Andrographis paniculata est une échinacée d’origine asiatique utilisée depuis plus de deux milles ans en médecine traditionnelle asiatique. Ses activités anti-bactériennes, anti-virales et hépatoprotectrices, entre autre, en ont fait un sujet d’étude important depuis les trente dernières années. Cette plante est principalement constituée de diterpènes de série ent-labdanes, dont le composé majoritaire est l’andrographolide, une lactone diterpénique, qui a montré des propriétés anti-inflammatoires sur la voie NFB. C’est dans le but de comprendre et d’améliorer les activités anti-inflammatoires des composés issus de la plante que nous avons envisagé l’étude de la famille des andrographolides. Une étude qualitative d’extraits issus de compléments alimentaires a permis d’obtenir trois composés naturels, qui ont été pharmacomodulés chimiquement. De plus, une méthode quantitative de quatre extraits bruts a permis de déterminer la concentration des trois composés naturels isolés dans chaque mélange. Les composés isolés, ceux modifés par hémisynthèse, ainsi que les mélanges de composition déterminée ont fait l’objet de tests d’activité anti-inflammatoire. Afin de modifier la structure de l’andrographolide sur des positions non accessibles par hémisynthèse, la synthèse totale et énantiosélective du produit naturel a été envisagée. Deux voies de synthèses ont été envisagées pour la préparation de ce composé : une voie biomimétique et une voie utilisant la cétone de Wieland-Miescher. Cette dernière voie est divisée en trois parties (cyclisation énantiosélective, fonctionnalisation du cycle A, fonctionnalisation du cycle B). Ainsi, nous avons pu mettre au point des méthodologies permettant de fonctionnaliser chaque cycle et d’accéder à la synthèse d’analogues de l’andrographolide. / Andrographis paniculata is an Asian Echinacea. This plant is widely used in traditionnal remedies and folkloric medicines for thousands years and is reported to possess a wide spectrum of biological activities. A large number of diterpenoid ent-labdanes have been isolated from this plant, where the major compound is andrographolide, a diterpenoid lactone, that exhibits interesting anti-inflammatory activities on the NFB way. In order to understand and improve the anti-inflammatory activity of these compounds, we implemented a program aimed at their study. Qualitative analysis of crude extracts from dietary supplements allowed to obtain three major natural compounds, which were in a second time chemically modified. These products, as well as the crude extracts, where the composition was determined by a quantitative method, were used for the study of anti-inflammatory activity. Our aim was also to modify andrographolide on some positions not accessible by hemisynthesis. So total and enantioselective synthesis of andrographolide was investigated. Two ways were studied: biomimetic pathway, and modification of a bicyclic precursor that already possessed the ent-labdane configuration (Wieland-Miescher ketone). Methodology using Wieland-Miescher ketone was divided in three parts (enantioselective cyclisation, cycle A functionalisation, cycle B functionalisation). By this way, the bicyclic core was successfully functionnalised and analogues of andrographolide were prepared.

Andrographis paniculata (AP) is a traditional herbal medicine which is widely used for the treatment of many diseases in Asia. Recently, various biological activities of AP extract or andrographolide (AND), such as immunostimulatory activity, anti-inflammatoryeffect, cytokine induction or deduction, a potential cancer therapeutic agent and T cell activation suppression, have been reported. However the potential ofAP extract or AND to stimulate the specific or adaptive immune response using microbial vaccines has not been determined. In this project, AND has been purifiedfrom AP and its identity confirmed by the melting point, colour test, TLC, UV absorption spectrum, ESI-MS and H-NMR. An aqueous and two ethanolic extracts ofAndrographis paniculata and AND, an active principle of Andrographis paniculata, were investigated for their antimicrobial activity against nine bacterial species in vitro using the disc diffusion method. It was discovered that neither the aqueous extract nor AND were bacteriostatic or bactericidal against S. typhimurium, E. coli, S. sonnei, S. aureus, P. aeruginosa, S. pneumoniae, S. pyogenes, L. pneumophila or B. pertussisbut the two ethanolic extracts of AP were bacteriostatic against L. pneumophila and B. pertussis. It was also observed that the ethanol extract of AP and AND stimulatedboth antibody and cell-mediated immunity (CMI) responses to a killed S. typhimurium vaccine. Mice were vaccinated with either one dose or two doses of killed S. typhimurium vaccine. They were fed two different quantities of an ethanol extract of AP or AND for 14 days in mice immunised with one dose of the vaccine, and for 28 days in mice immunised with two doses of vaccine, respectively. Both the extract and AND significantly increased the IgG antibody titres against S. typhimurium, with theincrease in antibody titres being statistically significant in the two dose vaccine group. Although not statistically significant, there was also a substantial increase in the IgG antibody titres in the one dose vaccine group. Splenocyte cultures from mice fromboth the immunisation groups treated with the extract or AND stimulated with the S. typhimurium lysate showed a significant increase in the production of IFN-γ in both14 and 28 day AP extract or AND treatment groups. The increase indicates the induction of a cell-mediated immune response. To confirm the immunomodulatory potential of AP extract and determine the immunomodulatory potential of AND, experiments were conducted using mouse salmonellosis as a model system. Mice were vaccinated with two doses of killed S. typhimurium vaccine by intraperitoneal(i/p) route and orally dosed with AP extract at 25 mg/kg bodyweight or AND at 4 mg/kg bodyweight for total 28 days, followed by oral challenge with virulent S. typhimurium. Both AP extract and AND substantially increased the survival rate by 50% after mice were challenged with 10 fold of a sublethal dose (1.5x10 6 cfu) of virulent S. typhimurium. They also promoted clearance of S. typhimurium from challenged mice by days 8 or 12 post-challenge with 1.5x10 cfu virulent S. typhimurium respectively. Sera IgG, IgA antibody titres against S. typhimurium and IFN-γ or IL-2 were detected after the mice were challenged by the oral route with a sublethal dose (1.5x10 cfu) of virulent S. typhimurium for 12 days. It was thus concluded that both Andrographis paniculata and AND not only elicited both humoral and cell-mediated immune responses in the mouse model, but also increasedthe protective efficacy against salmonellosis on mice vaccinated with inactivated S. typhimurium. Therefore the ability of Andrographis paniculata and AND to promoteacquired immunity, particularly in inducing CMI may be important in protection against intracellular pathogen infection.

Andrographis paniculata est une échinacée d'origine asiatique utilisée depuis plus de deux milles ans en médecine traditionnelle asiatique. Ses activités anti-bactériennes, anti-virales et hépatoprotectrices, entre autre, en ont fait un sujet d'étude important depuis les trente dernières années. Cette plante est principalement constituée de diterpènes de série ent-labdanes, dont le composé majoritaire est l'andrographolide, une lactone diterpénique, qui a montré des propriétés anti-inflammatoires sur la voie NFB. C'est dans le but de comprendre et d'améliorer les activités anti-inflammatoires des composés issus de la plante que nous avons envisagé l'étude de la famille des andrographolides. Une étude qualitative d'extraits issus de compléments alimentaires a permis d'obtenir trois composés naturels, qui ont été pharmacomodulés chimiquement. De plus, une méthode quantitative de quatre extraits bruts a permis de déterminer la concentration des trois composés naturels isolés dans chaque mélange. Les composés isolés, ceux modifés par hémisynthèse, ainsi que les mélanges de composition déterminée ont fait l'objet de tests d'activité anti-inflammatoire. Afin de modifier la structure de l'andrographolide sur des positions non accessibles par hémisynthèse, la synthèse totale et énantiosélective du produit naturel a été envisagée. Deux voies de synthèses ont été envisagées pour la préparation de ce composé : une voie biomimétique et une voie utilisant la cétone de Wieland-Miescher. Cette dernière voie est divisée en trois parties (cyclisation énantiosélective, fonctionnalisation du cycle A, fonctionnalisation du cycle B). Ainsi, nous avons pu mettre au point des méthodologies permettant de fonctionnaliser chaque cycle et d'accéder à la synthèse d'analogues de l'andrographolide.

No / Limited tumor penetrability of anti-cancer drugs is recognized as one of the major factors that lead to poor anti-tumor activity. SRJ09 (3,19-(2-bromobenzylidene) andrographolide) has been identified as a lead anti-cancer agent for colon cancer. Recently, this compound was shown by us to be a mutant K-Ras binder. In this present study, the penetrability of SRJ09 through the DLD-1 colon cancer multicell layer (MCL) was evaluated. The amount of SRJ09 that penetrated through the MCL was quantitated by utilizing high performance liquid chromatography (HPLC). Histopathological staining was used to visualize the morphology of MCL. A chemosensitivity assay was performed to assess the anti-cancer activity of SRJ09 in DLD-1 cells. SRJ09 was able to penetrate through DLD-1 MCL and is inversely proportional with the MCL thickness. The flow rates for SRJ09 through MCL were 0.90 ± 0.20 μM/min/cm2 and 0.56 ± 0.06 μM/min/cm2 for days 1 and 5, respectively, which are better than doxorubicin. Histopathological examination revealed that the integrity of the DLD-1 MCL was retained and no visible damage was inflicted on the cell membrane, confirming the penetration of SRJ09 was by diffusion. Short term exposure (1 h) in DLD-1 cells demonstrated SRJ09 had IC50 of 41 μM which was approximately 4-folds lower than andrographolide, the parent compound of SRJ09. In conclusion, SRJ09 successfully penetrated through DLD-1 MCL by diffusion and emerged as a potential candidate to be developed as a clinically viable anti-colon cancer drug.

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