DISTRIBUTION OF ANTi ~ JCYANINS
Bv ROBERT ROBINSON
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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. Advantage may be taken of this method to purify the monoglucosides occurring in plant extracts made with O·S% hydro chloric acid. Salt is added and the colour passed to amyl alcohol ; then on addition of light petroleum or benzene, the substance can be washed out with aqueous acid and thus shuttlecocked as often as desired. For example, this method of purification was used in the case of the anthocyanins of the leaves of the copper beech and by isolation it was later shown to be cyanidin-3-galactoside (idaein) first .obtained from the cranberry, common in Europe. The D.N. of many monoglucosides varies with the concentration due to the different degree of molecular association in the aqueous and alcoholic layers. This phenomenon has been to assist in the identifica tion of natural and synthetic monoglucosides ; two examples are oxycoccicyanin from the larger American cranberries and oenin from the skins of black grapes or the red cyclamin, or certain primulae; these are the 3-glucosides of peonidin and malviclin respectively. The amount of the variation mentioned afforded one criterion of agreement between natural and synthetic specimens. Foreign substances often have a large effect on the D.N. of the monoglucosides. Tannins and fl.avonols are the chief agents and if present in substantial amount may cause a monoglucoside to simulate a rhamnoglucoside or even a diglucoside. Washing the solutions with ethyl acetate followed by shuttlecock purification between water and butyl alcohol (NaCl present) is usually a remedy. . The D.N. (amyl alcohol) of the anthocyanidins is 95-100. ·special ffilXtures have been devised which enable the chief anthocyanidins to [ 6 J be differentiated by distribution tests. Tolucnc-cyclohcxanol (5: J) extracts a considerable proportion of pelargonidin and peonidin and is coloured by cyanidin. It does not extract malvidin, petunidin or delphinidin. A mixture of anisole and amyl ethyl ether containing picric acid completely extracts pelargonidin, peonidin, and malvidin, a good proportion of cyanidin (>50%), some petunidin (<20%) and no delphinidin. It will be seen that t~.ese tests alone suffice to establish the presence of malvidin and delpl{inidin. Other important tests are the ferric reaction, the caustic alkali stability test and aboye all the colour reactions in solutions of varying pH. The ferric reaction is a deep blue coloration produced in neutral or slightly acid solutions either aqueous, alcoholic or amyl alcoholic. It indicates free hydroxyls in positions -3' and -4' and is therefore positive for cyanidin, petunidin and delphinidin only. Petunidin and delphinidin, but no other anthocyanidins are at once destroyed by aerial oxidation in the presence of excess sodium hydroxide. This serves as a confirmatory test and distinguishes petunidin from cyanidin. A distinction between pelargonidin and peonidin can be made by the distribution method using toluene cyclohexanol (3: 1 and 4: 1) but as these anthocyanidins seldom occur together by far the best method is the colour reactions of the corresponding anthocyanins which are very different. The behaviour of the anthocyanidins and anthocyanins in solutions of varied pH depends on the number and position of the hydroxyl groups and is largely independent of the nature of other substituents. The oxonium salts themselves are orange-red to bluish-red and these colours in acid solutions give some guidance; orange or salmon-red, for example, nearly always denotes pelargonidin and a distinctly bluish-red denotes a delphinidin derivative (including the methyl ethers). On the other hand the colours may be made bluer, never yellower, by the presence of other substances termed co-pigments, and these must be removed. Frequently it suffices to heat the solutions, when the complex is dissociated and the true colour is seen. Near the neutral point, especially on the acid side, many antho cyanins are decolorizcd due to the formation of a pseudo-base; the colour is restored on the addition of acid. On the alkaline f!i~e of the neutral point, the colour-base, a quinonoid compound, is formed. This is usually red to violet in colour. As the alkalinity is increased metal salts of the colour-bases are produced and these are usually violet to blue in colour. There are of course exceptions to all these rules. The changes are illustrated in the case of cyanidin. Sometimes there may be two colour-base salts having different colours. Thus cyanidin and 3: 5-substituted cyanidins (e.g., cyanin chloride) give a pure blue coloured solution in aqueous sodium carbo- l 7 ] nate and this is not affected by tfie addition of sodium hydroxide (except that cyanin may be dccolorized if a great excess is used).
Cl
HO r-rocoC~~HOH co HO Cl-Io HO Cyanidin chloride (Red) Pseudo-base (Colourless) (in 0·5% HCl) (formed at about pH, 5·6) HoOO:?o HO CH 1-10 CH Colour-base (Violet) Sodium salt of Colour-base (in aqueous sodium acetate) (Blue) (in aqueous sodium carbonate)
3-Substituted-cyanidins (e.g., chrysanthemin and mccocyanin) give a violet solution in acqueous sodium carbonate and this becomes blue on the addition of sodium hydroxide. Obviously the pH of the cell sap must have an influence on fl0wer colour but it is not permissible to use the anthocyanins as indicators to estimate this pH . • The blue cornflower and the red rose both contain cyanin but the cornflower sap is actually more acid than that of the rose. This is almost certainly due to association of the pigment with a colloid in the cornflower. The colours are better seen than described and anyone who would like to indulge in the delightful hobby of identifying anthocyanins in plant material can easily acquire a list of suitable flower standards in which the anthocyanins are relatively homogeneous and the reactions are not obscured by foreign material. T. W. J. Taylor carried his 'research case' to the Galapagos Islands and in those inhospitable , regions was able to identify the anthocyanins of many species not readily accessible elsewhere. I should like to take this opportunity of acknowledging the enthusiastic collaboration of Lady Robinson in the work of identifying anthocyanins. In 1664 Boyle, in his 'Experiments and Considerations touching Colours' wrote: "Take good Syrrup of Violets, Impregnated with the Tincture of the flowers, drop a little of it upon a White Paper .... and j 8 ] \ on this Li
HO CO Quercetin
Anthoxanthins of this class are often excellent mordant dyestuffs; they occur in plants as glucosides etc., for the most part, but sometimes free. In recent years our knowledge of this group .has been greatly extended by investigations of Indian chemists and especially by Venkataraman and Seshadri. Turning from the methods to the results, the most striking point on the chemical side is the prevalence of the anthocyanins already mentioned and based on the three main types pelargoni~in, cyanidin and delphinidin. New combinations of known structures have been observed. Thus pelargonidin 3-rhamnoglucoside and ·a pelargonidin 3-bioside (sugar unidentified) occur in the scarlet gloxinia and orange scarlet nasturtium respectively. It would be worth while to isolate these new anthocyanins in substance. There are just a few exceptions to the rule of the three types. The anthocyanin of Gesnera fulgens (orange flowers) was clearly something quite unusual. It gave red solutions in alkalis and its acid solutions [ 9 ]
~ere bright orange-coloured. The anthocyanidin was soon recogniz:d a s one that had already been synthesized, and then the anthocyanm. itself was synthesized and proved to have the same properties as · Th's 's the first case noted of the occurrence of an antho- gesnenn. 1 1 . . . . cyanin with no substituent m posmon -3.
Cl
HO OH OH
(Glucose) CoH OH 11o,·o Gesneridin Chloride Gesnerin Chloride
Some years ago in collaboration with the late Professor A. G. Perkin a rare cosmetic pigment 'Carajura' used by the natives of the Orinoco was studied. The main constituent is carajurin the colour-base, beauti fully crystalline, of carajuridin chloride. This may originate from an anthocyanin similar to gesnerin.
Cl
HO OMe
HO
MeO Carajuridin Chloride
At least one new anthocyanidin occurs in certain poppy flowers but it is mixed with the usual ones and has not been obtained in a pure condition. A very important group, however, is that of the nitrogenous anthocyanins easily distinguished from the usual types by their quite different behaviour. The best known example is betanin in the common beet but the group is fairly well represented and similar substances occur in Amaranthus, Celosia, Bougainvillaea (bracts) etc. Little is known about the chemistry of these colouring matters but the composition of two of them suggests that they may be ordinary antho cyanins in which amino-acid residues are attached to the benzene rings by means of the amino-group. Synthetical compounds of this type have been made and show similar properties. The chief point of present interest is that the nitrogenous antho cyanins are found in fi\'c orders only, namely Caryophyllales, Chenopo diales, Lythrales, Thymelcales, and Cactales. Hutchinson (Families of Flowering Plants, 1926) considers that the first four are closely related but tentatively places the Cactales far from thern. However, Hutchinson [ 10 ] admits that the Cactales resemble the Ficoidaceae (e.g., Mesembryan themum), a family of the Caryophyllales and many botanists differ from him on morphological grounds in his placing of the Cactales. Similarly there are certain families which produce only one of the three main types, although there are many others in which only one occurs and still others in which pelargonidin, cyanidin (or peonidin) and delphinidin (or its methyl ethers) all ~~cur. The Rosaceae, for example, have a decided preference for cyaniqin (no blue rose), the Boraginaceae for delphinidin, whereas no family specializes in pelargonidin. The Amaryllidaceae have pigments based on either pelargonidin or malvidin, a common combination. The only generalization that can be put forward is that cyanidin predominates in the Archichlamydeae and pelargonidin and delphinidin arc together more common in the Metach lamydeae. The exceptional anthocyanins also occur in the latter near the end of the evolutionary development. This occurrence of pelar gonidin and delphinidin in the more differentiated plants suggests that the pigments themselves require more steps for their synthesis. It would be gratifying to be able to answer the question:, 'At what point in the progress from the lowest forms of vegetation to the higher plants do the anthocyanins appear?' I regret that this cannot be done as yet and it is an interesting topic for investigation. These colouring matters have not been found in bacteria, despite statements to the contrary. Scrutiny of the alleged instances has shown that the case has not been proven in any one of them. We have been able to examine a few specimens and have found no anthocyanins. It is also dol}btful whether anthocyanin occurs in algae; there are no clear cases up to the present. The usual anthocyanins are found in young fern fronds together with some of unusual constitution not yet identified. Up to the present dclphinidin derivatives have not been obtained from this source. This occurrence of anthocyanin in the Cryptogamae is of great interest for it disposes of the idea that these pigments are produced solely for the sake of colouring flowers. Anthocyanins also occur in the leaves and cones of Gymnospermae. Among flowering plants proper there are just a few families (no orders), or certain genera of families, which are reputed to be unable to produce anthocyanin. For example, the common cUcumber may re present the Cucurbitaceae among the dicotyledons and the Narcissus, the Amaryllidaceae among the monocotyledons. However, many genera of the latter family do produce anthocyanin, and in the order Cucur bitales of which the Cucurbitaccae are a family, there is another family, the Begoniaceae, which affords anthocyanin in plenty. The cause of these anomalies is unknown and would repay investigation, if that were feasible, [ ll 1
I cannot enter here into the vast subject of the morphological dis tribution of anthocyanin except to remark that it is a normal con stituent of green leaves and often, as in Coleus, a very noticeable permanent one. In so·me cases the you~g leaves alone. are heavily pigmented ; in others the colour appears 1~ old leaves or m aut~mnal reddening. Even in dense and shady tropical forests the col?rat10n of the under surfaces of the leaves if noteworthy. Also anthocyanm appears in many sites, as the result of dtbught or injury, in which it is normally absent. In such cases the anthocyanin product is always a derivative of cyani.din, that is, so far as our experience goes, and the identity of such anthocyanins has not been investigated by others. Permanently pigmented leaves contain pelargonidin, cyanidin or delphinidin deriva tives and autumnal reddening in the vast majority of cases is due to cyanidin glucosides. The percentage occurrence of cyanidin in the genera examined is the following: autumn leaves, 95; young leaves, 93; permanently pig mented leaves, SO; fruits, 69; flowers, 50. The anthocyanins .in fruits and flowers are of value to the plant and hence differentiation by selec tion has probably been at work. It appears that many plants have at hand a ready mechanism for the production of anthocyanin and it is incredible that this should have no deep physiological significance. It is equally clear that we do not yet know what this function is. None of the special theories is wide enough though they may indicate adaptations for particular purposes. Anthocyanin does not protect chlorophyll because the absorption spectra of the two colouring matters are largely complementary. l'lowers ferti lized by bees are usually blue, because these insects arc relat.{.vely insensi tive to red (or vice versa), but such accommodation is obviously a late event and throws no light on the real function of the anthocyanin, or its precursor, throughout the vegetable kingdom. One further point may be mentioned; anthocyanin often occurs where it cannot be seen, as for instance, in underground roots of the radish (a pelargonidin deri vative), growing root-tips of certain saxifrages, and underground stems of the potato. The coloured spots on potato-tubers contain a curious complex anthocyanin based on delphinidin. Different parts of the same plant often yield different anthocyanins. The petals of the St. Brigid anemone may be coloured by pelargonidin 3-bioside in the scarlet varieties but the deep blue anthers always yield a delphinidin derivative. A variety of Nasturtium (Tropaeolum Majus, Empress of India) was found to contain pelargonidin 3-bioside in the petals, cyanidin 3~bioside in the sepals and a delphinidin diglucoside in the leaves. These and many other instances of a like kind indicate that the three types are derived by modification of a common precursor. [ 12 ]
The conviction that this precursor is changed most naturally into cyanidin glucosides is based on the almost universal appearance of these under conditions of stress (injury, drought etc.) but it is also derivable fronr a statistical survey of the colours of flowers. The table gives the number of species etc. examined and the percentage in which the three main types appear ; cyanidin includes peonidin, and the delphinidin in- cludes its methyl ethers. t I Number examined. Pel.% Cy.% Delph.% Species 530 19 40 so Genera 299 22 52 49·5 Families 88 37·5 73 65 Orders 57 44 80 72
It should be noted that a process of selection has been applied to us; we naturally tested first the flowers readily available and the preference for blue and violet flowers shown by the public and fostered by the horticulturists is shown in the high figure for the delphinidin in species. But when we went further afield the predominance of cyanidin became more apparent and if leaves and fruits were included it would be overwhelming. The influence of climatic conditions is very marked ; there is a high proportion of delphinidin derivatives among alpine flowers and a pre ponderance of pelargonidin derivatives* in those growing in tropical and sub-tropical countries. Flowers coloured by delphinidin derivatives are not all 'Olue or violet. The red flax anthocyanin is a delphinidin derivative. Tn tropical countries the red delphinidin types are more numerous than the blue types and it is possible that red has a better survival value than blue under these conditions. A background of yellow or orange carotenoid emphasizes the red colour of many tropical flowers. . In the series of natural products related in structure to the antho cyanins, we also find the cyanidin type of structure to be the commonest. Allusion has already been made to the case of quercetin. The 3': 4'- 0ihydroxyphenyl group occurs in 220 of 268 cases examined (Gisvold and Rogers, 1938). A substance in which the central nucleus "is fully reduced is catechin, which is far more plentiful than any similar substance.
This was illustrated by the specimens available in Calcutta in January and used at the lecture. The great majority proved to be pelargonidin derivatives; cyanidin derivatives were also available, but no delphinidin types. Certainly some of the latter could be seen in the gardens but it was clear that they were not at all widely distributed. '[ 13 1
Finally, there is the very intert:sting class of leuco-anthocyanins which yield anthocyanidin on treatment with alcoholic hydrochloric acid in presence of oxygen. Rosenheim was one of the first to study this subject and he thought that the young leaves of the grap~ine
HOMHd~H WcH.OH HO CH. Catechin
contained a leuco-compound that gave rise to the grape-skin ambo cyanidin, namely malvidin. Actually the product was cyanidin. Lady Robinson has taken a particular interest in this topic and has shown that the distribution of these colourless substances is very wide indeed. They are nearly always to be found in woods, nutshells (cocoanut fibre), seed-coatings and the like, as well as in softer structures. Lignin which is probably a kind of natural Bakelite can very frequently be shown to contain a leuco-anthocyanin moiety. Most leuco-anthocyanins are convertible into cyanidin (84% of the cases) but some give pclargonidin and some delphinidin (banana skins, for example). Two substances that give rise to fiavylium salts by oxidation in acid solution have been isolated from woods and crystallized. These are cyanomaclurin (A. G. Perkin) and peltogynol (R. and R.). But the anthocyanidins obtained from these do not occur as anthocyanins. Hence this is a special class chemically related to catechin. ' HOCO:~J==>H HO CJ.i. \oH Cyanomaclurin (?)
We regard all these types, catechins, flavones, flavonols, leuco anthocyanins, and anthocyanins as different manifestations, or modi
fications of a proto-type, a C6 .C3 .C 6 structure. Reasons have been given elsewhere for the assumption that this proto-type is derived from two
molecules of hexose and one of triose. The C6.C3 intermediates are kno~n .in great variety and it has been pointed out that the assumption of lmkmg by aldol condensations requires one of the terminal nuclei to be less oxidized than the other and, in fact, cyanidin is the natural . result by dehydration without oxidation or reduction, ( 14 ]
The genetics of flower colour is a subject\ demanding full and separate treatment ; its study has been greatly stimulated by the possibility of determining anthocyanins in small quantities of material and"Dr. Scott-Moncrieff has been one of the pioneers in this chemica genetic field. In collaboration with W. J. C. Lawrence, she has suggested as the result of complete genetic study of Dahlia that the anthocyanins, in this group at least, produced from .two substances, one of which is always present in sufficient amount and the other in limited amount.
The relation of this to our C6 .C3 .C6 hypothesis is evident because the substance present in limited amount is presumably the c6.c3 inter mediate. The argument is too complex to summarize, involving as it does a consideration of the interaction of many identified genes and of the relations with the flavone pigments also. One clear outcome of this work is a definite indication of a relation ship between anthocyanin production and flavone production. If the limited component is used up to make much flavone it is not available for anthocyanin and vice versa. There are intermediate positions and this is important. To take an analogy. It might pass without notice if a very poor audience consisted of 20 men on one occasion and 20 women on the next similar occasion. It might be thought that some mutually exclusive principle was at work. But if on successive days every possible combination of men and women adding up to 20 were found to be present it would have to be assumed that there existed some limitation on the number rather than the kind of the audience, but what this might be I have not the least idea. The geneticist can do chemical experiments in vivo; by introducing a new gen~, for example, a methylation factor, he can observe its operation o-~, a new anthocyanin. This science is only in its infancy and it is probably from researches in this field that we shaU get the fullest knowledge of the chemical processes that modify the anthocyanin precursors or the anthocyanins themselves. We have collaborated with the John Innes Horticultural Institu tion in one such investigation, that relating to the flower colours of Sweet Peas and in this connexion performed hundreds of analyses. As a general rule all proceeded to our mutual satisfaction but on one ~r two occasions our findings were challenged and repetition showeJ that the geneticist was usually right. Possibly the labelling was at fault but an experience of this kind was impressive and disclosed to the chemist that at least one aspect of biology possesses the attributes of an exact science. It is possible to predict! Before closing this brief account attention should be drawn to the existence of complementary genes the presence of both of which is necessary for the development of anthocyanin. Punnett has identified these genes in the case of the Sweet Pea and denotes them as C and R, 15 ]
Flowers of plants contammg either C or R, bur not both, are white. On crossing them coloured flowers (C.R.) are obtained in the usual proportions. Two further complementary genes are necessary for ~he production of flavonols (anthoxanrhins). The chemical basis of these and similar phenomena is quite unknown. Finally I woiJld like to thank the members of this Association for giving me the present opportunity to review the resulrs of researches that were so pleasant to make. Much remains to be done and here in India there is a great opportunity for extended labour in this attractive field .
February 7th 1950.
Printed by P. C. Ray at Sri Gouranga Press, 5, Chintamani D