Elsevier

Fitoterapia

Volume 91, December 2013, Pages 113-124
Fitoterapia

Lipidated steroid saponins from Dioscorea villosa (wild yam)

https://doi.org/10.1016/j.fitote.2013.07.018Get rights and content

Abstract

Two groups of lipidated steroid saponins including seven new compounds (2, 3, 5, and 710) were isolated from the widely used botanical, wild yam (Dioscorea villosa), employing a fractionation protocol of metabolomic mining. This methodology recently led to the isolation of 14 diarylheptanoids from the same plant. Together with these lipidated steroid saponins, they establish additional new markers for D. villosa. The lipidation of steroids with analog long-chain fatty acids containing different degrees of unsaturation generates an entire series of compounds which are difficult to purify and analyze. The structures of the two series of lipidated steroid saponins (series A and B) were established by a combination of 1D and 2D NMR as well as GC–MS after chemical modification. Series A was determined to be a mixture of lipidated spirostanol glycosides (15), while series B (610) was proved to be a mixture of five lipidated clionasterol glucosides. The latter group represents the first derivatives of clionasterol to be found in D. villosa. The discovery of this specific structural type of aliphatic esters of steroid saponins expands the characterization of the secondary metabolome of D. villosa. It may also inspire biological studies which take into account the lipophilic character and significantly altered physiochemical characteristics of these otherwise relatively polar phytoconstituents.

Introduction

Species of the genus Dioscorea (family Dioscoreaceae) are widely used as botanical dietary supplements. These plants are well known for containing steroidal saponins, mainly belonging to the spirostanol and furostanol classes, and these have been used as chemical marker compounds for quality control of the botanical products [1]. Wild yam, the rhizomes and roots of Dioscorea villosa L., is an important source of diosgenin [2], a phytoestrogen that has been investigated thoroughly from both chemical and biological perspectives [3], [4], [5]. The methods aimed at the targeted isolation and purification of the major steroid saponins in D. villosa were developed by exploring various chromatographic techniques, such as high-speed countercurrent and centrifugal partition chromatographs as well as HPLC [1], [6], [7]. To date, based on a comprehensive literature survey, twelve steroidal saponins and two flavan-3-ol glycosides have been isolated as major secondary metabolites of wild yam [1], [2], [6], [7], [8]. Our group recently reported 14 diarylheptanoids [9]. These diarylheptanoids were first isolated and characterized from wild yam based on a new fractionation methodology using 1D qHNMR and 2D NMR profiles along the preparative fractionation pathway [9]. Primary fractionation of the methanolic extract using a MeOH/H2O solvent gradient on a preparative C18 solid phase extraction (SPE) cartridge effectively enriched the diarylheptanoids into three fractions (4–6), which were further purified by VLC, MPLC, and HPLC. Mining the 1H NMR spectral profiles of the last two fractions (10 and 11) led to the observation of similar patterns (i.e., signals of steroid glycosides) dominant in almost all of these relatively lipophilic fractions. Accordingly, normal phase TLC fraction monitoring was used throughout the separation process, and a polar solvent system of chloroform/methanol (8:1 v/v) was found to be most suitable for the last two fractions (10 and 11) to give TLC spots with Rf values around 0.5. However, the polarity of this solvent system is much higher than what would commonly be used for lipophilic fractions that elute from the C18 SPE cartridge with 90% and/or pure MeOH. Subsequent purification of these two fractions finally afforded two series of compounds, each of which showed a single spot on TLC in a variety of solvent systems. HPLC analysis using a C18 column resulted in no distinctive absorption at 210 nm with a UV detector, and remained undetected even in an ESI-LC–MS system. In addition to the characteristic resonances of steroid saponins, the 1H NMR spectra of these two series of compounds exhibited signals that were reasonably assigned to a mixture of homologous aliphatic residues, suggesting that both series were residual complex mixtures of lipidated steroid saponins containing, e.g., fatty acid residues. This feature assignment was also in line with the much more lipophilic chromatographic behavior of the compounds, in particular on the C18 absorbent. To elucidate their structures, 2D NMR and GC–MS were employed. The proportion of each constituent in each of the two samples was determined using a GC–MS method. This resulted in the characterization of two series of lipidated steroid saponins (110), including seven new compounds (2, 3, 5, and 710), which overall represents the first report of this metabolite class from D. villosa [10], [11], [12]. The occurrence of lipidated steroid saponins in the genus Dioscorea was first reported during the International Congress on Natural Products Research (ICNPR) 2012 and covered two of such compounds from Dioscorea cayenensis (1 and 4) [10]. Four additional analogs had been discovered in 2005 and 2010 from two other families, the Valerianaceae (Valeriana officinalis) and Brassicaceae (Sisymbrium irio) [11], [12]. Lipidated steroid saponins possess one polar sugar residue in the middle, and a non-polar long-chain aliphatic acid as well as a steroid residue on the two ends, which altogether explains their unusual chromatographic behavior in consideration to the general compound class. The present metabolomic mining approach yielded these lipidated steroid saponins and also the previously reported diarylheptanoids. Both findings widen the chemical profile of wild yam and other Dioscorea plants and facilitate the characterization of the residual complexity of D. villosa extracts. Discovery of new types of secondary metabolites also potentially offers new chemical leads for the development of standardization and quality control of wild yam and other Dioscorea botanical products. Herein, we present the details of the isolation methodology, the structure elucidation of the previously unreported lipophilic steroid saponin metabolites (2, 3, 5, and 710) of D. villosa, and their potential broader impact on the understanding of the biological mechanisms of action of yam botanicals in general.

Section snippets

General experimental procedures

NMR spectra were obtained on Bruker AVANCE-400 (5 mm broadband ATM probe), AVANCE-600 (5 mm TXI CryoProbe), or AVANCE-900 (5 mm TCI CryoProbe) NMR spectrometers (Bruker, Zürich, Switzerland) using pyridine-d5 (D 99.5%, Cambridge Isotope Laboratories, Inc., Andover, MA) as the solvent. The chemical shifts were referenced to the residual solvent signals (δH 8.740 and δC 150.35 for α-H and α-C, respectively). Offline NMR data processing was performed with the MestReNova software version 8.0.0-10524

New purification strategy and structural analysis

The purification scheme that yielded the lipidated steroid saponins started with the EtOAc partition of the MeOH extract of D. villosa roots/rhizomes (Scheme 1). A suspension of this material in MeOH-H2O (1:9, v/v) was loaded on a vacuum C18 SPE cartridge. Eleven primary fractions were collected by elution with a gradient of a MeOH-H2O from 0:10 to 10:0 v/v [12]. The most non-polar fractions, 10 and 11, were combined for further purification by VLC, MPLC, and HPLC. Purification and spectroscopic

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgments

The authors thank Dr. B. Ramirez from UIC-CSB for his expert support of the 600 and 900 MHz NMR spectrometers. The authors are also thankful to Dr. C. Simmler, a team colleague at UIC, for affording the figure of the graphical abstract. M. Totura is acknowledged for maintaining the Atkins Medicinal Plant Garden at UIC. This research was supported by grant P50AT000155 (UIC/NIH Botanical Center), co-funded by the National Center for Complementary and Alternative Medicine (NCCAM) and the Office of

References (29)

  • R.E. Marker et al.

    Steroidal sapogenins

    J Am Chem Soc

    (1947)
  • Aradhana et al.

    Diosgenin — a growth stimulator of mammary gland of ovariectomized mouse

    Indian J Exp Biol

    (1992)
  • K.D. Yoon et al.

    Preparative separation of dioscin derivatives from Dioscorea villosa by centrifugal partition chromatography coupled with evaporative light scattering detection

    J Sep Sci

    (2008)
  • P.Y. Hayes et al.

    Complete 1H and 13C assignments of the four major saponins from Dioscorea villosa (wild yam)

    Magn Reson Chem

    (2007)
  • Cited by (5)

    • Study on chemical profiles and metabolites of Allii Macrostemonis Bulbus as well as its representative steroidal saponins in rats by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry

      2016, Food Chemistry
      Citation Excerpt :

      Some were tentatively characterised as new compounds. According to the literature, acetylated steroidal saponins exist widely in the plants (Dong et al., 2013; Munafo & Gianfagna, 2011). Isolation and NMR experiments will be necessary to determine these new saponins.

    • A new acetylated spirostanol saponin and other constituents from the rhizomes of Dioscorea althaeoides R. Knuth (Dioscoreaceae)

      2016, Biochemical Systematics and Ecology
      Citation Excerpt :

      The occurrence of acetylated steroidal saponins in the genus Dioscorea (Dioscorea cayenensis) was first reported during the International Congress on Natural Products Research (ICNPR) 2012 and covered two of such compounds, 5-en-spirostanol-2′-O-α-rha-3-O-β-glucoside-6′-O-hexadecanoate and 5-en-spirostanol-2′-O-α-rha-3-O-β-glucoside-6′-O-9Z,12Z-octadecadienoate. Additionally, the above two saponins, together with three additional analogs were identified from Dioscorea villosa (native to North America) (Dong et al., 2013). Moreover, dioscin (1), gracillin (2), prosapogenin A of dioscin (6), and methyl protodioscin (10), along with diosgenin, were also obtained from D. cayenensis (Avula et al., 2014) and D. villosa (Sautour et al., 2006).

    • Combined scouring-bleaching of cotton fabric from wild yam root

      2022, Journal of Engineered Fibers and Fabrics
    • Dioscorea plants: A genus rich in vital nutra-pharmaceuticals-A review

      2019, Iranian Journal of Pharmaceutical Research

    Residual Complexity and Bioactivity, Part 21 (see S1, Supplementary data).

    View full text