L'objectif de ce travail de thèse était la caractérisation puis l'étude des applications potentielles dans le domaine des extraits naturels végétaux ou d'origine fermentaire d'un nouvel Extracteur de Partage Centrifuge (EPC) prototype. Ce but originel a été sensiblement infléchit et a conduit, au fil de l'avancement des études menées, à des développements différents et/ou complémentaires à ceux initialement imaginés. ce travail portait au départ sur l'extraction et la purification des métabolites issus principalement des biotechnologies blanches (acides succinique, itaconique,…), mais très rapidement, nous avons été amené à recentrer nos recherches sur d'autres métabolites naturels issus d'extraits de plantes (Glycyrrhiza glabra, Sinapis alba, et Anogeissus leiocarpus) et une seule cible fermentaire (l'acide itaconique). / The aim of this thesis was the characterization and study of potential applications of a new Centrifugal Partition Extractor (CPE) prototype, in the field of natural plant extracts and withe biotechnology. This work initially focused on the extraction and purification of metabolites mainly from white biotechnology (succinic acid, itaconic acid, ...), but very quickly, we have been led to focus our research on other metabolites from natural extracts plant (Glycyrrhiza glabra, Sinapis alba, and Anogeissus leiocarpus) and one target fermentation (itaconic acid).

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctorate of Philosophy. / Senecio serratuloides is widely used for wound healing in South Africa but minimal information regarding its efficacy is available. Furthermore toxic pyrrolizidine alkaloids may be present. The following investigation sought firstly to evaluate the efficacy and safety of Senecio serratuloides in a porcine wound model; secondly to assess for a potential mechanism and finally isolate and identify fractions in in-vitro assays. Assessment of Efficacy and Safety Materials and Methods: Deep partial thickness and full thickness wounds were created on 9 pigs. Treatment included an occlusive dressing (negative control), activated carbon, or the Senecio preparation. Wounds were monitored using photographic documentation, pH measurement and histological analysis (skin thickness and collagen content). Toxicity was monitored on blood and liver samples. Results and Discussion: Efficacy of Senecio serratuloides was established with a significantly thicker epidermis, maximal at day 7 post-operative, 2 days before the controls. Effects on collagen content was negligible with no toxicity detected. Mechanistic investigation Materials and Methods: Wound fluid was analysed for IL-10, IL-12, IL-1β, IL-6, IL-8, TNF-α using flow cytometry based assays. Tyrosine phosphorylation and cellular proliferation was assessed using dual immunofluorescence staining. Results and Discussion: IL-1β levels were significantly greater in the Senecio treatment. Tyrosine phosphorylation increased to day 9 post-operative where it stabilised in all groups. In the same period, cellular proliferation was sustained in the Senecio treated wounds but not in the controls. Keratinocyte proliferation was identified as the target for in-vitro assays. Extraction, Isolation and Partial Identification using In-vitro Proliferation Assays. Materials and Methods: The plant was fractionated using solid phase extraction cartridges. Keratinocytes were grown under standard conditions in 96-well plates. Cellular proliferation was assessed spectrophotometrically using a resazurin dye technique. Active fractions were analysed using gas chromatography and mass spectrometry. Results and Discussion: Identified fractions increased the rate of proliferation by 300- 400%. Potential lead compounds were identified. Importantly, pyrrolizidine alkaloids could not be detected. Conclusion Senecio serratuloides is efficacious in treating deep partial thickness wounds without inducing liver toxicity. Sustained keratinocyte proliferation linked to tyrosine phosphorylation may be an underlying mechanism. Although successful, in-vitro detection of active fractions requires further characterisation.