Isolation and identification of twelve metabolites of isocorynoxeine in rat urine and their neuroprotective activities in HT22 cell assay

Abstract

Isocorynoxeine, one of the major alkaloids from Uncaria Hook, shows the effects of lowering blood pressure, vasodilatation, and protection against ischemia-induced neuronal damage. In this paper, the metabolism of isocorynoxeine was investigated in rats. Twelve metabolites and the parent drug were isolated by using solvent extraction and repeated chromatographic methods, and determined by spectroscopic methods including UV, MS, NMR, and CD experiments. Seven new compounds were identified as 11-hydroxyisocorynoxeine, 5-oxoisocorynoxeinic acid-22-O-β-D-glucuronide, 10-hydroxyisocorynoxeine, 17-O-demethyl-16,17-dihydro-5-oxoisocorynoxeine, 5-oxoisocorynoxeinic acid, 21-hydroxy-5-oxoisocorynoxeine, and oxireno[18, 19]-5-oxoisocorynoxeine, together with six known compounds identified as isocorynoxeine, 18,19-dehydrocorynoxinic acid, 18,19-dehydrocorynoxinic acid B, corynoxeine, isocorynoxeine-N-oxide, and corynoxeine-N-oxide. Possible metabolic pathways of isocorynoxeine are proposed. Furthermore, the activity assay for the parent drug and some of its metabolites showed that isocorynoxeine exhibited a significant neuroprotective effect against glutamate-induced HT22 cell death at the maximum concentration. However, little or weak neuroprotective activities were observed for M-3, M-6, M-7, and M-10. Our present study is important to further understand their metabolic fate and disposition in humans.

Fig. 1

UHPLC-UV chromatograms of blank urine (A), a rat urine sample 0 to 24 h after oral administration of 40 mg/kg of ICN (B), and metabolite standards (C).

Fig. 2

Proposed metabolic pathways of isocorynoxeine.

Fig. 3

Effects of isocorynoxeine and some metabolites against 3 mM glutamate-induced HT22 cell death. HT22 cells were treated simultaneously with 3 mM glutamate. Cell viability was determined by calcein AM assay after 24 h exposure to the various samples. All data were normalized to percentage survival of control. Data are represented as mean ± SEM for n = 8; * p < 0.05, ** p < 0.01, and *** p < 0.001 versus glutamate-treated group alone. CN: corynoxeine; G: glutamate; ICN: isocorynoxeine; ZYC-26, 2-(1-adamantyl)-4-methylestrone.