THE COLOURING MATTER OF COTTON FI.OWERS. PART I l l . 145

XIK-The Co1ourin.g Matter of Cotton Flowers. Part irr.

By ARTHUR GEORGE PERKIN.

IN a former communication it was shown (T., 1909, 95, 2181) that the yellow flowers of the Egyptian cotton plant contain gossypitrin, a glucoside of gossypetin, and two quercetin glucosides, namely, quercimeritrin and isoquercitrin. The present work was undertaken to ascertain as far as possible if there was any chelmical distinction not only between thO Egyptian and ordinary Indian yellow cotton flower, Gossyp-urn herbaceurn, but also between these and the red, pink, and colourless petaIs of other varieties. As a side issue it was interesting to attempt t o determine if the red colouring matter present in both the red and yellow flowers was due t o the red oxidation products of either gossypetin or gossypitriii (T., 1913, 103, 565).

Owing to a failure to obtain a fresh supply of the Egyptian flowers the investigation of the first point cannot be considered

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146 PERKlN: THE COLOURING MATTER O F

complete, the results here given, which were finished fully eighteen months ago, having been hitherto held back on this account.

The author is indebted t o Mr. H. Martin h a k e , Economic Botanist for the Government of the United Provinces of Agra and Oudh, for the samples of flowers, these1 being obtained from special varieties of the plant which he has himself investigated (Proc. Roy. SOC., n11, [B], 83, 447). Among the types of cotton grown there are (a) red, ( b ) pink, ( c ) yellow, and (G?) white flowered plants. I n the offspring of a cross between (a ) and ( c ) there occur in the second and subsequent generations red and yellow flowered plants, which breed pure, whereas in the offspring of a cross betlween (a) and ( d ) all four forms occur which breed pure. The petals employed in this work were derived from such pure plants, occur- ring among the offspring of one o r other of these crosses.

The types were as follows: red flowered, G. arboreum, Linn.; pink, G. sanguineum, Harsk; yellow and white, two varieties of G. neglecturn, Tod, usually now treated as one species, but originally described as G. ueglectum and G. Tossrum. With rare exception little has been done as regards the examination of flavone consti- tuents in the flowers of plant varieties except in a haphazard manner, although mention may be made of the work of Wheldale and Bassett (Biochem. J., 1913, 7, 441; 1914, 8, 204) on the flower pigments of A ntirrhinum ma jus, in which certain varieties are shown to contain apigenin and others luteolin.

Flavones being present in general as glucosides which are colour- less or nearly so, they themselves can exert little or no yellow colour effect, although such is sometimes presumed to be the case, and it would indeed appear probable that where a yellow tint is due to their agency, this is t o be traced, as in the case of the yellow cotton flower, to their presence as a potassium or other salt.

That the anthocyanin of flowers is closely connected with the flavone glucoside also present was correctly surmised by Wheldale (f‘ Report to the Evolution Committee of the Royal Society,” 1909, 5, l), but her suggestion that the former represents an oxidation product of the latter is no longer tenable. Owing to the brilliant work of Willstatter with Everest (Annnlen, 1913, 401, 189) and others, the chemistry of many of these compounds is clear, and, indeed, they must now be regarded as the effect, of a reduction rather than an rlsidation on the flavone glucosides. As a reducing agent Everest (Proc. R o y . Soc., 1914, [ B ] , 88, 603, 326) has employed magnesium turnings with alcoholic hydrochloric acid, and there can be no doubt that flavone glucosides give anthocyanins in this manner which are identical with those naturally occurring in plants.

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COTTON FLOWERS. PART 111. 147

Wheldale and Bassett again (Zoc. c i t . ) sought in the case of A ntirrhinum ma jus t o isolate the anthocyanin (anthocyanidin) from the highly-coloured flower extract by hydrolysis with sulphuric acid. I n this manner they obtained magenta-reld and Indian-red powders, which with dilute alkali gave green solutions. The nature of these products, which bear some resemblance to compounds described later, is uncertain, for they do not agree in their general properties with those typical of the anthocyanidins described by Willstlatter and by Everest. Most probably they represent aheration products of the anthocyanidin a t first produced, and into which i t pawes by prolonged contact with the boiling dilute mineral acid.

EXPERIMENTAL.

Bed Cottoia Flowers, G. arboreum, Linn.

The flowers (1600 grams) were extracted with boiling alcohol, the rich red-coloured extract evaporated, diluted with hot water, and the alcohol evaporated. After agitation with ether t o remove wax and a trace of free1 colouring matter, the red liquid was boiled with lead acetate solution, which caused the separation of a greenish- brown precipitate. This was collected, washed, and, in the form of a thin cream, decomposed with hydrogen sulphide. The well- boiled mixture gave after filtration a deep maroon-coloured liquid, which when evaporated fo r several days in a vacuum deposited some quantity of a gelatinous mass possessing the same tint. This product (the filtrate from which is designated A ) after being washed with a little1 water, was dissolved in 25 C.C. of 40 per cent. alco'hol, and the red-coloured, semi-gelatinous deposit which sepa- rat'ed on cooling, was twice recrystallised from 10 C.C. of alcohol, and finally from dilute alcohol. The product (3 grams) consisted of small, pink needles, the red-coloured impurity remaining chiefly in the mother liquors which were added t o the filtrate A , but as the last traces of this latter adhered tenaciously t o the substance, f o r economy's sake a special process was adopted. This consisted in pouring a concentrated alcoholic solution into ether, which caused the immediate precipitation of the red substance, and this being rapidly renioved the ethereal liquid was agitated with water. The aquelous liquid gradually deposited a semi-gelatinous precipitate, which on boiling with a little' water became crystalline. It could be further purified by crystallisation from dilute alcohol containing a little pyridine, a trace of the pink impurity first separating out. The yield was 2.28 grams. (Found (dried a t 160°), C=53*96; H = 4.68. C,,H,,O,, requires C = 54.31 ; H = 4.31 per cent.)

After crystallisation from dilute alcohol the air-dried substance

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148 PERKIN: THE COLOURING MATTER ON

contains two molecules of water of crystallisation. (Found a t 160°, H,O = 7.04. C2~H20012,ZH20 requires H20 = 7.22 per cent.)

By hydrolysis with I per cent. boiling dilute sulphuric acid, the glucoside gave 64.8 per cent. of a yellow, crystalline product, the analysis and general properties of which coincided with those of quercetin. (Found, C = 59.59 ; H = 3.36. C,,H,,07 requires C=59.60; H=3.30 per cent,.) The amtyl compound melted a t

The sugar present in the acid filtrate from the quercetin was isolated in the usual manner, and proved to be dextrose, in that i t gave the osazone melting a t 204--205°.

The hydrolysis of the glucoside thus proceeds according t o the following equation :

which involves a yield of 65.09 per cent. of quercetin. Dyeing experiments employing mordanted woollen cloth and other com- parative tests proved that this glucoside is identical with the isoquercitrin which, together with quercimeritrin and gossypitrin, is present in the yellow Egyptian cotton flowers.

The red agueous filtrate A on evaporation in a vacuum gave a further deposit of isoquercitrin, and by careful examination the presence of a second flavone glueoside could not be detected in these flowers.

A sharp chemical distinction between these red cotton flowers and the yellow Egyptian flowers previously examined is thus obvious.

The final mother liquor from the isoquercitrin had a deep red colour and when highly concentrated in a vacuum deposited a red, viscous substance, which could not be' isolated in a crystalline condition. I n solution its colour, although slightly duller, was very similar to that of the original alcoholic extract, which thus appa- rently possemed considerable stability. That i t could scarcely represent a phobaphen o r partly decomposed tannin glucoside seemed certain, because although some cotton flowers, especially those which are yellow, do contain much phlobotannin, this is present in the form of a potassium or other salt, and is insoluble in alcohol. It has been previously shown that gossypetin is readily oxidised to the dull red quinone, gossypitone, this change taking place very readily even during the dyeing operation. I n the case of the cotton flowers i t thus appeared possible that the red colora- tion might not be duo in this case t o a typical anthocyanin, but rather to the oxidation product of the gossypetin glucoside. To determine this point, the red, viscous residue was submitted to the act'ion of boiling 1 per cent. sulphizric acid solution, causing the

194-196'.

C,1H,0012 + H2° C15H1007 $- Cf3H1206,

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COTTON FLOWERS. PART 111. 149

separation of a resinous precipitate. This was collected and dried, and in order to remove quercetin, which was present in some quantity, it was dissolved in pyridine and precipitated from this solution by means of ether, this operation being twice repeated. For analysis it was dried a t 160° f o r a long period. (Found, C=55*92; H=4*58.) It consisted of a dull red powder, very similar to gossypitone, with which also the analytical numbers -show some slight agreement. Again, wihh aluminium mosdanted calico it dyed, although not very readily, an olive shade, resembling that given by the latter substance. On the other hand, whereas sodium hydrogen sulphite solution rapidly converts gossypitone into gossypetin, thel boiling red-coloured solution of this product on addition of the reagent did not become yellow, but merely paler and somewhat browner. When neutralised with acid again, ether extracted from the) liquid but a, trace of flavone, which dissolved in alkali with a pure yellow colour. The evidence, therefore, of the presence of gossypitone o r its glucoside in the red flowers is thus of a negative character. Dilute alkalis dissolved the product with a greenish-yellow colour, whereas sulphuric acid gave a brown solution, passing to red on dilution with water.

The Pel lo w Flozoers, G . neglecturn.

The yellow flowers (1000 grams) were e,xtracted with alcohol and the alcoholic solution evaporated t o a small bulk. On cooling, but a trace of a dark brown-coloured deposit separated, ajnd a distinc- tion was thus a t first sight apparent between this extract and that of t*he yellow Egyptian flowers, which in these circumstances at once yield a heavy precipitate of the potassium salt of crude quer- cimeritrin. It was, however, subsequently observed that on keeping for a long period a similar separation in 'this case also gradually occurred. This was collected,* washed with alcohol, dissolved in hot water, and the filtered liquid trelated with lead acebate, causing the formation of a dull, redcoloured precipitate, which was decom- pwed with hydrogen sulplzide in the usual manner. The glucosidal solution on keeping for some days in a vacuum deposited a semi- gelatinous precipitate, which on recrystallisation from dilute alcohol formed warty nodules admixed with gelatinous matter. This product readily dissolved in hot water, but on boiling the concen- trated solution f o r a few seconds the hot liquid became semi-solid owing to the separation of fine needles. Theae were collected, washed with warm water, a*nd although now pure' could be

* Such a separation is not really necessary, and it is simpler to add water, evaporate the alcohol, remove wax, and precipitate the aqueous 1iqui.d.

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150 PERKIN: THE COLOURING MATTER OF

recrystallised by employing a very large volume of boiling water. (Found, C = 52.58, 52-26 ; H = 4.38, 4.39. C21H20013 requires C=52.50; H=4*17 per csnt.)

When prepared in this way the glucoside is devoid of water of crystallisation, melts a t 240---242O, dissolves in alkalis with a pale yellow colour, and gives with lead acetate a deep red precipitate. It is sparingly soluble in boiling alcohol, and almost insoluble in boiling acetone, a property which readily distinguishes i t from quercimeritrin, which is fairly soluble in the latter solvent.

When crystallised from acetic acid and water the air-dried product contains tlwo molecules of water of crystallisation, which it lows a t 160° but regains o a keeping in moist air. (Found, H20 = 7.02.

The glucoside (0.5162 gram) when suspended in boiling water was found t o be somewhat st'able towards the action of acid (boiling with 7 per cent. sulphuric acid for two hours), and in this respect i t resembles quercimeritrin (lor. cit.). The yield of yellow, crystal- line pro$duct (dried a t 1 6 0 O ) thus obtained was 66.6 per cent. This possessed all the properties of gossypetin, and yielded acetyl gossy- petin melting a t 226-228O. From the acid filtrate, the sugar was isolated in the usual manner, and identified as dextrose by means of the osazone, vhich melted a t 204-205O.

The hydrolysis of the glucoside occurs, theref ore, according tlo the following equation, which requires a yield of 66.25 per cent, of gossypetin :

C2,H2,0,3,2H20 requires H,O = 6.97 per cent.)

C,lH2?0013 + HZo = C15H1008 + C6H1206'

The acetyl derivative, C21H11013(C2H30)9, is readily produced by boiling with acetic anhydride for three hours. On cautious addi- tion of alcohol t o the solution a semi-solid, crystalline mass is produced, and the product can be recrystallised from acetic anhydride and alcohol in a similar manner. (Found, C=54*35, 54.32 ; H = 4-46, 4-52. Cs9H,,02, requires C = 54.54 ; H = 4-43 per cent.) It consisted of colourless needles melting a t 226--228O, almost insoluble in alcohol.

An acetyl determination by the acetic ether method gave: acetic acid=62*81, and gossypetin, 37.26; the numbers required by the above formula being respectively 62.93 and 37.06 per cent. It is accordingly an ennea-acetyl derivative.

During the examination of the Egyptian flowers (Zoc. c i t . ) a gossypetin glucoside, C,,H,00,3, termed gossypitrin, was isolated ; this melted a t 200-202°, and was obtaineld in small amount as the middle fraction between quercimeritrin and isoquercitrin. It appeared, theref ore', that gossypitrin and the glucoside present in the G. neglecturn melting a t 240-242O were distinct compounds,

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COTTON FLOWERS. PART 111. 151

for i t was unlikely that the former had been isolated in so impure a condition as its low melting point would indicate. No Egyptian flowers being available, the matter remained unoertain, until it was accidentally elucidateld as follows : The gossypetin glucoside melt- ing a t 240-242O was digested for some time with boiling acetone t,o determine i f quercimeritrin was present. Under this tre'atment the fine, hair-like needles were broken up into1 much finer needles, and thew when collected and dried a t looo now melted a t 200-202°. When dried at 160° 1 molecule of water of crystalha- tion was evolved, which was, however, reabsorbed a t the ordinary temperature in the presence of moist air. (Found H,O=3.72. Calc., H,O=3 61 per cent.) Curiously enough the anhydrous substance when plunged at once into hot sulphuric acid also melted a t 200-202°, so that this lower melting point does not arise from the) presence of water of crystallisation. By boiling with absolute alcohol f o r one hour the melting point of the undissolved portion is almost the same, but if boiling water is employed crystals quickly commence to separate from the hot solution, which now melt at 239-242O and remain anhydrous after exposure to moist air. The glucoside thus exists in two interchangeable modifications, and i t is accordingly certain that both Egyptian and Indian flowers contain the same substance, gossypitrin.

I n the mother liquors obtained during the isolation of gossypitrin from these flowers no certain evidence of the psewnce of querci- meritrin, so readily obtainesd from the Egyptian variety, could be detected. By further evaporation in a vacuum a second gelatinous deposit separated, but this was 09 so readily soluble a nature that by crystallisation from dilutel acetic acid and other methods but a trifling amount of an apparently pure product crystallising in yellow needles could be isolated. Although on hydrolysis these yielded quercetin, identified by means of its acetyl derivative melt- ing a t 194--196O, their ready solubility distinguished them in a marked manner from quercimeritrin. The final mother liquor, which possessed a reddish-brown colour, also contained much of a readily soluble quercetin glucoside, and is referred t o later ( B ) . The aqueous filtrate from the lead precipitate, on treatment now with basic lead acetate, gave some quantity of a yellow precipitate. By decomposition in the moist condition with hydrogen sulphide in the usual manner, this yielded a solution from which nodules sepa- rateld on keeping. These after recrystallisat'ion from dilute acetic acid consisted of pale yellow needles melting at 217--219O, and when hydrolysed gave quercetin. This glucocide proved t o be the same isoquercikin which is present in the Egyptian flowers (Zoc. c i t . ) and in the red petals of G. arboreurn.

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152 PEKKIN: THE COLOURIKG MATTER OF

An examination of the ordinary yellow Indian cotton flower, C. herbnceutii , available in small amount, gave the same results as those furnished by G'. i / e y l e c t u n ~ . So far, therefore, i t is certain that both the Egyptian and Indian yellow flowers contain the glucosides gossypitrin and isoquercitrin, although curiously enough quercimeritrin, an important constituent of the former, is appar- ently absent, or a t least present in but small amount in the latter. I t is impossible, however, to decide this point unless very much larger amounts of raw material are available than was here the case.

The mother liquor (B) , as above noted, possessed a reddish-brown colour, this t int being apparently due t o the fact that at the base of these petals a red spot or eye is present, which has the same shade as that of the' pet3als of G. arboreurn. On digestion with boiling dilute hydrochloric acid a precipitate containing much quercetin was deposited, and from this, by the same method as that employed previously, 0.15 gram of a dull red powder was isolated. (Found, C = 59.87 ; H = 3.63.) I n appearance and general properties it was indistinguishable from the product derived from G. arboreum, elxcept that its alkaline solution possessed a somewhat stronger green tint. The analytical figures are, however, interesting in that they differ but little frolm those given by quercetin itself.

Experiments with the white Indian cotton flowers, G. negZectum or rossrum, showed that these were devoid of dyeing property. One kilo. when extracted with boiling alcohol gave, by the method employed in the former cases, a nelarly colourless solution, from which by means of lead acetate a very small quantity of a buff- coloured precipitate separated. From the latter by decompcrsition with hydrogen sulphidei a minute amount of glucoside in the gela- tinous condition was isolated, which by hydrolysis with acid gave a trace of impure colouring matter. This, which consisted of a buff- coloured powder, in its general reactions closely resembled apigenin, and with litlls doubt consisted of this o r a very similar cosmpound. The pink flowers, G. sanguinezcm, gave also a disappointing result. Employing aluminium mordanted calico a faint green shade was produced, whereas the1 mere trace of flavone which could be isolated from them, although probably quercetin, could not be identified with certairty.

The Oxidat ion Product of Gossyp'tm'n.

It was previously pointed out that whe,reas gossypetin by means of alcoholic pbenzoquinone gives the quinone gossypibne, the glucoside gossypitrin yields also an apparently similar compound, which could not then be studied owing to lack of material. When a cold saturateld solution of gossypitrin in absolute alcohol is treated

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CO'I'TON FLOWERS. PART 111. 153

with an alcoholic solution of pbenzoquinone, the yellow liquid quickly becomes maroon-coloured and on gentle warming needlee commence to separate. These were collected and washed with alcohol. (Found, C = 52.52 ; H= 3-76. C21H18013 requires C = 52.72 ; H = 3 . 7 6 per cent,.)

This compound, for which the name gossypitrone is suggested, consists of maroon-coloured needles, which, however, possess a mom violet tint when during their preparation dilute alcohol is employed. When heated, i t gradually decomposes abolve 200° and does not possess a definite melting point, although fusion of the product occurs a t about 255--259*. Boiling water dissolves it somewhat sparingly to fo'rm a brownish-red solution, which with alkali becomes orange-yellow coloured, and gives with lead acetate a greenish- brown precipitate closely resembling that yielded by an extract of the red cotton petals with this reagent. I f gossypitrone suspended in warm water is treated with dilute sulphurous acid solution, a clear, yellow liquid rapidly results, and ultimately becomes semi- solid owing to the separation of needles. From an aqueous solution of these on boiling, crystals separated which melted a t 240-242O and had all the properties of gossypitrin. Gossypitrone is evidently, therefore, the quinone of gossypitrin.

Interesting is the fact that gossypitrone dyes aluminium mor- da'nted calicos pretty green shades, which are very similar t o those given by an extract of the red cotton petals. It is quite possible that the original shade may be blue, for gossypitrone undergoes reduction during the dyeing operation, and indeed the green-dyed portions ref erred to gradually become yellow on keeping. Experiments with mordanted woollen cloth gave shades which are identical with those produced by gossypitrin, and that these represent the dyeing property of the glucoside itself is clear owing t o their general similarity L80 those given by quercimeritrin in a similar manner :

Chromium. Aluminium. Tilt. Iron. Reddish-brown. Dull yellow. Bright orange. Dark olive-brown.

I n the earlier stages of the dyeing operation the shade produced on the aluminium mardanted wool possessed a decided green tint, but this gradually altered as the temperature of the dye-bath was raised. Whereas, therefore, when dyed gossype'tin passes into its quinone gossypitone, gossypitrone, the oxidation product of gossy- pitrin in these circumstances suffers the reverse change.

If a solution of gossypitrone in hot water is treated with 7 per cent. of sulphuric acid and heated to boiling, t8hel red liquid becomes first brighter and then subsequently paler in colour. On keeping, a pale brown, crystalline powder separates, which is readily solubIe VOL. CIX. H

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154 THE COLOURING MATTER OF COTTON FLOWERS. PART 111.

in alcohol, and from this solution, when concentrat,ed, ether deposits a hi ace of a reddisli-brown, amorphous substance resembling gossypitone. Here, again, reduction evidently occurs, the main product beling gossypetin, and this is unfortunate, f o r although gossypitrone, judging by its method of formation, would appear to be the glucoside of gossypitone, actual proof in this respect is Iacking.

The' dyeing properties of gossypitrin indicate that the sugar group is attached t o the tetrahydroxybenzene nucleus, it being somewhat analogous in this respect to quercimeritrin. The nature of this tetrahydroxybenmne nucleus is uncertain, although should this prove to be a diliydroxyquinol, a para-quinonoid grouping in gossypitroae is not impossible :

To determine this point, enzyme hydrolysis naturally suggests itself, which will be1 studied when an 0pportunit.y occurs. These experiments, however, indicate that although a certain resemblance exists between gossypitrone and the colouring matter of the red petals of the1 cotton flower, this is apparently but a coincidence. The behaviour of gossypitrone when hydrolysed by acid proves it to be in no way connected with the red, amorphous compounds here described, which would appear more probably t o consist of alter& tion products of the anthocyanin originally existing in the flower.

The author is indebt,ed t o Mr. Isaac Shulman for his assistance in the earlie,r stages of this work.

CLOTHWORKERS' RESEARCH LABORATORY, THE UNIVERSITY,

LEEDS. [Rece ived , December 18th, 1915.1

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