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Biochemical Systematics and Ecology 32 (2004) 937–941

www.elsevier.com/locate/biochemsyseco

Phytochemical investigation of Gentianadinarica

Dijana Krstic a, Teodora Jankovic a, Ivana Aljancic b,Katarina Savikin-Fodulovic c, Nebojsa Menkovic c,

Slobodan Milosavljevic d,�

a Institute for Biological Research ‘‘Dr. Sinisa Stankovic’’, 29 Novembra 142, 11000 Belgrade, Yugoslaviab Institute for Chemistry, Technology and Metallurgy, Njegoseva 12, 11000 Belgrade, Yugoslaviac Institute for Medicinal Plant Research ‘‘Dr. Josif Pancic’’, Tadeusa Koscuska 1, 11000 Belgrade,

Yugoslaviad Faculty of Chemistry, University of Belgrade, Studentski trg 16, P.O. Box 158, 11000 Belgrade,

Yugoslavia

Received 28 October 2003; accepted 20 March 2004

Keywords: Gentiana dinarica; Gentianaceae; Xanthone glycosides; Flavone-C-glycosides secoiridoids

1. Subject and source

Gentiana dinarica Beck. is a rare perennial plant species growing on carbonatesoils in subalpine and alpine regions (Tutin, 1972; Jovanovic-Dunjic, 1973). Plantmaterial (aerial parts and roots) was collected on the mountain Tara (~1300 m),west Serbia, in June 2001. A voucher specimen (accession number Gd072001) isdeposited in the herbarium at the Faculty of Biology, University of Belgrade-Herbarium, Code BEOU.

2. Previous work

The compounds isolated so far from Gentiana species are xanthones, secoiridoidsand flavone-C-glycosides (Hostettmann-Kaldas et al., 1981). Secoiridoid, gentio-picrin, and pyridine alkaloid, gentianine, the latter supposed to be an artifact

D. Krstic et al. / Biochemical Systematics and Ecology 32 (2004) 937–941938

formed by reaction between gentiopicrin and ammonia added in course of isolation

procedure, were found in G. dinarica (Floss et al., 1964).

3. Present study

Air-dried aerial parts (400 g) and roots (150 g) of G. dinarica were separately

extracted with methanol for 48 h at room temperature. The extracts (100 and 21 g,

respectively) were evaporated in vacuo to yield brown residues which were sus-

pended in water and reextracted with solvents of increasing polarity: ether, ethyl

acetate and n-butanol. The LC–DAD analysis of the ether (9.5 g), ethyl acetate

(1.5 g) and butanolic (31.6 g) extracts of the aerial parts indicated the presence of

the same secoiridoids and flavone-C-glycosides in all extracts, the highest concen-

tration of these compounds being in the butanolic extract. An aliquot of the buta-

nolic extract (10 g) was subjected to dry column flash chromatography using

CH2Cl2 with increasing amounts of MeOH (15–100%) to give six fractions (Frs

1–6). Silica gel column chromatography of Fr 3 (2.6 g), using CH2Cl2–MeOH

(100:0 to 80:20) as eluent, yielded secoiridoids swertiamarin (2, 95 mg) (Cornelis

and Chapelle, 1976), gentiopicrin (4, 160 mg) (Mpondo and Chulia, 1988) and a

mixture of 4 and sweroside (1) (200 mg), the latter identified by comparing its UV

spectrum and retention time under given HPLC conditions with our database of

UV spectra of standards. Fr 6 (1 g) was subjected to CC on polyamide SC6 start-

ing elution with water and gradually increasing the amounts of MeOH (0–70%)

to afford two C-glucoflavones, isoorientin (7, 10 mg) (Bellmann and Jacot-

Guillarmod, 1973) and isoorientin-40-O-glucoside (8, 27 mg) (Hostettmann et al.,

1973).HPLC screening of the ether (3.4 g) and ethyl acetate (1.2 g) extracts of the roots

indicated the presence of secoiridoids, while the butanolic extract (10.1 g) was rich

in xanthones and flavonoids, along with secoiridoids. Column chromatography on

polyamide SC6 of the ether extract, with water–MeOH (100:0 to 60:40) as eluent,

yielded the secoiridoid, amarogentin (3, 20 mg) (Inouye and Nakamura, 1971).

Butanolic extract (5 g) was subjected to CC on polyamide SC6 using water with

increasing amounts of MeOH (0–90%). Xanthone glycosides 5 (132 mg), and 6 (15

mg) were isolated, along with C-glucoflavones, 7 (12 mg) and 8 (6 mg). Secoir-

idoids 4 and 1 from the roots were identified by comparison of their UV spectra

and HPLC retention times with those in database.

3.1. Norswertianin-1-O-primeveroside (5) (Hostettmann et al., 1974)

Gum; 1H NMR (DMSO-d6) 200 MHz, d: 7.17 (d, J ¼ 8:9 Hz, H-6), 6.77 (d,

J ¼ 8:9 Hz, H-5), 6.63 (d, J ¼ 2 Hz, H-4), 6.36 (d, J ¼ 2 Hz, H-2), 13.31 (s, OH-

8), 4.91 (d, J ¼ 7:0 Hz, H-10, Glc), 4.21 (d, J ¼ 7:0 Hz, H-100, Xyl), 2.8–4.0 (10H,

Glc–Xyl). The 1H NMR spectrum was assigned by close similarity to that of nors-

wertianin-1-O-b-d-glucopyranoside (Ya et al., 1998).

939D. Krstic et al. / Biochemical Systematics and Ecology 32 (2004) 937–941

3.2. Norswertianin-8-O-primeveroside (6)

Gum; UV kmax, nm: (MeOH) 236, 262, 317, 374; (+MeONa) 244, 275, 349;

(+AlCl3) 233, 275, 332; (+AlCl3/HCl) 233, 275, 329; (+AcONa) 264, 352, 360;

(+AcONa/H3BO3) 263, 318; IR mfilmmax, cm�1: 3443, 1654, 1613, 1482, 1417, 1330,

1278, 1074, 1050; 1H NMR (DMSO-d6) 200 MHz, d: 7.38 (d, J ¼ 9:3 Hz, H-6),

7.28 (d, J ¼ 9:3 Hz, H-5), 6.26 (d, J ¼ 2:0 Hz, H-4), 6.12 (d, J ¼ 2:0 Hz, H-2),

13.04 (s, OH-8), 5.01 (d, J ¼ 7:6 Hz, H-10, Glc), 4.09 (d, J ¼ 7:0 Hz, H-100, Xyl);13C NMR (DMSO-d6), 50 MHz, d: 163.2 (C-1), 98.6 (C-2), 167.1 (C-3), 93.7 (C-4),

D. Krstic et al. / Biochemical Systematics and Ecology 32 (2004) 937–941940

157.1 (C-4a), 114.5 (C-5), 125.0 (C-6), 146.4 (C-7), 141.8 (C-8), 114.5 (C-8a), 180.2(C-9), 102.3 (C-9a), 149.7 (C-10a); Glc: 104.7 or 103.8 (C-10), 73.5 (C-20), 76.4(C-30), 69.7 (C-40), 76.4 (C-50), 68.1 (C-60); Xyl: 103.8 or 104.7 (C-100), 73.9 (C-200),76.4 (C-300), 69.7. (C-400), 65.7 (C-500); DCIMS (150 eV, isobutane) m=z 555(MþH, <0.5), 260 (C13H8O6, 100). The

1H and 13C NMR spectra were assignedby close similarity to that of norswertianin-8-O-b-d-glucopyranoside and 8-O-b-gentiobioside (Otsuka, 1999).

4. Chemotaxonomic significance

As pointed out by Jensen and Schripsema (2002), the secoiridoid glucosides, witha predominance of swertiamarin (2) and/or gentiopicrin (4) appeared to be presentin all species of Gentianaceae studied so far. Compounds 1, 2 and 4 together withtheir derivatives (e.g. 3) are the most characteristic iridoids in Gentianaceae.Xanthones are not universally present in the family. Hitherto, they were detected

in 121 species belonging to 21 genera (Jensen and Schripsema, 2002). The grade ofsubstitution of xanthones is characteristic for a genus, and the oxidation pattern isuniform within particular section (Meszaros, 1994). A detailed investigation ofxanthone distribution has been made in the genus Gentiana, in particular in sec-tions Gentiana L., Calathianae Froelich and Megalanthe Gaudin (Hostettmann andWagner, 1977). The classification of species into these sections is in accordancewith the more recent phylogenetic results based on molecular data (Gielly andTaberlet, 1996). Xanthones isolated from G. dinarica (section Megalanthe) showthe 1,3,7,8-oxygenation pattern characteristic for this section. It is noteworthy thatonly xanthone glycosides but not the aglycones were found in this species.C-glucoflavones, such as 7 and 8, are less variable than the iridoids and the xan-

thones. Only nine different compounds from nine genera (three belonging to Poto-liae and six to Gentianeae) have been reported so far, and further systematicinvestigation of other genera is desirable (Jensen and Schripsema, 2002).

Acknowledgements

The authors are grateful to Professor V. Stevanovic, Faculty of Biology, Univer-sity of Belgrade, for identification of the plant material. The authors also acknowl-edge their gratitude to Ministry of Science and Technology of Serbia for financialsupport.

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