The Colouring Mattem OJ Rhamnus Catharticus

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2350 OESCH AND PERKIN:

CCXVII1.-The Colouring Mattem OJ Rhamnus catharticus. By JOSEFOESCH and ARTHURGEORGE PERKIN. ACCORDINGto the work of Tschirch and Polacco (Arch. Yharm., 1900, 238, 459), tlie yellow, tinctorial constituents yielded by the berries of Bhamnus catharticus (Hungarian berries) are quite distinct from those given by tlhe berries of the various species of Rhamnus which constitute the Persian berry proper, and now well known to consist of rhamnetin, rhamnazin, and quercetin. Thus, according to these authors, in addition to rhamnoemodin and certain other allied non-tinctorial compounds, with which the present paper is not concerned, there could be isolated from the berries of R. catharticus four hitherto unknown yellow, crystalline substances, to which the names rhamnocitrin, P-rhamncrcitrin, rhamnochrysin, and rhamnblutin were assigned. Although the constitution of these compounds was not ascertained, it appeared likely that rhamnocitrin, Cl3HI0O5, was the triliydroxy-derivative of the dihydroxanthone, 0

and rhamnolutin a new tetrahydroxyflavone isomeric with luteolin and fisetin. Again, rhamnochrysin, CI3Hl0O7, was probably an oxidation product of rhamnocitrin, whereas the latter and P-rhamnocitrin, C13H1006, closely resembled the P-rhamnetin which

Published on 01 January 1914. Downloaded by UNIVERSITY OF NEBRASKA 17/10/2015 17:18:40. Schutzenberger describes as obtainable from Persian berries. 8-Rhamnocitrin is quite distinct from rhamnetin, C,,H,,O,, and, indeed, in all these compounds methoxy-groups were absent. It is somewhat remarkable that Tschirch and Polacco were unaware that the true formula of rhamnetin is C16H120, (Herzig, Mosaartsh., 1888, 9, 548; 1891, 12, 172), and that the so-called P-rhamnetin is with little doubt quercetin (T., 1895, 67, 502). Valiaschka and Krasowski (J. Buss. Yhys. C’hent. SOC.,1908, 40, 1502), and Krasowskj * (ibid., 1510), on tlie other hand, strongly criticise this work of Tschirch and Polacco, and contend that from the botanical relationship existing between the Persian berries and those of R. catharticus, and the similarity of their dyeing properties, it is only to be anticipated that the same colour-

* For R translation of this paper, which is written in ttre Russian language, we arc iodelrted to the kindiiess of Mr. T. 11. Pop. View Article Online

THE COLOURING MATTERS OF RHAMNUS CATHARTICUS 2351 ing matters will be yielded by both. As a matter of fact, the compounds of Tschirch and Polacco could not be isolated, although the presence of rhamnetin and quercetin was easily detected. Rhamnolutin is possibly rhamnetin, as the melting point of the acetyl derivative, 183--184O, would appear to suggest, whereas rhamnocitrin is exactly like quercetin and rhamnetin in its re- actions. Again, instead of rhamnochrysin, a mixture of quercetin and emodin was obtained. Further, xanthorhamnin, the glucoside of rhamnetin, is present in these berries. At the outset of this work we were unaware of Krasowski’s paper, our object being chiefly the study of the rhamnocitrin of Tschirch and Polacco, for although flavone derivatives are ex- tremely common throughout nature, the closely allied xanthone group of which this compound was apparently a member appears to be but poorly represented. Having, however, soon satisfied our- selves as to the nature and properties of this compound, a study of the other colouring matters which can be obtained from the berries has been carried out, with the results given in this paper. As it seemed possible that the divergent results of these former workers might have been occasioned in the one case or the other by the accidental use of berries not derived from R. catha?*ticus, special pains have been taken to ensure the botanical authenticity of the material here employed.

EXP E RIME NT AL. Four kilos. of the berries were extracted with eight litres of boiling water for three hours, the mixture was filtered, and the residue washed with 1.5 litres of hot water. Experiments were first carried out on the substances removed from this extract by Published on 01 January 1914. Downloaded by UNIVERSITY OF NEBRASKA 17/10/2015 17:18:40. agitation with ether, but this eventually proved to be unnecessary, and fortunately so, for not only did the mixture separate from the ether with difficulty, but very little colouring matter was removed thereby. The small quantity of the produccs isolated in this manner, however, closely resembled in property those described by Tschirch and Polacco. It was found better at once to hydrolyse the glucosides present in the aqueous solution, and for this purpose sulphuric acid to the extent of 1.5 per cent. was added, and the mixture boiled for two hours. A granular precipitate gradually separated, which in- creased in quantity on cooling, and this was collected and washed, and, when dry, consisted of 352 grams of a dark brown powder. In order to remove a trace of plant wax which was present, it was extracted with a little boiling benzene, only a minute amount of yellow coIouring matter being removed thereby. 702 View Article Online

2352 OESCH AND PERKIN:

The product in the finely powdered condition was now thoroughly extracted with boiling ether, and t’he extract evaporated, whereby a yellow, crystalline residue (A), weighing 102 grams, remained. The residue insoluble in ether, which possessed a dark brownish- black tint, was practically free from colouring matter, and was possibly largely a phlobaphen produced from a phobatannin by the action of the boiling acid. The crystalline residue (A) was now exhausted with boiling toluene, leaving 82.5 grams (B)undissolved, and on cooling overnight, 8 grams of yellow crystals (C) separated from the solution. These were extracted with 200 C.C. of 50 per cent. alcohol, the residue (D)being twice crystallised from toluene, and then from methyl alcohol, until the melting point was constant. Found, C = 63-77; H = 4.19. C,,H,,O, requires- C = 64.00 ; H = 4.00 per cent. It consisted of pale yellow leaflets, soluble in alkali with a yellow colour, and began to sintier at 210°, finally melting at 221-222O. With lead acetate it gave a yellow precipitate, and its solution in sulphuric acid possessed a deep green flkorescence. The amount obtained was exceedingly small. The acetyl compound, prepared in the usual manner, crystallised from alcohol in colourless needles, melting at 200-201°. The melting of this compound, and that of its acetyl compound, are practically the same as those given by Tschirch and Polacco for rhamnocitrin and acetylrhamnocitrin respectively, and the fact that its sulphuric acid solution is strongly fluorescent left no doubt as to ita identity. Tschirch and Polacco adopted the formula Cl3HlOO5 (C = 63.44 ; H = 4-06), and, curiously enough, tested for the presence of a methoxy-group, with a negative result. Feeling far from certain on this point, experiments were carried out by Published on 01 January 1914. Downloaded by UNIVERSITY OF NEBRASKA 17/10/2015 17:18:40. Zeisel’s method, with the result that this statement was found to be incorrect.* Found, Me = 4-92. C15H90,*OMerequires Me= 5-00 per cent.

* Manuiiig and Nierensteiu (Bey., 1913, 46, 3983) have stated that whcii acetic anhydride is employed in conjunction with Zeisel’s niethod, a prdcedure firat suggested as beneficial by Herzig (Monntsh., 1888, 9, 514), crroneous results art: obtained, because hydriodic acid and acetic anhydride aione give ill this way a weighable amount of silver iodide. Goldschmiedt (Ber., 1914, 47, 389) has pointed out that in the case of the ordinary apparatus, where bulbs containing amorphous phosphorus are employed, and when pure acetic anhydride is used, this statement of Manning and Nierenstein is incorrect. On tlie otlier hand, Goldschmiedt suggests as possible that the results of these authors had been obtained with tlie apparatus of I’erkin or of Hesse iii.which thesc bulbs are absent. Frequent practice with the former siiggrsted as improb ible that such an error occurred, but experiments were carried out with this apparatus employing pure acetic auhydride with “Zeisel View Article Online

THE COLOURING MATTERS OF RHAMNUS CATHARTICUS. 2353

The product from the action of the hydriodic acid, which was evidently preseut in the form of an oxonium salt, was poured into sodium hydrogen sulphite solution, the yellow precipitate collected, crystallised from dilute alcohol, and traces of iodine still present were removed by means of potassium iodide solution. Finally, it was crystallised from acetic acid, and obtained in yellow- needles, melting at 274O, which gave with sulphuric acid a fluorescent solution. The acetyl compound, prepared in the usual. manner and crystallised fro’m methyl alcohol, formed colourless needles, which melted at about 116O, gradually solidified as the temperature rose, and finally melted at 182O. Again all the properties of this colouring matter were identical with those of kaempferol, and from these results there could be no doubt that rhamnocitrin is not, as suggested by Tschirch and Polacco, an hydroxydihydroxanthone, but is in reality a monomethyl ether of kaempferol. The only substance of this constitution at present known is the kaempferide of Galanga root, A Zpinia oficinarurn, first isolated by Jahns (Ber., 1881, 14, 2385), who found its melting point to be 221-22207 and that of its acetyl derivative 188-189O. According, however, to the later work of Ciamician and Silber (Ber., 1899, 32, 861), and Testoni (Gazzetta, 1900, 30, ii, 327), kaempferide and acetylkaempferide melt, respectively, at 227-229O and 193-195O, and although in general respect rhamnocitrin very closely resembles kaempferide, it would appear, more especially from the difference in the melting point of their acetyl derivatives, that they are distinct compounds. In order to compare the two compounds, an ethereal extract of Galanga root was obtained by purchase, but this proved to be of no service, in that but a trace of kaempferide was present, and

Published on 01 January 1914. Downloaded by UNIVERSITY OF NEBRASKA 17/10/2015 17:18:40. further experiments could not be carried out owing to the fact that our joint work had to cease at an earlier period than had been anticipated. It is hoped, however, to subsequently deal with this matter. The main residue (D),82.5 grams, from which the rhamnocitrin had been removed, was now extracted, respectively, with boiling chloroform and boiling water, in the hope that some definite separation would be thereby effected. As this did not prove to be the case, the product was next digested with 350 C.C. of boiling alcohol, filtered [filtrate (E)],and the residue washed with 50 C.C. of hot alcohol. The latter, which weighed 11.5 grams, and wa.s of

hydriodicacid ” (D 1-7) nloiie. The results were as anticipated, no silver iodide being thns produced, from which it appe:trs that if Manning and Nierenstein employed Perkin’s apparatus their difficulties must have arisen from the use of impure acetic anhydride or of aoid of a strength greater than that known as ‘‘ Zeisel.” View Article Online

2354 OESCH AND PERICIN

a very sparingly soluble nature, was purified by extraction with 800 C.C. of boiling acetic acid, and the solution, on cooling, de- posited 3.7 grams of yellow crystals, melting at 280-282O [filtrate (F)]. On acetylation, colourless needlw were obtained, which, after re-acetylation, melted at 189O. A similar treatment of the residue not dissolved by the acetic acid indicated that this was almost entirely the same compound as the acetyl derivative melted at 190°. On hydrolysis of the latter, and subsequent re-acetylation, it was eventually obtained of the constant melting point 190--192O. Found, C = 59.16 ; H = 4.09. C16H80,Ac, requires c = 59-50; H = 4.13 per cent. Hydrolysis with hydrochloric acid in the usual manner indicated the presence of four acetyl groups. Found, C16H,20,= 65-02. C,,H200,1 requires C,,H,,O, = 65.29 per cent. An analysis of the colouring matter gave the following result: Found, C= 60.55 ; H =3*91. ClGHI20,requires C = 60.76 ; H = 3.79 per cent. It consisted of yellow needles, melting above 300°, soluble in alkaline solutions with a yellowish colour, and giving with alcoholic lead acebate an orange-red precipitate. This compound agreed in general properties with the P-rhamnocitrin of Tschirch and Polacco, who give the melting point of the acetyl compound as 190-191O. The method of isolation also adopted by these authors corresponds very closely with that given in this paper, but, on the other hand, the formula Cl3Hl0O5 which they have adopted requires C = 63.44 ; H = 4.06, much higher figures

Published on 01 January 1914. Downloaded by UNIVERSITY OF NEBRASKA 17/10/2015 17:18:40. than those here found. Again, it was observed by these chemist8 that 8-rhamnocitrin in some respects resembles rhamnetin, although mention is made in their paper that it is different from rhamnetin, and contains no methoxy-group. Krasowski (Zoc. cit.), however, states that he has isolated rhamnetin from the hydrolysed extract of these berries, but gives themelting point of the acetyl compound as 183--184O, which is practically identical with that given by Liebermann and Hormann (Ber., 1878, 11, 1618), 181--183O, and by Herzig (rMonatsh., 1888, 9, 548), 183--185O, but much lower than that of /3-rhamnocitrin, 190-192O. Examination showed that, contrary to the statement of Tschirch and Polacco, a methoxy-group is present in this substance. Found, Me = 3.03. C,,H,O,Ac, requires Me = 3-09 per cent. The demethylated product proved to be quercetin, in that it had View Article Online

THE COLOURING MATTERS OF RHAMNUS CATHARTICUS. 2355

all the properties of this substance, and gave the acetyl derivative, melting at 193-195O. (Found, C= 59-69; H = 3-47. C,,H,,O, requires C=59.60; H=3.31 per cent.) In order to determine if this compound was in reality rhamnetin, or a distinct monomethyl ether of quercetin, some quantity of rhamnetin was specially prepared from Persian berries and sub mitted to exhaustive purification. From this the acetyl compound, obtained in the usual manner, melted at 19O-19Zo, and was identical with that described above. It is evident, therefore, that this colouring matter of the berries of R. catharticus is rhamnetin, and Krasowski is correct in this respect, although his preparation cannot have been pure. On the other hand, there can be no doubt that, under the name of P-rhamnocitrin, Tschirch and Polacco obtained pure rhamnetin, but the statement that their compound (lid not contain a niethoxy-group, and their analytical results are difficult to explain. The acetic acid liquors (F) produced during the purification of rhamnetin were treated with water, and the yellow precipitate, which, when dry, weighed 2.9 grams, was extracted with alcohol, and the operation repeated until all traces of the less soluble rhamnetin had been removed. The combined extracts were evaporated to dryness, and the residue acetylated, by which means an acetyl compound, melting at 190°, was isolahd. By a re- digestion with boiling acetic anhydride and frequent recrystallisa- tion, this eventually melted at 193-194O. The free colouring matter (0.22 gram), obtained by hydrolysis, consisted of yellow needles, melting at 308--309O. (Found, C = 59-41; H = 3.49. C,,T3,,07 requires C = 59-60; H = 3.31 per cent.) It was evidently quercetin, and Krasowski's statement in this respect is thus

Published on 01 January 1914. Downloaded by UNIVERSITY OF NEBRASKA 17/10/2015 17:18:40. confirmed. Among the1 compound:; isolated by Tschirch and Polacco, there is none that corresponds in property with quercetin, and, indeed, the amount present in the hydrolysed berry extract is vexy' small, and minute in comparison with the quantity of this dyestuff which can be obtained from Persian berries. The alcoholic filtrate (E) obtj8ained during the isolation of the rhamnetin was treated with water, and the yellow precipitate, which, when dry, weighed 48 grams, was crystallised from 50 per cent. alcohol. It was now dissolved in alcohol, a trace of sparingly soluble substance removed, and the filtrate fractionally precipitated with water, employing quantities of the latter varying between 100 and 200 c.c., so that six distinct deposits were obtained. Of these, the first four, amounting to 27 grams, consisted almost entirely of the same substance, and this was crystallised from acetic acid until View Article Online

2356 THE COLOURING MATTERS OF RHAMSUS CATHARTICUS.

the melting point was constant. (Found, C = 62.86 ; H = 3.52. C1,H1006 requires C = 62.94 ; H = 3.49 per cent.) It consisted of yellow needles, melting at 275O, soluble in alkaline solutions with a yellow coloration, and giving with alcoholic lead acetate a yellow precipitate. The acetyl compound crystallised from methyl alcohol in colourless needles, which melted at 116O, resolidified at a higher temperature, and finally melted at 181O. (Found, C=60*79; H=3.93; C1,H1006= 63-25. C,,H606Ac, requires C = 60.79; H = 3.96; C15Hlo06= 62-99 per cent.) On fusion with alkali, this colouring matter gave phloroglucinol, melting at 21Z0, and p-hydroxybenzoic acid, melting at 209O, and there could, accordingly, be no doubt that it consisted of kaempferol. It is quite evident that this compound represents the rhamnolutin of Tschirch and Polacco, who isolated it in a pure condition, and determined its correct formula. They, however, erred in considering that it was a new flavone colouring matter, and were evidently unaware of the production of kaempferol from kaempferide, its methyl ether, by Gordin (Diss., Berne, 1897), and its isolation in the free condition by Perkin and Pilgrim (T., 1898, 73, 267) from the flowers of Delphinium zalil. Kaempferol is by far the main constitupt of the colouring matters derived from the berries of R. catharticus, and Krasowski, who goes so far as to deny its existence, and to criticise the work of Tschirch and Polacco in this respect, can at the most have made but a cursory examination of his product. As to the nature of Tschirch and Polacco's rhamnochrysin, C,,H,,O, (C=56.11; H=3-59), an opinion cannot be given, for no compound containing so low a percentage of carbon was

Published on 01 January 1914. Downloaded by UNIVERSITY OF NEBRASKA 17/10/2015 17:18:40. encountered during this work. Should any substance other than those isolated have escaped detection, the amount premnt could be but trifling, for the accumulated residues, apart from a trace of emodin, were found to consist chiefly of impure kaempferol. The shades given by the berries of R. catharticus on mordanted woollen cloth indicate that they possess much poorer dyeing properties than Persian berries. This is evident from the following table, which represents the dyeings obtained when equal weights of material were employed in each case:

Persian bewies. IiiL II yn ria n h err ics. Chronziwn ...... Dull orange red Dull olive yellow Alzrminium ...... Bright yellow Pale greenish yellow Copper ...... Dull olive yellow Dull puce Tin ...... Bright orange Pale orange yellow Iron., ...... Dull olive Pale dull olive It has long been known that Persian berries contain a peculiar View Article Online

THE METHYLATION OF CELLULOSE. PART 11. 23.57

ferment, now termed ‘‘rharnninase,” which at 40° in the presence of water hydrolyses the glucosides, with separation of the free colouring matters. This change naturally occurs when the dye- bath is slowly raised to boiling point, as in the foregoing experi- ment, the shades obtained being due to the free colouring matters, and not to the glucosides. On the other hand, if the berries are added to boiling water, the ferment is killed, and on dyeing with such an extract, colours of a much yellower character, due to the glucosides, are produced. By experimenting in a similar manner with the berries of R. catharticus, it was found that, after treatment with boiling water, shades similar to those described above, but of a much paler character, were obtained, indicating, apparently, the presence of a similar enzyme in this material. For reasons given above, attempts to isolate the latter have not as yet been made, Summary. The colouring matters rhamnolutin, rhamnocitrin, and 8-rhamnocitrin isolated from the berries of R. catharticus by Tschirch and Polacco consist, respectively, of kaempferol, a kaempferol methyl ether, and rhamnetin, but it is considered that their 8-rhamnochrysin is not a distinct substance. The statements of Krasowski that the two former compounds cannot be isolated from these berries, and that rhamnolutin is possibly rhamnetin, whereas rhamnocitrin is exactly like quercetin and rhamnetin, are obviously incorrect, although the results of this author in regard to the presence of rhamnetin and quercetin in the mixed colouring matters have been confirmed. The latter are, however, only present in small amount, the main constituent being kaempferol, and to this the dyeing property of these berries is chiefly due. Published on 01 January 1914. Downloaded by UNIVERSITY OF NEBRASKA 17/10/2015 17:18:40. CLOTRWORKERS’RESEARCH LABORATORY, TEE UNIVERSITY,LEEDB.