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1408 PERKTN : THUJIN.

CXXXIII.--Thujin. By ARTHURGEORGE PERKIN. INthe year 1858 Rochleder and Kawalier (Wien. Akad. Ber., 29, 10; J. pr. Chem., 74, 8) isolated from the green portions of the Arbor vitae (Thuja occidentalis) a thu jin, C20H22012, soluble in alkali solutions with a yellow colour. This substance, present in the plant only in minute quantity, by gentle with acid in alcoholic solution, yielded dextrose and a compound, thujigenin, C14H12O7, which on longer digestion with the acid combined with a of water, with the formation of thujetin, CI4H1408 : Published on 01 January 1914. Downloaded by University of California - San Diego 16/12/2016 23:13:26. c20H22012 + H2° == C6H7206 + C14H1207, C14H,207 + H2° = C14H1408* Thujigenin and thujetin are described as yellow, crystalline com- pounds, soluble in dilute alkali solutions with a green, and in alcoholic ammonia with a bluish-green, coloration. Thujetin gives a red precipitab with lead acetate solution, and by digestion with boiling baryta water was converted into1 thujetinic acid, C28H22013. From an alcoholic extract of 120 kilos. of the plant, these authors could isolate only a few grams of thujin, and it is probable on account of this very small yield that no further exhaustive investi- gation of the subject has been hitherto attempted. In many respects the description of this compound corresponds with that of a flavone glucoside, and as the result of a preliminary in- vestigation carried out several years ago, there could be little doubt, judging by the properties of the trace of yellow, crystalline colour- View Article Online

PERKIN : THUJIN. 1409

ing matter then isolated (I!.,1899, 75, 829), that this is the case.* Although tKe presence of flavone in plants is now known to be of common occurrence, the peculiar properties assigned to thujin were attractive, and it appeared desirable to clear up if possible the mystery with which it has been so long surrounded.

EXPERIMENTAL. It is known in certain caes that in the earlier stages of their growth the leaves of plants are richer in flavone glucoside than at a later period, and it therefore suggested itself that the younger leaves of the T. occidentaEis would on this account be most suitable for investigation. Twenty-five kilos. of this freshly gathered material, which on keeping for several days lost by evaporation of moisture approximately half its weight, were exhausted with alcohol, the extract evaporated, and the green, viscous residue gradually stirred into 6 litres of boiling water. When cold the pale brown aqueous liquid was decanted as far as possible from the semi- solid residue; the last traces were removed by filtration through paper, aq operation which occupied several hours. Addition of lead acetate solution in excess caused the separation of a dull, faintly yellow precipitate (A), which was collected after some time, the filtrate now giving with the basic acetate a much brighter yellow deposit (B). The procedure adopted appeared preferable to that of Rochleder and Kawalier, who merely employed neutral lead acetate as precipita_nt, extracting the product with dilute acetic acid, and after filtration reprecipitating with the basic compound. In the form of a thin cream with water (B) was treated with hydrogen sulphide, the mixture briskly boiled, and the lead sulphide removed by filtra- tion. The pale brown liquid on being kept in a vacuum deposited Published on 01 January 1914. Downloaded by University of California - San Diego 16/12/2016 23:13:26. at first a trace of yellow, semi-crystalline substance, which was of a non-glucosidal character ; this was removed, and subsequently crystals of the desired compound commenced to separate. The product was collected from time to time as a sufficient quantity accumulated, with the idea that some possible separation might thus be effected, and ultimately five distinct fractions were avail- able, the total weight of which in the air-dried condition was 2.95 grams. These samples, however, appeared to consist of one and the same substance, for by a recrystallisation from water, in four cases the glistening leaflets melted at about 183--185O, whereas the melting point of the fifth, namely, 176--178O, was only slightly lower. The final filtrate on further evaporation did not yield crystals, although dyeing experiments still indicated the preeence * The younger branches of the Chinese Arbor vik (7'. orimztnlis) are said to be used for dyeing yellow. View Article Online

1410 PERKIN : THUJln'.

of some quantity of glucoside. In order to hydrolyse this, the mixture was boiled with addition of acid, and the coloiwing matter (1'073 grams) isolated by meaiis of et Iier, aid wscrved for subse- quent examination (0). As the glucoside was possibly still impure, and contained a trace of free colouring matter, it was dissolved in a little boiling alcohol, and the solution slowly poured into a separating funnel, which contained one part of ether and two parts of water. After gectle agitation the aqueous solution was removed, boiled to expel ether and alcohol, and the crystals which separated on cooling were recrystallised from alcohol and water. The latter treatments, how- ever, caused no alteration in the melting point of the substance, which still melted at 183-185O. When dried in the air no lms was experienced by heating to looo (Found, C= 53.76 ; H =4-88. C21H2201zrequires C = 54.07 ; H = 4.72 per cent.). It was sparingly soluble in boiling water, dissolved in alkali solutions with a pale yellow colour, and gave with lead acetate a yellow precipitate. To determine the nature of this glucoside it was hydrolysed with boiling 1 per cent. sulphuric acid, and, after cooling, the yellow needles which had then separated were collected and dried at 160O. The yield was 65-11 per cent., and in a second experiment 64-89 per cent. (Found, C = 59.62 ; H = 3.58. Cl,Hl0O7 requires C = 59-60 ; H=3*31 per cent.). The acetyl compound prepared in the usual manner consisted of colourless needles melting at 191-195O (Found, C = 58-70; H = 3-95. C,,H,O,(C~H,O), requires C = 58.59 ; H'= 3-90 per cent.). This colouring matter had all the properties of except in respect of the colour of its alkaline solution, which possessed a slight green tint, and experiment indicated that it was in reality Published on 01 January 1914. Downloaded by University of California - San Diego 16/12/2016 23:13:26. this substance contaminated with a trace of a second compound. In order to characterise the sugar which was formed by the hydrolysis of this glucoside, the sulphuric acid present in the filtrate from the quercetin was removed by means of barium carbonate, the clear liquid evaporated, and the residue freed from mineral matter by extraction with alcohol. The product gave with phenylhydrazine bn osazone, which after purification melted at 179-181°, and was found to consist of -osazone. The hydrolysis of the glucoside could therefore be expressed as follows : c%Hzzo~z + Hzo = C,,H1oo, + C6H1,06J and this equation represents a yield of 64.81 per cent. of quer- cetin. From these results it seemed certain that the glucoside dried at looo was not anhydrous, and in reality possessed the View Article Online

PERRIN : THUJJN. 1411

formula C21H20011,H20,for anhydrous rhamnose is now known to be C6H,,05. This proved to be the case, and when dried at 160° the following analytical figures were obtained (Found, C= 55.90; H=4.58. C,,H,,O,, requires C =56*25; H=4.46 per cent.). The only simple rhamnoside of quercetin at present known is the of bark (Quercus tinctoria), and this in appearance and general properties possessed a striking resemblance to thujin. Again comparative dyeing experiments with the two substances, employing mordanted woollen cloth, further accentuated this similarity. On the other hand, as the melting point found by Herzig (Mouatsh., 1885, 6, 877), the latest worker on quercitrin, is 173O; it was still possible that thujin was a distinct substance. To settle this point some quercitrin was prepared from yellow flavine by the method previously described (T., 1913, 103, 1634) and recrystallised from dilute alcohol. In case a trace of quercetin was present the product was submitted to the ether treatment, detailed above in connexion with thujin, and then fractionally crystallised by the gradual addition of boiling water to its concen- trated alcoholic solution. The second fraction only was taken as pure, for Herzig, who recommends the latter procedure, has pointed out that any quercetin is contained in the first deposit. The air- dried product did not evolve water of crystallisation when heated to looo (Found, C = 54.11 ; R = 4.74. C;,H,,O,, requires C = 54.07 ; H=4*72 per cent.). At 160°, however, a molecule of water was given off (Found, H,O = 3-86, 3-75, 3-96. C,,H2,0,,,H20 requires H20= 3-86 per cent.), and on analysis the anhydrous compound gave: C=56*24; H=4.63. CzlHzOO1lrequires C = 56-25; H =4*46 per cent.

Published on 01 January 1914. Downloaded by University of California - San Diego 16/12/2016 23:13:26. The quercitrin obtained in this way melted at 183--185O, and was identical with the thujin prepared from Thuja occidentalis. The formula C,,H,,O,, previously assigned to quercitrin by Herzig, and correctly so in view of the temperature at which the specimens analysed by this author were dried, was based, as in the case of many other glucosides, on the assumption that rhamnose was C,H,,O, rather than C,H,,O,,H,O. It must now be replaced by C21H20011,a fact which has been conjectured for some time past. Quercitrin is given in Beilstein’s ‘‘ Handbuch ” as C,,H,,O1,,2H,O, which represents C,,H2,0,~3H20, whereas according to Hlasiwetz (L4~nalen,1859, 112, log), it is said to lose 3H,O in a vacuum over sulphuric acid, and when dried at looo, 1H20. The low melt- ing point, 173O, given by Rerzig is also peculiar, for there is at present no evidence of a second glucoside in quercitron bark. On one occasion, moreover, during this work a small preparation View Article Online

1412 PERKIN : THUJIN.

crystallising in minute prisms and melting at 172-173O was actually obtained, but although numerous attempts were made again to isolate this product, no further quantity could be prepared. Whether this wils an impure specimen, or a modification possibly containing more water of crystallisation, must remain in doubt. Attention was now directed to the lead precipitate (A), and this was decomposed with hydrogen sulphide in the usual manner. After boiling, the mixture was filtered, and on cooling a brown, tarry precipitate separated; this was removed, and by concentra- tion in a vacuum a dull, yellow powder consisting of free colouring matter admixed with but a trace of glucoside was gradually deposited. The lead sulphide on repeated extraction with boil- ing water .gave further quantities of a similar product amount- ing in all to 1.73 grams, which was soluble in alkali with a greenish- yellow coloration. For purification it was crystallised from dilute alcohol (Found, C = 59.50 ; H = 3.40). The acetyl compound melted st 190-195O, and there could be no doubt that in this case also the substance w5t9 a nearly pure qu eroetin . On examination the product (C) referred to above, and isolated by the hydrolysis of the thujin mother liquors, was observed to give a well-marked green coloration with dilute alkali, and with the hope of gaining some insight into the nature of this second compound, the acetyl compound was prepared and analysed by hydrolysis. Found : C,,H,,O, = 58.86. C15H507(C2H30)5requires C,,H,,07 = 58.98 per cent. No distinction could thus be observed between this compound and pure acetylquercetin, and it was accordingly evident that the

Published on 01 January 1914. Downloaded by University of California - San Diego 16/12/2016 23:13:26. amount of impurity was either too small to affect the analytical result, or that the latter was in reality a flavonol isomeric with quercetin itself. The colouring matter regenerated from the acetyl compound was practically unchanged in regard to its reaction with dilute alkali solutions. As apparently in no case was this green colour effect so marked as that assigned by Rochleder and Kawalier to their thujetin, it seems likely that the quantity of this second component may vary with the season of the year, or pmibly with the environment of the plant. These suggestions ars supported by the fad that during a preliminary examinaton of the Arbor vitE obtained by purchase in Leeds (T., 3899, 75, 829), the colouring matter isolated gave, not only a strong green coloration with dilute alkali, but a trace of an acetyl compound melting at 205-206O, results which appeared to indicate the presence of . , The composition View Article Online

PERKIN : THUJIN. 1413

C,,,H,,O,, assigned by Rochleder and Kawalier to thujin is also explainable if it be assumed that their preparation consisted of a mixture of quercetin with some myricetrin. With the hope of again repeating this earlier experiment 1.5 kilos. of the commercial fluid extract of the plant (Extractum thujae occidentalis liquidurn) were procured, and from this 0.62 gram of colouring matter was isolated by the method formerly employed. The result was, however, disappointing, for from this preparation, which was soluble in alkali solutions with a greenish- yellow colour, no acetyl compound melting higher than 195O could be obtained. Thu jin must accordingly be considered to consist of quercitrin admixed with a trace of a second glucoside, which appears also to belong to the flavonol group. The amount of this latter which is present in the plant would seem to be variable, but the circum- stances that affect. this increase have not yet been ascertained. Finally, it is evident that the thujigenin of Rochleder and Kawalier cannot be regarded as a definite. compound, whereas their tliu jetinic acid, C28H220,3(C = 59.36 ; H = 3-88 per cent.), would appear to consist mainly of quercetin (C = 59-60; H = 3-31 per cent.), the second component readily susceptible to oxidation having been to a large extent eliminated or converted into soluble products by the action of boiling baryta water in the presence of air,

The author is indebted to the Research Fund Committee of the Chemical Society for a grant which has been in part employed to defray the expenses of this investigation. CLOTHWORKERS'RESEARCH LABORATORY,

Published on 01 January 1914. Downloaded by University of California - San Diego 16/12/2016 23:13:26. THE UNIVERSITY,LEEDS.

VOL. cv. 42