Biotransformation and metabolic profile of anemoside B4 with rat small and large intestine microflora by ultra‐performance liquid chromatography–quadrupole time‐of‐flight tandem mass spectrometry

Pulsatilla chinensis (Bunge) Regel is commonly used in Asia, and anemoside B4 (AB4) is its major saponin, with diverse pharmaceutical effects. Previous studies showed that intestinal flora plays an important role in the metabolism of herbs administered orally. In this study, the metabolic profile of AB4 with microflora in rat small and large intestines in vitro was investigated. Gut microflora was collected from different intestinal segments and anaerobically incubated with AB4 at 37°C for 24, 48, 72 and 96 h, respectively. A total of 10 metabolites were detected and identified by ultra‐ performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry, involving the products of oxygenation and deglycosylation reactions. Gut microflora in the large intestine generated more comprehensive metabolic pathways, which appears to be attributable to the wider range of bacterial types and numbers of bacteria. Human cancer cell lines SMMC‐7721, Hela and MCF‐7 were treated with metabolite pools by MTT assay, together with M6 as the greatest deglycosylation product. As a result, M6 exhibited a reduction in cell viability of SMMC‐7721 with an IC₅₀ value of 22.28 ± 1.26 μg/mL. The present study provided scientific evidence for AB4 metabolism in small and large intestines, which is helpful to reveal the active forms of AB4 in vivo .     

Studies on metabolites and metabolic pathways of bulleyaconitine A in rat liver microsomes using LC‐MSn combined with specific inhibitors

Bulleyaconitine A (BLA) from Aconitum bulleyanum plants is usually used as anti‐inflammatory drug in some Asian countries. It has a variety of bioactivities, and at the same time some toxicities. Since the bioactivities and toxicities of BLA are closely related to its metabolism, the metabolites and the metabolic pathways of BLA in rat liver microsomes were investigated by HPLC–MSⁿ. In this research, the 12 metabolites of BLA were identified according to the results of HPLC‐MSⁿ data and the relevant literature. The results showed that there are multiple metabolites of BLA in rat liver microsomes, including demethylation, deacetylation, dehydrogenation deacetylation and hydroxylation. The major metabolic pathways of BLA in rat liver microsomes were clarified by HPLC‐MS combined with specific inhibitors of CYP450 isoforms. As a result, CYP3A and 2C were found to be the principal CYP isoforms contributing to the metabolism of BLA. Moreover, CYP2D6 and 2E1 are also more important CYP isoforms for the metabolism of BLA. While CYP1A2 only affected the formation rate of M11, its effect on the metabolism of BLA is very small. Copyright © 2014 John Wiley & Sons, Ltd.     

GC/MS characterization of urinary metabolites of doxylamine succinate: Identification of the aglycones formed from intestinal microflora metabolism of the polar glucuronide metabolites

This study describes the use of high performance liquid chromatography (HPLC) and capillary gas chromatography/mass spectrometry (GC/MS) in the characterization of polar glucuronide conjugates of doxylamine and their subsequent aglycones following deconjugation. Rat urinary extracts which contained doxylamine and both nonconjugated and conjugated doxylamine metabolites, were examined by HPLC before and after incubation with rat intestinal microflora. The subsequent deconjugated urinary metabolites and the nonconjugated products remaining in the urinary extracts were then isolated, acetylated, and assayed by GC/MS. Incubation with the intestinal microflora indicated that anaerobic bacteria were capable of effecting hydrolytic cleavage of these polar O-glucuronide metabolites of doxylamine and its demethylated products to their subsequent aglycones. GC/MS analysis was performed using a fused silica DB-5 GC column and was utilized for the identification of these deconjugated metabolites.     

Characterization of metabolites of sweroside in rat urine using ultra‐high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry and NMR spectroscopy

Sweroside, a major active iridoid in Swertia pseudochinensis Hara, is recognized as an effective agent in the treatment of liver injury. Based on previous reports, the relatively short half‐life (64 min) and poor bioavailability (approximately 0.31%) in rats suggested that not only sweroside itself but also its metabolites could be responsible for the observed hepato‐protective effect. However, few studies have been carried out on the metabolism of sweroside. Therefore, the present study aimed at identifying the metabolites of sweroside in rat urine after a single oral dose (100 mg/kg). With ultra‐high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UHPLC/Q‐TOF‐MS), the metabolic profile revealed 11 metabolites in rat urine, including phase I, phase II and aglycone‐related products. The chemical structures of metabolites were proposed based on accurate mass measurements of protonated or deprotonated molecules and their fragmentation patterns. Our findings showed that the aglycone of sweroside (M05) and its glucuronide conjugate (M06) were principal circulating metabolites in rats. While several other metabolic transformations, occurring via reduction, N‐heterocyclization and N‐acetylation after deglycosylation, were also observed. Two metabolites (M05 and M06) were isolated from the rat urine for structural elucidation and identifcation of reaction sites. Both M05 and M06 were characterized by ¹H, ¹³C and two‐dimensional nuclear magnetic resonance (NMR) spectroscopy. UHPLC/Q‐TOF‐MS analysis has provided an important analytical platform to gather metabolic profile of sweroside. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of the novel thiosemicarbazone anti‐cancer agent,Bp4eT,and its main phase I metabolites in plasma: Application to a pilot pharmacokinetic study in rats

Novel thiosemicarbazone metal chelators are extensively studied anti‐cancer agents with marked and selective activity against a wide variety of cancer cells, as well as human tumor xenografts in mice. This study describes the first validated LC‐MS/MS method for the simultaneous quantification of 2‐benzoylpyridine 4‐ethyl‐3‐thiosemicarbazone (Bp4eT) and its main metabolites (E/Z isomers of the semicarbazone structure, M1‐E and M1‐Z, and the amidrazone metabolite, M2) in plasma. Separation was achieved using a C₁₈ column with ammonium formate/acetonitrile mixture as the mobile phase. Plasma samples were treated using solid‐phase extraction on 96‐well plates. This method was validated over the concentration range of 0.18–2.80 μM for Bp4eT, 0.02–0.37 μM for both M1‐E and M1‐Z, and 0.10–1.60 μM for M2. This methodology was applied to the analysis of samples from in vivo experiments, allowing for the concentration–time profile to be simultaneously assessed for the parent drug and its metabolites. The current study addresses the lack of knowledge regarding the quantitative analysis of thiosemicarbazone anti‐cancer drugs and their metabolites in plasma and provides the first pharmacokinetic data on a lead compound of this class. Copyright © 2013 John Wiley & Sons, Ltd.     

In vitro metabolism of β‐lapachone (ARQ 501) in mammalian hepatocytes and cultured human cells

ARQ 501 (3,4‐dihydro‐2,2‐dimethyl‐2H‐naphthol[1,2‐b]pyran‐5,6‐dione, β‐lapachone) is an anticancer agent, currently in multiple phase II clinical trials as monotherapy and in combination with other cytotoxic drugs. This study focuses on in vitro metabolism in cryopreserved hepatocytes from mice, rats, dogs and humans using [¹⁴C]‐labeled ARQ 501. Metabolite profiles were characterized using liquid chromatography/mass spectrometry combined with an accurate radioactivity counter. Ion trap mass spectrometry was employed for further structural elucidation. A total of twelve metabolites were detected in the mammalian hepatocytes studied; all of which but one were generated from phase II conjugation reactions. Ten of the observed metabolites were produced by conjugations occurring at the reduced ortho‐quinone carbonyl groups of ARQ 501. The metabolite profiles revealed that glucuronidation was the major biotransformation pathway in mouse and human hepatocytes. Monosulfation was the major pathway in dog, while, in rat, it appears glucuronidation and sulfation pathways contributed equally. Three major metabolites were found in rats: monoglucuronide M1, monosulfate M6, and glucuronide‐sulfate M9. Two types of diconjugation metabolites were formed by attachment of the second glycone to an adjacent hydroxyl or to an existing glycone. Of the diconjugation metabolites, glucosylsulfate M10, diglucuronide M5, and glucuronide‐glucoside M11 represent rarely observed phase II metabolites in mammals. The only unconjugated metabolite was generated through hydrolysis and was observed in rat, dog and human hepatocytes. ARQ 501 appeared less stable in human hepatocytes than in those of other species. To further elucidate the metabolism of ARQ 501 in extrahepatic sites, its metabolism in human kidney, lung and intestine cells was also studied, and only monoglucuronide M1 was observed in all the cell types examined. Copyright © 2008 John Wiley & Sons, Ltd.     

Investigation of the interactions between Chrysanthemum morifolium flowers extract and intestinal bacteria from human and rat

Flos Chrysanthemi, dried flower of Chrysanthemum morifolium Ramat, has drawn much attention recently owing to its potential beneficial health effects for human. Flos Chrysanthemi products are usually taken orally and metabolized by intestinal microflora. However, there has been no investigation of the comprehensive metabolic profile of the Flos Chrysanthemi extract by intestinal flora owing to its chemical complexity and the limitations of analytical methods. In this paper, a rapid, sensitive and automated analysis method, ultra‐performance liquid chromatography/quadrupole time of flight mass spectrometry including MS^E technology and automated data processing Metabolynx? software, was developed and successfully applied for the biotransformation and metabolic profile of flavonoids in the Flos Chrysanthemi extract by intestinal flora from human and rat. A total of 32 metabolites were detected and tentatively identified in human and rat intestinal bacterial samples. These metabolites indicated that hydrolysis, hydroxylation, acetylation, methylation, hydrogenation and deoxygenation were the major conversion pathways of flavonoids in the Flos Chrysanthemi extract in vitro. Furthermore, the effects of the Flos Chrysanthemi extract on the growth of different intestinal bacteria were detected using an Emax precision microplate reader. Certain pathogenic bacteria such as Enterobacter, Enterococcus, Clostridium and Bacteroides were significantly inhibited by Flos Chrysanthemi, while commensal probiotics such as Lactobacillus and Bifidobacterium were moderately promoted. Our observation provided further evidence for the importance of intestinal bacteria in the metabolism and potential activity of the Flos Chrysanthemi extract. The results will also be helpful for the further pharmacokinetic study of Flos Chrysanthemi and to unravel how it works in vivo.     

Identification of metabolites of novel Anti‐MRSA fluoroquinolone WCK 771 in mouse,rat, rabbit,dog, monkey and human urine using liquid chromatography tandem mass spectrometry

WCK 771 is an l ‐arginine salt of levonadifloxacin (LND) being developed in intravenous dosage form and has recently completed a phase III trial in India. The pharmacokinetics of WCK 771, a novel anti‐MRSA fluoroquinolone, were examined in mice, rats, rabbits, dogs, monkeys and humans after systemic administration during pre‐clinical and clinical investigations. Urine and serum were evaluated for identification of metabolites. It was observed that LND mainly follows phase II biotransformation pathways. All of the species showed a different array of metabolites. In mice, rabbit and dog, the drug was mainly excreted in the form of O‐glucuronide (M7) and acyl glucuronide (M8) conjugates, whereas in rat and human major metabolite was sulfate conjugate (M6). Monkeys exhibited equal distribution of sulfate (M6) and glucuronide conjugates (M7, M8). In addition to these three major phase II metabolites; five phase I oxidative metabolites (M1, M2, M3, M4 and M5) were identified using liquid chromatography tandem mass spectrometry. Out of these eight metabolites M2, M3, M5, M7 and M8 are reported for the first time.     

Metabolic profiling of tenacigenin B,tenacissoside H and tenacissoside I using UHPLC‐ESI‐Orbitrap MS/MS

Marsdenia tenacissima, which is widely used as an anticancer herb in traditional Chinese medicine, has been shown to possess anticancer activity. However, its metabolic profile is poorly investigated. Tenacigenin B is the major steroidal skeleton of C‐21 steroids in M. tenacissima. Tenacissoside H and Tenacissoside I are detected at relatively high levels in M. tenacissima. Therefore, we studied their metabolic characteristics in human liver microsomes by ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry. Fourteen metabolites were tentatively identified by accurate mass measurement and MS/MS fragmentation behavior. It was found that hydroxylation reactions were the major metabolic pathway of Tenacissoside H and Tenacissoside I in human liver microsomes, whereas the metabolic pathway of Tenacigenin B involved dehydrogenation reactions. This is the first time that the metabolic profile of C‐21 steroids from M. tenacissima has been explored in human liver microsomes, which is of great significance for subsequent pharmacokinetic and interaction research. Biotransformation in vivo or in vitro may influence the structure of a compound and change its activity. Identification of their fragmentation behaviors and metabolites provides valuable and new information for further understanding the anti‐tumor activity of M. tenacissima. Copyright © 2016 John Wiley & Sons, Ltd.     

Metabolite identification of tanshinol borneol ester in rats by high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight mass spectrometry

Tanshinol borneol ester (DBZ) is a potential drug candidate composed of danshensu and borneol. It shows anti‐ischemic and anti‐atherosclerosis activity. However, little is known about its metabolism in vivo. This research aimed to elucidate the metabolic profile of DBZ through analyzing its metabolites using high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight mass spectrometry. Chromatographic separation was performed on an Agilent TC‐C₁₈ column (150 × 4.6 mm, 5.0 μm) with gradient elution using methanol and water containing 0.2% (v/v) formic acid as the mobile phase. Metabolite identification involved analyzing the retention behaviors, changes in molecular weights and MS/MS fragment patterns of DBZ and its metabolites. As a result, 20 potential metabolites were detected and tentatively identified in rat plasma, urine and feces after administration of DBZ. DBZ could be metabolized to O‐methylated DBZ, DBZ‐O‐glucuronide, O‐methylated DBZ‐O‐glucuronide, hydroxylated DBZ and danshensu. Danshensu, a hydrolysis product of DBZ, could further be transformed into 12 metabolites. The proposed method was confirmed to be a reliable and sensitive alternative for characterizing metabolic pathways of DBZ and providing valuable information on its druggability.     

Characterization of the metabolite of AdipoRon in rat and human liver microsomes by ultra‐high‐performance liquid chromatography combined with Q‐Exactive Orbitrap tandem mass spectrometry

AdipoRon is an orally active adiponectin receptor agonist. The aim of this study was to characterize the metabolites of AdipoRon in rat and human liver microsomes using ultra‐high performance liquid chromatography combined with Q‐Exactive Orbitrap tandem mass spectrometry (UPLC‐Q‐Exactive‐Orbitrap‐MS) together with data processing techniques including extracted ion chromatograms and a mass defect filter. AdipoRon (10 μm ) was incubated with liver microsomes in the presence of NADPH and this resulted in a total of 11 metabolites being detected. The identities of these metabolites were characterized by comparing their accurate masses and fragment ions as well as their retention times with those of AdipoRon using MetWorks software. Metabolites M1–M3, M6, and M8–M11 were identified for the first time. Metabolite M4, the major metabolite both in rat and human liver microsomes, was further confirmed using the reference standard. Our results revealed that the metabolic pathways of AdipoRon in liver microsomes were N‐dealkylation (M2), hydroxylation (M, M5–M9), carbonyl reduction (M4) and the formation of amide (M10 and M11). Our results provide valuable information about the in vitro metabolism of AdipoRon, which would be helpful for us to understand the mechanism of the elimination of AdipoRon and, in turn, its effectiveness and toxicity.     

Identification of metabolites of rupestonic acid in rat urine by liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Rupestonic acid, a potential anti‐influenza agent, is an important and characteristic compound in Artemisia rupestris L., a well‐known traditional Uighur medicine for the treatment of colds. In the present study, high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry was used to detect and identify the metabolites in rat urine after oral administration of rupestonic acid. A total of 10 metabolites were identified or partially characterized. The structure elucidations of the metabolites were performed by comparing the changes in accurate molecular masses and fragment ions with those of the parent compound. The results showed that the main metabolites of rupestonic acid in rat urine were formed by oxidation, hydrogenation and glucuronidation. A metabolism pathway was proposed for the first time based on the characterized structures. This metabolism study can provide essential information for drug discovery, design and clinical application of rupestonic acid. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolite profiling of ginsenosides in rat plasma,urine and feces by LC–MS/MS and its application to a pharmacokinetic study after oral administration of Panax ginseng extract

Panax ginseng is widely consumed as a functional food in the form of tea, powder, capsules, among others, and possesses a range of pharmacological activities including adaptogenic, immune‐modulatory, anti‐tumor, anti‐aging and anti‐inflammatory effects. The aim of this study was to identify and quantify the major ginsenosides and their metabolites in rat plasma, urine and feces after administration of P. ginseng extract using LC–MS/MS. We collected rat plasma samples at 0.5, 1, 2, 4, 8, 12, 24 and 48 h, and the amounts of urine and fecal samples accumulated in 24 h. Fourteen major ginsenosides and their metabolites were observed in fecal samples at high levels; however, low levels of 11 ginsenosides were detected in urine samples. The pharmacokinetics of the major ginsenosides and their metabolites was investigated in plasma. The results indicated that the maximum plasma concentration, time to maximum concentration and area under the curve of compound K were significantly greater than those of other ginsenosides. This study thus provides valuable information for drug development and clinical application of P. ginseng.     

Effect of pH on the Metabolism of Aconitine under Rat Intestinal Bacteria and Analysis of Metabolites Using HPLC/MS-MSn Technique

A semi‐quantitative method of mass spectrometry (MS) has been described for the analysis of metabolites of aconitine by rat intestinal bacteria at different pH. At pH 7.0, the rat intestinal bacteria exhibit optimal activity for the metabolism of aconitine. A high‐performance liquid chromatography‐electrospray ionization multiple‐stage mass spectrometry (HPLC/ESI‐MSⁿ) method has been applied to investigate the characteristic product ions of metabolites. Then, the logical fragmentation pathways of metabolites have been proposed. By comparing the retention time (t_R) of HPLC and the ESI‐MSⁿ data with the data of standard compounds and reports from literature, ten metabolites have been identified and a distinctive metabolite (15‐deoxyaconitine) has been deduced first time. The experimental results demonstrate that HPLC/ESI‐MSⁿ is a specific and useful method for the identification of metabolites of aconitine. Also, in the present paper, the HPLC‐MS method was introduced to determine the synthetical metabolite prior to the study of the toxicity by the method of Bliss.     

Effect of liquiritin on human intestinal bacteria growth: metabolism and modulation

Licorice is one of the oldest and most frequently used prescribed traditional Chimese medicines. However, the route and metabolites of liquiritin by human intestinal microflora are not well understood and its metabolites may accumulate to exert physiological effects. Therefore, our objective was to screen the ability of the bacteria to metabolize liquiritin and assess the effect of this compound on the intestinal bacteria. Finally, six strains, comprising Bacteroides sp. 22 and sp.57, Veillonella sp. 31 and sp.48, Bacillus sp. 46 and Clostridium sp. 51, were isolated and their abilities to convert liquiritin studied. A total of five metabolites were identified using ultra performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry in human incubated solution. The results indicated that hydrolysis, hydrogenation, methylation, deoxygenation and acetylation were the major routes of metabolism of liquiritin. On the other hand, effect of liquiritin on different strains of intestinal bacteria growth was detected using an Emax precision microplate reader. Growth of certain pathogenic bacteria, such as Enterobacter, Enterococcus, Clostridium and Bacteroides, was significantly repressed by liquiritin, while growth of commensal probiotics such as Lactobacillus and Bifidobacterium was less severely affected. Our observation provided further evidence for the importance of intestinal bacteria in the metabolism, and the potential activity of liquiritin in human health and disease. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification and quantitative investigation of the effects of intestinal microflora on the metabolism and pharmacokinetics of notoginsenoside Fc assayed by liquid chromatography with electrospray ionization tandem mass spectrometry

Notoginsenoside Fc, which is a protopanaxdiol‐type saponin isolated from the leaves of Panax notoginseng, exhibits an exceptional antiplatelet aggregatory effect. To study the modulating effect of gastrointestinal contents on the metabolic profile and pharmacokinetics, pseudo germ‐free rats were used to study the influence of the bacterial community structure on the metabolic profile. Glycosidase activities were measured using the spectrophotometric method. Biotransformations of notoginsenoside Fc in normal and pseudo germ‐free rat intestinal microflora were systematically investigated using ultra high performance liquid chromatography with tandem quadrupole/time‐of‐flight mass spectrometry. Moreover, a liquid chromatography with tandem mass spectrometry method was established for simultaneous determination of the notoginsenoside Fc prototype and its degradation products. Through an in vivo pharmacokinetic study, the pharmacokinetic characteristics were compared between normal rats and pseudo germ‐free rats. During the in vitro biotransformation, seven deglycosylated products were detected and identified after incubation in the intestinal bacteria of normal rats. In pseudo germ‐free rats, glycosidase activities were significantly decreased, and no obvious degradation occurred. In an in vivo study, the systemic exposure was significantly increased 40%, as evidenced by the area under the blood concentration–time curve from time zero to infinity value and half‐life value, which were prolonged more in the pseudo germ‐free group than in normal rats. The results demonstrate that patients who use intestinal bacteria‐metabolized herbs, such as panax notoginseng, should understand the profile of intestinal bacteria to ensure therapeutic efficacy.     

Studies on oral bioavailability and first‐pass metabolism of withaferin A in rats using LC–MS/MS and Q‐TRAP

Withaferin A (WA) is one of the major bioactive steroidal lactones with extensive pharmacological activities present in the plant Withania somnifera. The absolute oral bioavailability of WA remains unknown and human‐related in vitro data are not available. Therefore, in the present study, the absolute oral bioavailability of WA in male rats and the in vitro screening of absorption factors by Q‐trap and LC–MS/MS analysis were conducted to explore possible clinical properties of WA. The developed and validated analytical methods were successfully applied to the pharmacokinetic studies and in vitro measurement of WA. The oral bioavailability was determined to be 32.4 ± 4.8% based on intravenous (5 mg/kg) and oral (10 mg/kg) administrations of WA in male rats. The in vitro results showed that WA could be easily transported across Caco‐2 cells and WA did not show as a substrate for P‐glycoprotein. Moreover, the stability of WA was similar between male rat and human in simulated gastric fluid (stable), in intestinal microflora solution (slow decrease) and in liver microsomes (rapid depletion, with a half‐life of 5.6 min). As such, the first‐pass metabolism of WA was further verified by rat intestine‐liver in situ perfusion, revealing that WA rapidly decreased and 27.1% remained within 1 h, while the content of three major metabolites (M1, M4, M5) identified by Q‐trap increased. This perfusion result is consistent with the oral bioavailability results in vivo. The first‐pass metabolism of WA might be the main barrier in achieving good oral bioavailability in male rats and it is predicted to be similar in humans. This study may hold clinical significance.     

Structural elucidation of rat biliary metabolites of corynoxeine and their quantification using LC‐MSn

Corynoxeine (COR) is one of 4 bioactive oxindole alkaloids in Uncaria species. In this work two phase I metabolites, namely 11‐hydroxycorynoxeine (M1) and 10‐hydroxycorynoxeine (M2), and two phase II metabolites, namely 11‐hydroxycorynoxeine 11‐O‐β‐d ‐glucuronide (M3) and 10‐hydroxycorynoxeine 10‐O‐β‐d ‐glucuronide (M4), were detected in rat bile after oral dose of COR (0.105 mmol/kg), by optimized high‐performance liquid chromatography–tandem mass spectrometry (LC‐MSⁿ) with electrospray ionization in positive ion mode. Structures of M1–4 were determined by LC‐MSⁿ, nuclear magnetic resonance, circular dichroism and high‐resolution MS spectra. COR and its metabolites in rat bile were quantified by LC‐MSⁿ. The LC‐MSⁿ quantification methods for COR and its metabolites yielded a linearity with coefficient of determination ≥0.995 from 5.0 × 10^?10 to 5.0 × 10^?7 m . The recoveries of stability tests varied from 96.80 to 103.10%. Accuracy ranged from 91.00 to 105.20%. Relative standard deviation for intra‐day and inter‐day assay was <5.0%. After the oral dose 0.14% of COR was detected in rat bile from 0 to 8 h, in which in total 97.8% COR biotransformed into M1–4. M1 and M2 yielded 48.1 and 49.7%, which successively glucuronidated to M3 and M4 at 47.2 and 43.8%, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolic study of Angelica dahurica extracts using a reusable liver microsomal nanobioreactor by liquid chromatography–mass spectrometry

Highly active and recoverable nanobioreactors prepared by immobilizing rat liver microsomes on magnetic nanoparticles (LMMNPs) were utilized in metabolic study of Angelica dahurica extracts. Five metabolites were detected in the incubation solution of the extracts and LMMNPs, which were identified by means of HPLC‐MS as trans‐imperatorin hydroxylate (M1), cis‐imperatorin hydroxylate (M2), imperatorin epoxide (M3), trans‐isoimperatorin hydroxylate (M1′) and cis‐isoimperatorin hydroxylate (speculated M2′). Compared with the metabolisms of imperatorin and isoimperatorin, it was found that the five metabolites were all transformed from these two major compounds present in the plant. Since no study on isoimperatorin metabolism by liver microsomal enzyme system has been reported so far, its metabolites (M1′ and M3′) were isolated by preparative HPLC for structure elucidation by ¹H‐NMR and MS² analysis. M3′ was identified as isoimperatorin epoxide, which is a new compound as far as its chemical structure is concerned. However, interestingly, M3′ was not detected in the metabolism of the whole plant extract. In addition, a study with known chemical inhibitors on individual isozymes of the microsomal enzyme family revealed that CYP1A2 is involved in metabolisms of both isoimperatorin and imperatorin, and CYP3A4 only in that of isoimperatorin. Copyright © 2015 John Wiley & Sons, Ltd.