ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

74, 131-138 (1958)

On the Distribution of Caffeic Acid and the Chlorogenic Acid Isomers in Plants
Ernest Sondheimerl
From the New York State Agricultural Experiment Station, Cornell University, Geneva, New York Received June 26, 1957

Chlorogenic acid is perhaps the best known of the caffeylquinic acids, but other isomers, iso- (1) pseudo- (2), and neochlorogenic acids, (3), have also been reported. These compounds are of interest because of their possible role in plant disease resistance (4), as substrates for polyphenol oxidase (5), and in the non-enzymic browning of potatoes (6). Because of this and the lack of information on the distribution of the chlorogenic acid isomers in plant tissues, it seemed desirable to have a routine screening procedure amenable to quantitative interpretation. Silicic acid columns have been shown to possess high resolution power for many mixtures of organic acids (7). Caffeic acid and its depsides can be detected by titration of the carboxyl group or by measurement of the absorption peak near 325 rnp (8) (E = 18,500 for chlorogenic acid). It was hoped that separation of the acid mixture with a silicic acid column and detection both by titration and ultraviolet measurements would give a procedure with good resolving power and great specificity for the chlorogenic acids. The high extinction coefficients also are of value in increasing the sensitivity of the method.
EXPERIMENTAL

Separation of the Chlorogenic Acids

The silicic acid column and solvent schedule described by Bulen, Varner, and was adapted for this separation survey column Burrell (9) for the standard problem by doubling the quantities of silicic acid, 0.5 N sulfuric acid, and the sol1 Present address: Department of Chemistry, State University estry at Syracuse University, Syracuse 10, N. Y. 131 College of For-

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vents. The solvent schedule used was 200 ml. of 5%, 270 ml. of 15%, 200 ml. of 25%, 600 ml. of 35%, and 530 ml. of 50% n-butanol-chloroform saturated with 0.5 N sulfuric acid. The samples were applied with 2 g. silicic acid and 1 ml. of 0.5 N sulfuric acid. Ten-milliliter fractions were collected. Five-milliliter aliquots were titrated with 0.02 N sodium hydroxide in the presence of water to the bromothymol blue end point, and the rest was used for the absorption measurements at 330 rnp. Those samples which had an optical density above the upper limit of the Beckman spectrophotometer, model DU, were diluted with solvent of the same composition as the effluent. Similarly, the composition of the blanks used in the spectrophotometer was changed as the solvent composition of the fractions altered. The absorption between 310 and 340 rnp was determined at 5-rnp intervals for those fractions that contained the maximum amount of a given band. Only if an absorption peak was found at 330 rnp was it considered probable that a caffeic acid derivative was present. Caffeic acid, chlorogenic acid, isochlorogenic acid, and neochlorogenic acid were chromatographed singly and in mixtures, and the peak effluent volumes and recoveries were determined (Table I). The unknowns were chromatographed by the same procedure. With coffee, blueberry leaves, and sweet-potato peelings, 180 fractions were collected. Since no caffeic acid derivatives were detected in fractions 75-180, only 85 fractions were collected with the other samples. We have found it necessary to apply the caffeic acid to the column in solution and to keep the amount below 2 mg. for the 16 g. silicic acid column. Otherwise, a high proportion of the acid will be retained on the column until the 15% n-butano1 solvent is introduced, and it will be eluted with isochlorogenic acid. This factor is believed to explain the discrepancy between our results and those obTABLE Recovery of Ca$eic Acid and Chlorogenic
Acids Pe;&3h$nt
ml.

I Acid Column
Recovery Ultraviolet-light measurements
%

Acid Isomers from the Silicic

El% 1 cm. Titration
%

Caffeicb Isochlorogenic Chlorogenic Neochlorogenic

120 230 360-410 710

87OC 510d 460d 460h

105 108 1040 -

111 87 110 96

a The peak effluent volume was defined by Marvel and Rands (7) as that volume of effluent collected while a given compound moves from the top of the column to the bottom and is measured at the point at which the greatest concentration of the compound is eluted. b The caffeic acid concentration did not exceed 2 mg. for 16 g. silicic acid. c Measured at 325 rnp in n-butanol-chloroform (5:95 v/v). d Measured at 330 rnM in n-butanol-chloroform (15:85 v/v). e Calculated from a neutral equivalent of 580 (1). f Large amounts of chlorogenic acid tend to give higher peak effluent volumes. g Calculated from a neutral equivalent of 363. h Measured at 330 rnp in n-butanol-chloroform (25:75 v/v).

CAFFEIC

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ACIDS

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PLANTS

133

tained by Mabrouk and Deatherage (10). These authors also used the column described by Bulen et al. (9) but depended solely on titration for detection and used 5 mg. caffeic acid on an 8-g. column. Under these conditions they assigned the isochlorogenic acid band to caffeie acid and failed to detect isoehlorogenic acid, Band 510, and neochlorogenic acid in brewed, roasted coffee.

Preparation of Plant Materials

All the fruit samples with the exception of the coffee beans were frozen, thepits removed, and the flesh and skins freeze-dried. These preparations could be chromatographed without further treatments. One gram pulverized material, 2 g. silicic acid, and 1 ml. of 0.5 N sulfuric acid were ground together, and the mixture was transferred to the top of the column. The blueberry-leaf samples could be used without prior drying by grinding 0.5-g. samples with 1 g. sand, 1 g. silicic acid, and 1 ml. of 0.5 N sulfuric acid. To chromatograph the chlorogenic acid isomers from sweet potatoes and coffee, it was necessary to prepare extracts, since excessively wide bands were obtained otherwise. Freeze-dried sweet-potato peelings, 3.3 g., were pulverized and extracted twice with 40.ml. portions of 70% isopropyl alcohol for 2 hr. at room temperature. The extracts were combined and evaporated in vacuum. The residue was treated with 3.2 ml. water and acidified with sulfuric acid to pH 2 just before the chromatogram was started. The acids were chromatographed by placing 1 ml. of this preparation with 2 g. silicic acid on the top of the column. The coffee sample was prepared similarly from beans, variety Santos, which had been ground in a Wiley mill with a 20.mesh screen, but had not been freeze-dried. Although this procedure gave good results with all the fruits tested and with blueberry leaves, it could not be applied to the analysis of apple and peach leaves. With the latter materials only indistinct bands were obtained which did not have absorption peaks near 330 mp. Most of the fractions had relatively high absorption blanks at 330 rnp. Attempts to remove this interfering absorption in the leaf extracts before chromatographing were unsuccessful.

Isolation of Chlorogenic Acid from Blueberry Leaves

To 100 g. fresh blueberry leaves, 600 ml. 70y0 isopropyl alcohol was added and the mixture was ground in a Waring blendor. After storage at room temperature for 3 hr., the preparation was filtered and the insoluble fraction re-extracted with 600 ml. of 70% isopropyl alcohol. The combined extracts were concentrated in vacuum to 240 ml., cooled to 5, and filtered through Celite, and the filtrate was concentrated to 25 ml. Five milliliters of this extract was used for the isolation of the chlorogenic acid after acidification to pH 2 with sulfuric acid. The column was prepared from 96 g. silicic acid, 66 ml. of 0.5 N sulfuric acid, and 600 ml. developing solvent, n-butanol-chloroform, 15:85 (v/v), saturated with 0.5 N sulfuric acid. A column with a diameter of 5 cm. and a length of 75 cm. was used. A slurry of 5 ml. extract, 10 g. silicic acid, and 50 ml. developing solvent was placed on top of the column. Fifty-milliliter fractions were collected, and the absorption at 330 rng was determined. The bulk of the chlorogenic acid was found in fractions 18-26. These were combined, and the solvent was evaporated in vacuum yielding 180 mg. par-

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tially crystalline residue. The material was dissolved in 1 ml. absolute ethanol and precipitated as a crystalline solid on the addition of 29 ml. chloroform; yield 85 mg., m.p. 193-200. Two recrystallizations from water raised the m.p. to 292206, mixed melting point with authentic chlorogenic acid 20-205; the chlorogenie acid had been isolated from coffee as the potassium caffeine complex and had an m.p. 200-204. Anal. Calcd. for Cr~HrsO~.+H*O: C, 52.94; H, 5.27; neut. equiv. 363. Found: C, 52.7; H, 5.27; neut. equiv. 357.

Isolation of Band 610 from Green Co$ee Beans

Ground coffee beans, 100 g., were extracted twice with 600-ml. portions of 70% isopropyl alcohol. The combined extracts were concentrated to 50 ml. After acidification to pH 2 with sulfuric acid, 10 ml. of the extract was mixed with 20 g. silicic acid, and 100 ml. of the first development solvent and the slurry were added to the top of the column. The column consisted of 160 g. silicic acid, 110 ml. of 0.5 N sulfuric acid, and the first development solvent. Development was carried out with n-butanol-chloroform mixtures that had been equilibrated with 0.5 N sulfuric acid; 2700 ml. of 15y0 by volume n-butanol was followed by 1500 ml. of a 25$& nbutanol solution. One-hundred-milliliter fractions were collected. Ultraviolet measurements showed most of the Band 510 to be in fractions 32, 33, and 34. These were combined and concentrated in vacuum. A noncrystalline residue remained which could not be crystallized from various solvents nor by purification through its lead salt. On the standard survey column the substance was shown to have a peak effluent volume of 510 and to be 95.5% chromatographically pure; the chlorogenic, isochlorogenic, and caffeic acid bands had 1.7, 1.6, and 1.2% of the total absorption at 330 mp, respectively. The ultraviolet-absorption curve was similar to that of chlorogenic acid. The highest extinction observed was with a sample that had been purified by two conversions through the lead salt, E&. = 440 in n-butanol-chloroform (25:75 v/v) at 330 mp. Paper chromatography on Whatman No. 1 with n-butanol-acetic acid-water (4:1:5) gave one fluorescent spot with Rr = 0.59. Under these conditions chlorogenic acid had an R, value of 0.66. In aqueous solution the material is levorotatory; with one preparation [a]z4 = -55 (c 0.4 in water) was observed. Band 510 gives a yellow color with aqueous sodium hydroxide and a green color with ferric chloride. Analysis indicated 51.5% C and 6.04yc H. Evidence that the material is a caffeic acid derivative was obtained by hydrolysis of 50 mg. with 1 ml. of 1 N sodium hydroxide at room temperature for 16 hr. in the absence of oxygen. The addition of hydrochloric acid caused the immediate precipitation of yellow crystals. After storage at 0 for 4 hr., the crystals were collected by centrifugation, washed with water, and dried, yielding 16.5 mg. A second crop of 3.9 mg. was obtained by extraction of the supernatant and washings with ether, total yield 41%. The two crops were combined and recrystallized from water yielding 12.4 mg. crystals, m.p. 195-201 (decompn.). The mixed melting point with caffeic acid was 195-201 (decompn.). Anal. Calcd. for CcHsOd : C, 60.01; H, 4.48. Found: C, 59.67; H, 5.02. The water-soluble moiety obtained on alkaline hydrolysis has not as yet been identified.

CAFFEIC

AND

CHLOROGENIC

ACIDS

IN PLANTS

135

Band 510 was partitioned between 2.2 M phosphate buffer of pH 3.12 and ethyl acetate in a 24-plate Craig countercurrent machine. Ninty eight per cent of the material was obtained a.~ one band with a partition coefficient of 1.4. Two per cent of material was found in the last three tubes. At a pH of 5.8 Band 510 could not be partitioned due to its low solubility in the organic phase. RESULTS AND DISCUSSION

on the distribution of caffeic acid and of the chlorogenic acid Data isomers in a number of plants are summarized in Table II. Because the optical measurements allow detection of smaller amounts than with titration, the results were calculated from the ultraviolet absorption. The column resolved the ultraviolet-absorbing acids into five bands. Four of these have peak effluent volumes that coincide with caffeic, isochlorogenic, chlorogenic, and neochlorogenic acids. A fifth band with a peak eflluent volume of 510 and designated Band 510 was eluted between chlorogenic acid and neochlorogenic acid and has not as yet been completely identified. In its color reactions and ultraviolet- and infrared-absorption spectra, Band 510 resembles chlorogenic acid and its isomers. It has been isolated as a noncrystalline solid with the silicic acid column from green coffee beans. The preparation is optically active and yields caffeic acid in 41% yield on alkaline hydrolysis. The water-soluble moiety has not been identified as yet. When the isolated material was rechromatographed on paper or on the silicic acid column, no evidence for the presence of equilibrium components was obtained. This makes it unlikely that Band 510 is the cis isomer of any of the known chlorogenic acids. That Band 510 is not identical with pseudochlorogenic acid (2) was shown by the large difference in their partition coefficients. Urtani and Miyano (2) found a partitioncoefficient of 0.48 for pseudochlorogenic acid between phosphate buffer of pH 5.8 and ethyl acetate. At that pH the partition coefficient of Band 510 is so much in favor of the aqueous phase that very little is transferred to the ethyl acetate layer. In none of the plant samples examined in this study was pseudochlorogenic acid detected. However, this does not prove the absence of this substance since it is possible that the procedure used here did not separate pseudochlorogenic acid from the closely related isochlorogenic acid. Several conclusions can be drawn about the distribution of the caffeic acid and the chlorogenic acid isomers from the data in Table II. 1. The caffeic acid band was either absent or accounted only for a small percentage of the total ultraviolet-absorbing acids.

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TABLE Distribution
Plant source

II Acid Isomers

of (

ffeic Acid and Chlorogenic

T

i
Isochlorogenic acid band Chlorogenic acid band

Caff;~k.Gd

in Various
510

Plants

Band

Neochlorogenic acid band

W./l00

k!

%
3.: 5 1

ng./100

(:.I

%
11 6

m./ 100 g.

w./ 100 g

% 9 10

lg./loo g. 395 140

% 9.5 4

1. Coffee beansb (Santo) 2. Blueberry leavesb (Jersey) 3. Blueberry fruit (Jersey 4. Apples (McIn. tosh) 5. Pears (Covert) 6. Grapes (Steuben) 7. Sweet-potato peelings 8. Peaches (Late Rose) 9. Prunes (Imperial Epineuse) 10. Plums (Formosa) 11. Cherries (Emperor Francis)

140 40

480 2000

2850 2800

67 79

385 360

0 Tracec Tracec 0 11 0 0

25

10

190 105

76 84 79 77 32 40 7

25 20 15 20 53 0 40

10 16 9 11 5 0 4

1oc Trace 5 2c 48 40 850

4 3 1 5 3 8

1 0 0

15 2oc 603 5c 10

9 I1 57 7 1

135 140 335 30 65

0 0

0 0

10 2c

11 1

25 5

28 4

15 3%

17 26

40 95

4 9

a Concentrations are calculated from the ultraviolet-absorption data. b Concentrations are based on fresh weight; for the other samples the quantities are calculated on a dry weight basis. c There is some uncertainty about the presence of caffeic acid or the chlorogenie acid isomers in these bands because the fraction with the highest concentration did not have an absorption maximum between 310 and 340 rnp although the peak effluent volumes were normal.

2. Only in sweet-potato peelings was the isochlorogenic acid band a major constituent, but it seems to be widely distributed as a minor component in other plants. 3. In the members of the Prunus family examined here, samples 8-11, the neochlorogenic acid band was the predominent component.

CAFFEIC AND CHLOROGENIC ACIDS IN PLANTS

137

In samples 1-6, the chlorogenic acid band accounts for the greater proportion of these acids. 4. Band 510 seems to be widely distributed as a minor component. The silicic acid column technique was adapted with only minor modifications to the isolation of crystalline chlorogenic acid from blueberry leaves. It seems likely that this method can be applied to other problems involving isolation of chlorogenic acid and its isomers.
ACKNOWLEDGMENTS The author wishes to thank Mrs. Carole B. Karash for technical assistance and Dr. J. Gorse for a gift of neochlorogenic acid. SUMMARY

1. The silicic acid column technique described by Bulen et al. (9) has been used in a study of the distribution of caff eic acid and chlorogenic acid isomers in plants. Ultraviolet-absorption measurements and titration with alkali were used for the detection of these acids. 2. Resolution into five bands was achieved. Four of these bands had peak effluent volumes that coincided with caffeic acid, isochlorogenic acid, chlorogenic acid, and neochlorogenic acid. Pseudochlorogenic acid was not detected. Free caffeic acid appears to be absent or only a minor component in most plants. Of the eleven plant sources examined, six had the chlorogenic acid band as the major caffeic acid derivative. Only in sweet-potato peelings did the isochlorogenic acid band account for more than 50 % of the total. In all the members of the Prunus family studied, the neochlorogenic acid band comprised the greatest fraction of the chlorogenic acid isomers. 3. One band which was eluted between chlorogenic acid and neochlorogenic acid may be a new caffeic acid derivative and has been designated Band 510. This material was isolated from green coffee beans as a noncrystalline solid. 4. The silicic acid column has been used for the isolation of crystalline chlorogenic acid from blueberry leaves.
REFERENCES 1. BARNES, H. M., FELDMAN, J. R., AND WHITE, W. V., J. Am. Chem. Sot. 73, 4178 (1950). 2. URTANI, I., AND MIYANO, M., Nature 176, 812 (1955). 3. CORSE, J. W., Nature 173, 771 (1953).
4. Kuc, J., HENZE, R. E., ULL, A. L., AND QUACPENBUSH, F. W., J. Am. Chem. Sot. 78, 3123 (1956).

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5. OBATA, Y., AND SAKAMURA, A., J. Agr. Chem. Sot. Japan 27, 766 (19.53); C. A. 49, 16247 (1955). HERMANN, K., Naturwissenschaften 43. 109 (1956). 6. CHENG, R. C., AND HANNING, F., Food Research 20, 506 (1955). 7. MARVEL, C. S., AND RANDS, R. D., JR., J. Am. Chem. Sot. 72, 2642 (1950). 8. MOORES, R. G., MCDERMOTT, D. L., AND WOOD, T. R., Anal. Chem. 20, 620 (1948). 9. BULEN, W. A., VARNER, J. E., AND BURRELL, R. C., Anal. Chem. 24,187 (1952). 10. MABROUK, A. F., AND DEATHERAGE, F. E., Food Technol. 10, 194 (1956).