PPARγ Agonist from Chromolaena odorata - ACS Publications

Article pubs.acs.org/jnp

PPARγ Agonist from Chromolaena odorata Man-Li Zhang,*,† Dianne Irwin,‡ Xiao-Ning Li,§ Françoise Sauriol,⊥ Xiao-Wei Shi,† Yu-Fang Wang,† Chang-Hong Huo,† Li-Geng Li,† Yu-Cheng Gu,‡ and Qing-Wen Shi*,† †

School of Pharmaceutical Sciences, Hebei Medical University, 361 Zhongshan East Road, Shijiazhuang 050017, People's Republic of China ‡ Syngenta Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom § Department of Radiation Oncology, The Fourth Affiliated Hospital, Hebei Medical University, Shijiazhuang 050011, People's Republic of China ⊥ Department of Chemistry, Queen’s University, Kingston, Ontario, Canada, K7L 3N6 S Supporting Information *

ABSTRACT: A phytochemical investigation of Chromolaena odorata resulted in the isolation of five new compounds, 5aα,6,9,9aβ,10-pentahydro-10β-hydroxy-7-methylanthra[1,2-d][1,3]dioxol-5-one (1), 1,2-methylenedioxy-6-methylanthraquinone (2), 3-hydroxy-1,2,4-trimethoxy-6-methylanthraquinone (3), 3-hydroxy-1,2-dimethoxy-6-methylanthraquinone (4), and 7-methoxy-7-epi-medioresinol (5), together with 12 known compounds, odoratin (6), 3β-acetyloleanolic acid (7), ursolic acid (8), ombuin (9), 4,2′-dihydroxy-4′,5′,6′-trimethoxychalcone (10), (−)-pinoresinol (11), austrocortinin (12), tianshic acid (13), cleomiscosin D (14), (−)-medioresinol (15), (−)-syringaresinol (16), and cleomiscosin A (17). All the compounds were evaluated for their PPARγ transactivation activity, and compound 6 showed moderate activity with an EC50 value of 3.10 μM. whether its antihypertensive and anti-inflammatory activities are related to PPARγ and to identify the compounds responsible for these activities. Our extensive chemical investigation resulted in the isolation of five new compounds, 1−5, as well as 12 known compounds, from the dichloromethane extract of C. odorata. Bioassay data revealed that odoratin (6) has a significant transactivation effect on PPARγ. The cytotoxicities of all 17 compounds were evaluated, and compounds 4 and 7 were active. The details of the elucidation of the structures and biological activity of these compounds are reported herein.

Chromolaena odorata (L.) R.M. King & H. Rob. (Asteraceae, syn. Eupatorium odoratum L.) is a native perennial herb in North America. It is distributed widely from Florida and Texas to Mexico and the Caribbean and has been introduced to tropical Asia, West Africa, and parts of Australia. In China, this plant is found mainly in the south and southwest of the country, including Guangdong, Hainan, Guangxi, Yunnan, and Guizhou Provinces. In traditional medicine, its aqueous extract and decoctions of its leaves have been used to treat soft tissue wounds, burn wounds, and skin infections.1 It has also found other medicinal uses as an antidiarrheal, astringent, antispasmodic, antihypertensive, anti-inflammatory, and diuretic agent.2 A pharmacological study of an aqueous leaf extract showed antiinflammatory activity in rats that had been subjected to subchronic inflammatory conditions induced by formaldehyde.3 Ointments and gels formulated from the methanol extract of C. odorata have been evaluated for efficacy and safety.4 The ethanol extract of C. odorata leaves exhibited antiprotozoal and antimicrobial activity,5 and the whole-plant ethanolic extract exhibited cytostatic activity against MDA-MB-231, PC-3, MCF7, HT-29, 4T1, and RAW-267 cell lines in vitro.6 Its essential oil showed antibacterial activity against Bacillus cereus and antifungal activity against Aspergillus niger.7 Previous phytochemical studies of C. odorata led to the isolation of flavonoids, anthraquinones, alkaloids, triterpenoids, and steroids.8−13 Our interest in C. odorata was to establish © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

The dichloromethane extract of C. odorata was subjected to chromatographic separation and purification repeatedly and yielded one rare hydroanthranol (1), three new anthraquinones (2−4), one new furofuranoid lignan (5), and 12 known compounds (6−17) (Figure 1). Compound 1 was obtained as colorless crystalline needles (acetone). Its molecular formula was C16H16O4 based on analysis of the HRMS data, indicating an index of hydrogen deficiency of nine. Its 1H NMR spectrum (Table 1) included a pair of adjacent aromatic protons at δ 6.88 and 7.73 (1H each, Received: May 31, 2012

A

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Figure 1. Structures of compounds 1−17.

benzene ring. In the 1H−1H COSY spectrum, the olefinic proton H-8 (δ 5.45) correlated with a methylene proton at δ 2.77, thereby confirming the presence of a methylene group at C-9. The proton at δ 2.45 correlated with the CH2 group (δ 2.50 and 2.20) that constitutes C-6. The C-10 CH−OH group correlated with the C-9a hydrogen at δ 2.10, which correlates with the C-5a hydrogen at δ 2.45. In the HMBC spectrum, a methyl at δ 1.74 correlated with the olefinic C-7 and C-8 and the C-6 methylene group (δ 30.2); therefore, the methyl group is attached to C-7. The relative configuration of 1 was determined by the NOESY experiment and the 3J coupling constants. In the 1H NMR spectrum, H-10 displayed a large coupling constant with H-9a, indicating that H-10 and H-9a must be in a trans-diaxial orientation. H-10 did not exhibit an NOE correlation with H-9a but had a strong NOE correlation with H-5a; therefore, the ring junction must be trans, and 10OH is equatorial (Figure 2). Thus, compound 1 was characterized as 5aα,6,9,9aβ,10-pentahydro-10β-hydroxy-7methylanthra[1,2-d][1,3]dioxol-5-one. This compound is a rare hydroanthranol that has not been isolated previously from the genus Chromolaena.

Table 1. NMR Spectroscopic Data (500 MHz, CDCl3) for 1 position

δC, type

1 2 3 4 5 6

144.0, 152.0, 108.5, 123.7, 196.8, 30.2,

7 8 9a 9b 10 5a 9a 1a 4a 11 Me-7 OH-10

132.7, C 118.8, CH 30.3, CH2 71.8, 44.7, 41.7, 127.1, 125.8, 102.2, 23.4,

C C CH CH C CH2

CH CH CH C C CH2 CH3

δH (J in Hz)

HMBC

NOESY

6.88, d (8.3) 7.73, d (8.3)

1, 2, 1a, 4a 1, 2, 5, 10, 1a

2.50, m 2.20, m

7, 8, 5a, Me-7

5.45, 2.77, 2.07, 4.88, 2.45, 2.10,

9, 9a, Me-7 7, 8, 5a, 9a

9a, 9b, Me-7 9b, 8

1, 2, 1a, 4a 5, 6, 10, 9a 9, 10, 9a

5a, 9b, OH-10

br s br d (16.4) m d (9.4) m o

6.12, s 1.74, s 3.14, br s

4 3

1, 2 6, 7, 8

d, J = 8.3 Hz, H-3, 4), a methylenedioxy group at δ 6.12 (2H, s, H-11), an olefinic proton at δ 5.45 (1H, br s, H-8), an alcoholic hydroxy group at δ 3.14, and three methine groups at δ 4.88, 2.45, and 2.10. The 13C NMR data (Table 1) indicated the characteristic signals of a carbonyl (δ 196.8), six aromatic (δ 108.5−152.0), and two olefinic carbons (δ 118.8 and 132.7). Thus, based on the index of hydrogen deficiency of nine, the structure of 1 must contain four rings. The presence of three methylene, six methine, one methyl, five quaternary, and one carbonyl carbon was confirmed by the 13C NMR, DEPT, and HSQC data. The information above suggests that 1 has a hydroanthranol framework with a methylenedioxy group on the

Figure 2. HMBC and NOE correlations of compound 1. B

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Table 2. NMR Spectroscopic Data (500 MHz, CDCl3) for 2−4 2 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 (CH2) OMe-1 OMe-2 OH-3 OMe-4 Me-6 a

δC, type 146.0, 154.3, 112.3, 124.1, 127.6, 145.2, 134.7, 127.9, 183.2, 182.0, 133.6, 131.6, 116.9, 127.3, 103.8,

C C CH CH CH C CH CH C C C Ca Ca C CH

3 δH (J in Hz)

7.13, d (7.8) 7.97, d (7.8) 8.10, br s 7.57, dd (7.8, 1.2) 8.19, d (7.8)

δC, type 151.5, 146.2, 148.6, 145.0, 126.7, 144.3, 134.5, 126.8, 182.0, 182.7, 133.6, 132.1, 120.2, 122.6,

C C C C CH C CH CH C C C C C C

7.96, br s 7.52, dd (7.8, 1.2) 8.08, d (7.8)

δC, type 153.78, C 145.5, C 153.85, C 110.3, CH 126.9, CH 144.2, C 134.9, CH 127.3, CH 181.5, C 182.7, C 132.5, C 132.8, C 120.8, C 131.7, C

δH (J in Hz)

7.72, s 8.02, br s 7.57, br d (7.8) 8.16, d (7.8)

6.31, s 61.44, CH3 61.74, CH3

21.9, CH3

4 δH (J in Hz)

2.53, s

62.1, CH3 21.8, CH3

3.98, 4.09, 6.55, 4.01, 2.50,

s s br s s s

61.6, CH3 61.7, CH3

3.99, s 4.11, s 6.34, s

21.8, CH3

2.51, s

The shifts were obtained from the HMBC spectrum.

compounds of similar structures, compound 2 was identified as 1,2-methylenedioxy-6-methylanthraquinone.15 Compound 3 was obtained as an orange granular solid (acetone). HRMS data indicated a molecular formula of C18H16O6 based on the [M + H]+ ion at m/z 329.1020 (calcd for C18H17O6, 329.1025). The NMR spectrum included signals for one methyl group at δ 2.50 (s); three methoxy signals at δ 4.09, 4.01, and 3.98; and a phenolic hydroxy group at δ 6.55 (br s). In the A-ring, an ABX system was indicated by the three coupled protons at δ 7.52 (1H, dd, J = 7.8, 1.2 Hz), 8.08 (1H, d, J = 7.8 Hz), and 7.96 (1H, br s). The 13C NMR data confirmed the presence of four methyl, three methine, and 11 quaternary carbons, including two carbonyl carbons at δ 182.0 and 182.7. The 1H and 13C NMR data were similar to those of 4-hydroxy-1,2,3-trimethoxy-6-methylanthraquinone16 and differed only by a phenolic −OH group. HMBC correlations from the signals at δ 7.96 to C-7, C-10, C-12, and Me-6; 7.52 to C-5, C-12, and Me-6; and 8.08 to C-6, C-9, and C-11 confirmed the location of the methyl group at C-6. From its NOESY spectrum, the methoxy group at δ 3.98 was located at C-1, as indicated by its weak correlation with H-8 (δ 8.08); the methoxy at δ 4.09 at C-2, as indicated by its strong correlation with OMe-1 (δ 3.98); and the methoxy at δ 4.01 at C-4, as indicated by its correlation with the broad singlet H-5 (δ 7.96). Thus, compound 3 was identified as 3-hydroxy-1,2,4trimethoxy-6-methylanthraquinone. Compound 4 was obtained as an orange granular solid (acetone). The HRMS data indicated a molecular formula of C17H14O5 based on the [M + H]+ ion at m/z 299.0921 (calcd for C17H15O5, 299.0919). Its ESIMS gave ion peaks at m/z 299 [M + H]+, 284 [M + H − Me]+, and 269 [M + H − 2Me]+. The 1H NMR spectrum of 4 was similar to that of 3, except for the absence of a methoxy group in 4. The 1H NMR spectrum of 4 indicated the presence of four aromatic protons. ABX system signals were observed at δ 8.02 (1H, br s), 7.57 (1H, br d, J = 7.8 Hz), and 8.16 (1H, d, J = 7.8 Hz), and a proton singlet was located at δ 7.72 (1H, s). These findings indicated

Compounds 2−4 demonstrated similar spectroscopic characteristics. For example, the UV spectra of compounds 2−4 had absorbance maxima at 245 and 280 nm, typical of anthraquinone-type compounds.14 Their IR spectra included characteristic absorption bands of carbonyl groups in the range 1620−1675 cm−1 (compounds 2−4) and of free hydroxy groups in the range 3150−3600 cm−1 (compounds 3 and 4). Compound 2 was obtained as yellow needles (acetone). HRMS analysis indicated a quasimolecular ion [M + H]+ at m/ z 267.0652 (calcd for C16H11O4, 267.0657), corresponding to a molecular formula of C16H10O4. In the ESIMS spectrum, ions at m/z 267 [M + H]+, 221 [M + H − OCH2O]+, and 211 [M + H − 2CO]+ indicated that 2 might contain two carbonyl groups and one methylenedioxy group. The 1H and 13C NMR spectroscopic data (Table 2) revealed the typical pattern of anthraquinones. An AB system in the C-ring was indicated by a pair of aromatic proton doublets at δ 7.13 (1H, d, J = 7.8 Hz, H-3) and 7.97 (1H, d, J = 7.8 Hz, H-4), and the presence of an ABX system in the A-ring was confirmed by the three coupled protons at δ 7.57 (1H, dd, J = 7.8, 1.2 Hz, H-7), 8.19 (1H, d, J = 7.8 Hz, H-8), and 8.10 (1H, br s, H-5). The 1H NMR spectrum also showed one methyl group at δ 2.53 (3H, s) and one methylenedioxy group at δ 6.31 (2H, s). These assignments were further confirmed by the correlations in its 1 H−1H COSY spectrum. The 13C NMR spectrum indicated two carbonyls at δ 183.2 (C-9) and 182.0 (C-10), two oxygenated aromatic carbons at δ 146.0 (C-1) and 154.3 (C-2), one methylenedioxy carbon at δ 103.8 (C-15), one aromatic methyl carbon at δ 21.9 (Me-6), and various other aromatic carbon signals. HMBC correlations from the protons at δ 7.97 to C-2, C-10, and C-13; 8.10 to C-7, C-10, and C-12; and 8.19 to C-7, C-10, and C-12 confirmed the location of a methylenedioxy group at C-1 and C-2 and a methyl group at C-6. In the NOESY spectrum, H-3 (δ 7.13) correlated with H-4 (δ 7.97) and H-5 (δ 8.10), and H-7 (δ 7.57) correlated with Me-6 (δ 2.53) and H-8 (δ 8.19). On the basis of the information above and by comparison with the data from C

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compound 5 was similar to that of medioresinol (15),17 differing only in an additional methoxy signal at δ 2.96 and the absence of the doublet at δ 4.71. The methoxy signal at δ 2.96, which was much further upfield than the other methoxy signals, suggested that it was an aliphatic methoxy located at C-7. The 2D NMR spectrum revealed that 5 possessed a furofuranoid lignan skeleton.18 In the HMBC spectrum, the methoxy proton signal at δ 2.96 had a long-range correlation with an acetal carbon at δ 110.3, which was assigned to C-7 due to the presence of long-range correlations of C-7 with the aromatic proton signal at δ 6.72 (H-2 and H-6). Therefore, the aliphatic methoxy group was located at C-7. The relative configuration of 5 was deduced via the NOESY spectrum. Thus H-7′ (δ 4.46) had a strong NOE with H-9′α (δ 3.79) and H-9α (δ 4.08); H-8 (δ 3.28) had a strong NOE with H-8′ (δ 3.02); and H-9′β (δ 3.06) had a strong NOE with H-8′ and an NOE interaction with OMe-7 (δ 2.96). The relative configurations of C-7, C-8, C-8′, and C-7′ were determined as shown in Figure 3. Thus, the structure of compound 5 was defined as 7-methoxy-7-epimedioresinol.

an anthraquinone with one monosubstituted ring and one trisubstituted ring. The remaining signals in the 1H NMR spectrum included a broad singlet of a phenolic hydroxy proton at δ 6.34, two methoxy signals at δ 4.11 and 3.99, and a methyl singlet at δ 2.51. The 13C NMR data confirmed the presence of three O-methyl, four methine, and 10 quaternary carbons, including two carbonyl carbons at δ 181.5 and 182.7. The proton positions were confirmed by the HMBC spectrum, as both H-4 and H-5 (δ 7.72 and 8.02) correlated with C-10 (δ 182.7) and H-8 correlated with C-9 (δ 181.5). The methyl group at δ 2.51 was positioned at C-6 from its correlation with C-5 (δ 126.9), C-6 (δ 144.2), and C-7 (δ 134.9) in the HMBC spectrum. The methoxy group at δ 3.99 was positioned at C-1 from its correlation with C-1 (δ 153.78) in the HMBC experiment. The carbon at δ 145.5 correlated with the methoxy at δ 4.11 and with H-4. These correlations demonstrated that the methoxy group was located at C-2 or C-3. The chemical shift of C-2 indicated that it was more shielded than C-3 when C-2 is ortho to two OR groups. Therefore, the signal at δ 145.5 was attributed to C-2 and the methoxy at δ 4.11 was positioned at C-2. On the basis of this information and by comparison with similar compounds, compound 4 was determined to be 3hydroxy-1,2-dimethoxy-6-methylanthraquinone.16 Compound 5 was obtained as a colorless granular solid (acetone). Its HRMS data indicated a molecular formula of C22H26O8 based on [M + Na]+ at m/z 441.1517 (calcd for C22H26O8Na, 441.1520). The 1H NMR spectrum (Table 3) of Table 3. NMR Spectroscopic Data (500 MHz, CDCl3) for 5 position

δC, type

1 2

132.2, C 103.6, CH

3 4 5 6

146.9, 134.5, 146.9, 103.6,

7 8 9a

110.3, C 56.9, CH 69.6, CH2

C C C CH

9b 1′ 2′ 3′ 4′ 5′ 6′

132.8, 108.5, 146.7, 145.4, 114.2, 119.3,

C CH C C CH CH

7′ 8′

87.8, CH 52.8, CH

9′a 9′b

70.3, CH2

3, 5OMe 7-OMe 3′-OMe 4-OH 4′-OH

56.4, CH3 48.7, CH3 55.9, CH3

δH (J in Hz)

HMBC

6.72, br s

8, 3-OMe, 7OMe

6.72, br s

7, 7′, 9 7′

6.83, br s

1′, 3′, 4′, 6′, 7′

2.96 3.89 5.54 5.58

(s) (s) (s) (s)

Figure 3. NOE correlations of compound 5.

The known constituents 6−17 were identified as odoratin (6),19 3β-acetyloleanolic acid (7),20 ursolic acid (8),21 ombuin (9),22 4′,5′,6′-trimethoxy-4,2′-dihydroxychalcone (10),23 (−)-pinoresinol (11),24 austrocortinin (12),25 tianshic acid (13),26 cleomiscosin D (14),27 (−)-medioresinol (15),27 (−)-syringaresinol (16),28 and cleomiscosin A (17)27 by comparing their physical and spectroscopic data with reported data. The effects of compounds 1−17 on the activation of PPARγ were examined. Compound 6 increased transactivation activity in a dose-dependent manner within the range 1−50 μM, with an EC50 value of 3.10 μM (Figure 4). Rosiglitazone was used as a positive control, giving an EC50 value of 0.90 μM. This is the first report of an agonistic effect of compound 6 on PPARγ. We also evaluated all compounds against the human tumor cell lines HeLa, HOC-21, T-98, U251-SP, MCF-7, QG-56, PC-6, HLE, MM1-CB, and HMV-1 using the MTT assay (cisplatin was used as the positive control). Within the range of 1−100 μM, compound 4 had weak cytostatic activity (IC50 = 52.21 μM) against PC-6 cells, and compound 10 had weak cytostatic activity (IC50 = 92.72 μM) against U251SP cells. Previous phytochemical and bioactivity investigations of C. odorata described a prostaglandin-like compound, (9S,13R)-12oxophytodienoic acid, that activates PPARγ.29 Two flavonoid glycosides have been reported to show significant cytotoxicity against LLC and HL-60 cancer cell lines in vitro.30 In conclusion, five new compounds (1−5) have been isolated from C. odorata. Their structures were elucidated by analysis of

8, 3-OMe, 7OMe

3.28, q (9.0) 4.08, dd (9.0, 6.6) 4.03, dd (9.0, 1.8)

6.87, d (8.1) 6.81, br dd (8.1, 1.5) 4.46, d (6.8) 3.02, ddd (9.1, 6.8, 1.8) 3.79, t (9.1) 3.06, dd (9.1, 8.6) 3.91 (s)

NOESY

2, 6, 8′, 9′a,

7, 8, 7′, 8′

9, 1′, 2′, 6′, 8′

9b, 2′, 6′, 9′b 7, 9a, 2′, 6′

7, 7′,8′ 7, 8, 7′, 8′

8, 9′b 2, 6, 7′, 9′a

3, 5

2, 6

7 3′ 3, 4, 5 3′, 4′, 5′

2, 6, 9a 2′

D

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+120 (c 0.03, MeOH); UV (MeOH) λmax 237, 288, 316 nm; 1H and 13 C NMR data (see Table 1); ESIMS m/z 273 [M + H]+, 254 [M − H2O]+; HRESIMS m/z found 272.1042 [M]+ (C16H16O4 calcd 272.1049). 1,2-Methylenedioxy-6-methylanthraquinone (2): yellow needles; mp 270−272 °C; UV (MeOH) λmax 228, 257 nm; 1H and 13C NMR data (see Table 2); ESIMS m/z 267 [M + H]+, 221 [M + H − OCH2O]+, 211 [M + H − 2CO]+; HRESIMS m/z found 267.0652 [M + H]+ (C16H11O4 calcd 267.0657). 3-Hydroxy-1,2,4-trimethoxy-6-methylanthraquinone (3): orange granular solid; mp 232−234 °C; UV (MeOH) λmax 243, 278 nm; 1H and 13C NMR data (see Table 2); ESIMS m/z 329 [M + H]+, 311 [M−OH]+, 314 [M + H − Me]+, 299 [M + H − 2Me]+, 271 [M + H − 2Me − 2CO]+; HRESIMS m/z found 329.1020 [M + H]+ (C18H17O6 calcd 329.1025). 3-Hydroxy-1,2-dimethoxy-6-methylanthraquinone (4): orange granular solid; mp 205−206 °C; UV (MeOH) λmax 256, 280 nm; 1 H and 13C NMR data (see Table 2); ESIMS m/z 299 [M + H]+, 284 [M − Me]+, 269 [M − 2Me]+; HRESIMS m/z found 299.0921 [M + H]+ (C17H14O5 calcd 299.0919). 7-Methoxy-7-epi-medioresinol (5): colorless granular solid; mp 205−206 °C; [α]20D +90 (c 0.01, MeOH); UV (MeOH) λmax 233, 280 nm; 1H and 13C NMR data (see Table 3); ESIMS m/z 417 [M − H]+, 403 [M − Me]+, 385 [M − Me − H2O]+; HRESIMS m/z found 441.1517 [M + Na]+ (C22H26O8Na calcd 441.1520). Effects of 1−17 on Activation of PPARγ. Human hepatocyte L02 cells were cultured in RPMI-1640 medium supplemented with 10% FBS at 37 °C in a humidified 5% CO2 incubator and seeded into 96-well plates at a concentration of 3 × 104 cells/well one day before transfection. pBIND-PPARγ-LBD were cotransfected with the reporter vector pGL3-SV40-GAL4 at 90% confluence using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Test compounds diluted in DMSO, a negative control (0.1% DMSO), and a positive control (pioglitazone, PI) were added 6 h after transfection. Following incubation for 24 h, the cells were lysed, and luciferase activity was detected with the Luciferase Assay System (Promega, Madison, WI, USA).

Figure 4. Effect of odoratin (6) on the activation of PPARγ.

their 1D and 2D NMR spectra and by HRMS. Twelve known compounds (6−17) were identified, one of which, odoratin (6), showed moderate transactivation activity of PPARγ at concentrations in the range 1 to 50 μM.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a Autopol IV polarimeter (Rudolph Research Analytical). UV spectra were recorded with a Shimadzu UV-2201 spectrophotometer. ESIMS data were obtained on an Applied Biosystems 3200Q TRAP mass spectrometer. NMR spectra were obtained with a Bruker 500 spectrometer operating at 500 MHz for 1H and 125 MHz for 13C. HRESIMS data were obtained with a Thermo Scientific LTQ Orbitrap Discovery (Bremen, Germany). Column chromatography was performed with silica gel (Qingdao Haiyang Chemical Group Co., Ltd., China). TLC was performed on precoated silica gel 60 F254 plates (0.2 mm thick, Merck). Plant Material. Whole-plant samples of C. odorata were collected in August 2006 in Wenchang, Hainan Province, China. The taxonomic identification of plant material was conducted by Professor Jianhua Wang, Hebei Medicinal University, Shijiazhuang, China. A voucher specimen (No. 2006-8) was deposited in the Herbarium of the School of Pharmaceutical Sciences, Hebei Medical University, Shijiazhuang, China. Extraction and Isolation. Dried plants (1.2 kg) were extracted under reflux with EtOH (10 L × 3) for 2 h each time. Evaporation at reduced pressure yielded 150 g of crude extract. Water (1500 mL) was added, and lipids were removed by stirring the mixture with petroleum ether (3 × 500 mL). The aqueous phase was salted (NaCl) and extracted with CH2Cl2 (3 × 500 mL). The combined CH2Cl2 extracts were dried with anhydrous NaSO4, filtered, and evaporated to yield a dark green extract (17.5 g). The CH2Cl2 fraction (17.5 g) was dissolved in acetone, adsorbed onto 30 g of silica gel, and subjected to normal-phase column chromatography (silica gel 300−400 mesh, 550 g) using a mixture of petroleum ether and acetone as eluent (8:2 to 1:2, total of 7.2 L). Ten fractions, Fr1 to Fr10, were obtained. Fr2 (2.2 g) provided eight fractions (Fr2‑1 to Fr2‑8) after elution with petroleum ether and EtOAc (8:2) by normal-phase column chromatography. Fr2‑3 was subjected to preparative TLC developed with petroleum ether−acetone (7:3). The band at Rf = 0.40 was collected and gave compound 1 (10 mg), Fr2‑5 yielded compound 2 (8 mg). Fr3 (0.5 g) was subjected to preparative TLC developed with petroleum ether− acetone (3:2). The band at Rf = 0.50 was collected and gave compound 3 (15 mg). Fr4 (0.8 g) yielded compound 4 (12 mg) by the same preparative TLC method as Fr3. Fr8 (1.5 g) was further subjected to normal-phase column chromatography and eluted with a mixture of petroleum ether and acetone (3:4). Twelve fractions were obtained: Fr8‑1 to Fr8‑12. Fr8‑5 was further purified by preparative TLC and gave compound 5 (15 mg). 5aα,6,9,9aβ,10-Pentahydro-10β-hydroxy-7-methylanthra[1,2-d][1,3]dioxol-5-one (1): colorless needles; mp 204−205 °C; [α]20D

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ASSOCIATED CONTENT

* Supporting Information S

HR-MS and 1D and 2D NMR spectra for compounds 1−5 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-311-86265634. E-mail: [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Natural Science Foundation of China (81072551), Natural Science Foundation of Hebei Province, and Youth Science Foundation of Hebei Ministry of Education (2011172). We also wish to extend our sincere thanks for the financial support from Syngenta Ltd. (2008-Hebei Medical University-Syngenta02) and to Dr. John Clough for proofreading.

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REFERENCES

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