Structural characterization of minor metabolites and pharmacokinetics of ganoderic acid C2 in rat plasma by HPLC coupled with electrospray ionization tandem mass spectrometry

Abstract

The metabolites and pharmacokinetics of ganoderic acid C2 (GAC2), a bioactive triterpenoid in Ganoderma lucidum in rat plasma were investigated by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS). Totally, ten minor phase I metabolites of GAC2 were characterized after oral administration of GAC2, on the basis of their mass fragmentation pathways or direct comparison with authentic compounds by high-performance liquid chromatography coupled with diode array detection and electrospray ion trap tandem mass spectrometry (HPLC–DAD–ESI-MSⁿ), and liquid chromatography coupled with electrospray ionization hybrid ion trap and time-of-flight mass spectrometry (LC–ESI-IT-TOF/MS) methods. Moreover, a rapid and specific method for quantification of GAC2 in rat plasma after oral administration was developed by using a liquid–liquid extraction procedure and HPLC–ESI-MS/MS analysis. It is the first time to report the metabolites and pharmacokinetics of GAC2.

Introduction

Ganoderma lucidum (Leyss. ex Fr.) Karst, one of the oldest traditional Chinese medicine, has been used in Asia for centuries and called as “immortal herb” in ancient China. Nowadays, G. lucidum is widely used in the fields of life prolongation, brain ischemia, chronic viral hepatitis, chemotherapy-induced toxicity, antitumor immunostimulation, and diabetes mellitus [1], [2]. Highly oxygenated lanostane-type triterpenoids are one of the main bioactive constituents in G. lucidum. More than 140 triterpenoids have been isolated from the fruiting bodies, spores and mycelia of G. lucidum [2], [3], [4] with the significant and wide bioactivities such as cytotoxicity [5], neurotrophic effect [6], anti-inflammatory [7] and inhibition activities of aldose reductase [8] and cholinesterase [9]. Among them, ganoderic acid C2 (GAC2) is a major triterpenoid from the powder of G. lucidum, and generally exists in Ganoderma genus [10], [11], [12]. Previous studies indicated that GAC2 possessed the potential anti-tumor bioactivity [7], antihistamine [13], anti-aging [14] and cytotoxic effects [15]. The GAC2, as a potent aldose reductase inhibitor, may be used for the treatment of diabetic complications [16], [17]. Moreover, GAC2 has been used as the mark compound for quality control of G. lucidum and its related species and distinguishing the differences between species of Ganoderma [12], [18], [19].

In comparison with the reports of biological activities and quality evaluation, the studies regarding metabolic profile and pharmacokinetics in vivo of GAC2 are limited. Up to now, only mixtures containing three pure triterpenoids (ganoderic acid A, ganoderic acid T and ganoderiol F) from G. lucidum, were investigated on their metabolism and pharmacokinetics [20], [21], [22]. In the previous investigation, determination of GAC2 in rat plasma and urine after oral administration of G. lucidum extract was performed only by high-performance liquid chromatography (HPLC) methods [23], [24]. To our knowledge, there was been no report on the metabolites of GAC2 in vivo.

We previously reported the major compounds in rat plasma and bile after oral administration of total triterpenoids from G. lucidum [25], [26]. Our present work is focused on the metabolic and pharmacokinetic behaviors of GAC2 in rats. By HPLC–DAD–ESI-MSⁿ and LC–ESI-IT-TOF/MS methods, the trace metabolites of GAC2 in rat plasma after oral administration of GAC2 were structurally characterized. Furthermore, the pharmacokinetic parameters of GAC2 in rat plasma after oral administration of GAC2 were determined by a sensitive and rapid HPLC–ESI-MS/MS method.