Preparation and structural determination of four metabolites of senkyunolide I in rats using ultra performance liquid chromatography/quadrupole-time-of-flight tandem mass and nuclear magnetic resonance spectra

Background Senkyunolide I (SEI) is one of the most important bioactive phthalides of Ligusticum chuanxiong Hort. (Umbelliferae), a Traditional Chinese Medicine. Our previous studies suggested that it might be developed as a potential treatment for migraine. Methods In this paper, we aimed to isolate and characterize the main metabolites of SEI after gavage feeding in rats. Their structures were identified precisely on the basis of nuclear magnetic resonance (NMR) spectroscopy and UPLC/Q-TOF-MS spectrometry. We also established the main metabolic pathways of SEI in rats. Results Four metabolites (M1-M4) were isolated, for the first time, from bile samples of rats by preparative high-performance liquid chromatography. Their structures were determined as SEI-6S-O-β-D-glucuronide (M1), SEI-7S-O-β-D-glucuronide (M2), SEI-7S-S-glutathione (M3) and SEI-7R-S-glutathione (M4) on the basis of the molecular mass of the analytes, using ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry and 1D and 2D NMR. Conclusions The results demonstrated that glucuronide and glutathione conjugation were the major pathways of SEI metabolism in vivo, and the configuration at the 7th-position could be inverted during glutathione conjugation.


Background
Ligusticum chuanxiong Hort. (Umbelliferae) is a traditional Chinese herbal medicine that has been used to treat cardio-and cerebro-vascular disorders, such as angina pectoris, stroke and migraine, in China for thousands of years [1]. The chemistry and pharmacological effects of chuanxiong have been well documented.
Phthalides have been reported as the primary bioactive constituents in this herb. Various pharmacological activities of the main phthalides, such as ligustilide and senkyunolide A, have been revealed, and their contributions to the beneficial effects of the herb are supported by in vivo and in vitro studies [2][3][4][5].
Senkyunolide I (SEI, Fig. 1), a representative metabolite of ligustilide in vivo and in vitro [6,7], is one of the most important bioactive constituents of chuanxiong [6,[8][9][10][11]. Several studies have demonstrated that SEI could decrease hydrogen peroxide (H 2 O 2 )-induced oxidative damage in cultured human liver HepG2 cells [7] and PC12 cells [12]. SEI could also decrease the morphological damage to red blood cells induced by concanavalin A [13]. Our previous study demonstrated that SEI could treat migraines, although the mechanism is unclear [14]. In addition, SEI is more stably in vitro, and has higher solubility [15,16] and higher oral bioavailability [17,18] compared with ligustilide and senkyunolide A. These properties of SEI suggest that it has a better potential as a new drug than ligustilide and senkyunolide A.
Generally, drug metabolism has a significant impact on the safety and efficacy of a drug and is commonly investigated at early stage of new drugs development. The identification of the drug's metabolites is indispensable in this process [19]. Liquid chromatography (LC) coupled with tandem mass spectrometry has become a powerful tool to study drug metabolism because of its superior sensitivity and specificity [20,21]. Quadrupoletime-of-flight mass spectrometry (Q/TOF-MS) is very useful in the characterization of drug metabolites because the technique provides accurate masses of ions and reveals valuable structural information [22]. In our previous study [23], 18 metabolites were characterized on the basis of ultra performance liquid chromatography/Q/TOF-MS (UPLC/Q-TOF-MS) analysis. However, because there are two chiral carbons (6-C and 7-C) in the molecular structure of SEI, the chemical structures of SEI conjugates present in vivo have not been fully characterized using Q/TOF-MS. To confirm further the structures of the main metabolites, and to obtain the pharmacological and toxicological information on them, it is essential to obtain adequate reference standards. Therefore, as part of our research on SEI, we aimed to isolate and characterize the metabolites of SEI, and to confirm the principal pathways of SEI metabolism in vivo.
In this study, four main metabolites were isolated from bile samples after gavage feeding of 100 mg/kg of SEI to rats. Their structures were identified precisely on the basis of nuclear magnetic resonance (NMR) and UPLC/ Q-TOF-MS spectra. We also established the main metabolic pathways of SEI in rats.

Chemicals and reagents
SEI (purity >99.1%, as tested by HPLC-UV) was obtained from L. chuanxiong extracts in our laboratory. Its structure was confirmed by comparison of its MS and NMR profiles with that published in the literature [24,25]. Slices of L. chuanxiong (No. 20130419) were purchased from Bozhou Medicinal Materials Company (Anhui Province, China) and authenticated by Professor Zhi-li Zhao of the Shanghai University of Traditional Chinese Medicine (Shanghai, China). The ultrahigh purified water used in this study was prepared in a Milli-Q water purification system (Millipore Corp., Billerica, MA, USA). Methanol and acetonitrile (HPLC grade) were purchased from Merck KGaA (Darmstadt, Germany). MeOH-d4, with tetramethylsilane (TMS) as internal standard for NMR analysis, was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MS, USA). MCI Gel CHP 20P (75 μm-150 μm) for column chromatography was purchased from Mitsubishi (Tokyo, Japan). Sephadex LH-20 was obtained from GE Healthcare Bio-Sciences AB (Sweden). Other reagents and chemicals, including formic acid, were of analytical grade.

Animals and drug administration
Twenty-eight male Sprague-Dawley rats (275-300 g body weight) were provided by the Experimental Animal Centre, Shanghai University of Traditional Chinese Medicine, China. Animals were housed at an ambient temperature of 24 ± 2°C and 60 ± 5% humidity, with a 12 h dark-light cycle. They were kept in an environmentally controlled breeding room and given tap water and fed ad libitum until 12 h before the experiment. Animal experimental procedures and welfare were strictly in accordance with the Guide for the Care and Use of Laboratory Animals and the related ethics regulations of Shanghai University of TCM. Our animal protocol was approved by the Institutional Animal Care and Use Committee, Shanghai University of TCM (Shanghai, China). SEI was dissolved in deionized water (10 mg•ml-1) and administered by gavage at a dose of 100 mg•kg-1 body weight.

Samples collection and processing procedures
The 28 rats were intraperitoneally injected with urethane (1.0 g · kg −1 body weight). Under anesthesia, a polyethylene cannula was inserted into the bile duct. Bile samples were collected for 12 h (approximately 400 mL in total) after oral administration of SEI at a dose of 100 mg · kg −1 body weight. Blank rat bile was collected before oral administration. All the samples were stored at −20°C until further isolation and analysis.
Bile samples (100 μL) for HPLC-UV analysis were mixed with 400 μL of methanol for 60 s. They were then centrifuged at 13,000 rpm for 10 min, the supernatant was next transferred to a clear Eppendorf tube and then used 20 μLwas analysed using a HPLC-UV system.

HPLC-UV analysis conditions
Analytical HPLC-UV analysis was carried out on an Agilent 1200 Series analytical HPLC system (Agilent Technologies, USA). A 20-μL injection loop and a Grace C18 reversed-phase column (5 μm, 4.6 × 150 mm), protected by an Security Guard Cartridges C18 (5 μm, 4 × 3.0 mm) guard column, were used for analysis. The analytic HPLC conditions comprised: a flow rate of 1 mL•min -1 ; the mobile phase component A was water with 0.1% formic acid and B was acetonitrile; the column was eluted with a linear gradient of 4% B over 0-2 min, 4-8% B over 2-5 min, 8-28% B over 5-41 min, 28-4% B over 41-43 min and the composition was maintained 4% B for 2 min. The detector wavelength was set at 278 nm and column temperature was maintained at 25°C.

Chromatographic and mass spectrometric conditions
Chromatography was performed on an ACQUITY™ UPLC system (Waters Corp., Milford, MA, USA) with a conditioned auto sampler at 10°C. The chromatographic separation was carried out on an ACQUITY UPLC HSS T3 column (1.8 μm, i.d. 2.1 × 100 mm). The column temperature was maintained at 45°C. The analysis was achieved with gradient elution using A (aqueous 0.1% formic acid) and B (acetonitrile) as the mobile phase. The gradient condition of the metabolites analysis was 0-15 min 20% B. The injection volume was 5 μL. The electrospray ionization source was operated in positive ionization mode with the capillary voltage at 2.7 kV. The source and desolvation temperatures were set to 120°C and 400°C, respectively. The cone gas flow was 50 L.h −1 and the desolvation gas was set to 700 L/h.

NMR spectroscopy
A Broker AV 600 NMR spectrometer (Faellanden, Switzerland) was used to record 1H NMR (600 MHz) and 13C NMR (125 MHz) spectra in C5D5N at 25°C. Chemical shifts were expressed in parts per million (ppm), with tetramethylsilane as the standard.

Isolation and identification of SEI metabolites
As shown in Fig. 2, four main metabolites were found in bile samples of rats after oral administration of SEI, none of which had been isolated previously. The chemical structures of the metabolites were elucidated on the basis of UPLC/Q-TOF-MS and NMR spectra ( 1 H NMR, 13
The 1 H NMR and 13 C NMR data of M4 were similar to those of M3, and the same planar structure as that of M3 was deduced from the HSQC and HMBC data of M4, which suggested that M4 is a diastereoisomer of M3. In the 1 H NMR, the H-7 of M4 (δ H 3.59, s) was a single peak, different from H-7 (δ H 3.86, d, J = 3.6 Hz) in M3, which suggested that M4 is in the 6, 7-cisconfiguration [28]. Thus, the configurations of the 7-substituted groups were inverted during the metabolic process, and M4 was identified as SEI-7R-S-glutathione.

Discussion
In our previous study [18], SEI was rapidly absorbed and exhibited extensive distribution after oral administration. Its oral bioavailability was approximately 37.25%, but it was quickly eliminated from plasma, with a half life of less than 60 min. In a second metabolic experiment [23], the biotransformation of SEI was investigated using UPLC/Q-TOF-MS. Eighteen metabolites were identified and the result indicated that methylation, hydration, epoxidation, glucuronidation and glutathione conjugation were the major pathways of SEI metabolism in vivo. Based on the results above, the fast elimination from plasma could be explained by rapid and extensive metabolism. However, the chemical structures of SEI conjugates present in vivo have not been fully characterized.
In the present study, for the four main metabolites in bile samples, an elementary conclusion could be gained by analyzing the height and area of the peaks in HPLC-UV chromatograms. It suggested that SEI-6S-O-β-Dglucuronide (M1), SEI-7S-O-β-D-glucuronide (M2), SEI-7S-S-glutathione (M3) and SEI-7R-S-glutathione (M4) were the major metabolites in vivo. In our experiment, only trace metabolites of SEI were determined in the urine of rats, all of which suggested that the final excretion pathway in rats was bile. The metabolic pathways of SEI in rat bile mainly involved glucuronidation and glutathione conjugation during the phase II biotransformation pathway in rats because of the 6-and 7-hydroxyl groups in its structure, and the configurations of 7-substituted groups may be inverted during glutathione conjugation. Based on the structures of these metabolites, the proposed metabolic pathways of SEI are shown in Fig. 5.
Glutathione plays an important role in maintaining the redox state of cells via scavenging reactive oxygen species. Glutathione conjugation of xenobiotics is a detoxification pathway that inactivates electrophiles, which may covalently bind to endogenous proteins, resulting in potential detrimental effects [29]. This metabolic mechanism of SEI is indicated for its toxicity, and further work is needed to clarify the potential toxicity of SEI. In theory, medicines or metabolites are supposed to be excreted ultimately out of the body via faeces after bile excretion. However, we did not detect any metabolites, except for SEI, in fecal samples. Therefore, we suspect that the toxicity might be derived from its metabolites to a great extent. There are a few examples where phase II metabolism will produce significantly more biologically active or more toxic metabolites, including morphine [30] and certain heterocyclic aromatic amine compounds [31,32]. In this study, we identified only four main metabolites of SEI in vivo and further research will be carried out to identify other metabolites. Issues to be addressed comprise structure-activity relationship analysis, bioactivity evaluation, and the quantitative mass balance of these metabolites.

Conclusions
Our results demonstrated that glucuronide and glutathione conjugation are the major pathways of SEI metabolism in vivo, and the configuration at the 7th-position could be inverted during glutathione conjugation.