Journal of Natural Produos Vol. 47, No. 5 , pp. 809-814, Sep-Od 1984
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FLAVONOL GLYCOSIDES FROM DRYAS OCTOPETALA ORIEITASERVEITAZ, Dipartimento di Biologia, Univwsita di Milano, Via Celwia 26, 20133 Milano, Italy M . LAURACOLOMBO,
lstituto di Farmacologia e Fannarognosia, Univmita di Milano, Via Vanvitelli 32, 20129 Milano, Italy MARIADE BERNARDI,ELENAUBERTI,GIOVANNI VIDARI, and PAOM VITA-FINZI
Dipartinmito di Chimica Organica, Uniwrsita di Pavia, Via Taramelli 10, 27100 Pavia, Italy ABSTRACT.-six flavonol glycosides and enr-epicatechin were isolated from Dryas Dctopetala and their structures elucidated by chemical and spectroscopic methods. Two new flavonoids, 3-0-a-L-arabinofuranosyl-8-methoxyquercetin(2) and 3-O-P-galactopyranosyl-8methoxykaempferol (6),were identified along with 3-O-~-D-galactopyranosylquercetin (hyperin) (3), 3-0-a-L-arabinofuranosylquercetin(avicularin) (5), 3-0-P-L-arabinopyranosylquercetin (4),and 3-O-~-D-galactopyranosyl-8-methoxyquercetin (1).
Dryas octopetala L . (Rosaceae) is a creeping shrublet, widely distributed in the northern hemisphere from Alaska and Siberia southward. In Italy, it has a wide distribution on screes and rocky pastures in the pre-alpine and alpine regions (1). In France, Switzerland, and Austria, a powder, infusion, or extract of the leaves is used internally and externally as a digestive aid, astringent, and antidiarrhetic. In addition, various other healing properties have been attributed to this shrub (2-4),e.g., in Val Rendena (Trento) an infusion of the leaves is considered useful in curing heart ailments (5). The medicinal properties of this species are believed to depend on the amount of tannins present in the plant, which are reputed to be high (6). The only chemical study of D . octopetala reports that seven flavonol aglycones were isolated, but only after hydrolysis of the leaf extract (7). Because of these documented folk usages, an investigation of the biologically active substances of D . octopetala was undertaken. The leaves, collected on mountain valleys of the Dolomites, were extracted in two different ways: with hot H,O, following the folk procedure ofprepating a “tea” or infusion, and with a sequence of organic solvents. Both extracts showed similar flavonoid composition by tlc and hplc. From the first procedure, a brown residue containing chlorophyll, flavonoids, free sugars, and tannins was obtained after evaporation of H 2 0 in uacuo. Partitioning of the extract between solvents of differing polarity was performed to remove chlorophyll and tannins, but this was time consuming and gave poor results. W e found it best to extract the leaf material by percolating with solvents of increasing polarity (see Experimental section). A preliminary tlc screening of the chlorophyll-free extract on Si gel plates (CHC1,EtOH-H20, 98:20:2)revealed the presence of flavonoid 0-glycosides, but no flavonoid aglycones were detected. The similar mobilities of the flavonoid glycosides made their separation with standard chromatographic procedures quite difficult (8). Fdms analysis revealed that most of the fractions obtained by separation on column chromatography, using various substrates, were still mixtures of two or more flavonoids. Eventually, we found that pBondapak RP- 18 liquid chromatography, using various ratios of t-BuOH-H,O as a solvent, afforded the best separations on both an analytical and a preparative scale (9). Compounds 1-5were obtained from the second extract, while compounds 1 and 6 were isolated by cellulose and polyamide column chromatography of the first infusion extract.
8 10
Journal of Natural Products
1 2
3 4 5
6
R
R‘
CH,O CH,O H H H CH,O
OH OH OH OH OH H
{Vol. 47, No. 5
R” P-D-galactopyranosyl a-L-arabinofuranosyl P-D-galactopyranosyl 8-L-arabinopyranosyl a-L-arabinofuranosyl P-D-galactopyranosyl
Ent-epicatechin, isolated via polyamide column chromatography, was identified by ir spectrum (OH and aromatic bands, no CO bands) and the pmr data of mt-epicatechin acetate. the coupling constants of the pyrane ring protons being indicative of a syn-relationship between H-2 and H-3. Mp and specific rotation of the isolated compound corresponded to those of 2S, 3s-(+)-epicatechin (10) (ent-epicatechin). Pmr spectra of compounds 1 and 2 showed a similar pattern for the aromatic protons and had significant differences only for the sugar moiety, which suggested they were different 0-glycosides of the same aglycone. In fact, both of them gave corniculatusin (8-methoxy-quercetin) (ir pmr, ms) upon hydrolysis with trifluoroacetic acid. In addition, (+)-D-galactose and (+)-L-arabinose, identified by tlc and as trimethylsilyl ethers by gc, were obtained from 1 and 2, respectively. The bathochromic shifts observed for the uv absorption maxima of 1 and 2 by addition of the standard shifts reagents (8)and the mass fragmentation patterns of the corresponding permethylated and acetylderivatives were clearly indicative of 3-0-glycosides. The struc(1l), p-conture of 1 was thus assigned as 3-O-~-D-galactopyranosylcorniculatusin figuration being assigned to C- 1”on the basis of the coupling constant of the anomeric proton. Furthermore, co-chromatography with an authentic sample showed identical Rf values. In the case of 2, the small coupling constant of H-1” and H-2”, assigned by decoupling experiments, are typical of an a-furanoside (12). As expected, in the acetylderivative of 2 the H-2”, H-3“, and H-5“signals were shifted downfield compared to 2, while the chemical shift of H-4” was not affected significantly. Thus, 2 is 3-0-a-Larabinofuranosylcorniculatusin. Compound 3 was identified as hyperin (3-0-p-D-galactopyranosyl quercetin) on the basis of mp, ir, uv, ms, and nmr data. Hydrolysis of 3 gave quercetin and D-(+)galactose, and the structure of 3 was confirmed by co-chromatography with an authentic sample. Compounds 4 and 5 had identical mw 434 and the aromatic feature of quercetin (pmr), and both gave by hydrolysis quercetin and L-( +)-arabinose. Furthermore, the uv spectra with the standard shift reagents showed very similar absorption maxima, and ms data of permethylated derivatives indicated that 4 and 5 are isomeric 3-0arabinosyl derivatives of quercetin. Comparison of the analyzed pmr spectra of 4 and its eptaacetyl derivatives revealed that H-2”, H-3”, and H-4” are shifted downfield by acetylation, while H-5” was unaffected. The coupling constant of H-1” (J=7.0 Hz) is typical of a P-pyranoside ( 13). Thus, the structure of 3-0-P-L-arabinopyranosylquercetin was assigned to 4. By contrast, in the case of 5 , the H-2”, H-3”, and H-5” were shifted downfield by
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acetylation, suggesting a furanoside sugar, where the unaffected H-4" was on the acetylic carbon atom. The small coupling constant of H- 1" (J
