Metabolic profiling of five flavonoids from Dragon's Blood in human liver microsomes using high-performance liquid chromatography coupled with high resolution mass spectrometry

Abstract

Although much is known about the pharmacological activities of Dragon's Blood (DB, a traditional Chinese herb), its metabolism in human liver microsomes (HLMs) and the cytochrome P450 (CYP) enzymes has not been studied. This study aims to identify the metabolic profile of five flavonoids (loureirin A, loureirin B, loureirin C, 7,4′-dihydroxyflavone and 5,7,4′-trihydroxyflavanone) from DB in HLMs as well as the CYP enzymes that are involved in the metabolism of them. High-resolution mass spectrometry was used to characterize the structures of their metabolites and 10 cDNA-expressed CYP enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) were used to verify which isozymes mediate in the metabolism of the metabolites. Totally, 29 metabolites including 10 metabolites of loureirin A, 10 metabolites of loureirin B, 4 metabolites of loureirin C, 2 metabolites of 7,4′-dihydroxyflavone and 3 metabolites of 5,7,4′-trihydroxyflavanone were elucidated and identified on the basis of the high-resolution MSⁿ data. The metabolic profile of the five flavonoids in HLMs involved hydroxylation, oxidation and demethylation. Among them, hydroxylation was the predominant biotransformation of the five flavonoids in HLMs, occurring in combination with other metabolic reactions. Assay with recombinant P450s revealed that CYP2C9 and CYP2C19 played an important role in the hydroxylation of flavonoids in HLMs. To the best of our knowledge, this is the first in vitro evaluation of the metabolic profile of loureirin A, loureirin B, loureirin C, 7,4′-dihydroxyflavone and 5,7,4′-trihydroxyflavanone in HLMs.

Introduction

Dragon's Blood (DB) is one kind of a red resin, which derived from Dracaena cochinchinensis (Lour.) S. C. Chen (China) with Chinese name “Longxuejie” [1]. It has been used as a herbal medicine since ancient times in different cultures of world. This rare medicinal plant was mainly distributed in Guangxi and Yunnan provinces in China. It has been included in Guangxi Medicine Standards for clinical use [2]. In traditional Chinese medicine, DB is commonly prescribed to promote blood circulation for the treatment of blood stasis, traumatic injuries and pain. Our previous studies have demonstrated that DB could protect rat brain and gastrointestinal injury induced by different radiation sources [3], [4], [5], [6]. Recent pharmacological researches have shown that DB has anti-thrombotic [7], anti-cerebral ischemia [8], anti-bacterial [9], [10], [11], anti-inflammatory [12], [13], [14], anti-diabetic [15], [16], analgesic [17], [18], [19] and radio-protective activities [3], [4], [6], while it is also known to enhance immune function [20], promote blood circulation and stop bleeding [21], [22].

Flavonoids are the main constituents of DB and various compounds including loureirin A, loureirin B, loureirin C, 7,4′-dihydroxyflavone, 5,7,4′-trihydroxyflavanone and resveratrol have been isolated from the plant [2], [23], [24], [25]. As reported, total flavone extracted from DB is the effective fraction for the anti-inflammatory and analgesic effects of DB [26]. Among them, loureirin A, loureirin B and loureirin C were hypothesized to play a crucial role in promoting blood circulation and removing blood stasis [27]. Loureirin A was reported to exhibit analgesic activity [28] and had an inhibitory effect on platelet activation, perhaps through an impairment of PI3K/Akt signaling [29]. Loureirin B could modulate tetrodotoxin-sensitive and tetrodotoxin-resistant sodium currents in trigeminal ganglion neurons [30], [31] and inhibit scar formation through the ERK and JNK pathway [32]. 7,4′-Dihydroxyflavone could significantly inhibit adipocyte differentiation in 3T3-L1 cells [33].

So far, assays concerning the characterization and quantification for these marker compounds including loureirin A, loureirin B, loureirin C and 7,4′-dihydroxyflavone in bio-samples have been performed [34], [35], [36], [37], [38], [39]. However, the metabolism of DB in human liver microsomes (HLMs) has not been studied. Nowadays, HLMs and recombinant CYPs were widely used to investigate the in vitro biotransformation of drugs [40], [41], [42], [43], [44]. The compound was incubated with HLMs or human recombinant CYPs, and further analyzed by high performance liquid chromatography coupled with high resolution mass spectrometry (HPLC-HR-MS) to qualify and quantify its metabolites [45]. And HPLC-HR-MS has become a powerful and frequently used technique for metabolite identification owing to its accurate mass measurement and reliable metabolite identification [46], [47], [48]. The present study aimed to investigate the metabolic profile of loureirin A, loureirin B, loureirin C, 7,4′-dihydroxyflavone and 5,7,4′-trihydroxyflavanone (structures were shown in Fig. 1) in HLMs using HPLC-HR-MS. Furthermore, the specific CYP metabolizing enzymes responsible for the formation of each metabolite were characterized using 10 human cDNA-expressed CYPs. This study will be helpful for understanding the metabolic routes of the five flavonoids and provide important data for the safe clinical use of DB.