Phenolics as Chemosystematic Markers in and for the Genus Crepis ( Asteraceae , Cichorieae )

In contrast to the huge variety of sesquiterpene lactones reported, only a limited number of flavonoids and phenolic acids are known from the genus Crepis. Compounds detected in the course of this investigation encompass flavonoids luteolin, luteolin 7-O-glucoside, luteolin 7-O-gentiobioside, luteolin 7-Oglucuronide, and luteolin 4’-O-glucoside and caffeic acid derivatives chlorogenic acid, 3,5-dicaffeoylquinic acid, caffeoyltartaric acid, and cichoric acid. The spectrum of compounds found in the flowering heads of plants of the genus Crepis are useful chemosystematic markers both to differentiate between species and to characterize the genus and help delimiting it from morphologically similar genera within the Cichorieae tribe of the Asteraceae family.

In this investigation fifteen Crepis species, including two Mediterranean species, were analyzed by HPLC-DAD and HPLC-MS using a chromatographic system already successfully applied to the related genus Leontodon [8].

Results and Discussion
HPLC-DAD and HPLC-MS analyses revealed the presence of five luteolin derivatives (1-5) (Fig. 1) and four caffeic acid derivatives (6-9) (Fig. 2) in the flowering heads of the investigated Crepis flowering head samples.As only one sample per species was investigated, the quantification results are given in a semi-quantitative manner [9].These semi-quantitative results are summarized in Tab. 1.In Tab. 2 (Experimental part) the collection sites of the investigated samples are described.Fig. 3 shows an example of a Sci Pharm.2008; 76; 743-750.
chromatogram obtained with the analytical system used for quantifications (see experimental for details).
When comparing the results summarized in Tab. 1 with results obtained in earlier investigation of the genera Hieracium [10] and Leontodon s.l.[8], which also belong to the Cichorieae (syn.: Lactuceae) subtribe of the Asteraceae family, the array of caffeic acid derivatives of the three genera is of special interest.In Crepis and Leontodon caffeoyl tartaric acid and cichoric acid occur in most species.In contrast, these two compounds are absent from all species of Hieracium s.l.investigated so far.Crepis and Leontodon are also similar with regards to the spectrum of flowering head flavonoids as taxa of both genera predominantly contain luteolin and a limited number of simple derivatives of luteolin.
From the fifteen Crepis taxa investigated only one, Crepis alpestris, contains all detected compounds.Crepis conyzifolia and C. tingitana are characterized by the absence of luteolin 7-O-gentiobioside, caffeoyl tartaric acid and cichoric acid.The groupings based on flowering head phenolics are not congruent with the clades found in the recent molecular investigation by Enke and Gemeinholzer [3].Moreover, the results have to be interpreted with care as they are based on one sample per species only and it is well established that quantitative patterns of flowering heads phenolics in the Cichorieae are influenced by climatic factors of the growing site [6].

Plant Material
The origin of the plant material is summarized in Tab. 2. Per population 50 flowering heads were collected and air-dried.Nomenclature is in congruence with the "Katalog der Gefäßpflanzen Südtirols" [11] for plants occurring in Austria and the South Tyrol, follows Flora Europaea for C. tingitana [12] and Flora alpina for C. nemausensis [synonym: Crepis sancta (L.) Babc.subsp.nemausensis (Gouan) Babc.] [13].Voucher specimens of all investigated samples except C. rhaetica are deposited in the private herbarium of Christian Zidorn.Vouchers from C. rhaetica are kept in the herbarium of the Tiroler Landesmuseum Ferdinandeum in Innsbruck (IBF) and in the herbarium of the Botanical Garden Berlin-Dahlem (B) and doublet vouchers of C. tingitana are kept in the herbarium of the Botanical Garden Berlin-Dahlem (B).Scans of the vouchers representing the analyzed collections are included in the on-line version of this paper.500.0mg of dry plant material was mixed with 10.0 ml of a stock solution of quercetindihydrate [25.0 mg quercetin-dihydrate (Sigma, St Louis, USA) / 100 ml CH 3 OH/(CH 3 ) 2 CO/H 2 O (3/1/1)], then 15 ml of a mixture of CH 3 OH/(CH 3 ) 2 CO/H 2 O (3/1/1) was added and extracted for 5 min with an Ultra-Turrax apparatus at 24000 rounds/min and subsequently filtered; afterwards the plant material was again extracted for 5 min at 24000 rounds/min with 25 ml of a mixture of CH 3 OH/(CH 3 ) 2 CO/H 2 O (3/1/1).Then the plant material was extracted for 5 min at 24000 rounds/min with a mixture of CH 3 OH/H 2 O (1/1); the combined extracts were diluted to 100.0 ml with CH 3 OH/(CH 3 ) 2 CO/H 2 O (3/1/1).Finally 10.0 ml of this solution was brought to dryness in vacuo, re-dissolved in 2.00 ml of CH 3 OH/(CH 3 ) 2 CO/H 2 O (3/1/1), filtered and used for HPLC analysis.

HPLC analyses
Flavonoids and phenolic acids were identified as reported before [8].In crude extracts, phenolic acids and flavonoids were identified by their HPLC retention times, HPLC-MS and HPLC-DAD spectra in comparison with authentic reference substances.Amounts of the detected compounds in the investigated flowering heads were estimated by the ratio of the respective peak areas to the area of the internal standard quercetin.HPLC analyses for quantification were performed in triplicate.HPLC system 1 used for the HPLC/UV quantification of phenolic compounds; column: HP-ODS-Hypersil 2.

Fig. 3 .
Fig. 3. Chromatogram of an extract of C. paludosa L. flowering heads (960822a) obtained with HPLC system 1 at 350 nm (see experimental part for details).
Crepis samples