A CONTRIBUTION TO THE CHEMISTRY OF GRAPE

PIGMENTS.

II. CONCERNING THE ANTHOCYANS IN CLINTON GRAPES.

BY R. J. ANDERSON AND FRED P. NABENHAUER.

(From the Biochemical Laboratory, New York Agricultural Experiment Station, Geneva.)

(Received for publication, June 9, 1924.)

INTRODUCTION.

It has been shown in an earlier paper from this laboratory’ that the pigments occurring in two varieties of American grapes, viz. Norton (Vitis astivalis, labruscu) and Concord (Vitis labruscu), were identical and that they had the same chemical composition as the monoglucoside oenin isolated from Vitis vinz’jera by Will- st8tter and Zollinger.2 In continuation of the investigation of grape pigments we have examined the coloring matter that occurs in Clinton grapes. Clinton3 is said to be the first culti- vated variety of Vitis riparia, but it is believed that it also con- tains some blood of Vitis labrusca.

The pigment occurring in Clinton grapes was found to be identical with the anthocyanins isolated from Norton and Con- cord grapes and it was apparently identical with the anthocyanin isolated from Vitis riparia by WillstSitter and Zollinger.4 The anthocyanin consists principally of a monoglucoside. It was isolated as the picrate and the latter was converted into the chloride. The anthocyanin chloride could not be obtained in definitely crystalline form. It was precipitated by ether from its solution in methyl alcohol as amorphous flakes and it separated

1 Anderson, R. J., J. BioZ. Chem., 1923, Ivii, 795. * Willstltter, R., and Zollinger, E. H., Ann. Chem., 1915, cdviii, 83. 3 Hedrick, U. P., Grapes of New Tork, N. Y. Agric. Exp. Station, 1908. 4 Willstiitter, R., and Zollinger, E. H., Ann. Chem., 1916, cdxii, 195.

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98 Chemistry of Grape Pigments. II

slowly from a solution in a mixture of methyl and ethyl alcohol and dilute hydrochloric acid in the form of small globular particles. The anthocyanin picrate crystallized in beautiful bright red prisms and this salt was prepared and analyzed. It corresponds to the formula C23H,401,*CeH2(NOz)30H.

The anthocyanin from Clinton grapes has the same percenmgc composition as oenin, derived from Vz’tis vinifera, but it differs from this substance in that it does not form a crystalline chloride and also in that it gives in alcoholic solution an intense purple coloration with ferric chloride. The color reaction with ferric chloride is identical with that reported in our first paper’ and similar to that described by Willstatter and Zollingei4 for the pigment isolated from Vitis riparia.

The absorption spectrum is practically identical with that described in our first paper.’ It consists of one broad band extending from the yellow into the blue.

The anthocyanin chloride is easily hydrolyzed when boiled with 20 per cent hydrochloric acid, forming 1 molecule each of glucose and anthocyanidin chloride, the latter crystallizing from the hot solution in beautiful prisms.

The anthocysnidin chloride has the same percentage composi- tion as oenidin chloride, but it differs from this substance in that it contains a lower percentage of methoxyl. The amount of silver iodide obtained in the Zeisel determination corresponded more nearly to one methoxyl, but the values obtained were always high. It is probable that the substance is a mixture of mono- and di- methyl ethers of delphinidin. The reaction mixture from the Zeisel determination deposited, after the solution had cooled, small red prismatic crystals of delphinidin iodide. The delphinidin iodide was transformed into the chloride by the method described by Willst&tter and ZollingeP and this salt was analyzed.

We were able to isolate and identify phloroglucin as one of the decomposition products formed on fusing anthocyanidin chloride with potassium hydroxide, but we were unable to isolate any gallic acid. A derivative of the gallic acid part of the molecule was obtained by another method. The anthocyanidin chloride was boiIed with acetic anhydride and the resulting acetyl derivative was oxidized with neutral permanganate. The reaction mixture gave on extraction with ether an acetylated methyl ether of gallic

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R. J. Anderson and F. P. Nabenhauer 99

acid that contained nearly the same proportion of methoxyl as the anthocyanidin chloride itself. The amount of the gallie acid derivative was too small to permit of its identification, but the results indicate that the methyl groups arc attached to the g&c acid part of the molecule and not to the phloroglucin.

EXPERIMENTAL PART.

The skins were separated from the pulp by hand and pressed in a hydraulic press. The pressed skins, 13 kilos, were digested for about 20 hours in 13 liters of 0.5 per cent hydrochloric acid and they were then rubbed to a pulp in a mortar. The liquid was expressed in a hydraulic press and the residue was stirred up with about one-half of its weight of 0.5 per cent hydrochloric acid and again pressed. The solution thus obtained was filtered through a layer of paper pulp yielding an intensely deep red and perfectly clear filtrate that measured 22 liters. It was warmed on the water bath to about 45” and 225 gm. of finely powdered picric acid were added and the mixture was stirred until the picric acid was dissolved. The picrate separated slowly, after the solution had cooled, in bright red needle-shaped crystals. The crystals were colIected on a Biichner funnel and dried in a vacuum desic- cator over sulfuric acid and potassium hydroxide. The crude picrate obtained in this way weighed 64 gm.

The picrate was recrystallized from water and analyzed.

Analyses. 0.1912 gm. dry substance: 0.0689 gm. Hz0 and 0.3373 gm. coz.

Calculated anthocyanin picrate, C~OH~,O~QN~ (721). C 48.26, H 3.74 per cent.

Found. C 48.11, H 4.03 per cent.

The product lost 5.49 per cent of its weight on drying at 105” in a vacuum over phosphorus pentoxide, corresponding to slightly more than 2 molecules of water of crystallization.

Calculated for C&HlrOlpNS f 2 Hz0 (757). Hz0 4.76 per cent.

Conversion of Anthocyanin Picrate to the Chloride.

The picrate was converted to the chloride according to the method of WillstSitter and Zollinger. The crude picrate was dis- solved in 575 cc. of methyl alcohol, and 100 cc. of 20 per cent

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100 Chemistry of Grape Pigments. II

hydrochloric acid in methyl alcohol were added. The antho- cyanin chloride was precipitated by the addition of 5,200 cc. of &her. It was filtered and washed with ether until free from pic- ric acid. After drying in a vacuum desiccator it weighed 25 gm.

The anthocyanin chloride prepared in this way is a brownish red powder, easily soluble in water giving a brownish red solution which becomes bright red on addition of a.cid. The aqueous solu- tion of the pigment gives with a dilute solution of sodium acetate a purple color; carbonates produce a deep blue color, whereas sodium hydroxide gives a bluish green color which soon changes to brownish yellow. Alkali destroys the pigment since the red co101 cannot be restored by the addition of acid. The pigment, is com- pletely precipitated by lead acetate forming a deep blue amorphous precipitate. An aqueous solution of the pigment gives with 1 drop of very dilute ferric chloride a purple coloration which passes rapidly through wine-red to brown. In alcoholic solution a fairly permanent, violet coloration is produced with 1 drop of ferric chloride and a deep blue color is formed with 3 or 4 drops of the reagent.

Attqmpts to crystallize the glucoside by the method of Will- st&ter and Zollinger were unsuccessful. At first, a few hexngonal- shaped crystals separated from the solution, but for the most, part the substance separated, after the solution had stood for several days, in spherical granules. A sample of this precipitate was analyzed after it, had been dried at 105” in a vacuum over phos- phorus pentoxide. The loss in weight on drying was 5.98 pm cent,, corresponding nearly to 2 molecules of water of crystalliza- tion. Calculated for 2 HzO: Hz0 6.37 per cent.

Analyses. 0.2039 gm. substance: 0.0890 gm. H20 and 0.3945 gm. CO,. 0.2906 and 0.3142 gm. substance: 0.0941 and 0.1032 gm. AgCl.

Calculated for anthocyanin chloride, C&H?a01&1 (528.5). C 52.22, H 4.73, Cl 6.71 per cent.

Found. C 52.75, H 4.89, Cl 8.00, 8.11 per cent.

The percentages of carbon and chlorine were too high and the fact that, we found in the quantitative hydrolysis values too high for anthocyanidin chloride and too low for glucose makes it very probable that partial hydrolysis had occurred during the long con- tact, with the acidified alcohol.

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R. J. Anderson and F. P. Nabenhauer 101

Quantitative Hydrolysis of the Glucoside.

The substance used in this determination had been purified as. follows: The crude anthocyanin chloride was dissolved in water, acidified with a few drops of dilute hydrochloric acid, and precipi- tated by adding a warm saturated solution of picric acid. The picrate was filtered and recrystallized from water. The substance was filtered, dried, dissolved in methyl alcohol containing dry hydrochloric acid, and precipitated with ether. The amorphous- substance was filtered, washed free from picric acid with ether,, and dried in a vacuum desiccator over sulfuric acid. This amor- phous anthocyanin chloride, 0.4774 gm., was dissolved in 12 cc. of water, 13 cc. of concentrated hydrochloric acid were added, and the solution was boiled for 4 minutes. The crystals of antho- cyanidin chloride were filtered after the solution had cooled, washed with dilute hydrochloric acid, and allowed to dry in the air. The yield was 0.3275 gm. The small amount of pigment that re- mained dissolved in the filtrate was extracted with 3 portions of amyl alcohol. This extract was diluted to 250 cc. and compared in a calorimeter with a standard solution. It was found to con- tain 0.0183 gm. of pigment. The total amount of anthocyanidin chloride obtained, accordingly, weighed 0.3458 gm. The theo- retical amount of anthocyanidin chloride, calculated for C1,H,,O,CI + 1.5 HZO, is 0.3554 gm. The amount of pigment recovered is, therefore, 97.3 per cent.

The aqueous solution, mentioned above, that had been cx- tracted with amyl alcohol was used for the determination of glu- cose after first extracting the amyl alcohol with ether. In all these operations the extracting solvent was washed with water to minimize loss of glucose. The clear solution was neutralized with sodium hydroxide and boiled with GO cc. of Fehling’s solution. The cuprous oxide weighed 0.2859 gm. which is equivalent to 0.1314 gm. of glucose. This is 82 per cent of the theoretical amount, 0.1626 gm.

Identification of the Glucose.

The filtrate obtained after hydrolyzing 6 gm. of anthocyanin chloride was extracted with amyl alcohol until all the color was

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102 Chemistry of Grape Pigments. II

removed. The solution was treated with an excess of lead car- bonate and the lead salts were filtered off. The filtrate was treated with hydrogen sulfide to remove excess of lead and the lead sulfide was filtered off. The solution was neutralized with sodium hydroxide and concentrated under reduced pressure to about 30 cc. It was of a pale yellow color.

In a 1 dm. tube the angle of rotation was f2.76”. 3 cc. of the solution gave 0.3702 gm. of cuprous oxide on boiling with Feh- ling’s solution which is equivalent to 0.1724 gm. of glucose. Hence, [a]: = +48 .O”. This corresponds quite closely to the specific optical rotation of glucose which is +52.8’.

The balance of the solution, 6.4 cc., was diluted to 30 cc. with water and heated in a beaker of boiling water. To the hot solu- tion were added 3.5 cc. of an aqueous solution containing 0.67 gm. each of phenylhydrazine and glacial acetic acid. Crystals of the osazone began to separate after 11 minutes. These crystals had the characteristic appearance of phenylglucosazone and after the substance had been recrystallized from dilute alcohol it melted with decomposition at 206’ (uncorrected). The time of osazone formation and the melting point correspond closely to that of phenylglucosazone.

Preparation and Properties of Antho yanidin Chloride.

The sugar-free pigment is easily formed when the glucoside is boiled with about 20 per cent hydrochloric acid. The glucoside, 3 gm., was dissolved in 37 cc. of water and the solution was filtered. After adding 38 cc. of concentrated hydrochloric acid the solution was boiled for 4 minutes. The anthocyanidin chloride began to crystallize after the solution had been boiled about 2 minutes. After the solution had cooled the substance was filtered, washed with dilute hydrochloric acid, and dried in the air. The yield was about 1.2 gm.

In crystal form and in color the substance cannot be differen- tiated from the pigment isolated from Norton and Concord grapes as described in the first paper.’ The crystals dissolve in water giving a dull brownish red solution which becomes bright red when acidified and blue when made alkaline. An aqueous solution of the pigment is decolorized immediately by ferric chloride, but when

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R. J. Anderson and F. P. Nabenhauer 103

dissolved in ethyl alcohol the addition of a small quantity of ferric chloride produces a momentary purple color which changes quickly to blue, but in the course of a few hours the blue color fades leaving a nearly colorless solution. In this color reaction with ferric chloride the substance differs from the anthocyanidim from Norton and Concord grapes and the reaction is probably due to a difference in position of the methoxyl groups in the gallic acid part of the molecule.

The substance was dried for analysis at 105” in a vacuum over phosphorus pentoxide. The loss in weight corresponds to 1.5 molecules of water of crystallization.

Analyses. 0.1832 and 0.1845 gm. substance lost on drying 0.0131 and 0.0139 gm. 0.1701 and 0.1706 gm. substance: 0.0592 and 0.0603 gm. Hz0 and 0.3437 and 0.3463 gm. CO,. 0.2126 gm. substauce: 0.0799 gm. AgCl.

Calculated for C1,HljO#l (366.5). C 55.66, H 4.09, Cl 9.68 per cent. Found. C 55.11, 55.36; H 3.59, 3.95; Cl 9.29 per cent. Cnlculatcd for 1.5 HzO. Hz0 6.86 per cent. Found. H?O 7.15, 7.53 per cent.

These analyses correspond quite closely to the composition of a dimethyl ether of delphinidin, but it was found by the Zeisel method that there was present only slightly more than one methoxyl group.

Analyses. 0.2185, 0.1928, and 0.6140 gm. substance: 0.1591, 0.1455, and 0.4293 gm. AgI.

Calculated for 2 CH,O. CHIO 16.91 per cent. CL “1 “ “ 8.45 “ “

Found. CHaO 9.62, 9.97, 9.23 per cent.

Delphinidin Iodide and Chloride.

When the contents of the flasks used in the methoxyl determina- tions were allowed to cool, delphinidin iodide separated as shining prisms or whetstone-shaped crystals. The crystals were collected on a Riichner funnel, washed with ether, and allowed to dry in the air. The substance had a beautiful bronze luster. The iodide was converted to the chloride by shaking with silver chloride and hydrochloric acid in methyl alcohol solution as described by Will- stgtter and Zollinger.2 The residues from the three deter- minations yielded slightly more than 1 gm. of the iodide from which 0.53 gm. of delphinidin chloride was obtained.

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Chemistry of Grape Pigments. II

The delphinidin chloride dissolved easily in alcohol giving a beautiful red solution. When the alcoholic solution was diluted with water and warmed on the water bath the color gradually faded leaving finally a colorless solution. Evaporation of a small amount of this solution on a watch-glass left fine needles of the colorless base.

The delphinidin chloride was analyzed after it had been dried in a vacuum over phosphorus pentoxide at 105’. The loss in weight corresponded to 1.5 molecules of water of crystallization.

Analyses. 0.1985 and 0.1633 gm. substance lost on drying 0.0150 and 0.0124 gm. 0.1509 and 0.1469 gm. substance: 0.0479 and 0.0445 gm. Hz0 and 0.2948 and 0.2897 gm. CO*. 0.1827 gm. substance: 0.0770 gm. AgCI.

Calculated for Cts HI1 0, Cl (338.5). C 53.17, H 3.24, Cl 10.48 per cent. Found. C 53.28, 53.78;,H 3.55, 3.39; Cl 10.42 per cent. Calculated for 1.5 H?O. Hz0 7.38 per cent. Found. Hz0 7.55, 7.59 per cent.

Formation of Phloroglucin by Alkali Fusion.

1 gm. of anthocyanidin chloride was decomposed by heating with a solution of 10 gm. of potassium hydroxide in 3.5 cc. of water in a tube through which hydrogen was passed. The tube was heated in an oil bath and the pigment was added when the temperature had reached 160”. The heating was continued for 10 minutes during which time the temperature had risen to 200’. The color of the melt changed from brown to nearly black.

When the mixture had cooled it was dissolved in 100 cc. of 12.5 per cent hydrochloric acid. The solution was made slightly alkaline with sodium bicarbonate and the phenolic constituents were extracted with ether. The ether was evaporated and the residue was dissolved in water, decolorized with norit, filtered, and the solution was concentrated in a vacuum desiccator over sulfuric acid. Nearly colorless crystals of phloroglucin separated which, after being filtered and dried, melted between 210’ and 212”. It gave all the reactions noted in a previous paper.’

The bicarbonate solution was acidified and extracted with ether. On evaporation of the ether a dark colored residue remained from which no crystalline material could be obtained. It gave a dirty blue-black precipitate with ferric chloride.

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R. J. Anderson and F. P. Nabenhauer 105

Acetylation of Anthocyanidin Chloride and Oxidation.

2 gm. of anthocyanidin chloride were boiled for 4 hours with 25 cc. of acetic anhydride. The color changed to brown and a dark colored amorphous precipitate formed. The acetic anhydride was distilled off under reduced pressure and the residue was dissolved in 100 cc. of alcohol. The acetyl derivative was precipitated as a flocculent amorphous substance by the addition of 350 cc. of water. It could not be obtained in crystalline form from any of the usua1 solvents. The dried substance melted unsharply between 205O and 210”.

The crude substance was oxidized directly with neutral per- manganate. It was suspended in water and to it were added 10 gm. each of potassium permanganate and magnesium sulfate. The mixture was stirred at room temperature for several hours until the color of the permanganate had practically disappeared. The mixture was then acidified, treated with sodium bisulfite until colorless, filtered, and extracted with ether. The ether was evap- orated and the residue was dissolved in water and decolorized with norit. The solution was concentrated in a vacuum desiccator over sulfuric acid. The residue consisted of nearly colorless crys- tals mixed with some amorphous sticky substance. It was possi- ble to separate this material by extracting it with cold benzene which dissolved only the crystals. The benzene was evaporated and the residue was recrystallized from water. The white granu- lar substance weighed 0.2 gm. It melted between 144’ to 146“’ (uncorrected).

The aqueous solution of the substance showed an acid reaction to litmus. This solution gave no coloration with ferric chloride. After the substance had been hydrolyzed by boiling with concen- trated hydrochloric acid and the acid had been evaporated the aqueous solution gave with dilute ferric chloride a blue-black coloration similar to that given by gallic acid itself.

For analysis the substance was dried at 105’ in a vacuum over phosphorus pentoxide, but it did not lose in weight.

Analysis. 0.1092 gm. substance: 0.0459 gm. Hz0 and 0.2191 gm. COz. Found. C 54.72, H 4.70 per cent.

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106 Chemistry of Grape Pigments. II

It will be noticed that these values are intermediate between the calculated composition of a dimethyl ether of monoacetyl- gallic acid, C,lH1,Os (240), C 55 .OO, H 5.00 per cent, and a mono- methyl ether of diacetylgallic acid, * C,,H,,O, (268), C 53.73, H 4.47 per cent.

In the Zeiscl determination an intermediate value was also obtained as is indicated in the figures given below.

Analysis. 0.0875 gm. substance: 0.1108 gm. AgI. Found. OCHI 16.73 per cent. Calculated for 2 OCHI. O.CHI 25.83 per cent.

‘I “1 “ “ 11.56 “ “

The analyses indicate that the substance is a mixture consisting approximately of 40 per cent of a dimethyl ether of monoacetyl- gallic acid and 60 per cent of a monomethyl ether of diacetylgallic acid.

Identification of Gallic Acid.

The residue from the above mentioned Zcisel determination was diluted with water and treated with sodium bisulfite to remove any free iodine and it was then extracted with ether. The ether was evaporated and the residue was dissolved in water, decolorized with norit, and the solution was concentrated. Fine needles of gallic acid separated when the solution had cooled. It was recrystallized from water, filtered, and dried. The substance melted with decomposition at 243”. Some carefully recrystallized gallic acid melted with decomposition at the same temperature and in appearance both substances were identical. With ferric chloride the substance gave a blue coloration and later a blue-black precipitate formed. On adding a solution of potassium cyanide a pink color was obtained that gradually faded to yellow, but the pink color was restored when the solution was shaken. The sub- dance reduced Fehling’s solution. The melting point and the above mentioned reactions show that the substance was gallic acid.

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R. J. Anderson and F. P. Nabenhauer 107

Absorption Spectra of Anthoyanin and Antho yanidin Chloriok

The following absorption bands were observed.

Anthocyanin Chloride. 1 Molecule in 6,000 Liters.

Column.

mm.

2 3 5 7.5

10

Methyl alcohol. Ethyl alcohol.

575 555 -- . . . . . . 509 583 . 561 . . . . 577 559 . . . . 503 585 . . 566 . . 505 578 . . 562 . . 498 587 . . 571 . . 500 583 570 . . . . 488 590 . . 575 . . . . 493 587 . . 573 . . . 480 595 . . 584 . . . . 492

Anthocyanidin Chloride. 1 Molecule in 6,000 Liters.

Column.

mm.

1 2 3 5

10

Methyl alcohol. Ethyl alcohol.

575 . . 553 520 581 . . 562 . . . . 514 584 . . 567 . . . . 507 590 . . 574 . . . . 485 595 . . 577 - . . . .

581 . . 562 551 587 . . 567 . . . 527 590 . . 571 . . . . 509 594 . . 580 . . . . 494 596 . . 589 - . . . .

SUMMARY.

The pigment occurring in Clinton grapes consists principally of a monoglucoside, anthocyanin. The anthocyanin chloride, C23H25012C1, did not separate in definitely crystalline form, but the picrate, CWH24012.CRH2(N02)30H, crystallized in bright red. prisms or needles. The glucoside is easily hydrolyzed by boiling dilute hydrochloric acid yielding 1 molecule each of glucose and anthocyanidin chloride, C1,H,~07C1, the latter crystallizing from the hot solution in prisms.

The anthocyanidin chloride consists largely of a monomethyl ether of delphinidin, but the values for methoxyl were too high, indicating that it contained some dimethyl ether of delphinidin.

The absorption spectra of anthocyanin chloride and anthocyani- din chloride consist of one broad band extending from the yellow into the blue.

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R. J. Anderson and Fred P. NabenhauerCLINTON GRAPES

CONCERNING THE ANTHOCYANS IN II.CHEMISTRY OF GRAPE PIGMENTS:

A CONTRIBUTION TO THE

1924, 61:97-107.J. Biol. Chem. 

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