On the Distribution of Caffeic Acid and The

8/2/2019 On the Distribution of Caffeic Acid and The

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 74, 131-138 (1958)

On the Distribution of Caffeic Acid and theChlorogenic Acid Isomers in PlantsErnest Sondheimerl

From the New York State Agricultural ExperimentStation, Cornell University, Geneva, New York

Received June 26, 1957

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

EXPERIMENTALSeparation of the Chlorogenic Acids

The silicic acid column and solvent schedule described by Bulen, Varner, andBurrell (9) for the standard survey column was adapted for this separationproblem by doubling the quantities of silicic acid, 0.5 N sulfuric acid, and the sol-

1 Present address: Department of Chemistry, State University College of For-estry at Syracuse University, Syracuse 10, N. Y.

131


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132 SONDHEIMERvents. 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 Nsulfuric acid. The samples were applied with 2 g. silicic acid and 1 ml. of 0.5 Nsulfuric acid. Ten-milliliter fractions were collected. Five-milliliter aliquots weretitrated with 0.02 N sodium hydroxide in the presence of water to the bromothy-mol blue end point, and the rest was used for the absorption measurements at 330rnp. Those samples which had an optical density above the upper limit of the Beck-man spectrophotometer, model DU, were diluted with solvent of the same compo-sition as the effluent. Similarly, the composition of the blanks used in the spec-trophotometer was changed as the solvent composition of the fractions altered.The absorption between 310 and 340 rnp was determined at 5-rnp intervals forthose fractions that contained the maximum amount of a given band. Only if anabsorption peak was found at 330 rnp was it considered probable that a caffeicacid derivative was present. Caffeic acid, chlorogenic acid, isochlorogenic acid,and neochlorogenic acid were chromatographed singly and in mixtures, and thepeak effluent volumes and recoveries were determined (Table I). The unknownswere chromatographed by the same procedure. With coffee, blueberry leaves, andsweet-potato peelings, 180 fractions were collected. Since no caffeic acid deriva-tives were detected in fractions 75-180, only 85 fractions were collected with theother samples.We have found it necessary to apply the caffeic acid to the column in solutionand 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-buta-no1 solvent is introduced, and it will be eluted with isochlorogenic acid. Thisfactor is believed to explain the discrepancy between our results and those ob-

TABLE IRecovery of Ca$eic Acid and Chlorogenic Acid Isomers from the Silicic Acid Column

Acids Pe;&3h$nt El%RecoveryUltraviolet-light1 cm. Titration measurements

ml. % %Caffeicb 120 87OC 105 111Isochlorogenic 230 510d 108 87Chlorogenic 360-410 460d 1040 110Neochlorogenic 710 460h - 96

a The peak effluent volume was defined by Marvel and Rands (7) as that vol-ume of effluent collected while a given compound moves from the top of the col-umn to the bottom and is measured at the point at which the greatest concen-tration 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).


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CAFFEIC AND CHLOROGENIC ACIDS IN PLANTS 133tained by Mabrouk and Deatherage (10). These authors also used the column de-scribed by Bulen et al. (9) but depended solely on titration for detection and used5 mg. caffeic acid on an 8-g. column. Under these conditions they assigned the iso-chlorogenic acid band to caffeie acid and failed to detect isoehlorogenic acid,Band 510, and neochlorogenic acid in brewed, roasted coffee.

Preparation of Plant MaterialsAll 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 chro-matographed 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 mixturewas transferred to the top of the column. The blueberry-leaf samples could beused without prior drying by grinding 0.5-g. samples with 1 g. sand, 1 g. silicicacid, and 1 ml. of 0.5 N sulfuric acid. To chromatograph the chlorogenic acid iso-mers from sweet potatoes and coffee, it was necessary to prepare extracts, sinceexcessively wide bands were obtained otherwise. Freeze-dried sweet-potato peel-ings, 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 andevaporated in vacuum. The residue was treated with 3.2 ml. water and acidifiedwith sulfuric acid to pH 2 just before the chromatogram was started. The acidswere chromatographed by placing 1 ml. of this preparation with 2 g. silicic acidon 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, buthad not been freeze-dried.

Although this procedure gave good results with all the fruits tested and withblueberry 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 haveabsorption peaks near 330 mp. Most of the fractions had relatively high absorptionblanks at 330 rnp. Attempts to remove this interfering absorption in the leaf ex-tracts before chromatographing were unsuccessful.

Isolation of Chlorogenic Acid from Blueberry LeavesTo 100 g. fresh blueberry leaves, 600 ml. 70y0 isopropyl alcohol was added andthe 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 with600 ml. of 70% isopropyl alcohol. The combined extracts were concentrated in vac-uum to 240 ml., cooled to 5, and filtered through Celite, and the filtrate was con-centrated to 25 ml. Five milliliters of this extract was used for the isolation of thechlorogenic acid after acidification to pH 2 with sulfuric acid. The column wasprepared from 96 g. silicic acid, 66 ml. of 0.5 N sulfuric acid, and 600 ml. developingsolvent, n-butanol-chloroform, 15:85 (v/v), saturated with 0.5 N sulfuric acid. Acolumn 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 thecolumn. Fifty-milliliter fractions were collected, and the absorption at 330 rng wasdetermined. The bulk of the chlorogenic acid was found in fractions 18-26. Thesewere combined, and the solvent was evaporated in vacuum yielding 180 mg. par-


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134 SONDHEIMERtially crystalline residue. The material was dissolved in 1 ml. absolute ethanoland precipitated as a crystalline solid on the addition of 29 ml. chloroform; yield85 mg., m.p. 193-200. Two recrystallizations from water raised the m.p. to 292-206, mixed melting point with authentic chlorogenic acid 20-205; the chloro-genie acid had been isolated from coffee as the potassium caffeine complex andhad 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 BeansGround coffee beans, 100 g., were extracted twice with 600-ml. portions of 70%

isopropyl alcohol. The combined extracts were concentrated to 50 ml. After acidi-fication to pH 2 with sulfuric acid, 10 ml. of the extract was mixed with 20 g. silicicacid, and 100 ml. of the first development solvent and the slurry were added to thetop of the column. The column consisted of 160 g. silicic acid, 110 ml. of 0.5 Nsulfuric acid, and the first development solvent. Development was carried out withn-butanol-chloroform mixtures that had been equilibrated with 0.5 N sulfuricacid; 2700 ml. of 15y0 by volume n-butanol was followed by 1500 ml. of a 25$& n-butanol solution. One-hundred-milliliter fractions were collected. Ultravioletmeasurements showed most of the Band 510 to be in fractions 32, 33, and 34.These were combined and concentrated in vacuum. A noncrystalline residue re-mained which could not be crystallized from various solvents nor by purificationthrough its lead salt. On the standard survey column the substance was shownto 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% ofthe total absorption at 330 mp, respectively. The ultraviolet-absorption curve wassimilar to that of chlorogenic acid. The highest extinction observed was with asample 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 onWhatman No. 1 with n-butanol-acetic acid-water (4:1:5) gave one fluorescentspot with Rr = 0.59. Under these conditions chlorogenic acid had an R, value of0.66. 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 withaqueous sodium hydroxide and a green color with ferric chloride. Analysis indi-cated 51.5% C and 6.04yc H.

Evidence that the material is a caffeic acid derivative was obtained by hydroly-sis 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 immediateprecipitation of yellow crystals. After storage at 0 for 4 hr., the crystals were col-lected by centrifugation, washed with water, and dried, yielding 16.5 mg. A secondcrop of 3.9 mg. was obtained by extraction of the supernatant and washings withether, total yield 41%. The two crops were combined and recrystallized from wateryielding 12.4 mg. crystals, m.p. 195-201 (decompn.). The mixed melting pointwith 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 beenidentified.


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CAFFEIC AND CHLOROGENIC ACIDS IN PLANTS 135Band 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 ofthe material was obtained a.~ one band with a partition coefficient of 1.4. Twoper cent of material was found in the last three tubes. At a pH of 5.8 Band 510could not be partitioned due to its low solubility in the organic phase.

RESULTS AND DISCUSSIONData on the distribution of caffeic acid and of the chlorogenic acidisomers in a number of plants are summarized in Table II. Becausethe optical measurements allow detection of smaller amounts than withtitration, 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 witha peak eflluent volume of 510 and designated Band 510 was elutedbetween chlorogenic acid and neochlorogenic acid and has not as yetbeen completely identified. In its color reactions and ultraviolet- andinfrared-absorption spectra, Band 510 resembles chlorogenic acid

and its isomers. It has been isolated as a noncrystalline solid with thesilicic acid column from green coffee beans. The preparation is opticallyactive and yields caffeic acid in 41% yield on alkaline hydrolysis. Thewater-soluble moiety has not been identified as yet. When the isolatedmaterial 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 theknown chlorogenic acids.That Band 510 is not identical with pseudochlorogenic acid (2)was shown by the large difference in their partition coefficients. Urtaniand Miyano (2) found a partitioncoefficient of 0.48 for pseudochlorogenicacid between phosphate buffer of pH 5.8 and ethyl acetate. At that pHthe partition coefficient of Band 510 is so much in favor of the aqueousphase that very little is transferred to the ethyl acetate layer.In none of the plant samples examined in this study was pseudo-chlorogenic acid detected. However, this does not prove the absenceof this substance since it is possible that the procedure used here didnot separate pseudochlorogenic acid from the closely related iso-chlorogenic acid.Several conclusions can be drawn about the distribution of the caffeicacid and the chlorogenic acid isomers from the data in Table II.1. The caffeic acid band was either absent or accounted only for asmall percentage of the total ultraviolet-absorbing acids.


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136

Distribution of (Plant source

1. Coffee beansb(Santo)

2. Blueberryleavesb(Jersey)

3. Blueberryfruit (Jersey

4. Apples (McIn.tosh)

5. Pears(Covert)

6. Grapes(Steuben)

7. Sweet-potatopeelings

8. Peaches (LateRose)9. Prunes (Im-

perial Epi-neuse)

10. Plums (For-mosa)

11. Cherries(EmperorFrancis)

SONDHEIMER

TABLE IIffeic Acid and Chlorogenic Acid Isomers in Various Plants

Caff;~k.Gd

W./l00 k!14040

0TracecTracec

011

00

00

%3.:1

0

--1

00

00

TIsochlorogenicacid band

5ng./100 (

4802000

25

:.I

152oc

603

5c10

102c

i%116

10

9I157

71

111

Chlorogenicacid band

m./100 g. %2850 672800 79

190 76105 84135 79140 77335 32

30 4065 7

25 285 4

-1and 510 Neochlorogenicacid bandw./100 g385360

%9

10

lg./loo g.395140

25 10 1oc20 16 Trace15 9 520 11 2c53 5 48

0 0 4040 4 850

153%

1726

4095

%9.54

4-315

38

49

a Concentrations are calculated from the ultraviolet-absorption data.b Concentrations are based on fresh weight; for the other samples the quanti-

ties are calculated on a dry weight basis.c There is some uncertainty about the presence of caffeic acid or the chloro-

genie acid isomers in these bands because the fraction with the highest concen-tration did not have an absorption maximum between 310 and 340 rnp althoughthe peak effluent volumes were normal.

2. Only in sweet-potato peelings was the isochlorogenic acid band amajor constituent, but it seems to be widely distributed as a minorcomponent in other plants.3. In the members of the Prunus family examined here, samples8-11, the neochlorogenic acid band was the predominent component.


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CAFFEIC AND CHLOROGENIC ACIDS IN PLANTS 137In samples 1-6, the chlorogenic acid band accounts for the greaterproportion of these acids.

4. Band 510 seems to be widely distributed as a minor component.The silicic acid column technique was adapted with only minormodifications to the isolation of crystalline chlorogenic acid from blue-berry leaves. It seems likely that this method can be applied to otherproblems involving isolation of chlorogenic acid and its isomers.ACKNOWLEDGMENTS

The author wishes to thank Mrs. Carole B. Karash for technical assistance andDr. 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 chlorogenicacid isomers in plants. Ultraviolet-absorption measurements and titra-tion with alkali were used for the detection of these acids.2. Resolution into five bands was achieved. Four of these bands hadpeak effluent volumes that coincided with caffeic acid, isochlorogenicacid, chlorogenic acid, and neochlorogenic acid. Pseudochlorogenicacid was not detected. Free caffeic acid appears to be absent or onlya 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 accountfor more than 50 % of the total. In all the members of the Prunus familystudied, the neochlorogenic acid band comprised the greatest fractionof the chlorogenic acid isomers.3. One band which was eluted between chlorogenic acid and neo-chlorogenic acid may be a new caffeic acid derivative and has beendesignated Band 510. This material was isolated from green coffeebeans as a noncrystalline solid.4. The silicic acid column has been used for the isolation of crystallinechlorogenic acid from blueberry leaves.

REFERENCES1. 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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On the Distribution of Caffeic Acid and The