Drug Metabolism and Disposition Fast Forward
First published on June 6, 2007; DOI: 10.1124/dmd.107.015008
0090-9556/07/3509-1564-1573$20.00
DMD 35:1564-1573, 2007
Isolation and Identification of Phase 1 Metabolites of Demethoxycurcumin in Rats
Yongchi Zeng,
Feng Qiu,
Yuan Liu,
Gexia Qu, and
Xinsheng Yao
Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang, P. R. China
(Received February 1, 2007;
accepted June 5, 2007)
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Abstract
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Curcuminoids are a safe natural food coloring additive with anti-inflammatory, antioxidant, and anticarcinogenic activities. Although demethoxycurcumin is one of the major bioactive constituents of curcuminoids, knowledge about its metabolic fate is scant. In the present study, four new metabolites, 5-dehydroxy-hexahydro-demethoxycurcumin-A (M-1), 5-dehydroxy-hexahydro-demethoxycurcumin-B (M-2), 5-dehydroxy-octahydro-demethoxy-curcumin-A (M-3) and 5-dehydroxy-octahydro-demethoxycurcumin-B (M-4), were isolated from feces of male Wistar-derived rats and from urine; three new metabolites, 5-O-methyl-hexahydrodemethoxycurcumin-A (M-7), 5-O-methyl-hexahydro-demethoxycurcumin-B (M-8), and 5-dehydroxy-dihydro-demethoxycurcumin-B (M-9), and two known metabolites, hexahydro-demethoxycurcumin-A (M-5) and hexahydro-demethoxycurcumin-B (M-6), were isolated. Their structures were established by chemical and spectral methods. ll of them were reductive metabolites. Possibly of greater importance is that they occurred as pairs of isomers with a methoxyl group substituted on a different benzene ring. This finding in the metabolism of curcuminoids is reported here for the first time. In addition, the 5-dehydroxy or 5-O-methylated metabolites are also a novel finding. The fact that the metabolites occurred as pairs of the isomers suggests that demethoxycurcumin possibly undergoes tautomerization between 3-keto-5-enol (form A) and 3-keto-5-enol (form B) in rats. Based on the metabolite profiles, metabolic pathways of demethoxycurcumin in rats are proposed.
Curcuminoids are natural yellow pigments and food-coloring agents present in the rhizomes of the Asian tropical plant Curcuma longa, which has been used as a traditional medicinal herb for thousands of years. The dried rhizome of C. longa has been widely used as an aromatic stomachic, carminative, anthelmintic, laxative, as well as for liver ailments and as condiments in foods (Nurfina et al., 1997
). Curcuminoids are responsible for its biological actions. Curcuminoids consist mainly of three diarylheptanoids: curcumin, demethoxycurcumin, and bisdemethoxycurcumin (Govindaajan et al., 1980) (Fig. 1). These are recognized for their beneficial effects such as a choleretic (Ramprasad et al., 1956; Ammon and Martin, 1991), as antioxidants (Sharma, 1976; Unnikrishnan et al., 1995), anti-inflammatory agents (Arora et al., 1971; Ghatak et al., 1972), for treating human immunodeficiency virus infections (Mazumder et al., 1995; Eigner et al., 1999), and as anticarcinogens (Kuttan et al., 1985; Conney et al., 1991; Araujo et al., 2001; Duvoix et al., 2005). In recent years, their ability to protect neuronal cells from ßA insult (Kim et al., 2001; Park et al., 2002) has also attracted great attention. Demethoxycurcumin was found to be more effective in protecting PC12 and human umbilical vein endothelial cells from ßA insult than curcumin. Although numerous aspects of the pharmacology of curcuminoids, in particular their activity as a chemopreventive agent, have been studied, their metabolism in humans and experimental animals has not been fully characterized. The metabolism of curcumin has been studied mostly in rats in vivo and in vitro (Holder et al., 1978; Ravindranath et al., 1982; Asai et al., 1999; Pan et al., 2000; Ireson et al., 2001, 2002). More recently, information on the metabolism of curcumin in humans has been obtained from in vitro studies with hepatic and intestinal cells and subcellular fractions (Ireson et al., 2001, 2002), as well as from clinical studies in cancer patients (Cheng et al., 2001; Sharma et al., 2001, 2004; Garcea et al., 2004). The metabolism of demethoxycurcumin, which is the major active component in curcuminoids such as curcumin, has only been studied on one report. In that investigation, in vitro studies with tissue slices and subcellular fractions from rat liver were reported (Hoehle et al., 2006). No data have yet been published on the metabolism of demethoxycurcumin in vivo. Therefore, studies of the metabolic products of demethoxycurcumin in feces and urine after p.o. administration in male Wistar rats were undertaken. The isolation and identification of nine phase 1 reductive metabolites of demethoxycurcumin are described here.
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Materials and Methods
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Materials. Dry rhizomes of C. longa were collected from Gui Zhou province, China. A voucher specimen was identified by Qi-Shi Sun (CL200209) and deposited at the Department of Natural Products Chemistry, Shenyang Pharmaceutical University, China.
Demethoxycurcumin. Dry rhizomes of C. longa (2.5 kg) were pulverized and then extracted three times for 0.5 h/each time by ultrasound in an 8-fold volume (w/v) of 80% ethanol (EtOH). The EtOH solutions were combined and condensed to yield 362 g. Then the extract was chromatographed on a silica gel column using a CHCl3/methanol (MeOH) gradient solvent system to yield 17 fractions (Fr. A-Q). Fr. E (25.5 g) was further subjected to column chromatography on a silica gel with CHCl3/MeOH 50:1 to yield four fractions (Fr. F1-F4). Demethoxycurcumin (6.8 g) was obtained as a yellow-orange amorphous powder from Fr. F4 after repeated precipitation in MeOH. It had a purity of >98% according to high-performance liquid chromatography (HPLC) analysis. The structure of demethoxycurcumin was identified and confirmed by comparing the mass spectrometry (MS) and 1H and 13C NMR spectral data with those previously reported (Kiuchi et al., 1993).
Demethoxycurcumin is a yellow-orange amorphous powder; electrospray ionization (ESI)/MS: m/z 337 [M - H]-, 1H NMR (300 MHz, dimethyl sulfoxide-d6), and 13C NMR (75 MHz, dimethyl sulfoxide-d6).
Chemicals. The purity of MeOH for HPLC was 99.9% and was supplied by Jiangsu Hanbang Chemical Factory (Jiangsu, China); silica gel for column chromatography (200-300 mesh) and Silica Gel G60 for thin layer chromatography (TLC) (300-400 mesh), preparative TLC (300-400 mesh), and macroporous resin D101 were from Qingdao Marine Chemical Factory (Shandong, China); and reverse-phase preparatory TLC was from Merck (Darmstadt, Germany). Sephadex LH-20 and octadecylsilane (ODS) were from GE Healthcare (Little Chalfont, Buckinghamshire, UK) and YMC Co., Ltd. (Kyoto, Japan), respectively. Other chemicals were analytical grade and provided by Shenyang Chemical reagent factory (Shenyang, China).
Animals. Male Wistar-derived rats (200-250 g) were provided by the Institute of Jingfeng Medical Animal Center (Beijing, China). Subjects were judged to be in good health and housed in conditions of temperature (22 ± 2°C), humidity (55 ± 10%), and light (8:00 AM to 8:00 PM) in a controlled breeding room where they were acclimated for 7 days before study. Normal food and water were available ad libitum but withdrawn 24 h before intragastric administration of demethoxycurcumin. Demethoxycurcumin was administered p.o. as 30% aqueous 1,2-propylene glycol solution. Urine and feces were collected for 48 h from animals housed in stainless steel metabolism cages equipped with a urine and feces separator.
Preliminary Studies. For the sample group (four rats), a solution of demethoxycurcumin (50 mg/kg) was administered p.o. by direct stomach intubation in a volume of 10 ml/kg b.wt. For the control group (four rats), the solvent 30% aqueous 1,2-propylene glycol only was administered p.o. in rats by the same method. Pooled urine and feces of the sample group and those of the control group were simultaneously treated with parallel procedures. Urine was subjected to macroporous resin D101 chromatography and eluted with H2O, 50% EtOH, and 95% EtOH in turn after filtration. Each elution was concentrated to nearly 1.0 ml in vacuo and detected by TLC in CHCl3/MeOH (15:1) and CHCl3/MeOH/H2O (7:3:0.5) and spraying with 10% H2SO4. After heating, two metabolite spots [RF 0.45 (spot 1) and 0.50 (spot 2)] were observed in 50% EtOH and 95% EtOH fraction of the sample group in CHCl3/MeOH (15:1), and two metabolite spots were observed at RF 0.05 and 0.40 in 50% EtOH fraction of the sample group in CHCl3/MeOH/H2O (7:3: 0.5) but not in those of the control group. Feces were extracted twice with ethyl acetate (EtOAC) (100 ml) and then MeOH (100 ml) for 2 h. The combined EtOAC and MeOH extracts were concentrated to nearly 1.0 ml under vacuum and then detected by TLC in CHCl3/MeOH (15:1) and CHCl3/MeOH/H2O (7:3:0.5) and spraying with 10% H2SO4. After heating, the same metabolite spot [RF 0.57 (spot 3)] was observed both in EtOAC and MeOH extracts of the sample group, and one metabolite spot [RF 0.40 (spot 4)] was observed in MeOH extracts of the sample group in CHCl3/MeOH (15:1) but not in those of the control group. There was no other metabolite spot observed in CHCl3/MeOH/H2O (7:3:0.5).
Isolation of Metabolites. A solution of demethoxycurcumin (6 mg/ml) was administered p.o. at 50 mg/kg b.wt. to 80 rats and then repeated at 1-week intervals. The total administration of demethoxycurcumin was 3 g. The urine (approximately 10,000 ml in total) and feces (approximately 385 g) were treated by the same methods used in the preliminary tests. The results in TLC were essentially identical to those in the preliminary tests. The EtOAC (31.5 g) and MeOH (21.0 g) extracts of the feces were chromatographed, respectively, on silica gel columns using a CHCl3/MeOH gradient solvent system to yield 8 (FE 1-8) and 14 fractions (FM 1-14). FE 1 and FM 1 (containing spot 3) were combined and subjected to Sephadex LH-20 column chromatography (CHCl3/MeOH 1:1), followed by C18-ODS reverse-phase open-column chromatography (60% MeOH in water) and then purified by preparative TLC (CHCl3/MeOH 15:1) to yield spot 3 (the mixture of M-1 and M-2, 40.3 mg). FM 3 (containing spot 4) was applied to Sephadex LH-20 column chromatography (CHCl3/MeOH 1:1), followed by C18-ODS reverse-phase open-column chromatography (60% MeOH in water) and then purified using Sephadex LH-20 column chromatography eluting with MeOH to afford spot 4 (the mixture of M-3 and M-4, 3.1 mg). The 50% EtOH fraction (26 g) of the urine was dissolved in MeOH and then filtered. The filtrate (containing spots 1 and 2) was subjected to Sephadex LH-20 column chromatography, eluted with MeOH, followed by C18-ODS reverse-phase open-column chromatography (60% MeOH in water) to yield seven fractions (U01-07). Further purification of U05 (containing spots 1 and 2) was performed using C18-ODS reverse-phase preparative HPLC (55% MeOH in water, 277 nm) to afford five fractions (U051-U055). U055 was further subjected to C8-ODS reverse-phase preparative HPLC (50% MeOH in water, 284 nm) to yield M-9 (1.8 mg). U051 (containing spots 1 and 2) was applied to preparative TLC (CHCl3/MeOH 15:1) to yield spot 1 (the mixture of M-7 and M-8, 17.3 mg) and spot 2 (the mixture of M-5 and M-6, 67.6 mg).
Spectroscopic Methods. NMR spectra were measured on Bruker (Newark, DE) ARX-300 or AV-600 spectrometers using tetramethylsilane as an internal standard. ESI/MS was performed on an Agilent Technologies (Palo Alto, CA) 1100 Series LC/MSD Trap instrument whose mass range is 50 to 5000 (the mass was calibrated). The instrument was operated in both the positive and negative ion modes using nitrogen for nebulizing and dry gas. The ionization was performed applying the following parameters: dry gas temperature, 300°C; dry gas rate, 5 l/min; spray voltage, 4000 V, and atomization, 15 psi. Sample solutions were directly introduced into the ESI source at a flow rate of 3 µl/min by a syringe pump.
HPLC Instruments. Preparative HPLC was performed using a C8 column (C8, 250 x 20 mm, Inertsil Pak) and a C18 column (C18, 250 x 20 mm, Inertsil Pak) in a Waters (Milford, MA) 600 liquid chromatograph apparatus equipped with a Waters 490 UV detector. Analytical HPLC was performed using a C18 column (C18, 25 x 20 mm, Inertsil Pak) in a Waters 600 liquid chromatograph apparatus equipped with a Waters 996 UV detector.
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Results
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Metabolites M-1 and M-2 were obtained together as a viscous oil. The positive ESI/MS showed two quasi-molecular ion peaks at m/z 329 ([M + H]+) and 351 ([M + Na]+), and the negative ESI/MS gave a quasi-molecular ion peak at m/z 327 ([M - H]-) (Fig. 2). The 13C (Fig. 3) and 1H NMR spectra revealed two extremely similar groups of signals, which suggested that there was a pair of isomers with the same molecular formula of C20H24O4. For the two groups of signals, the stronger one corresponded to M-2, and the weaker one corresponded to M-1. The 13C and 1H NMR and heteronuclear multiple-quantum correlation (HMQC) spectra displayed two sets of 1,3,4-trisubstituted benzene ring signals [
6.62 (1H, brd, J = 8.0 Hz), 6.64 (1H, d, J = 1.5 Hz), 6.81 (1H, d, J = 8.0 Hz) and 6.62 (1H, brd, J = 8.0 Hz), 6.66 (1H, d, J = 1.5 Hz), 6.81 (1H, d, J = 8.0 Hz)], two sets of 1,4-bis-substituted benzene ring signals [6.74 (2H, d, J = 8.4 Hz), 6.99 (2H, d, J = 8.4 Hz) and 6.74 (2H, d, J = 8.4 Hz), 6.97 (2H, d, J = 8.4 Hz)], two methoxy groups [3.84 (3H, s, OCH3) and 3.82 (3H, s, OCH3)], two overlapped carbonyl signals (211.2), and 12 methylene groups, which suggested that M-1 and M-2 were the reduced metabolites of olefinic C-C double bonds of demethoxycurcumin. In the heteronuclear multiple-bond correlation (HMBC) spectrum of M-2 (Fig. 4), correlations from H-2', H-6' (6.99) to C-4' (154.1), C-3', and C-5' (115.3); H-3', H-5' (6.74) to C-1' (132.7) and C-4' (154.1) indicated the presence of 4'-hydroxyphenyl (group A). Correlations from H-2'' (6.64) to C-3'' (146.3), C-4'' (143.5), C-5'' (114.1), and C-6'' (120.8); H-5'' (6.81) to C-1'' (134.2), C-2'' (111.0), C-3'' (146.3), C-4'' (143.5), H-6'' (6.62) to C-2'' (111.0), and OCH3 (3.84) to C-3'' (146.3) suggested the existence of 3''-methoxy-4''-hydroxyphenyl (group B). Correlations from H-1 (2.80) to C-2 (44.5) and C-3 (211.2); H-2 (2.67) to C-3 (211.2); H-4 (2.39) to C-3 (211.2) and C-5 (23.3); H-5 (1.57) to C-3 (211.2), C-4 (42.9), and C-7 (35.3); H-6 (1.51) to C-4 (42.9), C-5 (23.3), and C-7 (35.3); and H-7 (2.50) to C-5 (23.3) and C-6 (31.1) confirmed the presence of the moiety of 3-heptanone (group C). In addition, correlations from H-2', H-6' (6.99) to C-1 (28.9) justified the connectivity between the group A and group C at C-1'/C-1, and correlations from H-2'' (6.64) and H-6'' (6.62) to C-7 (35.3) revealed the junction between group B and group C at C-1''/C-7 (Fig. 5). Thus, M-2 was elucidated as 1-(4'-hydroxyphenyl)-7-(3''-methoxy-4''-hydroxyphenyl)-3-heptanone (namely, 5-dehydroxy-hexahydro-demethoxycurcumin-B). By the same methods, the assignment of 3'-methoxy-4'-hydroxyphenyl moiety (group A) in M-1 was confirmed by HMBC correlations of H-2' (6.66) with C-3' (146.4), C-4' (143.8), C-5' (114.3), and C-6' (120.7); H-5' (6.81) with C-1' (132.9), C-2' (111.1), C-3' (146.4), C-4' (143.8); H-6' (6.62) with C-2' (111.1); and protons of methoxy (3.82) with C-3' (146.4). The assignment of 4'-hydroxyphenyl moiety (group B) in M-1 was supported by the HMBC correlations of H-2'', H-6'' (6.97) with C-4'' (153.8), C-3'', and C-5'' (115.1) and H-3'', H-5'' (6.74) with C-1'' (134.0) and C-4'' (153.8). The presence of 3-heptanone moiety (group C) in M-1 was suggested by the HMBC correlations of H-1 (2.80) with C-2 (44.6) and C-3 (211.2); H-2 (2.67) with C-3 (211.2); H-4 (2.39) with C-3 (211.2) and C-5 (23.2); H-5 (1.57) with C-3 (211.2), C-4 (42.9), and C-7 (34.7); H-6 (1.51) with C-4 (42.9), C-5 (23.2), and C-7 (34.7); and H-7 (2.50) with C-5 (23.2) and C-6 (31.1). Furthermore, the connectivity between group A and group CatC-1'/C-1 was justified by the HMBC correlations of H-2' (6.66) with C-1 (29.5), and the junction between group B and group C at C-1''/C-7 shown by the correlations of H-2'', H-6'' (6.97) with C-7 (34.7). Thus, M-1 was identified as 1-(3'-methoxy-4'-hydroxyphenyl)-7-(4''-hydroxyphenyl)-3-heptanone (namely, 5-dehydroxy-hexahydro-demethoxycurcumin-A). The full assignments of carbon and proton signals of M-1 and M-2 are summarized in Table 1 and Table 2, respectively.
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FIG. 2. ESI/MS spectra of [M - H]- ion of M-1 and M-2 (A), M-3 and M-4 (B), M-5 and M-6 (C), M-7 and M-8 (D), M-9 (E), and demethoxycurcumin (F).
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Metabolites M-3 and M-4 were obtained together as a viscous oil. The positive ESI/MS showed a quasi-molecular ion peak at m/z 353 ([M + Na]+), and the negative ESI/MS gave a quasi-molecular ion peak at m/z 329.0 ([M - H]-). The 13C and 1H NMR spectra also revealed two extremely similar groups of signals, which suggested that there was a pair of isomers with the same molecular formula of C20H26O4. For the two groups of signals, the stronger one corresponded to M-4, and the weaker one corresponded to M-3. The 13C and 1H NMR data of M-4 were similar to those of M-2 except for the upfield shifts of C-3 by 139.9 ppm and the appearance of the H-3 [3.62 (1H, m)], which showed that M-4 was the 3-hydroxy reductive product of M-2. In addition, the 13C and 1H NMR data of M-4 were nearly identical to those previously reported (Li et al., 2004). Thus, M-4 was established as 1-(4'-hydroxyphenyl)-7-(3''-methoxy-4''-hydroxyphenyl)-3-heptitol (namely, 5-dehydroxy-octahydro-demethoxycurcumin-B). The chemical shifts of M-3 were similar to those of M-1 except for those around C-3. In the 13C NMR spectrum, the signal of C-3 was shifted to a higher field (71.4) compared with that of M-1 (211.2). In the 1H NMR spectrum, the signal of H-3 [3.62 (1H, m)] occurred. These results suggested that M-3 was the 3-hydroxy reductive product of M-1. Therefore, M-3 was established as 1-(3'-methoxy-4'-hydroxyphenyl)-7-(4''-hydroxyphenyl)-3-heptitol (namely, 5-dehydroxy-octahydro-demethoxycurcumin-A).
Metabolites M-5 and M-6 were obtained together as a viscous oil. The positive ESI/MS showed a quasi-molecular ion peak at m/z 367 ([M + Na]+), and the negative ESI/MS gave a quasi-molecular ion peak at m/z 343 ([M - H]-). Similarly, the 13C and 1H NMR spectra also revealed two extremely similar groups of signals, which suggested that there was a pair of isomers with the same molecular formula of C20H24O5. For the two groups of signals, the stronger one corresponded to M-6, and the weaker one corresponded to M-5. In the 1H and 13C NMR spectra, the signal pattern of M-5 and M-6 was nearly identical to that of M-1 and M-2, except that the signals of C-5 and H-5 of M-5 were shifted to lower fields [C66.9 and H4.03 (1H, m)] compared with those of M-1 [C23.2 and H1.57 (2H, m)], and the signals of C-5 and H-5 of M-6 were shifted to lower fields [C66.9 and H4.03 (1H, m)] compared with those of M-2 [C23.3 and H1.57 (2H, m)], respectively. These results suggested that M-1 and M-2 were the 5-dehydroxylated products of M-5 and M-6. In addition, the MS, 1H NMR, and 13C NMR data of M-5 were nearly identical to those previously reported (Kikuzaki et al., 1991), and those of M-6 were the same as those previously reported (Shin et al., 2002). Thus, M-5 and M-6 were determined to be hexahydro-demethoxycurcumin-A and hexahydro-demethoxycurcumin-B, respectively. The full assignments of carbon and proton signals of M-5 and M-6 are summarized in Table 3 and Table 4, respectively.
Metabolites M-7 and M-8 were obtained together as a viscous oil. The positive ESI/MS showed a quasi-molecular ion peak at m/z 381 ([M + Na]+), and the negative ESI/MS gave a quasi-molecular ion peak at m/z 357 ([M - H]-). Similarly, the 13C and 1H NMR spectra also revealed two extremely similar groups of signals, which suggested that there was a pair of isomers with the same molecular formula of C21H26O5. For the two groups of signals, the stronger one corresponded to M-8, and the weaker one corresponded to M-7. The chemical shifts of M-7 were nearly the same as those of M-5 except for the following findings: the proton signal of H-5 (3.71) was shifted upfield by 0.32 ppm; the carbon signal of C-5 (76.7) was shifted downfield by 9.8 ppm; and the signals of methoxy [C56.9 and H3.31 (3H, s)] were present, which indicated that M-7 was the 5-O-methyl ether of M-5. Therefore, M-7 was elucidated as 5-O-methyl-hexahydro-demethoxycurcumin-A. By the same method, M-8 was determined to be the 5-O-methyl ether of M-6, and the MS, 1HNMR, and 13C NMR data of M-8 were nearly the same as those previously reported (Li et al., 2003). Therefore, M-8 was established as 5-O-methyl-hexahydro-demethoxycurcumin-B.
Metabolite M-9 was obtained as a yellow amorphous powder. The positive ESI/MS showed two quasi-molecular ion peaks at m/z 325 ([M + H]+) and 347 ([M + Na]+), and the negative ESI/MS gave a quasi-molecular ion peak at m/z 323 ([M - H]-), corresponding to the molecular formula C20H20O4, which was further supported by the 1H NMR and 13C NMR spectral data. The 13C and 1H NMR and HMQC spectra displayed a set of 1,3,4-trisubstituted benzene ring signals [6.76 (1H, d, J = 8.1 Hz), 6.96 (1H, brd, J = 8.1 Hz), 7.16 (1H, brs)], a set of 1,4-bis-substituted benzene ring signals [6.64 (2H, d, J = 8.1 Hz), 7.00 (2H, d, J = 8.1 Hz)], one pair of trans-conjugated olefinic protons [6.22 (1H, d, J = 15.5 Hz), 7.35 (1H, dd, J = 10.4, 15.4 Hz), and 6.93 (1H, dd, J = 10.2, 15.4 Hz), 6.96 (1H, d, J = 16.0 Hz)], a methoxy group [3.80 (3H, s, OCH3)], a carbonyl signal (199.2), and two methylene groups. The HMBC correlations of H-1 (2.71), H-2 (2.84), H-4 (6.22), and H-5 (7.35) with C-3 (199.2) suggested that H-1 and H-5 are three bonds away from the C-3 carbonyl group, whereas H-2 and H-4 are adjacent to the carbonyl group. The HMBC spectrum revealed H-2',6' (7.00) to be correlated with C-1 (29.0) and H-2'' (7.16) correlated with C-7 (141.9). Thus, M-9 was identified as 5-dehydroxy-dihydro-demethoxycurcumin-B. It is a 5-dehydroxy product with reduction of the double bond between C-1 and C-2 of demethoxycurcumin. The chemical shifts of M-9 were nearly identical to those previously reported (Li et al., 2004). The full assignments of carbon and proton signals of M-9 are summarized in Table 5.
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Discussion
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This is the first study on the metabolism of demethoxycurcumin in vivo. Nine phase 1 metabolites were obtained and identified by ESI/MS spectra and NMR spectroscopy including 1HNMR, 13C NMR, and two-dimensional NMR (HMQC, HMBC). Compared with earlier reports on the metabolism of demethoxycurcumin in vitro (Hoehle et al., 2006), an identical result was obtained in that reduction of the aliphatic moiety is the only pathway in phase 1 metabolism and no oxidative metabolites were discovered. However, there were two new discoveries in the present study: first, the existence of the dehydroxy or methylated metabolites was shown, and second, the existence of the isomers with a methoxy group substituted on a different benzene ring. In this study, the major metabolites of demethoxycurcumin in urine were hexahydro-demethoxycurcumins (M-5 and M-6) and the 5-O-methyl-hexahydro-demethoxycurcumins (M-7 and M-8) together with traces of 5-dehydroxy-dihydro-demethoxycurcumins (M-9), whereas the major metabolites in feces were 5-dehydroxy-hexahydro-demethoxycur-cumin (M-1 and M-2) and 5-dehydroxy-octahydro-demethoxycurcumin (M-3 and M-4). Combined with previous reports on the metabolism of curcuminoids (Holder et al., 1978; Wahlstrom et al., 1978; Ireson et al., 2001; Hoehle et al., 2006), it may be presumed that some demethoxycurcumin should initially undergo reduction to form dihydro-, tetrahydro-, hexahydro- (M-5 and M-6), and octahydro-demethoxycurcumin in a stepwise fashion (Ireson et al., 2002), followed by dehydroxylation (Feighner et al., 1980; Bokkenheuser et al., 1981; Kasahara et al., 1995) to form 5-dehydroxy-dihydro- (M-9), 5-dehydroxy-hexahydro- (M-1 and M-2), and 5-dehydroxy-octahydro-demethoxycurcumins (M-3 and M-4); on the other hand, some demethoxycurcumin may be initially methylated (Yang et al., 2005), followed by reduction to form 5-O-methyl-hexahydro-demethoxycurcumins (M-7 and M-8). Based on the metabolite profiles, the metabolic pathways of demethoxycurcumin in rats are proposed (Fig. 6).
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FIG. 6. Structures of demethoxycurcumin metabolites in rat urine and feces and possible metabolic pathways for their production.
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Based on present knowledge concerning the metabolism of curcuminoids, curcuminoids and their reduced metabolites appear to be easily conjugated in vivo and in vitro. The reported conjugates include monoglucuronides, monosulfates, and mixed sulfate/glucuronides (Holder et al., 1978; Ravindranath et al., 1982; Asai et al., 2000; Hoehle et al., 2006). In this study, seven new phase 1 metabolites were discovered, which provide new types of precursors for research on phase 2 metabolites of curcuminoids. In addition, because of the low bioavailability of curcumin, some previous research suggested that the pharmacological activities of curcumin were in part mediated by its metabolites (Ireson et al., 2001, 2002). This has been confirmed by recent experiments with the activities of the phase 1 metabolites (Leyon et al., 2004; Pari et al., 2004; Lee et al., 2005; Limtrakul et al., 2006; Murugan et al., 2006). Further studies of the nine phase 1 reductive metabolites should clarify whether they remain active.
From the comparison of the 13C NMR spectra of the four pairs of isomers, it was always found that there were two very similar groups of signals and one stronger than the other. These findings suggest that in rats demethoxycurcumin possibly undergoes tautomerization between 3-keto-5-enol (form A) and 3-keto-5-enol (form B) (Fig. 7) and that the forms of these two isomers were not equal; one form (form B) was the major one, and the other form (form A) was the minor one. In the HMQC spectrum of M-9, the proton signals H 7.40 (d, 8.6 Hz) associated with C 129.2 (Fig. 8) and H 3.76 with C 55.7 were observed in addition to the proton and carbon signals of M-9, which could be assigned to the isomer of M-9. The reason that the other correlative signals of the isomer were not entirely displayed in the 13C and 1H NMR spectra of M-9 might be because of the scant amount of the compound available.
M-5 and M-6 presumably would begin to transfer partly to M-7 and M-8 when placed in MeOH for more than 2 weeks. However, spot 1 (the mixture of M-7 and M-8) was observed in TLC in the preliminary study, and at that time the urine sample had not yet been dealt with MeOH. The data showed that M-7 and M-8 were the actual metabolites of demethoxycurcumin in rats. However, a portion of M-7 and M-8 probably transferred from M-5 and M-6 because a large quantity of MeOH was utilized during the course of the subsequent separation.
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, the structures of some metabolites deduced only from liquid chromatography/MS at stage n (LC/MSn) data may not be correct, especially in the case of the existence of isomerism of the metabolites. In this study, four groups of isomers (M-1 and M-2, M-3 and M-4, M-5 and M-6, M-7 and M-8) were obtained that have the same chromatographic behaviors and identical data in LC/MSn. Therefore, the findings could not be validated just by LC/MSn data (Hoehle et al., 2006). 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 for the identification of metabolites.
In summary, we have determined the definitive structures of nine phase 1 reductive metabolites of demethoxycurcumin by mass spectra and NMR spectroscopy. In the urine, the major reductive metabolites are the hexahydro-demethoxycurcumin and the methyl ether products of hexahydro-demethoxycurcumin. In the feces, the dehydroxy products of hexahydro- and octahydro-demethoxycurcumin are predominant. These results are important for the understanding of demethoxy-curcumin metabolism in rats and should provide information and reference for the further metabolic investigation of demethoxycurcumin in humans. Screening of the bioactivities of the novel metabolite is presently under study.
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Footnotes
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doi:10.1124/dmd.107.015008.
ABBREVIATIONS: EtOH, ethanol; MeOH, methanol; HPLC, high-performance liquid chromatography; MS, mass spectrometry; ESI, electrospray ionization; TLC, thin layer chromatography; ODS, octadecylsilane; EtOAC, ethyl acetate; HMBC, heteronuclear multiple-bond correlation; HMQC, heteronuclear multiple-quantum correlation; LC/MSn, liquid chromatography/mass spectrometry at stage n.
Address correspondence to: Feng Qiu, Department of Natural Products Chemistry, Shenyang Pharmaceutical University, No. 103 Road Wenhua, Shenyang, P. R. China 110016. E-mail: fengqiu20070118{at}163.com
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Abstract
Materials and Methods
C) corrrlations of M-1 and M-2.
