افرینش

^aLaboratoire de Pharmacognosie, Unité de Molécules d'Intérêt Biologique, UMIB UPRES-EA 3660, Faculté de Pharmacie, Université de Bourgogne, 7, Bd. Jeanne d'Arc, BP 87900, 21079 Dijon Cedex, France ^bGraduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

Received 7 January 2005; 

accepted 7 July 2005. 

Available online 8 September 2005.

Keywords: Dioscorea villosa; Dioscoreaceae; Steroidal saponin; Flavan-3-ol glycosides

Article Outline

1. Subject and source

2. Previous work

3. Present study

4. Chemotaxonomic significance

References

1. Subject and source

The underground parts (rhizomes) of Dioscorea villosa L. (Dioscoreaceae) were collected in Mexico in 2002. A voucher specimen (No. 1507) is deposited at the Herbarium of the Laboratory of Pharmacognosy, Dijon, France.

2. Previous work

The wild yam D. villosa was shown to be an important source of diosgenin (Benghuzzi et al., 2003 and Marker et al., 1940), a steroid precursor of progesterone. This yam is used in alternative medicines to minimize post-menopausal symptoms and for the treatment of low progesterone levels (Benghuzzi et al., 2003). The in vitro anticollagenase activity of D. villosa extract (Liviero and Allec, 2002) and the possibility of incorporation of sapogenins in cosmetics for the prevention of skin disorders (Picard, 2002) have also been reported. Nevertheless, no phytochemical investigation has been carried out on this plant.

3. Present study

Dried powdered rhizomes (280 g) of D. villosa were refluxed with MeOH:H₂O (7:3, 3 × 2 l), which after evaporation to dryness yielded 91 g of MeOH:H₂O extract. This was partitioned successively with hexane, CH₂Cl₂ and n-BuOH (each 3 × 200 ml) yielding after evaporation of the solvents the corresponding hexane (0.4 g), CH₂Cl₂ (0.2 g) and n-BuOH (29.7 g) fractions. Of the n-BuOH residue, 5 g was dissolved in MeOH and purified by precipitation with diethyl ether (3 × 300 ml), yielding a crude saponin mixture (3.7 g). This latter was submitted to vacuum liquid chromatography (VLC) on C₁₈ reversed-phase (12 × 3 cm) using as eluents H₂O (100 ml), MeOH:H₂O mixtures (5:5; 4:1, each 100 ml) and finally MeOH (100 ml). After evaporation of the solvents, three fractions were obtained: VLC-F1 (MeOH:H₂O, 4:1), VLC-F2 (MeOH) and VLC-F3 (MeOH:H₂O, 5:5).

VLC-F1 (0.74 g) was submitted to MPLC column chromatography (system a: Si gel (15–40 μm), CHCl₃:MeOH:H₂O (13:7:2, lower phase)) to give nine fractions (1–9). Fraction 8 was rechromatographed by MPLC (system a) yielding the pure compounds 1 (10 mg) and 2 (12 mg).

VLC-F2 (1.23 g) was submitted to MPLC (system a), yielding 10 fractions (1–10). Fraction 5 was rechromatographed by MPLC (system a) to give the pure compound 3 (15 mg). Fractions 4 and 3 afforded the pure compounds 4 (12 mg) and 5 (6 mg), respectively. Fraction 2 was concentrated to dryness yielding the pure compound 6 (8 mg).

VLC-F3 (0.49 g) was submitted to MPLC (system a) to give 10 fractions (1–10). Fractions 2 and 5 were concentrated to dryness to give the pure compounds 7 (25 mg) and 8 (7 mg), respectively.

The identification of all metabolites was achieved by FABMS on a Jeol X-102 spectrometer and by spectroscopic methods including 1D- and 2D-NMR experiments (COSY, TOCSY, HSQC and HMBC) on a Varian Inova instrument equipped with a Sun-4-L-X computer system as well as by comparison with literature values. They were identified as protodioscin (1) (Hu et al., 1996), methylprotodioscin (2) (Hu et al., 1997), parrisaponin (3) (Matsuda et al., 2003 and Sautour et al., 2004a), dioscin (4) (Hu et al., 1996), prosapogenin of dioscin (5) (Hu et al., 1996), progenin II (6) (Agrawal et al., 1985), epiafzelechin 5-O-β-d-glucopyranoside (7) (Sethi et al., 1984) and epiafzelechin 7-O-β-d-glucopyranoside (8) (Jin et al., 1999) (Fig. 1).

The antifungal activity of compound 6 was performed using the broth dilution test in comparison with a positive control of ketoconazole (Quiroga et al., 2001). It presented antifungal activity against Candida albicans (MIC = 12.5 μg/ml), Candida glabrata (MIC = 12.5 μg/ml) and Candida tropicalis (MIC = 25 μg/ml). These results confirm our previous work concerning the antifungal activity of spirostane-type saponins isolated from Dioscorea cayenensis (compounds 3–5) (Sautour et al., 2004a M. Sautour, A.C. Mitaine-Offer, T. Miyamoto, A. Dongmo and M.A. Lacaille-Dubois, Planta Med. 70 (2004), p. 90. View Record in Scopus | Cited By in Scopus (27)Sautour et al., 2004a and Sautour et al., 2004b).

4. Chemotaxonomic significance

The saponins found in D. villosa rhizomes belong to the furostan-type (1–2) and spirostan-type skeletons (3–6), the most common aglycones found in the Dioscoreaceae family (Tang and Eisenbrandt, 1992).

Concerning the phenolic compounds (7–8), the presence of flavan-3-ol such as (+) catechin or (−) epicatechin and procyanidin dimers B-1 and B-3 have been reported in Dioscorea cirrhosa (Hsu et al., 1985), Dioscorea alata, D. cayenensis, Dioscorea dumetorum, Dioscorea rotundata (Ozo et al., 1984) and Dioscorea bulbifera (Ozo et al., 1984 and Gao et al., 2002). Furthermore, procyanidin trimers and tetramers were also found in D. cirrhosa (Hsu et al., 1985) and D. alata. The latter was also reported to contain cyanidin glycosides such as cyanidin-3-glucoside (Ozo et al., 1984), cyanidin-3,5-diglucoside, cyanidin-3-rhamnoglucoside (Rasper and Coursey, 1967) and cyanidin-3-gentiobioside acylated with ferulic acid (Imbert and Seaforth, 1968) but to our knowledge, the present work is the first report of flavan-3-ol glycosides in Dioscorea species.