RESUMEN
Angular-type pyranocoumarins (APs), the derivatives of khellactone, are widely documented as the main active constituents in Peucedani Radix (Chinese name: Qian-hu). Owing to the natural occurrence of chiral centers, enantiomers of APs are extensively distributed in the original plant, and enantioselective performances have been definitely demonstrated for these enantiomers. In current study, the chemical characterization of the major and minor APs in Peucedani Radix was performed using ultra high performance liquid chromatography coupled with diode array detector and hybrid ion trap-orbitrap mass spectrometry. On the other hand, a heart-cut two-dimensional achiral-chiral liquid chromatography combining triple quadropole-linear ion trap mass spectrometry system (2D LC-MS/MS) was developed for simultaneous enantiospecific quantification of eighteen coumarins, including seven pairs of enantiomers. Eleven APs (1-11) were recruited to propose UV absorption characteristics and electrospray ionization fragmentation patterns of APs. A total of 42 components were categorized into APs based on their UV spectral properties and identified according to the proposed mass fragmentation pathways, while two linear-type furanocoumarins (12-13) were unambiguously assigned by further purification. A Capcell core RP-C18 column was employed in the primary LC dimension to achieve efficient racemic separation for the main chemical constituents (1-9 and 12-13) in Peucedani Radix, while a Chiralpak AD-RH column was utilized in the secondary dimension to contribute enantioselective separation for seven enantiomerically enriched components (1, 3 and 5-9). Collectively, the results provided the chemical evidences for revealing the material basis of the therapeutic effects of Peucedani Radix, and the developed 2D LC-MS/MS system in the present study is expected to be an ideal tool for the quality control of Peucedani Radix as well as a reliable technique for complex matrices containing both achiral and chiral components.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Medicamentos Herbarios Chinos/química , Piranocumarinas/análisis , Espectrometría de Masas en Tándem/métodos , Piranocumarinas/aislamiento & purificación , EstereoisomerismoRESUMEN
(±)-Praeruptorin A (PA) is the major component of Peucedani Radix. The present study investigated stereoselectivity in PA metabolism in liver microsomes of rats (RLMs) and humans (HLMs), for the first time. PA was enantioseparated by semi-preparative chiral HPLC. Metabolic profiles of the dextrorotatory (dPA) and the levorotatory (lPA) forms in HLMs and RLMs were determined using LC-MS/MS. (-)-cis-Khellactone (D1) prepared from basic hydrolysis of dPA, and (3'R, 4'R)-4'-angeloyl-khellactone (L8) and (3'R, 4'R)-3'-angeloyl-khellactone (L9) isolated from a scale-up incubation of lPA with rat plasma were unambiguously identified by LC-MS/MS and NMR analysis. Other metabolites were tentatively identified using LC-MS/MS. In the absence of NADPH-regenerating system, dPA remained intact, however, lPA yielded L8 and L9 via a carboxylesterase(s)-mediated process. In the presence of NADPH-regenerating system, lPA produced 9 (L1-9) metabolites in both species, while dPA generated 12 (D1-12) and 6 (D1-3, 6, 9 and 10) metabolites in RLMs and HLMs, respectively. Hydrolysis, oxidation and acyl migration were demonstrated to be the predominant pathways for both enantiomers. Both enantiomers were eliminated faster in RLMs than in HLMs, while lPA showed greater species difference. PA enantiomers exhibited stereoselective metabolism in RLMs and HLMs, implying chiral discrimination in their actions.
Asunto(s)
Apiaceae/química , Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/metabolismo , Cumarinas/química , Cumarinas/metabolismo , Medicamentos Herbarios Chinos/química , Microsomas Hepáticos/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Cumarinas/aislamiento & purificación , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Redes y Vías Metabólicas , NADP/metabolismo , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Estereoisomerismo , Factores de TiempoRESUMEN
(±)-Praeruptorin A (dl-PA) is one of the main pyranocoumarins of Peucedani Radix and the chemical marker for quality control of the herb in China. This study investigated the transport and metabolism of dl-PA, for the first time, in Caco-2 cell monolayers. PA enantiomers of dl-PA in the transport study were simultaneously determined using a simple and rapid enantio-selective high performance liquid chromatography-UV method. Both dextrorotatory (d-PA) and levorotatory (l-PA) enantiomers traversed Caco-2 monolayer rapidly in both directions (absorptive P(app): 2.01-3.03 × 10(-5) cm/s; secretory P(app): 1.58-1.96 ×10(-5) cm/s) mainly via passive diffusion. Higher transport rates of dPA were observed in both directions. PA enantiomers were incompletely recovered after the transport study. Nonspecific binding to the Transwell inserts, irreversible binding to cellular components and metabolism within the cells accounted for the loss. dl-PA was partially hydrolyzed in Caco-2 monolayers and yielded two stereoisomers via removal of the acetyl group from C-4' position. Both phenylmethylsulphonyl fluoride (a nonspecific esterase inhibitor) and bis(p-nitrophenyl) phosphate sodium salt (a specific inhibitor of carboxylesterases) completely abolished dl-PA hydrolysis. In summary, PA enantiomers were rapidly transported across Caco-2 cells and partially hydrolyzed by carboxylesterases during permeation. These findings provide mechanistic understanding of in vivo pharmacokinetic properties of dl-PA.