Horse Chestnut Saponins-Escins, Isoescins, Transescins, and Desacylescins.

Escins constitute an abundant family of saponins (saponosides) and are the most active components in Aesculum hippocastanum (horse chestnut-HC) seeds. They are of great pharmaceutical interest as a short-term treatment for venous insufficiency. Numerous escin congeners (slightly different compositions), as well as numerous regio-and stereo-isomers, are extractable from HC seeds, making quality control trials mandatory, especially since the structure-activity relationship (SAR) of the escin molecules remains poorly described. In the present study, mass spectrometry, microwave activation, and hemolytic activity assays were used to characterize escin extracts (including a complete quantitative description of the escin congeners and isomers), modify the natural saponins (hydrolysis and transesterification) and measure their cytotoxicity (natural vs. modified escins). The aglycone ester groups characterizing the escin isomers were targeted. A complete quantitative analysis, isomer per isomer, of the weight content in the saponin extracts as well as in the seed dry powder is reported for the first time. An impressive 13% in weight of escins in the dry seeds was measured, confirming that the HC escins must be absolutely considered for high-added value applications, provided that their SAR is established. One of the objectives of this study was to contribute to this development by demonstrating that the aglycone ester functions are mandatory for the toxicity of the escin derivative, and that the cytotoxicity also depends on the relative position of the ester functions on the aglycone.

Mass spectrometry analysis of saponins.

Saponins are amphiphilic molecules of pharmaceutical interest and most of their biological activities (i.e., cytotoxic, hemolytic, fungicide, etc.) are associated to their membranolytic properties. These molecules are secondary metabolites present in numerous plants and in some marine animals, such as sea cucumbers and starfishes. Structurally, all saponins correspond to the combination of a hydrophilic glycan, consisting of sugar chain(s), linked to a hydrophobic triterpenoidic or steroidic aglycone, named the sapogenin. Saponins present a high structural diversity and their structural characterization remains extremely challenging. Ideally, saponin structures are best established using nuclear magnetic resonance experiments conducted on isolated molecules. However, the extreme structural diversity of saponins makes them challenging from a structural analysis point of view since, most of the time, saponin extracts consist in a huge number of congeners presenting only subtle structural differences. In the present review, we wish to offer an overview of the literature related to the development of mass spectrometry for the study of saponins. This review will demonstrate that most of the past and current mass spectrometry methods, including electron, electrospray and matrix-assisted laser desorption/ionization ionizations, gas/liquid chromatography coupled to (tandem) mass spectrometry, collision-induced dissociation including MS3 experiments, multiple reaction monitoring based quantification, ion mobility experiments, and so forth, have been used for saponin investigations with great success on enriched extracts but also directly on tissues using imaging methods.

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

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.

Microwave-Assisted Desulfation of the Hemolytic Saponins Extracted from Holothuria scabra Viscera.

Saponins are plant and marine animal specific metabolites that are commonly considered as molecular vectors for chemical defenses against unicellular and pluricellular organisms. Their toxicity is attributed to their membranolytic properties. Modifying the molecular structures of saponins by quantitative and selective chemical reactions is increasingly considered to tune the biological properties of these molecules (i) to prepare congeners with specific activities for biomedical applications and (ii) to afford experimental data related to their structure-activity relationship. In the present study, we focused on the sulfated saponins contained in the viscera of Holothuria scabra, a sea cucumber present in the Indian Ocean and abundantly consumed on the Asian food market. Using mass spectrometry, we first qualitatively and quantitatively assessed the saponin content within the viscera of H. scabra. We detected 26 sulfated saponins presenting 5 different elemental compositions. Microwave activation under alkaline conditions in aqueous solutions was developed and optimized to quantitatively and specifically induce the desulfation of the natural saponins, by a specific loss of H2SO4. By comparing the hemolytic activities of the natural and desulfated extracts, we clearly identified the sulfate function as highly responsible for the saponin toxicity.

Phytostimulant properties of highly stable silver nanoparticles obtained with saponin extract from Chenopodium quinoa.

BACKGROUND: Quinoa (Chenopodium quinoa Willd) is an Andean original pseudocereal with high nutritional value. During quinoa processing, large amounts of saponin-rich husks byproducts are obtained. Quinoa saponins, which are biologically active, could be used for various agriculture purposes. Silver nanoparticles have increasingly attracted attention for the management of crop diseases in agriculture. In this work, silver nanoparticles are synthesized by a sustainable and green method, using quinoa husk saponin extract (QE) to evaluate their potential for application in agriculture as biostimulants. RESULTS: Quinoa extract was obtained and characterized by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Sixteen saponin congeners were successfully identified and quantified. The QE obtained was used as a reducing agent for silver ions to synthesize silver nanoparticles (QEAgNPs) under mild conditions. The morphology, particle size, and stability of Ag nanoparticles were investigated by transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-visible), energy-dispersive X-ray (EDS), zeta potential, and Fourier transform infrared spectroscopy with attenuated total reflection (FTIR-ATR). Ultraviolet-visible spectroscopy measurements confirmed the formation of silver nanoparticles in the presence of QE, with estimated particle sizes in a range between 5 and 50 nm. According to the zeta potential values, highly stable nanoparticles were formed. The QE and QEAgNPs (200-1000 µg/mL) were also tested in radish seed bioassay to evaluate their phytotoxicity. The seed germination assays revealed that QEAgNPs possessed a phytostimulant effect on radish seeds in a dose-dependent manner, and no phytotoxicity was observed for both QE and QEAgNPs. CONCLUSION: Silver nanoparticles obtained by a so-called 'green' method could be considered as good candidates for application in the agricultural sector for seed treatment, or as foliar sprays and plant-growth-promoters. © 2020 Society of Chemical Industry.

Enhancing the Membranolytic Activity of Chenopodium quinoa Saponins by Fast Microwave Hydrolysis.

Saponins are plant secondary metabolites. There are associated with defensive roles due to their cytotoxicity and are active against microorganisms. Saponins are frequently targeted to develop efficient drugs. Plant biomass containing saponins deserves sustained interest to develop high-added value applications. A key issue when considering the use of saponins for human healthcare is their toxicity that must be modulated before envisaging any biomedical application. This can only go through understanding the saponin-membrane interactions. Quinoa is abundantly consumed worldwide, but the quinoa husk is discarded due to its astringent taste associated with its saponin content. Here, we focus on the saponins of the quinoa husk extract (QE). We qualitatively and quantitively characterized the QE saponins using mass spectrometry. They are bidesmosidic molecules, with two oligosaccharidic chains appended on the aglycone with two different linkages; a glycosidic bond and an ester function. The latter can be hydrolyzed to prepare monodesmosidic molecules. The microwave-assisted hydrolysis reaction was optimized to produce monodesmosidic saponins. The membranolytic activity of the saponins was assayed based on their hemolytic activity that was shown to be drastically increased upon hydrolysis. In silico investigations confirmed that the monodesmosidic saponins interact preferentially with a model phospholipid bilayer, explaining the measured increased hemolytic activity.

Discrimination of Regioisomeric and Stereoisomeric Saponins from Aesculus hippocastanum Seeds by Ion Mobility Mass Spectrometry.

Modern mass spectrometry methods provide a huge benefit to saponin structural characterization, especially when combined with collision-induced dissociation experiments to obtain a partial description of the saponin (ion) structure. However, the complete description of the structures of these ubiquitous secondary metabolites remain challenging, especially since isomeric saponins presenting small differences are often present in a single extract. As a typical example, the horse chestnut triterpene glycosides, the so-called escins, comprise isomeric saponins containing subtle differences such as cis-trans ethylenic configuration (stereoisomers) of a side chain or distinct positions of an acetyl group (regioisomers) on the aglycone. In the present paper, the coupling of liquid chromatography and ion mobility mass spectrometry has been used to distinguish regioisomeric and stereoisomeric saponins. Ion mobility arrival time distributions (ATDs) were recorded for the stereoisomeric and regioisomeric saponin ions demonstrating that isomeric saponins can be partially separated using ion mobility on a commercially available traveling wave ion mobility (TWIMS) mass spectrometer. Small differences in the ATD can only be monitored when the isomeric saponins are separated with liquid chromatography prior to the IM-MS analysis. However, gas phase separation between stereoisomeric and regioisomeric saponin ions can be successfully realized, without any LC separation, on a cyclic ion mobility-enabled quadrupole time-of-flight (Q-cIM-oaToF) mass spectrometer. The main outcome of the present paper is that the structural analysis of regioisomeric and stereoisomeric natural compounds that represents a real challenge can take huge advantages of ion mobility experiments but only if increased ion mobility resolution is attainable.

Triterpenoids in Echinoderms: Fundamental Differences in Diversity and Biosynthetic Pathways.

Echinoderms form a remarkable phylum of marine invertebrates that present specific chemical signatures unique in the animal kingdom. It is particularly the case for essential triterpenoids that evolved separately in each of the five echinoderm classes. Indeed, while most animals have Δ5-sterols, sea cucumbers (Holothuroidea) and sea stars (Asteroidea) also possess Δ7 and Δ9(11)-sterols, a characteristic not shared with brittle stars (Ophiuroidea), sea urchins (Echinoidea), and crinoids (Crinoidea). These particular Δ7 and Δ9(11) sterols emerged as a self-protection against membranolytic saponins that only sea cucumbers and sea stars produce as a defense mechanism. The diversity of saponins is large; several hundred molecules have been described in the two classes of these saponins (i.e., triterpenoid or steroid saponins). This review aims to highlight the diversity of triterpenoids in echinoderms by focusing on sterols and triterpenoid glycosides, but more importantly to provide an updated view of the biosynthesis of these molecules in echinoderms.

Ion mobility mass spectrometry of saponin ions.

RATIONALE: Saponins are natural compounds presenting a high structural diversity whose structural characterization remains extremely challenging. Ideally, saponin structures are best established using nuclear magnetic resonance experiments conducted on isolated molecules. However, saponins are also increasingly characterized using tandem mass spectrometry (MS/MS) coupled with liquid chromatography, even if collision-induced dissociation (CID) experiments are often quite limited in accurately determining the saponin structure. METHODS: We consider here ion mobility mass spectrometry (IMMS) as an orthogonal tool for the structural characterization of saponin isomers by comparing the experimental collisional cross sections (CCSs) of saponin ions with theoretical CCSs for candidate ion structures. Indeed, state-of-the-art theoretical calculations perfectly complement the experimental results, allowing the ion structures to be deciphered at the molecular level. RESULTS: We demonstrate that ion mobility can contribute to the structural characterization of saponins because different saponin ions present significantly distinct CCSs. Depending on the nature of the cation (in the positive ion mode), the differences in CCSs can also be exacerbated, optimizing the gas-phase separation. When associated with molecular dynamics simulations, the CCS data can be used to describe the interactions between the cations, i.e. H+ , Na+ and K+ , and the saponin molecules at a molecular level. CONCLUSIONS: Our work contributes to resolve the relationship between the primary and secondary structures of saponin ions. However, it is obvious that the structural diversity and complexity of the saponins cannot be definitively unraveled by measuring a single numerical value, here the CCS. Consequently, the structural characterization of unknown saponins will be difficult to achieve based on IMMS alone. Nevertheless, we demonstrated that monodesmosidic and bidesmosidic saponins can be distinguished via IMMS.

Tackling saponin diversity in marine animals by mass spectrometry: data acquisition and integration.

Saponin analysis by mass spectrometry methods is nowadays progressively supplementing other analytical methods such as nuclear magnetic resonance (NMR). Indeed, saponin extracts from plant or marine animals are often constituted by a complex mixture of (slightly) different saponin molecules that requires extensive purification and separation steps to meet the requirement for NMR spectroscopy measurements. Based on its intrinsic features, mass spectrometry represents an inescapable tool to access the structures of saponins within extracts by using LC-MS, MALDI-MS, and tandem mass spectrometry experiments. The combination of different MS methods nowadays allows for a nice description of saponin structures, without extensive purification. However, the structural characterization process is based on low kinetic energy CID which cannot afford a total structure elucidation as far as stereochemistry is concerned. Moreover, the structural difference between saponins in a same extract is often so small that coelution upon LC-MS analysis is unavoidable, rendering the isomeric distinction and characterization by CID challenging or impossible. In the present paper, we introduce ion mobility in combination with liquid chromatography to better tackle the structural complexity of saponin congeners. When analyzing saponin extracts with MS-based methods, handling the data remains problematic for the comprehensive report of the results, but also for their efficient comparison. We here introduce an original schematic representation using sector diagrams that are constructed from mass spectrometry data. We strongly believe that the proposed data integration could be useful for data interpretation since it allows for a direct and fast comparison, both in terms of composition and relative proportion of the saponin contents in different extracts. Graphical Abstract A combination of state-of-the-art mass spectrometry methods, including ion mobility spectroscopy, is developed to afford a complete description of the saponin molecules in natural extracts.