Metabolic profiles of neotuberostemonine and tuberostemonine in rats by high performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Neotuberostemonine (NS) and tuberostemonine (TS), a pair of stereoisomers, are the active components contained in Stemona tuberosa, an antitussive herbal medicine in China. Two isomers have different pharmacological efficacies, which will be related with their in vivo disposition. However, the metabolic fates of NS and TS remain unknown. A method of high performance liquid chromatography/quadrupole time-of-flight mass spectrometry coupled with mass detect filter technique was established to investigate the metabolites in rat plasma, bile, urine, and feces after oral administration of the equal doses of NS and TS. The results showed that NS produced 48 phase I metabolites, including NS, 3 hydrolyzed, 14 hydroxylated, 20 monohydrolyzed+hydroxylated and 10 dihydrolyzed+hydroxylated metabolites. The number of detected NS metabolites was 11, 39, 22 and 30 in plasma, bile, urine and feces. TS yielded 23 phase I metabolites, including TS, 3 hydrolyzed, 7 hydroxylated, 9 monohydrolyzed+hydroxylated and 3 dihydrolyzed+hydroxylated metabolites. Besides, TS yielded 9 phase II metabolites, including 1 glucuronic acid and 2 glutathione conjugates, and the later further degraded and modified into cysteine-glycine, cysteine and N-acetylcysteine conjugates. The number of detected TS metabolites was 9, 24, 24 and 15 in plasma, bile, urine and feces. Different metabolic patterns may be one of the main reasons leading to different pharmacological effects of NS and TS.

Rab8A regulates insulin-stimulated GLUT4 translocation in C2C12 myoblasts.

Rab proteins are important regulators of GLUT4 trafficking in muscle and adipose cells. It is still unclear which Rabs are involved in insulin-stimulated GLUT4 translocation in C2C12 myoblasts. In this study, we detect the colocalization of Rab8A with GLUT4 and the presence of Rab8A at vesicle exocytic sites by TIRFM imaging. Overexpression of dominant-negative Rab8A (T22N) diminishes insulin-stimulated GLUT4 translocation, while constitutively active Rab8A (Q67L) augments it. In addition, knockdown of Rab8A inhibits insulin-stimulated GLUT4 translocation, which is rescued by replenishment of RNAi-resistant Rab8A. Together, these results indicate an indispensable role for Rab8A in insulin-regulated GLUT4 trafficking in C2C12 cells.

Pharmacokinetics, biodistribution and excretion studies of neotuberostemonine, a major bioactive alkaloid of Stemona tuberosa.

Neotuberostemonine is a potent antitussive alkaloid extracted from Stemona tuberosa. However, the pharmacokinetics, tissue distribution and excretion of pure neotuberostemonine have not been reported. The present study was aimed to investigate the pharmacokinetic parameters of neotuberostemonine by developing an ultra-high performance liquid chromatography-tandem mass spectrometry method. Neotuberostemonine and tetrahydropalmatine (internal standard, IS) in bio-samples were extracted by protein precipitation with methanol and successfully separated on a Zorbax Extend C18 column by using a mobile phase of acetonitrile and a mixture of 0.1% formic acid and 5mM ammonium acetate. The detection was performed by using positive ion electrospray ionization in multiple reaction monitoring mode. The MS/MS ion transitions were monitored at m/z 376.1→302.0 for neotuberostemonine and 355.8→192.0 for IS. After oral administration of neotuberostemonine in rats, the Cmax and AUC0-∞ were 11.37ng/mL and 17.68ng·h/mL at 20mg/kg and 137.6ng/mL and 167.4ng·h/mL at 40mg/kg, and the t1/2 were 2.28 and 3.04h at 20 and 40mg/kg, respectively. The high neotuberostemonine concentrations were found in intestine, stomach and liver, and there was no long-term accumulation of neotuberostemonine in tissues. Total recoveries of neotuberostemonine were only 0.90% (0.19% in bile, 0.05% in urine and 0.66% in feces), which might be resulted from the intestine and liver first-pass effects, indicating that neotuberostemonine may be mainly excreted as its metabolites. All above results would provide helpful information for the further pharmacological and clinical studies of neotuberostemonine and the crude drug.