Botanical Journal of the Linnean Society ( 1984), 89: 3 13-3 19.

Flavoaoids and other phenolics of Cichoriurn and related members of the Lactuceae (Compositae)

SARAH REES AND JEFFREY HARBORNE

Department o f Botany, Plant Science Laboratories, University o f Reading, Whiteknights, Reading RG6 2AS

Received June 1984, accepted for publication October 1984

REES, S. & HARBORNE, J. B., 1984. Flavonoids and other phenolics of Cichoorium and related members of the Lactuceae (Compositae). Extraction of the leaves of chicory, Cichorium intybus, revealed the presence for the first time of the 3-glucuronides of kaempferol, quercetin and isorhamnetin, the 3-glucoside of kaempferol and the 7-glucuronide of luteolin. The same compounds appear to occur throughout the genus. An aglycone survey of the leaves of 240 other members of the Lactuceae failed to reveal any other source of isorhamnetin, although quercetin and kaempferol were found as occasional constituents in 12 other genera. In Lactuca, a survey of 12 species showed eight with quercetin present. In general, though, flavonols are more uncommon than flavones and thus have more potential as taxonomic markers within the tribe. The coumarins cichoriin and aesculin and the caffeoyl-tartaric acid ester, chicoric acid, were also found to be present widely in the tribe.

KEY WORDS:-Chemotaxonomy ~ chicory - cichoriin - flavone glycosides - flavonol glycosides ~

isorhamnetin.

CONTENTS

Introduction . . . . . . . . . . . Material and methods . . . . . . . . .

Plant material . . . . . . . . . Phenolic analysis . . . . . . . . .

Results . . . . . . . . . . . .

Flavonoids of Lactuca . . . . . . . . Other flavonol-containing members of the Lactuceae

Discussion . . . . . . . . . . . .

References. . . . . . . . . . . .

Flavonoids of chicory leaves . . . . . .

Acknowledgement . . . . . . . . . .

. . . . . . . . 313

. . . . . . . . 314

. . . . . . . . 314

. . . . . . . . 314

. . . . . . . . 315

. . . . . . . . 315

. . . . . . . . 317

. . . . . . . . 317

. . . . . . . . 318

. . . . . . . . 319

. . . . . . . . 319

INTRODUCTION

As part of an analysis of the defence chemistry of the chicory plant, a detailed study was undertaken of the flavonoids and related phenolics in Cichorium intybus L. and related Cichorium species. This work also forms part of a continuing

3 I 3 0024/85/080313 + 07 $03.00/0 0 1984 The Linnean Society of London

314 S. REES AND J. B. HARBORNE

chemotaxonomic investigation of members of the Lactuceae (or Cichorieae), the tribe to which Cichorium belongs, for their flavonoid constituents (Harborne, 1977a, 1978).

Cichorium is a small genus of nine species of European and Mediterranean origin, two of which are cultivated, C. intybus and C. endivia L. Previous flavonoid studies have shown the presence of apigenin 7-arabinoside, luteolin 7-glucoside, quercetin 3-galactoside and quercetin 3-rhamnoside in the leaves (Dem’yanenko & Dranik, 1973). The anthocyanins of the petals (Harborne, 1977b) and the leaves (Bridle et al., 1984) have also been identified. The related salad plant, C. endivia, has been analysed and the 3-glucoside and 3-glucuronide of kaempferol have been isolated (Woeldecke & Herrmann, 1974). Lactuca sativa L., the lettuce plant, which is chemically related to chicory in having the same sesquiterpene lactones (lactucin and lactupicrin) (Gonzalez, 1977), is reported to have some of the same constituents, namely the 3-glucoside and 3- glucuronide of quercetin, quercetin 3-malonyl-glucoside and luteolin 7- glucuronide (Woeldecke & Herrmann, 1974).

Otherwise, relatively little systematic work has been carried out on the flavonoids of the Lactuceae (Gonzalez, 1977) and the present survey of Cichorium, Lactuca and 34 other genera is the first to establish the basic pattern in this tribe.

MATERIAL AND METHODS

Plant material

Dried leaf samples were taken from specimens in the Herbarium, Reading University, and these have been so labelled. Fresh material of Cichorium intybus (seed obtained from the Botanischer Garten der Philipps, Universitat Marburg) was grown in the Experimental Garden of the Botany Department, Reading University. Leaves of species of Lactuca of authentic origin were kindly donated by the staff of the Royal Botanic Gardens, Kew.

Phenolic analysis

Alcoholic extractions and two-dimensional paper chromatograms were made, developed and analysed according to standard procedures. Cichoriin and aesculin were identified by co-chromatography with authentic standards. Chicoric acid was isolated by preparative paper chromatography and hydrolysed in alkali to yield tartaric and caffeic acids. Flavonoid aglycones were identified after acid hydrolysis and co-chromatography against standards. Isorhamnetin, which runs close to quercetin or kaempferol in many of the standard TLC solvents, was clearly separated from them by TLC in chloroform- acetic acid-water (2 : 1 : 1) and this method was routinely used in all surveys (Harborne, 1984). The sugars of the flavonoids were analysed after acid hydrolysis by paper chromatography in four solvents and the position of attachment deduced by the ultraviolet spectral measurements. Glycoside identifications were confirmed in almost all cases by co-chromatography with authentic markers in at least four solvents.

FLAVONOIDS OF COMPOSITAE 3 1 5

RESULTS

Flavonoids o f chicory leaves

Two-dimensional paper chromatography of leaf extracts of Cichorium intybus revealed the presence of two major flavonoid components, one corresponding to a flavone glycoside fraction and the other to a flavonol glycoside fraction. The first fraction, on purification, gave a single compound, readily identified as luteolin 7-glucuronide. By contrast, the second fraction was found to be a mixture of several closely related glycosides, which, on purification in other solvents, yielded five substances: the 3-glucuronides of kaempferol, quercetin and isorhamnetin, the 3-glucoside of kaempferol and the 3-galactoside of quercetin. All these flavonoids were identified by standard hydrolytic and spectral procedures and by direct comparison with authentic markers.

Of the six compounds reported (Table l ) , only one, quercetin 3-galactoside, was described in an earlier study of chicory leaves (Dem’yanenko & Dranik,

Table 1. Flavonoids of Chicory Leaves

Dem’yanenko & Dranik, 1973 This paper

Apigenin 7-arabinoside

Luteolin 7-glucosidr Luteolin 7-glucuronide

Quercetin 3-galactoside Quercetin 3-galactoside

Quercetin 3-rhamnoside Quercetin 3-glucuronide Kaempferol 3-glucoside* Kaempferol 3-glucuronide* Isorhamnetin 3-glucuronide

*Reported by Woeldecke & Herrmann (1974) in Cichorium end&.

1973). Two further compounds, kaempferol 3-glucoside and 3-glucuronide, were reported in the closely related C. endivia (Woeldecke & Herrmann, 1974), so their occurrence in C. inQbus is to be expected. What is surprising is that three compounds reported earlier in this plant, namely apigenin 7-arabinoside, luteolin 7-glucoside and quercetin 3-rhamnoside, could not be detected, although authentic markers were available for comparison. The reason for this discrepancy is not clear.

Furthermore, a survey of several accessions of cultivated and wild C. intybus and of three other Cichorium species (C. endivia, C. pumilum Jacq. and C. spinosurn L.) indicated that the flavonoid pattern in the leaves was the same throughout the genus (Table 1). Thus, all taxa gave isorhamnetin, kaempferol, quercetin and luteolin on acid hydrolysis and the pattern of glycosidic spots on 2D chromatograms was identical throughout to that of C. intybus. On most 2D chromatograms, there were traces of other flavonoid components, but these occupied a position usually taken up by C-glycosylflavones and from their behaviour on acid treatment, it is reasonable to assume that they were probably C-glycosides. Insufficient material was available for further characterization.

Tab

le 2

. Fla

vono

id p

atte

rns

in l

eave

s of

Lac

tuca

Spec

ies

2D p

atte

rn

Agl

ycon

es*

Cou

ntry

Fl

avon

e Fl

avon

ol

of o

rigi

n 7-

gl y

cosid

es

3-gl

ycos

ides

C

icho

riin

Q

uerc

etin

L

uteo

lin

Api

geni

n

L. v

irosa

L.

Bri

tain

+

+ +

+ +

+ L.

alta

ica F

ische

r &

C. A

. Mey

er

Bri

tain

+

+ +

trac

e +

L. ta

taric

a (L

.) C

. A. M

eyer

B

rita

in

+ +

+ tr

ace

+ +

L. sa

tiua

L.

Bri

tain

+

+ +

+ +

L. sa

ligna

L.

Fran

ce

+ +

+ tr

ace

+ L.

serr

iola

L.

Fran

ce

+ +

+ +

+ +

L. tr

zque

tra B

enth

. & H

ook

f. C

ypru

s +

+ +

L. gr

aeca

Boi

ss.

Gre

ece

+ +

+ +

+ L.

acan

thifo

lia (

Will

d.) B

oiss

. T

urke

y +

+ +

+ +

L. te

nem

'ma

Pour

ret

Spai

n &

Afr

ica

+ -

+ -

+ +

L. pe

renn

is L.

M

aced

onia

+

+ +

L. a

culea

ta B

oiss

. Kot

schy

ex

Boi

ss.

Iraq

+

-

+ -

+ +

-

-

-

-

+ -

- - -

+ -

9

z U

FLAVONOIDS OF COMPOSITAE 3 1 7

Flauonoids o f Lactuca

In view of the chemical similarities in sesquiterpene lactone content between Cichorium and Lactuca, it was of interest to compare their flavonoid patterns. A survey of 12 Lactuca species (Table 2 ) showed that luteolin was consistently present, and that quercetin and apigenin were also common. 2D chromatography of direct leaf extracts indicated that the two flavones were usually present as 7-monoglycosides and that quercetin was present as 3- glycosides. Some yellow-fluorescing components, probably of a flavonoid nature, were also cornrnonly present but could not be identified further. The characteristic coumarin of Cichorium, cichoriin, was also almost always present in the Lactuca samples examined (Table 2).

As can be seen from Table 2, the presence or absence of quercetin 3-glycosides in Lactuca shows some correlation with the geographical distribution of the species. These compounds are mainly restricted to European mainland species and do not occur in the species from the more southerly and easterly parts of the Mediterranean. Preliminary studies of two further African species and two Asian species suggest that flavonols are more generally absent from non- European Lactuca but further surveys are needed to establish this geographical correlation.

Other Jiauonol-containing members o f the Lactuceae

The presence of flavonols throughout both Cichorium and Lactuca (see above) is of taxonomic significance, since the predominant flavonoid type reported earlier in the leaves of Lactuceae is flavone, and particularly the presence of luteolin (Harborne, 1977a, b, 1978). A survey of a selected sample of some 230 members of the tribe was therefore initiated and flavonols were recorded in major amounts in some 27 further taxa (Table 3) . The other 203 species lacked flavonols, having only flavones (usually luteolin) . Interestingly, no further records of isorhamnetin were found, so that this compound appears to be a distinctive marker for the genus Cichorium.

In general, the flavonols quercetin and kaempferol appear to be relatively uncommon as major leaf constituents in the tribe, their frequency of occurrence being 16%. In addition to Cichorium and Lactuca (Table 1, 2), they appear to be regular constituents in only two other genera of those surveyed, in 10 species of Scorzonera and in five species of Tragopogon (Table 3) . However, quercetin, has been recorded earlier in two species of Agoseris, in two species of Microseris and in Krigia uirginica (L,.) Willd. (Harborne, 1977a). In some of the genera in which flavonols occur (,Table 3) , they are relatively rare in their appearance. For example, 30 species of Sonchus were surveyed, and only S. pustulatus showed the presence of quercetin, in addition to the ubiquitous luteolin. The same is true in Cicerbita, where the only flavonol-positive species appears to be C. plumieri. Four other species surveyed (C. bourgaei Beauverd, C. haimmania Beauverd, C. macrophylla (Willd.) Wallr. and C. tenerrima (Pourret) Beauverd) contained luteolin and apigenin. In this case, the result might be of taxonomic significance, since this and other species of Cicerbita have in the past been placed alternatively in the genera Sonchus or Mulgedium. This chemical distinction would indicate that C. plumieri might need separation from other members of Cicerbita, as

318 S. REES AND J. B. HARBORNE

Table 3. Species of Lactuceae with flavonols as major leaf constituents

Aglycones present Species Quercetin Kaempferol Luteolin Apigenin

- - + Cicerbita plumieri (L.) Kirsch. -

Crepis divaricata Boiss. & Meidr. C. pygmea L. + Hyoseris radicata L. + Koelpinia linearis Pallas + Rhagadiolus stellatus (L.) Gaertner + Rothmaleria granatensis (Boiss. ex DC.) Font Quer + Scariola tetrantha (Burtt & Davis) Sojak + Scolymus hispanicus L. + + S . maculata L. - Scorzonera graminifha L. + S. austriaca Willd. var. angustifolia DC. + + S. crispatula (Boiss.) Boiss. + S. hirsuta L. + S . hispanica L. + S. laciniata L. + + + S. mollis Bieb. + S. pseudolanata Grossh. + S. pusilla Pallas + Sonchus pustulatus Willk. + + + Tragopogon crocijolius L. + 7. hybridus L. + 7. longirostris Bischoff ex Sch. Bip. T. porrijolius L. + 7. pratensis L. + + + Urospermum picroides (L.) Scop. ex F. W. Schmidt

- + + +

- + - - - -

- - - - - -

- - - - + -

- - - - +

- + +

+ +

- - -

- - -

- - - -

- + +

- - - -

- - - - - -

- + - - - - + - - - - - - - +

~ ~~

In addition, glycosylflavones were detected in some species of Scorzonera, Sonchus and Tragopogon.

constituted at present (Tutin et al., 1976), but other relevant taxa need to be analysed before a judgement can be made.

It was confirmed during this survey that the major leaf flavonoids in members of the Lactuceae are indeed flavones, usually luteolin in 7-glycosidic combination. C-glycosylflavones based on luteolin and apigenin have been recorded earlier in Catananche and Tragopogon (Gonzalez, 1977) and were noted during these analyses to be present also in Andyala , Koelpinia, Scorzonera and Sonchus. In fact, they appear to be fairly widespread, being more common in those genera in which flavonols are recorded (Table 3). The widespread, almost universal occurrence of the simpler phenolics cichoriin, aesculin and cichoric acid in leaf extracts was also observed in the 230 or more taxa surveyed.

DISCUSSION

As a result of the present studies, the flavonols kaempferol and quercetin have been identified as being common in at least four genera, Cichorium, Lactuca, Scorzonera and Tragopogon. They have also been recorded in 10 other genera, several of which are monotypic. The relative infrequency of flavonols within the Lactuceae, which contains over 70 genera, suggests that they have more potential as taxonomic markers than have the more common flavones. For example, the two generic pairs with flavonols, namely Cichorium and Lactuca on one hand and Scorzonera and Tragopogon on the other, are usually classified close

FLAVONOIDS OF COMPOSITAE 119

together in most systems of classification (Tomb, 1977). In Cichorium these flavonols occur as 3-glucuronides, as well as 3-glucosides and 3-galactosides, so that there is some interesting variation in glycosidic type deserving of further exploration within the tribe.

The Aavone pattern in the leaves of the Lactuceae seems to be more uniform, being based on the widespread presence of apigenin and luteolin, as 0- and/or C-glycosides. There is the distinctive occurrence noted earlier (Harborne, 1978) of the flavone isoetin (5,7,2',4',5'-pentahydroxyflavone) in leaves of subtribes Leontodinae and Crepidinae and the presence of scutellarein in many of the same plants. Otherwise, the leaf flavones show few other variable features within the tribe and, similarly, the occurrence of simple phenylpropanoids such as cichoriin seems to be almost universal. Further chemotaxonomic exploration of the phenolics of the Lactuceae would seem to be more profitably directed towards the floral constituents and work along these lines is in progress.

ACKNOWLEDGEMEN'I

This study wa:; funded by SERC in collaboration with Messrs Tate & Lyle Limited.

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HARBORNE, J. B.. 1978. The rare flavone isoetin as a yellow pigment in Heyzroodielln olzgocephala and in othcr

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