Epicuticular Flavonoid Aglycones in the Genus Cistus, Cistaceae

Epicuticular Flavonoid Aglycones in the Genus Cistus, Cistaceae'~)

I) Botanisches lnstitut der Universitat zu Kein, Gyrhofstr. 15, D·5000 Koln 41, Bundesrepublik Deutschland 2) Institur ftir Pharmazeurische Biologie, Technische Universitat Braunschweig, Mendelssohnstr. 1, D-3300 Braunschweig, Bundesrepublik Deutschland

Received January 1, 1987 . Accepted April 22, 1987

Summary The epicuticular leaf resins of 16 species and 3 subspecies of the genus Cistm were analysed with regard to theif flavonoids. A total of 51 different flavonoids including flavanones, flavones, and flavonnis could be idemified. Many of these compounds are common and wide-

spread flavonoid methyl ethers, some 6- and 8-0-methylated flavonols, several myricetin

methyl ethers found, however, are rare natural products. In addition to the flavonoids two coumarin derivatives were identified in the leaf resins of several species. The flavonoid patterns elucidated are more or less species specific. Based on the flavonoid pattern we are able to comment on several difficult imer- and intraspecific taxonomic problems in the genus Cistus.

Key words: Cistus, Cist.aceae, epicuticuJar flavonoid aglycones. chemot.axonomy. Introduction The mediterranean macchias and garigues are the predominant habitats for members of the genus Cistus. Cistus plants are widespread throughout the whole mediterranean region from the Iberian peninsula up to the eastern part of Turkey. Sixteen different species are known in this region (Warburg, 1968). In addition, two Cistus species are endemic to the Canary Islands (Kunkel, 1980). Cistus taxa are woody evergreen shrubs. In the Cistaceae they are characterized by their white to purplishred colour of always five petals as well as their fruit capsules consisting of five, six, or ten valves (Warburg, 1968). The leaves of Cistus plants are covered with hairs and glandular trichomes. The trichomes secrete a resin, which mainly consists of terpenoids (Pascual Teresa et aI., 1972, 1986; Giilz et aI., 1984). Only recently flavonoid aglycones have been found as constituents of this resin (Wollenweber and Mann, 1984; Proksch and GUlz, 1984; Vogt et aI., 1987). There have been some reports on several flavonoid aglycones detected in Cistus, some years ago, but localisation of these substances was not quite clear (Pascual Teresa et ai., 1974, 1977, 1978, 1979, 1982, 1983 a, 1983 b). We now want to give a summary of epicuticular flavonoids of *) Dedicated to Prof. Dr. H. Reznik on the occasion of his 65th birthday.

Abbreviations: he, kaempferol; que, quercetin; myr, myricetinj ap, apigeninj lut, luteolinj

ce, column chromatography;

TLC, thin layer chromatography; UV, ultraviolet; MS. massspectrometry; Tol, toluene; HOAc, acetic acid; MeCOEt, methylethylketon;MeOH, methanol.

J. Plant Piry>iol. Vol. 131.pp. 25-36 {1987}

26

THOMAS

VOGT,

PETER PROKSCH,

and

P AUL-GERHARD

GOLZ

sixteen species and three subspecies of the genus. In addition to our analytical interest we tried to apply the elucidated flavonoid patterns as chemotaxonomic characters (Harborne, 1977), in comparing our results with those obtained previously by Poetsch and Reznik (1972, Poetsch 1973) on the flavonoid glycosides of Cistus. Materials and Methods Cistus plants, all grown from seeds collected at their natural habitats, were cultivated in the field of the Botanical Institute of Cologne. C osbeckiae/olius was obtained as dried material from Teneriffa. The plants were harvested in August 1984, buds and faded leaves removed and the resinous material was obtained by dipping the leafy twigs in CHCh. Yields of resin per dry weight are listed in Table 1. To remove the waxes from the resinous material the extracts were dissolved in warm MeOH (50- 60 OC), 1 g extract/laO ml MeOH. Subsequently the solutions were cooled down to -20 °C over night. This resulted in the precipitation of the MeOH unsoluble wax from the soluble resin, which contained the flavonoids. After evaporation of the MeOH the crude extract was directly chromatographed over Sephadex (l00 g Sephadex LH-20, columns 1 m length, 2.5 cm diameter), to separate the flavonoids from the terpenoids that eluted first. Isolation of individual compounds was achieved by the usual procedures of CC on Polyamid SC-6 with Tal and increasing amounts of MeCOEt and MeOH as eluants, TIC on

Poly.mid DC·6 and DC-ll solvent system ToIIMeCOEt/MeOH 10/2/1, 13/5/3 and 6/4/3,

on Cellulose, solvent system 20% and 40% HOAc as well as on silica gel 60 precoated plates (Merck, Darmstadt), solvent system CHCh/MeOH 9/1 and Tol/Dioxan/HOAc 90125/4. Different application and combination of these chromatographic systems resulted in the isolation of individual compounds, which were finally purified over Sephadex LH-20 prior to UV analysis.

Table 1: Amounts of epicuticular leaf extracts and their relative amounts of flavonoids in the genus Cistus L. specIes

extract

relative amounts of flavonoids

sympbyti/olim osbeckiaefolius albidm incanus ssp. villosus incanus ssp. tauricus incanus ssp. corsicus incanus ssp. creticus crispus parviflorm monspeliensis psilosepalus albanicus C salvifolius C populi/alius C. laurifolius C. ladani/er C. paihinhae C. clusii C. libanotis

13.4

++++ ++ ++ + ++ ++ ++ ++ ++ +++++ +++ +++

C. C C. C C C C C. C. C C. C

J.PlantPhysiol. Vol. 13I.pp. 25-36 {1987}

(% of dry W(.) 1.4 1.6-2.3 0.6 3.7 2.0 2.4 4.0 2.0 15.5 4.7 6.9 0.5 9.0 8.1 10.8 13.3 10.2-10.8 7.3

+++ ++++ ++++ ++++ ++++ ++++

Epicuticular flavonoid agJycones in Cistus

27

Identification of the isolated compounds was achieved by co-chromatography with authentic samples, Rf-values, colour reactions with and without Naturstoffreagenz A (NA), as well as by and MS-analysis. The MS-data were recorded with a Varian MAT, EI-mode, 70eV. Sources of authentic samples were available in most cases (Wollenweber and Mann, 1984; Proksch and Giilz, 1984; Vogt et aI., 1987). The coumarins were detected by their intense blue fluorescence (UV-350nm) and identified by UV- and MS-data and by direct comparison with authentic samples. Data of the rare 6- and 8-0-methylated flavonols in Cistus aibanicus and C parviflorus have already been published (Vogt et aI., 1987). Data of the rare myricetin methyl ethers and the coumarins are as follows. Myricetin·J,J',4',5'.tetrametyl ether; deep purple (brown with NA) on Polyamid DC-ll (Toll

uv-

MeCOEt/MeOH 13/5/3) Rf 0.61; UV Amax: MeOH 268, 306 (shoulder), 344; +NaOMe 277, 290 (sh), 374; +AlCl, 276, 306, 354, 402 (sh); +AlCI, +HCI276, 307, 354, 402 (sh), +NaOAc 276,304 (sh), 354; + NaOAc +H,BO, 271, 304, 344; MS: mlz (tel. int.) 374 (M)+ (100); 359 (M· 15)+ (40); 331 (M-43)+ (35); (Stivasta"" aI., 1981). Mymetin-3,7,4'·trimethyl ether: deep purple with NA; on Polyamid DC-II (ToI/MeCOEtl MeOH 13/5/3) Rf 0.54; UV Amax: MeOH 264, 304 (sh), 345; + NaOMe 266, 372; + AlCl, 275, 304 (sh), 350, 396; +AlCI, +HCl 277, 304 (sh), 343, 396; +NaOAc 263, 347; +NaOAc +H,BO, 264, 346; MS: mh. (eel. int.) 360 (M)+ (100); 345 (M-15)+ (15); 317 (M-43j+ (18) 167 (AI + H) + (20) (Hen,ick and Jeffeties, 1964). Myricetin·J,7,Y·trimethyl ether: purple (orange-red with NA): on Polyamid DC-ll (ToIlMe-

COEt/MeOH 13/513) Rf 0.43; UV Amax: MeOH 253, 263 (sh), 303 (sh), 359; +NaOMe 263, 406; +AlCl, 274, 314 (sh) 442; +AlCI,+HCI274, 308, 367, 403; + NaOAc 258, 298 (sh) 390; +NaOAc + H)BO) 258, 295 (sh) 379 (Krishna Kumari et al., 1984).

Myricetin-3',5'-dimethyl ether: yellow (green with NA); on Polyamid DC-ll (ToIlMeCOEtl MeOH 13/5/3) Rf 0.24; UV Amax: MeOH 259 305 (sh) 364; +NaOMe 265, 325 (sh), 405 +AlCI, 272, 307, 353, 419; +AlCl, +HCI272, 307, 350, 420; +NaOAc 265, 370; +NaOAc +H,BO, 262, 300 (sh), 365; MS: mlz (reI. int.) 346 (M)+ (100); 331 (M-15) + (60); 300 (M-46)+ (20) (Wollenweber, 1974). Myricetin-3 ',4 '-dimethyl ether: yellow (green with NA); on Polyamid DC-II (ToIlMeCOEtl MeOH 13/5/3) RI 0.1 I; UV ~max: MeOH 256 (sh), 262, 306 (sh), 363; +NaOMe 278, 328 (sh), 413; +AlCl, 272, 298 (sh), 350, 420; +AlCl, +HCI272, 298 (sh), 350, 420; +NaOAc 277, 387; +NaOAc +H,BO, 266, 305 (sh), 365; MS, mlz (reI. inc.) 346 (M)+ (100); 331 (M-15)+ (45); 300 (M-46)+ (20); 153(A,+H)' (IO)(Ay.noglu er aI., 1981). Scopoletin (6-0-methyl-7-0H
MeOH 224, 252, 344; + NaOMe 283, 295 (sh), 344; MS: mlz (reI. int.) 190 (M)+ (100); 172 (M18) + (85). Authentic sample was synthesized hy Wollenweber after a method described by Tal

and Robeson (1986). Data of the other isolated flavonoids are in accordance to the literature, which was reviewed by Wollenweber and Dietz in 1981.

Results The flavonoid composition of all Cistus species is listed in Tables 2 and 3. With the exception of C. salvifolius all Cistus species were found to secrete flavonoid aglycones. The total amount of flavonoids was in accordance to the quantity of resin obtained. In plants which secreted great amounts of resin the flavonoid content was much higher than in species characterized by a small amount of resin (Table 1). The number

of different flavonoid aglycones, however, was not equivalent to the quantity of

J. Plant Phy,iol. Vol. 131. pp. 25-36 (1987)

'""' ill~

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Table 2: Flavonoids in the leaf resin of the genus Cistus L., kaempferol, quercetin and myricetin derivatives.

j

.g

U

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Flavonoid

+ ++ ++ ++ ++ ++ ++ ++

++ ++ ++ ++ +

+ ++ + ++

++

++

Kaempferol Kae-l-methyl ether Kae-7-methyl ether Kae-4' -methyl ether Kae-3,7-dirnethyl ether Kae-J,4'-dimethyl ether .K.ae-7,4'-dimethyl ether Kae-l,7 ,4'-trimethyl ether Quercetin Que-l-methyl ether Que-l'-methyl ether Que-3,7-d.imethyl ether Que-3,J'-dimethyl ether Que-7,3'-dimethyl ether Que-3,7,3'·trimethyl ether Que-3,l' ,4'-trimethyl ether Que-7,3' ,4'-trimethyl ether Que-l,7,3' ,4'-tetramethyl ether Myr-3 ' ,4'-dimethyl ether Myr-3 ' , 5'-dimethyl ether Myr-3,7,3'.trimethyl ether Myr-3,7,4 '-trimethyl ether Myr-3,3' ,4' -trimethyl ether Myr-7,3' ,4'-trimethyl ether Myr-3,7 ,3' ,4'-tetramethyl ether Myr-3,)','" ,5' -tetramethyl ether

( +) in trace amounts, + minor flavonoid., + + major flavonoid.

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Table 3: Flavonoids in the leaf resin of the genus Cistus L, further components.

,~

;g,

Flayonoid + ++ ++ ++

i

Apigenin Ap-7-methyl ether ether ethe r Ap~4'-methyl

Ap~7,4J~dimethyl

Luteolin Lut-7-methyl ether Lut-Y-mcthyl ether Lut-7,3 '-dimethyl ether Lut-7,Y ,4' -trimethyl ether +

E 0

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it (j

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Naringenin Nar-7-met hyl ether Eriodictyol-7-methyl ether Pinocembrin Chrysin Toctochrysin &.OH-K.ae-3,6-dimethyl ether &'OH-Kae-J,6,4'-trimethyi ether

+ +

+

++

+ + + + +

Herbacetin-3,8-dimethyl ether Hcr-3,8,4 '-trimethyl ether Hu-3.7,8,4' -tetramethyl ether Quercetagetin-3,6-dimethyl ether Queg-3,6,3'-trimethyl ether Gossypetin-J,8,JI-trimethyl ether Gos-3,S,3' ,4' ~tetramethyl ether Gos-3,7.8 ,3' .4'-pentamethyl ether

( +) in trace amounts, + minor flavonoid, + + major flavonoid.

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30

THOMAS VOCT, PETER PROKSCH,

and

PAUL-GEllHARD GULl

OH 0 Kaem~ferol Quercetin

R,

OH H Rs H RJ' H ,,"' OH R, ' H

'"

OH H H OH OH H

Mrricelin

A~isenin Lut~lin ~ercetall,('tin

OH H H OH OH OH

H H H H OH H

H H H OH OH H

OH OH H OH OH H

GossrE!:tin OH H OH OH OH H

Hcrbacetin Ch!!sin

OH H OH H OH H

H H H H H H

HO OH 0 R, R, ' R.'

Pinocembrin

Naringenin

Eriodictyol

H, H H

H, H OH

H, OH OH

Fig. t: Structures of flavonoids.

resin. The structures of the isolated flavonoids and coumarins are illustrated in Figs. 1 and 2. C symphytiJolius, endemic to the Canary Islands, has been investigated for the first time for its flavonoid aglycone pattern. It also proved to be one of the most interesting Cistus species with regard to the epicuticular flavonoid aglycones. Beside some methylated apigenin, kaempferol and quercetin derivatives different rare methyl ethers of myricetin could be identified. Most interesting is the occurrence of myricetin-3',4'-dimethyl ether and of myr-3,3 I ,4 1,5 '-tetramethyl ether, which have been identified only once before as a natural flavonoid (Ayanoglu et al., 1981; Srivastava e[ aI., 1981). C. osbeckia/folius is [he second Cistus species, which is endemic to the Canary Islands. To our knowledge it has never been investigated previously for its chemical

J.

Plant P/rysiol. Vol. 131. pp. 25-36 (1987)

Epicuticular flavonoid aglycones in C'istus

HO

31

0 0 vyVy

CH30~

Scopoletin

Fig. 2: Structures of coumarins.

constituents. By comparison with authentic samples rather common flavones and flavonols, with kaempferol-7-methyl ether as the dominating compound were found. This is the only Cistus species, in which the flavanone eriodictyol-7-methyl ether could be detected as a resin constituent. The flavonoid pattern of C albidus is characterized by the secretion of the flavanone naringenin beside kaempferol, quercetin and apigenin methyl ethers (Pascual Teresa et aI., 1978; Wollenweber and Mann, 1984). We studied two individual plants from different localities and both were found to secrete exactly the same flavonoids. C. incanus is subdivided into different subspecies. The taxonomy of these subspecies is very uncertain because of the variety of hybrids in this species. The plants analysed in this report descended from seeds collected at typical and well defined places, characteristic for each subspecies (Warburg, 1968). C. incanus ssp. incanus (formerly villosus) (seeds from Tunesia) secretes only small amounts of three methylated derivatives of kaempferol. C. incanus ssp. incanus (formerly tauricus) (seeds from Taurus mountains) exhibits a flavonoid pattern, quite different from C. incanus ssp. creticus (seeds from Creta) and C. incanus ssp. corsicus (seeds from Corsica). Otherwise the flavanone naringenin-7methyl ether is the major compound in all three subspecies. The results obtained in [he case of C. incanus ssp. creticu5 were not in accordance with results obtained by Wollenweber and Mann in 1984. C. crispus like the C. incanus ssp. secretes only small amounts of flavonoids. Characteristic for these flavone and flavonol methyl ethers seems to be the lack of methylation in position 4' of the flavonoid skeleton. C. albanicus and C parviflorus have been discussed recently in detail with special hints to the rare 6- and 8-0-methylated flavonols (Vogt et aI., 1987). Flavones are completely missing. C. monspeliensis secrets very large amounts of flavonoids mainly flavonols with quercetin-3,7~dimethyl ether as the main compound. Unique in the genus Cistus the rare myricetin-3,7,Y-trimethyl ether could be detected in addition to two other myricetin derivatives (Pascual Teresa et aI., 1979; Wollenweber and Mann, 1984). C. psilosepalus has been found to secrete a heterogenous pattern of methylated flavunes and f1avonols with quercetin-3,3'-dimethyl ether and que-3,7,3'-trimethyl ether as the main flavonoids (Pa.·Kual Teresaa et al., 1977; Wollenweber and Mann, 1984). j. Plant P/ry
32

THOMAS VOGT, PETE.1t PR.O~SCH,

and PAUL-GERHARD GOLZ

C. salvifolius from five different origins was studied, but in contrast to other results no trace of any epicuticular flavonoid could be detected (Wollenweber and Mann, 1984). C. populi/olius was found to contain great amounts of different methyl ethers of kaempferol and quercetin and in addition some apigenin and luteolin derivatives. The flavanone pinocemhrin and the flavones chrysin and tectochrysin (both unique to C. populi/olius) were also reported (Pascual Teresa et al., 1977). C. laurifolius secretes large amounts of flavonoids with quercetin-3-methyl ether and que-3,7-dimethyl ether as the main components. As in C. populi/olius pinocembrin was also a resin component. The results obtained are largely in accordance with the results of Pascual Teresa et al. (1979) and Wollenweber and Mann (1984). C palhinhae and C. ladanifer secrete a flavonoid pattern confirming the results from Pascual Teresa et al. (1974, 1983 b), Wollenweber and Mann (1984) and Proksch and Giilz (1984). In C. elusii higher methylated flavonoids dominate. Two individual plants analysed, originating from different populations, exhibit the same flavonoid pattern (Pascual Teresa et aI., 1983 a). C. libanotis yielded mostly well-known kaempferol, quercetin and apigenin derivatives. Some of them were already detected by Pascual Teresa et aI., in 1982. In addition to the flavonoids two coumarin derivatives were well detectable as resin constituents by their intense blue fluorescence in UV-light. Scopoletin (6-0H-7-0methyl coumarin) could be detected in nearly all Cistus species so far analysed. With the exception of C. ladani/er and C. palhinhae, probably even in C. salvi/olius, scopoletin is a resin constituent in the genus Cistus. A more apolar very rare coumarin, ayapin, (6,7-methylene dioxy coumarin), presumable directly biosynthesized from scopoletin (Tal and Robeson, 1986) could be verified as a constituent of at least four Cistus species. Originally it was mentioned by Wollenweber and Mann (1984) in the leaf resin of C. monspeliensis. In addition we found C. !aun/olius (in very large amounts of up to 0.2 % of the crude resin), C. sympbyti/olius and C. psilosepalus to contain this coumarin derivative. Some phenolic epicuticular substances which were isolated in small amounts could not be identified so far. One bright yellow fluorescent flavonoid with a molecular weight of 330 is assumed to be a S-O-methylated-quercetin derivative as suggested by the UV- and MS-data, but the data are not sufficient for final structure determination. In trace amounts some quite polar flavonoids with lTV-data indicating compounds esterified with cinnamic acids, are present in C. laurifolius, C. psilosepalus and C. clusii.

Discussion The pattern of Cistus epicuticular flavonoid aglycones is characterized by methyl ethers of the common flavonols kaempferol quercetin and myricetin and the flavones apigenin and luteolin. Nevertheless the methylation pattern of these compounds in some cases is rather unusual and very rare natural substances like the myricetin methyl ethers of C. sympbyti/olius could be detected. With the exception of C. salvi-

J. Plant Pbysiol. Vol. 131. pp. 25 -36 (1987)

Epicuticular flavonoid aglycones in Cistus

33

falius which did not yield any f1avonoids at all, kaempferol derivatives have been found in all species and subspecies, quercetin derivatives in twelve taxa and myricetin methyl ethers are restricted to only foue Cistus species. The flavone derivative with tei-O-substituted B-ring, tricetin, does not occur at all. Flavanones are of very limited occurrence and are restricted with at most one derivative to some C. incanus ssp., to C. albidus, C. asbeckiMfolius, C. launfalius and C. papuli/alius (pinocembrin). The f1a· vonoid pattern of the genus seems to be more or less species specific. Some flavonoid aglycones already detected by Pascual Teresa et aJ. (1974, 1977, 1978, 1979, 1982, 1983 a, 1983 b) could be corroborated as major components in the species analysed previously by the Spanish scientists. In contrast to our investigations, Pascual Teresa et al. analysed whole plant extracts. It is not clear if these flavonoids are located extraceJlularly (on the plant cuticula) or intracellularly (in the vacuoles). Their high methylation increases the Jjpophility and favours these flavonoids to be part of the epicuticular substances. In most cases, with the exception of C. salvi/olius, our results are in accordance to the flavonoid pattern reported by Wollenweber and Mann (1984), who already inves· tigated some Cistus species for epicuticular flavonoids. Because of this documented consistency of the Cistus flavonoids their use for systematic purposes seems plausible. Systematic treatments in the genus Cistus have been problematic for the last hundred years (Grosser, 1903; Janchen, 1925; Dansereau, 1939; Bolanos and Guinea, 1949; Warburg, 1968). A great potency of hybridisation within this genus as well as between Cistus and the related genus Halimium is one of the main reasons for this difficulty. Based on morphological and anatomical criteria, Warburg neglected the subdivision of the genus into several subgenera as proposed by Grosser in 1903, and divided Cistus into sixteen species, the Canary Islands endemics C. symphytiJolius and C. osbeckiaefalius not included. Chemotaxonomic studies based on flavonoids are generally well established and often provide a powerful tool in plant systematics (Harborne, 1975). Chemotax· onomic studies of the genus Cistus, based on intracellular flavonoid aglycones were carried out some ten years ago by Poetsch (1973) and Poetsch and Reznik (1972). The aglycones of the glycosides have been shown to consist only of kaempferol, quercetin and myricetin. The detected semiquantitative differences of single compounds, however, only allowed conclusions related to the systematic problems in the genus. The pattern of the epicuticular flavonoids is of a far greater variety than that of the glycosides encountered. The endemic species C. symphytifolius is well characterized by its rare myricetin methyl ethers. Along with the large amounts of flavonols, the dominating occurrence of compounds with a trihydroxylated B-ring (myricetin methyl ethers) shows this species to be of a rather primitive evolutional status (Harborne) 1977). The woody appearence and the endemism support this judgement. The more herbaceous character of the other endemic, C. osbeckiae/olius, is in accordance with the apomorphic flavonoid pattern like for example encountered in C albidus. The flavonoid patterns of these two species are characterized by a lack of compounds with a trihydroxylated B-ring, the predominance of 3.methylated fla}. Plant Pry,iol. Vol. 131. pp. 25 - 36 {1987}

34

THOMAS VOGT, PETER P:R.OJ::SCH,

and PAUL-GERHARD GOLZ

vonols and the occurrence of flavanones. Based on the flavonoids there is thus very

little support for a closer systematic relationship of the two Canary Islands endemics. C monspeliensis and C laurifolius appear to be similar to C sympbytifolius as far as their flavonoid character is concerned. A great quantity of di- and trihydroxylated Bring flavonoids (C monspeliensis) as well as the predominance of flavonols, especially those with a free 3-0H-group, support the woody and rather primitive evolutional character of these two species. The species C. incanus is divided into several subspecies. From our point of view C incanus ssp. corsicus and C. incanus ssp. creticus. even though well differentiated in anatomical characteristics exhibit an almost identical epicuticular flavonoid pattern. On the other hand C. incanus Ssp. incanus (formerly tauricus) and C incanus ssp. incanus

(formerly villosus) differ from each other and from C incanus ssp. corsicus and C in· canus ssp. creticus, based on their flavonoids. Although there are some correlations (3O-methyl flavonols, naringenin-7-methyl ether as the major component) of C. in· canus ssp. incanus (formerly tauricus) to those subspecies, there seems to be no resemblance of C incanus ssp. incanus (formerly villosus) compared to the rest of the analysed subspecies as far as the epicuticular flavonoids are concerned. These data again question the subdivision of the species C. incanus and the true taxonomic status of the taxa currently recognized as subspecies. The very small amounts of flavonoids and total resin extracted from C. incanus ssp. incanus (formerly villosus) point to a

possible hybrid origin of this taxon as being derived from C salvifolius and C. in· canus. Loss of flavonoid secretion in accordance with the herbaceous character reveals these taxa to be more progressive forms (Swain, 1975) in the genus Cistus. C. ladanifer and C. palhinhae have been discussed to be of a close relationship (Proksch and Giilz, 1984). Basically these results could be verified although C. palhinhae was found to secret no apigenin derivatives as formerly reported. The C. palhinhae specimen analysed by Proksch and Giilz (1984) has been shown to be most likely a hybrid between C. ladanifer and C. palhinhae. C elusii and C. libanotis show close systematic relationship as far as anatomical and morphological features are concerned. Although there are some identical epicuticu-

lar flavonoids, both can be distinguished easily by a very different pattern of luteolinand quercetin derivatives. C. elusii is characterized by a large quantity of 3-0H-quercetin methyl ethers and luteolin derivatives, whereas C. libanotis shows a more progressive flavonoid pattern, characterized by 3-0-methyl flavonols and apigenin methyl ethers as dominating flavonoids.

The isolated position of C. alhanicus and C. paruiflOTU~ which both contain a unique pattern of rare 6- and 8-0-methyl flavonols has been discussed recently (Vogt et al., 1987). It is remarkable, that all their 6- and 8.methoxy flavonols are quite uncommon in the Dilleniidae, to which the family of the Cistaceae belongs, and related orders of the plant kingdom. These facts support the extremely isolated position of these two species within the genus. In ·summary almost all taxa of the genus Cistus are characterized by an abundant secretion of lipophilic, mostly methylated flavonoids and to a lesser degree of coumarins. The chemical patterns encountered are usually complex and seem more or

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Epicmicuiar flavonoid aglycones in CistU5

35

less species specific with the exception of C. albanicus and C. paruiflorus, the two isolated and probably closely related taxa. Application of the lipophilic flavonoid pattern to systematic studies of Cistus seems more promising than that of the uniform flavonoid glycosides, but still holds problems compared to the currently valid treatment of the genus (Warburg, 1968). The flavonoids appear helpful in further studying the C. incanus complex, that has repeatedly been subdivided into several subspecies. Flavonoid analysis suggests that the currently acknowledged subdivision of C. In· canus does not reflect the natural system.

Acknowledgements The authors wish to thank Prof. Dr. E. Wollenweber for several flavonoid standards as well as for his help in flavonoid and coumarin identification. We thank Prof. Dr. Wildpret for plant material of C. osbeckiaefolius. P. P. thanks the DFG for support.

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