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The brown pigments of autolyzed tobacco leaves
A R C H I V E S OF
BIOCHEMISTRY AND BIOPHYSICS
93,580-590 (1961)
The Brown Pigments of Autolyzed Tobacco Leaves I. Isolation and Characterization JAY S. JACOBSON 1 From the Department el Botany, Columbia University, New York, New York
Received January 9, 1961 The isolation and partial purification of the brown pigments of autolyzed shadegrown tobacco leaves have been aecomplished. The solubilities of these substances, their ultraviolet and infrared absorption spectra, and their elementary composition are reported. Some of the products of chemical hydrolysis, as identified by paper chromatography, were found to be identical to the products of hydrolysis of chlorogenie acid and rutin. A large variety of amino acids was also found after hydrolysis. The results of these experiments indicate that the brown pigments may consist of a combination of polyphenols with protein.
INTRODUCTION The browning of tobacco leaves under autolytic conditions (i.e., during curing) is a matter of considerable biochemical and economic interest. Only in recent years, however, have the underlying questions begun to receive the attention that their importance merits. The foundation of a systematic investigation of browning reactions in tobacco leaves was laid by Carl Neuberg. With Kobel (1) he found that tobacco contains an o-diphenolic glycoside, rutin, in appreciable amount. Further, by the use of model experiments he reported t h a t rutin m a y be oxidized with a mixture of a crude enzyme preparation obtained from fresh tobacco and hydrogen peroxide to yield brown pigments in vitro. The suggestion was made that turin m a y be an important source of brown pigmentation in autolyzing tobacco leaves. Koenig and D f r r (2) isolated a second o-diphenol from tobacco leaves, namely, Cigar Manufaeturers Association Fellow, 195559. Present address: Boyce Thompson Institute for Plant :Research, 1086 North Broadway, Yonkers, N. Y.
the depside ehlorogenic acid. Dawson and Wada (3) observed that chlorogenic acid occurs even more abundantly in cigar tobacco types than does rutin. Further, they reported that a crude enzyme preparation of fresh tobacco leaves will oxidize chlorogenie acid to brown pigments using molecular oxygen and without the requirement for hydrogen peroxide. Rutin alone was not oxidized by this enzyme. Model experiments indicate that browning reactions involve an oxidative step. Since autolysis generally is regarded as a process of extensive hydrolysis and oxidation of cellular constituents, it is reasonable to assume t h a t browning reactions in rive may also be oxidative. The enzymic basis of browning is supported by the observations t h a t heating Io-baceo leaves to very high temperatures will prevent the disappearance of phenols (4, 5) and the appearance of brown pigments (6-8). Further support for the hypothesis that the brown pigments are the result of the enzymic oxidation of phenols comes from comparative biochemistry. The occurrence of brown pigments from the enzymic oxidation of phenols throughout the
580
PIGMENTS OF TOBACCO LEAVES. I plant and animal kingdoms has been described in a review by Mason (9). We have a t t e m p t e d to ascertain whether or not the brown pigments of autolyzed tobaceo leaves contain detectable fragments or residues of the polyphenols chlorogenic acid and rutin. To do this it was necessary to devise procedures for the extraction, fraetionation, and purification of these pigments. Such procedures are described herein. The results of color reactions and of chemical hydrolyses have indicated t h a t the pigmerits m a y contain residues of chlorogenic acid and of rutin. Evidence is also presented which indicates t h a t the phenols are coupled with proteins to form at least two m a j o r components of the brown pigment complex of autolyzed tobacco leaves. During the course of our work, the results of Wright et al. (10) were published. T h e y found t h a t some of the brown pigments of Burley tobacco m a y be derived from protein, rutin, ehlorogenie acid, and iron. The possibility t h a t proteins might be involved in browning had been suggested earlier b y F r a n k e n b u r g (11). EXPLORATORY" WOUK The results of model experiments (unpublished data) indicated t h a t a variety of other compounds could enter into secondary reactions with ehlorogenic acid during the enzymic oxidation of the latter. Various intensities and shades of color could be obtained with a number of compounds known to be present in mature, green tobacco leaves. Much of this information has since been published by others (12-15). Our observations on the relative simplicity of model systems giving rise to brown pigments, the work on the condensation products of quinones and amines by Beevers and J a m e s (16), the work on the isolation o f ' a crystalline condensation product of quinones and amines by Jackson and Kendall (17), and the work of Hess (15) led to the assumption t h a t the products of browning reactions in tobacco leaves m a y have relatively low molecular weights. We thus a t t e m p t e d the extraction, separation, and characterization of the pigments using methods adapted for substances of lower molecular weight. Our initial efforts in this
581
direction resulted in the separation both by paper and column c h r o m a t o g r a p h y of several brown pigmented zones. One of these gave typical protein color reactions while the others gave typical phenol color reaetions. As the work progressed, it became clear t h a t the substances in question were labile and not readily purified by fraetionation with the usual organic solvents (only one fraction was soluble in solvents other than water). These and other experiences led to the realization t h a t the brown pigments were not substances of relatively low molecular weight. The appearance of the work of Wright et at. (10) on the pigments of Burley tobacco gave further indication t h a t the brown pigments of Connecticut Shade tobacco might also have high moleeular weights. As a result, the problems of extraction, separation, and characterization of the pigments were re-examined from this changed viewpoint. MATERIALS AND METHODS ISOLATION OF I)IGMEI~'TS A modification of the procedure of Wright et al. (10) gave virtually complete extraction of the brown pigments. Weighed aliquots of dried Connecticut Shade No. 49 tobacco leaf powder were extracted with absolute acetone, ether, and ethanol, in that order, in glass-stoppered Erlenmeyer flasks at room temperature with mechanical agitation. The volume of solvent used in each extraction cycle was 60 ml., and the extracts were centrifuged after overnight agitation. The particulate material was returned to the flasks, fresh solvent added, and the procedure repeated until the eentrifugate was colorless or almost so. A total of about 2 1. of the above solvents was suNcient to extract a 10-g. sample of leaf powder at room temperature. The powders were then extracted with sodium dihydrogen phosphate buffer of about 2 M concentration adjusted to pH 7 with sodium hydroxide. The extractions were carried out at room temperature in exactly the same manner as above. A few drops of chloroform and toluene were used to inhibit growth of microorganisms. A total of about 1 1. of buffer solution in 60-ml. portions was used to extract a 10-g. sample of leaf powder. Additional brown pigments were then extracted with 0.1 and 1.0 N sodium hydroxide solutions. The residue, after complete extraction, was buff-colored and appeared to have lost most of its pigmentation.
582
JACOBSON 1.25
1.00
Z IJJ
,75
_J ~[
o
,50
o. o .25
0
, 400
I 500 WAVELENGTH
,
I 600
, 700
My
FIG. 1. Reflectance spectra of disks prepared from tobacco powders at various stages of extraction. A, before extraction; B, after extraction with ether, acetone, and ethanol; C, after extraction with ether, acetone, ethanol, and water; D, after extraction with preceding soNents and final extraction with sodium hydroxide solutions. At each stage of extraction, the powders were dried in a vacuum desiccator over calcium chloride for a standard period of time and weighed, and the reflectance spectra (Fig. 1) were taken. The latter show a decrease in color intensity with each succeeding extraction. They were obtained by pressing the dry leaf powders into l-in. in diameter pellets at 9000 lb./sq, in. and taking readings in a Beckman D U spectrophotometer following the procedure of Dawson et al. (18). A pellet of magnesium carbonate was used as a standard. The p H 7 buffer extracts were concentrated to one-quarter volume in a rotating evaporator at about 35°C. using ethyl acetate to prevent foaming. This solution was then centrifuged to free it of any particulate matter, and enough solid c.F. ammonium sulfate was added to saturate the solution. After standing overnight in the cold, the solution was again centrifuged and the dark, gummy precipitate redissolved in buffer. Both this solution and the colored supernate were dialyzed separately in Visking cellophane tubing against running distilled water for 2 days with stirring. A few drops of chloroform were placed in each dialysis bag to prevent the growth of microorganisms. The dialyzed solutions were evaporated to dryness by allowing a fan to blow air over the dialysis
bags. The brown to black materials were then weighed. The 0.1 and 1.0 N sodium hydroxide extracts were pooled, and enough glacial acetic acid was added to lower the pH to about 8. The solutions were then evaporated to about one-quarter volume using a few drops of oleic acid to inhibit foaming. Fractionation was again accomplished by salt precipitation, and dialysis was carried out on a slurry of the precipitate in distilled water. CHARACTERIZATION OF PIGMENTS
Ultraviolet Spectra Ultraviolet spectra of the pigment fractions were taken in dilute sodium hydroxide, dilute hydrochloric acid, distilled water and dilute sodium hydrosulfite solution from 700 to 210 m~ where possible. A Beckman D U speetrophotometer was employed, and the solvents were used as controls. Insoluble materials were removed by centrifugation before the spectra were taken.
Infrared Spectra Infrared spectra were taken in Nujol and hexachlorobutadiene mulls in a Perkin-Elmer Spectrocord recording spectrophotometer from 2.5 to 16 ~.
P I G M E N T S OF TOBACCO LEAVES. I
Elementary Analyses Elementary analyses were obtained on the powders at different stages of extraction and on the pigment fractions. These included ash content, per cent carbon, per cent hydrogen and per cent nitrogen?
Analysis o/ Pigment Fractions for the Presence o] Low Molecular Weight Impurities I n order to find out whether small amounts of low molecular weight substances were present in the pigment fractions, 700 mg. buffer-soluble, dialyzed, a m m o n i u m sulfate precipitate was extracted with ethanol with mechanical agitation. After centrifugation, the ethanol solution was evaporated to small volume and chroma~ographed in n-butanol-acetic acid-water (12:3:5) (10). No spots were found under ultraviolet light or by the use of spray reagents for quinic acid, amino acids, and reducing sugars. The possibility t h a t small molecules could be adsorbed onto the surface of the pigments was not precluded by these findings.
Homogeneity o] Pigment Fractions The only m e t h o d for detecting separation of components in the pigment fractions t h a t was available to us was paper chromatography. Portions of the powdered pigments were triturated in a n u m b e r of different solvents and the resultant solutions chromatographed. In addition to concentrated acid, concentrated base, and sodium hydrosulfite solutions, the pigments were treated as above with ethylenediaminetetraacetic acid on the hypothesis t h a t the phenol-protein a t t a c h m e n t is provided by metal-organic chelation. Subsequent paper chromatography showed no separation of components except for the appearance of small amounts of free amino acids.
Hydrolysis of Pigments A large n u m b e r of hydrolyses were carried out on pure chlorogenic acid and pure rutin to determine the best methods for splitting these molecules into their respective components. These methods were then used on the pigment fractions in a search for components identical with those obtained from the hydrolysis of ehlorogenie acid and rutin. Most of the procedures used were variations of the following : Alkaline Hydrolysis (19). The powdered material was dissolved in distilled water and, after a pre-evacuation to avoid foaming, was placed in a T h u n b e r g tube. From 1 to 2 mh of a sodium hyThe nitrogen analyses were carried out by the Schwarzkopf Microanalytical Laboratories, WoodMde, N. Y.
583
droxide solution was placed in the side arm, and the T h u n b e r g tube was evacuated with a water aspirator and filled with nitrogen gas. The evacuation and replacement with nitrogen was repeated at least four times before the T h u n b e r g tube was closed off and tipped to allow mixing of the solutions. Hydrolysis was allowed to continue at room temperature. After a period of time, the solution was neutralized by the addition of sulfuric acid. The slightly acidic solution was extracted with six 20-ml. aliquots of ether, and the ether extracts were concentrated to dryness and taken up in ethanol. The aqueous solution was concentrated to dryness and taken up in acetone, ethanol, or water. The two extracts were then chromatographed in the downward direction. This hydrolysis procedure, using 1 N sodium hydroxide for a period of 15 hr., was found best for obtaining caffeic and quinic acids from chlorogenic acid. Sul]uric Acid Hydrolysis (20). The powdered material was refluxed with sulfuric acid, and the solution was extracted with six 20-ml. portions of ether. The ether extract was concentrated to dryness, and the residue was taken up in methanol or ethanol. The aqueous solution was neutralized by the addition of barium hydroxide, and the barium sulfate was centrifuged off. The clear, slightly acidic supernatant was concentrated to dryness and taken up in acetone, ethanol, or water. This procedure, using 1 N sulfuric acid and refluxing for 30 rain., was found best for obtaining quereetin, rhamnose, and glucose from the hydrolysis of turin. Hydrochloric Acid Hydrolysis (10). The powdered material was refluxed with hydrochloric acid and extracted with six 20-ml. portions of ether. T h e ether solution was taken to dryness, and the residue was taken up in ethanol. T h e aqueous solution was evaporated to dryness several times with distilled water to remove the hydrochloric acid, and the residue was taken up in ethanol. The amino acids were obtained by refiuxing with 6 N hydrochloric acid for 24 hr. Most of the hydrolyses performed on the pigments were variations of the above procedures either in duration of hydrolysis or in concentration of acid or base.
Identification of Hydrolysis Products Comparison of chromatogrammed spots found after hydrolysis was made with authentic compounds as to mobility and color reactions in two or more solvents. Due to the variability of R r values (probably caused by differences in salt concentrations), cochromatography was employed as a further means of identification. For the identification of quinic acid, Rr values
584
JACOBSON
in n-butanol-acetic acid-water (12:3:5), 2% acetic acid and isopropyl alcohol-ammonium hydroxidewater (20:1:4) were used (10). Quinic acid was located by its yellow color with metaperiodatenitroprusside-piperazine spray reagents (21). For the identification of rhamnose and glucose, R ~ values in n-butanol-acetic acid-water (12: 3 : 5), isopropyl alcohol-n-butanol-water (7:1:2) and isopropyl alcohol-ammonium hydroxide-water (20: 1:4) were used (10). The sugars were located by the development of brown colors with aniline acid phthalate spray reagent (22). For the identification of caffeic acid, Rr values in 15% acetic acid and n-butanol-acetic acidwater (12:3:5) were used (10). Caffeie acid was located by its fluorescence under long-wave ultraviolet illumination. The results of hydrolysis of authentic chlorogenie acid and rutin are summarized in Table I. Note that in hydrolysis 2, two yellow spots, in addition to the one at Rr 0.33, appeared with the quinic acid spray reagent at R / s 0.56 and 0.65. In hydrolysis 3, the two additional spots appearing
with the quinic acid spray reagent had R / s of 0.22 and 0.56.
Acetylation When it became clear that hydrolysis of the pigments gave only trace amounts of caffeic acid and no quercetin, we attempted to improve yields by acetylation. If phenols were being split off the pigments, it might be possible to prevent their further reaction by making the acetylated derivatives. Since, to our knowledge, no one had attempted to aceylate the brown pigments before, we tried a number of different procedures. In order to have R r values for comparison, we attempted to acetylate caffeic acid, chlorogenic acid, quercetin, and rutin. Caffeic acid was acetylated according to the procedures of Pacsu and Stieber (23). The white fakes obtained had a melting point of 202-204°C. (uncorr.). Paper chromatograms showed a dark fluorescent spot under short-wave ultraviolet light. No fluorescence appeared under longwave ultraviolet illumination unless the paper was treated with ammonia fumes. The R r values
TABLE I HYDROLYSIS OF P U R E CItLOROGENIC ACID AND I~UTIN Pure compound hydrolyzed
Procedurea and amount hydrolyzed
1. Chlorogenic acid 2. Chlorogenic acid
A
(lO rag.) B (20 mg.)
B
3. Chlorogenic acid 4. l~utin
(20 nag.) B (20 rag.)
5. R u t i n
6. Rutin
C (20 rag.) l
C
I (20 rag.)
Reagent and length of hydrolysis
1 N NaOH 15 hr. 0.1 N HC1 35 rain.
6 N HC1 12 hr. 0.1 N HC1 20 rain. or 6 N tIC1 5 rain. 1 N H~S04 30 rain. 1 N H2SO4
3 hr.
Compounds found by paper chromatography
R f values b
Method of developmentC
Colorsd
Caffeic acid Quinie acid Caffeic acid Chlorogenic acid Quinic acid ~ Quinie acid ~
0.81 0.43 0.78 0.64
UV fl. MNP UV fl. UV ft.
Deep blue Yellow Deep blue B1, NHa-Y-G
0.33 0.34
MNP MNP
Yellow Yellow
Unknown A Quereetin Rhamnose Glucose Quercetin Rhamnose Glucose Unknown A Quercetin Rhamnose Glucose
0.92 0.71 0.44 0.25 0.75 0,41 0,21 0.91 0.71 0.45 0.25
UV ft. UV ft. AAP AAP UV ft. AAP AAP UV fl. UV ft. AAP AAP
Light blue Y, NH~-B. O-Y Dark brown Dark brown Yellow D a r k brown Dark brown Light blue Yellow Dark brown Dark brown
A = alkaline hydrolysis, B = hydrochloric acid hydrolysis, C = sulfuric acid hydrolysis. For procedures see text. Chromatographic solvent : n-butanol-acetic acid-water (12:3:5). UV fl, = ultraviolet fluorescence, M N P = metaperiodate-nitroprusside-piperazine spray reagents (54), AAP = aniline-acid phthalate spray reagent (22). a B1 = blue, Y-G = yellow-green, B.O-Y = bright orange-yellow. See text.
PIGMENTS OF TOBACCO LEAVES. I
585
and fluorescent colors of acetylated and nonacetylated caffeic acid are given in Table II. Quercetin was acetylated according to the procedure of HSrhammer et al. (24). Chromatography of the reaction mixture gave one fluorescent spot whose Rr and fluorescent color are given in Table II. Chlorogenic acid was acetylated according to the procedure of Gorter (25). Two fluorescent spots were found, one of which corresponded to the product of acetylation of caffeic acid. Chromatography of the other spot assumed to be an acetylated derivative of chlorogenic acid is described in Table II. The above procedures were applied to the water-soluble, ammonium sulfate-precipitated pigment using from 25 to 30 rag. material. Also attempted were reductive acetylations using zinc dust and stannous chloride (26). An acetylation was performed using acetyl chloride; another acetylation was followed by hydrolysis with 6 N hydrochloric acid. None of the above procedures gave fluorescent spots corresponding to the acetylated derivatives of caffeie acid, quercetin, or chlorogenic acid.
the results of an experiment in which p H 7 buffer was used instead of distilled water. The amount of material recovered in the nondialyzable pigment fractions is given in Table V. Thus, in a typical case, 4.1% of the original weight of leaf powder was isolated as buffer-soluble, nondialyzable pigment (Table V). This material was found to contain no free chlorogenic acid, rutin, the hydrolysis products of these compounds, or amino acids by paper chromatography. This material was obtained from a fraction in which 33% of the original weight of leaf powder was extracted (Table IV). Thus, about one-eighth of the material extracted by buffer was isolated as the nondialyzable pigment fraction. The results of elementary analysis of the leaf powders at various stages of extraction (Table VI) show a progressively declining carbon content.
RESULTS
All the pigment fractions isolated gave yellow to dark red-brown solutions depending on concentration. When dry, the buffer precipitates appeared almost coal-black, the sodium hydroxide-extracted pigments brown-black. T h e y were completely soluble in their original solvents of extraction. The buffer nondialyzable pigment precipitate was completely soluble in distilled water, partially soluble in 1 N hydrochloric acid, and completely soluble in sodium hydroxide. The latter solvent gave a darker solution than did distilled water.
ISOLATION
OF PIGMENTS
The effectiveness of the extraction solvents in removing the pigmented materials is evidenced by the observation t h a t the tobacco leaf powders, after complete extraction, were almost white. The amount of m a terial extracted by each solvent is given in Table I I I for an experiment in which the solvent for extraction of the brown pigments was distilled water. Table I V summarizes
CHARACTERIZATION OF PIGMENT FRACTIONS
TABLE I I Rf VALUES AND FLUORESCENCE OF COMPOUNDS BEFORE A N n AFTER Chromatographic solvent: n-butanol-acetic acid-water (12:3:5) (10). Compound
Caffeic acid Quercetin Chlorogenic acid
Treatment
-Acetylated -Acetylated -Acetylated
Ry values 0.81 0.86 0.72 0.83-0.87 0.64 0.79
ACETYLATION
Ultraviolet fluoresent color Without NH~
With NH3
Bright blue Dark (SW) ~ Yellow Blue-or.-yel. b Blue-green Light blue
Blue-white Deep blue Bright yellow Bright yellow Yellow-green Brt. c blue-green
Acetylated caffeic acid showed quenching of fluorescence in short-wave ultraviolet light only. b Blue-or.-yel. = blue-orange-yellow. Some deacetylation of quercetin appears to have taken place. c Brt. = bright.
586
JACOBSON TABLE I I I
FRACTIONAL WEIGHT OF CONNECTICUT SHADE TOBACCO LEAF POWDER EXTRACTED B Y SOLVENTS Plant sample I
Residue (by difference)
III
% 4.8 4.2 4.7 3.5 3.5 3.7 2.1 2.3 2.3 42 42 41 0.79 0.98 Average : 12%~ %
Ether extract Acetone extract Ethanol extract Distilled water extract 90% acetone extract Sodium hydroxide extracts
II
26
%
25
26
The specific extinction values of the sodium hydroxide solutions of the pigments were higher than the specific extinction values of the distilled water and acid solutions. The buffer-soluble pigment fraction consistently showed higher specific extinction values than the corresponding sodium hydroxide-soluble pigment fraction. The infrared spectra of these pigment fractions showed a strong peak at about 3 ~ and another at 6.1 ~. H e a v y absorption continued to 15 ~. Weak peaks were visible at about 7 and 7.2 ~, with broad areas of absorption at 8 and 9 ~. A sample of colorless squash seed globulin showed almost the same spectrum. The only exception was less general absorption between 8.7 and 9.9 ~.
a Due to mixing of powders, individual figures could not be obtained.
TABLE V
TABLE IV
DRY WEIGHT xffIELDS OF NONDIALYZABLE PIGMENT FRACTIONS
DRY WEIGHT EXTRACTED FROM CONNECTICUT SHADE TOBACCO LEAVES BY SOLVENTS
Pigment Fractions
Plant sample I
Plant sample Plant sample Plant sample I II III
Lipid extract Buffer extract NaOH extract
So 7.5 32.7 36.7
%4 8.3 33.5 37.2
%~ 7.3 53. lb 27.6
Buffer-extracted NaOK-extracted Totals
Plant sample II
Plant sample III
%~
%~
%a
4.11 4.50
4.14 6.52
4.51 4.72
8.61
10.66
9.23
Calculated as per cent of original dry weight. 76.9
79.0
88.0
Calculated as per cent of original dry weight. b Bacterial contamination. The sodium hydroxide-soluble, nondialyzable pigment precipitate was partially soluble in distilled water. I t was slightly soluble in hydrochloric acid but yielded, in part, a fibrous white or light yellow precipitate. The results of elementary analysis of the pigment fractions (Table -VI) show a relative enrichment in carbon content as compared to the whole leaf. This compares well with the decline in carbon content of the leaf powders. The pigment fractions also possessed very high ash contents. Absorption spectra of the buffer and sodium hydroxide-soluble, nondialyzable ammonium sulfate precipitates under acidic, basic, neutral, and reducing conditions showed increasing absorption toward the ultraviolet region with no significant peaks.
TABLE VI ELEMENTARY ANALYSES OF RADIOACTIVE TOBACCO POWDERS AND PIGMENT FRACTIONS Carbon Hydrogen
Ash
Plant sample II Unextracted powder Buffer precipitate Buffer-extracted powder NaOH precipitate NaOlq extracted powder
%
%
%
33.17 41.14 29.26 33.57 25.17
5.11 6.21 5.42 6.26 4.59
19.38 9.65 28.19 11.85 34.2
Plant sample I I I Unextractedpowder 34.43 Buffer precipitate 41.37 Buffer-extracted powder ~ 20.81 NaOH precipitate 37.15 NaOH extracted powder 14.15 a Bacterial contamination.
5.24 23.07 5.95 8.07 4.37 6.10 13.46 2.78 65.28
PIGMENTS OF TOBACCO LEAVES. I Thus the absorption spectra of the pigments were of little value in their characterization. The appearance of small amounts of caffeic acid by alkaline hydrolysis of the pigments was confirmed b y paper chromatography (Table V I I ) . I n hydrolysis 7, Table V I I , two brown-yellow zones appeared at R / s 0.54 and 0.67 with the quinic acid spray reagents in addition to the RI 0.30 zone. A blue fluorescent substance with RI between 0.91 and 0.93 appeared in m a n y hydrolyzates of the pigments (e.g., hydrolyzate 8, T a b l e V I I ) . This substance could be eluted with ether or methanol. A substance having a similar fluorescence and R I value was found in hydrolyzates of pure turin (Unknown A, Table I ) . Whether these two substances are identical is not known. The sixteen or more amino acids found in hydrolysis 9 (Table V I I ) were separated by two-dimensional chromatography, secB u t a n o l - 3 % a m m o n i u m hydroxide (5:3) was run twice in the first direction and secbutanol-formic a c i d - w a t e r (15:3:2) (10) was run once in the second direction. No individual amino acids were identified. The presence of quinic acid, rhamnose, and glucose in the pigments was confirmed
587
by the appearance of these substances after sulfuric acid hydrolysis (Table V I I I ) . The unknown found in hydrolysis 12 appeared on development with aniline acid phthalate, A red-brown color appeared which is indicative of a pentose sugar. I t was not further investigated. Elementary Analysis The per cent carbon, hydrogen, and ash for typical preparations of the labeled powders and pigment fractions are compared in Table VI. The nitrogen content of a sample of the nondialyzable buffer-soluble a m monium sulfate precipitate was 6.01% while the nitrogen content of a similarly derived fraction of the sodium hydroxide extract was 10.18%. DISCUSSION The extraction procedure t h a t we used has two important advantages. I t effects an almost complete extraction of the colored materials of autolyzed tobacco leaves, and it is conducted at room temperature. Avoidance of high temperatures seems desirable, despite the tediousness of the procedure, because of (a) the ease of oxidation of polyphenols and (b) the possibility of the occurrence of other reactions in the highly
TABLE VII l~ESULTS OF A L K A L I N E AND ACID HYDROLYSIS OF THE NONDIALYZABLE AMMONIUM SULFATE PRECII~ITATES Hydrolysis procedurea and amount hydrolyzed
Reagent and length of hydrolysis
Compounds found by paper chromatography
7. Buffer-soluble
A (27 rag.)
1 N NaOH 14.5 hr.
8. Buffer-soluble
A (40 rag.)
9. Buffer-soluble
B
10. NaOH-soluble
(50 rag.) B (50 rag.)
5 N NaOH 45.5 hr. 6 N HC1 24.5 hr. 6 N HC1 24 hr.
Unknown B Caffeic acid Quinic acid b Unknown B Caffeic acid Unknown B Amino acids Unknown B Amino acids
Pigment fraction hydrolyzed
Method of R / v a l u e s c development d
0.93 0.74 0.30 0.93
0.78 0.91 0.91
UV ft. UV fl. MNP UV ft. UV 1t. UV ft. NHD UV ft. NHD
Colorse
Light blue Light blue Yellow Light blue Light blue Light blue R, p, bl, y Light blue R, p, bl, y
A = alkaline hydrolysis, B = hydrochloric acid hydrolysis. For details see text. b See text. c Chromatographic solvent : n-butanol-acetic acid-water (12: 3:5). a UV ft. = ultraviolet fluorescence, MNP = metaperiod,.te-nitronrusside-piperazine spray reagents, NHD = ninhydrin spray reagent. e R = red, p = purple, bl = blue, y = yellow.
588
JACOBSON T A B L E VIII I:~ESULTS OF HYDROLYSIS OF THE NONDIALYZABLE AMMONIUM SULFATE I°I%ECIPITATES
Pigment fraction hydrolyzed
11. Buffer-soluble
12. Buffer-soluble
13. Buffer-soluble
14. Buffer-soluble
Procedurea and amour hydrolyzed
Reagent and length of hydrolysis
C (25 mg.)
1 N H2S0t 30 rain.
C (30 mg.)
2 hr.
C (25 rag.)
1 N H2SO4 3 hr.
C (50 rag.)
1 N H~SO4
1 N H2804 2.5 hr.
R$ values in solvents b Found on paper chromatograms _
Unknown B Rhamnose Control Unknown C Glucose Control Quinic acid Rhamnose Unknown C Glucose Control Quinic acid Unknown B Rhamnose Quinic acid Unknown C Glucose l%hamnose Unknown C Glucose
1
2
3
4
0.95 0.53 0.45 0.37 0.30 0.23 0.15 0.41 0.26 0.19
0.56
0.38 0.29 0.20 0.90
0.92 0.50 0.40 0.35 0.28 0.39 0.25 0.15
a C = sulfuric acid hydrolysis. For procedure see text. Solvent 1 : n-butanol-acetic acid-water (12: 3: 5) ; solvent 2 : isopropyl alcohol-ammonium hydroxide-water (20:1:4); solvent 3: isopropyl alcohol-n-butanol-water (7:1:2); solvent 4: 2% acetic acid (lO).
complex mixture of substances in the extracts. The possibility still remains, however, t h a t some changes m a y t a k e place during the extraction of the brown pigments even under such mild conditions. I t is to be noted t h a t Wenusch (27) was the first to discover t h a t some of the brown pigments of tobacco are soluble in pyridine and t h a t precipitation of some of the brown pigments can be accomplished with ammonium sulfate. However, we have found no indication of the involvement of chlorophyll degradation products in the formation of the brown pigments as was suggested by Wenusch (28) and by Bgbler (12). The results of spectrophotometry are similar to the results of Wright et at. (10) on the brown pigments of Burley tobacco. The importance of the increase in general absorption between 8.7 and 9.9 /, has not been established. I t m a y represent the formation of a new functional grouping characteristic of these pigments. I t is evident,
however, that infrared spectra are of little value in analyzing such large molecules. Table I X presents the elementary analyses of two pigment fractions and of certain known compounds for purposes of comparison. The elementary composition of the two pigment fractions is quite similar except t h a t the per cent nitrogen in the sodium hydroxide-soluble pigment is significantly lower. Note t h a t the per cent carbon in the pigments is over 10% lower than those figures given in the literature for melanins and proteins. Compared with ehlorogenic acid and rutin, the pigment fractions have about a 10% lower carbon content and a 2% higher hydrogen content. I f we assume, for the moment, t h a t the pigments were derived from plant protein, then the buffersoluble pigment fraction would contain about 42% protein and the sodium hydroxide-soluble pigment fraction about 66% protein, corrected for ash content. This would leave 58 and 34%, respectively, of
PIGMENTS
OF TOBACCO
TABLE IX COMPARISON OF ELEMENTAL COMPOSITION PIGMENT FRACTIONS AND SOME POSSIBLE PRECURSORS
CT Buffer ppt. a CT NaOtt ppt. b "Melanins" (32) "Proteins" (33) Chlorogenic acid Rutin Glucose
Carbon
nydr( gen
44 43 57 50-55 54.3 52.5 40.0
% 7 7 3.5 6-7 5.1 4.9 6.7
Other c
OF
Nitrogen
% % 42 7 39 11 30.5 9 20-23 12-19 40.6 42.6 53.3
Nondialyzable buffer-soluble precipitate. b The same as in footnote a but sodium hydroxide-soluble pigment fraction. c Since these values were obtained by difference, they are valid estimates of the oxygen content of ehlorogenic acid, rutin, and glucose only. the pigment fractions to be accounted for as polyphenols and, perhaps, other substances. If we consider condensation with proteins to have taken place, we find that the per cent carbon would still be about 10% above that of the pigments isolated, the per cent hydrogen would be slightly lower, and the per cent nitrogen lowered toward that of the pigments isolated. If we then consider that carbohydrates make up the remaining portion of the pigment fractions, this would provide a source of low carbon and relatively high hydrogen content needed to bring the elementary composition close to that found. This might also explain the presence of the third sugar in hydrolyzates of the pigments. In further support of this, Wright et al. (10) found large quantities of a polysaeeharide contaminating their Burley pigment fractions. Of particular interest is the mode of attachment between the polyphenols and the protein. After numerous attempts at separating the two, we have come to the conclusion that the mode of attachment must be a fairly strong and stable one. If mere physical absorption were involved one would expect separation of the polyphenols or oxidized polyphenols and protein to have taken place under the conditions used. It, thus, appears likely, after considering simi-
LEAVES.
I
589
lar situations in the plant and animal kingdoms, that a chemical union approximating covalent bond formation has occurred, probably between the oxidized polyphenols (quinones) and the amino groups of the proteins. The possibility of chelation as suggested by Wright still remains. The results obtained support the conclusion that ehlorogenie acid and rutin do participate in the formation of the pigment relatively unchanged. Our inability to find quereetin and appreciable quantities of eaffeie acid suggest, by analogy with the known reactions of phenols, that it is, indeed, the phenolic moieties which are involved in the reactions leading to pigment formation. The absence of quinic acid in hydrolyzates of the sodium hydroxide-soluble pigment is not surprising. If ehlorogenic acid were present in this pigment, the extraction solvent, 0.1 N sodium hydroxide, would have been sufficient to liberate quinie acid by hydrolysis. It would then have been lost during the subsequent dialysis. In 1953, in their review on proteins, Wildman and Jagendorf (29) reported that solutions of extracted cytoplasmic proteins were always brownish. They concluded that the color moiety was either a part of the protein or very tightly adsorbed, since it was not removed during prolonged dialysis, it was removed on isoeleetrie or trichloroacetic acid precipitation, and it moved with the protein during eleetrophoresis and ultraeentrifugation. In 1956, Cohen et al. (30) reported that browning of plant extracts could be completely prevented by performing the extraction and fractionation in an atmosphere of high purity nitrogen. The ability of extracts to brown was retained for 3 weeks under anaerobic conditions and lost after 60 hr. of anaerobic dialysis. This coincides with what is now known of enzymic browning. The enzyme and the reactants in the form of phenols and molecular oxygen must be present. Either the absence of oxygen or the loss of polyphenolie reactants by dialysis meant the loss of the ability to brown. In addition, the work of Wright et al. (10) and of the author indicates that much of the enzymic oxidative browning of both autolyzing leaf tissues and fresh plant extracts may involve con-
590
JACOBSON 15. HEss, E. It., Arch. Biochem. Biophys. 74, 198 (1958). I6. BEEVERS,H., ANDJAMES,W. 0., Biochem. J. 43, 636 (1948). 17. JACKSON, I-I., AND KENDAL,L. P., Biochem. J. 44, 477 (1949). 18. DAWSON,R. F., SOLT,M., ANDWADA,E., "Progress Report on Cigar Manufacturers Association Research Grant." Columbia University, New York, 1955. 19. BARNES, H. M., FELDMAN,J. R., AND WHITE, W. V., J. Am. Chem. Soc. 72, 4178 (1950). 20. NAGHSKI, J., FENSKE, C. S., JR., AND CoucI{~ REFERENCES J. F., J. Am. Pharm. Assoc. Sci. Ed. 60, 613 NEUBERG,C., AND KOBEL, M., Enzymologia 1, (1951). 177 (1936). 21. CARTWRIGHT,R. A., AND ROBERTS, E. A. H., KOENI~, P., AND DSRR, W., Biochem. Z. 263, Chem. & Ind. (London) 1955, 230. 295 (1933). 22. BLOCK, R. J., LESTRANGE,R., AND ZWEIG, G., DAWSON, R. F., AND WADA,E., Tobacco 144, "Paper Chromatography," p. 81. Academic 18 (1957). Press, New York, 1952. ROBERTS,E. A. It., Biochem. J. 35, 1289 (1941). 23. PACSU, E., AND STIERER, C., BeT. 62(3), 2974 WEYBREW,J. A., AND GREEN,P. E., JR., Science (1929). l l S , 466 (1952). 24. H6aHAMMER, L., ENDRES, L., WAGNER,H., AND RICHTHAMMER, F., Arch. Pharm. 290, 342 WENUSCH, A., Z. Lebensm.-Untersuch. u(1957). Forsch. 80, 61 (1940). FRANKENBURG,W. G., Advances in Enzymol. 25. GORTEE,K., Ann. 379, 110 (1911). 26. FIESER, L. F., "Experiments in Organic Chem6, 309 (1946). istry," 3rd ed., p. 176. D. C. Heath & Co., NAGASAWA,M., Bull. Agr. Chem. Soc. Japan Boston, 1952. 22, 21 (1958). MASON, It. S., Advances in Enzymol. 16, 105 27. WENUSCH, A., Z. Lebensm.-Untersuch. uForsch. 82, 34 (1941); cf. C. A. 38, 2074 (1955). (1944). WI~IGI~T,H. E., JR., BURTON,W. W., ANDBLURRY, R. C., JR., Arch. Biochem. Biophys. 86, 94 28. WENUSCH, A., Z. Lebensm.-Untersuch. uForsch. 81, 134 (1941). (1960). FRANKENBURO,W. G., Arch. Biochem. 14, 157 29. WILDMAN,S. G., AND JAGENDORF,A. T., Ann. Rev. Plant Physiol. 3, 131 (1952). (1947). 30. COHEN, M., GINOZA,W., DORNER,R. W., HUDB.~BLER, S., "On the Browning of Tobacco." SON, W. R., AND WILDMAN, S. G., Science Dissertation, Consolidated Technical Uni124, 1081 (1956). versity, Zurich; Publ. No. 2735, Juris-Ver- 31. WHITE, T., J. Soc. Leather Trades' Chemists lag, Zurich, 1957. 40, 78 (1956). REID, W. W., "The Chemistry of Yegetable 32. LERNER,A. B., AND FITZPATRICK,T. B., Physiol. Tannins," pp. 75-86. Symposium, Society of Revs. 30, 91 (1950). Leather Trades' Chemists, Croyden, 1956. 33. FR~:TON,J. S., AND SIMMONDS,S., "General BioWADA,E., AND IItIDA, M., Arch. Biochem. Biochemistry," p. 30. J. Wiley and Sons, New phys. 71, 393 (1957). York, 1953.
secutive reactions allied to those connected with the t a n n i n g of leather (31). I t will be of considerable interest to learn whether the a p p e a r a n c e of water-insoluble nitrogenous c o m p o u n d s during the f e r m e n t a t i o n of P e n n s y l v a n i a tobacco involves a similar interaction between soluble nitrogenous compounds and phenols. I f it does, the original suggestions of F r a n k e n b u r g (11) will have been substantiated.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12.
13.
14.
BIOCHEMISTRY AND BIOPHYSICS
93,580-590 (1961)
The Brown Pigments of Autolyzed Tobacco Leaves I. Isolation and Characterization JAY S. JACOBSON 1 From the Department el Botany, Columbia University, New York, New York
Received January 9, 1961 The isolation and partial purification of the brown pigments of autolyzed shadegrown tobacco leaves have been aecomplished. The solubilities of these substances, their ultraviolet and infrared absorption spectra, and their elementary composition are reported. Some of the products of chemical hydrolysis, as identified by paper chromatography, were found to be identical to the products of hydrolysis of chlorogenie acid and rutin. A large variety of amino acids was also found after hydrolysis. The results of these experiments indicate that the brown pigments may consist of a combination of polyphenols with protein.
INTRODUCTION The browning of tobacco leaves under autolytic conditions (i.e., during curing) is a matter of considerable biochemical and economic interest. Only in recent years, however, have the underlying questions begun to receive the attention that their importance merits. The foundation of a systematic investigation of browning reactions in tobacco leaves was laid by Carl Neuberg. With Kobel (1) he found that tobacco contains an o-diphenolic glycoside, rutin, in appreciable amount. Further, by the use of model experiments he reported t h a t rutin m a y be oxidized with a mixture of a crude enzyme preparation obtained from fresh tobacco and hydrogen peroxide to yield brown pigments in vitro. The suggestion was made that turin m a y be an important source of brown pigmentation in autolyzing tobacco leaves. Koenig and D f r r (2) isolated a second o-diphenol from tobacco leaves, namely, Cigar Manufaeturers Association Fellow, 195559. Present address: Boyce Thompson Institute for Plant :Research, 1086 North Broadway, Yonkers, N. Y.
the depside ehlorogenic acid. Dawson and Wada (3) observed that chlorogenic acid occurs even more abundantly in cigar tobacco types than does rutin. Further, they reported that a crude enzyme preparation of fresh tobacco leaves will oxidize chlorogenie acid to brown pigments using molecular oxygen and without the requirement for hydrogen peroxide. Rutin alone was not oxidized by this enzyme. Model experiments indicate that browning reactions involve an oxidative step. Since autolysis generally is regarded as a process of extensive hydrolysis and oxidation of cellular constituents, it is reasonable to assume t h a t browning reactions in rive may also be oxidative. The enzymic basis of browning is supported by the observations t h a t heating Io-baceo leaves to very high temperatures will prevent the disappearance of phenols (4, 5) and the appearance of brown pigments (6-8). Further support for the hypothesis that the brown pigments are the result of the enzymic oxidation of phenols comes from comparative biochemistry. The occurrence of brown pigments from the enzymic oxidation of phenols throughout the
580
PIGMENTS OF TOBACCO LEAVES. I plant and animal kingdoms has been described in a review by Mason (9). We have a t t e m p t e d to ascertain whether or not the brown pigments of autolyzed tobaceo leaves contain detectable fragments or residues of the polyphenols chlorogenic acid and rutin. To do this it was necessary to devise procedures for the extraction, fraetionation, and purification of these pigments. Such procedures are described herein. The results of color reactions and of chemical hydrolyses have indicated t h a t the pigmerits m a y contain residues of chlorogenic acid and of rutin. Evidence is also presented which indicates t h a t the phenols are coupled with proteins to form at least two m a j o r components of the brown pigment complex of autolyzed tobacco leaves. During the course of our work, the results of Wright et al. (10) were published. T h e y found t h a t some of the brown pigments of Burley tobacco m a y be derived from protein, rutin, ehlorogenie acid, and iron. The possibility t h a t proteins might be involved in browning had been suggested earlier b y F r a n k e n b u r g (11). EXPLORATORY" WOUK The results of model experiments (unpublished data) indicated t h a t a variety of other compounds could enter into secondary reactions with ehlorogenic acid during the enzymic oxidation of the latter. Various intensities and shades of color could be obtained with a number of compounds known to be present in mature, green tobacco leaves. Much of this information has since been published by others (12-15). Our observations on the relative simplicity of model systems giving rise to brown pigments, the work on the condensation products of quinones and amines by Beevers and J a m e s (16), the work on the isolation o f ' a crystalline condensation product of quinones and amines by Jackson and Kendall (17), and the work of Hess (15) led to the assumption t h a t the products of browning reactions in tobacco leaves m a y have relatively low molecular weights. We thus a t t e m p t e d the extraction, separation, and characterization of the pigments using methods adapted for substances of lower molecular weight. Our initial efforts in this
581
direction resulted in the separation both by paper and column c h r o m a t o g r a p h y of several brown pigmented zones. One of these gave typical protein color reactions while the others gave typical phenol color reaetions. As the work progressed, it became clear t h a t the substances in question were labile and not readily purified by fraetionation with the usual organic solvents (only one fraction was soluble in solvents other than water). These and other experiences led to the realization t h a t the brown pigments were not substances of relatively low molecular weight. The appearance of the work of Wright et at. (10) on the pigments of Burley tobacco gave further indication t h a t the brown pigments of Connecticut Shade tobacco might also have high moleeular weights. As a result, the problems of extraction, separation, and characterization of the pigments were re-examined from this changed viewpoint. MATERIALS AND METHODS ISOLATION OF I)IGMEI~'TS A modification of the procedure of Wright et al. (10) gave virtually complete extraction of the brown pigments. Weighed aliquots of dried Connecticut Shade No. 49 tobacco leaf powder were extracted with absolute acetone, ether, and ethanol, in that order, in glass-stoppered Erlenmeyer flasks at room temperature with mechanical agitation. The volume of solvent used in each extraction cycle was 60 ml., and the extracts were centrifuged after overnight agitation. The particulate material was returned to the flasks, fresh solvent added, and the procedure repeated until the eentrifugate was colorless or almost so. A total of about 2 1. of the above solvents was suNcient to extract a 10-g. sample of leaf powder at room temperature. The powders were then extracted with sodium dihydrogen phosphate buffer of about 2 M concentration adjusted to pH 7 with sodium hydroxide. The extractions were carried out at room temperature in exactly the same manner as above. A few drops of chloroform and toluene were used to inhibit growth of microorganisms. A total of about 1 1. of buffer solution in 60-ml. portions was used to extract a 10-g. sample of leaf powder. Additional brown pigments were then extracted with 0.1 and 1.0 N sodium hydroxide solutions. The residue, after complete extraction, was buff-colored and appeared to have lost most of its pigmentation.
582
JACOBSON 1.25
1.00
Z IJJ
,75
_J ~[
o
,50
o. o .25
0
, 400
I 500 WAVELENGTH
,
I 600
, 700
My
FIG. 1. Reflectance spectra of disks prepared from tobacco powders at various stages of extraction. A, before extraction; B, after extraction with ether, acetone, and ethanol; C, after extraction with ether, acetone, ethanol, and water; D, after extraction with preceding soNents and final extraction with sodium hydroxide solutions. At each stage of extraction, the powders were dried in a vacuum desiccator over calcium chloride for a standard period of time and weighed, and the reflectance spectra (Fig. 1) were taken. The latter show a decrease in color intensity with each succeeding extraction. They were obtained by pressing the dry leaf powders into l-in. in diameter pellets at 9000 lb./sq, in. and taking readings in a Beckman D U spectrophotometer following the procedure of Dawson et al. (18). A pellet of magnesium carbonate was used as a standard. The p H 7 buffer extracts were concentrated to one-quarter volume in a rotating evaporator at about 35°C. using ethyl acetate to prevent foaming. This solution was then centrifuged to free it of any particulate matter, and enough solid c.F. ammonium sulfate was added to saturate the solution. After standing overnight in the cold, the solution was again centrifuged and the dark, gummy precipitate redissolved in buffer. Both this solution and the colored supernate were dialyzed separately in Visking cellophane tubing against running distilled water for 2 days with stirring. A few drops of chloroform were placed in each dialysis bag to prevent the growth of microorganisms. The dialyzed solutions were evaporated to dryness by allowing a fan to blow air over the dialysis
bags. The brown to black materials were then weighed. The 0.1 and 1.0 N sodium hydroxide extracts were pooled, and enough glacial acetic acid was added to lower the pH to about 8. The solutions were then evaporated to about one-quarter volume using a few drops of oleic acid to inhibit foaming. Fractionation was again accomplished by salt precipitation, and dialysis was carried out on a slurry of the precipitate in distilled water. CHARACTERIZATION OF PIGMENTS
Ultraviolet Spectra Ultraviolet spectra of the pigment fractions were taken in dilute sodium hydroxide, dilute hydrochloric acid, distilled water and dilute sodium hydrosulfite solution from 700 to 210 m~ where possible. A Beckman D U speetrophotometer was employed, and the solvents were used as controls. Insoluble materials were removed by centrifugation before the spectra were taken.
Infrared Spectra Infrared spectra were taken in Nujol and hexachlorobutadiene mulls in a Perkin-Elmer Spectrocord recording spectrophotometer from 2.5 to 16 ~.
P I G M E N T S OF TOBACCO LEAVES. I
Elementary Analyses Elementary analyses were obtained on the powders at different stages of extraction and on the pigment fractions. These included ash content, per cent carbon, per cent hydrogen and per cent nitrogen?
Analysis o/ Pigment Fractions for the Presence o] Low Molecular Weight Impurities I n order to find out whether small amounts of low molecular weight substances were present in the pigment fractions, 700 mg. buffer-soluble, dialyzed, a m m o n i u m sulfate precipitate was extracted with ethanol with mechanical agitation. After centrifugation, the ethanol solution was evaporated to small volume and chroma~ographed in n-butanol-acetic acid-water (12:3:5) (10). No spots were found under ultraviolet light or by the use of spray reagents for quinic acid, amino acids, and reducing sugars. The possibility t h a t small molecules could be adsorbed onto the surface of the pigments was not precluded by these findings.
Homogeneity o] Pigment Fractions The only m e t h o d for detecting separation of components in the pigment fractions t h a t was available to us was paper chromatography. Portions of the powdered pigments were triturated in a n u m b e r of different solvents and the resultant solutions chromatographed. In addition to concentrated acid, concentrated base, and sodium hydrosulfite solutions, the pigments were treated as above with ethylenediaminetetraacetic acid on the hypothesis t h a t the phenol-protein a t t a c h m e n t is provided by metal-organic chelation. Subsequent paper chromatography showed no separation of components except for the appearance of small amounts of free amino acids.
Hydrolysis of Pigments A large n u m b e r of hydrolyses were carried out on pure chlorogenic acid and pure rutin to determine the best methods for splitting these molecules into their respective components. These methods were then used on the pigment fractions in a search for components identical with those obtained from the hydrolysis of ehlorogenie acid and rutin. Most of the procedures used were variations of the following : Alkaline Hydrolysis (19). The powdered material was dissolved in distilled water and, after a pre-evacuation to avoid foaming, was placed in a T h u n b e r g tube. From 1 to 2 mh of a sodium hyThe nitrogen analyses were carried out by the Schwarzkopf Microanalytical Laboratories, WoodMde, N. Y.
583
droxide solution was placed in the side arm, and the T h u n b e r g tube was evacuated with a water aspirator and filled with nitrogen gas. The evacuation and replacement with nitrogen was repeated at least four times before the T h u n b e r g tube was closed off and tipped to allow mixing of the solutions. Hydrolysis was allowed to continue at room temperature. After a period of time, the solution was neutralized by the addition of sulfuric acid. The slightly acidic solution was extracted with six 20-ml. aliquots of ether, and the ether extracts were concentrated to dryness and taken up in ethanol. The aqueous solution was concentrated to dryness and taken up in acetone, ethanol, or water. The two extracts were then chromatographed in the downward direction. This hydrolysis procedure, using 1 N sodium hydroxide for a period of 15 hr., was found best for obtaining caffeic and quinic acids from chlorogenic acid. Sul]uric Acid Hydrolysis (20). The powdered material was refluxed with sulfuric acid, and the solution was extracted with six 20-ml. portions of ether. The ether extract was concentrated to dryness, and the residue was taken up in methanol or ethanol. The aqueous solution was neutralized by the addition of barium hydroxide, and the barium sulfate was centrifuged off. The clear, slightly acidic supernatant was concentrated to dryness and taken up in acetone, ethanol, or water. This procedure, using 1 N sulfuric acid and refluxing for 30 rain., was found best for obtaining quereetin, rhamnose, and glucose from the hydrolysis of turin. Hydrochloric Acid Hydrolysis (10). The powdered material was refluxed with hydrochloric acid and extracted with six 20-ml. portions of ether. T h e ether solution was taken to dryness, and the residue was taken up in ethanol. T h e aqueous solution was evaporated to dryness several times with distilled water to remove the hydrochloric acid, and the residue was taken up in ethanol. The amino acids were obtained by refiuxing with 6 N hydrochloric acid for 24 hr. Most of the hydrolyses performed on the pigments were variations of the above procedures either in duration of hydrolysis or in concentration of acid or base.
Identification of Hydrolysis Products Comparison of chromatogrammed spots found after hydrolysis was made with authentic compounds as to mobility and color reactions in two or more solvents. Due to the variability of R r values (probably caused by differences in salt concentrations), cochromatography was employed as a further means of identification. For the identification of quinic acid, Rr values
584
JACOBSON
in n-butanol-acetic acid-water (12:3:5), 2% acetic acid and isopropyl alcohol-ammonium hydroxidewater (20:1:4) were used (10). Quinic acid was located by its yellow color with metaperiodatenitroprusside-piperazine spray reagents (21). For the identification of rhamnose and glucose, R ~ values in n-butanol-acetic acid-water (12: 3 : 5), isopropyl alcohol-n-butanol-water (7:1:2) and isopropyl alcohol-ammonium hydroxide-water (20: 1:4) were used (10). The sugars were located by the development of brown colors with aniline acid phthalate spray reagent (22). For the identification of caffeic acid, Rr values in 15% acetic acid and n-butanol-acetic acidwater (12:3:5) were used (10). Caffeie acid was located by its fluorescence under long-wave ultraviolet illumination. The results of hydrolysis of authentic chlorogenie acid and rutin are summarized in Table I. Note that in hydrolysis 2, two yellow spots, in addition to the one at Rr 0.33, appeared with the quinic acid spray reagent at R / s 0.56 and 0.65. In hydrolysis 3, the two additional spots appearing
with the quinic acid spray reagent had R / s of 0.22 and 0.56.
Acetylation When it became clear that hydrolysis of the pigments gave only trace amounts of caffeic acid and no quercetin, we attempted to improve yields by acetylation. If phenols were being split off the pigments, it might be possible to prevent their further reaction by making the acetylated derivatives. Since, to our knowledge, no one had attempted to aceylate the brown pigments before, we tried a number of different procedures. In order to have R r values for comparison, we attempted to acetylate caffeic acid, chlorogenic acid, quercetin, and rutin. Caffeic acid was acetylated according to the procedures of Pacsu and Stieber (23). The white fakes obtained had a melting point of 202-204°C. (uncorr.). Paper chromatograms showed a dark fluorescent spot under short-wave ultraviolet light. No fluorescence appeared under longwave ultraviolet illumination unless the paper was treated with ammonia fumes. The R r values
TABLE I HYDROLYSIS OF P U R E CItLOROGENIC ACID AND I~UTIN Pure compound hydrolyzed
Procedurea and amount hydrolyzed
1. Chlorogenic acid 2. Chlorogenic acid
A
(lO rag.) B (20 mg.)
B
3. Chlorogenic acid 4. l~utin
(20 nag.) B (20 rag.)
5. R u t i n
6. Rutin
C (20 rag.) l
C
I (20 rag.)
Reagent and length of hydrolysis
1 N NaOH 15 hr. 0.1 N HC1 35 rain.
6 N HC1 12 hr. 0.1 N HC1 20 rain. or 6 N tIC1 5 rain. 1 N H~S04 30 rain. 1 N H2SO4
3 hr.
Compounds found by paper chromatography
R f values b
Method of developmentC
Colorsd
Caffeic acid Quinie acid Caffeic acid Chlorogenic acid Quinic acid ~ Quinie acid ~
0.81 0.43 0.78 0.64
UV fl. MNP UV fl. UV ft.
Deep blue Yellow Deep blue B1, NHa-Y-G
0.33 0.34
MNP MNP
Yellow Yellow
Unknown A Quereetin Rhamnose Glucose Quercetin Rhamnose Glucose Unknown A Quercetin Rhamnose Glucose
0.92 0.71 0.44 0.25 0.75 0,41 0,21 0.91 0.71 0.45 0.25
UV ft. UV ft. AAP AAP UV ft. AAP AAP UV fl. UV ft. AAP AAP
Light blue Y, NH~-B. O-Y Dark brown Dark brown Yellow D a r k brown Dark brown Light blue Yellow Dark brown Dark brown
A = alkaline hydrolysis, B = hydrochloric acid hydrolysis, C = sulfuric acid hydrolysis. For procedures see text. Chromatographic solvent : n-butanol-acetic acid-water (12:3:5). UV fl, = ultraviolet fluorescence, M N P = metaperiodate-nitroprusside-piperazine spray reagents (54), AAP = aniline-acid phthalate spray reagent (22). a B1 = blue, Y-G = yellow-green, B.O-Y = bright orange-yellow. See text.
PIGMENTS OF TOBACCO LEAVES. I
585
and fluorescent colors of acetylated and nonacetylated caffeic acid are given in Table II. Quercetin was acetylated according to the procedure of HSrhammer et al. (24). Chromatography of the reaction mixture gave one fluorescent spot whose Rr and fluorescent color are given in Table II. Chlorogenic acid was acetylated according to the procedure of Gorter (25). Two fluorescent spots were found, one of which corresponded to the product of acetylation of caffeic acid. Chromatography of the other spot assumed to be an acetylated derivative of chlorogenic acid is described in Table II. The above procedures were applied to the water-soluble, ammonium sulfate-precipitated pigment using from 25 to 30 rag. material. Also attempted were reductive acetylations using zinc dust and stannous chloride (26). An acetylation was performed using acetyl chloride; another acetylation was followed by hydrolysis with 6 N hydrochloric acid. None of the above procedures gave fluorescent spots corresponding to the acetylated derivatives of caffeie acid, quercetin, or chlorogenic acid.
the results of an experiment in which p H 7 buffer was used instead of distilled water. The amount of material recovered in the nondialyzable pigment fractions is given in Table V. Thus, in a typical case, 4.1% of the original weight of leaf powder was isolated as buffer-soluble, nondialyzable pigment (Table V). This material was found to contain no free chlorogenic acid, rutin, the hydrolysis products of these compounds, or amino acids by paper chromatography. This material was obtained from a fraction in which 33% of the original weight of leaf powder was extracted (Table IV). Thus, about one-eighth of the material extracted by buffer was isolated as the nondialyzable pigment fraction. The results of elementary analysis of the leaf powders at various stages of extraction (Table VI) show a progressively declining carbon content.
RESULTS
All the pigment fractions isolated gave yellow to dark red-brown solutions depending on concentration. When dry, the buffer precipitates appeared almost coal-black, the sodium hydroxide-extracted pigments brown-black. T h e y were completely soluble in their original solvents of extraction. The buffer nondialyzable pigment precipitate was completely soluble in distilled water, partially soluble in 1 N hydrochloric acid, and completely soluble in sodium hydroxide. The latter solvent gave a darker solution than did distilled water.
ISOLATION
OF PIGMENTS
The effectiveness of the extraction solvents in removing the pigmented materials is evidenced by the observation t h a t the tobacco leaf powders, after complete extraction, were almost white. The amount of m a terial extracted by each solvent is given in Table I I I for an experiment in which the solvent for extraction of the brown pigments was distilled water. Table I V summarizes
CHARACTERIZATION OF PIGMENT FRACTIONS
TABLE I I Rf VALUES AND FLUORESCENCE OF COMPOUNDS BEFORE A N n AFTER Chromatographic solvent: n-butanol-acetic acid-water (12:3:5) (10). Compound
Caffeic acid Quercetin Chlorogenic acid
Treatment
-Acetylated -Acetylated -Acetylated
Ry values 0.81 0.86 0.72 0.83-0.87 0.64 0.79
ACETYLATION
Ultraviolet fluoresent color Without NH~
With NH3
Bright blue Dark (SW) ~ Yellow Blue-or.-yel. b Blue-green Light blue
Blue-white Deep blue Bright yellow Bright yellow Yellow-green Brt. c blue-green
Acetylated caffeic acid showed quenching of fluorescence in short-wave ultraviolet light only. b Blue-or.-yel. = blue-orange-yellow. Some deacetylation of quercetin appears to have taken place. c Brt. = bright.
586
JACOBSON TABLE I I I
FRACTIONAL WEIGHT OF CONNECTICUT SHADE TOBACCO LEAF POWDER EXTRACTED B Y SOLVENTS Plant sample I
Residue (by difference)
III
% 4.8 4.2 4.7 3.5 3.5 3.7 2.1 2.3 2.3 42 42 41 0.79 0.98 Average : 12%~ %
Ether extract Acetone extract Ethanol extract Distilled water extract 90% acetone extract Sodium hydroxide extracts
II
26
%
25
26
The specific extinction values of the sodium hydroxide solutions of the pigments were higher than the specific extinction values of the distilled water and acid solutions. The buffer-soluble pigment fraction consistently showed higher specific extinction values than the corresponding sodium hydroxide-soluble pigment fraction. The infrared spectra of these pigment fractions showed a strong peak at about 3 ~ and another at 6.1 ~. H e a v y absorption continued to 15 ~. Weak peaks were visible at about 7 and 7.2 ~, with broad areas of absorption at 8 and 9 ~. A sample of colorless squash seed globulin showed almost the same spectrum. The only exception was less general absorption between 8.7 and 9.9 ~.
a Due to mixing of powders, individual figures could not be obtained.
TABLE V
TABLE IV
DRY WEIGHT xffIELDS OF NONDIALYZABLE PIGMENT FRACTIONS
DRY WEIGHT EXTRACTED FROM CONNECTICUT SHADE TOBACCO LEAVES BY SOLVENTS
Pigment Fractions
Plant sample I
Plant sample Plant sample Plant sample I II III
Lipid extract Buffer extract NaOH extract
So 7.5 32.7 36.7
%4 8.3 33.5 37.2
%~ 7.3 53. lb 27.6
Buffer-extracted NaOK-extracted Totals
Plant sample II
Plant sample III
%~
%~
%a
4.11 4.50
4.14 6.52
4.51 4.72
8.61
10.66
9.23
Calculated as per cent of original dry weight. 76.9
79.0
88.0
Calculated as per cent of original dry weight. b Bacterial contamination. The sodium hydroxide-soluble, nondialyzable pigment precipitate was partially soluble in distilled water. I t was slightly soluble in hydrochloric acid but yielded, in part, a fibrous white or light yellow precipitate. The results of elementary analysis of the pigment fractions (Table -VI) show a relative enrichment in carbon content as compared to the whole leaf. This compares well with the decline in carbon content of the leaf powders. The pigment fractions also possessed very high ash contents. Absorption spectra of the buffer and sodium hydroxide-soluble, nondialyzable ammonium sulfate precipitates under acidic, basic, neutral, and reducing conditions showed increasing absorption toward the ultraviolet region with no significant peaks.
TABLE VI ELEMENTARY ANALYSES OF RADIOACTIVE TOBACCO POWDERS AND PIGMENT FRACTIONS Carbon Hydrogen
Ash
Plant sample II Unextracted powder Buffer precipitate Buffer-extracted powder NaOH precipitate NaOlq extracted powder
%
%
%
33.17 41.14 29.26 33.57 25.17
5.11 6.21 5.42 6.26 4.59
19.38 9.65 28.19 11.85 34.2
Plant sample I I I Unextractedpowder 34.43 Buffer precipitate 41.37 Buffer-extracted powder ~ 20.81 NaOH precipitate 37.15 NaOH extracted powder 14.15 a Bacterial contamination.
5.24 23.07 5.95 8.07 4.37 6.10 13.46 2.78 65.28
PIGMENTS OF TOBACCO LEAVES. I Thus the absorption spectra of the pigments were of little value in their characterization. The appearance of small amounts of caffeic acid by alkaline hydrolysis of the pigments was confirmed b y paper chromatography (Table V I I ) . I n hydrolysis 7, Table V I I , two brown-yellow zones appeared at R / s 0.54 and 0.67 with the quinic acid spray reagents in addition to the RI 0.30 zone. A blue fluorescent substance with RI between 0.91 and 0.93 appeared in m a n y hydrolyzates of the pigments (e.g., hydrolyzate 8, T a b l e V I I ) . This substance could be eluted with ether or methanol. A substance having a similar fluorescence and R I value was found in hydrolyzates of pure turin (Unknown A, Table I ) . Whether these two substances are identical is not known. The sixteen or more amino acids found in hydrolysis 9 (Table V I I ) were separated by two-dimensional chromatography, secB u t a n o l - 3 % a m m o n i u m hydroxide (5:3) was run twice in the first direction and secbutanol-formic a c i d - w a t e r (15:3:2) (10) was run once in the second direction. No individual amino acids were identified. The presence of quinic acid, rhamnose, and glucose in the pigments was confirmed
587
by the appearance of these substances after sulfuric acid hydrolysis (Table V I I I ) . The unknown found in hydrolysis 12 appeared on development with aniline acid phthalate, A red-brown color appeared which is indicative of a pentose sugar. I t was not further investigated. Elementary Analysis The per cent carbon, hydrogen, and ash for typical preparations of the labeled powders and pigment fractions are compared in Table VI. The nitrogen content of a sample of the nondialyzable buffer-soluble a m monium sulfate precipitate was 6.01% while the nitrogen content of a similarly derived fraction of the sodium hydroxide extract was 10.18%. DISCUSSION The extraction procedure t h a t we used has two important advantages. I t effects an almost complete extraction of the colored materials of autolyzed tobacco leaves, and it is conducted at room temperature. Avoidance of high temperatures seems desirable, despite the tediousness of the procedure, because of (a) the ease of oxidation of polyphenols and (b) the possibility of the occurrence of other reactions in the highly
TABLE VII l~ESULTS OF A L K A L I N E AND ACID HYDROLYSIS OF THE NONDIALYZABLE AMMONIUM SULFATE PRECII~ITATES Hydrolysis procedurea and amount hydrolyzed
Reagent and length of hydrolysis
Compounds found by paper chromatography
7. Buffer-soluble
A (27 rag.)
1 N NaOH 14.5 hr.
8. Buffer-soluble
A (40 rag.)
9. Buffer-soluble
B
10. NaOH-soluble
(50 rag.) B (50 rag.)
5 N NaOH 45.5 hr. 6 N HC1 24.5 hr. 6 N HC1 24 hr.
Unknown B Caffeic acid Quinic acid b Unknown B Caffeic acid Unknown B Amino acids Unknown B Amino acids
Pigment fraction hydrolyzed
Method of R / v a l u e s c development d
0.93 0.74 0.30 0.93
0.78 0.91 0.91
UV ft. UV fl. MNP UV ft. UV 1t. UV ft. NHD UV ft. NHD
Colorse
Light blue Light blue Yellow Light blue Light blue Light blue R, p, bl, y Light blue R, p, bl, y
A = alkaline hydrolysis, B = hydrochloric acid hydrolysis. For details see text. b See text. c Chromatographic solvent : n-butanol-acetic acid-water (12: 3:5). a UV ft. = ultraviolet fluorescence, MNP = metaperiod,.te-nitronrusside-piperazine spray reagents, NHD = ninhydrin spray reagent. e R = red, p = purple, bl = blue, y = yellow.
588
JACOBSON T A B L E VIII I:~ESULTS OF HYDROLYSIS OF THE NONDIALYZABLE AMMONIUM SULFATE I°I%ECIPITATES
Pigment fraction hydrolyzed
11. Buffer-soluble
12. Buffer-soluble
13. Buffer-soluble
14. Buffer-soluble
Procedurea and amour hydrolyzed
Reagent and length of hydrolysis
C (25 mg.)
1 N H2S0t 30 rain.
C (30 mg.)
2 hr.
C (25 rag.)
1 N H2SO4 3 hr.
C (50 rag.)
1 N H~SO4
1 N H2804 2.5 hr.
R$ values in solvents b Found on paper chromatograms _
Unknown B Rhamnose Control Unknown C Glucose Control Quinic acid Rhamnose Unknown C Glucose Control Quinic acid Unknown B Rhamnose Quinic acid Unknown C Glucose l%hamnose Unknown C Glucose
1
2
3
4
0.95 0.53 0.45 0.37 0.30 0.23 0.15 0.41 0.26 0.19
0.56
0.38 0.29 0.20 0.90
0.92 0.50 0.40 0.35 0.28 0.39 0.25 0.15
a C = sulfuric acid hydrolysis. For procedure see text. Solvent 1 : n-butanol-acetic acid-water (12: 3: 5) ; solvent 2 : isopropyl alcohol-ammonium hydroxide-water (20:1:4); solvent 3: isopropyl alcohol-n-butanol-water (7:1:2); solvent 4: 2% acetic acid (lO).
complex mixture of substances in the extracts. The possibility still remains, however, t h a t some changes m a y t a k e place during the extraction of the brown pigments even under such mild conditions. I t is to be noted t h a t Wenusch (27) was the first to discover t h a t some of the brown pigments of tobacco are soluble in pyridine and t h a t precipitation of some of the brown pigments can be accomplished with ammonium sulfate. However, we have found no indication of the involvement of chlorophyll degradation products in the formation of the brown pigments as was suggested by Wenusch (28) and by Bgbler (12). The results of spectrophotometry are similar to the results of Wright et at. (10) on the brown pigments of Burley tobacco. The importance of the increase in general absorption between 8.7 and 9.9 /, has not been established. I t m a y represent the formation of a new functional grouping characteristic of these pigments. I t is evident,
however, that infrared spectra are of little value in analyzing such large molecules. Table I X presents the elementary analyses of two pigment fractions and of certain known compounds for purposes of comparison. The elementary composition of the two pigment fractions is quite similar except t h a t the per cent nitrogen in the sodium hydroxide-soluble pigment is significantly lower. Note t h a t the per cent carbon in the pigments is over 10% lower than those figures given in the literature for melanins and proteins. Compared with ehlorogenic acid and rutin, the pigment fractions have about a 10% lower carbon content and a 2% higher hydrogen content. I f we assume, for the moment, t h a t the pigments were derived from plant protein, then the buffersoluble pigment fraction would contain about 42% protein and the sodium hydroxide-soluble pigment fraction about 66% protein, corrected for ash content. This would leave 58 and 34%, respectively, of
PIGMENTS
OF TOBACCO
TABLE IX COMPARISON OF ELEMENTAL COMPOSITION PIGMENT FRACTIONS AND SOME POSSIBLE PRECURSORS
CT Buffer ppt. a CT NaOtt ppt. b "Melanins" (32) "Proteins" (33) Chlorogenic acid Rutin Glucose
Carbon
nydr( gen
44 43 57 50-55 54.3 52.5 40.0
% 7 7 3.5 6-7 5.1 4.9 6.7
Other c
OF
Nitrogen
% % 42 7 39 11 30.5 9 20-23 12-19 40.6 42.6 53.3
Nondialyzable buffer-soluble precipitate. b The same as in footnote a but sodium hydroxide-soluble pigment fraction. c Since these values were obtained by difference, they are valid estimates of the oxygen content of ehlorogenic acid, rutin, and glucose only. the pigment fractions to be accounted for as polyphenols and, perhaps, other substances. If we consider condensation with proteins to have taken place, we find that the per cent carbon would still be about 10% above that of the pigments isolated, the per cent hydrogen would be slightly lower, and the per cent nitrogen lowered toward that of the pigments isolated. If we then consider that carbohydrates make up the remaining portion of the pigment fractions, this would provide a source of low carbon and relatively high hydrogen content needed to bring the elementary composition close to that found. This might also explain the presence of the third sugar in hydrolyzates of the pigments. In further support of this, Wright et al. (10) found large quantities of a polysaeeharide contaminating their Burley pigment fractions. Of particular interest is the mode of attachment between the polyphenols and the protein. After numerous attempts at separating the two, we have come to the conclusion that the mode of attachment must be a fairly strong and stable one. If mere physical absorption were involved one would expect separation of the polyphenols or oxidized polyphenols and protein to have taken place under the conditions used. It, thus, appears likely, after considering simi-
LEAVES.
I
589
lar situations in the plant and animal kingdoms, that a chemical union approximating covalent bond formation has occurred, probably between the oxidized polyphenols (quinones) and the amino groups of the proteins. The possibility of chelation as suggested by Wright still remains. The results obtained support the conclusion that ehlorogenie acid and rutin do participate in the formation of the pigment relatively unchanged. Our inability to find quereetin and appreciable quantities of eaffeie acid suggest, by analogy with the known reactions of phenols, that it is, indeed, the phenolic moieties which are involved in the reactions leading to pigment formation. The absence of quinic acid in hydrolyzates of the sodium hydroxide-soluble pigment is not surprising. If ehlorogenic acid were present in this pigment, the extraction solvent, 0.1 N sodium hydroxide, would have been sufficient to liberate quinie acid by hydrolysis. It would then have been lost during the subsequent dialysis. In 1953, in their review on proteins, Wildman and Jagendorf (29) reported that solutions of extracted cytoplasmic proteins were always brownish. They concluded that the color moiety was either a part of the protein or very tightly adsorbed, since it was not removed during prolonged dialysis, it was removed on isoeleetrie or trichloroacetic acid precipitation, and it moved with the protein during eleetrophoresis and ultraeentrifugation. In 1956, Cohen et al. (30) reported that browning of plant extracts could be completely prevented by performing the extraction and fractionation in an atmosphere of high purity nitrogen. The ability of extracts to brown was retained for 3 weeks under anaerobic conditions and lost after 60 hr. of anaerobic dialysis. This coincides with what is now known of enzymic browning. The enzyme and the reactants in the form of phenols and molecular oxygen must be present. Either the absence of oxygen or the loss of polyphenolie reactants by dialysis meant the loss of the ability to brown. In addition, the work of Wright et al. (10) and of the author indicates that much of the enzymic oxidative browning of both autolyzing leaf tissues and fresh plant extracts may involve con-
590
JACOBSON 15. HEss, E. It., Arch. Biochem. Biophys. 74, 198 (1958). I6. BEEVERS,H., ANDJAMES,W. 0., Biochem. J. 43, 636 (1948). 17. JACKSON, I-I., AND KENDAL,L. P., Biochem. J. 44, 477 (1949). 18. DAWSON,R. F., SOLT,M., ANDWADA,E., "Progress Report on Cigar Manufacturers Association Research Grant." Columbia University, New York, 1955. 19. BARNES, H. M., FELDMAN,J. R., AND WHITE, W. V., J. Am. Chem. Soc. 72, 4178 (1950). 20. NAGHSKI, J., FENSKE, C. S., JR., AND CoucI{~ REFERENCES J. F., J. Am. Pharm. Assoc. Sci. Ed. 60, 613 NEUBERG,C., AND KOBEL, M., Enzymologia 1, (1951). 177 (1936). 21. CARTWRIGHT,R. A., AND ROBERTS, E. A. H., KOENI~, P., AND DSRR, W., Biochem. Z. 263, Chem. & Ind. (London) 1955, 230. 295 (1933). 22. BLOCK, R. J., LESTRANGE,R., AND ZWEIG, G., DAWSON, R. F., AND WADA,E., Tobacco 144, "Paper Chromatography," p. 81. Academic 18 (1957). Press, New York, 1952. ROBERTS,E. A. It., Biochem. J. 35, 1289 (1941). 23. PACSU, E., AND STIERER, C., BeT. 62(3), 2974 WEYBREW,J. A., AND GREEN,P. E., JR., Science (1929). l l S , 466 (1952). 24. H6aHAMMER, L., ENDRES, L., WAGNER,H., AND RICHTHAMMER, F., Arch. Pharm. 290, 342 WENUSCH, A., Z. Lebensm.-Untersuch. u(1957). Forsch. 80, 61 (1940). FRANKENBURG,W. G., Advances in Enzymol. 25. GORTEE,K., Ann. 379, 110 (1911). 26. FIESER, L. F., "Experiments in Organic Chem6, 309 (1946). istry," 3rd ed., p. 176. D. C. Heath & Co., NAGASAWA,M., Bull. Agr. Chem. Soc. Japan Boston, 1952. 22, 21 (1958). MASON, It. S., Advances in Enzymol. 16, 105 27. WENUSCH, A., Z. Lebensm.-Untersuch. uForsch. 82, 34 (1941); cf. C. A. 38, 2074 (1955). (1944). WI~IGI~T,H. E., JR., BURTON,W. W., ANDBLURRY, R. C., JR., Arch. Biochem. Biophys. 86, 94 28. WENUSCH, A., Z. Lebensm.-Untersuch. uForsch. 81, 134 (1941). (1960). FRANKENBURO,W. G., Arch. Biochem. 14, 157 29. WILDMAN,S. G., AND JAGENDORF,A. T., Ann. Rev. Plant Physiol. 3, 131 (1952). (1947). 30. COHEN, M., GINOZA,W., DORNER,R. W., HUDB.~BLER, S., "On the Browning of Tobacco." SON, W. R., AND WILDMAN, S. G., Science Dissertation, Consolidated Technical Uni124, 1081 (1956). versity, Zurich; Publ. No. 2735, Juris-Ver- 31. WHITE, T., J. Soc. Leather Trades' Chemists lag, Zurich, 1957. 40, 78 (1956). REID, W. W., "The Chemistry of Yegetable 32. LERNER,A. B., AND FITZPATRICK,T. B., Physiol. Tannins," pp. 75-86. Symposium, Society of Revs. 30, 91 (1950). Leather Trades' Chemists, Croyden, 1956. 33. FR~:TON,J. S., AND SIMMONDS,S., "General BioWADA,E., AND IItIDA, M., Arch. Biochem. Biochemistry," p. 30. J. Wiley and Sons, New phys. 71, 393 (1957). York, 1953.
secutive reactions allied to those connected with the t a n n i n g of leather (31). I t will be of considerable interest to learn whether the a p p e a r a n c e of water-insoluble nitrogenous c o m p o u n d s during the f e r m e n t a t i o n of P e n n s y l v a n i a tobacco involves a similar interaction between soluble nitrogenous compounds and phenols. I f it does, the original suggestions of F r a n k e n b u r g (11) will have been substantiated.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12.
13.
14.