| In this dissertation, a high-through put off-line centrifugal ultrafiltration LC-MS-MS assay were used to screen COX-2 ligands from crude chloroform-soluble ginger extract that was prepared from ginger dietary supplement. In total, ten gingerol-related compounds, including 10-gingerol, 12-gingerol, 8-shogaol, 10-shogaol, 6-paradol, 8-paradol, 6-gingerdione, 8-gingerdione, 10-gingerdione and 6-dehydro-10-gingerol, were identified as ligands of COX-2. In order to further evaluate the inhibitory activities of several COX-2 ligands, a mass spectrometry based prostaglandin E2 (PGE2) quantitation assay was developed and validated. As the results demonstrated, the chloroform extract of ginger and COX-2 ligands (10-shogaol, 8-shogaol and 10-gingerol) are found to be selective COX-2 inhibitors; while those non COX-2 ligands 6-gingerol, 8-gingerol, and 6-shogaol are not COX-2 inhibitors. Compared with other in vitro COX inhibition assays, the combination of the current ultrafiltration LC-MS-MS assay and LC-MS-MS based PGE2 quantitation assay can enhance the productivity of the discovery of selective COX-2 inhibitors from botanical dietary supplements as well as other natural products.;To address the need for quality control and standardization of ginger dietary supplements, a sensitive, accurate and precise assay based on liquid chromatography-tandem mass spectrometry (LC-MS-MS) with negative ion electrospray was developed and validated for the quantitative analysis of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol, 8-shogaol, 10-shogaol in three different ginger dietary supplements.;In an effort to investigate the efficacy and safety of ginger dietary supplements, human hepatocytes and pooled liver microsomes were used for the evaluation of the in vitro metabolism of several abundant ginger constituents, including 6-, 8-, and 10-gingerol and 6-shogaol. All of the metabolites formed from these compounds were characterized using liquid chromatography-mass spectrometry (LC-MS) or LC-MS-MS. The gingerols and 6-shogaol were substrates for CYP450-catalyzed mono-hydroxylation, dehydrogenation, and O-demethylation during incubation with liver microsomes. Less extensive phase I metabolism was observed after the incubation of these compounds with human hepatocytes, and glucuronic or sulfate conjugates of the unmetabolized gingerols and 6-shogaol were found to be the major phase II metabolites. Furthermore, an off-line ultrafiltration LC-MS-MS assay was used to investigate the in vitro bioactivation of 10-gingerol by human liver microsomes. Seven glutathione (GSH) adducts for 10-gingerol were detected using LC-MS-MS. This case study demonstrated an example that the gingerol-related compounds could be bioactivated by human liver microsomes. |
