LXXXV. NATURAL ANTHOCYANIN PIGMENTS. I. THE MAGENTA FLOWER PIGMENT OF ANTIRRHINUM MAJUS. BY ROSE SCOTT-MONCRIEFF. From the Biochemical Laboratory, Cambridge. (Received May lst, 1930.) FROM her study of the Mendelian factors for flower eolour in Antirrhinum majus, Wheldale [1907, 1914] showed that, on crossing white with yellow or ivory varieties, plants bearing red or magenta flowers are produced. This led her to suggest that the red and magenta anthocyanin pigments might be derived from the yellow and ivory flavones, and that the white varieties, from which these pigments are absent, might carry the factors which act on the flavones to form anthocyanin. With a view to testing this hypothesis, Wheldale [1913] commenced an investigation of the chemistry of these four pigments. With Bassett [1913, 1914, 1] she isolated the yellow and ivory flavone pigments and identified them as glucosides of luteolin and apigenin. The yellow flowers contained glucosides of both these pigments, while apigenin alone was present in the ivory variety. From the red and magenta flowers they isolated two amorphous pigments of high molecular weight and uncertain constitution, which were, apparently un- combined with any acid [Wheldale and Bassett, 1914, 2]. At this time Willstatter and Everest [1913] published an account of the isolation of the blue anthocyanin from Centaurea cyanus (cornflower). In this classical paper, they revealed the constitution and true nature of the antho- cyanins, showing that they could be isolated by means of their stable crystal- line oxonium salts. Unfortunately, this communication, which revolutionised the methods employed in the extraction and isolation of these pigments, was published too late to be of any use to Wheldale and Bassett, and the question of the relationship between these co-existing flavones and anthocyanins remained unanswered. More recent research has still left the question open [Onslow, 1925] as to whether anthocyanins are derived directly from flavones or not: it is possible for instance that they are formed independently from the same parent sub- stances. With a view to throwing more light upon the interrelationships between flavones and anthocyanins in the same varieties, a further attempt has been made to isolate and identify the red and magenta pigments of Antirrhinum majus. The pure crystalline chloride of the magenta pigment antirrhinin has been isolated by a process based on the methods of Willstiitter and his co-workers. The preparation of the red pigment is to be attempted next. If there were any evidence for the direct origin of anthocyanins from Biochem. 1930 xxIiv 48 754 R. SCOTT-MONCRIEFF flavones in the plant, the genetical relationships would point to apigenin as the parent substance of this magenta pigment. On crossing an ivory variety with a white of certain origin, a magenta offspring is produced. In this case the flavone apigenin is the only pigment present in either parent, and only this flavone and magenta anthocyanin are to be found in the offspring. If Whel- dale's original hypothesis had been correct, some simple relationship should be apparent between the structures of these two pigments. Wheldale had originally suggested that the reaction occurring on the formation of antho- cyanins would be one of oxidation. After the elucidation of the constitution of the anthocyanins, and the production by reduction, in vitro, of the antho- cyanidin cyanidin from the flavone quercetin [Willstiitter and Mallison, 1914], Everest [1918] supported the view that the process in vivo was one of reduction. If this second theory is correct, the magenta anthocyanin should prove to be apigeninidin, a pigment which has not yet been found to occur naturally. C1 0 _ HO < OH HO -/ OH HO 10 Apigeriin H Apigeninidin chloride The analysis of antirrhinin chloride, however, has shown it to be a 3-rham- noglucoside of-cyanidin chloride, which is closely related, structurally, to the flavonol quercetin and not to apigenin. C1 0 OH O OH HO .-/-\OH HO ) \ OH -OH OH HO Quercetin HO Cyanidin chloride Since cyanidin contains one more molecule of oxygen than apigenin, the connection between these two pigments cannot be one of reduction, nor can one explain their relationship by any simple oxidative process. Wheldale and Bassett did not identify the sugar residues attached to the flavones of Antir- rhinum, so that it is not yet known whether there is any correlation between the carbohydrate portions of these co-existing pigments. The determination of the identity of antirrhinin therefore throws no further light upon the nature of a relationship, either of oxidation or reduction, be- tween flavones and anthocyanins, which has been indicated by the study of the Mendelian factors. In only four other cases have the constitutions of the co-existing flavones and anthocyanins been determined. The red rose contains cyanin [Willstiitter and Nolan, 1914], together with the corresponding flavone quercetin [Karrer and Schwarz, 1928], while the author (unpublished work) has recently identified a similar anthocyanin in PIGMENT OF ANTIRRHINUM MAJUS 755 brown Cheiranthus cheiri (wallflower), from which quercetin and its mono- methyl ether, isorhamnetin, have already been isolated [Perkin and Hummel, 1896]. Thus in these two flowers the constitution of the pigments favours the theory of their interconversion in vivo. The significance of the results in the case of Viola tricolor is at present not very clear, since Everest [1919] claims that myricetin is present in this flower together with the quercetin and delphinidin identified by Mandelin, and later by Perkin [1902] and by Willstatter and Weil [1916]. His claim, however, is not based on analytical results. The flavone kaempferol [Perkin and Wilkinson, 1902] and the anthocyanin delphinin [Willstiitter and Mieg, 1914] have been isolated from Delphinium consolida: thus here, as in Antirrhinum, no close relationship is apparent. The information derived from the study of the constitution of these flavones and anthocyanins is therefore conflicting. There is no conclusive evidence that the plant produces the latter from the former, or that they are both formed by separate but parallel syntheses from the same parent substance. The flowers used for this investigation were those of the deep crimson varieties, Purple King (Carter), Purple Queen (Ryder), and Giant Crimson and Dwarf Crimson (Sutton). They all presumably contained the magenta antho- cyanin together with luteolin and apigenin. The plants, which were grown on a plot of land kindly lent to me by Miss E. R. Sanders of Newnham College, were planted and tended by the staff of the Cambridge Botanic Gardens. The isolation of the pigment was carried out by a modification of Willstatter and Zollinger's [1916] method for the preparation of keracyanin chloride from the cherry. Dilute methyl alcoholic hydrochloric acid was used for the first extraction, since glacial acetic acid extracts contained large amounts of flavone which formed a gel, and made filtration very difficult. Owing to the flavone present even in the alcoholic extracts, great difficulty was experienced at first in obtaining the chloride of the pigment in a pare enough state for crystallisation, but this was at length achieved. Purification of the pigment by means of the crystalline picrate was not possible in this case, owing to the extreme solubilitv of this salt. Use was there- fore made of the insoluble lead salt. This was precipitated from the crude alcoholic extracts, and then dissolved in glacial acetic acid, in which the lead salts of flavones and other contaminating substances are not soluble. The yield of pure pigment was small in proportion to the large amount of material used, but, as flowers were not lacking, it was found to be more economical in labour and reagents to start with large quantities, and subse- quently to discard mother-liquors which still retained appreciable amounts of pigment. The pure chloride of antirrhinin was finally isolated in four hydrated forms. These differed in colour and water of crystallisation, and each of them possessed a characteristic crystalline form. The chloride of the sugar-free pigment was identified as cyanidin 48-2 756 R. SCOTT-MONCRIEFF chloride by means of its appearance, general properties and reactions, com- bustion analyses and products of alkali fission. In all these respects, perfect agreement was found with the account of this pigment given by Willstiitter and Everest [1913]. It has also been possible to compare the pigment from Antirrhinum with a sample of pure synthetic cyanidin chloride which was very kindly sent to me byiProf. R. Robinson. Theyshowed a close resemblance both in their absorption spectra and behaviour in a range of buffered solutions. This latter method, which has been introduced by Robertson and Robinson [1929], is an excellent and reliable means of comparing and characterising anthocyanins and antho- cyanidins. The identification of the sugar residue as a combination of a molecule each of rhamnose and giucose was determined by means of the distribution number, and qualitative and quantitative analyses of the sugars produced on hydrolysis. The position of the sugar residue at position 3 in the antirrhinin molecule is indicated by the violet colour reaction given with sodium carbonate. If the substitution were at 5 or 7, the colour reaction would be either pure blue or violet blue, according to whether the hydroxyls at 7, 3, 3' and 4', or at 5, 3, 3' and 4', were unsubstituted. This decision is strengthened by the fact that, after hydrolysis of the glucoside, one obtains a pure blue reaction with the re- sulting cyanidin chloride from which the rhamno-glucosidal residue at position 3 has been removed. The validity of these colour reactions as a test for the position of the sugar residue has been studied by Robinson and his co-workers during their extensive syntheses, first of pyrylium compounds [Pratt and Robinson, 1925], and later of substituted anthocyanidins, and even of antho- cyanins themselves [Robertson and Robinson, 1927, 1928].