Five new eudesmane-type sesquiterpenoid lactones biotransformed from atractylenolide I by rat hepatic microsomes

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Ying Li, Xiu-Wei Yang ⁎ State Key Laboratory of Natural and Biomimetic Drugs (Peking University), Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University Health Science Center, Peking University, Beijing 100191, PR China a r t i c l e i n f o a b s t r a c t Article history: Received 12 December 2012 Accepted in revised form 28 December 2012 Available online 13 January 2013 The work presented here is the first study performed on the biotransformation and/or metabolism of atractylenolide I (1) as a valuable anti-inflammatory and chemopreventive agent, using liver microsomes from rats pre-treated with sodium phenobarbital. Two known eudesmane-type sesquiterpenoid lactones, namely 1β-acetoxyatractylenolide I (2) and 1β-hydroxyatractylenolide I (3), and five new ones, namely 3β-hydroxy-atractylenolide I (4), 1β,13-dihydroxyatractylenolide I (5), 1β,2α-dihydroxy-atractylenolide I (6), 1β,3α-dihydroxy-atractylenolide I (7), and 1β,3β-dihydroxy-atractylenolide I (8) were obtained. Their chemical structures were unambiguously established by both 1D and 2D NMR as well as mass spectroscopic techniques. The result indicated that the parent prototype compound 1 could be specifically oxidized at C-1, C-2 and C-3 of A-ring, suggesting that the oxidizable of 1 may contribute to its in vivo anti-inflammatory and chemopreventive effects. And the result also provided valuable information for further investigation of relationship among metabolic activation and liver microsomal cytochrome P450 enzyme isoforms. © 2013 Elsevier B.V. All rights reserved. Keywords: Atractylodes macrocephala Koidz. Compositae Atractylenolide I Sesquiterpenoids Rat hepatic microsomes Biotransformation 1. Introduction Atractylenolide I (1, chemical structure shown in Fig. 1) is a eudesmane-type sesquiterpenoid lactone and one of the main principles of the rhizomes of Atractylodes macrocephala Koidz. (family: Compositae) used in China, Korea and Japan for the treatment of splenic asthenia, anorexia, oedema, excessive perspiration and abnormal fetal movement [1,2]. The previous study has suggested that 1 exhibited in vivo and in vitro anti-inflammatory activity [3–6], protective effect against immunological liver injury [7], and dose-dependent inhibitory effect on the proliferation of cultured human tumor cell lines such as A549 (non-small-cell lung carcinoma), SK-OV-3 (ovary), SK-MEL-2 (melanoma), XF498 (central nerve system), and HCT 15 (colon) in vitro [8]. At the time, it was also reported that 1 was a potential inhibitor of aromatase [9], which is a molecular target in the treatment of estrogen receptor sensitive breast cancer. In addition, 1 also exhibited positive modulatory activities on γ-aminobutyric acid type A receptor in vivo resulting in sedation or anxiolysis [10]. In clinical trials, 1 can be beneficial for treating cancer cachexia [11]. These reports suggested that 1 may be valuable anti-inflammatory and chemopreventive agents. In new drug research and development (R & D), absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of drug are important criteria for assessing druglikeness of candidates. ADMET evaluation at the early stage of drug design and development is be helpful to improve the success rate and reduce costs, and further access to safe, effective drugs. As a part of R & D of 1, intestinal permeability and transport of 1 has been examined using the human Caco-2 (human colon adenocarcinoma cell line) cell monolayer model in our research group [12]. Absorption kinetics of 1 in intestines of rats has also been reported [13]. The result indicated that 1 Fitoterapia 85 (2013) 95–100 ⁎ Corresponding author at: No.38, Xueyuan Road, Haidian District, Beijing, 100191, PR China. Tel.: +86 10 82805106; fax: +86 10 82802724. E-mail addresses: girlinred@126.com (Y. Li), xwyang@bjmu.edu.cn (X.-W. Yang). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2012.12.033 Contents lists available at SciVerse ScienceDirect Fitoterapia journal homepage: www.elsevier.com/locate/fitote had high permeability and was shown to be well-absorbed compound [14], suggesting that 1 should be able to enter liver by bloodstream. As is well known, after drug was absorbed into bloodstream, liver is the most important metabolic and/or biotransformation process location. Thus, the study presented here was to examine the biotransformation of 1 in liver microsomes from rats pre-treated with sodium phenobarbital, a potent cytochrome P450 (CYP)-inducing agent. The biotransformation products were characterized by various methods including optical, infra-red (IR), ultra-violet (UV) and nuclear magnetic resonance (NMR) spectroscopies, and mass spectrometry (MS). 2. Experimental 2.1. General Optical rotation was measured on an Autopol III polarimeter with MeOH as solvent. IR spectra were recorded on a Nexus 470 FT–IR spectrometer as KBr disks. UV spectra were acquired on a Varian Cary 300 ultraviolet–visible spectrophotometer in methanol (MeOH) solution. Mass spectra were recorded on a Finnigan TRACE 2000 mass spectrometer (for EI– MS) and a Bruker Daltonics APEX IV Fourier Transform ICR high-resolution mass spectrometer (for HR–ESI–MS). 1D and 2D NMR spectra were run on a Bruker AV 400 spectrometer (400 MHz for 1H NMR and 100 MHz for 13C NMR) with TMS as internal standard. A mode HZS–H thermostatic water bath oscillator was used for biosample incubation. Open column chromatography (CC) separation was carried out using silica gel (200–300 mesh; Qingdao Marine Chemical Co., Qingdao, China). Thin layer chromatography (TLC) was conducted on silica gel GF254 plates (Merck, Darmstadt, Germany). Glucose-6-phosphate (G-6-P), glucose-6-phosphate dehydrogenase (G-6-PDH), reduced nicotinamide adenine dinucleotide (NADH) and β-nicotinamide adenine dinucleotide phosphate (NADP) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Sodium phenobarbital (PB) was purchased from Peking University Third Hospital (Beijing, China). Deionized water (H2O) was purchased from Wahaha Co., Ltd. (Hangzhou, China). Milli-Q grade H2O was used for assay. Other reagents were of analytical grade and purchased from Beijing Fine Chemicals (Beijing, China). Reference compound 1 was isolated and purified from the rhizomes of A. macrocephala by our research group and was identified on the basis of spectral data, including optical rotation, IR, UV, NMR, and MS. The purity of 1 was over 99% by HPLC–UV analysis. 2.2. Preparation of rat liver microsomes Adult male Sprague–Dawley (SD) rats weighing 200–220 g were obtained from Department of Laboratory Animal Science, Peking University Health Science Center (Peking University, China). The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of the Peking University (SYXK [Jing] 2006–0025). The rats were maintained in a 12 h light/12 h dark cycle, a temperature-controlled environment (22±1 °C) and allowed free access to feedstuff and water until experiment. In order to induce CYP enzymes, PB was given intraperitoneally in physiological saline at a single dose of 60 mg/kg daily for 3 days. The rats were sacrificed on the 4th day after fasting for 12 h. The livers were removed as quickly as possible and the blood residue was rinsed with 4 °C physiological saline, and then cut into small pieces and thoroughly homogenized in three-volumes of 4 °C phosphate buffer solution (PBS) (50 mM, pH 7.4) using an F6/10 superfine homogenizer (Fluko equipment, Shanghai, China). The homogenate was centrifuged at 15,000 g for 20 min by a GL-20B centrifuge (Anke equipment, Shanghai, China) and the supernatant was then transferred into an LB-80M ultracentrifuge (Beckman instrument, CA, USA) to get ultracentrifuged at 105,000 g for 60 min. The precipitation (liver microsomes) was collected and resuspended in 4 °C PBS (50 mM, containing 20% glycerol, pH 7.4). The entire process of preparation was carried out under ice bath and the microsomal suspension was immediately stored at −80 °C until used. The content of the microsomal protein was determined by the method of Lowry–Folin [15]. CYP concentrations were determined according to the method developed by Omura and Sato [16]. The microsome suspension was found to contain 28.8 mg protein per mL and 1.85 nmol CYP per mg protein. O O H 1 O O H 2 OAc O O CH3 H 3 O O H 4 HO O O CH3 H CH2 5 HO HO O O CH3 H 6 HO HO O O CH3 H 7 HO HO O O CH3 H 8 HO HO A B C 1 2 3 4 5 6 8 7 9 10 11 12 13 14 15 HO H H H H H H H H H Fig 1. Structures of 1–8 and key correlations in HMBC spectra of 3–8. 96 Y. Li, X.-W. Yang / Fitoterapia 85 (2013) 95–100 2.3. Biotransformation of AI by rat liver microsomes The biotransformation incubation system was made up by 2.5 g microsomal proteins and 500 mL PBS (100 mM, pH 7.4) and an NADPH generating system (0.5 mM NADH, 1.0 mM NADP, 10 mM G-6-P, 1.0 IU/mL G-6-PDH and 4.0 mM MgCl2). After preincubation at 37 °C for 5 min, 400 mg of 1 dissolved in 5 mL of acetone was added into the incubation system as substrate (the final concentration of acetone no more than 1.0%). Incubation was carried out at 37 °C for 80 min with continuous shaking (100 rpm) in a Dubnoff incubator, and a mixed gas of O2/CO2 (95:5) was supplied for 90 s per 20 min. Reactions were terminated by adding ice-cold ethyl acetate (EtOAc; 500 mL), followed by centrifugation at 15,000 g for 10 min at 4 °C. The supernatant was extracted with EtOAc (500 mL) for six times. The combined EtOAc phases were concentrated under reduced pressure at 38 °C to afford a residue (2.4 g). 2.4. Extraction and isolation of biotransformation products of 1 The above-mentioned residue (2.4 g) was chromatographed over silica gel and eluted with petroleum (PE)–EtOAc mixtures of increasing polarities (15:1→5:1→5:2→2:1→3:2→1:1→ 1:2; v/v) to obtain 43 fractions. Fractions 3–5, eluted with PE– EtOAc (15:1), were combined and evaporated in vaccum to afford parent prototype compound 1. Fractions 8 and 16, eluted with PE–EtOAc (5:1), were evaporated in vaccum to obtain biotransformation products 2 (8 mg) and 3 (53 mg). Fraction 22, eluted with PE–EtOAc (2:1), was evaporated in vaccum to give biotransformation product 4 (0.8 mg). Fractions 26–30, eluted with PE–EtOAc (3:2), were combined and further separated by silica gel CC eluted with CHCl3–MeOH mixtures of increasing polarities (80:1→60:1; v/v) to afford biotransformation product 5 (1.6 mg). Fractions 35 and 36, eluted with PE–EtOAc (1:1), were combined and purified according to same manner with 5 to afford 6 (2.3 mg). Fractions 39–43, eluted with PE–EtOAc (1:2), were combined and further purified by silica gel CC eluted with CHCl3–MeOH mixtures of increasing polarities (100:1→100:1.2→100:1.5→100:2→ 100:3; v/v) to give 7 (5 mg) and 8 (2 mg). 1β-Acetoxyatractylenolide I [1β-Acetoxy-eudesma-4(15), 7(11),8(9)-trien-8,12-olide; 2): White powder; [α]D 20+4.6 (c 0.65, MeOH); UV λmax (MeOH) nm (log ε): 274 (0.49); IR (KBr) ν max 3437, 2933, 2851, 1769, 1737, 1652, 1376, 1250, 1062, 1016, 973, 914, 894, 835, 753 cm−1; EI–MS m/z 288 [M]+, 246, 228, 213, 185, 105, 91, 57; 1H NMR (400 MHz, CDCl3), see Table 1; 13C NMR (100 MHz, CDCl3), see Table 2. 1β-Hydroxy-atractylenolide I [1β-Hydroxy-eudesma-4(15), 7(11),8(9)-trien-8,12-olide; 3): White powder; [α]D 20+11.0 (c 1.25, MeOH); UV λmax (MeOH) nm (log ε): 276 (1.17); IR (KBr) ν max 3480, 2939, 2871, 1770, 1749, 1665, 1650, 1443, 1381, 1317, 1278, 1230, 1117, 1092, 1067, 1022, 967, 890, 858, 757, 730 cm−1; EI–MS m/z 246 [M]+, 228, 213, 200, 185, 162, 91, 77, 53; 1H NMR (400 MHz, CDCl3), see Table 1; 13C NMR (100 MHz, CDCl3), see Table 2. 3β-Hydroxy-atractylenolide I (3β-Hydroxy-eudesma-4(15), 7(11),8(9)-trien-8,12-olide; 4): White powder; [α]D 20+1.3 (c 0.3, MeOH); UV λmax (MeOH) nm (log ε): 276 (0.89); IR (KBr) ν max 3445, 2934, 2857, 1767, 1652, 1450, 1377, 1240, 1109, 1067, 1027, 903, 760 cm−1; EI–MS m/z 246 [M]+, 228, 213, 199, 185, 174, 162, 157, 142, 129, 115, 105, 91, 77, 53; HR–ESI– MS m/z 247.13266 [M+H]+ (calcd for C15H19O3, 247.13287); 1H NMR (400 MHz, CDCl3), see Table 1; 13C NMR (100 MHz, CDCl3), see Table 2. 1β,13-Dihydroxy-atractylenolide I (1β,13-Dihydroxyeudesma-4(15),7(11),8(9)-trien-8,12-olide; 5): White powder; Table 1 1H NMR spectral data for 1–8 (δppm; J/Hz). No. 1a 2a 3a 4a 5b 6b 7b 8b 1α 1.65, 1H, td (13.3, 4.3) 4.80, 1H, dd (11.4, 4.8) 3.65, 1H, dd (11.6, 4.9) 1.73, 1H, td (13.2, 3.5) 3.58, 1H, dd (11.6, 4.8) 3.27, 1H, d (8.8) 3.95, 1H, dd (11.8, 4.6) 3.63 (1H, dd, 11.7, 4.8) 1β 1.67, 1H, dt (13.3, 3.6) 1.77, 1H, ddd (13.2, 5.1, 2.7) 2α 1.74, 1H, m 2.03, 1H, m 1.94, 1H, m 2.10, 1H, qd (13.2, 5.2) 1.89, 1H, m 2.06, 1H, ddd (13.8, 4.6, 2.6) 2.23, 1H, ddd (16.2, 4.8, 4.8) 2β 1.70, 1H, m 1.62, 1H, qd (12.6, 5.1) 1.64, 1H, qd (12.8, 5.1) 1.60, 1H, qd (11.2, 5.2) 1.63, 1H, qd (11.2, 4.9) 3.60, 1H, ddd (12.4, 8.8, 5.6) 1.80, 1H, ddd (13.8, 11.8, 4.8) 1.60, 1H, ddd (16.2, 11.8, 11.7) 3α 2.08, 1H, td (13.7, 5.3) 2.21, 1H, td (13.8, 5.1) 2.16, 1H, td (13.8, 5.1) 4.10, 1H, dd (11.2, 5.6) 2.19, 1H, td (13.8, 5.1) 2.14, 1H, dd (13.2, 12.4) 4.09, 1H, dd (11.8, 4.8) 3β 2.38, 1H, ddd (13.7, 5.3, 3.0) 2.40, 1H, ddd (14.3, 5.1, 2.0) 2.39, 1H, ddd (13.8, 5.1, 1.8 2.38, 1H, ddd (13.8, 5.1, 2.1) 2.66, 1H, dd (13.2, 5.6) 4.35, 1H, dd (4.8, 2.6) 5α 2.40, 1H, dd (13.6, 3.9) 2.36, 1H, dd (13.2, 4.1) 2.27, 1H, dd (12.8, 4.2) 2.30, 1H, dd (13.0, 4.2) 2.34, 1H, dd (13.5, 4.0) 2.39, 1H, dd (13.2, 4.0) 2.83, 1H, dd (13.1, 4.0) 2.22, 1H, dd (13.2, 4.2) 6α 2.71, 1H, dd (16.8, 3.9) 2.75, 1H, dd (16.9, 4.1) 2.74, 1H, dd (17.0, 4.2) 2.72, 1H, dd (17.0, 4.2) 3.04, 1H, dd (17.2, 4.0) 2.85, 1H, dd (17.0, 4.0) 2.79 (1H, dd (17.6, 4.0) 2.82, 1H, dd (17.4, 4.2) 6β 2.55, 1H, ddd (16.8, 13.6, 1.6) 2.59, 1H, ddd (16.9, 13.2, 1.2) 2.60, 1H, ddd (17.0, 12.8, 1.2) 2.62, 1H, ddd (17.0, 13.0, 1.2) 2.77, 1H, ddd (17.2, 13.5, 1.2) 2.69, 1H, ddd (17.0, 13.2, 1.6) 2.73, 1H, ddd (17.6, 13.1, 1.2) 2.76, 1H, ddd (17.4, 13.2, 1.2) 9 5.63, 1H, s 5.72, 1H, s 6.18 1H, s 5.65, 1H, s 6.23, 1H, s 6.14, 1H, s 6.12, 1H, s 6.10, 1H, s 13 1.93, 3H, d (1.6) 1.91, 3H, d (1.2) 1.91, 3H, d (1.2) 1.94, 3H, d (1.2) 4.37, 2H, (1.2) 1.90, 3H, d (1.6) 1.91, 3H, d (1.2) 1.91, 3H, d (1.2) 14 0.96, 3H, s 1.00, 3H, s 0.92, 3H, s 0.96, 3H, s 0.91, 3H, s 0.91, 3H, s 0.89, 3H, s 0.90, 3H, s 15a 4.65, 1H, d (1.2) 4.73, 1H, br s 4.71, 1H, br s 4.85, 1H, br s 4.74, 1H, br s 4.84, 1H, br s 4.90, 1H, br s 4.92, 1H, br s 15b 4.94, 1H, d (1.2) 4.99, 1H, br s 4.97, 1H, br s 5.30, 1H, br s 4.96, 1H, br s 5.05, 1H, br s 5.19, 1H, br s 5.33, 1H, br s Acc 2.12, 3H, s a Determined in CDCl3. b Determined in CD3OD. c Acetoxyl group. Y. Li, X.-W. Yang / Fitoterapia 85 (2013) 95–100 97 [α]D 20+2.0 (c 0.75, MeOH); UV λmax (MeOH) nm (log ε): 223 (0.22), 276 (0.49); IR (KBr) νmax 3423, 2931, 2867, 1765, 1650, 1447, 1382,1256, 1142,1067,1018, 938,900 cm−1; HR–ESI–MS m/z 263.12803 [M+H]+ (calcd for C15H19O4, 263.12779); 1H NMR (400 MHz, CD3OD), see Table 1; 13C NMR (100 MHz, CD3OD), see Table 2. 1β,2α-Dihydroxy-atractylenolide I (1β,2α-Dihydroxyeudesma-4(15),7(11),8(9)-trien-8,12-olide; 6): White powder; [α]D 20+2.9 (c 1.5, MeOH); UV λmax (MeOH) nm (log ε): 276 (0.78); IR (KBr) νmax 3700, 2974, 2933, 2884, 1762, 1732 cm−1; HR–ESI–MS m/z 263.12751 [M+H]+ (calcd for C15H19O4, 263.12779); 1H NMR (400 MHz, CD3OD), see Table 1; 13C NMR (100 MHz, CD3OD), see Table 2. 1β,3α-Dihydroxy-atractylenolide I (1β,3α-Dihydroxyeudesma-4(15),7(11),8(9)-trien-8,12-olide; 7): White powder; [α]D 20+6.2 (c 1.0, MeOH); UV λmax (MeOH) nm (log ε): 276 (1.14); IR (KBr) νmax 3448, 2927, 1767, 1652, 1437, 1321, 1221, 1118, 1066, 1018, 967, 911, 760 cm−1; EI–MS m/z 262 [M]+, 244, 226, 216, 201, 187, 173, 162, 145, 134, 115, 105, 91, 77, 65, 53; HR–ESI–MS m/z 263.12769 [M+H]+ (calcd for C15H19O4, 263.12779); 1H NMR (400 MHz, CD3OD), see Table 1; 13C NMR (100 MHz, CD3OD), see Table 2. 1β,3β-Dihydroxy-atractylenolide I (1β,3β-Dihydroxyeudesma-4(15),7(11),8(9)-trien-8,12-olide; 8): White powder; [α]D 20+5.92 (c 1.25, MeOH); UV λmax (MeOH) nm (log ε): 276 (0.71); IR (KBr) νmax 3452, 2927, 2857, 1766, 1651, 1444, 1105, 1059, 1018, 903, 761, 736 cm−1; EI–MS m/z 262 [M]+, 244, 226, 216, 211, 201, 187, 173, 162, 145, 134, 119, 105, 91, 77, 69, 55; HR–ESI–MS m/z 263.12758 [M+H]+ (calcd for C15H19O4, 263.12779); 1H NMR (400 MHz, CD3OD), see Table 1; 13C NMR (100 MHz, CD3OD), see Table 2. 3. Results and discussion Microsomal preparation of male SD rat liver was used to examine 1 biotransformation. After the experimental procedure (microsomes and NADPH, reaction times and concentrations of 1) was optimized in accordance with previously reported method [17,18], EtOAc extract of the biotransformation products of 1 was subjected to silica gel CC to give seven biotransformation products (2–8) and the parent prototype compound 1. Their structures were established on the basis of optical rotation, UV, IR, NMR (including 2D NMR) and MS data, and by comparison with authentic compounds and literature data as shown in Fig. 1. The products 2 and 3 were identified as known compounds 1β-acetoxyatractylenolide I [19] and 1β-hydroxy-atractylenolide I [20],respectively, whereasthe 13C NMR spectroscopic data of 2 had not been reported before. The products 3 – 8 were new compounds. The HR–ESI–MS of 4 indicated the molecular formula C15H18O3, m/z 247.13266 [M+H]+ (calcd for C15H19O3, 247.13287). An extra oxygen atom added to the molecular in comparison with the parent prototype compound 1. Peaks at m/z 246 [M]+ and 228 [M−H2O]+ in the EI–MS suggested the presence of a hydroxyl group. The 1H NMR spectrum of 4 showed a double doublets one proton signal at δ 4.10 (J=11.2, 5.6 Hz) (Table 1), whereas it was lack in 1. In the 13C NMR spectra (Table 2), the signal for C-3, which occurred at δ 36.2 in 1, was replaced by a signal at δ 72.7 in 4, indicated that C-3 was substituted by a hydroxyl group in 4. Complete unambiguous assignments for the 1H and 13C NMR signals were made by combination of COSY, HSQC and HMBC spectra. The H-3 was α-oriented based on the NOESY correlation between H-3 and H-5α [δH 2.30 (1H, dd, J=13.0, 4.2 Hz)] (Fig. 2). Thus, 4 was determined to be 3β-hydroxy-eudesma-4(15),7(11),8(9)-trien- 8,12-olide, namely 3β-hydroxy-atractylenolide I. The molecular formula of 5 was established as C15H18O4 by its HR–ESI–MS, m/z 263.12803 [M+H]+ (calcd for C15H19O4, 263.12779), which was two oxygen atom more than 1 and was an oxygen atom more than 3. The 1H NMR spectrum of 5 was similar to that of 3, which occurred at δH 1.91 (3H, d, J=1.2 Hz, H3-13) in 3, was replaced by a signal at δH 4.37 (2H, d, J=1.2 Hz, −CH2OH) in 5, indicated a hydroxymethyl group situated at C-11. This conclusion was further supported by the correlations between H-13 (δH 4.37) and C-7 (δC 152.1), C-12 (δC 171.7) in the HMBC experiment Table 2 13C NMR spectral data for 1–8 (δppm). No. 1a 2a 3a 4a 5b 6b 7b 8b 1 39.1 t 76.6 d 74.6 d 37.0 t 75.2 d 80.3 d 69.6 d 73.4 d 2 22.7 t 27.5 t 31.4 t 32.4 t 32.2 t 72.2 d 37.6 t 41.4 t 3 36.2 t 33.2 t 33.6 t 72.7 d 34.7 t 43.7 t 72.1 d 70.6 d 4 148.3 s 145.5 s 146.1 s 150.1 s 148.1 s 146.2 s 148.5 s 150.8 s 5 48.4 d 46.1 d 46.0 d 46.5 d 47.3 d 47.4 d 40.4 d 44.4 d 6 23.0 t 22.0 t 22.2 t 22.6 t 23.4 t 23.0 t 21.4 t 22.9 t 7 148.1 s 149.0 s 147.7 s 147.7 s 152.1 s 150.2 s 148.3 s 150.2 s 8 148.0 s 146.9 s 148.5 s 148.2 s 150.0 s 149.5 s 148.8 s 149.8 s 9 119.2 d 113.7 d 115.7 d 118.0 d 119.3 d 117.0 d 115.5 d 116.7 d 10 38.1 s 41.3 s 42.9 s 37.8 s 44.0 s 43.4 s 42.9 s 43.9 s 11 120.5 s 121.2 s 120.5 s 120.9 s 124.1 s 121.7 s 120.2 s 121.6 s 12 171.4 s 171.0 s 171.6 s 171.3 s 171.7 s 173.3 s 171.8 s 173.3 s 13 8.5 q 8.5 q 8.5 q 8.5 q 54.8 t 8.3 q 8.6 q 8.2 q 14 18.6 q 14.2 q 12.8 q 18.6 q 13.0 q 14.4 q 10.9 q 13.1 q 15 107.5 t 109.2 t 108.7 t 104.7 t 108.9 t 110.4 t 110.3 t 106.0 t Acc 170.5 s Acc 21.2 q C-multiplicities were established by a HSQC experiment. s: C; d: CH; t: CH2, q: CH3. a Determined in CDCl3. b Determined in CD3OD. c Acetoxyl group. 98 Y. Li, X.-W. Yang / Fitoterapia 85 (2013) 95–100 (Fig. 1). Accordingly, 5 was determined to be 1β,13-dihydroxyeudesma-4(15),7(11),8(9)-trien-8,12-olide and was given the trivial name 1β,13-dihydroxy-atractylenolide I. The product 6 had the same molecular formula (C15H18O4) as 5, which was established by HR–ESI–MS at m/z 263.12751 [M+H]+ (calcd for C15H19O4, 263.12779). The 1H NMR spectrum (Table 1) of 6 exhibited signals for two methyl groups and one terminal double bond, as in 3. In the HMBC experiment (Fig. 1) of 6, Me-14 (δH 0.91) showed 2J correlation to C-10 (δC 43.4) and 3J correlations to C-9 (δC 117.0), C-5 (δC 47.4), and C-1 (δC 80.3), indicated a hydroxyl group situated at C-1. Furthermore the carbon signal at δC 80.3 correlated with proton signal at δH 3.27 (1H, d, J = 8.8 Hz, H-1) in the HSQC experiment and H-1 showed 2J correlation to C-10, C-2 (δC 72.2) as well as 3J correlations to C-9, C-5, C-3 (δC 43.7) in the HMBC experiment, indicated another a hydroxyl group situated at C-2. This conclusion was also supported by the correlation between H-1 and H-2 [δH 3.60 (1H, ddd, J=12.4, 8.8, 5.6 Hz)] in the 1H–1H COSY experiment. In the NOESY spectrum of 6, the observation of the correlations between H-1/H-5α [δH 2.39 (1H, dd, J = 13.2, 4.0 Hz)] and H-2/Me-14β revealed that H-1 was α-oriented and H-2 was β-oriented, as well as consequently the hydroxyl groups at C-1 and C-2 were β-oriented and α-oriented, respectively. The above data evidenced the structure of 6 to be 1β,2α-dihydroxy-eudesma- 4(15),7(11),8(9)-trien-8,12-olide, trivially named 1β,2α- dihydroxy-atractylenolide I. The product 7 had the same molecular formula (C15H18O4) as 6, which was established by HR–ESI–MS at m/z 263.12769 [M+H]+ (calcd for C15H19O4, 263.12779). Peaks at m/z 262 [M]+, 244 [M - H2O]+ and 226 [M - 2H2O]+ in the EI–MS suggested the presence of two hydroxyl groups. HSQC analysis allowed the assignment of all protonated carbons observed in the 13C NMR spectrum. A hydroxyl group was located at C-1 based on the HMBC correlations from Me-14 (δH 0.89) to C-1 (δC 69.6), C-9 (δC 115.5), and C-10 (δC 42.9), as in 3. Another a hydroxyl group was located at C-3 by the HMBC experiment which displayed the cross peaks between a terminal double bond proton signals at δH 4.90 (1H, br s, Ha-15), 5.19 (1H, br s, Hb-15) and the tertiary carbon (δC 72.1, C-3), as in 4. Because of displayed NOE correlations between H-3 [δH 4.35 (1H, dd, J=4.8, 2.6 Hz)] and Me-14β (δH 0.89), as well as H-1 [δH 3.95 (1H, dd, J=11.8, 4.6 Hz)] and H-5α [δH 2.83 (1H, dd, J=13.1, 4.0 Hz)], H-3 should be β-orientated and H-1 should be α-orientated. Therefore, the structure of 7 was determined to be 1β,3α- dihydroxy-eudesma-4(15),7(11),8(9)-trien-8,12-olide, trivially named 1β,3α-dihydroxy-atractylenolide I. The molecular formula of 8 was determined to be C15H18O4 on the basis of HR–ESI–MS at m/z 263.12758 [M+H]+ (calcd for C15H19O4, 263.12779). Peaks at m/z 262 [M]+, 244 [M−H2O]+ and 226 [M−2H2O]+ in the EI–MS suggested the presence of two hydroxyl groups. The 1H NMR spectrum (Table 1) of 8 was similar to that of 7, the differences consisting mainly in the chemical shifts of protons in A-rings. The NOE experiments shown in Fig. 2, indicated that 7 and 8 were C-3 epimers. Finally, the structure of 8 was unambiguously confirmed to be 1β,3β- dihydroxy-eudesma-4(15),7(11),8(9)-trien-8,12-olide, trivially named 1β,3β-dihydroxy-atractylenolide I. Although the tissue distribution and pharmacokinetics of 1 in rats after the oral administration has been reported [21,22], its biotransformation involving in liver microsomes has not been characterized. The present study obtained seven biotransformation products of 1 by rat liver microsomes model in vitro for the first time and it was proved that 1 could be specifically oxidized at the C-1, C-2 and C-3 of A-ring. Previous studies showed that 1 exhibited anti-inflammatory and chemopreventive effects. Reactive oxygen species (ROS) react with biological target molecules and destroy the structure of cells and eventually cause free radical-induced disease such as inflammation and cancer [23–25], suggesting that the anti-inflammatory and chemopreventive properties of 1 may be mediated by its oxidizable to accept ROS, namely 1 may react preponderantly with ROS to prevent and/or attenuate further oxidative damage caused by ROS to biological target molecules. In addition, the metabolism and/or biotransformation is one of the most important factors that can affect an overall pharmacokinetics profile of drug [26]. Because the CYP enzyme isoforms are involved in a significant number of events associated with drug metabolism and/or biotransformation [27], also, further studies are needed to clarify the relationship among metabolic activation and liver microsomal CYP enzyme isoforms. Acknowledgments This research was partly supported by the National Key Technology R & D Program of China (2011BAI07B08) and the O O H 3 O O H 4 HO O O H 5 OH O O CH3 H 6 OH HO O O CH3 H 7 OH HO O O CH3 H 8 OH HO OH H H CH 3 CH3 CH3 OH H H H H H H H H H H H H H H H H H H H Fig. 2. Key NOESY correlations of 3–8. Y. Li, X.-W. Yang / Fitoterapia 85 (2013) 95–100 99 Program of Improving the Standard of Pharmacopoeia of the People's Republic of China (2015). References [1] Liu YQ, Cai Q. HPLC determination of atractylenolide I and atractylenolide III in 50 batches crude drugs and slices of Atractylodes macrocephala Koidz. from different sources. Chin J Pharm Anal 2012;32:1249–52. [2] Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China, volume I. Beijing: China Medical Science and Technology Press; 2010. p. 95–6. [3] Sin KS, Kim HP, Lee WC, Pachaly P. Pharmacological activities of the constituents of Atractylodes rhizomes. Arch Pharm Res 1989;12:236–8. 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