Promoteur: Pascal Gerbaux

Co-promoteur: Patrick FLAMMANG

Résumé de la dissertation : Saponins are abundant secondary metabolites present in plants and in marine invertebrates. Even if the saponin family of molecules is characterized by a huge chemical diversity, the saponin structure invariably associates two distinct moieties; an apolar steroidic or triterpenoidic aglycone and a polar oligosaccharidic part, named the glycone. Saponins are mostly considered as toxic compounds and their cytotoxicity is strongly associated to their membranolytic properties. Indeed, saponins may deleteriously incorporate into cell membranes inducing their lysis and the cell death. In this context, the present thesis tried to afford pieces of relevant information regarding the Structure-Activity Relationship (SAR) of saponins.
We selected two natural sources, namely the quinoa husk and the horse chestnut seeds. Actually, both biomasses have been selected due to the lack of applications making them just considered as natural waste.
The first part of the work is related to the saponins from the quinoa husk. These saponins are bidesmosidic molecules in which the two sugar residues are anchored by two different linkages on the aglycone, namely an acetal and an ester functions. These saponins were submitted to a microwave-assisted alkali hydrolysis to quantitatively and selectively produce their monodesmosidic analogs upon specific hydrolysis of the ester bond. The optimal reaction conditions have been defined using mass spectrometry analyzes. It was further demonstrated that the cytotoxicity of the saponins is significantly increased upon hydrolysis in agreement with the associated structural modifications. Afterwards, in the context of optimizing the MS-based methods for the natural molecule characterization, we successfully associated ion mobility to collision-induced experiments to achieve the straightforward distinction between isomeric saponins.
The second part of the thesis concerns the saponins extracted from the horse chestnut (HC) seeds. The HC saponins are triterpenoid saponins, named Escins. They are mainly distinguished by the nature and the position of the acyl chains on the aglycone, generating stereoisomeric and regioisomeric saponins. Using ion mobility experiments, we succeeded in discriminating these isomeric saponins, provided a high resolution is available. We tested for the first time for saponin ions cyclic ion mobility mass spectrometry in partnership with the R&D Department of Waters UK. Using state-of-the-art mass spectrometry experiments, including MALDI imaging experiments, we also established the inter and intra-organ distributions of saponins within the different HC parts, with a special attention paid to the seeds. Based on the MALDI-imaging experiments, we confirmed the defensive role often associated to saponins. Microwave-assisted reactions conducted in specific solvent conditions allowed to investigate the influence of the presence and position of the aglycone acyl chains on the cytotoxicity. We optimized experimental conditions to either quantitatively hydrolyze the acyl chains or rather induce intramolecular transesterification reactions. Doing so, we were able to prepare a new family of escin molecules, isomers to the natural molecules. We shown based on hemolytic tests that the loss of the acyl chains nearly suppresses the membranolytic activity whereas the displacement of the acyl chains affects it.
The antifungal activity of all the natural and modified saponins, from the quinoa husk and the HC seeds, was then evaluated against pathogenic fungi for humans. We obtained interesting data revealing that saponins, alone or in combination with common antifungal drugs, may be considered in the future for topical applications on the skin. Indeed, saponins presenting antifungal activities are also the most hemolytic ones. However, the observation that saponins associated to antifungal drugs may exert a synergic effect leading to a net decrease of the antifungal drug concentrations seems to us really promising.
As a final conclusion, in the present study, the combination of state-of-the-art mass spectrometry methods including imaging methods, specific chemical modifications of saponins and biological assays affords relevant data that may contribute to the knowledge of the Structure-Activity relation of saponins.