Impact of the Hydrolysis and Methanolysis of Bidesmosidic Chenopodium quinoa Saponins on Their Hemolytic Activity

Abstract

Saponins are specific metabolites abundantly present in plants and several marine animals. Their high cytotoxicity is associated with their membranolytic properties, i.e., their propensity to disrupt cell membranes upon incorporation. As such, saponins are highly attractive for numerous applications, provided the relation between their molecular structures and their biological activities is understood at the molecular level. In the present investigation, we focused on the bidesmosidic saponins extracted from the quinoa husk, whose saccharidic chains are appended on the aglycone via two different linkages, a glycosidic bond, and an ester function. The later position is sensitive to chemical modifications, such as hydrolysis and methanolysis. We prepared and characterized three sets of saponins using mass spectrometry: (i) bidesmosidic saponins directly extracted from the ground husk, (ii) monodesmosidic saponins with a carboxylic acid group, and (iii) monodesmosidic saponins with a methyl ester function. The impact of the structural modifications on the membranolytic activity of the saponins was assayed based on the determination of their hemolytic activity. The natural bidesmosidic saponins do not present any hemolytic activity even at the highest tested concentration (500 µg·mL^-1). Hydrolyzed saponins already degrade erythrocytes at 20 µg·mL^-1, whereas 100 µg·mL^-1 of transesterified saponins is needed to induce detectable activity. The observation that monodesmosidic saponins, hydrolyzed or transesterified, are much more active against erythrocytes than the bidesmosidic ones confirms that bidesmosidic saponins are likely to be the dormant form of saponins in plants. Additionally, the observation that negatively charged saponins, i.e., the hydrolyzed ones, are more hemolytic than the neutral ones could be related to the red blood cell membrane structure.

Keywords: chemical modification; hemolytic activity; mass spectrometry; saponins; structure-activity relationship.

Figure 1

Specific chemical modifications of the bidesmosidic saponins extracted from Chenopodium quinoa husk: (i) hydrolysis of Saponin O to Saponin O^hydro, and (ii) transesterification of Saponin O to Saponin O^alkyl.

Figure 2

General structure of the bidesmosidic saponins extracted from the quinoa husk. R₁ corresponds to the C3-attached glycan as detailed in Table 1. R₂ and R₃ functions are specific to the aglycone moiety as shown in the presented aglycones. The C28-glucose is highlighted in red since this residue will be involved in the chemical modifications targeted, i.e., hydrolysis and methanolysis.

Figure 3

MALDI mass spectrometry analysis of three different saponin extracts: (a) the natural extract (NE); (b) the microwave-assisted alkaline hydrolysis (pH 10—150 °C—5 min) reaction products; and (c) the transesterification using MeOK (MeOH_anh—N₂—60 °C—60 min) reaction products. The saponin ion assignment was performed using the [x + y] symbolism, where x and y stand for the number of monosaccharide residues at C3 and C28, respectively. [x + Me] indicates that the C-28 glucose residue has been substituted by a methoxy group. Please note that the monodesmosidic [2 + 0] and [3 + 0] saponin ions detected in the NE (a) are produced during the MALDI processes from the corresponding bidesmosidic saponins (see text).

Figure 4

Mass spectrometry qualitative and quantitative analysis of the (a) natural, (b) hydrolyzed, and (c) transesterified saponin extracts: the saponin relative proportions (%) correspond to the molar proportions as determined by LC-MS signal integration. Please note that the relative proportions of Saponins X and Saponins Y correspond to the sum of the proportions of saponins G, 32 and 61 and the proportions of saponins N, 4, Q, H, 19 and F, respectively. M.W. stands for microwave activation.

Figure 5

LC-MSMS analysis of the (a) natural extract, (b) hydrolyzed extract, and (c) transesterified extract. Collision-induced dissociation (CID) mass spectra of the (a) m/z 973, (b) m/z 811 and (c) m/z 825 precursor ions, respectively, corresponding to the [M + H]⁺ ions of (a) Saponin B, (b) hydrolyzed Saponin B, and (c) transesterified Saponin B.

Figure 6

Mechanistic proposal for the hydrolysis (HO⁻) and transesterification (CH₃O⁻) reactions undergone by the bidesmosidic saponins extracted from the quinoa husk: addition–elimination mechanism specifically occurring at the ester function.

Figure 7

Cytotoxicity evaluation of the three saponin extracts: hemolytic activity comparison between the natural bidesmosidic saponins, the hydrolyzed saponins, and the transesterified saponins. Experiments were performed in triplicate, using a 2% erythrocytes suspension from bovine blood. Hemolytic activities are expressed in % of the activity of the referent, a 500 µg·mL⁻¹ solution of A. hippocastanum saponins.