Bioavailability of apocynin through its conversion to glycoconjugate but not to diapocynin

Abstract

Apocynin (4-hydroxy-3-methoxyacetophenone) is a major active ingredient from the rhizomes of Picrorhiza kurroa, a botanical plant used as an herbal medicine for treatment of a number of inflammatory diseases. Recently, apocynin is regarded as a specific inhibitor for NADPH oxidase in cell and animal models. In vitro studies indicated conversion of apocynin to diapocynin in the presence of peroxidases, e.g., myloperoxidase, posing the possibility that diapocynin also contributes to the anti-oxidative action of apocynin. The objectives of this study are to examine the bioavailability of apocynin to plasma, liver and brain tissue after intraperitoneal (i.p.) injection, and to examine whether apocynin is converted to diapocynin in vivo. Diapocynin was chemically synthetized and characterized by NMR and IR. Apocynin (5mg/kg body wt) was injected i.p. to adult male Sprague-Dawley rats and plasma, liver and brain were collected at different times (30min, 1 and 2h) after injection. Samples were treated with beta-glucuronidase to hydrolyze the glycosyl linkage and analyzed by HPLC/MS. At 30min and 1h after injection, approximately 50% of apocynin was converted to its glycosyl derivative and was distributed in plasma, liver and brain. No diapocynin was detected in any samples. These results indicate rapid glycosylation of apocynin and its transport to blood and other organs but no apparent conversion to diapocynin in vivo.

Fig. 1

Reaction for conversion of apocynin to diapocynin.

Fig. 2

Analysis of apocynin and diapocynin standards by HPLC. See text for details of the analysis.

Fig. 3

(A) Proton-Decoupled ¹³C-NMR Spectrum of Diapocynin in DMSO-d₆. (B) APT Spectrum of Diapocynin in DMSO-d₆. Positive peaks are due to C and CH₂, negative peaks are due to CH and CH₃.

Fig. 4

LC-MS and UV analysis of diapocynin.

Fig. 5

Representative chromatographs showing apocynin in plasma at different times after i.p. injection (5 mg/kg). Arrow points to apocynin peak.

Fig. 6

Levels of apocynin in plasma with or without β-glucuronidase treatment.

Fig. 7

Apocynin in plasma, liver and brain comparing with control and 30 min after i.p. injection (5 mg/kg). Samples were treated with β-glucoronidase prior to analysis by HPLC.