DISTRIBUTION of ANYHOCYANINS R. ROBINSON IACS.Pdf

DISTRIBUTION OF ANTi ~ JCYANINS Bv ROBERT ROBINSON "" . \ , , ,l 061(540J\ R 566 'D ~.s· 9'2-98 1.} / ' .. ·,i F.-I j . Special Publication No. 14 · Dr, Bimala Churn Lriw Gold Medal Lecture for 1945 Indian Association for the Cultivation of Science THE DISTRIBUTION OF ANTHOCY ANINS BY SIR ROBERT ROBINSON, F.R.S., N.L. President of the Royal Society of England CALCUTTA 1950, -THE DISTRIBUTION OF ANTHOCYANINS When I was honoured by your invitation to deliver th.is lecture my thoughts turned to the an~ho~yani'jf' partly because. bright flowers make such an important contnbuuon t\· colourful lnd1a, but <\lso because my own work in the field owes so ~uch to Indian collaborators. Undoubtedly a botanist with a smattering of chemistry could shed more light on this topic than can a chemist with even less than a smattering of botany, but the chemical aspect is at "least fundamental and it must be developed before much progress can be wade. When a botanist of the older school wrote "anthocyan" he impLed a red or blue water-soluble colouring matter and he was not much concerned with either the homogeneity or the identity of the pigment. Indeed it must be admitted that no technique had been developed which could have been used to investigate these points even had it been desired to do so. The necessary information was acquired between 1914 and 1934 and we are now in a position to identify, within certain limits, the anthocyanins occurring in plant material. Quite small specimens, a few petals for example, suffice if we are satisfied with the limitations of the methods. Full knowledge of the constitution of a particular antho cyanin is only obtained by its isolation in substance and by its degrada tion and synthesis. The most important of the pioneers was R. Willstatter (1913-1916) who isolated many of these colouring matters in a pure condition and proved that they are glucosides of the colour nuclei, the anthocyanidins. Willstiitter has placed on record his debt to the stimulus provided by the botanist Molisch (1905) who not only greatly amplified earlier observations on the occasional occurrence of anthocyan crystals in plants (e.g., Bohm, 1857, noticed such crystals in berries of Passifiora) but was actually the first to show that they could be crystallized outside the plant. One experiment of Molisch is the following. A few scarlet pelargonium petals are rolled with a glass rod on a microscope slide so as to break up the structures to some extent. Place a few drops of 75% acetic acid, and then a cover glass, on the petals. On keeping for a week or so well-formed crystals are sure to have developed in the extract, especially round the edges. It is not surprising that one of the first pigments isolated by Willstatter was pelargonin. My own interest in the subject was due iQ the first instance to the fact that in 1907, when working in Perkin's laboratory in Manchester, a synthetic method was discovered of quite general applicability to the whole group of benzopyrylium salts, of which the anthocyanins [ 2 ] were later shown to be members. Many difficulties had to be overcome before this method could be adapted to the end in view, but eventually we were able to synthesize all the anthocyanidins and even the more important anthocyanins. In this work I was fortunate in having a succession of zealous and effective collaborators including D. G. Pratt, W. Bradley, A Robertson, L. F. Levy, T. R. Seshadri .and A. R. Todd. It is worthy of note that all the of erations in the actual synthesis of anthocyanidins and anthocyanin¥ from the intermediates are now carried out at the room temperature or lower. I do not intend to discuss the technical details of Willstatter's methods for the isolation of the anthocyanins, or those of the later syntheses, but a short summary of the results is necessary. The anthocyanins are isolated in the form of oxonium salts, usually the chloride, and the salt function resides in a pyrylium nucleus of aromatic character. Cl Cl 0 0NR Benzene. A Pyridinium Chloride. Pyrylium Chloride. On hydrolysis with hot 10-15% hydrochloric acid they break down into anthocyanidin chlorides and glucose or other sugars (galactose,. rha~ nose, pen.toses); occasionally also acids such as p-hydroxybenzmc actd are produced. The structures of the three chief anthocyanidin chlorides are: Cl HO HO OH HO HO Pelargonidin Cyanidin Cl Ho HO Delphinidin [ 3 ] In addition a methyl ether of cyanidi'n and three methylated delphi nidins are known. Cl Cl Ho OH uo Peonidin Petunidin Cl Cl MoO OH HO 1-10 Malvidin Hirsutidin Of these four, malvidin is much the most frequently, and hirsutidin the least frequently encountered. We call the unsubstituted oxonium chloride, fla·uylium chloride, numbered according to the scheme Cl 4' s Flavylium Chloride The anthocyanins may be monoglucosidic and in that case the sugar is always attached to the hydroxyl in the pyrylium nucleus (position-3). The monoglucosides of pelargonidin, cyanidin and malvidin are callistephin, chrysanthemin and oenin respectively. The 3-galacto side of cyanidin is idaein. The diglucosidic anthocyanins may be 3-biosides, e.g., mecocyanin is cyanidin 3-gentiobioside and antirrhinin is cyanidin 3-rhamnoglucoside. In an important group, glucose rests are attached to different hydroxyls ; these include the 3: 5-dimonosides. The 3: 5-diglucosides of pelar gonidin, cyanidin, delphinidin, and malvidin are palargonin, cyanin, delphin, and malvin respectively. Acyl groups may be attached to the sugar groups or to the phenolic hydroxyls but nothing is known in z [ 4 J detail about the structure of acylatcd anthocyanins apart from the nature and number of the acid residues in one or two examples only. The properties of the individual anthocyanins and anthocyanidins are highly characteristic and distinctive and the possession of pure synthetic samples afforded an essential basis of comparison. This applies, not only ·to the recognition of known anthocyanins in plant material but also to the study of ~)y anthocyanin exhibiting unusual behaviour. The reason is that syn:'h.esis has covered a very wide range of substituted flavylium salts and we know what influence the number and combinations of positions of hydroxyl and neutral groups will have on the properties. I counted up to 200 synthesized flavylium salts possessing such reference value and then gave it up; the actual number must approach 300. The absorption spectra of anthocyanins and anthocyanidins have been used to identify natural and synthetic products and in the case of the anthocyanins the rotatory powers (due to the sugar group) have also been employed. The most generally useful properties* are, however, the 'Distribu tion Numbers' between immiscible solvents, and various colour reactions, especially because the study of these does not always necessitate the isolation of the pigments in a pure condition. The Distribution Number for a particular solvent is the percentage of the colouring matter that passes into it, when shaken with an equal volume of aqueous acid solution. O·S% hydrochloric acid is often used and the organic solvent and acid are previously equilibrated SO that no change OCCUtS OJ?. mixing. • Ordinary amyl alcohol is one of the most useful solvents and serves to distinguish at once the various classes. Diglucosides have a very low D.N. and the alcoholic layer is very pale-coloured. The number is frequently <1 and rarely exceeds 3. This makes the colori metric determination unreliable and therefore n-butyl alcohol is employed for more accurate work. For example, mecocyanin chloride from Papaver Rhoeas was recognized as a cyanidin 3-bioside, the biose being composed of glucose units. This conclusion is drawn from the results of degradation (-?-cyanidin chloride+2 glucose, or -?-chrysan themin chloride+ 1 glucose) coupled with the observation that the colour reactions of mecocyanin are identical with thos~ of chrysan themin which had already been synthesized. If the second glucose molecule were attached to any other position in the ffavylium nucleus, we knew from other models that this identity of behaviour could not · The lecture was illustrated by demonstrations of the more important of these properties and the spoken word was built up round these . experi~ents. Hence the present account is largely supplementary to the matenal delivered. The lecturer expresses his warmest thanks to the Officers of the Section and to Professor R. N. Chakravarti and Miss D. Mukherji for assistance in preparing the experiments. [ 5 ] be anticipated. Accordingly various 3-biosides of cyanidin chloride were synthesized and their Distribution N um~crs (butyl alcoh~l) ;ve~e: cellobioside (23 ·1) ; maltoside (28·6); lactos1de (30·6); gentwb1os1de (17·0). The D.N. of natural mecocyanin was 16·7. A careful com parison then showed that mecocyanin wa~ .identical with cyanidi~ 3- gentiobioside in all other respects; solub1hty, crystal form, rotatwn, formation of derivatives such as tlfe ferrocyanide, ~nd .power to ino~u late super-saturated solutions. Til~ last observatiOn IS coupled wnh the inability of the other synthesized biosides to induce crystallization. Rhamnoglucosides and pentoseglucosides have somewhat higher D.N.'s (amyl alcohol) but they may be distinguished from diglucosides by a simple method. At a low concentration the D.N. is near zero, as judged by the eye, but on saturating the aqueous layer with salt, a large propor:rion of the colour passes into the amyl alcohol. This does not occur with the diglucosides. · The monoglucosides have D.N.s (amyl alcohol) from 12-50 and 80-100 on adding salt. They are distinguished from the rhamno glucosides by the fact that a considerable D.N. is obtained even at very low concentrations.

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