Doctor of Philosophy
Chemical and Biochemical Engineering
Dr. Paul A. Charpentier
North American ginseng is a unique medicinal plant which is believed to show several biological activities including: anti-stress, anti-angiogenic, immunosuppressive, and anti-oxidant activity. Components previously isolated from North American ginseng include ginsenosides, polysaccharides, peptides, polyacetylenic alcohols, and fatty acids. The biological and pharmacological effects of ginseng are mainly related to the ginsenoside components, making their extraction and characterization of interest in order to identify them, and study their biological activities.
This thesis focused on the extraction of ginsenosides from North American ginseng by an ultrasonication method with methanol and DMSO as solvents and their aqueous mixtures. Quantitative analysis of individual ginsenosides from the extracts was measured by HPLC, which demonstrated that ultrasonication significantly enhanced the extraction efficiency, with the best efficiency found using 80% solvent (methanol, and DMSO) and 20% water. Immunosuppressive activity of these ginseng extracts was tested in LPS-induced macrophage cells showing that the 80% DMSO and 80% methanol extracts gave significant potency toward immunosuppressive activity in a dose-dependent manner. Moreover, significant quantities of 6”-O-acetylginsenoside Rb1 were obtained using DMSO as the extraction solvent during ultrasonication, and identified using MS, FTIR, and 1D (1H and 13C) and 2D (gCOSY, gHSQC, and gHMBC) NMR. Also, subsequent bioassay experiments confirmed that acetyl ginsenoside Rb1 demonstrated additional immunosuppressive activity towards inhibiting the production of nitric oxide (NO) and tumor necrosis factor (TNF)-α in LPS-induced macrophage cells in a dose-dependent manner using murine macrophages. In addition, acetyl ginsenoside Rb1 gave significant anti-angiogenic activity and exhibited enhanced potency towards inhibiting tube-like structure formation of endothelial cells.
Supercritical fluid chromatography (SFC) using supercritical carbon dioxide which is considered as a “green” separation method and believed as a promising technique for separation, isolation, and identification of herbal and medicinal plants, was used to separate and isolate ginseng extracts obtained by supercritical CO2 extraction (SFE). The effect of temperature and pressure on the separation of ginsenosides was studied with methanol being added to the CO2 mobile phase. Acidic, basic, and ionic additives were introduced to the mobile phase, respectively, to study their effect on the separation of ginsenosides. The best separation condition was obtained by adding 0.05% v/v trifluoroacetic acid in methanol. A high-concentration component in the extracts from the supercritical fluid extraction of North American ginseng was isolated by SFC and identified as sucrose using NMR, HPLC, and ESI-MS.
Because of it's unique biological activities, development of a suitable delivery system for acetyl ginsenoside Rb1 (ac-Rb1) was investigated for the first time in this research. PLGA microspheres were used to encapsulate ac-Rb1,examining both a double emulsion and a microfluidic technique. The size and morphology of the ac-Rb1 loaded PLGA microspheres were characterized by SEM and ZEISS light microscopy, showing unimodal 50-65 µm size diameters, respectively using the microfluidic technique. Also, another delivery system of PLGA in gelatin hydrogel was prepared in order to achieve a localized delivery method, overcoming drawbacks such as PLGA removal by macrophages and a high initial burst effect from gelatin hydrogel that can damage tissues around the injection site. The ac-Rb1 loaded microspheres were incorporated into the gelatin hydrogels to form a new delivery system examining gluteradlehyde crosslinking concentrations from 10-100µl. FTIR, DSC and TGA confirmed the formation and chemical stability of the gelatin encapsulated composites. Release profiles were studied and quantified by UV-Vis spectrophotometry with the results showing that the release of ac-Rb1 from the unimodal microspheres prepared by the microfluidic technique showed a lower initial burst effect than those from the double emulsion method. The burst effect was followed by a slow release profile which can be used for long term drug delivery applications to maintain the ginsenoside concentration for an extended time period. It should be mentioned that although the large burst effect could release a therapeutic agent relatively fast, it can also damage tissues around the treatment site. Hence, a combination delivery system was developed using cross-linked gelatin. The release of ac-Rb1 from the cross-linked gelatin encapsulated microspheres was effected by the pH of the releasing medium as well as the crosslinker concentration. Then, the in vitro cumulative release data of the core and core-composite systems was analyzed using empirical equations in Matlab. The results showed that the in vitro release kinetics data followed Fickian diffusion with the best fit observed using the Weibull model, for all investigated cases. Moreover, the released ac-Rb1 from delivery systems showed a significant immunosuppressive effect on LPS-induced macrophages indicating the novel delivery systems for ac-Rb1 have potential for next-generation biomedical agents in drug-release devices.
Samimi, Raziye, "Acetyl Rb1 Ginsenoside from North American Ginseng: Extraction and Application" (2014). Electronic Thesis and Dissertation Repository. 2005.