Komatsu, Shigeru

Nodzu, Ryuzaburo

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RIGHT:URL:CITATION:AUTHOR(S):ISSUE DATE:TITLE:On the Chemistry of JapanesePlants, VII : The PhytochemicalStudy of the Yamamomo-Fruit(Myrica rubra S. and Z.)Komatsu, Shigeru; Nodzu, RyuzaburoKomatsu, Shigeru ...[et al]. On the Chemistry of Japanese Plants, VII : The PhytochemicalStudy of the Yamamomo-Fruit (Myrica rubra S. and Z.). Memoirs of the College of Science,Kyoto Imperial University. Series A 1925, 8(4): 223-2291925-05-10http://hdl.handle.net/2433/256712On the Chemistry of Japanese Plants, VI I. The Phytochemical Study of the Yamamomo-F ru it (Azyrica rubra S and Z) By Shigeru Komatsu and Ryuzaburo Nodzu. (Received February 5, 1925) The plant pigments of red, blue or violet colour which occur in flowers, fruits or leaves, were regarded by chemists to have an intimate connection, from the chemical and physiological points of view, with the flavons of the yellow colour found in plant tissues. The extensive research by A. E. Everest1 on the isolation of the glucoside of myricetin and violanin-a glucoside of delphinidin-in the same petals of purple black violas, was highly esteemed in these respects, with that by R. Willstitttcr and H. Mallison2 on the conversion of quercetin into cyanidin chloride by chemical means. The present phytochemical study of the Yamamomo-fruit was under-taken to afford some strong evidence for the feeling of investigators thal: the anthocyans are directly derived in plant tissues from the flavons. The Yamamomo-tree grown in the southern parts of Japan, was favoured in those districts as its bark has afforded a raw material. for a mordant dye3 due to the presence of myricetin, and also its beautiful reddish or white juicy fruits gave a fresh sour taste. Hitherto nothing was known of the chemical nature of these red fruits, or of the substances to which their beauty and taste were due. Myricetin in the Barks of Yamamomo-Tree. 800 grm. of the air-dried bark were extracted with boiling water, and crude myricitrin was obtained on cooling the hot extract, which was I Proc. Roy. Soc., [BJ; 90, 251 (1918). 2 Sitzber. d. Kg!. Preuss. Akad, d. Wiss., 769 (1914). 3 K. Shirai: The plants for dyestuff (in Japanese), p. 89 (1918). 224 Shticru Komatsu and Ryuzaburo Nod:m. purified by recrystalJization from dilute alcohol. The yield was 80 grm. It melts at 199°, and gave on analysis: C = 5 1 ·02 ; H = 4•92; theory requires C= 50-40; H=4•80 for C21H20O13,2H2O. 5 gnn. of the substance by hydrolysis with 0-25 per cent. sulphuric acid, yielded 3•1 grm. of myricetin and rhamnose M. p. 180-181° (its phenyl osazone M. p. 95-97°), and on analysis the former gave C=53-60; H=3-72; theory requires C=53-56; H=3·6o for C15H 10O3,Hp. The chemical properties of our myricetin and its rhamnoside agree with those of substances mentioned previously by A. G. Perkin1 and S. Sato2• Anthocyan in the Fruits. The fruits used in this investigation, were collected 111 quantity (23 Kilo) at the mature period, 7th of July 1924. from the trees grown at Nakabatake, Hyogoken, from which the raw material of myricetin was obtained, through the courtesy of Prof. T. Tanaka of the College of Agriculture, Kyushu Imp. University. The edible part of the fruit separated by hand from the stones, had the juice pressed out, and 2 Kilo. of the mare, thus obtained, were treated with 4 litres of 2 per cent. methyl alcohol hydrochloric acid for 3 hours to extract the pigment according tc) the method used by Willstiitter3 in extracting the petals' pigments, the solution was filtered and the mare washed with the acidified alcohol. The filtrate combined together with the washings, and 5 litres of combined bluish-red coloured solution, mixed with I 5 litres of ether and the mixture was allowed to stand over night. The dark red aqueous layer was deposited on the bottom of the vessel, separated from the upper one, and concentrated to a thick syrup in a vacuum over sulphuric acid. The syrup thus obtained was dissolved in methyl alcohol and again precipitated with ether. The precipitate was dissolved completely in methyl alcohol and half of its volume of IO per cent. hydrochloric acid was added to the solution whereby the pigment separated slowly in the form of brownish-red granular p:uticles which separated from the solution and dried in a vacuum over sulphuric acid and potassium hydroxide. It weighed 1 • 1 grm. The crude pigment thm obtained could not be extracted completely with amylalcohol fro:n a 0-5 per cent. hydrochloric acid solution, indicating the presence of some diglucoside4. The purplish-red amyblcohol solution was treated repeatedly with 0· 5 per cent. 1 J. Chem. Soc., 81, 204 (1902). 2 Kogiokagakuzasshi, 17, 23 (1914). 3 Lieb. Ann., 412, 178 (1916). 4 R. Willstatter u. E. H. Zollinger: Lieb. Ann., 412, 200 (1917). On tlte Clze111istrJ1 of ]<1p,111ese Plants, VII. 225 hydrochloric acid, whereby a large part of the colouring matter was passed into the dilute acid solution, but it was impossible to remove all the pigment from the amylalcohol solution, since, as stated by Willstatter1, the free anthocyanidins remain in the amylacohol in this treatment. Thus, our pigment was regarded as being constituted mainly of monoglucoside accompanied by a small amount of diglucoside and free anthocyanidin. This crude pigment gave a dull red solution with water which readily decolourised. It dissolved in very dilute hydrochloric acid, and 7 per cent. sulphuric acid, and on adding water or an alkali solution to the acid solution the colour changes from clear brownish-red to blue. It also was easily soluble in methyl and ethyl alcohols giving a beautiful bluish-red solution. When ferric chloride was added to the alcoholic solution, a fine blue colour was produced, which on the addition of water changed to violet. Without further purification of this pigment, it was hydrolysed. It was dissolved in 0-05 per cent. hot hydrochloric acid, the insoluble matter formed filtered off, and concentrated hydrochloric acid was added, and then the mixture was boiled for 2} minutes, whereby the antho-cyanidin chloride crystallized. After standing the acid solution in an ice box over night, the crystals were filtered, washed with cold 20 per cent. hydrochloric acid and dried in vacuo over sulphuric acid and potassium hydroxide. The yield was O· 1 gnn. from o• 5 grm. of the crude sample. It was dissolved in a small volume of alcohol and half of its volume of 10 per cent. hydrochloric acid was added. On allowing the alcohol to evaporate, the anthocyanidin chloride separated out as crystalline aggregates, filtered and washed with 20 per cent. hydrochloric acid and dried on a porcelain tile. The anthocyanidin chloride thus obtained was dark red in colour, having a green reflex. When it was treated with a little water, it gave a dull brownish-red solution. It was fairly soluble in dilute hydrochloric acid (0-5-1 per cent.) and cold 7 per cent. sulphuric acid, giving a brownish-reel solution which turned to a blue colour by adding water, sodium hydroxide or sodium carbonate, and with lead or copper acetate it gave a. fine blue or a reddish violet precipitate. In methyl and ethyl alcohols it dissolved easily with a bright bluish-red colour. On addition of ferric chloride to the alcoholic solution, it gave a fine intense blue colouration which, diluted with water, changed to a violet. It was confirmed by the Zeisel method that the methoxyl group was present in the pigment, accordingly it was demethylated by means of I R. Willstatter u. E. H. Zollinger: Lieb. Ann., 412, 200 ( 1917 ). Shigeru Komatsu and Ryuz,1l>11ro Nodzu. hydroiodic acid, and anthocyanidin iodide was obtained in dark red crystals, which were converted into the chloride. The demethylated anthocyanidin chloride thus obtained was soluble in methyl and ethyl alcohols, water and dilute hydrochloric acid, and sparingly in 7 per cent. sulphuric acid. The alcoholic solution with ferric chlorid.:: and the acidic solution with sodium carbonate gave equally a blue colouration. The solubilities and colour reactions above-cited are similar to those of dclphinidin chloride described by Willstiitter and Mieg1• For the identification, the absorption spectrum of 00800 grm. sub-stance in I litre ethyl alcohol ( r mol. in 4,687 litres as with delphinidin chloride: C1,HuCl-2H20.) was examined with the following results: Column 2·5 mm .... 532 " 5·0 ,, 526 ,, IO·O ,, 500 ,, 15-0 " 495 The results, showing the characteristic b:ind in the region of the green spectrum, agree fairly well with those of dclphinidin chloride.2 Although the analytical data of the pure sample were lacking, due to the small quantity of the material, it must be admitted that the dcmethylated anthocyanidin chlorid..: is no doubt delphinidin chloride, and accordingly that a methyl ether of clelphinidin occurs in the pigment isolated from the fruit. The sugar solution obtained as one of the cleavage products from the anthocyan by hydrolysis was concentrated to 25 c.c., aftcr clarified with lead carbonate, and its optical rotatory and reducing powers were determined with the following results: a =0-80° in a IO cm. tube, whence 0-38 grm. cl-glucose in the solution which fairly agree with the results calculated from the reducing power of the Fehling's solution (0•39 grm. of the reducing sugar calculated as cl-glucose). Phenylglucosazone M. p. 204-205° was isolated in a pure state from the above solution. It wa3 concluded from these experimental results that the sugar combined with the anthocyanidin was cl-glucose. When the rhamnoside of myricetin which occurs in the bark of the Yamamomo-tree, transported to the sap of the fruit, so far as we have studied, where the conversion of methyl pento:.;c into d-glucose-hexose-on the one side, and on the other, the methylation of delphinidin which was regarded as resulting from the reduction of myricetin, took place. 1 Lieb. Ann., 408, 125 (1915). 2 ]bid., 75. On the Chemistry of Japanese Plants, VII. n7 The Sugars in the Fruit. The chemical study of the sugars in the fruit, therefore, attracted attention and 13•5 litres of the pressed juice separated from the mare were used for this purpose and also for the isolation of some acids. The juice after being clarified with basic lead acetate contains 7·7 per cent. of the reducing sugar as cl-glucose. Cane sugar, pentose or methyl pentose, could not be proved by the inversion or phloroglucinol test to be present in the sugar solution before and after fermentation with yeast. The reducing power of Fehling's solution and the measurement of the optical rotatory power indicated the glucose-fructose ratio in the juice as I : 1•25. cl-Glucose was actually isolated from the clarified solution by the usual method, using glacial acetic acid as solvent, and from the mother liquor separated from the crystals of d-gluc03e, cl-fructose was also obtained in a pure state ; both sugars were confirmed by the determina-tion of the melting point and the mutarotation of the aqueous solution : d-gluc03e M. p. 145-146.0 cl-fructose M. p. 98-104.0 [a JH = + 5·01 x 100 + 100-6° after 1 5 min. - 2,4888 X 2 + 2·61 X 100 r a]11 _ ----~= + 52-4° constant. L - D - 2•4888 X 2 [,.,]01t __ -8,31 X 100 o "" -~~---- -92•2 constant. C-C)9 X l Non-Volatile Acids in the Fruit. The fruit affords a refreshing and delicious flavour which is owing to the harmony of the organic acids and sugars present in it. What organic acids occur in this fruit which ripens in the hot season in the southern parts of Japan, was a very interesting question from the view point of p'.ant physiology, since organic acids were mually recognized by chemists as formed by the metabolism of the sugars in the plant tissues. The pres~ed juice had sp. gr. 1 -043, and acidity O· 1 3 N. titrated with clecinormil solution of sodium hydroxide, using the anthocyan in the juice as an indicator, and one part of which was used previously for the isolation of the sugars, and to our intere3t no volatile free acid could be found in it. Citric Acid. 5 litres of the juice were treated with an exce3s of basic lead acetate, the precipitate (A) there formed was separated from the solution and Shigcru J{omntsu mzd R)'u:m!mro l\'o-1:w. washed with water. The precipitate (A) suspencled in water was then ckcomposed with hydrogen sulphiclc gas, and the acid solution, separated from the lead sulphide, w;is concentrated into a small volume under reduced pressure, this containecl 65 grm. of acid calculated to be citric acid, and was optically inactive and did not clecolourize bromine water. On standing the concentrated solution, citric acid was deposited in fine crystals which melted at r 5 2.0 On analysis of the anhydrous substance, it gave C=37-55; H=4·42; while theory requires C=37-49; H=4-r9 for C6H80 7• Oxalic Acid. The mother liquor separated from the crystals of citric acid, was converted into calcium salts and concentrated to a small volume whereby precipitate (B) consisting of calcium citrate together with a small quantity of calcium mnlate was separated, in which no tartaric acid1 could be found. The ox1.late was confirmed by analysis of the purified sample ; Ca0=37,85, while theory requires Ca0=38-38 for C,P~Ca-Hp. Calcium citrate was also confirmed by the well-known colour reaction with vaniline and sulphuric acid. Malic Acid. The filtrate sqx:trated from the precipitate (B) after confirming that no succinic acid occurs in it, was acidified with cone, sulphuric acid, treated with ether to extract malic acid, if present, and the ethereal solution was evaporated to a small volume after neutralizing with potassium hydroxide (indicating o• 1 24 grm. m:1lic acid present), whereby potas:-;ium malate was' sep:1ratcd, which was confirmed by the method of L. Rosenthaler. ~ Lactic Acid. 1 I Litres of the juice were neutralized with barium hydroxide, the barium citrate separated, filtrated off and the filtrate was concentrated under reducecl pressure to small volume, acidified with sulphuric acid and the lactic acid was extracted with ether. The acid was converted into the zinc salt and analysed : ZnO = 27 • 1 5 ; water of crystallization= r 8, 34 ; theory requires Zn0=27-27; HtO= 18,18 for (C,1H,,0,1t Zn,3Hp. The analytical 1-..:sults and the optic:11 measurement of the salt a::;rec well with those of inactive lactic acid, I II. Schmalfuss u. K. Keitel: Zs. physiol. Chem., 138, 156 (1924). 2 Chem. Centrall,l., IT, 754 (1912). On the Chemistry ef Japanese Plants, VII. 229 Summary. I. Myricitrin was isolated from the bJ.rk of the Yamamomo-tree, and was confirmed to b~ a rhamnoside of myricetin. 2. The red pigment of the fruit is composed mainly of monoglucoside accompanied by a small quantity of the diglucoside of the anthocyanidin and some free pigment. 3. The demethylated anthocyanidin by Zeisel's method was ascertained to be dclphinidin by the colour reactions and spectroscopic method. 4. The sugar group associated with the anthocyanidin was d-glucose. 5. The sugars in the fruit were composed of d-glucose and cl-fructose with ratio of I: 1-25. 6. The non-vo'.atile free acids in the fruit consist principally of citric acid with a small qu:mtity of malic, oxalic and inactive lactic acids. Dec. 1924. Laboratory of Organic- and Bio-Chemistry. 

On the Chemistry of Japanese Plants, VII : The Phytochemical Study of the Yamamomo-Fruit (Myrica rubra S. and Z.)

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On the Chemistry of Japanese Plants, VII : The Phytochemical Study of the Yamamomo-Fruit (Myrica rubra S. and Z.)

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