XLYIIL-Robinin, Viozaquercetin, Myrticolorin, and 0Syyitrin

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ROBININ, VIOLAQUERCETIN, MPRTICOLORIN, AND OSYRITRIN. 473

XLYIIL-Robinin, VioZaquercetin, Myrticolorin, and 0syyitrin. By ARTHURGEORGE PERKIN, F.R.S.E. Robinin. INa previous communication (Trans., 1900, 77, 430), it was shown that the leaves of Robinia Psezcdacacia contain acacetin, a monomethyl ether of apigenin, an interesting point in view of the discovery by Zwenger and Dronke (Annalen Xuppl., 1861,1,263) that the flowers of the same plant contain a quercetin glucoside, robinin. AS these flowers, however, are practically devoid of dyeing properties, it seemed doubtful whether a quercetin compound was present, and reference to the work of these chemists made ib probable this suspicion was well founded. At the time of Zwenger and Dronke's investigation, the obstinacy with which these colouring matters retain water of crystallisation was not fully appreciated, and results accurate in themselves frequently received a wrong interpretation. The analyses of their " quercetin " were made with material dried at looo, a temperature at which it is rarely rendered anhydrous; again, it was unlikely that picric acid mould result from the action of nitric acid on quercetin. Some difficulty wa8 at first experienced in procuring raw material for this investigation, but ultimately this was overcome by the kindness of Dr. J. van Rijn, of Maastricht, who was good enough to superintend the gathering and drying of some quantity of the flowers. For isolating robinin, Zwenger and Dronke digested the flowers with boiling water, subsequently evaporating the extract and treating the

Published on 01 January 1902. Downloaded by McMaster University 23/10/2014 18:52:43. residue with alcohol. The following method is more rapid, and is suit- able for dealing with small quantities of raw material. The flowers were digested with ten times their weight of boiling alcohol for 4 hours, the mixture strained through calico, and the residue well pressed and again treated in a similar manner. The pale green extract, which deposited a wax on cooling, was concentrated by evaporation, poured into water, and the mixture extracted with ether, the alcohol contained in the aqueous liquid being removed by distilla- tion ; on standing overnight, this solution deposited crystals which were collected and washed with a mixture of alcohol and chloroform until the washings were colourless. The residue was then purified by two or three crystallisations from water with the aid of animal char- coal, One hundred and ninety grams of the flowers gave 1.76 grams, or 0.82 per cent., of robinin in the crude condition, this being reduced approximately one-Ealf on purification. As thus obtained, it consisted of extremely pale yellow needles sinter- VOL. LXXXI. KK View Article Online

474 PERKIN : ROBININ, VTOLAQUERCETIN,

ing at 190' and melting at 196-19'7' (2. and D. 195O), and had the general properties mentioned by these authors, When dried at looo i 0.1103 gave 0.3123 GO, and 0.0550 H,O. C = 52-49 ; H= 5.54. C,,H,2020requires C = 52.24 ; H = 5.54 per cent. Zwenger and Dronke assign to robinin at 100' the formula C,,H,oO,G (C = 51.19 ; H = 5.1 l),and with this their analytical figures closely agree. This discrepancy suggested that our preparation might contain a trace of free colouring matter which would raise the percentage com- position. An alcoholic solution of a second preparation was therefore poured into ether (in which the colouring matter is soluble, but not the glucoside) and the precipitated product collected, again crystallised from water, and dried at 100' : 0*1131gave 0.2165 CO, and 0,0575 H20. C=52*20; H=5*64. The glucoside was thus evidently homogeneous. Determinations of the water of cry stallisation contained in the glucoside dried at the ordinary temperature, employing two distinct preparations, gave the following results : 1.6790, at looo, lost 0.2450 H,O. H20=1551. 1'6079, ), ,, 0.2606 H,O. H20=15.58. --- C,3H420,0,8H,0requires H,O = 15-97 per cent,

C,3H420,(),7frH20 99 H,O= 15*11 39 Air-dried robinin thus crystallises with 8H,O. These results are not in agreement with those of Zwenger and Dronke, who found 14-53 per cent. of water, agreeing with the amount requiFed for the formula C,,Hao0,,,5~H20, or according to the above, C,3~42020,7H,0.This discrepancy is curious, although the lower figures these authors give Published on 01 January 1902. Downloaded by McMaster University 23/10/2014 18:52:43. for robinin dried at 100' suggest the possibility that their product still contained some water of crystallisation, hCO!rr?,p08itiO9?,with Acid,-The dried glucoside was boiled with dilute sulphuric acid * for at least 2 hours, and after standing overnight, the liberated colouring matter was collected, washed, and dried at 100' : 0.6110, at looo, gave 0.2330 colouring matter. Found, 38.13. 0.9804, ,, ,, 0.3'745 9, ,, 38.11). la2266, ,) ,, 004650 7, ,, 37.92. Zwenger and Dronke, on the other hand, found that air-dried robinin gave 37.96 per cent. of '' quercetin " dried at loo', an amount considerably in excess of that given a.bove. Thus, air-dried robinin, C,,H,20,0,SH,0, should give 33.70 per cent. of the colouring matter C,,H,oO,,H,O, or but 31-70 per cent. of the anhydrous substance. In their paper, they state that robinin is " extremely easily " decom- * 750 C.C.of 1 per cent. solution for each gram of glucoside. View Article Online

MPRTfCOLORTN, AND OSYRITRIN. 475

posed by boiling dilute sulphuric or hydrochloric acids, but with acid of 1 per cent. strength this was not found to be the case, for with a short digestion, 39-54 per cent. of “colouring matter ” was obtained, a fact suggesting that it was contaminated with unaltered glucoside. The CoZouring Mattey.- For analysis, the product was recrystallised from dilute alcohol and dried at 160’ : 0.1122 gave 0.2589 CO, and 0-0355 H,O. C= 62.93 ; H = 3.51. C,,H,,O, requires C = 62.93 ; H = 3.49 per cent. Zmenger and Dronke found that this colouring matter, dried at loo’, gave C=59*31, H=4.49, numbers in close agreement with those required for the formula C:,,H,,O,,H,O (C = 59.2, H = 3’94). Prepared as above, it crystallised in slender, yellow needles melting at 276-278’, readily soluble in boiling alcohol, and soluble in alkaline solutions with a pale yellow colour, For additional proof that it was not quercetin, comparative dyeing trials were carried out employing woollen cloth mordanted with chromium, aluminium, tin, and iron. Chromium. Aluminium, Tin. Iron. Quercetin .....,....,...,... Red-brown. Brown-orange- Bright Olive-black. yellow. orange. Robinin colouring matter Brownbyellow. Full golden- Lemon-yellow. Deep olive- yellow. brown. With mineral acids in the presence of acetic acid, it yielded crystalline compounds, and to confirm its molecular weight the sulphuric acid derivative was analysed. 0&1556gave 0.2657 CO, and 0.0457 H20, C = 46.57 ; H = 3.26. Cl,H,,06,H,S0, requires C = 46-87 ; H = 3.12 per cent.

Published on 01 January 1902. Downloaded by McMaster University 23/10/2014 18:52:43. Alcoholic potassium acetate yielded a monopotassium salt, but owing to lack of material this was not fully investigated. The acetyl derivative, prepared in the ueual manner, crystalliaed from methyl alcohol in colourless needles : 0.1093 gave 0,2438 CO, and 0*0400H20. C = 60.83 ; H = 4a06. C,,H,O,(C,H,O), requires c = 60.79 ; H = 3.96.per cent. When fused with alkali, the colouring matter gave p-hgdroxybenxoic acid (m. p. 208-210°) and phloroglucinol. These facts, together with a comparative dyeing trial, conclusively proved that the colouring matter derived from robinin is identical with that isolated from the flowers of the Delphinum ConsoZida (Proc., 1900, IS, 182). This similarity was corroborated by the peculiar behaviour of their acetyl derivatives when heated ;thus, while some preparations melted at 180--182O, others became liquid at about 116O, resolidified as the temperature rose, and finally melted at 180-182’. The colour- KK2 View Article Online

476 PERKIN : ROBININ, VIOLAQUERCETIN,

ing matter is in reality hmpherol, first obtained by aordin (Diss. Berne) by the decomposition of its monomethyl ether karnpheride, which is contained in galanga root (AZpinia oficinarum). This, as Kostan- ecki suggests (Ber., 1901, 34, 3723), has in all probability the con- stitution

and may be considered as the connecting link between apigenin and yuercetin. Kampherol, and not guercetin, is the colouring matter produced when robinin is hydrolysed with acid. The Sugars.-The acid filtrates formed by the decomposition of robinin were neutralised with barium carbonate, filtered, and evaporated to a small bulk. The product yielded an osazone which after three crystallisations from dilute alcohol was obtained as a spongy mass of yellow needles sintering at 165' and melting at 178-180°. As a further treatment in this manner did not yield a homogeneous substance, it was dissolved in alcohol, the solution poured into ether, and well washed with water. On slow evaporation, a small quantity of crystalline matter separated, and this was collected, washed with ether, and recrystallised from alcohol. It melted at 204-205O and resembled glucosaxone. The filtrate which contained the main bulk of the osazone, on spontaneous evaporation, deposited crystals which were extracted with benzene, washed with traces of ether, and recrystallised from dilute alcohol, In the preliminary notice (Proc., 1901, 17, 87), it was Published on 01 January 1902. Downloaded by McMaster University 23/10/2014 18:52:43. considered probable that this substance was galac tosazone, but fermentation experiments kindly carried out for me by Dr. Turnbull, of the Leather Industries Department, did not corroborate this view. Employing the sugar solution, as obtained in the above manner from robinin, a s1igb.t fermentation did indeed occur, but the main bulk was unattacked and gave an osazone meeting at 180-182°. This was found to be identical with rhamnosaxone prepared from pure rhamnose, thus harmonising with the. results of Zwenger and Dronke. Whether the trace of the glucose derivative simultaneously isolated is an ingredient of >robininitself or is derived from 8 second glucoside OF kampherol also present in minute quantity must, although it appears unlikely, remain a. matter for conjecture. Very large quantities of ram material would be required to decide this point. According to these views, the formula of robinin is consequently either C33H44020or C33H42020,and its decomposition by acid may be represented as follows : View Article Online

MYRTICOLORIN, AND OSYRITRIN. 477

C33H44020 f QH20= C15H,oO6 -k 3C6H1406. C33H42020 f 4H20= C15H10064- 2c6H1406 4- C6HI2O6. It is thus a most interesting glucoside, as it appears to be the first known substance of this class to contain three sugar nuclei. The above equations require respectively a yield of 37.63 and 37.73 per cent. of kampherol, whereas that actually obtained was 38-13, 38.19, and 37.92 per cent. Dyeing sxperiments carried out in the usual manner with woollen cloth showed that robinin is almost devoid of tinctorial properties ; this was to be expected from the preliminary examination of the flowers.

The Identity of Osyritrin, Violaquercitrin and Myrticolorin. Jn a previous communication (Trans., 1897, 71, 1133), it was shown that the leaves of the CoZpoon compressurn contain a quercetin glucoside, osyritrin, to which, dried at 130°, the formula C2'7H30017was assigned. At that time, the raw material available yielded little more than 1 gram of the pure substance, but subsequently, through the kindness of Mr. R. H. Coaton, of Wellington, Cape of Good Hope, a larger supply was obtained, and it became possible to investigate it more fully. Water of Crystallisation.-For determining the loss of weight which the air-dried glucoside undergoes, it was exposed for about three weeks over sulphuric acid in a desiccator. As the product on exposure to the atmosphere rapidly assumed its original weight, the analysis was performed indirectly in this manner. 1,3345, dried as above, gained 0.0770 H,O. Found 5.76. C27H30017,2H20requires H20= 5.44 per cent. Published on 01 January 1902. Downloaded by McMaster University 23/10/2014 18:52:43. At 130°, employing an oil-bath jacketed with amyl alcohol (b. p. 130-131"), it suffered a further loss of 0*0200H,O, or 1.49 per cent. ($H,O requires 1.43) and finally, at 160" (oil-bath jacketed with 'tur- pentine), 0.0220 gram H20was evolved, or 1.67 per cent. ($H,O = 1.47). Osyritrin, therefore, crystallises from water with 3 mols. of water of cry stallisation. Anhydrous osyritrin is remarkably hygroscopic, thus, on exposure for 1 hour in the air of the room, it completely regains its water of crystallisation. On analysis :

0.1170 gave 0.2275 CO, and 0.0537 H20. C = 53.03 ; H = 5.09.

C27H28016requires C =) 53.28 ; H = 4.60 per cent. Decomposed with dilute sulphuric acid: 0*8400gave 0.4137 C15H1007. Found 49.25. C27H2,0,,requires Cl5HI0O7= 49.67 per cent. View Article Online

478 PEREIN : ROBININ, VIOLAQUERCETIN, The osazone of the sugar melted at 204-2305', as previously found, and was evidently glucosazone. Osyritrin dried at 160' has thus the formula C27H28016,aqd at 130' (C27H28016)2,H20, and not C27HR0017as formerly suggested. Its hydro- lysis with acid is analogous to that of rutin, which in a similar manner gives quercetin and rhamnose (Schunck, Trans., 1888, 53, 264) : C27H2,ol6 + 3H20 = C15Hlo07+ 2C6H120,. C27H32016 4- 3H20 = C,,Hl00, 4- 2C6H#6. Rutin.

Vio 2ccquercitrim. Mandelin (Jahresber., 1883, 1369) isolated this glucoside from the flowers of the Viola tricolor uariensis, and assigned to it the formula C,,H42024. It has been previously pointed out that this is more correctly C27H,601,,as the true molecular weight of quercetin was not known at that time. For its preparation from the flowers, a method identical with that employed for the preparation of osyritrin (Zoc. cit.) gave an excellent result : 0.1071, dried at 130°, gave 0,2036 CO, arid 0.0510 H20. C = 51-84 ; H = 5.29 per cent. 0.1144, dried at 160', gave 0.2230 CO, and 0-0535 H20. C = 53.16 ; H = 5.19 per cent. As Mandelin employed water for the isolation of his substance, it seems possible that the result he obtained was due to the contamination of his product with a trace of quercetin. Water of CvystaZZiscction.-When dried over sulphuric acid, 0.970 2 Published on 01 January 1902. Downloaded by McMaster University 23/10/2014 18:52:43. gained, on exposure to the atmosphere, OgO580 H,O. Found H20= 5.64 ; 2H20 requires 5-44 per cent. 1.0280, at 130°, lost 0.070 H20. H20= 6.80. 2.5H20 requires H,O = 6.79 per cent. 1.0280, at 160°, lost 0.085 H20. H20= 8.26. 3H20 requires H20= 8.1 6 per cent. Violaquercitrin thus crystallises with 3H20, When dried at 160' and decomposed with acid, it gave 49.35 per cent. of quercetin, which is in accordance with the following equation (49.67 per cent.) : C27H2,0164- 3H20 = cl5Hl007 4- 2C6H120p The osazone of the sugar melted at 204-205' and was evidently glucosazone. Violaquercitrin melts at 186' when slowly and at 190° when rapidly heated, and is undoubtedly identical with osyritrin. View Article Online

MYRTICOLORIN, AND OSYRITRIN. 479

Ngrticolorin. This quercetin glucoside was isolated by H. G. Smith (Trans., 1898, 73, 697) from the leaves of the Ezccalyptue mcworhynaha, who assigned to it the formula C27H28016,and represented its decomposition with acid by the equation

C2!7H28016 f.3H20 = C,,H,,07 + 2c6H1206* Smith at first considered the sugar thus produced (Zoc. cit.) to be galactose, but more reoently, in a private communication, he informs me that this is not the case, as he has obtained from it glucosazone, (m. p. 204-205O). It is worthy of note that he (Zoa. cit.) calls atten- tion in his paper to the remarkable similarity of myrticolorip to o syrit rin. The raw material employed mas some commercial myrticolorin, for which I am indebted to the kindness of Mr. Smith. After purification, a sample dried at 160' gave C = 53.03 and H = 5-09 per cent,, and on decomposition with acid 49.25 per cent. of quercetin : Driedoversulphuric acid,on exposure to air it gained H20= 5.76 percent.

9, ,, and heated at 130' it lost H20= 1.49 per cent, Dried at 130° and heated at 160° it lost H20=1.67 per cent, Myrticolorin, therefore, crystallises from water with 3H20, and this may be fractionally removed by methods identical with those employed in the case of osyritrin. As the melting points, general reactions, and dyeing properties also agree, there can be no doubt that the azlbstances am identical, moreover, it was previously shown (Trans., 1899,75,433) that osyritrin, violaquercitrin, and myrticolorin give, by means of potassium Published on 01 January 1902. Downloaded by McMaster University 23/10/2014 18:52:43. acetate, monopotassium derivatives (found K = 6-21). There is no doubt, therefore, that these also must be regarded as one of the same compound, represented by the formula C27H27OI6 (K = 6.03 per cent.). Certain glucosides, as ruberythric acid and the purpurin glucoside contained in madder are decomposed during the dyeing operation by means of the mordant, which combines with the alizarin or purpurin thus liberated. Such, however, I 6nd is not the case with the known glucosides of the quercetin group, which are dyestuffs of themselves, and give shades differing considerably in most cases from those yielded by the colouring matters from which they are derived.* A simple experiment with Persian berries clearly illustrates this point. This dye contains glucosides of rhamnetin, rhamnazin, and quercetin, and * Nietzki (" Chemistry of Organic Dye-stuffs," 1892, 255) considers it probable that in dyeing the quercitrin splits up, and that the shades obtained are due to the formation of quercetin lakes. View Article Online

480 PERMAN: THE INFLUENCE OF SALTS ON THE

there is also present a ferment which at about 40' in the presence of water hydrolyses these compounds ; if, therefore, the dye-bath be raised slowly to the boiling point, this change occurs, and the resulting shade is due to the free colouring matters and not to the glucosides. On the other hand, if the berries be plunged into boiling water, the activity of the ferment is at once destroyed; and the tinctorial property of this extract is now due to the glucosides as the shade indicates. This difference is very similar to that shown between quercetin and its glucosides, which gave the following results with woollen cloth mordanted in the usual manner : Chromium. Aluminium. Tin. Iron. Quercitrin .. . Full brown-yellow. Full golden-yellow. Lemou-yellow. Deep olive. Quercetin ... Red-brown. Brown-orange, in- Bright orange. Olive-black. clining to red. Osyritrin ... Brown-yellow. Full golden-yellow. Lemon-yellow. Dull brown. Rntin...... Brown-yellow. Full golden-yellow. Lemon-yellow. Dull brown. It is interesting to observe that rutin and osyritrin have identical tinctorial properties, which points to the fact that the two sugar nuclei of each (in the one case rhamnose and the other dextrose) are similarly attached to the quercetin residue. As regards their actual disposition, satisfactory proof is at present wanting, but it is highly probable that one at least is attached to the catechol group, as the dyeing properties suggest the absence of o-hydroxyl groups. Their behaviour with potassium acetate is an indication that they contain intact the hydroxyl of the pyrone ring, for it is found that galangin, by this method, forms a monopotassium salt. The properties as a whole would harmonise closely with those of a compound containing both sugar nuclei attached to the catechol group, but further evidence is needed before this conclu-

Published on 01 January 1902. Downloaded by McMaster University 23/10/2014 18:52:43. sion can be adopted.

CLOTHWORKERB' RESEARCHLABORATORY, DYEINGDEPARTMENT, Y ORKSHIRE COLLEGE.