Determination of Saponins in Aerial Parts of Barrel Medic (Medicago truncatula) by Liquid Chromatography−Electrospray Ionization/Mass Spectrometry

Abstract

Triterpene saponins from aerial parts of Medicago truncatula cv. Jemalong A-17, M. truncatula Gaertn.var. longispina Urb., and M. truncatula Gaertn. var. truncatula were profiled and quantified using reverse-phase liquid chromatography with on-line photodiode array detection and electrospray ionization mass spectrometry (LC-PDA/ESI/MS/MS). The determination was based on standard curves obtained for the 18 available saponin standards, previously isolated from Jemalong A-17. Aerial parts of all three subspecies contained 17 saponins previously identified and also a substantial amount of astragaloside VIII (3-GlcA-Xyl-Rha soyasapogenol B), not previously reported in M. truncatula. The compositions of saponin mixtures were very similar in the three subspecies with three dominant groups, recognized as zanhic acid, medicagenic acid, and soyasapogenol glycosides. Relative proportions of these three groups were also similar in the three subspecies: var. longispina had 49.5, 48.1, and 2.4%; var. truncatula, 41.5, 53.4, and 5.1%; and Jemalong A-17, 42.1, 56.6, and 1.3% of zanhic acid, medicagenic acid, and soyasapogenol glycosides, respectively. Jemalong A-17 had 30% lower total content of saponins as compared to M. truncatula var. longispina and M. truncatula var. truncatula; in relation to the dry matter, var. longispina contained 0.22%, var. truncatula, 0.22%, and Jemalong A-17, 0.15% dry matter of saponins. If one takes into consideration that this determination was performed on spring-collected samples, it can be concluded that the concentration of saponins in M. truncatula is similar to the concentration in alfalfa (Medicago sativa); the proportions of the three groups of saponins in these species are slightly different from those found in alfalfa, having a higher content of zanhic acid glycosides.

Genomic and Coexpression Analyses Predict Multiple Genes Involved in Triterpene Saponin Biosynthesis in Medicago truncatula

“…Medicagenic acid conjugates are major constituents in the aerial parts of M. truncatula, whereas soyasapogenol conjugates are major constituents in roots Kapusta et al, 2005a). However, it is likely that a single b-AS is responsible for production of most triterpene saponins in this species.…”

“…Six different saponins have been identified in M. truncatula root extracts according to previously published work (Huhman and Sumner, 2002;Huhman et al, 2005;Kapusta et al, 2005aKapusta et al, , 2005b, and mass data for some are provided in Supplemental Figure 8 online. Levels of Rha-Hex-Hex-Hex-hederagenin, Hex-Hex-Hex-bayogenin, 3-Glc-28-Glc-medicagenic acid, 3-Glc-Ara-28-Glc hederagenin, and Hex-Hex-Hex-soyasapogenol E were significantly (P value <0.05) reduced in mutant lines compared with controls, by around threefold overall ( Figures 7A and 7C).…”

“…All identified metabolites accumulated significantly differently in controls compared with mutant plants with a P value of <0.05. Identification of saponins and isoflavonoids was based on previously published work (Huhman and Sumner, 2002;Huhman et al, 2005;Kapusta et al, 2005aKapusta et al, , 2005bFarag et al, 2007). Explanations of masses for saponins are provided in Supplemental Figure 8 online.…”

Metabolite Profiling of Triterpene Saponins in Medicago truncatula Hairy Roots by Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

“…A remarkable observation was the absence of zanhic acid glycosides in the list of identified saponins. In previous studies, zanhic acid was shown to be one of the major aglycone moieties in aerial parts of M. truncatula, 8,9 and it was also reported to be present in other M. truncatula plant tissues, including roots. 7 In a more recent study, however, zanhic acid could not be detected in M. truncatula roots.…”

“…7 In a later study, 12 additional saponins from the aerial parts of M. truncatula were characterized via spectroscopic methods, and their fragmentation behavior under negative ionization was reported. 8,9 Nevertheless, M. truncatula was postulated to synthesize many more triterpene saponins than identified to date. 2 For identification purposes, LC-MS-based metabolomics studies benefit from the use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS).…”