Drug Metabolism and Disposition Fast Forward
First published on January 11, 2005; DOI: 10.1124/dmd.104.001958
0090-9556/05/3304-555-562$20.00
DMD 33:555-562, 2005
ISOLATION AND IDENTIFICATION OF SEVEN GLUCURONIDE CONJUGATES OF ANDROGRAPHOLIDE IN HUMAN URINE
Liang Cui,
Feng Qiu, and
Xinsheng Yao
Department of Natural Products Chemistry (L.C., F.Q., X.Y.), Shenyang Pharmaceutical University, Shenyang, China; and Traditional Chinese Medicines & Natural Products Research Center Shenzhen (X.Y.), Shenzhen, China
(Received August 24, 2004;
accepted January 7, 2005)
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Abstract
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Andrographolide is one of the principal components of a famous traditional Chinese herbal medicine, Andrographis paniculate (Burm) Nees, and has been widely used in the clinic for the treatment of infectious diseases. In this paper, metabolites of andrographolide in the urine of eight healthy volunteers after oral administration were further investigated. Building on previous findings, an additional seven phase II metabolites were isolated by liquid-liquid extraction, open-column chromatography, medium-pressure liquid chromatography, and, finally, preparative high-performance liquid chromatography. Structural elucidation was carried out by mass spectra and NMR spectroscopy including 1H NMR, 13C NMR and two-dimensional NMR (distortionless enhancement by polarization transfer, heteronuclear multiple-quantum correlation, heteronuclear multiple-bond correlation, 1H-1H correlated spectroscopy, and nuclear Overhauser enhancement spectroscopy). All of the metabolites were characterized as glucuronide conjugates, and the structures were determined to be andrographolide-19-O-ß-D-glucuronide (M-1), isoandrographolide-19-O-ß-D-glucuronide (M-2), 14-deoxy-12-hydroxy-andrographolide-19-O-ß-D-glucuronide (M-3), andrographolide-19-O-[6'-methyl-ß-D-glucuronide] (M-4), 14-deoxy-12(13)-en-andrographolide-19-O-ß-D-glucuronide (M-5), 14-deoxyandrographolide-19-O-ß-D-glucuronide (M-6), and 3-oxoandrographolide-19-O-ß-D-glucuronide (M-7), respectively.
Andrographolide is one of the principal constituents of a famous traditional Chinese herbal medicine, Andrographis paniculate (Burm) Nees. Its chemical structure is 3-[2-[decahydro-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methylene-1-napthalenyl] ethylidene] dihydro-4-hydroxy-2(3H)-furanone. Andrographolide has many bioactivities, such as anti-inflammatory (Shen et al., 2000
, 2002), anti-platelet aggregation (Amroyan et al., 1999), antihyperglycemic (Yu et al., 2003), hepatoprotective (Shukla et al., 1992), antitumor (Rajagopal et al., 2003; Kumar et al., 2004), and anti-human immunodeficiency virus activities (Handa and Sharma, 1990; Basak et al., 1999). This compound has been widely used in the clinic for the treatment of fever, cold, inflammation, diarrhea, and other infectious diseases.
Pharmacokinetic studies showed that andrographolide was quickly absorbed and extensively metabolized in rats and humans (Panossian et al., 2000). Ten andrographolide metabolites, mainly as sulfonic acid adducts and sulfate compounds, have been isolated and identified in urine, feces, and contents of small intestine after the drug was orally administrated to rats (He et al., 2003a,b,c). One of the metabolites, 14-deoxy-12(R)-sulfo-andrographolide, was found to be identical to an anti-inflammatory drug (Lianbizhi) currently being used in the clinic as an injection in China (Meng, 1981). Recently, we reported the structural identification of four new urinary metabolites of andrographolide in humans, three of which were characterized as 3-O-sulfate conjugates and the remaining one as a 3-O-sulfate-12-S-cysteine conjugate (Cui et al., 2004). To gain additional insight into its metabolism, we further examined the biotransformation of andrographolide in urine after oral administration in humans. The present study describes the isolation and identification of seven glucuronide conjugates of andrographolide (Fig. 1).
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FIG. 1. Structures of andrographolide metabolites in human urine and possible metabolic pathways for their production.
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Materials and Methods
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Materials. Andrographolide tablets (50-mg tablets, purity >98.5%) were supplied by Huipushen Pharmaceutical Inc. (Hainan, China). Methanol was HPLC grade and water was double distilled in our laboratory. All other analytic reagents were analysis grade and purchased from Shenyang Chemical Company (Shenyang, China). Silica gels G60 and GF254 for thin-layer chromatography were products of Qingdao Marine Chemical Factory (Qingdao, China). Normal-phase and reverse-phase preparatory thin-layer chromatography was performed using products from Merck (Darmstadt, Germany). Macroporous resin D101 was purchased from the Chemical Plant of NanKai University (Tianjin, China). Sephadex LH-20 and ODS were the products of Pfizer, Inc. (New York, NY). Kedde and Legal reagents were prepared according to the reference (Stahl, 1969).
Subjects and Dosing Procedure. Eight healthy volunteers aged 21 to 28 years and weighing 50 to 80 kg (all males) participated in this study. Subjects were judged to be in good health based on a medical history, physical examination, and laboratory profiles that were performed within 2 weeks before the study. Each subject was given orally three tablets, three times per day for 2 days, and the urine was collected between 0 and 72 h.
Isolation of Metabolites. The urine samples (approximately 50,000 ml in total) were concentrated to almost dryness in vacuo after collection. The residue was suspended in water and partitioned with ethyl acetate and n-butanol three times, respectively. The water layer was concentrated in vacuo to remove the solvent, and the residue was dissolved in water. The solution was then subjected to macroporous resin D101 and eluted with H2O/EtOH in a stepwise manner. The 10% EtOH elution was subjected to a silica gel chromatography column with a CHCl3-MeOH solvent system (10:1, 9:1, 8:2, 7:3, 6:4, 5:5, 3:7). The fractions eluted by CHCl3-MeOH (7:3) and CHCl3-MeOH (6:4) were subjected to Sephadex LH-20 and RP-18 silica gel column chromatography with a MeOH-H2O solvent system (0–50%), respectively. The fractions containing the metabolites were further purified by preparative HPLC. The fraction eluted from the D101 resin by 50% EtOH was directly subjected to Sephadex LH-20 and RP-18 silica gel column chromatography with a MeOH-H2O solvent system (0–50%), and the fractions containing the metabolites were also further purified by preparative HPLC.
Purification by HPLC. Preparative HPLC was performed with an ODS column (C-8, 25,020 mm; Inertsil Pak) in a Waters 600 liquid chromatograph apparatus equipped with a Waters 490 UV detector (Waters, Milford, MA). The usual detection wavelength was 200 nm. Elution was carried out with MeOH-H2O containing 0.05% trifluoroacetic acid at a flow rate of 10 ml/min. Elution with MeOH-H2O (30:70) yielded M-1 (36 min), M-2 (43 min), and M-3 (30 min). Elution with MeOH-H2O (40:60) yielded M-7 (42 min). Elution with MeOH-H2O (50:50) yielded M4 (60 min), M5 (40 min), and M-6 (48 min).
Spectroscopic Methods. Electrospray ion trap mass spectrometry in a multistage full scan mode was performed on a Bruker Esquire 2000 instrument (Bruker, Newark, DE) having a mass range of 25 to 2200 (the mass was calibrated). The instrument was operated in the negative ion mode, using nitrogen for nebulizing and dry gas. The collision-induced dissociation of [M - H]- was achieved with helium as the collision gas. The ionization was performed applying the following parameters: capillary temperature, 250°C; capillary voltage, -4.0 kV. Sample solutions were directly introduced into the ESI source at a flow rate of 3 µl/min by a syringe pump.
Optical rotations were measured using a P-1020 digital polarimeter (Jasco, Tokyo, Japan). Infrared spectra were determined on a Shimadzu FT/IR-8400 spectrometer (Shimadzu, Kyoto, Japan) in KBr pellets. UV spectra were measured on a Shimadzu UV-2201 spectrometer. HRSI-MS was recorded with a Bruker second ionization mass spectrometer. NMR spectra were measured on Bruker ARX-400 spectrometer, and chemical shifts are given in ppm from tetramethylsilane as an internal standard. All compounds were dissolved in CD3OD.
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Results
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Isolation and Structure Elucidation of Andrographolide Metabolites. By the above methods, seven glucuronide conjugates of andrographolide were obtained: M-1 (105.5 mg), M-2 (20.2 mg), M-3 (18.0 mg), M-4 (16.5 mg), M-5 (25.6 mg), M-6 (10.5 mg), and M-7 (3.0 mg). The structures of the seven metabolites are shown in Fig. 1.
Metabolite M-1 (andrographolide-19-O-ß-D-glucuronide), white amorphous powder, [
]D22 -89.8 (MeOH, C = 0.85), was positive for the Legal and Kedde reactions, suggesting the presence of an ,ß-unsaturated lactone. The infrared spectrum showed the presence of hydroxyl (3417 cm-1), ester carbonyl (1739 cm-1), and exo-methylene (898 cm-1) groups in the molecule. The HRSI-MS showed the quasimolecular ion [M + H]+ at m/z 527.2490 (calculated 527.2492), corresponding to the molecular formula C26H38O11 (Table 1), which was further supported by the 1H NMR and 13C NMR spectral data. M-1 was 176 mass units higher than that of andrographolide, suggesting that it was a glucuronide conjugate of andrographolide. In the negative ESI-MS3 spectrum, the [M - H]- ion m/z 525 of M-1 eliminated 18 mass units (H2O) to give m/z 507, and the ion m/z 507 eliminated 176 mass units (glucuronic acid - H2O) to give m/z 331 (Fig. 2). The 13C NMR data clearly showed the existence of a glucuronic acid [
73.6 (C-4'), 74.8 (C-2'), 76.1 (C-5'), 77.8 (C-3'), 105.0 (C-1'), 176.7 (C-6')]. The 13C NMR data of the genin part of M-1 were similar to those of the parent drug andrographolide, except for the downfield shifts of C-19 by 7.1 ppm (Fig. 3). In the HMBC spectrum (Fig. 4), the signals of H-19 ( 4.35, 1H, d, J = 10.1 Hz; 3.34, 1H, o) correlated with C-1' ( 105.0), and the anomeric proton of glucuronide ( 4.18, 1H, d, J = 7.8 Hz) had correlations with C-19 ( 72.0). These correlated peaks indicated that the linkage site of the glucuronic acid moiety was at C-19. The ß-form anomeric configuration of the glucuronic acid was judged from its coupling constant of the anomeric proton (J = 7.8 Hz). Based on the above data, M-1 was determined to be andrographolide-19-O-ß-D-glucuronide. The full assignments of carbon and proton signals are summarized in Table 2.
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FIG. 2. Tandem mass specrtometry spectra of [M - H]- ion of metabolite M-1, andrographolide-19-O-ß-D-glucuronide (A), metabolite M-4, andrographolide-19-O-[6'-methyl-ß-D-glucuronide] (B), metabolite M-5, 14-deoxy-12(13)-en-andrographolide-19-O-ß-D-glucuronide (C), and metabolite M-7, 3-oxoandrographolide-19-O-ß-D-glucuronide (D).
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Metabolite M-2 (isoandrographolide-19-O-ß-D-glucuronide), white amorphous powder, was positive for the Legal and Kedde reactions, suggesting the presence of an ,ß-unsaturated lactone. The infrared spectrum showed the presence of hydroxyl (3417 cm-1), ester carbonyl (1739 cm-1), and exo-methylene (898 cm-1) groups in the molecule. In the HRSI-MS, a quasimolecular peak [M + Na]+ at m/z 549.2311 (calculated 549.2312) was observed, and, thus, the molecular formula of C26H38O11 was derived. The assignment of this formula was further supported by the 1H NMR and 13C NMR spectral data. The molecular formula of M-2 was 176 mass units higher than that of andrographolide, and the negative ESI-MS3 spectrum of M-2 and M-1 was almost the same, suggesting that M-2 was an isomer of M-1. The 13C NMR data clearly showed the existence of a glucuronic acid [ 73.5 (C-4'), 74.7 (C-2'), 76.2 (C-5'), 77.8 (C-3'), 105.1 (C-1'), 175.8 (C-6')]. The 1H NMR and 13C NMR data of M-2 were very similar to those of M-1 except for the following findings. The proton H-11 ( 2.87, 2H, m) signal was shifted downfield by 0.27 ppm, the H-12 ( 6.51, 1H, m) signal was shifted upfield by 0.33 ppm, and the carbon signal of C-14 ( 69.8) was shifted downfield by 3.2 ppm. In the NOESY spectrum, the cross peak was observed from H-12 to H-14 ( 4.70, 1H, m) in M-2, whereas it was absent in M-1, which indicated that M-2 was the geometric isomer of M-1 at the 12(13) double bond (Matsuda et al., 1994). Based on the above analyses, M-2 was determined to be isoandrographolide-19-O-ß-D-glucuronide except for the absolute configuration at C-14. The full assignments of carbon and proton signals are summarized in Table 2.
Metabolite M-3 (14-deoxy-12-hydroxy-andrographolide-19-O-ß-D-glucuronide), white amorphous powder, was positive for the Legal and Kedde reactions, suggesting the presence of an ,ß-unsaturated lactone. In the infrared spectrum, the absorption at 3444 cm-1 was the absorption of the hydroxyl group, whereas the absorption at 1747 cm-1 was the absorption of the ester carbonyl group. The HRSI-MS showed the quasimolecular ion [M + H]+ at m/z 527.2483 (calculated 527.2492), which corresponds to the molecular formula C26H38O11 by combining the 1H NMR and 13C NMR spectral data. The molecular formula of M-3 was 176 mass units higher than that of andrographolide, and the negative ESI-MS3 spectrum of M-3 was almost the same as those of M-1 and M-2, suggesting that M-3 was also an isomer of M-1 and M-2. The 13C NMR data clearly showed the existence of a glucuronic acid [ 73.6 (C-4'), 74.8 (C-2'), 76.1 (C-5'), 77.8 (C-3'), 105.0 (C-1'), 176.8 (C-6')]. 13C NMR of M-1 and M-3 showed similarities except for the chemical shifts at C-9 and C-11 to C-16, whereas the other carbons were almost the same, suggesting that the difference of M-3 occurred at the side chain of the lactone. The structure of the side chain was determined from the 1H-1H COSY and HMBC results. In the 1H-1H COSY spectrum, a stepwise coupling was observed from H-9 ( 2.10, 1H, m) through H-12 ( 4.40, 1H, d, J = 10.2 Hz), mediated by H-11 ( 1.93, 1H, overlapped; 1.53, 1H, overlapped), whereas the signal of H-14 ( 7.47, 1H, m) showed correlations with one proton signal of H-15 ( 4.87, 1H, overlapped). In the HMBC spectrum (Fig. 5), one proton signal of H-15 ( 4.87, 1H, overlapped) had peaks correlated with C-13 ( 139.2) and C-14 ( 147.4), whereas the signal of H-12 had peaks correlated with C-11 ( 32.0), C-13 ( 139.2), and C-14 ( 147.4). These correlated peaks indicated that the double bond at 12(13) of M-1 had changed to the 13(14) double bond, by which the double bond transferred from the outside to the inside of the lactone ring and the hydroxyl at C-14 of M-1 changed to C-12 in M-3. In the UV spectrum, the maximal absorption of M-3 was at 202 nm, which was different from that of andrographolide at 225. The hypsochromic shift in UV spectrum also supported the change of the conjugated system. From the above evidence, M-3 was determined to be 14-deoxy-12-hydroxy-andrographolide-19-O-ß-D-glucuronide except for the absolute configuration at C-12. The full assignments of carbon and proton signals are summarized in Table 3.
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FIG. 5. Partial HMBC correlations of metabolite M-3, 14-deoxy-12-hydroxy-andrographolide-19-O-ß-D-glucuronide (A), metabolite M-5, 14-deoxy-12(13)-enandrographolide-19-O-ß-D-glucuronide (B), metabolite M-6, 14-deoxyandrographolide-19-O-ß-D-glucuronide (C), and metabolite M-7, 3-oxoandrographolide-19-O-ß-D-glucuronide (D).
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Metabolite M-4 (andrographolide-19-O-[6'-methyl-ß-D-glucuronide]), white amorphous powder, was positive for the Legal and Kedde reactions, suggesting the presence of an ,ß-unsaturated lactone. The infrared spectrum showed the presence of hydroxyl (3421 cm-1), ester carbonyl (1747 cm-1), and exo-methylene (906 cm-1) groups in the molecule. The HRSI-MS showed the quasimolecular ion [M + H]+ at m/z 541.2667 (calculated 541.2649). The molecular formula was determined to be C27H40O11 in combination with the 1H NMR and 13C NMR spectral data. The negative ESI-MS3 spectrum and the 13C NMR spectrum also showed the existence of a glucuronic acid. The molecular formula of M-4 was 14 mass units higher than that of M-1, suggesting that M-4 was methylated M-1. The 13C NMR data of M-4 and M-1 were very similar except for the chemical shift at C-6' of the glucuronic acid, which was shifted upfield by 5.5 ppm. Moreover, an additional oxygen-bonding methyl carbon signal at 52.8 was observed. Correspondingly, a new signal of three protons derived from the methoxy group was observed at 3.76 (3H, s) in the 1H NMR spectrum. The location of the methyl group was determined by the observation of the cross-peak in the HMBC spectrum between methyl protons and C-6' of the glucuronic acid. From the above evidence, M-4 was determined to be andrographolide-19-O-[6'-methyl-ß-D-glucuronide]. The full assignments of carbon and proton signals are summarized in Table 3. M-4 might be an artifact of M-1 during the purification process.
Metabolite M-5 (14-deoxy-12(13)-en-andrographolide-19-O-ß-D-glucuronide), white amorphous powder, was positive for the Legal and Kedde reactions, suggesting the presence of an ,ß-unsaturated lactone. The infrared spectrum showed the presence of hydroxyl (3417 cm-1), ester carbonyl (1739 cm-1), and exo-methylene (898 cm-1) groups in the molecule. The positive high-resolution ESI-MS showed the quasimolecular ion [M + Na]+ ion at m/z 533.2388 (calculated 533.2363), which corresponds to the molecular formula C26H38O11 by combining the H NMR and 13C NMR spectral data. The negative ESI-MS3 spectrum and the 13C-NMR spectrum also showed the existence of a glucuronic acid. The molecular formula of M-5 was 16 mass units lower than that of M-1, suggesting that M-5 was a deoxygenated glucuronide conjugate of andrographolide. Comparison of the 13C NMR spectrum of M-5 with that of M-1 showed similarities except for the chemical shifts at C-12 to C-16, in which the hydroxyl-linked carbon signal at 66.6 (C-14) of M-1 disappeared and a new carbon signal of methylene at 26.0 (C-14) was observed in M-5. In the 1H-1H COSY spectrum, a stepwise coupling was observed from H-9 ( 1.90, 1H, overlapped) through H-12 ( 6.60, 1H, m), mediated by one proton signal of H-11 ( 2.27, 1H, m), whereas the signal of H-14 ( 2.91, 2H, m) showed correlations with the signal of H-15 ( 4.39, 2H, overlapped). In the HMBC spectrum (Fig. 5), the signals of H-11 ( 2.40, 1H, o; 2.27, 1H, m) had peaks correlated with C-13 ( 126.6), C-12 ( 142.9), and C-9 ( 57.3), and the signal of H-14 ( 2.91, 2H, m) had peaks correlated with C-16 ( 173.7), C-13 ( 126.6), and C-12 ( 142.9), whereas the signal of H-15 ( 4.39, 2H, o) had peaks correlated with C-16 ( 173.7), C-14 ( 26.0), and C-13 ( 126.6). Thus, the new carbon signal of methylene at 26.0 was designated to C-14 and the structure of the side chain was determined. Based on the above analyses, M-5 was determined to be 14-deoxy-12(13)-en-andrographolide-19-O-ß-D-glucuronide. The full assignments of carbon and proton signals are summarized in Table 4.
Metabolite M-6 (14-deoxyandrographolide-19-O-ß-D-glucuronide), white amorphous powder, was positive for the Legal and Kedde reactions, suggesting the presence of an ,ß-unsaturated lactone. The infrared spectrum showed the presence of hydroxyl (3402 cm-1), ester carbonyl (1747 cm-1) and exo-methylene (902 cm-1) groups in the molecule. The positive high-resolution ESI-MS showed the quasimolecular ion [M + Na]+ at m/z 533.2385 (calculated 533.2363), corresponding to the molecular formula C26H38O10, which was further supported by the 1H NMR and 13C NMR spectral data. The molecular formula and the negative ESI-MS3 spectrum of M-6 were almost the same as those of M-5 except for some carbon signals in the 13C NMR spectrum, suggesting that M-6 was an isomer of M-5. Comparison of the 13C NMR data of M-6 with those of M-5 showed wide variation in the chemical shifts at C-11 to C-15, whereas the other carbons were almost the same, suggesting that the difference of M-6 occurred at the side chain of lactone. In the 1H-1H COSY spectrum, a stepwise coupling was observed from H-9 ( 1.64, 1H, overlapped) through one proton signal of H-12 ( 2.08, 1H, overlapped) mediated by one proton signal of H-11 ( 1.76, 1H, overlapped), whereas the signal of H-14 ( 7.32, 1H, d, J = 1.4Hz) showed correlations with the signal of H-15 ( 4.80, 2H, overlapped). In the HMBC spectrum (Fig. 5), one proton signal of H-12 ( 2.08, 1H, d) had peaks correlated with C-16 ( 176.9), C-14 ( 147.6), and C-13 ( 134.7), and the signal of H-14 ( 7.32, 1H, d, J = 1.4Hz) had peaks correlated with C-16 ( 176.9), C-15 ( 72.0), C-13 ( 134.7), and C-12 ( 25.4), whereas the signal of H-15 ( 4.80, 2H, o) had peaks correlated with C-16 ( 176.9), C-14 ( 147.6), and C-13 ( 134.7). These correlated peaks indicated that the double bond at 12(13) of M-5 had changed to the 13(14) double bond, by which the double bond transferred from the outside to the inside of the lactone ring in M-6. In the UV spectrum, the maximal absorption of M-6 was at 203 nm, which was different from that of andrographolide at 225. The hypsochromic shift in UV spectrum also supported the change of the conjugated system. From the above evidence, M-6 was determined to be 14-deoxyandrographolide-19-O-ß-D-glucuronide. The full assignments of carbon and proton signals are summarized in Table 4.
Metabolite M-7 (3-oxoandrographolide-19-O-ß-D-glucuronide), white amorphous powder, was positive for the Legal and Kedde reactions, suggesting the presence of an ,ß-unsaturated lactone. The ESI-MS showed an [M - H]- ion at m/z 523.3 and an [M + 2Na-H]+ ion at m/z 569.3, which corresponds to the molecular formula C26H38O10 by combining the H NMR and 13C NMR spectral data. Comparison of the 1H NMR and 13C NMR data of M-7 with those of M-1 showed wide variation in the chemical shifts at C-1
C-5, whereas the other carbons were almost the same, suggesting that the difference of M-7 occurred at the ring A. In the HMBC spectrum (Fig. 5), the signal of H-18 ( 1.19, 3H, s) had peaks correlated with C-19 ( 73.5), C-5 ( 58.4), C-4 ( 54.5), and C-3 ( 217.4), and one proton signal of H-2 ( 2.14, 1H, o) had peaks correlated with C-3 ( 217.4). Moreover, the signals of H-19 ( 4.56, 1H, d, J = 9.7Hz; 3.30, 1H, o) had peaks correlated with C-18 ( 20.8), C-4 ( 54.5), and C-3 ( 217.4). These correlated peaks indicated that the hydroxyl at C-3 of M-1 had been oxygenated to carbonyl in M-7. From the above evidence, M-7 was determined to be 3-oxoandrographolide-19-O-ß-D-glucuronide. The full assignments of carbon and proton signals are summarized in Table 4.
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Discussion
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Recently, we reported the structural identification of four sulfate conjugates of andrographolide obtained from urine of eight healthy volunteers after oral administration. The current study was our continuing effort to explore the metabolic fate of andrographolide in human urine, and seven glucuronide conjugates were obtained. Their unambiguous structures were elucidated by mass spectra and NMR spectroscopy including 1H NMR, 13C NMR, and two-dimensional NMR (DEPT, HMQC, HMBC, 1H-1H COSY, and NOESY), although the absolute configuration at C-14 of M-2 and at C-12 of M-3 was not established. Among these glucuronide conjugates, M-1 and M-2, M-5 and M-6 are two pairs of geometric isomers, whereas M-3 is an isomer of M-1 and(or) M-2 occurring in the position of the 12(13) double bond and the linked position of hydroxyl group at C-14. On the basis of the metabolite profiles, we propose the metabolic pathways of andrographolide in Fig. 1.
Structural elucidation of metabolites is an important task in drug metabolism studies. In recent years, comparisons of ESI-MSn data and retention times in HPLC with synthesized standards are usually used to identify the structures of metabolites. However, when the standards are difficult to synthesize, some metabolites' structures deduced only from LC/MSn data may not be correct, especially in the case of the existence of isomeric metabolites. In our study, two groups of isomers (M-1, M-2, and M-3; M-5 and M-6) were obtained, and they have similar chromatographic behaviors and identical data in LC/MSn. Therefore, their structures could hardly be identified only by LC/MSn data. In these cases, preparation of metabolites and further identification based on NMR data must be done. Of course, the direct isolation of the metabolites from urine, bile, or feces of humans or animals has difficulties, but it is the most reliable method in the identification of metabolites.
In summary, we have determined the definitive structures of seven glucuronide conjugates of andrographolide by mass spectra and NMR spectroscopy. These results are important to understand its in vivo metabolic fate and disposition of andrographolide in humans.
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Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.104.001958.
ABBREVIATIONS: HPLC, high-performance liquid chromatography; EtOH, ethanol; MeOH, methanol; DEPT, distortionless enhancement by polarization transfer; HMQC, heteronuclear multiple-quantum correlation; COSY, correlated spectroscopy; HMBC, heteronuclear multiple-bond correlation; NOESY, nuclear Overhauser enhancement spectroscopy; HRSI-MS, high-resolution second ionization-mass spectrometry; ESI-MS, electrospray ionization-mass spectrometry; LC/MSn, liquid chromatography-mass spectrometry at stage n.
Address correspondence to: Xinsheng Yao, Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang, P.R. China 110016. E-mail: yaoxinsheng{at}hotmail.com
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Abstract
Materials and Methods
