In vitro studies of absorption and metabolism of natural products using mass spectrometry

Early predictions of metabolism and pharmacokinetic properties of drug candidates are essential to streamline drug development, and mass spectrometry has become an essential analytical tool throughout this process. Natural products are receiving renewed attention as sources of new drug candidates. Therefore, liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS-MS) were used to investigate the in vitro intestinal absorption and metabolism and hepatic metabolism of various natural products.; As an in vitro model of human intestinal permeability and metabolism, incubations of natural products were carried out with Caco-2 cell monolayers followed by LC-MS and LC-MS-MS analysis. For resveratrol, passive diffusion occurred rapidly across the Caco-2 cell monolayers. However, extensive phase II conjugation was observed that might reduce its bioavailability. Next, the intestinal permeabilities of red clover (Trifolium pratense ) phytoestrogens genistein, daidzein, biochanin A, and formononetin were investigated. These isoflavones passively diffused across the Caco-2 cell monolayers rapidly but were converted to sulfates and glucuronides to some extent. The cancer chemotherapeutic agent betulinic acid was evaluated using the Caco-2 cell monolayers and showed poor intestinal permeability. In another set of Caco-2 cell monolayer experiments, the intestinal permeability of potentially active compounds in black cohosh (Cimicifuga racemosa ) was investigated. These results indicate that caffeic acid derivatives do not readily cross the intestinal epithelium but some triterpenoids have sufficient permeability. Therefore, this new application of Caco-2 cell monolayers is useful to guide bioassay-directed fractionation.; In addition, drug candidates brassinin, oxomate, sulforamate, 4 '-bromoflavone, and 8-prenylnarigenin were assayed using Caco-2 cell monolayers. Except for 4'-bromoflavone which demonstrated moderate permeability, all of these compounds showed high permeability.; Furthermore, incubations of these five compounds were conducted with human liver microsomes and hepatocytes as hepatic metabolism models. Metabolites were characterized and in many cases identified using LC-MS, LC-MS-MS, and comparison to synthetic standards. Pathways for the hepatic metabolism of the five compounds were proposed. One significant finding was that brassinin was metabolized to additional active compounds. Moreover, 8-prenylnarigenin was found to undergo extensive phase II metabolism. Therefore, its bioavailability will probably be low due to extensive first-pass metabolism.