Isolation and Identification of Phase 1 Metabolites of Curcumol in Rats

Transcription

1 DMD Fast This article Forward. has not been Published copyedited on and August formatted. 3, The 2010 final version as doi: /dmd may differ from this version. Isolation and Identification of Phase 1 Metabolites of Curcumol in Rats Yan Lou, Hui Zhang, Hao He, Kaifeng Peng, Ning Kang, Xingchuan Wei, Xuegai Li, Lixia Chen, Xinsheng Yao and Feng Qiu Department of Natural Products Chemistry Y.L., H.Z., H.H., K.P., X.W., X.L., L.C., X.Y., F.Q., School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, People s Republic of China; Key Laboratory of Structure-Based Drug Design Discovery of Ministry of Education Y.L., H.Z., H.H., K.P., X.W., X.L., L.C., X.Y., F.Q., Shenyang Pharmaceutical University, Shenyang, People s Republic of China; Department of Biochemistry and Molecular Biology N.K., School of Life Sciences & Biopharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang, People s Republic of China 1 Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics.

2 a) Running title: Metabolites of Curcumol in Rats b) Corresponding author: Feng QIU Address: Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, No.103 Road Wenhua, Shenyang, People s Republic of China. Post Code: Telephone: Fax: fengqiu @163.com c) text pages:24; tables: 6; figures: 2; references: 21; the number of words in the Abstract: 178; the number of words in the Introduction: 207; the number of words in the Discussion: 694. d) Abbreviations: HPLC, high-performance liquid chromatography; ODS, octadecylsilane; TLC, thin layer chromatography; R f, retention factor; EtOH, ethanol; MeOH, methanol; (CD 3 ) 2 CO, deuterated acetone; CDCl 3, deuterated chloroform; CD 3 OD, deuterated methanol; C 5 D 5 N, deuterated pyridine; TMS, tetramethylsilane; 2D NMR, two-dimensional nuclear magnetic resonance; HMQC, heteronuclear multiple-quantum correlation; HMBC, heteronuclear multiple-bond correlation; ESI-MS, electrospray ionization-mass spectrometry; HRFAB-MS, high resolution fast atom bombardment-mass spectrometry. 2

3 Abstract Curcumol is one of the major components of the essential oil of Curcuma wenyujin with the structure of a guaiane-type sesquiterpenoid hemiketal. It exhibits clear anti-tumor, anti-hepatic fibrosis, antioxidant and antimicrobial activities. In this paper, the metabolism of curcumol in rats was investigated by characterizing metabolites excreted into urine. Sixteen phase 1 metabolites of curcumol were isolated from the urine of rats after an oral dose of 40 mg.kg -1, and their structures were elucidated on the basis of spectroscopic data. The metabolites were characterized as 2α-hydroxycurcumol (M-1), (11αH)-3α-hydroxy-9-en-8,12-epoxycurcumol (M-2), (11αH)-14- hydroxy-9-en-8,13-epoxycurcumol (M-3), (11βH)-14-hydroxy-9-en-8,12-epoxycurcumol (M-4), 10α,14-dihydroxy-(1αH,7βH)-guai-4-en-3,8-dione (M-5), 10β,14-dihydroxy-(1αH,7βH)-guai-4- en-3,8-dione (M-6), 10β-hydroxy-(1αH,7βH,11αH)-guai-8(13),8(14)-diepoxy-4-en-3-one (M-7), 10β-hydroxy-(1αH,7βH,11βH)-guai-8(12),8(14)-diepoxy-4-en-3-one (M-8), 10α-hydroxy-(1αH, 7βH,11αH)-guai-8(13),8(14)-diepoxy-4-en-3-one (M-9), 10α-hydroxy-(1αH,7βH,11βH)-guai- 8(12),8(14)-diepoxy-4-en-3-one (M-10), 10α,14,15-trihydroxy-(1αH,7βH)-guai-4-en-3,8-dione (M-11), 10β-hydroxy-(1αH,7βH)-guai-4-en-3,8-dioxo-13-oic acid (M-12), (1αH,7βH)- guai-4,10(14)-dien-3,8-dioxo-13-oic acid (M-13), 5β,10β-dihydroxy-(1αH,7βH,11αH)-guai- 8(13),8(14)-diepoxide (M-14), 10β,14-dihydroxycurcumol (M-15), and 5β,10β,14-trihydroxy- (1αH,7βH)-guai-8-one (M-16). All were newly identified compounds, among which M-3 and M-4; M-5 and M-6; M-7, M-8, M-9 and M-10 are three groups of epimers. Based on the metabolite profile, the possible metabolic pathways of curcumol in rats are proposed. This is the first study of the metabolites of guaiane-type sesquiterpene in animals. 3

4 Introduction Curcumol is a representative index component for the quality control of the essential oil of Curcuma wenyujin which is currently used as an anti-cancer and anti-virus treatment, and included in the Pharmacopoeia of the People s Republic of China (2005). Pharmacological studies have suggested that curcumol has anti-tumor(xu et al., 2005), anti-hepatic fibrosis(jiang et al., 1993), antioxidant(mau et al., 2003), and antimicrobial(phan et al., 2000) activities. In China, this compound has also been used as a pesticide for rodent control due to its contraceptive effect (Liu et al.,2006; Hou et al., 2007 ). The structure of curcumol has been characterized on the basis of chemical and spectral data ( Hikino et al., 1965), and its absolute stereostructure was determined by X-ray analysis (Inayama et al., 1984). There are so far only two research reports on its metabolism which indicated that absorption of curcumol from gastrointestinal tract in rats was rapid and complete (Zhang R. et al., 2007), and that the kidney seemed to be the main route of clearance (Su et al., 1980). In order to characterize its biotransformation in mammals, curcumol was given orally to rats, and its urinary metabolites were investigated. The present paper mainly describes the isolation and identification of these metabolites. Materials and Methods Curcumol. Curcumol (purit\ 99% in HPLC) was isolated from the essential oil of Curcuma wenyujin, and characterized by chemical and spectral methods as described in the literature (Li et al., 1988). Chemicals. Ethyl acetate, ethanol, n-butanol and methanol of analytical grade were obtained from Tianjin Bodi Chemical Factory (Tianjin, China) while methanol of HPLC grade was obtained from Jiangsu Hanbang Chemical Factory (Jiangsu, China). Water was double distilled in our laboratory; Silica gel (Polygosil 40-63µm) was provided by Nippon Rensui Co. (Tokyo, Japan); Sephadex 4

5 LH-20 was provided by GE Healthcare (Little Chalfont, Buckinghamshire, UK), RP-18 (10-75 µm) silica gel was purchased from Merck Chemical Ltd (Darmstadt, Germany), and macroporous resin D101 was obtained from Qingdao Marine Chemical Factory (Shandong, China). Spectroscopic Methods. IR spectra were recorded on a Bruker IFS 55 spectrophotometer (KBr) (Bruker, Newark, DE). NMR spectra ( 1 H-NMR, 13 C-NMR, DEPT, 1 H- 1 H-COSY, HMQC, HMBC and NOESY) were recorded on a Bruker AVANCE-400 ( 1 H-NMR, 400 MHz; 13 C-NMR, 100 MHz) (Bruker, Newark, DE) or AV-600 spectrometer ( 1 H-NMR, 600 MHz; 13 C-NMR, 150 MHz) and chemical shifts were recorded in ppm using TMS as an internal standard. ESI-MS spectra were measured on a Bruker Esquire 2000 (Bruker, Newark, DE) instrument in both positive and negative modes. HRFAB-MS spectra were recorded using a Bruker APEX II instrument (Bruker, Newark, DE). HPLC Instruments. Preparative HPLC was performed using a C8 column (C8, mm, Inertsil Pak) and a C18 column (C18, mm, Inertsil Pak) in a Waters (Milford, MA) 600 liquid chromatograph equipped with a Waters 490 UV detector. Analytical HPLC was performed using a C18 column (C18, mm, Inertsil Pak) in a Waters 600 liquid chromatograph equipped with a Waters 996 UV detector. Animals. Male Wistar rats ( g) were provided by the Institute of Jingfeng Medical Animal Center (Beijing, China). The animals were judged to be in good health and housed under standard conditions of temperature (22 ± 2 ), humidity (55 ± 10%), and light (8:00-20:00) in a controlled breeding room where they were allowed to acclimatize for 7 days prior to the study. Normal food and water were available ad libitum, but withdrawn 24 hr prior to intragastric administration of curcumol. Curcumol was orally administered as a 50% aqueous 1,2- propylene glycol solution. 5

6 Urine and feces were collected for 48 hr from animals housed in stainless-steel metabolism cages equipped with a urine and feces separator. Preliminary Studies. For the sample group (five rats), a solution of curcumol (4 mg/ml) was administered orally by direct stomach intubation in a volume of 10 ml/kg body weight. For the control group (five rats), only the solvent, 50% aqueous 1,2-propylene glycol, was given orally to the rats by the same method. Pooled urine and feces of the sample group and those of the control group were simultaneously treated using parallel procedures. Urine was extracted with EtOAc after filtration to give an EtOAc layer and an H 2 O layer. The H 2 O layer was subjected to macroporous resin D101 chromatography and eluted sequentially with H 2 O (YW-1), 50% EtOH (YW-2), and 95% EtOH (YW-3) after filtration. Each eluate was concentrated to nearly 1.0 ml in vacuo at 40 C and examined by TLC in cyclohexane:acetone (2:1), CHCl 3 :MeOH (2:1) and CHCl 3 :MeOH:H 2 O (65:36:5), and spraying with a 1% solution of vanillin in concentrated sulfuric acid. After heating, three major distinctive metabolite spots [Rf 0.38 and 0.26 and 0.18] were observed in the EtOAc layer of the sample group in cyclohexane: acetone (2:1), and two [Rf 0.48 and 0.36] were observed in the YW-2 (50% EtOH eluate) in CHCl 3 : MeOH: H 2 O (65:36:5), but not in the control group. Feces were extracted ultrasonically with EtOAc (100ml) then twice with MeOH (100ml) for 20 min. The combined EtOAc and MeOH extracts were concentrated to nearly 1.0 ml in vacuo, and examined by TLC by the same procedures, but there were no metabolite spots observed in both extracts of the sample and control groups. Isolation of Metabolites. A solution of curcumol (4 mg/ml) was orally administered at a dose of 40 mg/kg body weight to eighty rats and then repeated after an interval of one week. The total amount of curcumol administered was 2.6 g. Urine was collected every day and extracted with EtOAc after filtration. Then each layer was treated by the same method as used in the preliminary test. The TLC 6

7 results were essentially identical to those obtained in the preliminary test. Urine (1500 ml/day) was collected and extracted each day with EtOAc (1500 ml/time) three times at room temperature after filtration to give an EtOAc layer and an H 2 O layer. The EtOAc layer was evaporated to dryness in vacuo at 40 C, and the residue was dissolved in water. This solution was then subjected to ODS column ( mm) chromatography and eluted with a linear gradient of MeOH:H 2 O (0.5:9.5, 1:9, 3:7, 5:5, 7:3, 10:0) to obtain six fractions (Fr. E1-6). Fr. E3 was subjected to Sephadex LH-20 chromatography with CHCl 3 -MeOH (1:1) to give four major sub-fractions (Fr. E31-34). The fractions (Fr. E32-33) containing metabolites were then purified by preparative HPLC using MeOH-H 2 O (30:70) to give M-5 (99.8 mg), M-6 (33.8 mg), M-7 (68.1mg), M-8 (86.9 mg), M-9 (45.1 mg), M-10 (13 mg), and M-15, M-16 (38 mg). Fr. E4 was applied to a Sephadex LH-20 column and eluted with CHCl 3 -MeOH (1:1) to give four major sub-fractions (Fr. E41-44). The fractions (Fr. E42-43) containing the metabolites were then purified by preparative HPLC using MeOH-H 2 O (40: 60) to obtain M-1 (3.9 mg), M-2 (66.8 mg), M-3 (33.4 mg), and M-4 (143.9 mg). The H 2 O layer was subjected to macroporous resin D101 chromatography and eluted with H 2 O (Fr. W1), 50% EtOH (Fr. W2), and 95% EtOH (Fr. W3) in turn. The 50% EtOH fraction (Fr. W2) of the urine was applied to a moderate pressure liquid chromatography column (ODS, mm) and eluted with MeOH:H 2 O (0:10, 1:9, 3:7, 5:5, 7:3, 10:0) to obtain 6 sub-fractions (Fr. W21-26). Fr. W22 was then applied to a Sephadex LH-20 column and eluted with MeOH: H 2 O (0:1, 1:1, 1:0), followed by preparative HPLC to obtain M-14 (7.0 mg). Fr. W24 was subjected to Sephadex LH-20 column chromatography and eluted with MeOH: H 2 O (0:1, 1:1, 1:0), followed by preparative HPLC using MeOH: H 2 O (25:75) to obtain M-11 (11.0 mg). Fr. W25 was chromatographed on a Sephadex LH-20 column and eluted with MeOH: H 2 O (0:1, 1:1, 1:0), followed by preparative HPLC using MeOH:H 2 O (30:70) to obtain M-12 (13.0 mg), M-7 (20.0 mg), 7

8 and M-13 (25.0 mg). Results Isolation and Structure Elucidation of Curcumol Metabolites. Using the above methods, sixteen metabolites were isolated after oral administration of curcumol. All the metabolites were characterized as new compounds and their structures were identified on the basis of ESI-MS, 1 H-NMR, 13 C-NMR and 2D-NMR techniques. M-1 was obtained as a white powder. The positive ESI-MS of M-1 showed an [M + Na] + ion peak at m/z 275 (see Fig.1), indicating the molecular formula of C 15 H 24 O 3. This conclusion was further supported by HRFAB-MS at m/z [M + H] + (calcd for C 15 H 25 O 3, ). The molecular formula had one more oxygen atom than that of the parent molecule(curcumol), suggesting that M-1 was a hydroxy metabolite of curcumol. In the 1 H-NMR spectrum, an additional signal assigned to H-2 was observed at δ 4.24 (1H, m) in contrast to that of curcumol. The 13 C-NMR spectrum of M-1 exhibited an additional oxygen-bearing methine signal at δ 74.2 (C-2), further suggesting the presence of a hydroxyl group in the molecule. Its location was deduced to be at C-2 from the correlations of H-3 (δ 1.92, 1H, m; δ 1.98, 1H, m) and H-1 (δ 2.09, 1H, d, J = 8.5 Hz) to C-2 in the HMBC spectrum. Meanwhile, the signals of C-1 and C-3 had shifted downfield from δ 54.5 to δ 62.9 and from δ 30.9 to δ 40.2, while the signals of C-4 and C-10 had shifted upfield from δ 39.4 to δ 36.9 and from δ to δ 142.3, respectively, in comparison with that of curcumol. The α-configuration of 2-OH was deduced from the presence of the correlations between Me-15 and H-3β, H-2 and H-3β in the NOESY spectrum. Thus, M-1 was characterized as 2α-hydroxy curcumol. M-2, isolated as a white powder, showed a [M + H] + ion at m/z (calcd for C 15 H 23 O 3, 8

9 ) in the HRFAB-MS, corresponding to the molecular formula of C 15 H 22 O 3. The 1 H- and 13 C-NMR and heteronuclear multiple-quantum correlation (HMQC) spectrum showed three methyls, three methylenes (one oxygenated), six methines (one oxygenated, one olefinic carbon), three quaternary carbons (one acetal carbon, one oxygenated, one olefinic carbon). In the HMBC spectrum, correlations of H-15 (δ 1.11, 3H, d, J = 6.9 Hz), H-4 (δ 1.77, 1H, m) and H-2β (δ 1.98, 1H, m) with C-3 (δ 78.1) indicated the location of the hydroxyl group at C-3, and correlations of H-14 (δ 1.65, 3H, br.s), H-1 (δ 2.19, 1H, dd, J = 9.5, 9.6 Hz) and H-2β with C-10 (δ 139.5), and H-1 and H-14 with C-9 (δ 124.7) revealed that the double bond has been transferred from C-10 (14) in curcumol to C-9 (10) in M-2 and C-14 (δ 20.7) formed a methyl group. The HMBC correlations of H-11 (δ 2.12, 1H, m), H-12 (δ 1.00, 3H, d, J = 6.1 Hz) and H-13 (δ 4.38, 1H, dd, J = 8.0, 7.6 Hz) with C-7 (δ 57.2), and H-13 (δ 3.67, 1H, dd, J = 8.0, 8.3 Hz; δ 4.38, 1H, dd, J = 8.0, 7.6 Hz)with C-8 (δ 113.4) suggested the formation of a new tetrahydrofuran between C-8 and C-13 (δ 78.6). In the NOESY experiment, the correlation of H-3 (δ 4.14, 1H, ddd, J = 13.0, 8.2, 4.8 Hz) and H-15 suggested an α-orientation of OH-3, and correlation of H-9 (δ 5.68, 1H, s) and H-11 indicated a β-orientation of Me-12. So, the relative configuration of H-11 was suggested to be α. Therefore, the structure of M-2 was identified as (11αH)-3α-hydroxy-9-en-8,13-epoxy curcumol. M-3 was obtained as a colorless oil. Its positive ESI-MS displayed an [M + Na] + ion at m/z 273, implying a molecular formula of C 15 H 22 O 3, which was confirmed by the HRFAB-MS. The 1 H- and 13 C-NMR and heteronuclear multiple-quantum correlation (HMQC) spectrum displayed two methyls, five methylenes (one oxygenated), five methines (one olefinic carbon), and three quaternary carbons (one ketal carbon, one oxygenated, one olefinic carbon). The 13 C-NMR data of M-3 were very similar to those of M-2, except for C-3 (δ 31.2) and C-14 (δ 64.4) due to the hydroxylation of these two carbons. This was further supported by the HMBC data: H-15 (δ 1.04, 9

10 3H, d, J = 5.6 Hz), H-2 (δ 1.55, 1H, m; δ 1.90, 1H, m) and H-4 (δ 1.84, 1H, m) correlated with C-3, H-2, H-1 (δ 2.11, 1H, dd, J = 9.1, 8.3 Hz) and H-14 (δ 4.05, 2H, br.s) correlated with C-10 (δ 143.8), H-1 and H-9 (δ 5.96, 1H, s) correlated with C-14, H-11 (δ 2.18, 1H, m), H-12 (δ 1.01, 3H, d, J = 6.5 Hz) and H-13 (δ 3.70, 1H, dd, J = 7.5, 7.8 Hz; δ 4.41, 1H, dd, J = 7.5, 7.0 Hz) correlated with C-7 (δ 57.3), and H-12 correlated with C-8 (δ 113.2). The α-configuration of H-11 was established by the NOESY correlations between H-11 and H-9. Thus, the structure of M-3 was identified as (11αH)-14-hydroxy-9-en-8,13-epoxycurcumol. M-4 was obtained as colorless crystals. Its positive ESI-MS showed a quasi-molecular ion peak at m/z 273 ( [M + Na] + ) and HRFAB-MS gave an [M + H] + ion peak at m/z (calcd for C 15 H 23 O 3, ), indicating a molecular formula of C 15 H 22 O 3, which is the same as that of M-3. The 13 C-NMR spectrum was similar to that of M-3, except that the signals of C-7 and C-11 had shifted upfield ( δ 51.8, δ 33.8), compared with those of M-3 ( δ 57.3, δ 40.6), which indicated that M-4 might be a diastereomer of M-3. This deduction was further supported by the fact that the HMBC correlations were identical to those of M-3. In the NOESY spectrum, H-11 (δ 2.40, 1H, m) correlated with H-7 (δ 2.68, 1H, dd, J = 9.8, 4.1 Hz), indicating the β-orientation of H-11, which confirmed the above deduction. Therefore, the structure of M-4 was concluded to be (11βH)-14-hydroxy-9-en-8,12-epoxycurcumol. M-5 was obtained as a white powder. Its positive ESI-MS ([M + Na] + at m/z 289) and the HRFAB-MS ( [M + H] + at m/z , calcd for C 15 H 23 O 4, ), revealed the molecular formula to be C 15 H 22 O 4, with 5 unsaturated degrees in the molecule. The 1 H- and 13 C-NMR and heteronuclear multiple-quantum correlation (HMQC) spectrum displayed three methyls, four methylenes (one oxygenated), three methines, and five quaternary carbons (two ketone carbonyls, one oxygenated, two olefinic carbons). In the HMBC experiment, H-2 (δ 2.39, 1H, dd, J = 18.9,

11 Hz; δ 2.53, 1H, dd, J = 18.9, 6.8 Hz) correlated with C-1 (δ 52.3), C-3 (δ 207.5) and C-5 (δ 169.0), H-15 (δ 1.72, 3H, d, J = 1.6 Hz) correlated with C-3, C-4 (δ 139.7) and C-5, and H-1 (δ 3.22, 1H, m) correlated with C-5, indicating the five-member ring substructure of 4-methyl-4-en-3-cyclopentanone. The HMBC correlations of H-9 (δ 2.63, 1H, d, J = 11.8 Hz; 3.03, 1H, d, J = 11.8 Hz) with C-1, C-8 (δ 211.9), and C-10 (δ 75.3), H-2 and H-14 (δ 3.24, 1H, d, J = 11.6 Hz) with C-10, H-6 (δ 2.94, 1H, dd, J = 13.0, 4.6 Hz; δ 2.57, 1H, br.d, J = 13.0 Hz) with C-8, C-7 (δ 57.7), C-4 and C-5, and H-12 (δ 0.98, 3H, d, J = 6.6 Hz) and H-13 (δ 1.02, 3H, d, J = 6.7 Hz) with C-7 suggested the structure of 7-isopropyl-10-hydroxyl-10-hydromethyl-8-cycloheptanone. The above planar structure indicated cleavage of the hemiketal structure. The relative stereochemistry of M-5 was confirmed by a NOESY experiment: the correlations between H-14 and H-2β, between H-1α and 2α, and between H-1α and H-9α indicated the β-orientation of CH The structure of M-5 was concluded to be 10α,14-dihydroxy-(1αH,7βH)-guai-4-en-3,8-dione. M-6 was obtained as a white powder. Its positive ESI-MS showed an [M + Na] + ion peak at m/z 289, and the HRFAB-MS showed an [M + H] + ion peak at m/z (calcd for C 15 H 23 O 4, ), indicating the molecular formula of C 15 H 22 O 4, which was the same as that of M-5. The 13 C-NMR data were almost identical to those of M-5, except for the following findings: the signal of C-14 (δ 69.2) had shifted downfield by 4.7ppm, and the signal of C-1 (δ 49.8) and C-9 (δ 47.0) had shifted upfield ( δ -2~3 ppm). In the NOESY spectrum, the correlations of H-6α (δ 2.36, 1H, dd, J = 13.0, 12.2 Hz) and H-9α (δ 3.17, 1H, d, J = 11.9 Hz), H-1α (δ 2.85, 1H, br.d, J = 6.4 Hz) and H-9α, H-1α and H-6α, H-1α and H-2α (δ 2.28, 1H, dd, J = 18.3, 6.8 Hz), and H-1α and H-14 (δ 3.59, 1H, d, J = 10.9 Hz) suggested the α-configuration of CH On the basis of the above analysis, M-6 was identified as 10β,14-dihydroxy-(1αH,7βH)-guai-4-en-3,8-dione. M-7 was obtained as a white powder. Its negative ESI-MS showed an [M-H] - ion peak at m/z 11

12 263 indicating the molecular formula of C 15 H 20 O 4, with 6 unsaturated degrees in the molecule, which was further supported by the HRFAB-MS ([M-H] - at m/z , calcd for C 15 H 19 O 4, ). The 1 H- and 13 C-NMR and heteronuclear multiple-quantum correlation (HMQC) spectrum displayed two methyls, five methylenes (two oxygenated), three methines, and five quaternary carbons (one ketone carbonyl, one oxygenated, two olefinic carbons, one ketal carbon). Comparison of the 13 C-NMR data of M-7 with those of M-6 suggested the ketone carbonyl group in the seven-member ring was missing, and a new ketal carbon and oxygenated methylene were found. The HMBC correlations of both H-13 (δ 4.06, 1H, dd, J = 8.1, 8.1 Hz; δ 3.30, 1H, dd, J = 9.3, 8.1 Hz) and H-14 (δ 3.98, 1H, d, J = 8.3 Hz; 4.11, 1H, d, J = 8.3 Hz) with C-8 (δ 116.5) suggested the formation of two new epoxy rings between C-8 and C-13 (δ 75.5), and between C-8 and C-14 (δ 82.0). In the NOESY spectrum, the correlation between H-14 and H-1α (δ 3.21, 1H, br.s) indicated the α-orientation of CH The correlations between H-9 (δ 1.84, 1H, d, J = 12.5 Hz) and H-7β (δ 1.26, 1H, dd, J = 11.7, 3.2 Hz), and between H-12 (δ 1.08, 3H, d, J = 6.6 Hz) and H-7β suggested the α-orientation of H-11 (δ 2.21, 1H, m). Based on the above evidence, the structure of M-7 was concluded to be 10β-hydroxy-(1αH,7βH,11αH)-guai-8(13),8(14)-diepoxy-4-en-3-one. M-8 was purified as a white amorphous powder. The molecular formula C 15 H 20 O 4 was deduced from the ESI-MS and HRFAB-MS analysis, which was the same as that of M-7. The 13 C-NMR data were very similar to those of M-7, except that the signal of C-7 had shifted upfield from δ 57.4 to δ 52.5, and the signal of C-10 (δ 79.6) and C-8 (δ 116.6) had shifted downfield ( δ 1~2 ppm). In the NOESY spectrum of M-8, the correlations between H-11 (δ 2.45, 1H, m) and H-7β (δ 1.78, 1H, m), and between H-1α (δ 3.22, 1H, br.s) and H-14 (δ 4.01, 1H, d, J = 8.2 Hz; 4.10, 1H, d, J = 8.2 Hz) indicated the β-orientation of H-11 and the α-orientation of H-14. On the basis of the aforementioned information, the structure of M-8 was determined as 12

13 10β-hydroxy-(1αH,7βH,11βH)-guai-8(12),8(14)-diepoxy-4-en-3-one. M-9 was obtained as a white powder. Its negative ESI-MS showed a quasi-molecular ion peak at m/z 263 ( [M - H] - ), and the HRFAB-MS afforded an [M - H] - ion peak at m/z (calcd for C 15 H 19 O 4, ), indicating the molecular formula of C 15 H 20 O 4, which was the same as that of M-7 and M-8. Its 13 C-NMR data, compared with those of M-7, showed there was a great deal of similarity except that C-14 had shifted upfield from δ 82.0 in M-7 to δ 71.2 in M-9, and C-9 had shifted downfield from δ 43.4 to δ 48.0, implying that M-9 was an isomer of M-7. This deduction was further supported by the HMBC correlations identical with those of M-7. In the NOESY spectrum, the correlations between H-9α (δ 2.57, 1H, dd, J = 13.0, 0.7 Hz) and H-1α (δ 3.09, 1H, m), and between H-2β (δ 2.23, 1H, dd, J = 19.2, 1.7 Hz) and H-14 (δ 3.31, 1H, dd, J = 8.6, 0.9 Hz) suggested the β-orientation of CH 2-14; the correlations between H-15 (δ 1.66, 3H, dd, J = 1.9, 0.5 Hz) and H-6β (δ 3.04, 1H, dd, J = 12.6, 5.6 Hz), and H-11 (δ 1.98, 1H, m) and H-6α (δ 2.17, 1H, dd, J = 12.6, 12.6 Hz) indicated the α-orientation of H-11. Based on the above analyses, the structure of M-9 was determined as 10α-hydroxy-(1αH,7βH,11αH)-guai-8(13),8(14)-diepoxy-4-en-3-one. M-10, isolated as a white amorphous powder, had a molecular formula of C 15 H 20 O 4 as determined by HRFAB-MS at m/z [M - H] - (calcd for C 15 H 19 O 4, ), which is the same as that of M-9. Comparison of the 1 H- and 13 C-NMR data with those of M-9 suggested M-10 might be an isomer of M-9, which was further supported by the HMBC correlations identical with those of M-9. In the NOESY spectrum, the correlations between H-14 (δ 3.38, 1H, d, J = 8.8 Hz) and H-7β (δ 2.27, 1H, m), and between H-9α (δ 2.49, 1H, d, J = 13.3 Hz) and H-1α (δ 3.01, 1H, br.d, J = 6.5 Hz) indicated the β-orientation of CH 2-14, The correlation between H-13 (δ 1.00, 2H, d, J = 7.1 Hz) and H-1α indicated the α-orientation of H-13. Based on the above evidence, the structure of M-10 was determined to be 10α-hydroxy-(1αH,7βH,11βH)-guai-8(12),8(14)-diepoxy-4-en-3-one. 13

14 M-11 was obtained as a white powder. Its positive ESI-MS showed m/z: 305 [M + Na] +, 587 [2M + Na] +, 321 [M + K] +, 283 [M + H] + and the negative ESI-MS showed m/z: 281 [M - H] -, 317 [M + Cl] -. The HRFAB-MS showed an [M - H] - ion peak at m/z (calcd for C 15 H 21 O 5, ), indicating the molecular formula of C 15 H 22 O 5, with 5 unsaturated degrees in the molecule. Comparison of the 1 H- and 13 C-NMR spectra with those of M-5 showed that they were very similar except for CH 3-15 which was oxidized to CH 2 OH. This deduction was further supported by the HMBC and NOESY data. In the HMBC experiment, the correlations of H-2 (δ 3.09, 1H, dd, J = 18.0, 2.2 Hz; δ 2.58, 1H, dd, J = 18.0, 6.6 Hz) with C-1 (δ 50.3), C-3 (δ 208.1), C-4 (δ 143.0), and C-5 (δ 175.6), and H-15 (δ 4.68, 1H, d, J = 13.2 Hz; 4.72, 1H, d, J = 13.2 Hz) with C-3, C-4 and C-5 established the structure of 4-hydroxymethyl-4-en-3-cyclopentanone; also, the correlations of H-14 (δ 3.92, 1H, d, J = 10.8 Hz; 4.00, 1H, d, J = 10.8 Hz) with C-1, C-9 (δ 53.1) and C-10 (δ 75.5), H-11 (δ 2.42, 1H, m) with C-6 (δ 28.0), C-7 (δ 57.1) and C-8 (δ 212.8), and H-6 (δ 3.56, 1H, dd, J = 12.0, 3.0 Hz; δ 2.74, 1H, dd, J = 12.0, 8.4 Hz) with C-1, C-5, C-7, C-8, and C-11 (δ 32.0), together with H-12 (δ 0.90, 3H, d, J = 7.2 Hz) and H-13 (δ 1.10, 3H, d, J = 7.2 Hz) with C-7 and C-11 suggested the structure of 7-isopropyl-10-hydroxyl-10-hydromethyl-8-cycloheptanone. In the NOESY spectrum, the correlations between H-1α (δ 3.63, 1H, br.d, J = 6.6 Hz) and H-2α (δ 2.58, 1H, dd, J = 18.0, 6.6 Hz), between H-6α (δ 2.74, 1H, dd, J = 12.0, 8.4 Hz) and H-9α (δ 3.82, 1H, d, J = 12.6 Hz), between H-14 and H-2β as well as H-14 and H-9β (δ 3.23, 1H, d, J = 12.6 Hz), indicated the β-orientation of CH Based on the above evidence, the structure of M-11 was assigned as 10α,14,15-trihydroxy-(1αH,7βH)-guai-4-en-3,8-dione. M-12 was obtained as a white powder. Its molecular formula, C 15 H 20 O 5, was determined on the basis of the HRFAB-MS at m/z [M - H] + (calcd for C 15 H 19 O 5, ). The 13 C-NMR 14

15 spectrum was similar to that of M-6 except that CH 3-12(or 13) had been oxidized to a carboxyl group and CH 2-14 has been replaced by a methyl group, which was supported by the HMBC correlations of H-14 (δ 1.22, 3H, br.s) with C-1 (δ 53.5), C-9 (δ 42.6), and C-10 (δ 72.5), and H-7 (δ 1.66, 1H, o) with C-13 (δ 173.2). In the NOESY spectrum, H-2α (δ 2.77, 1H, m) correlated with H-1α (δ 1.69, 1H, m), and H-14 correlated with H-2α, suggesting the α-configuration of CH On the basis of the above analysis, M-12 was identified as 10β-hydroxy- (1αH,7βH)-guai-4-en-3,8-dioxo-13-oic acid, except for the configuration of C-11. M-13, obtained as a white powder, was assigned the molecular formula C 15 H 18 O 4, as deduced from its ESI-MS and HRFAB-MS. The 1 H- and 13 C-NMR and heteronuclear multiple-quantum correlation (HMQC) spectra displayed two methyls, four methylenes (one olefinic carbon), three methines, and six quaternary carbons (three carbonyl carbons, three olefinic carbons). Comparison of the 13 C-NMR spectrum with that of M-6 suggested that the CH 3-12(or 13) in M-6 had been oxidized to a carboxyl group and a double bond at C10(14) was formed, which was supported by the HMBC data. In the HMBC experiment, correlations of H-2 (δ 3.09, 1H, br.d, J = 12.6 Hz; δ 2.29, 1H, dd, J = 13.2, 12.6 Hz) with C-3 (δ 207.2), and C-5 (δ 171.1), H-1 (δ 3.50, 1H, m) with C-3, and H-15 (δ 1.88, 3H, s) with C-3, C-4 (δ 138.1) and C-5 suggested the structure of 4-methyl-4-en-3-cyclopentanone. Also, correlations of H-14 (δ 5.07, 1H, s; 5.12, 1H, s) with C-1 (δ 49.4) and C-9 (δ 50.9), H-12 (δ 1.43, 3H, d, J = 7.2 Hz) with C-7 (δ 53.6), C-11 (δ 40.5) and C-12 (δ 14.3), together with H-1 with C-9, C-10 (δ 142.1), and C-14 (δ 116.7) established the structure to be 7-isopropionyloxy-10(14)-en -8-cycloheptanone. On the basis of the above analysis, the structure of M-13 was concluded to be (1αH,7βH)-guai-4,10(14)-dien-3,8-dioxo-13-oic acid, except for the configuration of C-11. M-14 was obtained as a white powder. The positive ESI-MS (m/z 291 [M + Na] +, 559 [2M + 15

16 Na] + ) analysis showed the molecular formula was C 15 H 24 O 4 in combination with the HRFAB-MS data, which showed an [M - H] - ion peak at m/z (calcd for ). The 1 H- and 13 C-NMR and heteronuclear multiple-quantum correlation (HMQC) spectra displayed two methyls, six methylenes (two oxygenated), four methines, and three quaternary carbons (two oxygenated, one ketal carbon). Comparison of the NMR data of 14 with those of 10 showed that the two molecules were structurally closely related, the main differences being the resonances corresponding to C-3, C-4, and C-5. From the upfield shift of C-1 (δ 58.7) to C-5 (δ 79.2), it was evident that M-14 retained the five-member ring substructure of curcumol. The HMBC correlations of H-1 (δ 1.91, 1H, m) with C-4 (δ 44.0), H-3 (δ 1.48, 1H, m; 1.87, 1H, m) with C-2 (δ 21.9), C-4, and C-5, and H-15 (δ 0.93, 3H, d, J = 6.6 Hz) with C-3 (δ 28.6), C-4, and C-5 further confirmed the above assignment. In the NOESY spectrum, the correlations between H-7β (δ 1.98, 1H, m) and H-12 (δ 0.96, 3H, d, J = 6.1 Hz) indicated the β-orientation of H-12. The correlation of H-1 and H-6α (δ 1.08, 1H, m), H-15 and H-6β (δ 2.17, 1H, m) suggested the β-orientation of OH-5. The correlations of H-1 and H-9α (δ 2.20, 1H, d, J = 11.2 Hz) indicated the α-orientation of H-14 (δ 3.98, 1H, br.d, J = 8.2 Hz). Based on the above evidence, the structure of M-14 was identified as 5β,10β-dihydroxy-(1αH,7βH,11αH)-guai-8(13),8(14)-diepoxide. Metabolites M-15 and M-16 were obtained together as a colorless oil. The positive ESI-MS showed two quasi-molecular ion peaks at m/z 293 [M + Na] +, 309 [M + K] +, and the negative ESI-MS showed quasi-molecular ion peaks at m/z 269 [M - H] -, 305 [M + Cl] -, and the HRFAB-MS showed an [M - H] - ion peak at m/z (calcd for ), indicating the molecular formula to be C 15 H 26 O 4. The 13 C and 1 H-NMR spectra revealed two groups of signals, the stronger one corresponded to M-16, and the weaker one corresponded to M-15. Because of the same molecular formula, the two metabolites should be isomers of each other. 16

17 The 1 H-NMR and 13 C-NMR spectra of M-15 were similar to those of 10α,14-dihydroxy curcumol (Zhang H. et al., 2007) which was a microbial transformation product of curcumol, except that the signal of C-14 had shifted upfield from δ 70.9 to δ 67.5, and the signal of C-3 (δ 35.5) and C-6(δ 41.1) had shifted to downfield ( δ 4~5 ppm), and the signal of C-1 (δ 53.3) and C-9 (δ 40.7) had shifted upfield ( δ -1 ppm), indicating that M-15 might be a diastereomer of 10α,14-dihydroxy curcumol. In the NOESY spectrum, the correlation of H-14 (δ 3.30, 2H, br.s) and H-1α (δ 1.90, 1H, m) indicated the α-orientation of CH Therefore, M-15 was identified as 10β,14-dihydroxy curcumol. Comparison of the NMR data of 16 and 15 revealed that the hemiketal substructure in 15 was cleaved in 16 to form a carbonyl carbon (C-8) and a hydroxy group at C-5(5-OH). The HMBC correlations of H-4 (δ 1.80, 1H, m) with C-3 (δ 30.2), C-5 (δ 84.1), and C-10 (δ 75.9), H-14 (δ 3.30, 1H, d, J = 10.7 Hz; 3.41, 1H, d, J = 10.7 Hz) with C-1 (δ 54.8), C-9 (δ 52.7), and C-10, together with H-9 (δ 2.52, 1H, d, J = 11.3 Hz; 3.10, 1H, d, J = 11.3 Hz) with C-1, C-7 (δ 57.1), C-8 (δ 216.4), C-10 and C-14 (δ 69.9) further confirmed the planar structure of M-16. In the NOESY spectrum, the correlation between H-14 and H-1α (δ 2.05, 1H, dd, J = 10.6, 9.3 Hz) indicated the α-orientation of CH The correlation between H-6α (δ 1.53, 1H, d, J = 14.6 Hz) and H-1α suggested that OH-5 must have a β-orientation. From the above evidence, the structure of M-16 was identified as 5β,10β,14-trihydroxy-(1αH,7βH)-guai-8-one. Discussion This is the first report of the in vivo urinary metabolites of guaiane-type sesquiterpenes. Sixteen phase 1 metabolites of curcumol were obtained and identified by ESI-MS, HRFAB-MS and NMR spectroscopy including 1 H-NMR, 13 C-NMR, HMQC, HMBC, NOESY. 17

18 Based on the identified metabolites, we found that hydroxylation, epoxidation, alcohol oxidation, dehydration, condensation and epoxy-ring opening were the main phase 1 metabolic pathways of curcumol in rats. It could be presumed that C-2 or C-3 or C-15 in curcumol is hydroxylated in vivo following the biocatalysis of hydroxylase (M-1, M-2, M-11) and 3-OH was further oxidized to form a carbonyl (M-5~13). In addition, the carbon-carbon double bond at C-10 (14) could easily undergo hydration (M-12) followed by dehydration to generate a new double bond at C-9 (10) (M-2~4) or undergo epoxidation followed by hydration (M-5, M-6, M-11, M-15 and M-16). The formation of a cyclic ether in M-2~4 might be due to dehydration between 8-OH and 12-OH or 13-OH after the hydroxylation of C-12 or C-13. The hemiketal structure of curcumol could be transformed into a hydroxyl at C-5 (M-14) and a carbonyl at C-8 (M-16), and then dehydrated to form a double bond at C-4 (5) (M-5~13), and the carbonyl at C-8 could be easily condensated with 12-OH (or 13-OH) after hydroxylation of C-12 (or C-13), followed by cyclization between C-8 and C-14 to form two substituted tetrahydrofuran rings (M-7~10, M-14). CH 3-12 (or 13 ) could be oxidized to form a carboxylic group (M-12, M-13). The possible pathways for the formation of all the metabolites are shown in Fig. 2. In this study we identified ten metabolites with newly formed tetrahydrofuran rings, which may not be derived from an enzyme-mediated metabolic reaction. The 8-ketone carbonyl group, formed after decomposition of the hemiketal substructure in curcumol, is easily condensated with the 12 or 13 -hydroxy group to form a five-member ring, which is relatively stable in aqueous solution ( Bruyn et al., 1975; Funcke et al., 1979; Dais et al., 1985), and the newly formed hemiketal hydroxy group at C-8 can be further cyclized with the 14- hydroxy group to form another five-member ring. The structural elucidation of metabolites is important in drug metabolism studies. In recent years, 18

19 comparisons of ESI-MS n data and retention times in HPLC with synthetic standards are usually used to identify the structures of metabolites. However, some structures of the metabolites deduced only from LC/MS n data might not be correct, especially in the case of the existence of isomeric metabolites (He et al., 2003; Cui et al., 2004; Cui et al., 2005; Xia et al., 2007; Zeng et al., 2007). In our study, three groups of epimers (M-3 and M-4; M-5 and M-6; M-7, M-8, M-9 and M-10) were obtained and they had similar chromatographic behaviors and identical LC/MS n data. In these cases, preparation of metabolites and further identification based on NMR data is necessary. Of course, the direct isolation of the metabolites from urine, bile or feces of humans or animals is difficult, but it is the most reliable method for the identification of metabolites. In general, NOESY spectrum could be used only for determination of the relative stereochemistry. However, the absolute stereostructure of the parent compound curcumol has been characterized and some positions, such as C-1, C-4 and C-7 in curcumol, remain unchanged in all the metabolites. As a result, the absolute configurations of the newly formed chiral centers in the metabolites were consequently established by means of the stepwise correlations with H-1, H-4 or H-7 in the NOESY spectrum. In summary, we have determined the definitive structures of sixteen phase 1 metabolites of curcumol by mass spectra and NMR spectroscopy analysis. All of them were characterized as new compounds, among which, M-2~M-4, M-7~M-10, and M-14 are the first examples of guaiane-type sesquiterpenoid ketals with two substituted tetrahydrofuran rings. Baesd on the amounts of the obtained metabolites, the total recovery was calculated to be more than 25%, especially M-2, M-4, M-5, M-7, M-8 and M-9 seems to be the major metabolites. These results are important for the understanding of curcumol metabolism in rats and provide useful information and acts as a reference for further metabolic investigations of curcumol in humans. 19

20 Identification of the Metabolites 2α-hydroxycurcumol (M-1). Colorless powder (methanol). IR ν max (KBr): 3433, 3073, 2968, 1646, 1460, 1367, 1333, 1281, 1131, 1053, 989, 893, 799, 613 cm -1. ESI-MS m/z: 275 [M + Na] +. HRFAB-MS m/z [M + H] + (calcd for C 15 H 25 O 3, ). 1 H NMR (400 MHz, CDCl 3 ) and 13 C-NMR (100 MHz, CDCl 3 ) see Table 1 and Table 5, respectively. (11αH)-3α-hydroxy-9-en-8,13-epoxycurcumol (M-2). Colorless powder (methanol). IR ν max (KBr): 3414, 2958, 2872, 1663, 1457, 1215, 1125, 1055, 968, 861, 570 cm -1. ESI-MS m/z: 273 [M + Na] +. HRFABMS m/z [M + H] + (calcd for C 15 H 23 O 3, ). 1 H NMR (400 MHz, CDCl 3 ) and 13 C-NMR (100 MHz, CDCl 3 ) see Table 1 and Table 5, respectively. (11αH)-14-hydroxy-9-en-8,13-epoxycurcumol (M-3) Colorless oil (methanol). IR ν max (KBr): , 2898, 1663, 1461, 1227, 1139, 1058, 973, 858, 603 cm -1. ESI-MS m/z: 273 [M + Na] +. HRFAB-MS m/z [M + H] + (calcd for C 15 H 23 O 3, ). 1 H NMR (400 MHz, CDCl 3 ) and 13 C-NMR (100 MHz, CDCl 3 ) see Table 1 and Table 5, respectively. (11βH)--14-hydroxy-9-en-8,12-epoxycurcumol (M-4). Colorless crystal (methanol), IR ν max (KBr): 3436, 2967, 2875, 1659, 1459, 1378, 1303, 1217, 1150, 1063, 982, 840 cm -1. ESI-MS m/z: 273 [M + Na] +. HRFAB-MS m/z [M + H] + (calcd for C 15 H 23 O 3, ). 1 H NMR (400 MHz, CDCl 3 ) and 13 C-NMR (100 MHz, CDCl 3 ) see Table 1 and Table 5, respectively. 10α,14-dihydroxy-(1αH,7βH)-guai-4-en-3,8-dione (M-5). Colorless powder (methanol), IR ν max (KBr): 3414, 2959, 1693, 1642, 1386, 1346, 1184, 1051, 918, 595 cm -1. ESI-MS (m/z): 289 [M + Na] +. HRFAB-MS m/z [M + H] + (calcd for C 15 H 23 O 4, ). 1 H NMR (400 MHz, CDCl 3 ) and 13 C-NMR (100 MHz, CDCl 3 ) see Table 1 and Table 5, respectively. 10β,14-dihydroxy-(1αH,7βH)-guai-4-en-3,8-dione (M-6). Colorless powder (methanol), IR ν max (KBr): 3425, 2923, 1686, 1633, 1463, 1342, 1272, 1049, 954, 873, 617 cm -1. ESI-MS (m/z): 289 [M 20

21 + Na] +. HRFAB-MS m/z [M + H] + (calcd for C 15 H 23 O 4, ). 1 H NMR (400 MHz, CDCl 3 ) and 13 C-NMR (100 MHz, CDCl 3 ) see Table 2 and Table 5, respectively. 10β-hydroxy-(1αH,7βH,11αH)-guai-8(13),8(14)-diepoxy-4-en-3-one (M-7). Colorless powder (methanol), IR ν max (KBr): 3447, 2881, 1697, 1639, 1462, 1339, 1214, 1016, 987, 869, 775 cm -1. ESI-MS (m/z): 263 [M - H] -. HRFAB-MS m/z [M - H] - (calcd for C 15 H 19 O 4, ). 1 H NMR (400 MHz, CD 3 COCD 3 ) and 13 C-NMR (100 MHz, CD 3 COCD 3 ) see Table 2 and Table 5, respectively. 10β-hydroxy-(1αH,7βH,11βH)-guai-8(12),8(14)-diepoxy-4-en-3-one (M-8). Colorless powder (methanol), IR ν max (KBr): 3437, 2924, 2879, 1681, 1629, 1451, 1339, 1295, 1015, 972, 868, 774 cm -1. ESI-MS (m/z): 263 [M - H] -. HRFAB-MS m/z [M - H] - (calcd for C 15 H 19 O 4, ). 1 H NMR (400 MHz, CD 3 COCD 3 ) and 13 C-NMR (100 MHz, CD 3 COCD 3 ) see Table 2 and Table 5, respectively. 10α-hydroxy-(1αH,7βH,11αH)-guai-8(13),8(14)-diepoxy-4-en-3-one (M-9). Colorless powder (methanol), IR ν max (KBr): 3440, 2926, 1698, 1639, 1461, 1116, 1044, 1012 cm -1. ESI-MS (m/z): 263 [M - H] -. HRFAB-MS m/z [M - H] - (calcd for C 15 H 19 O 4, ). 1 H NMR (400 MHz, CD 3 COCD 3 ) and 13 C-NMR (100 MHz, CD 3 COCD 3 ) see Table 2 and Table 5, respectively. 10α-hydroxy-(1αH,7βH,11βH)-guai-8(12),8(14)-diepoxy-4-en-3-one (M-10). Colorless powder (MeOH), ESI-MS (m/z): 263 [M - H] -. HRFAB-MS m/z [M - H] - (calcd for C 15 H 19 O 4, ). 1 H-NMR (600 MHz, CDCl 3 ) and 13 C-NMR (150 MHz, CDCl 3 ) see Tables 2 and 6, respectively.. 10α,14,15-trihydroxy-(1αH,7βH)-guai-4-en-3,8-dione (M-11). Colorless oil (MeOH/H 2 O) ESI-MS (positive) m/z:305[m + Na] +, 587[2M + Na] +, 321[M + K] +, 283[M + H] +, 281[M - H] - ; HRFAB-MS m/z: [M - H] - (calcd for C 15 H 21 O 5, ); 21

22 1 H-NMR (600 MHz, C 5 D 5 N) and 13 C-NMR (150 MHz, C 5 D 5 N) see Table 3 and Table 6, respectively. 10β-hydroxy-(1αH,7βH)-guai-4-en-3,8-dioxo-13-oic acid (M-12) Colorless oil (MeOH/H 2 O), ESI-MS m/z: 279.1[M - H] - ; HRFAB-MS m/z: [M - H] - (calcd for C 15 H 19 O 5, ). 1 H-NMR (600 MHz, CDCl 3 ) and 13 C-NMR (150 MHz, CDCl 3 ) see Table 3 and Table 6, respectively. (1αH,7βH)-guai-4,10(14)-dien-3,8-dioxo -13-oic acid (M-13) Colorless oil (MeOH/H 2 O), ESI-MS m/z: 285 [M + Na] +, 301 [M + K] +, 263 [M + H] +, 261 [M - H] - ; HRFAB-MS m/z: [M - H] - (calcd for C 15 H 17 O 4, ). 1 H-NMR (600 MHz, C 5 D 5 N) and 13 C-NMR (150 MHz, C 5 D 5 N) see Table 3 and Table 6, respectively. 5β,10β-dihydroxy-(1αH,7βH,11αH)-guai-8(13),8(14)-diepoxide (M-14) Colorless oil (MeOH/H 2 O), ESI-MS m/z: 291 [M + Na] +, 559 [2M + Na] +, 267 [M - H] -. HRFAB-MS m/z: [M - H] - (calcd for C 15 H 23 O 4, ); 1 H-NMR (600 MHz, CDCl 3 ) and 13 C-NMR (150 MHz, CDCl 3 ) see Table 3 and Table 6, respectively. 10β,14-dihydroxycurcumol (M-15) 5β,10β,14-trihydroxy-(1αH,7βH)-guai-8-one (M-16) Colorless oil (MeOH/H 2 O), ESI-MS m/z: 293 [M + Na] +, 309 [M + K] +, 269 [M - H] -, 305 [M + Cl] +. HRFAB-MS m/z: [M - H] - (calcd for C 15 H 25 O 4, ). 1 H-NMR (600 MHz, CD 3 OD) and 13 C-NMR (150 MHz, CD 3 OD) see Table 4 and Table 6, respectively. Acknowledgements We wish to thank Dr. David Jack (United Kingdom) for the language check and the editorial assistance. 22

23 References de Bruyn A, Anteunis M, Verhegge G (1975) A l H-n.m r. study of D-fructose in D 2 O. Carbohyd Res 41: Cui L, Qiu F, Wang NL, Yao XS (2004) Four new andrographolide metabolites in human urine. Chem Pharm Bull 52: Cui L, Qiu F, Yao XS (2005) Isolation and identification of seven glucuronide conjugates of andrographolide in human urine. Drug Metab Dispos 33: Dais P, Perlin AS (1985) Stabilization of the β-furanose form, and kinetics of the tautomerization of D-fructose in dimethyl sulfoxide. Carbohyd Res 136: Funcke W, Sonntag CV, Triantaphylides C (1979) Detection of the open-chain forms of D-fructose and L-sorbose in aqueous solution by using 13C-n.m.r. spectroscopy.carbohyd Res 75: He XJ, Li JK, Gao H, Qiu F, Hu K, Cui XM, Yao XS (2003) Four new andrographolide metabolites in rats. Tetrahedron 59: Hikino H, Meguro K, Sakurai Y, Tsunematsu T (1965) Structure of Curcumol. Chem Pharm Bull 13: Hou XM, Wen X, Li WM, Li SJ, Tang JW, Sun ZQ, Wang FP, Fang ZC, Wu SY (2007) The trial report of curcumol, a female infertility agent to control grassland rodents. Qinghai Cao Ye 16(4): Inayama S, Gao JF, Harimaya K, Kawamata T, Iitaka Y, Guo YT (1984) The absolute stereo-structure of curcumol isolated from Curcuma wenyujin. Chem Pharm Bull 32: Jiang Y, Li ZS, Jiang FS, Deng X, Yao CS, Nie G (2005) Effects of different ingredients of zedoary on gene expression of HSC-T6 cells. World J Gastroentero 11: Li X, Wu LJ, Ji ZZ, Harigaya Y, Konda Y, Iguchi M, Takahashi H, Onda M (1988) Curcumol and its 23

24 one-step formation from curdione. J Heterocyclic Chem 25: Liu DH, Zheng Q, Li ZH, Zhao RL, Liu SQ, Zhang L, Wang ZM, Lin CG 2006 The trial of curcumol bait on the rodent control for forest protection. Nong Ye Ke Xue Yu Guan Li 27(2): Mau JL, Lai EYC, Wang NP, Chen CC, Chang CH, Chyau CC (2003) Composition and antioxidant activity of the essential oil from Curcuma zedoaria. Food Chem 82: Phan MG, Van NH, Phan TS (2000) Antimicrobial activity of sesquiterpene constituents from some Curcuma species of Vietnam. Tap Chi Hoa Hoc 38: Su CY, Liu JY, Xu HX (1980) The metabolism of 3 H-curcumol in normal rats and tumor bearing mice. Yao Xue Xue Bao 15: The State Pharmacopoeia Commission of P. R. China (2005) Pharmacopoeia of the P. R. China, Chemical Industry Press vol.1, pp 279. Xia HJ, Qiu F, Zhu S, Zhang TY, Qu GX, Yao XS (2007) Isolation and identification of ten metabolites of breviscapine in rat urine. Biol Pharm Bull 30: Xu LC, Bian KJ, Liu ZM, Zhou J, Wang GK (2005) The inhibitory effect of the curcumol on women cancer cells and synthesis of RNA. Zhong Liu 25: Zeng YC, Qiu F, Liu Y, Qu GX, Yao XS (2007) Isolation and Identification of Phase 1 Metabolites of Demethoxycurcumin in Rats. Drug Metab Dispos 35: Zhang H, Qiu F, Yao XS, QU GX (2007) Microbial transformation of curcumol by Cunninghamella blakesleana. J Asian Nat Prod Res 9: Zhang R, Wang BJ, Zhao HL, Li XL, Wei CM, Guo RC (2007) Determination of curcumol in plasma by HPLC-MS/MS method and its pharmacokinetics in Beagle dogs. Yao Xue Xue Bao 42:

25 Footnotes This work was supported by the National Natural Science Foundation of China [Grant ]; and the Liaoning Natural Science Foundation [Grant ]. The authors Yan Lou and Hui Zhang contributed this work equally. 25

26 Figure legends Fig. 1. ESI-MS spectra of M-1, M-2, M-5, M-7, M-11, M-12, M-13, M-14 and M-15 Fig. 2. Structures of curcumol metabolites in rats and possible metabolic pathways for their production. a: epoxidation; b: hydration; c: dehydration; d: hydroxylation; e: alcohol oxidation; f: epoxy-ring opening reaction; g: condensation; h: cyclization 26

27 Table 1. 1 H NMR data of metabolites 1 to 5. NO M-1 M-2 M-3 M-4 M (d, 8.5) 2.19(dd,9.5,9.6) 2.11(dd,9.1,8.3) 2.18(dd, 9.4, 8.9) 3.22(m) (m, β) 1.81(m, α) 1.55(m, β) 1.56(m, β) 2.39(dd,18.9,2.2,β) 1.98(m, β) 1.90(m, α) 1.92(m,α) 2.53(dd,18.9,6.8, α) (m, α) 1.63(m, α) 1.63(m,α) 4.14(ddd,13.0,8.2,4.8) 1.98(m, β) 1.87(m, β) 1.87(m, β) (m) 1.77(m) 1.84(m) 1.93(m) (dd,12.7,6.7,α) 1.78(m, α) 1.83(m) 2.10(dd,13.2,4.1, α) 2.57(br.d,13.0,α) 2.15(dd,12.7,12.4,β) 1.90(dd, 12.9, 7.6, β) 1.83(m) 1.67(dd,13.2,9.8, β) 2.94(dd,13.0, 4.6,β) (m) 2.12(m) 2.18(m) 2.68(dd,9.8, 4.1) 2.17(m) (br.d,14.8,β) 2.63(d,11.8, β) 5.68(s) 5.96(s) 6.00(s) 2.65(br.d,14.8,α) 3.03(d,11.8, α) (m) 2.12(m) 2.18(m) 2.40(m) 2.23(m) (d, 6.4) 1.00(d,6.1) 1.01(d,6.5) 3.79(dd,8.3,8.4, α) 4.27(dd,8.3, 8.0, β) 0.98(d,6.6) (d,6.5) 3.67(dd,8.0, 8.3, β) 3.70(dd,7.5,7.8, β) 0.99(d,7.1) 4.38(dd,8.0, 7.6, α) 4.41(dd,7.5,7.0,α) 1.02(d, 6.7) (s, β) 3.24(d 11.6,a) 1.65(br.s) 4.05(br.s) 4.06(br.s) 5.00(s, α) 3.41(d 11.6,b) (d,6.9) 1.11(d,6.9) 1.04(d,5.6) 1.05(d,6.7) 1.72(d 1.6) Recorded at 400MHz in CDCl 3. δ in ppm, J in Hz. 27

28 Table 2. 1 H NMR data of metabolites 6 to 10. NO M-6 a M-7 c M-8 c M-9 c M-10 b (br.d,6.4) 3.21(br.s) 3.22 (br.s) 3.09 (m) 3.01 (br.d, 6.5) (dd,18.3,6.8,α) 2.57 (br.d,18.3,β) 2.43 (br.s) 2.42 (br.s) 2.44 (d, 1.0) 2.43 (d, 2.1) 2.23(dd,19.2,1.7,β) 2.35(dd,19.2,6.4,α) 2.32 (m, β) 2.45 (dd, 6.5,19.5, α) (dd,13.0,12.2,α) 2.13 (dd, 12.0, 11.7,β) 2.32(dd, 11.9, 12.0,α) 2.17(dd,12.6,12.6,α) 2.80 (dd,4.8,12.0, β) 3.02(dd,13.0, 5.1,β) 2.92 (dd, 12.0, 3.3,α) 2.87 (dd, 11.9, 3.1,β) 3.04(dd,12.6, 5.6, β) 2.22 (br.d, 12.0, α) (m) 1.26 (dd, 11.7, 3.2) 1.78 (m) 1.70 (dd, 11.9, 5.6) 2.27 (m) (d, 11.9,β) 3.17(d, 11.9,α) 1.84 (d, 12.5,β) 2.13(dd,12.5, 1.5,α) 1.82 (d, 12.4,α) 2.15 (d, 12.4,2.4, β) 2.19 (d, 13.0,β) 2.57(dd,13.0,0.7,α) 2.38 (d, 13.3, β) 2.49 (d, 13.3, α) (m) 2.21 (m) 2.45 (m) 1.98 (m) 2.32 (m) (d,6.9) 1.08 (d, 6.6) 3.58 (dd, 8.3, 3.2,α) 4.07 (dd, 8.3, 7.9,β) 1.03 (dd, 6.4, 2.6) 4.03 (dd, 4.3, 8.4, β) 3.64 (br.d, 8.4, α) (d, 6.9) 3.30 (dd, 9.3, 8.1,α) 4.06 (dd, 8.1, 8.1,β) 1.12 (d, 7.3) 3.37(dd, 10.6, 7.9,α) 1.00 (d, 7.1) 3.88 (dd, 7.9, 7.0,β) (d,10.9) 3.59(d,10.9) 3.98 (d, 8.3,α) 4.11 (d, 8.3,β) 4.01 (d, 8.2,α) 4.10 (d, 8.2,β) 3.31(dd, 8.6, 0.9,β) 3.50(dd, 8.6, 1.3,α) 3.38 (d, 8.8, β) 3.57 (d, 8.8, α) (s) 1.67 (d, 2.1) 1.68 (d, 1.9) 1.66 (dd, 1.9, 0.5) 1.70 (d, 1.6) a Recorded at 400MHz in CDCl 3. b Recorded at 600MHz in CDCl 3. c Recorded at 600MHz.in (CD 3 ) 2 CO. δ in ppm, J in Hz. 28

29 Table 3. 1 H NMR data of metabolites 11 to 14. NO M-11 a M-12 b M-13 c M-14 b ( br.d, 6.6) 1.69 (m) 3.50 (m) 1.91 (m) (dd, 18.0, 6.6, α) 2.28 (m, β) 2.29(dd,13.2,12.6,α) 1.71 (m, α) 3.09(dd, 18.0, 2.2, β) 2.77 (m, α) 3.09 (br. d, 12.6, β) 1.87 (m, β) (m, β) 1.88 (m, α) (m, α) (dd, 12.0, 8.4, α) 2.71(dd,15.2,12.2, α) 3.56(dd, 12.0, 3.0, β) 2.62(dd,12.2,6.8, β) 2.38 (br.d, 18.6, α) 2.73 (dd, 18.6, 6.0, β) 1.08 (m, α) 2.17 (m, β) (dd, 8.4, 3.0) 1.66 ( 1H, o) 3.24 (m) 1.98 (m) (m) (d, 12.6, β) 3.82 (d, 12.6, α) 2.07 (1H, m, α) 2.61 (1H, d, 14.5, β) 3.29 (s, α) 3.29 (s, β) 2.20 (d, 11.2) 2.15 (d, 11.2) (m) 2.27 (1H, m) 3.39 (m) 1.90 (m) (d, 7.2) 1.04 (d, 6.5) 1.43 (d, 7.2) 0.96 (d, 6.1) (d, 7.2) 3.56 (dd, 8.0, 2.0, β) 3.97 (dd, 8.0, 6.6, α) (d, 10.8, a) 5.12 (s,b) 3.64 (dd, 8.2, 1.2, β) 1.22 (br. s) 4.00 (d, 10.8, b) 5.07 (s,a) 3.98 (br.d, 8.2, α) (d, 13.2, a) 1.88 (s) 0.93 (d, 6.6) 1.66 (s) 4.72 (d, 13.2, b) a Recorded in C 5 D 5 N. b Recorded in CDCl 3. c Recorded in C 5 D 5 N. Recorded at 600MHz. δ in ppm, J in Hz. 29

30 Table 4. 1 H NMR data of metabolites 15 and 16. NO. M-15 b M-16 b (m) 2.05(dd,10.6, 9.3) (m, α) 1.75 (m, α) 1.58 (m, β) (m, α) 1.92 (m, β) 1.36 (m, α) 1.35 (m, β) 1.85 (1H, m, β) (m) 1.80 (m) (dd,14.4,7.2, α) 1.53 (d,14.6,α) 2.24(dd, 16.2, 14.4, β) 2.23(d,14.6, β) (m) 2.39 (m) (d, 14.4, α) 3.10 (d, 11.3, α)) 1.78 (m, β) 2.52 (d, 11.3, β) (m) 1.90 (m) (d,6.0) 0.92 (d, 7.2) (d, 6.4) 0.99 (d, 6.6) (br.s) 3.30 (br.s) 3.30 (d, 10.7,a) 3.41 (d, 10.7,b) (d, 6.4) 0.91 (d, 7.2) b Recorded in CD 3 OD. Recorded at 600MHz. δ in ppm, J in Hz. 30