Dereplication of Bioactive Spirostane Saponins from Agave macroacantha

Abstract

A dereplication strategy using UPLC-QTOF/MS^E, the HMAI method, and NMR spectroscopy led to the identification of five main steroidal saponins (1-5), including three previously unknown compounds named macroacanthosides A-C (3-5), in a bioactive fraction of Agave macroacantha. The major saponins were isolated, and some of them together with the saponin-rich fraction were then evaluated for phytotoxicity on a standard target species, Lactuca sativa. The inhibition values exhibited by the pure compounds were confirmed to be in agreement with the phytotoxicity of the saponin-rich fraction, which suggests that the saponin fraction could be applied successfully as an agrochemical without undergoing any further costly and/or time-consuming purification processes. The NMR data of the pure compounds as well as of those corresponding to the same compounds in the fraction were comparable, which indicated that the main saponins could be identified by means of this replication workflow and that no standards are required.

Figure 1

Structural features of A. macroacantha saponins as determined by the HMAI method. OH, hydroxy group; DB, double bond; CO, carbonyl group; OGlc, glucopyranosyloxy group.

Figure 2

Structures of the main saponins of A. macroacantha.

Figure 3

Selected 1D TOCSY NMR spectra (mixing time 100 ms) of the major anomeric signals and the methyl deoxyhexapyranose signal in the saponin-rich fraction of A. macroacantha. All the signals displayed were assigned using the 1D TOCSY subspectra corresponding to an acquisition array that included 15, 30, 55, 70, 100, and 150 ms as mixing times.

Figure 4

Selected ROE correlations (arrows) of the sugar chain (SC). MS/MS losses were determined according to the MS^E analysis.

Figure 5

Conformation calculated for compound 2 and NOE effects observed between the different sugar units and the aglycone.

Figure 6

Conformation calculated for compound 4. The areas highlighted in red and blue colors represent deshielding and shielding of the chemical shifts of the sugar chain compared to compound 2 (which has no hydroxy group at C-2). The dotted line represents the hydrogen bridge between the hydroxy group at C-2 and the galactopyranose oxygen.