Xxxi. a Survey of Anthocyanins. Iii

XXXI. A SURVEY OF ANTHOCYANINS. III. NOTES ON THE DISTRIBUTION OF LEUCO-ANTHOCYANINS. BY GERTRUDE MAUD ROBINSON AND ROBERT ROBINSON. From the Dyson Perrins Laboratory, Oxford. (Received December 31st, 1932.) IN the course of an investigation of the pigment of young vine leaves, Rosen- heim [1920] noted the presence in the leaves of a water-soluble colourless substance which yielded a colouring matter regarded as anthocyanidin on treatment with hot 20 % hydrochloric acid. This important discovery threw light on the cause of some unexplained observations of Willstiitter and Nolan [1915] who found that a methyl alcoholic hydrogen chloride extract of Rosa gallica showed a gradual increase of tinctorial intensity to about twice its original value on keeping; this change occurred in the absence of oxygen. Rosenheim called the new class of colourless generators of anthocyanidins "leuco-anthocyanins " and advanced the hypothesis that they are glucosides of the pseudo-bases of the anthocyanidins. He found that leuco-anthocyanin occurs in the skins of white grapes and that the formation of anthocyanidin on hydrolysis is just as rapid in a stream of carbon dioxide as in air. The present authors, having meanwhile found that colourless progenitors of anthocyanidin occur in almost every kind of plant material, enquired of Dr Rosenheim as to whether a further publication was contemplated. The reply was, respecting the immediate future, in the negative; but Dr Rosenheim kindly communicated that "he has followed up the relative distribution of leuco- anthocyanins, anthocyanins and flavones in a vine during a whole year from spring to autumn. He examined the young and mature (pigmented) leaves, the ripe and unripe fruit, the seeds, bark, wood and roots; he has also isolated a white powder containing leuco-anthocyanin from the skins of muscat grapes and noted the occurrence of leuco-anthocyanin in various parts of rose trees." Although we propose to retain the name leuco-anthocyanin for the colourless substances which we have found in a variety of woods, seeds and other parts of plants, it is unfortunate in one respect in that the prefix usually connotes reduction of a dyestuff and in this case it is probable that the leuco-anthocyanin and the anthocyanidin are in the same state of oxidation. The leuco-anthocyanins are normally stable in the presence of 10-15 % aqueous hydrochloric acid in the cold, although alcoholic hydrogen chloride of similar concentration, or even weaker, brings about gradual formation of anthocyanidin. This degree of stability to the action of a mineral acid excludes, in our opinion, the hypothesis of Rosenheim that the leuco-anthocyanins are saccharides of the anthocyanidin (or substituted anthocyanidin) pseudo-bases. Substitution of the hydrogen of the carbinol hydroxyl by alkyl groups does not protect the pseudo- bases derived from flavylium salts against the action of even quite weak acids. LEUCO-ANTHOCYANINS 207 Thus dilution of an alcoholic solution of O-pentamethylcyanidin chloride by absolute alcohol causes slow decoloration and such a solution must contain the ethyl ether of the pseudo-base (I or II). Reconstitution of the anthocyanidin 0 GEt OMe 0 ~~OMe MeO Me oMe MeO OMe |COMe CH(OEt) MeO MeO (I) (II) colour-salt is rapidly brought about by the addition of 5 % aqueous hydrochloric acid. Other anthocyanidins and also anthocyanins behave similarly, but the naturally occurring members of the group are not easily changed to pseudo- bases in pure alcohol; they tend to give rather stable colour-bases. In order to avoid this difficulty we suggest that the leuco-anthocyanins contain the group -CH(OH) . CH(OH)- at position (3: 4) in the pyran ring. Thus the immediate precursor of cyanidin chloride is assumed to be (III), ring-chain tautomeric with (IV); conversion to the anthocyanidin then demands dehydra- tion (loss of hydroxyl at 4 and hydrogen at 3) and as the hydroxyl at 4 is in the O OH OH OH OH HO / \OH HO co- OH 6H(OH) I|CH(OH) YH(OH) HEH/(OH) (III) (IV) f-position to a carbonyl group this process will certainly proceed in the direction leading to flavylium salt formation rather than in that leading to a flavone. In the actual leuco-anthocyanins any of the hydroxyl groups may bear carbo- hydrate residues or be acylated. Although the greater number of the leuco-anthocyanins are readily water- soluble, a certain number of them are obtained in the first instance in colloidal solution and after flocculation cannot be redissolved. We suggest that in these cases the potential anthocyanidin molecule is attached to a polysaccharide (see Eucalyptu8 mnrgincata). The actual details will be found in the experimental section and the plan adopted has been the identification of the anthocyanidins obtained from the leuco-anthocyanins, the methods being those of Part I [1931], and Part II [1932] of this series. In order to compare the properties of a new anthocyanidin from the wood of Peltogyne spp. and from the wood of Copaifera pubiflora with authentic speci- mens, new syntheses of rhamnetinidin chloride (V) and 5-0-methylcyanidin chloride (VI) have been effected. C1 C' O OH 0 _OH- MeO OH HO OH OH OVH HO Me (V) (VI) 208 G. M. ROBINSON AND R. ROBINSON The condensation of 4-methoxy-2: 6-dihydroxybenzaldehyde and W-3: 4- trihydroxyacetophenone with the help of hydrogen chloride gave a product (V) having exactly the same properties as the rhamnetinidin chloride which Robert- son and Robinson [1927] prepared by the reduction of rhamnetin. Hence the statement of Malkin and Nierenstein [1930] that rhamnetinidin chloride is a bisflavylium salt cannot be accepted. We have long been of the opinion that the major (sparingly soluble or insoluble) products of the reduction of flavonols in acid solution are produced from pinacols but these substances are accompanied by true crystallisable anthocyanidins in smaller relative amount. Thus the pre- paration of cyanidin chloride from quercetin by reduction under the conditions prescribed by Willstatter and Mallison [1914] is reproducible and, as in the case of rhamnetinidin, we have identified cyanidin chloride made in this manner with cyanidin chloride synthesised by way of benzoylcyanidin chloride [Robertson and Robinson, 1928]. The remarkably wide distribution of leuco-anthocyanins opens up many interesting questions of biogenesis and possible physiological function, but it is too early to speculate even on the nature of the relation between the leuco- anthocyanins and the anthocyanin pigments of flowers. This is certainly not so simple and direct as it appears at first sight. EXPERIMENTAL. Extraction of leuco-anthocyanins. In many cases the leuco-anthocyanin can be extracted by means of distilled water, various woods yielding acid solutions. Boiling 1 % hydrochloric acid has, however, usually been employed because this also extracts, preserves and indicates free anthocyanin or anthocyanidin. Should free anthocyanin be present it is possible to effect the complete hydrolysis of the glucosidal pigment before attacking the leuco-anthocyanin. The anthocyanidin is then removed by means of amyl alcohol and the separated aqueous solution, on concentration by boiling, affords further anthocyanidin, the result of decom- position of the leuco-anthocyanin. Extraction by means of alcohol or alcoholic hydrogen chloride was always more effective than the use of water. In many cases the anthocyanidins required drastic purification, and removal of impurities by extraction with ethyl acetate was frequently practised. When ethyl alcohol or ethyl acetate was employed especial care was taken to remove every trace of these solvents before carrying out distribution-ratio experiments with the anthocyanidin solutions. Summary of results. Aesculus hippocastanum. The crushed endocarp was extracted with boiling 1 % hydrochloric acid, the ffitered solution made up to 10 % hydrochloric acid, boiled for 2 minutes and the anthocyanidin purified. This is the standard process unless otherwise stated and in this case the anthocyanidin proved to be cyanidin. Arachis hypogaea. The testa is rich in leuco-anthocyanin affording cyanidin. Baphia nitida. Barwood contains a leuco-anthocyanin which gives rise to an anthocyanidin not conforming to the usual tests and requiring further examination. Bertholletea excelsa. The endocarp (shell of brazil nut) yields a little leuco-anthocyanin to boiling 1 % hydrochloric acid but treatment with hot 15 % alcoholic hydrogen chloride results in the formation of a relatively large amount of cyanidin chloride. Cae8alpinia granadillo. Results similar to those with Baphia nitida from the heartwood. Since this is a well-known redwood containing brazilin it is possible that the anomalous results are due to contamination with isobrazilein salts. LEUCO-ANTHOCYANINS 209 Castanea sativa. Endocarp. Leuco-A. -->cyanidin. Cocos nucifera. The fibres of the coconut contain much leuco-A. -+ cyanidin. Copaifera pubiflora. The 1 % hydrochloric acid extract of the ground heartwood contains already some anthocyanidin of undetermined nature and this is removed by the minimum of amyl alcohol. In this case the leuco-anthocyanin can be almost completely extracted from the aqueous layer by means of ethyl acetate. It may therefore be non-glucosidic or at most monoglucosidic. The substance may be rendered to 1 % hydrochloric acid after addition of light petroleum to the ethyl acetate and, on boiling with 10 % hydrochloric acid, it affords an anthocyanidin tallying in every detail with peltogynidin (see Peltogyne below). Diospyros sp. Ebony of W. African origin extracted by means of alcoholic hydrogen chloride gave anthocyanidin having the same reactions as that from Caesalpinia granadillo. Entandrophragema macrophyllum. The leuco-A. of mahogany affords mainly delphinidin with indications of admixed methylated derivative or cyanidin. Erica gracili8. The flowers, stem-wood and roots contain (apparently) the same leuco-A. yielding a complex (i.e. acylated) anthocyanidin which is Fe +. It is unusually difficult to hydrolyse by means of alkalis but, on prolonged treatment, cyanidin is produced.

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