitatively from the aqueous layer, and they cannot be removed from the amyl alcohol by washing with aqueous acid.
Willstätter and Zollinger have given the name "Distribution Number" (Verteilungszahl) to the percentage of the total pigment present which is taken up into the amyl alcohol layer when the above test is carried out under certain conditions. These numbers are of considerable use in characterising a pure pigment of this series.
For the purpose of estimating the "Distribution Number" of an anthocyan, 0.01 gr. of the pigment is dissolved in 50 c.c. of 0.5 per cent, hydrochloric acid. In the original description of this method (Annalen, 1916, 412, 208) this is erroneously stated as 0.05 per cent. HCl, but the various references in the context, p. 208 and p. 209, make it obvious that 0.5 per cent, is meant. It is stated also that a more dilute acid allows of some isomerisation, whilst in more concentrated acids the pigments are less soluble (hence the choice of 0.5 per cent). The solution is shaken twice with amyl alcohol (free from pyridine), using 50 c.c. amyl alcohol for each shaking. The small quantity of acid that passes to the alcoholic layer is sufficient to prevent isomerisation of the pigment in that layer.
The amyl alcoholic extracts are then quantitatively estimated by comparison (colorimetrically) with standard solutions of the pure pigment in amyl alcohol also containing a small quantity of acid to prevent isomerisation. As -the anthocyan pigments are not very soluble either in aqueous acid or in amyl alcohol, it is necessary to work with dilute solutions so that both layers shall remain unsaturated.
By these means Willstätter and Zollinger have obtained the following values:
| Pigment | Type | Distribution Number (Mean). |
| Malvin chloride | Diglucoside | 1.6 |
| Cyanin " | " | 1.8 |
| Salvinin " | " | ca.1-2 |
| Keracyaninen " | Rhamno-glucoside | 6.8 |
| Prunicyanin " | " | 9.7 |
| Ampelopsin " | Mono-glucoside | 9.8 |
| Oenin " | " | 10.4 |
| Myrtillin " | " | 10.8 |
| Salvianin " | ? Complex anhydro-di-glucoside | ca.50 |
| Salvin " | ? Anhydro-diglucoside | 57 |
| Cyanidin " | Non-glucoside | 100.0 |
| Oenidin " | " | 100.0 |
At one time it appeared as if diglucoside anthocyans had a number ca. 1-2, the monoglucosides ca. 10-11, and the nonglucosides 100, but the above table shows that this does not hold good now that further pigments have been investigated.
The discovery by Willstätter and Zollinger that certain diglucoside anthocyans have distribution numbers of the same order as those of the monoglucosides, prevents the full use of this test in the direction in which they were developing it viz. to characterise the three types of anthocyan pigments: non-glucosides, mono-, and disaccharides. Indeed, except in such cases where it is known that disaccharide pigments which have numbers similar to those of the monosaccharides are absent, it remains much in the position where Willstätter and Everest left it; i.e. that sugar-containing pigments either do not pass to the amyl alcohol layer, or, if they do so to some extent, they may be completely removed again by frequent shaking with fresh aqueous acid, whereas the sugar-free pigments pass quantitatively to the alcoholic layer and cannot be removed from it by such treatment.
With this method there are a few peculiarities that concern the sugars attached to the pigments, and which appear to show that these are of considerable importance in deciding the distribution number of these pigments.
Comparison of the values given in the table above, for disaccharide pigments, and of the products of their hydrolysis, shows that those which are rhamno-glucosides yield numbers that approximate to those of the monosaccharides,
Another very interesting case is that met with in the colouring matters of the salvia (scarlet). Here the flower pigment "Salvianin" appears to be rather more complex than most anthocyans (in this it resembles delphinin) in that on hydrolysis it yields pelargonidin, 2 molecules of glucose, and also malonic acid (proportion not yet determined), but by partial hydrolysis a compound "Salvin" is obtainable which appears to have the formula C27H26O13, i.e. that of a pelargonidin diglucoside less 2 molecules of water, whilst a third pigment "Salvinin -" a true diglucoside of pelargonidin - is also obtained by partial hydrolysis of the original salvianin. Now, whilst salvinin, which is isomeric with pelargonin, behaves as a normal disaccharide anthocyan, i.e. gives a low distribution number, both salvianin - the original flower pigment - and salvin give extraordinarily high numbers, the former well over fifty, the latter almost exactly fifty. Willstätter ascribes these abnormal numbers to the diminution in the number of OH groups present in the sugar molecules attached to the pigment as is indicated by analysis, and by peculiarities observed during the carrying out of quantitative hydrolyses of these pigments.
By means of the test described above it has been shown that in almost every case investigated the anthocyan pigments are present in plants solely as glucosides; in a few instances, however, sugar-free pigments appear to be present to a small extent also. Thus in black grapes (North Italian or hot-house grown) Willstätter and Zollinger (Annalen, 1916, 412, 206) found that the sugar-free pigment, oenidin, was present, but to the extent only of a few per cent, of the total pigment. In this respect a very interesting exception was met with by them in specimens of "Black Alicante" grapes, which were gathered at the Kgl. Gartner-lehranstalt, Berlin-Dahlem, towards the end of November, and which had light brown-violet berries; in these they found (by colorimetric estimation) as much as 12 per cent, of the colour present as the non-glucoside pigment.
In 1914 Miss Wheldale (Biochem. Jour., 1914, 8, 204) published further work from which she concluded that the fact that she failed to obtain a crystalline pigment, and that her product had no melting-point, was evidence of the high molecular weights of the anthocyan pigments. By comparison with the case of cyanidin chloride, the melting-point evidence collapses at once, and it appears as though the non-crystalline condition of her pigment was either due to the presence of a small quantity of impurity, or that she did not ascertain the conditions for its necessary crystallisation.
That luteolin and morin give red pigments on reduction in acid alcoholic solution by means of sodium amalgam has been known for many years (cf. Rüpe, "Die Chemie der natürlichen Farbstoffe," vol. i., pp. 77 and 85). Watson and Sen (Chem. Soc. Trans., 1914, 389) obtained a red pigment from quercetin in like manner, whilst R. Combes (Comptes rend., 1913, 1002) produced a red pigment, identical with that which he had obtained from the red leaves of Ampdopsis hederacea, by reducing in the same way the yellow pigment obtained from the green leaves of the same plant. A further paper of Combes (Comptes rend., 1913, 1454) described the reverse change, viz. from red to yellow by means of oxidation with hydrogen peroxide. In each case he obtained crystalline compounds and compared their melting-points; he did not, however, give analyses, nor state whether he worked with glucosides or not.
That a series of red pigments whose properties coincide in every way with those of the anthocyanidins may be produced by reduction of the flavonol derivatives by various methods, the best of which appears to be treatment of the pigment dissolved in a mixture of five volumes absolute alcohol and one volume concentrated hydrochloric acid, with magnesium, has been confirmed by Everest (Proc. Roy. Soc., 1914, B., 87, 444), who, moreover, in the same paper described the production, by the same means, of anthocyanins from the glucoside flavonol derivatives present in various flowers, thus showing the direct formation of red glucoside pigments from the yellow flavonol glucosides without intermediate hydrolysis. This important observation makes the hypothesis of Miss Wheldale and others, in which hydrolysis of the flavonol glucoside is an essential factor, unnecessary, and moreover shows that reduction, not oxidation, is necessary for the passage from flavonol to anthocyan.
The results obtained, coupled with those of Willstätter and Everest in their investigation of the corn-flower pigment, led Everest to suggest a scheme to represent the passage from a typical flavonol to the corresponding anthocyan, and to put forward a structural formula for the anthocyans (see below). The correctness of these has been confirmed by the more recent work upon the natural pigments carried out by Willstätter and his collaborators.
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