chlorogenic acid- springer

C H L O R O G E N I C A C I D S A N D R E L A T E D D E P S I D E S

ERNEST SONDHEIMER S t a t e U n i v e r s i t y C o l l e g e o [ F o r e s t r y x

S y r a c u s e U n i v e r s i t y

S y r a c u s e , N e r o Y o r k

Introduction ................................................................................................................................................................. 667

Isolation and Chemistry ................................................................................................................................ 668 General Isolation Procedures ...................................................................................................... 668 The Chlorogenic Acids ........................................................................................................................ 671 Additional Quinie Acid Depsides .......................................................................................... 677 Other Phenylpropenoyl Depsides ............................................................................................. 680 Transesterifieation Reactions in Quinir Acid Depsides ................................. 681 The Chlorogenic Acid-Caffeine Complex ..................................................................... 682

Distribution of the Chlorogenic Acids and Related Depsides ........................... 683

Biosynthesis .................................................................................................................................................................. 6 8 5 Biosynthesis of Phenylpropenoic Acids ........................................................................... 685 Role of D-Quinie Acid in Higher Plants ..................................................................... 688 Biosynthesis of Phenylpropenoid Depsides ..................................................................... 689 Factors Affecting the Formation of Chlorogenie Acids ................................. 691

Properties of Chlorogenie Acid and Related Depsides ......................................... 692 Properties of Economic Significance ................................................................................. 692 Oxidation of Chlorogenie Acid ................................................................................................ 695 In vitro Activation and Inhibition of Enzymes by Mono- and

Dihydric Phenolies ...................................................................................................................... 697 Do the Chlorogenic Acids Have any Physiological Functions in

Higher Plants? ............................................................................................................................... 701

Literature Cited ...................................................................................................................................................... 705

Addendum ..................................................................................................................................................................... 712

I N T R O D U C T I O N

Chlorogenic acid has attracted continuous attention since it was first

detected in coffee by Robiquet and Boutron in 1837 (95) . It is extremely widely distributed among higher plants and in some cases is found in surprisingly high concentrations. For example, the chlorogenic acids content in unroasted Brazilian coffee beans is 6.3 percent of the total weight; yet the reducing sugars and sucrose account for only one

and 6.4 percent, respectively (114) . An additional reason for the sus-

tained interest is that a great many closely related depsides (esters of

hydroxy acids and aromatic acids) have been discovered recently, and

that this group of substances has a myriad of effects on biological

~This work was supported by a grant from the Council for Tobacco Research U.S.A.

667

668 THE BOTANICAL REVIEW

reactions in vitro. Tantalizingly enough, the role (s) of these compounds in vivo is still very controversial.

Previous reviews concerning chlorogenic acids have been published by Herrman (46) and Politis (89) who compiled extensive lists of plants in which chlorogenic acids are found. A review by Farkas and Kiraly (14) on the role of phenolic compounds in the physiology of plant diseases and disease resistance touches on the chlorogenic acids and offers an excellent resum6 of the work in this area. In this article the isolation, characterization, biosynthesis, and biological properties of these substances will be discussed. The caffeoylglucose esters and related glycosides will not be covered.

ISOLATION AND CHEMICAL PROPERTIES

GENERAL ISOLATION PROCEDURES

Because of their low cost and high chlorogenic acids content, unroasted coffee beans are a particularly convenient source for the isolation of these substances. Chlorogenic acid itself is generally isolated from coffee as the caffeine complex of the potassium salt (71). On acidification and extraction with chloroform, the free acid crystallizes from aqueous solution as the hemihydrare. The other isomers are not precipitated by caffeine under these conditions, although they do form caffeine complexes in solution (118). The chlorogenic acids can also be precipitated with neutral lead acetate as yellow solids, from which the original compounds can be regenerated with hydrogen sulfide. The advent of partition techniques has gready simplified the detection and isolation of these substances and has at the same time established their nearly ubiquitous occurrence in higher plants. Although one- dimensional paper chromatography has been used, it does not generally give good resolution. Better results are obtainable with two- dimensional systems. Common solvents are n-butanol, acetic acid, water mixtures, and aqueous acetic acid. Williams (135) showed that in the latter solvent, chlorogenic acid and other cinnamic acid derivatives give two spots due to the separation of the cis- and trans-isomers. Although the trans-isomer is the more stable, the e/s-derivative can be detected under these conditions, due to its larger Rf value and a hypochromic shift of the ultraviolet absorption maximum. The fluo- rescence of these compounds with ultraviolet light, particularly after exposure to ammonia vapors, affords a very convenient and sensitive method of detection.

CHLOROGENIC ACIDS AND RELATED DEPSIDES 669

Paper chromatography of quinic acid has also led to the appearance of multiple spots (54), attributable, in part at least, to the formation of the 3-1actone. The observation by Butler and Siegelman (7) that caffeic acid (3-4-dihydroxycinnamic acid) is partially oxidized to esculetin (I) during paper chromatography points to another possible source of artifact formation.

I

Akhough countercurrent distribution and column procedures are more time-consuming than paper chromatography, their greater re- solving power and the possibility of obtaining the purified material in appreciable quantity give these techniques a distinct advantage. Countercurrent distribution has been used for the isolation of neochlorogenic acid (11) and 3-feruloyl-D-quinic acid (12). Cellulose columns (129), thin hyer chromatography (107) and electrophoresis (87) have also been employed. Ion exchange chromatography on the conventional polystyrene resins is unsuited for the fractionation of chlorogenic acids beacuse of poor recoveries. Apparently the phenolic moiety is very strongly bound to the resin, and a certain amount of irreversible adsorption occurs. These shortcomings can probably be overcome by the use of modified cellulose ion exchange materials, e.g., diethylaminoethylcellulose and carboxymethylcellulose. Silica gel columns are particularly well adapted for work with chlorogenic acids. Using dilute aqueous sulfuric acid as the stationary phase and a chloroform-butanol mixture in which the butanol content was increased step- wise, Sondheimer separated the chlorogenic acids into four distinct bands (115). This procedure has yielded quantitative information on the distribution patterns of the chlorogenic acids in different plants (Table I), and has also been used in the isolation of chlorogenic acid and related compounds from unroasted coffee beans (117). A refine- ment of this method, employing a linear solvent gradient with cyclohexane, t-butyl alcohol and chloroform, has been described by Hanson and Zucker (37). As will be discussed subsequently, there is danger of rearrangements because of transesterifications during the isolation

670 T H E B O T A N I C A L REVIEW

i,,,,4

~ .u I ~

Z

~ ~.~

~ �9

~ ~

0

z 0

oo to ~o

=o


of the chlorogenic acids. This seems to be more apt to occur with bases than under acidic conditions. Experiments in the author's laboratory have not revealed any evidence for artifact production under the mildly acidic conditions, 0.5 N sulfuric acid at room temperature, utilized in the separations on silica gel columns.

THE CHLOROGENIC ACIDS

CHLOROGENIC ACID. In addition to the physical properties and chromatographic data listed in Table II and Table III, other characteristics are worthy of note. Chlorogenic acid, together with other caffeoyl derivatives, gives a greenish yellow color above pH 7, which changes slowly to brown in the presence of air. With ferric chloride a deep green is obtained. The copper complexes have also been studied (125). The extinction coefticients for chlorogenic acid samples may differ according to their previous history as a result of the varying amounts of "oxidized form" that they contain. Sisler and Evans (112) found that the absorption at 326 m~ of chlorogenic acid in aqueous solu-

TABLE II PHYSICAL CONSTANTS FOR CHLOROGENIC ACID

References

Melting Point 208~ laid =~ - 37 ~ (c 3% in H20) 4 pK1 (carboxylic acid) 3.59 125 pK2 (first phenol) 8.59 125 Cmax. 322 m~ in H20 18500 4 ~max. 324 m~ pH6.8 at 20 ~ 19230 128a ~m~x. 324 m~ pH3.5 at 20 ~ 20600 128a ~m,x. 330 m~ in n-butanol-

chloroform 15:85 v/v 16700 115

Solubility g/100ml solution at 20 ~ 128a methanol 15.2 ethanol 6.2 water 0.59 ether 0.12 ethyl acetate 0.06 chloroform 0.0059 benzene 0.0033


TABLE III

CHROMATOGRAPHY DATA FOR CHLOROGENIC ACIDS

Solvents Rf References

ONE-DIMENSIONAL PAPER

n-PrOH, concd. NH4OH (7:3) 0.10 1331 EtOAc, 2N HC1 (1:1) 0.31 133 10% HOAc 0.64 133 iso-PrOH, H20 (2:3) 0.75 133 benzyl alcohol, HOAc, H20 aqueous phase (4:1 : 5 ) 0.76 133 n-BuOH, HOAc, H20 aqueous phase (4:1:5) 0.78 133 iso-PrOH, H20 (1:4) 0.81 133 H20 0.89 133

2% HOAc 0.56 (main spot) 135 0.75 (minor-ds compd.)

COUNTERCURRENT DISTRIBUTION

K

2M phosphate pH3.0 - EtOAc 1.40 112

SILICIC ACID COLUMN

Peak effluent

volume

n-BuOH, CHCIa 360 ml. 115 ~

1. All Rt values were determined on Whatman number 1 paper at 21• 0.5~ solvent proportions are v/v.

2. The distribution constant K is the ratio of the concentration of ehlorogenie acid in the lighter to that in the heavier phase.

3. The peak effluent volume is defined as that volume of effluent collected while a given compound moves from the top of the column to the bottom and is measured in the fraction of highest concentration. The column consists of 16 g. silicic acid and 11 ml. 0.SN H~SO~; the elution solvent is 200 ml of 5% n-BuOH in CHCh followed by 15% n-BuOH in CHCI3 v/v.


tion can sometimes be raised by addition of ascorbic acid. This was attributed to the reducing properties of ascorbic acid. The infrared spec- trum of chlorogenic acid in a Nujol mull has been published (4).

The presence of an ester group in chlorogenic acid makes acid, base or enzymatic (63) hydrolysis a convenient means of degradation. The hydrolysis products are caffeic acid and D-quinic acid. The structure of the latter substance was established by Fischer and Dangschat (16) as D-trans-l,4,5-ds-3-tetrahydroxycyclohexane carboxylic acid. Additional degradative studies (17) as well as synthesis (85) have established the structure of chlorogenic acid as 3-0-caffeoyl-D-quinic acid (II).

, 2' 'pH

H 0 o~"C'H=C H ~ I'I

I I

Hanson (35) has pointed out that in many textbooks and journal articles the structures of quinic acid derivatives are represented either by ambiguous formulae or as the mirror images of their established structures. This, however, does not reflect any uncertainty about the configuration of quinic acid, since stereochemical studies by Grewe and Lorenzen (32) clearly establish that the isomer with [~]D -----44 ~ in water is D-quinic acid. No work has been reported on the confor- mations of chlorogenic acid. In the absence of large polar interactions in the cyclohexane series, that chair conformation in which the largest number of the bulky groups are in equatorial positions is the most stable one. Therefore, structure III is expected to be the predominant conformer of quinic acid in solution. This is confirmed by the observation that platinum-catalyzed dehydrogenation of quinic acid in water gives 5-dehydroquinic acid in accord with the observation that only axial hydroxyl groups are attacked by this reagent (49). Chlorogenic acid and other quinic acid derivatives, in which the large groups can


HOOC ~ H

III

be accommodated in the equatorial position, would also have this conformation. On the other hand, quinic acid lactone must have structure IV, since the carboxyl group has to be axial.

IV

Reduction with sodium amalgam yields dihydrochlorogenic acid, m.p. 167-168 ~ (30). With acetone and dry hydrogen chloride the diisopropylidene derivative (V) is obtained, m.p. 196-197 ~ (17).

cHi

I /

Acetylation yields the totally substituted 1,4,5,3',4'-pentaacetyl derivative, m.p. 181 ~ which can be partially deacetylated to 1,4,5-tri- acetylchlorogenic acid, m.p. 150-152 ~ (30). A type of conversion that is not fully understood is the reaction of chlorogenic acid with aqueous ammonia in the presence of oxygen. The reaction product (s) has been named viridic acid, but little structural work has been carried out.


Geisman (25) suggested that this reaction may be similar to the oxidative decarboxylation of alpha amino acids by ninhy&in. In this connection it is of interest that Oparin (82) observed that in air chlorogenic acid and glycine react to yield green pigments and carbon dioxide.

ISOCHLOROGENIC ACID. Suspicion that chlorogenic acid is not the only caffeoyl derivative in coffee beans was aroused by the observation that the crystallizable chlorogenic acid-caffeine complex never exceeds about two-thirds of the total caffeic acid derivatives present. This belief was confirmed in 1950 by the isolation from coffee beans of a fraction called isochlorogenic acid (4). Isochlorogenic acid can be separated from chlorogenic acid by extraction into butyl acetate from 2M phosphate buffer, pH 4.7. Barnes et al. (4) were of the opinion that isochlorogenic acid is isomeric with chlorogenic acid and that the caffeoyl group is substituted on the 5-OH group of quinic acid. They ascribed the lack of crystallinity of their sample and the fact that the neutral equivalent is about 40 percent higher than the molecular weight to a mobile equilibrium between the free acid and the 3-1actone. These conclusions have been criticized on a number of points. Thus Bean and Corse (5) point out that an enchancement of conductance of a chlorogenic acid isomer in boric acid solution can be due to either the presence of vicinal hydroxyls or to alpha hydroxy carboxylic acid groups. Therefore the claim by Barnes et al. (4) that this test is evidence for unsubstituted 3,4 hydroxyls on the quinic acid moiety of isochlorogenic acid is not proven. Interpretation of the periodate uptake is also ambiguous, since the reaction does not reach completion in the time used to calculate the ratio of uptake of reagent to amount of oxidized substance. Titration experiments and infrared absorption spectra of isochlorogenic acid have failed to support the claim for a mobile equilibrium between an open acid form and a lactone (117). Also, evidence has become available that isochlorogenic acid, as purified by the procedure of Barnes et al., is still heterogeneous and can be subdivided by chromatographic techniques into several fractions (37, 107).

PSEUDOCHLOROGE~Ir ACID. Uritani and Miyano (127) reported the isolation of several phenols from the sound portion of sweet potato adjacent to a site attacked by Ceratostomella/~mbriata, Ell. & Halst., the organism that causes black rot. One of these, a non-crystalline ma-


terial, was considered a new chlorogenic acid isomer and given the name pseudochlorogenic acid. Its described properties are extremely similar to those of isochlorogenic acid. The major difference would appear to be in the distribution pattern obtained on countercurrent frac- donation. Unquestionably, the same reservations cited above for isochlorogenic acid also hold for pseudochlorogenic acid. In all likeli- hood pseudochlorogenic acid does not represent a new isomer but is merely a somewhat different mixture from that encountered in isochlorogenic acid.

NEOCHLOROGENIC ACID. This compound was first isolated by Corse (11) from peaches by countercurrent distribution and can also be obtained readily from unroasted coffee beans through silica gel column chromatography (117). Hydrolysis data, as well as physical constants, show this compound to be a monocaffeoyl quinic acid. Corse (11) reports a m.p. of 177-179 ~ (decomp.) and [aid ---- --5.4 ~ (50% ethanol), while Scarpati and Esposito (107) give m.p. 204-206 ~ (decomp.) laiD ---- +3.1 ~ Scarpati and Esposito (107) report the preparation of the following derivatives of neochlorogenic acid: lactone, m.p. 222- 225 ~ infrared gamma lactone band, 1790 cm-1; lactone dimethyl ether obtained by heating the Iactone in acetone with methyl iodide and potassium carbonate, m.p. 178-180~ dimethyl ether obtained from above lactone by warming with dilute acetic acid, m.p. 183-185~ methyl 3', 4'-dimethoxycinnamoylquinate obtained by treatment of neochlorogenic acid with diazomethane, m.p. 83-85 ~ No elemental analyses of these derivatives were presented. On the basis of the periodate consumption of the dimethoxy derivative and the chromatographic separation of neochlorogenic acid from synthetic 1-0-caffeoylquinic acid, Scarpati and Esposito (107) suggest that neochlorogenic acid is 5-0-caffeoyl-D-quinic acid.

"BAND 510". Sondheimer (115) isolated a chromatographically homogeneous, non-crystalline caffeoyl derivative, [~]D 24 -"-55 ~ (C 0.4 in water), from coffee that appeared to be an isomer of chlorogenic acid. This material was designated "Band 510" due to its elution volume from a standardized silica gel column. The partition coefficient of "Band 510" between 0.5 N sulfuric acid and butanol-chloroform is somewhat more in favor of the aqueous phase than it is for chlorogenic acid, but less so than for neochlorogenic acid. Quantitative hydrolysis and optical rotation studies have shown "Band 510" to be a mono-


caffeoyl derivative of D-quinic acid (Unpublished work from the author's laboratory) and therefore a true isomer of chlorogenic acid. From the lack of periodate consumption of the diazomethane treated "Band 510", 8carpad and Esposito have suggested that this compound is 4-0-caffeoylquinic acid (107).

1-0-CAFFEOYL-D-QUINIC ACID. Synthesis of this compound has been reported by Scarpati et al. (105), but definitive information on its occurrence in plants is not available. Scarpati and Esposito (107) list the following properties for the compound [~]D = --8.3~ Re 0.65 on Schleicher and Schiill paper 2043 with 2% aqueous acetic acid; Rt 0.27 on silica gel G thin layer chromatography with ethyl acetate- acetone 8:2.

HAUSCHILD'S SUBSTANCE. Hauschild (43) isolated a material, m.p. 235-236 ~ which seemed to be related to chlorogenic acid from leaves of Ilex paraguaryensis St-Hil that had been treated for the preparation of mat& Badin et al. (3) have confirmed this isolation and suggest that the compound is the lactone of neochlorogenic acid produced as an artifact from neochlorogenic acid during manufacture of mat& The following physical constants were reported: m.p. in capillary, sin- tering from 232 ~ 237-239~ [~]D = + 4 9 ~ "-'> +21.9 ~ (24 hr.) (c.0.29 in ethanol); chromatography on S & S 2043 A paper: 0.33 N acetic acid R~ 0.36; butanol, acetic acid, water (4:1: 5) R~ 0.78; butyl acetate, acetic acid, water (4:1:5) Rf 0.48. Scarpati and Esposito (107) gave a m.p. 222-225 ~ for the lactone of neochlorogenic acid.

ADDITIONAL QUINIC ACID DEPSIDES

CYNARIN. Cynarin is the name given to a compound isolated from artichoke leaves through lead acetate precipitation and shown by degradation (84) and synthesis (85) to be 1,4-0,0-dicaffeoylquinic acid. The compound melts at 227-228 ~ (decomp.) and [a]D = -59 ~ (c.4% in methanol). It gives a hexaacetyl derivative, m.p. 168-172 ~ and a tetrahydro derivative, m.p. 134-140 ~ Methylation of cynarin with dimethyl sulfate and potassium carbonate in acetone yields methyl di-3',4'-dimethoxycinnamoyl -1,4-quinate, m.p. 160-161 ~ The proof of structure is based primarily on the isolation of 3,5-0,0-dimethylquinic acid from the hydrolysate of exhaustively methylated cynarin.

3-0-FERULOYL-D-QUINIC ACID. This substance which is the 3'-methyl ether of chlorogenic acid was isolated from unroasted Robusta coffee


beans by counter-current distribution, m.p. 196-197~ [~]D ~--42.8 ~ (ethanol); r at 325 m~ in ethanol is 19,200 (12). The compound has a partition coefficient of 2.7 in 10% sodium chloride pH 2.0 buffer-ethyl acetate; Rf 0.61 in 2% acetic acid and 0.74 in n-butanol, acetic acid, water (20:5:11) on Whatman No. 1 paper. The proof of structure rests on nuclear magnetic resonance spectral studies, conversion of the compound to methyl 0-pentamethylchlorogenate and 1,4,5-0,0,0-trimethylquinic amide.

3-0-p-COUMAROYL-D-QUINIC ACID. Williams (136) isolated a 10- coumaroylquinic acid from immature cider apples which he believed to be the 3-isomer. The material was obtained from a t-butanol ex- tract. After evaporation of the solvent the catechol derivatives were removed by precipitation with lead acetate, phlorizin was separated by extraction of the neutral filtrate with isopropyl acetate. Extraction of the acidified aqueous phase with the isopropyl acetate yielded a residue containing at least two coumaroyl derivatives, one of which could be obtained in a crystalline form after countercurrent distribution, m.p. 247-248 ~ [~]D ------53.5~ r at 250 m~ in 95% ethanol is 14,000. Comparison of the isolated substance with synthetic 3-0-10- coumaroyl-D-quinic acid (40) established the identity of the two preparations. Chromatographic procedures have shown that this compound also occurs in other plants. Hydrogenation of the coumaroyl derivative yielded 3-0-p-hydroxyphenylpropionyl-D-quinic acid, m.p. 201 ~ [~]D -- -44.0 ~ (c. 0.32 in ethanol).

Schiitte and Langenbeck (108) isolated from the white flowers of an Antirrhinum majus, L., mutant a crystalline substance which on hydrolysis gave p-coumaric and quinic acids but is not identical with 3-0-p-coumaroyl-D-quinic acid; m.p. 212-214~ [~]D = -28 ~ (in methanol); R~ 0.76 with butanol, acetic acid and water (4:1:5); and R~ 0.66 with 2% acetic acid.

Sutherland and Gormer (122) detected a substance in the vegeta- tive portions of pineapple plants which gave p-coumaric acid and quinic acid on hydrolysis, but was contaminated by small amounts of a ferulic acid derivative. They tentatively suggest that this substance might be a 1,4-0,0-di-p-coumaroyl ~ quinic acid. The following R~ values were obtained for ascending chromatograms on Whatman No. 1 paper: n-butanol, acetic acid, water (4:1:5) 0.86; phenol water (73:27 W/V) 0.30; acetic acid, concentrated hydrochloric acid, water (30: 3:10) 0.89.


GALLOYLQUINIC ACIDS. Roberts and Myers (94) obtained an amorphous substance, theogallin, from green tea that on hydrolysis yielded gallic acid and D-quinic acid in a 1:1 ratio. Although the four mono- galloylquinic acids now been synthesized (42), the question of the position of the ester bond in theogallin has not yet been resolved. The synthetic Compounds are all amorphous, but the lactone of 1-0-galloyl- D-quinic acid has been obtained as needles from water, m.p. 258-260 ~ [~]n = -19.2 ~ (c. 1.0 in acetone). The physical properties and chromatographic data for the galloyl-D-quinic acids are summarized in Table IV.

A polygalloylquinic acid has been shown to be the major component of tara tannins (41). This material is obtained from the fruit pods of the South American shrub Caesalpinia spinosa, L., and was identified as a 3,4,5-tri-0-galloyl-D-quinic acid derivative, to which two or three galloyl groups are linked, most likely having structure VI.

v'r

CINNAMOYL-D-QUINIC ACIDS. The procedures developed for the isolation of chlorogenic acids are not well suited for the detection of cinnamoyl derivatives. Therefore, even if these compounds are normal plant constituents, they might easily have escaped notice. It has been claimed by Levy and Zucker (63) that a cinnamoylquinic acid was present in potato tuber disks as a result of floating these preparations on 0.05 M L-phenylalanine or 0.01 3/I trans-dnnamate. This claim has been questioned by Runeckles (99) who suggests that Levy and Zucker may have mistaken the cis-isomer of a coumaroylquinic acid for the cinnamoyl derivative.


TABLE IV PHYSICAL PROPERTIES OF GALLOYL-D-QUINIC ACID DERIVATIVES

Quinic acid RI Values 1 Derivatives [a]n A B

1-0-galloyl- -13.6 ~ (c 0.77 in water) 0.69 0.34 3-0-galloyl- -41.3 ~ (c 0.73 in water) 0.62 0.46 4-0-galloyl- -37 ~ (c 0.95 in acetone) 0.68 0.42 5-0-galloyl- -15.4 ~ (c 1.5 in water) 0.72 0.28 4,5-di-0-galloyl- -58.2 ~ (c 0.93 in acetone) 0.51 0.44 3,4,5-tri-0-galloyl- -130 ~ (c 1.8 in water) 0.32 0.52 1,3,4,5-tetra-0-

galloyl- -177 ~ (c 0.77 in water) 0.22 0.42

1. Chromatography was carr ied out at 20 ~ ___ 3 ~ on W h a t m a n No. 2 filter paper. Solvent A was 6% acetic acid; solvent B, 2-butanol, acetic acid and water (14:1:5). (42)

The four possible mono-0-cinnamoyl-D-quinic acids have been pre- pared by Hanson (36). The 1- and 3-isomers were synthesized by direct procedures and the 4- and 5-substituted products were obtained through base catalyzed transesterification of the 1-isomer, separation of the mixture, and structure assignment on the basis of periodate uptake. The melting points reported for the 1-; 3-; 4-; and 5- mono-0- cinnamoyl-D-quinic acids are 195 ~ 166 ~ 157 ~ and 204 ~ respectively.

OTHER PHENYLPROPENOYL DEPSIDES

In addition to the quinic acid depsides, esters of phenylpropenoic acid with other hydroxy acids have also been found. The information on the distribution of these compounds is very meager.

SHIKIMIC ACID DEPSIDES. Goldschmid and Hergert (27) reported chromatographic evidence for the presence of shikimic acid esters of caffeic, ferulic and coumaric acids in the cambial layer of western hemlock. Cambial extracts from other coniferous species failed to yield these depsides. Hanson and Zucker (37) detected similar compounds in potato tuber disks after floating these on solutions containing phenylalanine and quinic acid. Maier et al. (67) isolated 3-0- caffeoylshikimic acid, m.p. 224-225 ~ [~]D -- -124 ~ (c. 1 in 62.5% ethanol) by the use of silica gel column chromatography from fresh green dates. The compound which was named dactylifric acid has


also been synthesized by this group. Additional substances in green dates have been separated as bands by means of silica gel column chromatography and tentatively identified as the 4- and 5-0-mono- caffeoylshikimic acids.

DICAFFEOYL-D-TARTARIC ACID. This compound, m.p. 206 ~ [~]D-- +383.5 ~ (c. 1.555 in methanol), has been isolated from chicory (Ci- ehorium intybus, L.) by Scarpati and Oriente (104). The name chicoric acid has been given to it. The authors have synthesized this substance as well as the rneso and L-tartaric acid derivatives.

CAFFEOYL-L-MALIC ACID. Scarpati and Oriente (106) isolated this substance as a yellow hygroscopic material, [~]D 2~ ---- +28.3 ~ (c. 4.36 in water) from the leaves of kidney beans (Phaseolus vulgaris, L.). The structure was established through degradation and synthesis. The substance has been named phaselic acid.

2-0-CAFFEOYL-3- (3,4-DIHYDROXYPHENYL) -D-LACTIC ACID. This substance, named rosmarinic acid, m.p. 204 ~ (decompn.), [~]D 2~ -- + 145 ~ has been isolated in 2-3% yield from Rosmarinus o~dnalis, L. by Scarpati and Oriente (103). 3-(3,4-dihydroxyphenyl)-lactic acid, [~]i~ ~7 -- +22 ~ has been shown to have the D-configuration.

TRANSESTERIFICATION REACTIONS IN QUINIC ACID DEPSIDES

The presence of unsubstituted hydroxyl groups in sterically favorable positions to the acyl groups of the quinic and shikimic acid depsides suggests that transesterification might be observed. That this is indeed the case has been indicated in a number of recent publications. Badin et al. (3) obtained evidence for the partial isomerization of neochlorogenic acid to "Band 510" as well as hydrolysis to cafl[eic acid in 0.033N sodium hydroxide in 33% ethanol during heating at 55 ~ for one hour. Scarpati and Esposito (107) indicate that boiling of chlorogenic acid, neochlorogenic acid and "Band 510" for 30 minutes in pH 7 buffer leads in each case to the formation of mixtures of all three in about equal amounts. Haslam et al. (42) found that a galloyl group migrates readily from the 1- m the 3- position of quinic acid in sodium bicar- bonate solutions and that the 4- and 5-galloyl groups interchange to some extent in hot acetic acid. Claims that chlorogenic acid isomerizes (54) to two additional isomers during 15 minute treatment at 20 ~ in 5 N sulfuric acid could not be confirmed (117), nor is there any evidence for rearrangements during chromatography of chlorogenic


acids on silica gel columns containing 0.5 N sulfuric acid as the stationary phase (117).

Hanson (35) observed that treatment of 1-0-monocinnamoylquinic acid with one equivalent of barium hydroxide leads to the formation of all four possible isomers plus cinnamic acid and the lactone of the 1-isomer. If the isomerization is carried out in the presence of C 14- quinic acid, none of the rearrangement products are radioactive and the isomerization must, therefore, proceed exclusively by intramolecu- lar processes. Hanson suggests that the wandering of the acyl substituents of quinic acid derivatives occurs in the position sequences 1 ~ 5; 5 ~.-~- 4; 4 ~.~- 3.

Although all of these observations are of a preliminary nature and require more extensive study, they do indicate the possibility of artifact formation during isolations and syntheses, particularly under alkaline conditions.

THE CHLOROGENIC ACID-CAFFEINE COMPLEX

Since 1907, when Gorter (28, 29) showed that potassium chlorogenate crystallized with caffeine in a 1:1 complex, very little additional work has been done in this area. Thus the interesting questions concerning the nature of the attractive force and the possible physiological significance of the complex have not been fully answered.

It has been shown that in the presence of excess caffeine the 322 ml~ peak of an aqueous solution of chlorogenic acid is shifted to higher wavelengths (118). From these spectral studies and partition data it was concluded that a 1:1 complex exists in solution, and an equilibrium constant of 44 • 4 liters moles -1 at 25 ~ was calculated. The state of ionization of the carboxyl group of chlorogenic acid has no effect on the amount of complex in solution and therefore the requirement for the potassium salt must be due to the solubility or crystallizability of the complex. Scarpati and Esposito (107) obtained a crystalline 1:1 complex of neochlorogenic acid and caffeine, m.p. 128-130 ~ Complex formation decreased drastically as the pH was raised to the point where ionization of the phenolic hydroxyl groups occurred. In fact, when excess sodium hydroxide is added to a concentrated solution of the complex, caffeine precipitates.

There seems to be very little difference in the complexing ability of the different chlorogenic acid isomers and caffeic acid in aqueous solution; the benzene ring, the conjugated double bond, and the phe-


nolic groups all contribute to complex stability. The problem of the nature of the attractive force has not yet been settled. The fact that methylation of the phenolic hydroxyls and elimination of the carboxyl group in model compounds does not lead to a diminution of complex strength shows that hydrogen bonding is not the major attractive force. Major contributions from charge transfer interactions have also been eliminated on various grounds. Thus far the complex has been detected only in aqueous solvents.

Reports that potassium chlorogenate decreases the toxicity and increases the diuretic effect of caffeine in rabbits (23) and inhibits the action of caffeine on frog muscle (120) indicate that this type of complex may have physiological significance. From the published equilibrium constant (118) for aqueous solutions, one can calculate the percentage of chlorogenic acid and caffeine complexed at 25~ if it is assumed that the compounds are freely accessible to each other. Under these conditions, in coffee beans that contain 5% chlorogenic acid and 1.5% caffeine, 43% of the chlorogenic acid and 78% of the caffeine would be present as the complex. Since caffeine is not the only compound that will complex with chlorogenic acid (for example, chlorogenic acid and riboflavin also form complexes in aqueous solution (118)), this type of complexation must be considered in the evaluation of the physiological role of chlorogenic acid in coffee beans and possibly also other plants.

DISTRIBUTION OF THE CHLOROGENIC ACIDS AND RELATED DEPSIDES

Although the detection procedures used by different investigators have varying degrees of reliability, there is ample evidence for the wide distribution of this class of compounds in higher plants. Exten- sive lists of plants in which caffeoyl derivatives have been detected have been published by Politis (89) and Herrmann (46). Laurent detected chlorogenic acids and p-coumaroyl derivatives in the prothallia of ferns (61), showing that these substances are not limited to the spermatophytes. Some data on the quantitative distribution of chlorogenic acids are summarized in Table I. From these and other studies it can be stated that free caffeic acid is either absent from, or only a minor constituent in, most plants. Improvements in fractionation procedures continue to reveal an ever increasing number of depsides in the same plant. For example, Hanson and Zucker (37)


showed that white potato tuber contains at least seven caffeic acid derivatives. Also, the four homogeneous depsides that have thus far been isolated from untoasted coffee beans represent only a minimum figure, since further work with the isochlorogenic acid fraction will undoubtedly reveal the presence of additional related compounds.

From the available data, which at this stage are still highly frag- mentary, it would appear that those esters which have substituents on the 3-position of quinic acid and shikimic acid occur at higher concentrations than the other isomers. The evidence for this statement is better for the caffeoyl than for the p-coumaroyl or feruloyl derivatives. However, this distribution pattern is not universal, since in all representatives of the genus Prunus listed in Table I, neochlorogenic acid is the predominant isomer. Up to now, no compounds have been detected in which the carboxylic acid group of quinic acid is esteri- fled with a phenolic hydroxyl of an aromatic acid. But there are frag- mentary reports alluding to the existence of additional types of compounds related to the chlorogenic acids. On the basis of extremely meager evidence, Yunoshev (140) suggests the presence of a glycoside of chlorogenic acid in the tobacco variety Trapenzond 1867. Al- though it seems likely that the substance in question is a glycoside of caffeic acid, not of chlorogenic acid, Yunoshev's report does raise an interesting possibility. In this connection it may be of significance that Kiermeier and Rickerl (56) claimed to have observed a tenfold increase in chlorogenic acid content after steaming freshly harvested potatoes. If this report can be confirmed, it would indicate the presence of substances that are converted to chlorogenic acids under ex- ceptionally mild conditions.

Large variations are known to exist in the relative concentrations in which chlorogenic acids occur in different plants. In addition to

uaroasted coffee beans, flue-cured tobacco is .a particularly rich source, and concentrations as high as 8% on a dry weight basis are obtainable (134). Blueberry leaves and lettuce seed (8) contain 3.54% and 2%, respectively (Table I). Examples of material with a low but easily detectable quantity of chlorogenic acids are apple (McIntosh) and peach (Late Rose), fruits which on a dry weight basis contain 0.13% and 0.08%, respectively (Table I).

The fact that none of the above precedures give any information on the localization of the phenolics within the intact plant tissue makes the semi-quantitative histochemical studies of Reeve (90) on


peach fruit most welcome. According to Reeve, the nitrous acid test can be made fairly selective for catechol derivatives. Stable red colors are obtained with chlorogenic acids and catechol,' while coumaric acids and some methyl coumarates give only fleeting red colors which change to brown. In young peaches the endocarp gives a more intense red color than the mesocarp. The color increase in the endocarp was found to continue until the pits were well hardened. After cessation of mitosis and the beginning of enlargement of the parenchyma cells, a pronounced increase in the "catechol tannins" was detected. On a per cell basis it was found that during the first four weeks of cell enlargement -when cell size increases approximately four-fold--a 10- to 20- fold increase in the red color-yielding compounds occurs. In the later stage of fruit growth, when cell size increased only about 1.5-fold, there was a 50% decrease in "catechol tannins" per cell.

BIOSYNTHESIS It is always easier to establish that a compound can participate in

a given biosynthetic sequence than to prove that the substance is an obligatory intermediate. This is particularly true in studies on the biosynthesis of chlorogenic acid and is one of the main obstacles to rapid progress in this field. However, sufficient work has been done to establish that the biosynthesis of the chlorogenic acids is closely connected to what has become known as the "shikimic acid pathway". It also seems reasonable to assume that this group of compounds is derived through condensation of a phenylpropenoid derivative with D-quinic acid or a related substance. However, when these reactions are considered in more detail, it becomes apparent that a great deal of information concerning specific intermediates, individual steps, enzyme and cofactor requirements is still missing.

BIOSYNTHESIS OF PHENYLPROPENOIC ACIDS

Biosynthesis of benzenoid compounds in higher plants has been reviewed by Neisch in 1960 (77) and will therefore not be discussed in detail here. A very much abbreviated map showing the biosynthesis of phenylalanine and tyrosine in Aerobacter aerogenes (Kruse) is shown in Fig. 1.

It will be observed that D-quinic acid is not directly on the pathway of phenyhlanine or tyrosine synthesis in Aerobacter, although microorganisms that have a quinic dehydrogenase can utilize the sub-


I OOH

Phosphoenol pyruvic acid

HO o.O' D-Qulnic Acid

D-Erythroae 5-Dehydro-D- phosphate quinic acid

o=•OON H2 COOH

HOOC~H D-Shikimic

I -- I Acid

/

Phenylpyruvic acid

Phenylalanine

Prephenic acid

~-Hydr oxyphenylpyruvic acid

H~Hr~H'C~DOH N~

~jrosine

Fig. 1. Biosynthesis of Phenylalanine and Tyrosine in /lerobacter /lerogenes.


stance when it is supplied exogenously (70). At the present time the most attractive scheme for the synthesis of cinnamic and p-coumaric acids in a number of higher plants involves direct deamination of phenylalanine and tyrosine. Koukol and Conn (58) isolated an enzyme, phenylalanine deaminase, from sweet clover that catalyzes the formation of cinnamic acid from phenylalanine. Neish (78) has found a comparable enzyme, tyrase, which acts on tyrosine to yield p-coumaric acid. The enzyme was found in acetone powders from etiolated and light-grown seedlings of sorghum, wheat, corn, barley, oats, rice and sugar cane, but could not be detected in peas, white lupin or white sweetclover.

A number of investigators have shown that phenylalanine and cinnamic acid are converted by plants to caffeic acid derivatives. For

R -- phenyl R v -- p-hydroxyphenyl

R-CH2CH ( OH ) -COOH Rv-CH2 -CH (OH) -COOH

R-CH2-CO-COOH< Shikimic acid >Rv-CH=-CO-COOH

Jt R-CHu-CH (NH=) -COOH Rv-CH2 -CH (NHu) -COOH

Phenylalanine ~ deaminase ~ tyrase

p-hydroxylation R-CH--CH-COOH > Rv-CH =CH-COOH

Fig. 2. Formation of Cinnamic Acids from Shikimie Acid.

example, L-phenylalanine is oxidized to caffeic acid without rearrangement of the carbon skeleton in Nieotiana tabaeum (L). (24). McCalla and Neish (66) found that L-phenyhlanine, (-)-phenyllactic acid, cinnamic acid and p-coumaric acid are all good precursors of catteic acid derivatives in Salvia splendens (Ker-Gawl.). Hanson and Zucker (37) have reported the oxidation of 3-0-p-coumaroylquinic acid to chlorogenic acid in 3% yield with an enzyme fraction from potato cortex. The same enzyme fraction also catalyzed the oxidation of a p-coumaroylshikimic acid to a caffeoylshikimic acid. Identification of


both oxidation products was based mainly on chromatographic and spectral evidence.

Although it is not yet certain that phenylalanine or tyrosine is an obligatory intermediate in the biosynthesis of caffeic acid derivatives, all tracer experiments, including conversion of alpha hydroxy acids to phenylpropenoic acids, can be explained through the participation of these amino acids. The outline shown in Fig. 2 has been suggested by Neish (78) as a general scheme for the interrelation of these substances.

Finkle and Nelson (15) have obtained a meta-O-methyl transferase from cambial layers of apple trees and the woody shrub Pittosporum crassifolium (Cunn.) that catalyzes the methylation of caffeic acid to ferulic acid, and Fales et al. (13) have shown the prescence of a para- O-methyl tranferase in bulbs of Nerine bowdeni (Herb.) S.-Adeno- sylmethionine is required for these reactions.

ROLE OF D-QUINIC ACID IN HIGHER PLANTS

Since quinic acid is an integral part of the chlorogenic acid mole- cule, its biosynthesis and function in higher plants must also be considered. Tracer experiments as well as work with cell-free systems suggest that the shikimic acid pathway functions in higher plants and leads to the synthesis of aromatic compounds. But the role of quinic acid in this scheme is still uncertain, and the possibility remains that it may not be identical with that found in Aerobacter and other microorganisms. In Aerobacter the interconversion of quinic acid and 5- dehydroquinic acid (Fig. 1) is mediated by quinic dehydrogenase (70). However, no such enzyme could be detected in spinach or peas, although the presence of 5-dehydroquinase and 5-dehydroshikimic reductase in these plants indicates that the shikimic acid pathway is operating (70). Attempts by Szymanski (123) to detect quinic dehydrogenase in etiolated wheat seedlings were also unsuccessful, although several other dehydrogenase and shikimic reductase activities were readily demonstrated. The lack of detectable quinic dehydrogenase in these seedlings is particularly noteworthy because of the large changes in the quinic acid content. The quinic acid level, which ~s very low in the seeds before germination, reaches a maximum of 0.6 mg/g. fresh weight after six clays and then declines rapidly to approximately 0.1 mg/g. During this time the changes in the concentration of shikimic acid are much less striking. Large changes in the quinic


add levels during the development of wheat had been shown pre- viously by Caries and Lattes (9). If the synthesis of quinic acid in higher plants occurs by the same general process at it does in microorganisms, then 5-dehydroquinic acid must be reduced to quinic acid. Until an enzyme that catalyzes this reaction is found, it will be premature to conclude that the biosynthesis of quinic acid in higher plants is identical to that of bacteria.

Tracer studies by Weinstein and co-workers (130, 131, 132) on the conversion of quinic acid to aromatics in higher plants have also failed to indicate the exact position of quinic acid with regard to the shikimic acid pathway. Uniformly CX4-1abeled D-quinic acid was converted to shikimic acid, phenylalanine and tyrosine, with relatively high specific counts (131, 132). However, with some plants the amount of respiratory C1402 produced (as high as 24.2% of the total C 14 absorbed in Kalancho~ leaves) was much higher than could be accounted for by the decarboxylation of prephenic acid. Also the specific activity of the phenylalanine isolated from rose petals 2.5 hours after incorporation of CI4-labeled quinic acid was 7.8 times as high as that of the isolated shikimic acid (132). This has led Weinstein et al. (132) to suggest that in the rose, shikimate may not be in the direct pathway between quinate, tyrosine and phenylalanine.

BIOSYNTHESIS OF PHENYLPROPENOID DEPSIDES

Tracer experiments by Reid (91) showed that in Nicotiana tabacum phenylalanine is incorporated into the chlorogenic acid fraction. Very little label was found in the quinic acid portion, and the dilution of the specific activity was much less with phenylalanine than with acetate or phenylacetate. Reznik and Urban claimed that ferulic acid can be converted to chlorogenic acid in wheat and corn leaves (92) and in red cabbage seedlings (93). However, Runeckles (100) showed that with leaf disks of Nieotiana tabacum in light, ferulic acid does not yield chlorogenic acid but is incorporated into feruloylquinic acid. Since the separation techniques used by Reznick and Urban do not resolve feruloyl and caffeoylquinic acids, Runecldes suggests that they may also have been dealing with feruloylquinic acids. Zucker and co-workers (36, 63) have studied chlorogenic acid synthesis by floating disks cut from Kennebec potato tubers in shallow layers of solutions. When cultured with appropriate compounds, chlorogenic acid and related compounds build up to much higher concentrations


than in the intact tuber. With this system Levy and Zucker (63) confirmed Reid's finding that phenylahnine is incorporated into the caffeoyl portion of chlorogenic acid and showed that quinic and cinnamic acids stimulate chlorogenic acid synthesis. Randomly labeled glucose-C 14 was incorporated into chlorogenic acid with only a two- fold dilution in radioactivity on a carbon basis, and 65% of the activity was in the quinic acid moiety. Quinic acid markedly stimulated chlorogenic acid production but did not decrease the specific activity in the caffeoyl portion when randomly labeled C14-phenyhhnine was also present. Phenylpyruvate, phenylhctate, p-coumarate and shikimate did not stimulate chlorogenic acid synthesis. The toxicity of caffeic acid to these tissues prevented its evaluation in this system. Trans- cinnamic acid was less effective than phenyhlanine, but in the presence of quinic acid the two aromatics stimulated chlorogenic acid production to the same extent. These results are readily reconcilable with the hypothesis that phenyhhnine is converted to cinnamic acid through the mediation of phenyhhnine deaminase (58). Furthermore, it lends support to the belief that chlorogenic acid is formed by condensation of a phenylpropenoid unit with quinic acid or a related derivative.

In their 1960 paper Levy and Zucker (63) chimed to have obtained evidence for synthesis of cinnamoylquinic acid in potato disks bathed with 0.005 M trans-cinnamic acid and 0.05 M sodium quinate. They also suggested that this ester is a direct precursor in the synthesis of chlorogenic acid via p-coumaroylquinic acid. However, di~cukies are being encountered in attempts to verify this claim. Runeckles (99) found no clear-cut evidence that p-coumarolyquinic acid is an intermediate in chlorogenic acid synthesis in tobacco, although this possibility was not ruled out. Furthermore, attempts by Runeckles to detect a cinnamoylquinic acid in both tobacco leaf and potato tuber after the administration of radioactive cinnamic acid were unsuccessful (99). The author suggests that the compound believed to be cinnamoylquinic acid by Levy and Zucker may be the eis-isomer of p-coumaroylquinic acid. This is based on similarities of the chromatographic behavior in 5% acetic acid and of the spectral properties of these two substances.

Future progress will depend to a great extent on whether cell-free systems can be found in which the formation of these depsides can be studied. Of particular interest would be information on the mechanism of esterificarion. Does the aromatic compound add as an activated


carboxylic acid derivative, e.g., as a coenzyme A ester, or does the quinic acid moiety become activated? Do the aromatic glucose esters play a role in chlorogenic acid synthesis, as has been suggested by Harbone and Corner (38)? Another intriguing question concerns the interrelationship between the chlorogenic acid isomers. Are they synthesized individually or are they interconverted through enzyme- catalyzed transesterification processes? Grifliths (33) found a decrease in chlorogenic and p-coumaroylquinic acids during growth of young leaves of Theobroma cacao ( L ) . Concomitant with this decrease was the appearance of the two substances, one of which might be neochlorogenic acid, suggesting the possibility of interconversions. One can also speculate that compounds like cynarin, i.e., dicaffeoylquinic acid derivatives, may play a role in the equilibration of various isomers. If this is the case it would be analogous to the equilibration of glucose-1 and 6-phosphate by phosphoglucomutase which is known to require catalytic amounts of glucose-l,6-diphosphate. There is also a possibility that variations in the ratio of the different chlorogenic acid isomers in different plants do not reside in differences in their rates of synthesis but in the rate at which they are destroyed or utilized.

FACTORS AFFECTING THE FORMATION OF CHLOROGENIC ACIDS

The fact that chlorogenic acids and other aromatics build up in plant tissues as a result of attack by fungi, bacteria, viruses ,or mechani- cal injuries, has been well documented by Farkas and Kir~ly (14). Frey-Wyssling and Babler (20) and Lott (65) found that the chlorogenic acids content of tobacco grown in greenhouses is very low but can be increased to a level even higher than that of the field-grown crop if the normal greenhouse illumination is supplemented with ultraviolet light. The effect of the leaf age of Theobroma cacao on the relative concentration of chlorogenic acids and i0-coumaroylqttinic acid has been investigated by Griltiths (33). Both decreased in amount during growth and disappeared at maturity. Wolf (138) found that the concentration of neochlorogenic acid decreased during ripening of sweet cherries. Hanson and Zucker (37) believe that synthesis of phenolic substances in the intact potato tuber may be limited by lack of substrates as well as lack of oxygen. Increases in the rate of respiration of potato disks brought on by aeration is accompanied by new protein synthesis (10) and by accumulation of chlorogenic acids (63). Zucker (143) showed that the enhancement of chlorogenic acid ac-


cumulation in potato disks, brought on by light with transmission maximum of 525m~, is also accompanied by new protein synthesis. Compounds such as ethionine and chloramphenicol, which inhibit protein synthesis, also lower chlorogenic acid levels. In view of these findings, Zucker suggested that the high capacity for phenolic synthesis results from a light-induced development of chloroplasts in the slices.

In a number of cases where infection led to accumulation of phenolics, a concomitant increase in pentose phosphate shunt activity was observed (110). Increased activity of glucose-6-phosphate and 6-phos- phogluconate dehydrogenases has been found in cell-free extracts of Phytophthora-infected potato (97) and in rust-infected wheat (57). Farkas and Kir~ly (14) have discussed the possible relation between pentose phosphate shunt activity and enhanced synthesis of phenolics more fully.

PROPERTIES OF CHLOROGENIC ACID AND RELATED DEPSIDES

PROPERTIES OF ECONOMIC SIGNIFICANCE

PHARMACOLOGICAL PROPERTIES. Chlorogenic acids do not appear to have pronounced pharmacological action. Their possible modifying effects of the physiological action of caffeine (23, 120) have already been mentioned. Panizzi and co-workers (84) have shown that most or all of the choleretic and cholesterinolytic action of artichoke (Cynara) group is attributable to cynarin. Mancini et al. (68) confirmed the hypocholesterolemic action of cynarin with 23 arteriosclerotic patients. They showed that use of cynarin decreased the average cholesterol content from 256 to 192 rag/100 ml. serum. The fine dust from raw coffee beans is known to be the cause of bronchial asthma, rhinitis and dermatitis of sensitized persons. Freedman et al. (18) have suggested that the major allergen in green coffee beans, to which sensitive workers react, is chlorogenic acid. The substance can cause the same bronchial asthma, rhinitis and dermatitis as the coffee beans. Intradermal in- jection of one microgram of chlorogenic acid in the skin sites of passively sensitized normal persons produced large wheals and flares. Although caffeic and quinic acid alone were non-allergic, large doses of a mixture of the two acids gave the allergic symptoms at a low response level. Evidence is presented that chlorogenic acid acts as a hapten in human skin or mucous membrane. Siddiqi and Freedman


(111) also suggest that part of the allergenic activity of castor bean and orange is attributable to their chlorogenic acid content.

PROPERTIES OF INTEREST IN" THE FOOD AND TOBACCO INDUSTRIES. Wilkinson et al. (134) have suggested that the chlorogenic acid content of tobacco be used as a grading criterion, since the leaves with the higher chlorogenic acid content gave the more desired color on processing. Zucker ~rld Stinson (142) found a direct correlation between the concentration of chlorogenic acid in the fresh leaf and of brown pigments in the cured leaf. No direct chemical role in the browning reaction could be assigned to the plastid pigments. In variegated leaves of Connecticut shade tobacco, the chlorogenic acids level, which was higher in the green portions, accounted for the variegated pattern of browning in the cured leaf. Jacobson (53) obtained similar results on feeding radioactive chlorogenic acid and rutin to ex- cised shade-grown tobacco leaves and letting them brown. Evidence for the participation of chlorogenic acid as well as rutin in brown pigments formation was found.

During roasting of coffee, extensive decomposition of chlorogenic acid occurs, and the formation of dark brown pigments is attributable to chlorogenic acid decomposition. Lenmer and Deatherage (62) found that between 32 and 52 percent of the chlorogenic acid was destroyed during roasting.

There is general agreement that the chlorogenic acids play a domi- nant role in the polyphenol oxidase-catalyzed browning of apples (45), Fears (133), green pods of broad beans (75), tea leaves, sour cherries, peaches and others. Weurman and Swain (133) found three fluorescent degradation products when chlorogenic acid was oxidized with polyphenol oxidase from apples. Although the nature of the oxidation products is not precisely known, it does seem likely that orthoquinones form first and that these then polymerize or decompose, possibly with further aerobic oxidation, to brown pigments. Rogachev and co- workers (96) showed that the darkening of potato tubers observed on sterilization with ionizing radiation is partially due to polyphenol oxidase-catalyzed oxidation of chlorogenic acid.

Non-enzymatic discoloration of steamed potatoes has been attributed to a reaction between chlorogenic acid and ferrous salts (56). Oxy- gen is taken up during the color change. The degree of discoloration


was found to parallel the amount of added ferrous salts and is inten- sified by a high pH but not by copper or manganese salts.

THE ROLE OF CHLOROGENIC ACIDS IN PLANT DISEASES AND DISEASE RESISTANCE. This topic has been reviewed recently by Farkas and K/filly (14), and no attempt will be made here to cover this area in detail. Frequently one finds an increase in the concentration of polyphenolics during fungal and viral infection of many plants irrespective of whether or not the variety is resistant or susceptible. The claims that the chlorogenic acid concentration before infection can be cor- related with the degree of resistance in a number of plant diseases has been questioned. What does seem to be correlatable is the speed with which polyphenols accumulate in infected tissue, resistant reactions being associated with faster accumulation (57). In addition, susceptible combinations of host and parasite accumulate ascorbic acid, whereas in resistant species the concentration of ascorbic acid decreases during infection. These observations can be understood on the assumption that the oxidation products of chlorogenic acids and other ortho-dihydric phenols are the actual fungistatic or antiviral substances. If these oxidation products can be produced fast enough, then the infection will be localized. The decrease in ascorbic acid in resistant tissue may very direcdy lead to the higher rate of oxidation of the polyphenolics. In this connection it is very suggestive that resistance of rice plants to Cochliobolus miyabeanus (Ito & Kuribayashi) Drechsler ex Dasmr. can be broken with reducing agents such as ascorbic acid or glutathione (81). Generally, although chlorogenic acid or caffeic acid may have some weak bacteriostatic (119), fungistatic (70) and antiviral (139) activity, the quinones produced during oxidation exhibit a much higher degree of inhibition (14). Evidence is also available which shows that once oxidation has advanced to the brown pigment stage, the fungistatic properties are lower than in the intermediary quinone stage (14).

It is well established that changes in the concentration and metabolism of phenolics play a significant role in the production of disease symptoms following invasion of higher plants by fungi, bacteria or viruses. However, the specific role that can be assigned to any one compound, for example, chlorogenic acid, is still highly problematic. There are two major reasons for this: first, sufficient data on the changes in the concentration of phenolics are not available; secondly,


not enough is known concerning the function of specific phenolics in the activation and inhibition of the enzymes.

OXIDATION OF CHLOROGENIC ACID

Chlorogenic acid and related compounds are very readily oxidized, either aerobically in an alkaline pH region, or enzymatically with polyphenol oxidase. Ingraham and Corse (52) found that the initial rates of oxidation over a pH range of 7.49 to 8.74 are directly pro- portional to the chlorogenic acid concentration and to the partial pressure of oxygen, and vary inversely with the hydrogen ion activity. The activation energy for autoxidation is 13.6--+ I Kcal/mole. The inverse hydrogen ion relationship shows that the monoionized pheno- late is very probably the reactive species in this pH range. Since polyphenol oxidases contain copper, the effect of the addition of cupric ions on the rate of autoxidation was studied. However, the rate of the autoxidation of chlorogenic acid was unaffected at pH 7.7 by cupric ions in the range of 0.30 to 7.6 rag/liter. Addition of a copper- complexing agent, thenoyltrifluoracetone, aIso was without effect. It could also be shown that the oxidation is not light-sensitive.

In the presence of oxygen, catechol derivatives are oxidized by polyphenol oxidase to o-quinones. The latter are highly reactive chemical species which can be oxidized further by secondary reactions to poorly characterized brown polymers. Paper chromatography of the chlorogenic acid after treatment with polyphenol oxidase from apples showed three fluorescent oxidation products (133). The chlorogenic acid oxidation products formed early in the reaction can be reduced again to starting material by ascorbic acid (112). However, if the oxidized product is allowed to stand until brown pigments are present, the reversal by ascorbic acid is incomplete. The polyphenol oxidases after purification are still associated with varying cresolase activity and therefore these enzyme preparations are usually active toward a wide range of monophenolic and o-diphenolic substrates. Evidence is available that plants contain more than one polyphenol oxidase. Thus, Sisler and Evans (113) found that the ratio of the specific activity of mushroom and tobacco extracts toward chlorogenic acid and catechol changed as a function of purification. With tobacco (113) and potato (79,2) extracts the oxidation of chlorogenic acid was more rapid than that of catechol, while the reverse prevailed in mushroom preparations (113). Enzymatic oxidation of chlorogenic acid results in a


large decrease in light absorption between 280 and 370 mlz. It has been suggested that the absorbance change at 326 m~ be used as a direct assay for chlorogenic acid oxidase activity (112).

The long argument concerning the role of polyphenol oxidase and chlorogenic acid in aerobic respiration of higher plants seems to be finally resolved. The concensus is that cytochrome-c-oxidase is the most important terminal oxygen acceptor and that the role of the polyphenol oxidases is at most a secondary one. Evidence for this view has been supplied by many studies, but the work by Nakabayashi (76) is particularly germane. Oxygen uptake by apple slices increases only temporarily after addition of chlorogenic acid, catechins or care- chol, the normal substrates for polyphenol oxidase. The respiration of slices is not inhibited by p-nitrophenol or p-nitrocatechol, both of which are known inhibitors of apple polyphenol oxidase. On the other hand, respiration is inhibited by carbon monoxide and this inhibition is reversed by light. This argues strongly for participation of cytochrome-c-oxidase in respiration instead of polyphenol oxidase, since inhibition of the latter by carbon monoxide is not reversed by illumination. One experimental fact which argues against participation of the typical cytochrome system in all plant tissues is that respiration is frequently unaffected, or may even be promoted, by cyanide and carbon monoxide (34). For example, in potato tuber slices the rate of oxygen consumption increases approximately fourfold, respiration becomes relatively insensitive to the above inhibitors, and chlorogenic acid levels rise (143). However, since carbon monoxide and cyanide are potent inhibitors of polyphenol oxidase, the increase in the rate of respiration cannot be attributed to this enzyme, It seems quite probable that cytochrome-b enzymes are responsible for this modified type of respiration (34).

The rate of succinic acid oxidation by mitrochondrial fractions from apple peel can be greatly enhanced by addition of polyvinylpyrrolidone to the extraction medium (55). While it appears likely that the mode of action of the polyvinylpyrrolidone is a removal of inhibition due to certain phenolic compounds, chlorogenic acid, caffeic acid, phlorid- zin and o-coumaric acid do not appear to be involved. Addition of 0.001 M solutions of these phenolics to the isolated particles caused at most a 10% decrease in the activity of the succinic acid oxidase. At higher concentrations (0.02 M) chlorogenic acid brought about


an uptake of oxygen about equal to succinate, and when the two were added together the oxygen consumption was almost additive.

IN VITRO ACTIVATION AND INHIBITION OF ENZYMES BY MONO- AND DIHYDRIC PHENOLICS

Studies on the oxidation of reduced pyridine nudcotides, NADPH and NADH, and on indolcacetic acid, IAA, attest to the fact that the activity of peroxidases is strongly influenced by phenolics (1, 22, 31, 72). As a general rule, those phenols which are di~cultly oxidized to quinones act as activators or are inert, while ortho and para dihydric phenolics may exert inhibitory influences. However, the concentration at which a compound is tested and the presence of other activators may be of critical importance. For example, Gormer and Kent (31) found that in the absence of other activators, fcrulic acid stimu- Iares the oxidation of IAA by IAA or2dase from pineapple. However, if 10-coumaric acid was present, ferulic acid at a somewhat higher concentration had an inhibitory effect. One possible explanation for this behavior is that competitive binding of the cofactors by the enzyme is involved.

Comparison of the characteristics of the peroxidases from horseradish, pea epicotyls and spruce shoots indicates that the oxidizing systems for IAA and the reduced pyridine nuclcotides may be identical (22). Gamborg et al. (22) showed that hydrogen peroxide, manganese ions and a phenolic activator are essential for the oxidation of NADH by peroxidases and that the oxidation of NADH required one half mole oxygen per mole substrate. Omission of hydrogen peroxide increased the length of the lag phase without affecting the final maximum oxidation rate. This is attributable to the ability of peroxidase to generate hydrogen peroxide in the presence of Mn ++. However, Mn ++ also appears to have a direct role in the oxidation of NADH, since hydrogen peroxide alone is not sufficient to obtain maximum oxidation. Of the phenolics evaluated by Gamborg et al. for the oxidation of NADH, p-coumaric acid was the most potent activator. Its activation effects were inhibited competitively by ferulic acid and hydroquinone. Other compounds that can be oxidized by the peroxidase- manganese-phenolic system are NADPH, reduced glutathione, cytochrome c (1), cysteine, ascorbic acid (22), and IAA (72).

Extracts from many different plants have been shown to catalyze


the oxidation of IAA and are, therefore, said to have IAA oxidase activity. Akhough the pH optima of these preparations differ frequently, reported values ranging from 3.5-7.0, these variations may be caused by the assay conditions, since addition of Mn ++ or the use of citrate buffer lowers the pH optima (137). Hinman et al. (51) have shown that, in the absence of added hydrogen peroxide, the principal oxidation product obtained from IAA with crystalline horseradish peroxidase is 3-methyleneoxindole (VII). Since many of the IAA oxidase prepa-

VII

rations are very crude, it is not always necessary to add Mn ++ or a phenolic activator for the demonstration of activity. However, it is believed that these substances are required by all systems, and in at least one instance, the extracellular enzyme(s) of crown-gaU from Parthenocissus tricuspidata (Sieb. and Zucc.) Planch., the oxidation of IAA does not occur unless Mn ++ and a phenolic activator are added (137, 64).

Gormer and Kent (31) have evaluated the ability of a large number of aromatic compounds to serve as cofactors or inhibitors of IAA oxidase from pineapple. These substances were tested with crude enzyme that had been freed from naturally occurring inhibitors and coenzymes. The solutions contained 0.005 M Mn ++ and were at pH 4.0. p-Coumaric acid, alpha- and beta-naphthols were the most effective activators; 20 m~zmoles of p-coumaric acid were as effective as 300 m~zmoles of 2,4-dichlorophenol, of 400 m~moles of scopoletin, 700 m~- moles phenol and 2000 m~moles of resorcinol, o-Coumaric acid, p-phy- droxybenzaldehyde and tyrosine, as well as such substances as 2,4- di- chlorophenoxyacetic acid, alpha naphthylacetic acid, gibbereUic acid and kinetin, were inert. Ferulic acid, as stated above, has activator properties when tested in the absence of other phenolics, but acts as an inhibitor in the presence of p-coumaric acid. The ability of ferulic acid to suppress IAA oxidation by the enzyme also depends on the


IAA substrate levels. Evidence was obtained that in cases where apo- enzyme is limited but p-coumaric acid is present, ferulic acid seems to compete with IAA for the same site on the enzyme. Where IAA is present in appreciable quantities but p-coumaric acid is limited, ferulic acid shows competitive inhibition with p-coumaric acid. However, this was true only for relatively narrow concentration ranges.

Gormer and Kent (31) found that a large number of phenols have an inhibitory effect on the IAA oxidase from pineapple in the presence of p-coumaric acid; o-dihydric phenols were the most strongly inhibitory. Since 3,4-dihydroxyphenylalanine, dihydrocaffeic acid and caffeic acid were equally inhibitory, the conjugated double bond of caffeic acid is not a requirement for inhibition of pineapple IAA oxidase. On a molar basis and under the assay conditions used by Gormer and Kent (31), chlorogenic acid is three times as effective as caffeic acid, eight times as effective as hydroquinone or 5-hydroxytryptophan, 130 times as effective as guaiacol and 3200 times as effective as scopoletin. Similar results were obtained with IAA oxidase from other sources, an exception being 2,4-dinitrophenol which inhibits the enzyme from pineapple (31), stimulates that from Lens culinaris (Medik.) (88) and is without effect on the enzyme from peas (26). The activity of the IAA oxidase from peas is also very strongly influenced by the relative concentration of p-coumaric and chlorogenic acids (116). As little as 1 tzg/ml of either p-coumaric or chlorogenic acid can produce large changes in the rate of IAA oxidation with the enzyme preparation from etiolated peas. The chlorogenic acid isomers and "isochlorogenic acid" were as effective as chlorogenic acid, while caffeic acid was somewhat less effective (116).

A number of additional phenolics have been isolated from higher plants which influence the in vitro activity of IAA oxidase. The buds of etiolated pea plants which were exposed to red light prior to harvest were shown to contain a kaempferol p-coumaroyl triglucoside which has IAA oxidase-inhibitory properties (73). Additional studies in this area have been carried out by Furuya et al. (21) and Mumford et al. (74).

Mechanisms which would seem to account for at least some of the facts concerning the activation and inhibition of peroxidase by phenolics have been proposed by Akazawa and Conn (1), Gamborg et al. (22), and Gormer and Kent (31). The initial steps are believed to be concerned with the aerobic formation of hydrogen peroxide and


the oxidation of the phenolic activator, possibly to an enzyme-bound, reactive free radical, RO'. The latter species would then serve as the oxidant of NADH and in the process become reduced again. These reactions, in summarized form, are ( 1 ) :

Mn + +, H202 2 ROH + 02 > 2 RO* + H202

peroxidase

peroxidase H202 + 2ROH ) 2 RO" + 2H20

4RO" + 2 N A D H + 2H + > 4 ROH + NAD +

These reactions account for the observed stoichiometry and for the functioning of Mn ++ and the phenolic activators in catalytic amounts. Although the nature of the oxidized phenolic product(s) RO" is unknown, it has been shown that resorcinol (1) and coniferyl alcohol (19) are oxidized by peroxidase in the presence of hydrogen peroxide. Since ferulic and p-hydroxyphenylpyruvic acids have intermediate activating effects when tested alone, their inhibitory activity in the presence of p-coumaric acid may be due to competitive binding at a common site on the enzyme (31). The oxidation of IAA is assumed to proceed by a similar mechanism to that proposed for the reduced pyridine nucleotides.

The reason for the inhibition of NADH or IAA oxidation in the presence of ortho or para dihydric phenols is not at all clear. It is conceivable that this phenomenon is causally connected with the ease with which these substances are oxidized to quinones, but there is little experimental basis fo~ this conjecture. There is some evidence that the inhibition is connected with eithel the generation or utiliza- tion of hydrogen peroxide. Witham and Gentile (137) reported that in the presence of added hydrogen peroxide, chlorogenic acid does not inhibit the oxidation of IAA by either horseradish peroxidase or enzymes from crown-gall tissue slices. Sacher (101) found that if the inhibitor is added before the enzyme, a lag in the oxidation is produced, but at the end of the lag phase the oxidation rate is comparable to that in the absence of the inhibitor. This seems to argue for destruction of the inhibitor during the lag period. It has been suggested that the inhibitors act as free radical traps which are destroyed in the course of the reaction.


The activity of several other enzymes is also influenced by phen- holies. Schwimmer (109) found that potato phosphorylase is inhibited by chlorogenic acid; a concentration of 10 -a M produced approximately 50 percent inhibition under his assay conditions. Braunstein (6) had pointed out earlier that transaminases are inhibited by para and ortho qulnones as well as certain dihydric phenols. Methionine (69) and 3,4-dihydroxyphenylalanine decarboxylases (39) are known m be inhibited by chlorogenic acid. Since all of the above enzymes contain pyridoxal phosphate, Schwimmer's suggestion that the chlorogenie acid inhibition of potato phosphorylase may be connected with the activity of this coenzyme merits further study. Krogmann and Stiller reported that chlorogenic acid, caffeic acid, quercetin, quercitrin and catechin enhance photosynthetic phosphorylation in spinach chloroplasts (59). Addtional activating effects for chlorogenic acid have been reported by Herzmann for the oxidation of 3,4-dihydroxyphenylalanine by an enzyme from bean leaves (47), for the oxidation of adrenaline by horseradish peroxidase (48) and by Sakamura and Obata (102) for the oxidation of pelargonidin 3-monoglucoside by an anthocyanase from eggplant.

Do THE CHLOROGENIC ACIDS HAVE ANY PHYSIOLOGICAL FUNCTIONS IN HIGHER PLANTS. )

There appear to be two major roles for which the chlorogenic acids might be considered; they may serve as mobilizable reserve materials and they may function in growth regulation. Their activity in various aspects of plant diseases, although well established, is not considered a physiological response, since it is usually accompanied by extensive loss of cellular integrity.

CHLOROGENIC ACIDS AS MOBILIZABLE RESERVE SUBSTANCES. In a number of plant tissues, particularly seeds, the chlorogenic acids content is quite high, and the possibility that these phenols act as reserve substances deserves discussion. Studies of the changes in the chlorogenie acid levels during germination of sunflower (98) and lettuce seeds (8) have shown large differences. During germination of sunflower seeds in the dark at 23 ~ the chlorogenic acids levels, expressed on a per seedling basis, decreased by a factor of 6 to 7 during the first five days. After this period a plateau was reached. On the other hand, in the germination of lettuce seeds no changes in the chlorogenic acids


were detected during the first two weeks. The dry seeds contain two percent chlorogenic acids, and after the two-week germination period very little, if any, changes in the chlorogenics acid level had occurred in the cotyledons and very little, if any, was translocated into the stems or roots. In the William pear (126), leaves of Theobroma cacao (33) and in sweet cherries (138) the chlorogenic acids levels decrease with age.

Isotope competition studies by Higuchi and Brown (50) and earlier work by this group showed that p-coumaric, caffeic and ferulic acids can participate in lignification. But it is not known whether the p- coumaroylquinic acids, chlorogenic acids and feruloylquinic acids or their shikimic acid analogues represent mobilizable reserve pools for these phenylpropenoic acids. Goldschmid and Hergert (27) found no evidence to support the hypothesis that the phenylpropenoylshikimic or quinic acid derivatives serve as precursors in lignin biosynthesis in conifers. Thus it has been established that in some, but not all, of the plants examined the levels of the chlorogenic acids change. No valid information on the turnover numbers of these compounds has been reported. And although the phenylpropenoic acids can be incorporated into lignin, it is not known whether their quinic and shikimic acid depsides serve as mobilizable pools. Finally, it must a l so be stated that no information is available on the possible metabolic functions of the quinic and shikimic acid moieties of these depsides.

DO THE CHLOROGENIC ACIDS AND RELATED PHENOLICS INFLUENCE

GROWTH IN HIGHER PLANTS? Vendrig and Buffel (128) found that caffeic acid at a concentration of 1 ~g/ml or less greatly enhances elongation of Avena coleoptile sections, and they suggested "that this substance may be a very important growth regulator, not less important than IAA." Thimann et al. (124) have questioned this interpretation and proposed that the observed effects are due to synergistic action of the caffeic acid with IAA. They found that in the absence of exogenous IAA, caffeic acid ( 3 X 1 0 -6 M to 1 X 1 0 -5 M) and 1 X 1 0 -0 M chlorogenic acid, catechol and hydroquinone caused no promotion of Arena coleoptile elongation over the water control. However, in the presence of a level of IAA (25 ~g/liter) which by itself had no effect, these substances produced small but significant growth incre- ments, about 10-47%, above those of the IAA controls. Thimann et al. suggested that these actions probably rest on inhibition of the IAA


oxidizing system. Henderson and Nitsch (44) and Nitsch and Nitsch (80) have extended these studies, using the Avena first internode test which also involves a response to exogenous auxins. Chlorogenic acid, caffeic acid, "isochlorogenic acid", protocatechuic acid, quercetin, quercitrin, rutin and alpha-tocopherol all promoted the elongation of the first internode sections above that of IAA, but had little or no effect with alpha-naphthylacetic acid or with gibbereflic acid. p-Coumaric acid strongly inhibited the promotion of mesocotyl elongation by exogenous IAA, but had much smaller effects on the elongation produced by alpha-naphthylacetic acid or gibberellic acid. Mso, t0-coumaric acid obliterated the chlorogenic acid effect in the presence of IAA, and chlorogenic acid reduced the loss of IAA from the incubation solutions in the presence of first-intemode Sections. No inhibition of elongation could be demonstrated for 10 -4 M p-coumaric acid in the controls that did not contain exogenous IAA. The behavior of the phenolics in the growth responses can be explained on the basis of their regulatory action on IAA oxidase. Those phenols which inhibit IAA oxidase in in vitro systems have a sparing effect on the exogenously supplied IAA and therefore promote growth. Phenols which enhance the in vitro IAA oxidase activity inhibit the promotion of mesocotyl elongation by exogenous IAA.

The effect of phenolics on the rate of decarboxylation of carboxyl- Cl~-labeled IAA by dark-grown Avena coleoptile was investigated by Zenk and Miiller ( 141 ). For the tests involving the phenolics they used 3-mm long dark-grown Avena coleoptile sections that were shaken gently at 21 ~ in solutions containing IAA and phenolics. Only where decarboxylation of IAA in the absence of phenolics was studied did these authors use 2-cm long coleoptile that had been dipped into col- lodion at the cut end to avoid wound effects. The rate of IAA decomposition was followed by collecting carbon dioxide at stated intervals from mixtures containing coleoptile sections floating in IAA solution. From these data the authors draw conclusions concerning the in vivo role of IAA oxidase. In view of the lack of proof that all of the IAA decarboxylation detected in the presence of phenolics actually occurred within the coleoptiles and not in the solutions, these conclusions seem to rest on dubious grounds. Short coleoptile sections with two cut surfaces may quite possibly release IAA oxidase into the solutions during 12 hours equilibration. In fact, evidence has already been presented (121) that tissue cultures of EDhedra release IAA oxidase into


the nutrient medium and into aqueous washings, and that IAA is destroyed exogenously during these conditions. Thus, until it is proven that all of the radioactive carbon dioxide comes from IAA that has been transported into the coleoptile sections and that the carbon dioxide release is actually the result of IAA oxidase activity instead of some other enzyme, e.g., a non-oxidative decarboxylase, the conclusions of Zenk and M/filer concerning an in vivo role for IAA oxidase seem premature.

Zenk and Miiller (141) also calculate "IAA uptake in mtmaoles/ g/12 hr" from the radioactivity of hot ethanol extracts of the coleoptile secdons. From these data the authors infer a cause and effect relationship between the amount of radioactive IAA in the tissue and the rate of decarboxylation of the IAA in the presence of various phenolics. While their data do support this contention in a number of cases they violate it in others. In fact, of the 13 phenolics examined, only with six is there a clear relation between IAA decarboxylation rates and the level of the radioactive IAA in the coleoptile sections. For example, the concentration of radioactive IAA found in coleop- dies that had floated in 10 -5 M IAA without phenols, IAA plus 10 -4 M caffeic acid and IAA plus 10 -4 M ferulic acid was 46.0, 46.0 and 45.6 m~moles IAA/g/12 hrs., respectively. Yet caffeic acid inhibited the rate of IAA decarboxylation by 47% and ferulic acid enhanced it by 41% for the twelve-hour period. Conversely, in the presence of cinnamic acid and tyrosine, which were without effect on the IAA decarboxylation rate, the radioactive IAA concentration within the sections was lower than in the IAA controls (39.9 and 40.0 m~moles/ g/12 hrs., respectively).

In summary, it has been demonstrated that exogenously supplied phenolics can affect elongation growth of Arena sections. According to Thimann et al. (124) and Nitsch et al. (44, 80), these growth effects are observed only in the presence of exogenously supplied IAA and therefore these phenolics are considerd to act synergistically with IAA. This non-additive growth effect is attributed to activation or inhibition of IAA oxidase by these phenolics. Vendrig and Buffel (128) do not accept this view. The arguments advanced by Zenk and M0.11er (141) that their data support an in vivo role for IAA oxidase seem to this reviewer to have inadequate experimental support. It would appear that in none of the reports in which the role of the phenolics on elongation has been evaluated has the possibility of exo-


genous IAA oxidase activity been exclude& If it were found that I A A

oxidase is released from the coleoptile sections into the growth medium, then the response produced by the phenols could be explained simply

on the basis of a change in the exogenous IAA levels. The reviewer

therefore feels that these experiments do not permit any conclusions

concerning a growth-regulatory function for phenols in intact plants

in the absence of exogenously supplied IRA.

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A D D E N D U M

Haslam, et al., have synthesized and characterized the 1-, 4-, and 5-0-p coumaroylquinic acids. The structure of neochlorogenie acid has been confirmed by this group as 5-0-caffeoylquinic acid through synthesis. Haslam, E., Makinson, G. K., Naumann, M. O., and Cunnlngham, Jill. 1964. Synthesis and properties of some hydroxycinnamoyl esters of quinic acid. Jour. Chem. Soc. 1964: 2137-2146.

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Earlier claims (18, 111) that chlorogenic acid has allergenic properties and is involved in the atopic hypersensitivity to unroasted coffee, castor beans and oranges have been questioned. Layton, L. L., Greene, F. C., Corse, J. W., and Panzani, R. 1964. Pure chlorogenic acid not alergenic in atopy to green coffee: a specific protein probably is involved. Nature 2113: 188.

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