Metabolites identification and multi-component pharmacokinetics of ergostane and lanostane triterpenoids in the anticancer mushroom Antrodia cinnamomea

https://doi.org/10.1016/j.jpba.2015.04.010Get rights and content

Highlights

  • We studied the DMPK of triterpenoids in Antrodia cinnamomea, an anticancer mushroom.

  • In total 18 triterpenoids and 8 metabolites were identified by UHPLC/qTOF-MS.

  • Two hydrogenated metabolites were obtained by semi-synthesis as new compounds.

  • PK of 14 unchanged triterpenoids and 2 metabolites were studied by LC/MS/MS.

  • High-polarity ergostanes are major plasma-exposed components of A. cinnamomea.

Abstract

Antrodia cinnamomea is a precious medicinal mushroom popularly used for adjuvant cancer therapy in Taiwan. Its major bioactive constituents are ergostane and lanostane triterpenoids. Although clinical trials for A. cinnamomea have been recently initiated, its metabolism remains unclear. The present study aims to elucidate the metabolism and pharmacokinetics of A. cinnamomea in rats. After oral administration of an ethanol extract, 18 triterpenoids and 8 biotransformed metabolites were detected in rats plasma by UHPLC/qTOF-MS. Four of the metabolites were prepared by semi-synthesis and fully identified by NMR, while the others were tentatively characterized by comparing with the metabolites of single compounds (antcins B, C, H and K). Furthermore, a multi-component pharmacokinetic study of A. cinnamomea was carried out to monitor the plasma concentrations of 14 triterpenoids (ergostanes 13, 58, 1416; lanostanes 9, 10, 17, 19) and 2 metabolites (M5, M6) by LC/MS/MS in rats after oral administration of the ethanol extract (1.0 g/kg). The results showed that ergostanes and Δ7,9(11) lanostanes, but not Δ8 lanostanes, could get into circulation. The low-polarity ergostanes (antcins B and C) undertook hydrogenation (C-3 or C-7 carbonyl groups) or hydroxylation to produce polar metabolites. High-polarity ergostanes (antcins H and K) and Δ7,9(11) lanostanes were metabolically stable. We also discovered that ergostanes and lanostanes showed remarkably different pharmacokinetic patterns. The ergostanes were generally absorbed and eliminated rapidly, whereas the lanostanes remained in the plasma at a low concentration for a relatively long time. The results indicate that high-polarity ergostanes are the major plasma-exposed components of A. cinnamomea, and may play an important role in its therapeutic effects.

Introduction

Antrodia cinnamomea (Antrodia camphorata, Polyporaceae family) is a precious medicinal mushroom [1]. Known as the traditional medicine Niu-Chang-Chih, it has been popularly used to treat cancer, intoxication and inflammation for a long history in Taiwan [2], [3]. According to a recent survey, 12% cancer patients in Taiwan use A. cinnamomea as adjuvant therapeutic agent or nutrient during cancer treatment [4]. Preclinical studies have revealed that A. cinnamomea could inhibit tumor growth by 42% and 80% in mice bearing H22 liver tumor and MDA-MB-231 breast tumor xenograft, respectively [5], [6]. A. cinnamomea is also widely used by healthy people as a dietary supplement to promote physical strength. Clinical trials for A. cinnamomea have been initiated recently [7], [8]. However, little is known on in vivo metabolism and pharmacokinetics of this multi-component traditional medicine, so far.

A. cinnamomea contains abundant ergostane and lanostane tetracyclic triterpenoids, accounting for 30% of its methanol extract [3]. Ergostanes (C-4 α-CH3, Δ24,28) are its characteristic constituents. Although lanostanes are widely distributed in medicinal fungi such as Ganoderma lucidum (Ling-Zhi) and Poria cocos (Fu-Ling), most lanostanes from A. cinnamomea have lower degree of oxygenation (1–3 –OH/double bondO groups) [9]. Many of these triterpenoids show anti-cancer and anti-inflammatory activities [10], [11]. For example, dehydroeburicoic acid (19, 10 μg/g dosage) could significantly inhibit tumor growth (0.16 g vs 0.29 g, p < 0.05) in HL 60 cell xenograft mice; antcin C (5/6) could protect 2,2-azobis(2-amidinopropane) dihydrochloride-induced mice liver damage via Nrf2 pathway [12], [13]. Thus, triterpenoids are generally considered as the major bioactive constituents of A. cinnamomea, and are used as chemical markers for its quality control [14]. Metabolic studies of G. lucidum and P. cocos showed that lanostane triterpenoids were orally bioavailable, and mainly underwent hydrogenation, hydroxylation, and dehydrogenation metabolic reactions in vivo [15], [16]. However, the in vivo metabolism of ergostanes and lanostanes in A. cinnamomea has never been reported. Furthermore, most triterpenoids in A. cinnamomea have poor solubility in water and even ethanol or acetone. Their absorption after oral administration, as well as their in vivo metabolic pathway and plasma concentrations warrants to be clarified.

The present work studied the metabolism and pharmacokinetics of A. cinnamomea in rats. After oral administration of an ethanol extract, 18 triterpenoids and 8 metabolites were detected in rats plasma by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC/qTOF-MS). The metabolites were identified by comparing with reference standards, or by comparing to metabolites of single compounds (antcins B, C, H and K). A multi-component pharmacokinetic study was then conducted by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to simultaneously monitor 14 triterpenoids (13, 510, 1417, 19) and 2 metabolites (M5, M6). A brief workflow of this study is depicted in Fig. 1.

Section snippets

Chemicals and reagents

Acetonitrile, methanol, and formic acid (Mallinkrodt Baker, Phillipsburg, NJ, USA) were of HPLC grade. De-ionized water was obtained from a Milli-Q system (Millipore, MA, USA). High-purity nitrogen (99.9%) and helium (99.99%) were purchased from Gas Supplies Center of Peking University Health Science Center (Beijing, China). Reference compounds 110, 1317, 19 and 20 were isolated from A. cinnamomea by the authors [10]. Reference compounds 11, 12, M5 and M6 were synthesized in this study. Their

Metabolites identification for Antrodia cinnamomea in rats

After oral administration of 1.5 g/kg A. cinnamomea extract (equivalent to 6.0 g/kg of crude drug), the rats plasma samples were analyzed by UHPLC/qTOF-MS. The total ion current chromatogram for a 1.5-h plasma sample is shown in Fig. 3, as it contained more metabolites than the 0.5-h or 12-h samples (Supplemental Fig. 1). A total of 26 triterpenoids could be detected. All the 20 major triterpenoids in A. cinnamomea were present in the plasma, except for the two Δ8 lanostanes 13 and 20 (compound 9

Discussion

Although A. cinnamomea is widely used as adjuvant therapeutic agent and dietary supplement, its metabolism has rarely been revealed. Only the plasma distribution of two maleic anhydride derivatives (antrodin B and C) had been reported [22]. Triterpenoids accounted for around 10% of the drug materials, and should be its major bioactive constituents. In this work, we used UHPLC/qTOF-MS and LC/MS/MS to elucidate the metabolism and pharmacokinetics of A. cinnamomea in rats. Due to its complicated

Conclusions

The metabolism and pharmacokinetics of A. cinnamomea in rats was studied. A total of 26 triterpenoids and metabolites were detected in rats plasma after oral administration. The ergostanes and Δ7,9(11) lanostanes, but not the Δ8 lanostanes, could get into circulation. Different types of triterpenoids showed remarkably different pharmacokinetic patterns. The ergostanes were generally absorbed and eliminated rapidly, whereas the lanostanes remained in the plasma at a low concentration for a

Acknowledgements

This work was supported by National Natural Science Foundation of China (no. 81222054, no. 81303294), and the Program for New Century Excellent Talents in University from Chinese Ministry of Education (no. NCET-11-0019).

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