Characterization of Steroidal Saponins in Crude Extracts from Dioscorea Species using Liquid Chromatography/Electrospray Ionization Time-of-Flight Mass Spectrometry

Yam (Dioscorea spp.) is an important tuber plant for edible and medicinal use to promote health and longevity in Chinese traditional medicne [1]. Steroidal saponins were reported to be the major physiologically active constituents in yam [2]. Furostanol and spirostanol glycosides, two main steroidal saponins, have been found in many types of yams (Dioscorea species) [3]. A steroidal saponin molecule consists of an aglycone and any of several glycosyl moieties. The common sugars present in steroidal saponins from Dioscoreaceae are hexose (glucose) and 6-deoxyhexose (rhamnose), and generally, glucosyls are connected with the hydroxyl groups at C-3 and/or C-26 positions of steroidal aglycones [3].

The application of TOF-MS can yield empirical chemical formulas based on the accurate masses of molecular ions and detailed fragmentation information, which eliminates ambiguities in the interpretation of spectra, confirms the identities of the fragment ions and facilitates structural elucidation. In this work, the structural characteristics of the steroidal saponins in the methanolic extracts from the dried rhizomes of Dioscorea species (D. villosa L., D. cayenensis Lam., D. rotundata Poir., D. nipponica Mark., D. opposita L. and D. caucasica Lipsky) have been identified using LC/TOF-MS in both negative and positive ion modes. The fragmentation patterns of reference standards were investigated and the steroidal saponins in the extracts were identified or tentatively characterized by their retention times and MS data. This approach also provides an excellent method for rapid screening of steroidal saponins in plant extracts. Dioscin was used as an example to discuss the fragmentation patterns in detail. In (-)-ESI-MS, dioscin gave [M-H]^- ions at m/z 867.47 and due to presence of formic acid in the mobile phase, the standard also gave [M+HCOO]^- ions at m/z 913.47. In (+)-ESI-MS, the mass spectrum of dioscin gave [M+H]⁺ ions at m/z 869.5. The fragment ions at m/z 723.4314 [M-Rha+H]⁺, 577.3733 [M-2Rha+H]⁺, 415.3206 [M-Glu-2Rha+H]⁺, 397.3104 [M-Glu-2Rha-H₂O+H]⁺, 271.2046 [M-Glu-2Rha-C₈H₁₆O₂+H]⁺ and 253.1922 [M-Glu-2Rha-C₈H₁₆O₂-H₂O+H]⁺ resulted from the protonated molecular ion [M+H]⁺ (m/z 869.4898) [Fig.1]. The fragment ion at m/z 415.3 [aglycone+H]⁺ resulted from the loss of one hexose and two deoxyhexose. The fragments were formed from the characteristic cleavage of glycosidic bonds, and the fragmentation pattern directly provided the detailed structural information about the sequence of sugars. Based on previous publications, twenty-five saponins were identified or tentatively characterized from the crude extracts of Dioscorea species. In addition, four previously unknown steroidal saponins were tentatively elucidated. However, for definite identification of these unknown saponins, further investigation is required. This may provide a model for the rapid screening and structural characterization of bioactive constituents from plant extracts. Dioscin was identified from samples of D. villosa, D. cayenensis, D. nipponica and D. caucasica; however it was not detected in two other species (D. opposita and D. rotundata) samples.

Fig.1: Proposed fragmentation pathway for Dioscin

Acknowledgements : This research is supported in part by Science Based Authentication of Dietary Supplements funded by the Food and Drug Administration grant No. 1U01FD004246–01; by NIH grant entitled “Botanical Identification, Characterization, Quality Assurance and Quality Control” (NIH Prime award number 1P50AT006268–01); and the Global Research Network for Medicinal Plants (GRNMP), King Saud University. The authors would like to thank Annette Ford for extraction of the plant samples. References: [1] Liu SY, et al. (1995)J Chin Med 6: 111–126. [2] Hu K, et al. (1996) Planta Med 62: 573–575. [3] Zhu J, et al. (2010)J Pharm Biomed Anal 53: 462–474.