Eight Pairs of Epimeric Triterpenoids Involving a Characteristic Spiro-E

Jan 2, 2015 - Department of Phytochemistry, Second Military Medical University, ... University of Traditional Chinese Medicine, Fuzhou, Fujian 350108,...
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Eight Pairs of Epimeric Triterpenoids Involving a Characteristic SpiroE/F Ring from Abies faxoniana Guo-Wei Wang,† Chao Lv,‡,§ Xin Fang,‡ Xin-Hui Tian,‡ Ji Ye,‡ Hui-Liang Li,‡ Lei Shan,‡ Yun-Heng Shen,*,‡ and Wei-Dong Zhang*,†,‡ †

School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China Department of Phytochemistry, Second Military Medical University, Shanghai 200433, People’s Republic of China § School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350108, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Five pairs of new epimeric lanostane-type triterpenoids, abiespirones A−D (1−4) and G (7), two pairs of new epimeric cycloartane-type triterpenoids, abiespirones E and F (5, 6), and a pair of new epimeric 7(8→9)abeo-spirolanostane abiespirones H (8) with spiro-B/C and -E/F ring systems were isolated from Abies faxoniana as inseparable mixtures of C-23 epimers in a specific proportion. The HPLC plots showed that each purified isomer rapidly equilibrated with the C-23 epimer in solution. The structures of compounds 1−8 were elucidated by analysis of the NMR spectra and single-crystal X-ray diffraction. Compound 6 showed cytotoxicity against three hepatoma cell lines, namely, HepG2, Huh7, and SMMC7721, with IC50 values of 9.8, 7.5, and 10.7 μM, respectively, but exerted low cytotoxicity on normal QSG7701 hepatic cells, indicating its selective cytotoxicity for hepatoma cells. Compound 6 arrests the cell cycle at G2/M and induces cell apoptosis in Huh7 cells. In addition, the generation of reactive oxygen species (ROS) was detected in Huh7 cells when treated with compound 6, and a ROS scavenger partly blocked the effects of compound 6-induced Huh7 cell death, suggesting that compound 6-induced apoptosis is associated with elevated levels of ROS in Huh7 cells.

T

4) and G (7), two pairs of new epimeric cycloartane-type triterpenoids, abiespirones E and F (5, 6), a pair of new epimeric 7(8→9)abeo-spirolanostane abiespirones H (8) with spiro-B/C and E/F ring systems, and 11 known triterpenoids (9−19) were isolated from the branches and leaves of Abies faxoniana (Figure 1). Compounds 1−8 were obtained as inseparable C-23 epimeric mixtures in specific proportions, and the C-23 epimerization of the purified isomers for compounds 1 and 2 were observed by HPLC (see Supporting Information). Here, the isolation and structural elucidation of compounds 1− 8, the in vitro cytotoxicity against three human hepatoma cell lines (HepG2, Huh7, and SMMC7721) and one normal hepatic cell line (QSG7701), and a preliminary study on the mechanism of action underlying the in vitro antitumor effect of compound 6 are described.

he genus Abies of the Pinaceae family has approximately 50 species, 19 of which occur exclusively in China.1 In recent decades, Abies plants have been studied extensively because of their interesting, structurally diverse, and biologically important terpenoid constituents, resulting in the isolation and characterization of more than 250 terpenoids with different chemical profiles. These Abies terpenoids exhibited a wide range of biological activities, particularly antitumor activities against various human tumor cell lines.2−13 Since 2005, our continuous investigation of Abies species has led to the isolation of approximately 150 terpenoids from 10 species that are distributed across China, of which a unique sesquiterpenoid, cycloabiesesquine A,14 two tetraterpenoids, abiestetranes A and B,15 and a sesquiterpenoid spirolactone with a 6/6/5 ring, abiespiroside A,16 have been found to exhibit potent antiinflammatory, cytotoxic, and antitumoral activities. In further investigations of indigenous Abies species, five pairs of new epimeric lanostane-type triterpenoids, abiespirones A−D (1− © 2015 American Chemical Society and American Society of Pharmacognosy

Received: August 31, 2014 Published: January 2, 2015 50

DOI: 10.1021/np500679s J. Nat. Prod. 2015, 78, 50−60

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Figure 1. Structures of compounds 1−8.

■

RESULTS AND DISCUSSION Compounds 1a and 1b were assigned the molecular formula C30H44O4 based on 13C NMR data and the m/z 469.3244 [M + H]+ ion in the positive HRESIMS, indicating nine indices of hydrogen deficiency. The IR spectrum suggested the presence of carboxyl (1772 cm−1), carbonyl (1720 cm−1), and olefinic bonds (1647 cm−1). The 1H, 13 C, and DEPT NMR spectroscopic data of 1a and 1b (Tables 1 and 3) indicated 30 carbon signals including five methyl singlets, two methyl doublets, nine methylenes, five methines, five quaternary carbons, a ketocarbonyl, a lactone carbonyl, a dioxygenated secondary carbon (C-23), and an oxygenated tertiary carbon (C-17), which revealed the presence of a lanostane skeleton. Comparison of the 13C NMR spectrum of compounds 1a and 1b to those of neoabieslactone C showed a difference in the double-bond position.5 The HMBC correlations from H-7 to C-5, C-6, and C-9 and from H3-30 to C-8 indicated that there was a Δ7,8 double bond in 1a and 1b (δH 5.63/5.66; δC 121.6/ 121.9 and 148.6/149.1) instead of the Δ8,9 double bond in neoabieslactone C (Figure 2). According to the indices of hydrogen deficiency and signals for a carboxylic carbon (δC 178.5/179.3, C-26), an acetal secondary carbon (δC 111.6/ 112.9, C-23), and an oxygenated sp3 tertiary carbon (δC 100.6/ 100.7, C-17), the planar structures of 1a and 1b had to contain

an oxaspirolactone moiety. In the NOESY spectrum, H3-30 correlated with H3-21, suggesting a (17R*) relative configuration of 1a and 1b (Figure 2). Although the NOESY correlation could be used to deduce the relative configuration, the absolute configuration was assigned via Cu Kα X-ray crystallographic analysis. Colorless orthorhombic crystals of 1a were obtained from MeOH and subjected to Cu Kα X-ray crystallographic analysis, which confirmed the absolute configuration (Figure 3). The C-16 signal for the (23S)-isomer (δC 34.1) was more shielded than that of the (23R)-isomer (δC 34.3) because of the shielding effect of the lactone oxygen atom.17 The 21-Me resonance of the (23R)-isomer (δH 0.95) was more deshielded by the anisotropic effect of the lactone oxygen atom than that of the (23S)-isomer (δH 0.94).17 A NOESY correlation between H3-21 and H-24 was observed in the (23S)-isomer but not in the (23R)-isomer (Figure 2). Therefore, the assignments of all the atoms in the (23R)- and (23S)-epimers were established by the above spectroscopic features. The structures of compounds 1a and 1b were deduced to be 17β,23-epoxy-3-oxo-9β-lanosta-7-en-26,23R-olide and 17β,23-epoxy-3-oxo-9β-lanosta-7-en-26,23S-olide, respectively, and they were named abiespirones A1 and A2. The molecular formula C30H42O5 was established for compounds 2a and 2b on the basis of 13C NMR data and 51

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Table 1. 1H (500 MHz) NMR Spectroscopic Data of Abiespirones A−D in CDCl3 no. 1 2 3 5 6 7 8 9 11 12 15 16 18 19 20 21 22 24 25 27 28 29 30

1a 1.61 1.93 2.40 2.56

m m m m

1b 1.61 1.94 2.40 2.56

m m m m

1.65 dd (14.5, 3.0) 1.78 m 1.97 m 5.63 m

1.65 dd (14.5, 3.0) 1.78 m 1.97 m 5.66 m

1.77 1.62 1.92 1.38 2.23 1.37 1.78 1.78 1.97 0.94 0.94 2.27 0.95 1.78 2.75 2.02 2.48 2.97 1.23 1.06 1.08 1.32

1.77 1.62 1.92 1.38 2.23 1.37 1.78 1.78 1.97 0.94 0.94 2.27 0.94 1.90 2.79 2.07 2.51 2.94 1.23 1.06 1.08 1.32

m m m m m m m m m s s m d (7.2) m m m m m d (7.2) s s s

m m m m m m m m m s s m d (7.2) m m m m m d (7.2) s s s

2a 1.80 1.95 2.55 2.73

m m m m

1.85 dd (14.5, 3.0) 1.69 m 1.84 m 2.36 m 2.41 m

2.52 m 2.74 m 1.86 m 1.94 m 1.95 m 2.05 m 0.83 s 1.09 s 2.44 m 1.02 d (7.2) 2.32 m 2.53 m 2.36 m 2.55 m 2.97 m 1.22 d (7.2) 1.06 s 0.91s 1.31 s

2b 1.81 1.95 2.55 2.73

3a

m m m m

1.85 dd (14.5, 3.0) 1.70 m 1.84 m 2.36 m 2.41 m

2.52 2.73 1.86 1.94 1.95 2.04 0.84 1.10 2.44 1.01 2.32 2.53 2.36 2.54 2.94 1.21 1.07 0.91 1.32

m m m m m m s s m d (7.2) m m m m m d (7.2) s s s

the m/z 483.3017 [M + H]+ ion in the positive HRESIMS, indicating 10 indices of hydrogen deficiency. The IR spectrum showed absorption bands that were characteristic of carbonyl (1773, 1720, and 1711 cm−1) and olefinic bonds (1651 cm−1). Their NMR spectra were similar to those of 1a and 1b, except for the presence of an α,β-unsaturated carbonyl group [δC 161.9/163.1 (C-8), 139.3/139.6 (C-9), and 197.6/197.9 (C11)] in 2a and 2b instead of a Δ7,8 double bond in 1a and 1b. This assumption was determined by HMBC correlations from H3-30 to C-8 (δC 161.9/163.1), from H3-19 to C-9 (δC 139.3/ 139.6), and from H-12 to C-11 (δC 197.6/197.9). Accordingly, the structures of compounds 2a and 2b were elucidated as 17β,23-epoxy-3,11-dioxolanosta-8-en-26,23R-olide and 17β,23epoxy-3,11-dioxolanosta-8-en-26,23S-olide, which were named abiespirones B1 and B2, respectively. Compounds 3a and 3b were assigned the molecular formula C30H46O4 based on 13C NMR data and the m/z 471.3393 [M + H]+ ion in the positive HRESIMS, indicating eight indices of hydrogen deficiency. The IR absorptions showed the presence of hydroxy (3383 cm−1), carboxylic (1772 cm−1), and olefinic groups (1646 cm−1). The NMR spectroscopic data were closely related to those of 1a and 1b, with the only difference being the presence of a C-3 hydroxy group (δC 79.1/79.2) in 3a and 3b instead of a carbonyl moiety (δC 218.8/219.1) in 1a and 1b. In the NOESY spectrum, H-3 was correlated to H3-19, which

3b

1.63 m 1.94 m 2.40 m 2.55 m 3.19 m 1.65 dd (14.5, 3.0) 1.79 m 1.95 m 5.55 m

1.63 m 1.94 m 2.40 m 2.55 m 3.20 m 1.65 dd (14.5, 3.0) 1.79 m 1.94 m 5.58 m

1.77 1.63 1.91 1.40 2.23 1.38 1.78 1.77 1.97 0.95 0.95 2.33 0.95 1.77 2.75 2.02 2.49 2.97 1.22 1.08 1.09 1.30

1.77 1.63 1.91 1.40 2.23 1.38 1.78 1.77 1.97 0.95 0.95 2.33 0.94 1.79 2.79 2.07 2.50 2.94 1.22 1.08 1.09 1.30

m m m m m m m m m s s m d (7.2) m m m m m d (7.2) s s s

m m m m m m m m m s s m d (7.2) m m m m m d (7.2) s s s

4a 1.60 1.93 2.39 2.56

4b

m m m m

1.60 1.94 2.39 2.56

1.65 dd (14.5, 3.0) 1.77 m 1.97 m 1.78 m 1.98 m 1.94 m 1.49 m 1.62 m 1.92 m 1.36 m 2.22 m 1.37 m 1.78 m 1.78 m 1.97 m 0.94 s 0.94 s 2.27 m 0.95 d (7.2) 1.78 m 2.75 m 2.02 m 2.48 m 2.97 m 1.23 d (7.2) 1.06 s 1.08 s 1.31 s

m m m m

1.65 dd (14.5, 3.0) 1.77 m 1.97 m 1.78 m 1.98 m 1.94 m 1.49 m 1.62 m 1.92 m 1.36 m 2.22 m 1.37 m 1.78 m 1.78 m 1.97 m 0.94 s 0.94 s 2.27 m 0.94 d (7.2) 1.90 m 2.79 m 2.06 m 2.51 m 2.94 m 1.23 d (7.2) 1.06 s 1.08 s 1.32 s

established a 3α-OH group in 3a and 3b. Consequently, the structures of compounds 3a and 3b were defined as 17β,23epoxy-3α-hydroxy-9β-lanosta-7-en-26,23R-olide and 17β,23epoxy-3α-hydroxy-9β-lanosta-7-en-26,23S-olide, which were named abiespirones C1 and C2, respectively. Compounds 4a and 4b were assigned the molecular formula C30H46O4 based on 13C NMR data and the m/z 469.3411 [M − H]− ion in the negative HRESIMS, indicating eight indices of hydrogen deficiency. The IR spectrum suggested the presence of carboxylic (1770 cm−1) and carbonyl (1722 cm−1) functionalities. The 1H and 13C NMR spectroscopic data exhibited 30 carbon signals, which was consistent with those of 1a and 1b, except that the Δ7,8 double bond (δC 121.6/121.9 and 148.6/149.1) was saturated. Analysis of the HMBC spectrum facilitated definition of the structures of compounds 4a and 4b as 17β,23-epoxy-3-oxo-9β-lanosta-26,23R-olide and 17β,23-epoxy-3-oxo-9β-lanosta-26,23S-olide, and they were named abiespirones D1 and D2, respectively. Compounds 5a and 5b had the molecular formula C30H46O4 based on 13C NMR data and the m/z 471.3401 [M + H]+ ion in the positive HRESIMS, indicating seven indices of hydrogen deficiency. The IR absorptions showed the presence of hydroxy (3343 cm−1) and carboxylic (1770 cm−1) functionalities. The 1 H NMR spectrum showed two doublets (J = 4.1 Hz) at δH 0.37 and 0.50, and the 13C NMR two sp3 quaternary (δC 26.5/ 52

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Table 2. 1H (500 MHz) NMR Spectroscopic Data of Abiespirones E−H in CDCl3 no. 1 2 3 5 6 7 8 11 12 15 16 18 19 20 21 22 24 25 27 28 29 30 2′ PhH

5a 1.60 m 1.93 m 2.39 m 2.56 m 3.46 m 1.65 dd (14.5, 3.0) 1.77 m 1.97 m 1.78 m 1.98 m 1.94 m 1.62 m 1.92 m 1.36 m 2.22 m 1.37 m 1.78 m 1.78 m 1.97 m 0.83 s 0.37 d (4.1) 0.50 d (4.1) 2.20 m 1.01 d (7.2) 1.75 m 2.71 m 2.02 m 2.49 m 2.97 m 1.25 d (7.2) 0.91 s 1.06 s 1.30 s

5b 1.60 m 1.94 m 2.39 m 2.56 m 3.46 m 1.65 dd (14.5, 3.0) 1.77 m 1.97 m 1.78 m 1.98 m 1.94 m 1.62 m 1.92 m 1.36 m 2.22 m 1.37 m 1.78 m 1.78 m 1.97 m 0.83 s 0.37 d (4.1) 0.50 d (4.1) 2.20 m 1.00 d (7.2) 1.75 m 2.71 m 2.02 m 2.50 m 2.97 m 1.25 d (7.2) 0.91 s 1.06 s 1.30 s

6a 1.60 m 1.93 m 2.39 m 2.56 m 4.68 m 1.65 dd (14.5, 3.0) 1.77 m 1.97 m 1.78 m 1.98 m 1.94 m 1.62 m 1.92 m 1.36 m 2.22 m 1.37 m 1.78 m 1.78 m 1.97 m 0.84 s 0.36 d (4.1) 0.49 d (4.1) 2.20 m 1.01 d (7.2) 1.75 m 2.71 m 2.02 m 2.49 m 2.97 m 1.25 d (7.2) 0.91 s 1.06 s 1.30 s 2.07 s

6b

7a

1.60 m 1.94 m 2.39 m 2.56 m 4.68 m 1.65 dd (14.5, 3.0) 1.77 m 1.97 m 1.78 m 1.98 m 1.94 m 1.62 m 1.92 m 1.36 m 2.22 m 1.37 m 1.78 m 1.78 m 1.97 m 0.84 s 0.36 d (4.1) 0.49 d (4.1) 2.20 m 1.00 d (7.2) 1.75 m 2.71 m 2.02 m 2.50 m 2.97 m 1.25 d (7.2) 0.91 s 1.06 s 1.30 s 2.07 s

1.60 1.93 2.39 2.56

m m m m

7b 1.60 1.94 2.39 2.56

m m m m

8a 1.61 1.94 2.42 2.58

8b

m m m m

1.61 1.94 2.42 2.58

m m m m

1.65 dd (14.5, 3.0) 1.97 m 2.10 m 5.34 m

1.65 dd (14.5, 3.0) 1.97 m 2.10 m 5.34 m

1.85 dd (14.5, 3.0) 1.61 m 2.30 m 4.39 m

1.85 dd (14.5, 3.0) 1.61 m 2.30 m 4.39 m

1.75 2.05 1.44 2.12 1.37 1.78 1.78 1.97 0.94 1.02

m m m m m m m m s s

1.75 2.05 1.44 2.12 1.37 1.78 1.78 1.97 0.94 1.02

m m m m m m m m s s

1.62 1.93 1.72 2.06 1.60 1.90 1.70 1.95 0.72 1.49

m m m m m m m m s s

1.62 1.93 1.72 2.06 1.60 1.90 1.70 1.95 0.72 1.49

m m m m m m m m s s

2.20 1.01 1.75 2.71 2.02 2.49 2.97 1.25 1.06 1.08 1.31 3.60 7.27

m d (7.2) m m m m m d (7.2) s s s s m

2.20 1.00 1.75 2.71 2.02 2.50 2.97 1.25 1.06 1.08 1.32 3.60 7.27

m d (7.2) m m m m m d (7.2) s s s s m

2.20 1.00 1.73 2.72 2.04 2.42 2.90 1.13 0.88 0.95 1.19

m d (7.2) m m m m m d (7.2) s s s

2.20 0.99 1.73 2.72 2.04 2.42 2.90 1.13 0.88 0.95 1.19

m d (7.2) m m m m m d (7.2) s s s

basis of the NOESY correlation of H-3 with H-19 (Figure 4). Thus, the structures of compounds 6a and 6b were defined as 17β,23-epoxy-3α-acetoxy-9,19-cyclo-9β-lanosta-26,23R-olide and 17β,23-epoxy-3α-acetoxy-9,19-cyclo-9β-lanosta-26,23Solide, and they were named abiespirones F1 and F2 , respectively. Compounds 7a and 7b were assigned the molecular formula C38H50O7 based on 13C NMR data and the m/z 619.3569 [M + H]+ ion in the positive HRESIMS, indicating 14 indices of hydrogen deficiency. The IR absorptions showed the presence of aromatic rings (1585 and 1506 cm−1) and carboxylic groups (1770, 1769, and 1720 cm−1). According to the hydrogen deficiency indices and signals for two oxygenated sp3 tertiary carbons (δC 70.5/70.6 and 69.6/69.7), 7a and 7b were deduced as having an 8,9-epoxide moiety; its position was determined by the HMBC correlations of H3-30 to C-8 and of H3-19 to C-9 (Figure 5). H3-19 was correlated to Hβ-12 in the NOESY experiment, which established the α-orientation of the epoxide unit. A comparison of the 1H, 13C, and DEPT NMR data between 7a/7b and 1a/1b showed the following major differences: the Δ7,8 double bonds in 1a and 1b were absent in 7a and 7b, and phenylacetoxy groups were present in 7a and 7b. The HMBC correlations of H-7 (δH 5.34) to C-5, C-6, C-8,

26.3 and 20.0/20.0) and one methylene (δC 28.5/28.6) carbon characteristic of the presence of a cyclopropane ring. These signals and the remainder of the 1H and 13C NMR spectra indicated the presence of a cycloartane triterpenoid skeleton. The hydroxy group in 5a and 5b could be located at C-3 because of the HMBC correlations from H3-28 and H3-29 to the oxymethine at δC 77.0/77.2. The NOESY correlation of H3 with H-19 indicated that the 3-OH group was α-oriented. The structures of compounds 5a and 5b were thus established as 17β,23-epoxy-3α-hydroxy-9,19-cyclo-9β-lanosta-26,23Rolide and 17β,23-epoxy-3α-hydroxy-9,19-cyclo-9β-lanosta26,23S-olide, and they were named abiespirones E1 and E2, respectively. Compounds 6a and 6b were found to possess the molecular formula C32H48O5 based on 13C NMR data and the m/z 513.3506 [M + H]+ ion in the positive HRESIMS, indicating nine indices of hydrogen deficiency. The IR absorptions suggested the presence of two carboxylic groups (1772 and 1770 cm−1). The NMR data of 6a and 6b showed overall similarities to those of 5a and 5b, except for the presence of an acetoxy moiety (δC 20.7/20.9 and 170.84/170.85) at C-3 in 6a and 6b instead of a hydroxy group (δC 77.0/77.2) in 5a and 5b. The α-disposition of the acetoxy group was established on the 53

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Table 3. 13C (125 MHz) NMR Spectroscopic Data of Abiespirones A−D in CDCl3 no.

1a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

34.0 32.7 218.8 46.9 52.5 22.9 121.6 149.1 45.1 35.8 20.1 27.1 51.1 48.8 31.3 34.3 100.6 23.0 14.9 38.9 21.2 45.9 111.6 44.2 35.7 179.3 19.7 27.7 25.6 28.0

1b t t s s d t d s d s t t s s t t s q q d q t s t d s q q q q

33.7 33.2 219.1 46.8 53.1 22.7 121.9 148.6 44.7 35.9 19.6 27.1 49.9 49.7 31.5 34.1 100.7 23.0 16.2 37.0 20.8 45.7 112.9 40.6 36.1 178.5 18.3 27.1 26.3 28.3

2a t t s s d t d s d s t t s s t t s q q d q t s t d s q q q q

34.3 31.5 214.4 49.2 50.2 23.9 23.9 163.1 139.3 39.4 197.9 35.5 46.6 47.1 31.3 37.1 98.9 19.1 19.2 37.0 17.8 45.7 111.9 44.5 35.6 179.3 14.9 24.9 21.3 25.1

2b t t s s d t t s s s s t s s t t s q q d q t s t d s q q q q

34.2 32.2 214.3 50.0 50.1 23.6 24.6 161.9 139.6 39.3 197.6 35.5 45.6 47.1 31.5 37.0 99.0 18.1 20.0 36.0 17.9 45.7 112.9 40.7 35.7 178.4 16.3 24.9 21.3 25.2

3a t t s s d t t s s s s t s s t t s q q d q t s t d s q q q q

33.1 27.9 79.2 38.7 48.7 23.0 121.6 149.1 48.0 35.8 22.2 27.9 52.0 48.5 31.6 35.4 100.8 24.4 14.9 37.8 19.6 45.7 111.5 44.2 35.8 179.5 16.3 28.8 26.8 30.8

3b t t d s d t d s d s t t s s t t s q q d q t s t d s q q q q

33.7 29.6 79.1 38.8 49.2 22.9 122.2 148.7 47.9 35.9 22.0 28.2 50.9 49.5 31.9 35.3 101.0 24.5 16.3 36.7 18.2 45.6 112.8 40.7 36.1 178.7 16.4 28.8 27.7 30.6

4a t t d s d t d s d s t t s s t t s q q d q t s t d s q q q q

34.0 32.8 219.1 46.9 52.5 22.9 28.1 46.9 45.0 35.8 20.1 27.0 51.1 48.3 31.2 34.2 100.6 22.9 14.9 38.1 21.1 45.8 111.7 44.1 35.8 178.4 19.7 27.6 25.7 28.1

4b t t s s d t t d d s t t s s t t s q q d q t s t d s q q q q

33.6 33.2 218.9 46.8 53.0 22.7 29.6 45.5 44.6 35.9 19.7 27.1 49.7 49.9 31.4 34.1 100.6 22.4 16.2 37.1 20.9 45.7 113.0 40.6 36.0 179.4 18.4 27.3 26.3 28.4

t t s s d t t d d s t t s s t t s q q d q t s t d s q q q q

Figure 2. Key 1H−1H COSY, HMBC, and NOESY correlations for 1a and 1b.

Figure 4. Key 1H−1H COSY, HMBC, and NOESY correlations for 6b.

C-9, and the carboxylic carbon (δC 169.94/169.95) indicated that the phenylacetoxy group was attached to C-7. In the NOESY spectrum, H-7 was correlated to H3-30, revealing that the phenylacetoxy group was assigned a β-orientation in 7a and 7b. Thus, the structures of compounds 7a and 7b were defined

Figure 3. ORTEP plot of 1a.

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trien-26,23-olide (18),20 and 3-oxo-9βH-lanosta-7-en-23,26olide (19).21 The two epimers 2a and 2b occur concurrently in the branches and leaves of A. faxoniana. The HPLC plots showed that both compounds were isomerized to afford a mixture of compounds 2a and 2b at a ratio of ca. 2:1. As illustrated in Figure S63 (Supporting Information), there was no obvious interconversion of 2a and 2b in less than 15 min. After approximately 75 min, the proportion of the two epimers was not changed. In addition, the mutual epimerization between compounds 1a and 1b with a proportion of ca. 3:1 was also confirmed by semipreparative and analytical HPLC (Figure S62, Supporting Information). All isolates were tested for in vitro antitumor activity against three human hepatoma cell lines (HepG2, SMMC7721, and Huh7) and one normal hepatic cell line (QSG7701) by MTT assay (Table 5) with doxorubicin as a positive control. Among these isolates, the mixture of abiespirones F1 and F2 (6a and 6b) showed the strongest inhibitory activity against three hepatoma cell lines. In these three hepatoma cell lines, the Huh7 cells were more sensitive to treatment with compound 6 than HepG2 and SMMC7721 cells; the 50% inhibitory concentrations of cell viability were 7.5 (Huh7), 9.8 (HepG2), and 10.7 μM (SMMC7721) after 24 h of treatment. Normal hepatic cells QSG7701 were less sensitive to the inhibitory effects of the compound than three human hepatoma cell lines, indicating its selective cytotoxicity for cancer cells (Figure 7A). Although the MTT assay provided a general view of the cytotoxicity of the compound, the mechanisms of action are still not clear. Cell cycle arrest and apoptosis are considered to be the major mechanisms for reducing the initiation and progression of cancers. To elucidate the mechanisms responsible for compound 6-induced cell death, we investigated the effects of compound 6 on apoptosis and cell cycle progression in Huh7 cells. To determine if compound 6 arrested cell cycle progression, Huh7 cells were exposed to various concentrations of compound 6 for 24 h, and the distribution of cells in the cycle was determined by flow cytometric analysis. As shown in Figure 7B and C, there was a progressive increase in the percentage of cells in the G2/M phase, whereas there was a significant decrease in the number of cells in the G0/G1 and S phases. Moreover, compound 6 induced a dose-dependent increase in Huh7 cells undergoing apoptosis (Figure 8A and B). These results confirmed that the cytotoxicity of compound 6 to Huh7 cells is accomplished through the arrest of G2/M cell cycle progression and apoptosis induction. Many studies have shown that reactive oxygen species can induce apoptotic cell death in various types of cancer cells after treatment with anticancer drugs.22−27 Therefore, we next investigated whether compound 6-induced apoptosis is associated with elevated ROS levels in Huh7 cells. When Huh7 cells were treated with compound 6 for 24 h, the DCFHDA fluorescence shifted to a higher intensity, indicating an increase in ROS levels (Figure 9A). Furthermore, we pretreated Huh7 cells with the ROS scavenger NAC at 10 mM for 1 h followed by compound 6 (20 μM) treatment for an additional 24 h. As shown in Figure 9B, the viability of the Huh7 cells was rescued by the ROS scavenger NAC. These data suggested that intracellular ROS is involved in compound 6-induced apoptotic cell death in Huh7 cells.

Figure 5. Key 1H−1H COSY, HMBC, and NOESY correlations for 7a.

as 8α,9α;17β,23-diepoxy-7β-phenylacetoxy-3-oxolanosta26,23R-olide and 8α,9α;17β,23-diepoxy-7β-phenylacetoxy-3oxolanosta-26,23S-olide, and they were named abiespirones G1 and G2, respectively. Compounds 8a and 8b were assigned the molecular formula C30H44O6 based on 13C NMR data and the m/z 501.3124 [M + H]+ ion in the positive HRESIMS, indicating nine indices of hydrogen deficiency. The IR spectrum suggested the presence of carboxylic groups (1772, 1720, and 1710 cm−1) and a hydroxy group (3401 cm−1). The 1H and 13C NMR data of 8a and 8b resembled those of the known compound spiroveitchionolide except that the C-17 side chain was changed to an oxaspirolactone moiety (δC 99.8/100.0, 111.6/112.7, and 178.7/179.1).3 The key HMBC correlations from H3-19 to C-9 (δC 63.6/63.7 s), from H3-30 to C-8 (δC 217.7/218.1 s) and C-14, and from H-7 to C-6, C-8, C-9, and C-10, together with the COSY correlation from H-7 to H-6, indicated the presence of a 7(8→9)abeo-spiro-B/C ring (Figure 6). The β-

Figure 6. Key 1H−1H COSY, HMBC, and NOESY correlations for 8a.

orientation of the C-7 hydroxy moiety was deduced from the NOESY correlation of H-7 with H-5α. In the NOESY spectrum, H-7 correlated with H3-30, indicating that the C-9 spiro carbons in compounds 8a and 8b were R*-configured. Hence, the structures of compounds 8a and 8b were identified as 17β,23-epoxy-7β-hydroxy-3,8-dioxo-7(8→9)abeo-lanosta26,23R-olide and 17β,23-epoxy-7β-hydroxy-3,8-dioxo-7(8→9) abeo-lanosta-26,23S-olide, which were named abiespirones H1 and H2, respectively. Eleven known triterpenoids were also isolated from the branches and leaves of A. faxoniana. By comparing observed and reported 1H and 13C NMR and MS data, the known compounds were identified as spiroveitchionolide (9),3,18 neoabiestrines A (10) and C (11),6 abiesatrines D (12) and E (13),19 neoabieslactones A (14) and E (15),5 3α-hydroxy-9βlanosta-7,24-dien-26,23R-olide (16),10 3-oxo-9β-lanosta-7,24dien-26,23R-olide (17),10 (22Z)-3-oxo-9β-lanosta-7,22,2455

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Table 4. 13C (125 MHz) NMR Spectroscopic Data of Abiespirones E−H in CDCl3 no.

5a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′

35.9 29.7 77.0 39.5 43.1 21.0 25.8 48.9 20.0 26.5 25.6 27.4 49.0 49.6 25.3 36.6 99.7 14.8 28.5 41.0 20.5 45.0 113.4 45.0 35.6 179.4 18.2 25.8 21.1 20.5

5b t t d s d t t d s s t t s s t t s q t d q t s t d s q q q q

36.0 29.7 77.2 39.4 41.6 21.0 25.7 49.2 20.0 26.3 25.5 27.7 49.5 49.8 25.3 36.5 99.6 16.6 28.6 41.1 20.8 45.3 113.8 45.1 35.7 178.8 17.1 25.7 21.2 20.9

6a t t d s d t t d s s t t s s t t s q t d q t s t d s q q q q

35.9 29.6 79.0 38.7 43.1 20.9 25.8 49.0 20.1 26.3 25.5 26.1 49.0 49.5 25.2 36.6 99.6 14.9 28.1 42.1 20.5 45.0 113.4 44.9 35.6 179.4 18.2 25.4 21.3 20.6 170.8 20.7

6b t t d s d t t d s s t t s s t t s q t d q t s t d s q q q q s q

35.3 29.8 77.4 37.6 42.8 21.0 25.7 49.3 20.1 26.3 25.7 26.1 49.2 49.8 25.3 36.4 100.8 17.1 28.7 42.2 20.4 45.2 113.8 43.5 35.7 179.5 18.3 25.4 21.3 20.5 170.9 20.9

7a t t d s d t t d s s t t s s t t s q t d q t s t d s q q q q s q

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Table 5. Cytotoxic Activity of Triterpenoids from A. faxoniana against Human Hepatoma Cell Lines

HepG2

Huh7

SMMC7721

QSG7701

1a and 1b 2a and 2b 3a and 3b 4a and 4b 5a and 5b 6a and 6b 7a and 7b 8a and 8b 9 10 11 12 13 14 15 doxorubicin

14.1 31.2 >50 >50 21.1 9.8 >50 15.4 20.6 >50 >50 24.0 >50 >50 16.7 0.5

13.5 >50 16.4 18.4 16.0 7.5 >50 12.3 35.1 >50 >50 >50 >50 40.1 20.9 0.7

20.7 >50 19.1 28.3 25.2 10.7 >50 10.1 >50 >50 >50 >50 >50 34.6 13.4 0.3

15.5 26.8 17.6 >50 >50 >50 >50 8.2 >50 42.4 >50 >50 >50 37.0 10.1 2.9

7b t t s s d t d s s s t t s s t t s q q d q t s t d s q q q q s t s d d d d d

34.7 34.0 217.0 45.8 45.0 20.8 71.4 70.5 69.7 36.0 24.5 24.5 50.8 46.2 27.7 30.5 100.9 19.6 20.1 37.6 16.2 45.5 111.6 44.1 35.6 179.9 15.1 26.6 21.9 20.3 169.6 42.3 133.3 129.5 128.7 127.4 128.6 129.5

8a t t s s d t d s s s t t s s t t s q q d q t s t d s q q q q s t s d d d d d

29.0 24.4 214.0 46.3 50.8 34.9 79.1 218.1 63.6 51.0 29.6 34.1 48.2 59.4 29.6 29.7 99.8 19.4 19.1 37.0 19.6 45.6 111.6 44.2 35.6 179.1 14.8 29.4 22.3 20.0

8b t t s s d t d s s s t t s s t t s q q d q t s t d s q q q q

29.1 25.0 213.3 46.5 50.9 34.8 79.1 217.7 63.7 51.7 29.6 34.2 48.3 59.4 29.6 29.6 100.0 18.0 18.0 36.7 19.5 45.6 112.7 40.5 35.8 178.7 16.3 29.4 22.2 19.8

t t s s d t d s s s t t t s t t s q q d q t s t d s q q q q

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined using a Buchi 510 capillary melting point apparatus. Optical rotations were obtained with a JASCO P-2000 polarimeter. UV spectra were obtained on a Shimadzu UV-2550 spectrometer. IR spectra were recorded on a Bruker FTIR Vector 22 spectrometer using KBr pellets. 1D and 2D NMR spectra were determined with a Bruker Avance 500 spectrometer. ESIMS were acquired on an Agilent LC/ MSD Trap XCT mass spectrometer. HRESIMS were measured using an Agilent 6520 Accurate-Mass Q-TOF LC/MS. Column chromatography (CC) was performed on silica gel (100−200, 200−300 mesh, Shandong, China), Sephadex LH-20 (GE Healthcare, Sweden), and YMC 50 μm ODS-A (Milford, MA, USA). Preparative TLC (0.4−0.5 mm, 20 × 20 cm) was conducted with glass plates precoated with silica gel GF254 (Yantai, Shandong, China). A semipreparative column (Agilent ZORBAX SB-C18, 5 μm, 9.4 × 250 mm) and an analytical column (Agilent ZORBAX Extend-C18, 5 μm, 4.6 × 250 mm) were used for HPLC (Shimadzu LC-2010A HT). HPLC grade MeOH was purchased from Thermo Fisher Scientific. Water was purified by a Millipore Milli-Q system (Bedford, MA, USA). Other reagents were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Cell culture supplies and media, phosphate-buffered saline (PBS), fetal

IC50 (μM) compounds

34.7 33.8 216.9 45.9 44.9 21.1 71.5 70.6 69.6 35.7 24.6 24.6 50.9 46.1 27.8 30.6 100.8 19.6 20.1 37.3 16.0 45.6 111.3 44.2 35.7 179.1 14.8 26.5 21.9 20.2 169.9 42.3 133.3 129.4 128.6 127.3 128.6 129.4

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Figure 7. (A) Cell viability results after treating Huh7 and QSG7701 cells with 5, 10, and 20 μM compound 6 for 24 h. The results are the means ± SEM from three independent experiments. (B) Effect of compound 6 on the cell cycle in Huh7 cells. The DNA content at each cell cycle phase was analyzed by flow cytometry. The flow cytometric histograms are from Huh7 cells incubated with different concentrations of compound 6. (C) Cell cycle effects are presented as the percent distribution of specific phases for Huh7 cells. The data are expressed as the means ± SEM of three independent experiments. *p < 0.05; **p < 0.01 vs the control. bovine serum (FBS), sodium pyruvate, N-acetyl cysteine (NAC), nonessential amino acids (NEAA), and penicillin−streptomycin were obtained from Sigma (St. Louis, MO, USA). Plant Material. The branches and leaves of A. faxoniana were collected from Li County, Sichuan Province, in August 2009 and authenticated by Prof. Han-Ming Zhang in the Department of Pharmacognosy, Second Military Medical University. A voucher specimen (20090813001) was deposited at the Herbarium of the School of Pharmacy, Second Military Medical University, Shanghai, China. Extraction and Isolation. The air-dried branches and leaves of A. faxoniana (5.3 kg) were powdered and extracted with 80% EtOH (3 × 15 L) under reflux. The solvent was evaporated to dryness under reduced pressure to afford a crude EtOH extract (635 g), which was suspended in H2O and partitioned successively with petroleum ether (PE), DCM, EtOAc, and n-BuOH, respectively. The DCM extract (100.0 g) was subjected to silica gel CC (⦶ 8 × 100 cm; 100−200 mesh, 1000 g) eluted with gradient PE/EtOAc (50:1 to 1:1) to give 10 fractions (Fr.1−Fr.10) based on TLC analysis. Fr.2 (35.0 g) was chromatographed (⦶ 4.5 × 60 cm) on silica gel (200−300 mesh, 700 g) eluted with a gradient of PE/EtOAc (20:1 to 1:1) to afford five subfractions (Fr.2-1−Fr.2-7). Subfraction Fr.2-2 was subjected to semipreparative HPLC (MeOH/H2O, 40:60) to give 10 (60.8 mg), 12 (40.5 mg), and 14 (30.0 mg). Fr.4 (25.5 g) was subjected to silica gel CC (⦶ 4.5 × 60 cm; 200−300 mesh, 500 g) eluted with gradient PE/ EtOAc (5:1 to 1:1) to give 11 subfractions (Fr.4-1−Fr.4-11). From subfraction Fr.4-5 (2.9 g), compounds 2a/2b (80.9 mg) and 8a/8b (23.0 mg) were isolated after CC over Sephadex LH-20 (⦶ 4.0 × 150 cm; MeOH) followed by semipreparative HPLC (MeOH/H2O, 45:55). Fr.4-7 (3.3 g) was chromatographed on silica gel (⦶ 2.5 × 30 cm; 50 g, 200−300 mesh) eluted with gradient PE/EtOAc (5:1 to 1:1) followed by semipreparative HPLC (MeOH/H2O, 40:60) to give 3a/ 3b (120.0 mg) and 11 (30.4 mg). Compounds 13 (50.0 mg) and 1a/

1b (2.0 g) were crystallized (MeOH) from Fr.4-8 (1.5 g) and Fr.4-10 (1.9 g), respectively. Fr.4-10 (2.0 g) was chromatographed on a silica gel column (⦶ 2.5 × 30 cm; 50 g, 200−300 mesh) eluted with gradient DCM/MeOH (1:0 to 1:1) to give four subfractions (Fr.410a−Fr.4-10d). Fr.4-10d was subjected to semipreparative HPLC (MeOH/H2O, 45:55) to give 15 (15.0 mg) and 19 (14.0 mg) and further eluted with (MeOH/H2O, 60:40) to yield 7a/7b (80.0 mg) and 4a/4b (90.0 mg). Compounds 17 (18.0 mg), 18 (20.0 mg), and 9 (10.0 mg) were isolated after CC over silica gel (⦶ 2.5 × 20 cm; 30 g, 200−300 mesh) followed by semipreparative HPLC (MeOH/H2O, 55:45) from Fr.4-11 (0.9 g). Fr.6 (10.5 g) was subjected to semipreparative HPLC (MeOH/H2O, 40:60) to give 5a/5b (30.3 mg), 16 (21.5 mg), and 6a/6b (35.6 mg). Compounds 1a and 1b: colorless, orthorhombic crystals (methanol); mp 185−187 °C; [α]20D +101 (c 1, CHCl3); UV (MeOH) λmax (log ε) 202.9 (2.96) nm; IR (KBr) νmax 2965, 1772, 1720, 1647, 1308, 1203, 1166, 1084, 884 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS (positive) m/z 469.3244 [M + H]+ (calcd for C30H44O4, 469.3240). Compounds 2a and 2b: amorphous powder; [α]20D +54 (c 1, CHCl3); UV (MeOH) λmax (log ε) 252.0 (3.10) nm; IR (KBr) νmax 2970, 1773, 1720, 1711, 1651, 1383, 1216, 910 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS (positive) m/z 483.3017 [M + H]+ (calcd for C30H42O5, 483.3032). Compounds 3a and 3b: amorphous powder; [α]20D −38 (c 1, CHCl3); UV (MeOH) λmax (log ε) 203.9 (3.18) nm; IR (KBr) νmax 3383, 1772, 1646, 1383, 906, 881 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS (positive) m/z 471.3393 [M + H]+ (calcd for C30H46O4, 471.3396). Compounds 4a and 4b: amorphous powder; [α]20D +98 (c 1, CHCl3); UV (MeOH) λmax (log ε) 203.1 (3.01) nm; IR (KBr) νmax 1770, 1722, 1380, 1301, 1011, 874 cm−1; 1H and 13C NMR data, see 57

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Figure 8. Compound 6-induced apoptosis in Huh7 cells. (A) The apoptotic cells were determined by flow cytometry with the annexin V and PI double staining kit. The horizontal and vertical axes represent annexin V and PI, respectively. The flow cytometric pictures were taken with Huh7 cells that had been incubated with different concentrations of compound 6. (B) Compound 6 induced apoptosis in Huh7 cells in a dose-dependent manner. The data are expressed as the means ± SEM of three independent experiments with similar results. *p < 0.05; **p < 0.01 vs the control.

Figure 9. ROS involvement in compound 6-induced Huh7 cell apoptosis. (A) Huh7 cells were treated with different concentrations of compound 6, and then DCFH-DA fluorescence intensity was detected by flow cytometry. The increased fluorescence of DCFH-DA was determined as increased intracellular ROS accumulation. (B) Huh7 cells were pretreated with ROS scavenger NAC at 10 mM for 1 h and incubated with 20 μM compound 6 for 24 h, and the cell viability was determined by MTT assay. *p < 0.05; **p < 0.01 vs the control. Tables 1 and 3; HRESIMS (negative) m/z 469.3411 [M − H]− (calcd for C30H46O4, 469.3396). Compounds 5a and 5b: amorphous powder; [α]20D −20 (c 1, CHCl3); UV (MeOH) λmax (log ε) 205.0 (3.63) nm; IR (KBr) νmax 3343, 2963, 1770, 1281, 1078, 856 cm−1; 1H and 13C NMR data, see Tables 2 and 4; HRESIMS (positive) m/z 471.3401 [M + H]+ (calcd for C30H46O4, 471.3396). Compounds 6a and 6b: amorphous powder; [α]20D −65 (c 1, CHCl3); UV (MeOH) λmax (log ε) 202.0 (2.97) nm; IR (KBr) νmax 1772, 1770, 1382, 1209, 916, 878 cm−1; 1H and 13C NMR data, see

Tables 2 and 4; HRESIMS (positive) m/z 513.3506 [M + H]+ (calcd for C32H48O5, 513.3502). Compounds 7a and 7b: amorphous powder; [α]20D +70 (c 1, CHCl3); UV (MeOH) λmax (log ε) 203.9 (3.21) nm; IR (KBr) νmax 1770, 1769, 1720, 1585, 1506, 1156, 987, 861 cm−1; 1H and 13C NMR data, see Tables 2 and 4; HRESIMS (positive) m/z 619.3569 [M + H]+ (calcd for C38H50O7, 619.3557). Compounds 8a and 8b: amorphous powder; [α]20D +31 (c 1, CHCl3); UV (MeOH) λmax (log ε) 203.9 (3.06) nm; IR (KBr) νmax 3401, 2878, 1772, 1720, 1710, 1390, 1103, 986 cm−1; 1H and 13C 58

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NMR data, see Tables 2 and 4; HRESIMS (positive) m/z 501.3124 [M + H]+ (calcd for C30H44O6, 501.3138). X-ray crystallography for compound 1a: orthorhombic crystal of C30H44O4, M = 468.65, space group P2(1)2(1)2(1), a = 6.9472(14) Å, α = 90°; b = 17.137(3) Å, β = 90°; c = 22.894(5) Å, γ = 90°, V = 2725.6(9) Å3, Z = 4, Dcalcd = 1.142 mg/m3, crystal size 0.42 × 0.16 × 0.13 mm, Cu Kα (λ = 1.541 78 Å), F(000) = 1024, T = 296(2) K, Flack parameter = 0.10 (14). The final R values were R1 = 0.0576, and wR2 = 0.1656, for 12 523 observed reflections (θ = 3.86° to 65.97°) [I > 2σ(I)]. Crystallographic data for 1a have been deposited at the Cambridge Crystallographic Data Centre with the deposition number CCDC 1021527. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44-1223-336033 or e-mail: [email protected]]. Cell Lines and Cell Culture. The human hepatoma cell lines (HepG2, Huh7, and SMMC7721) as well as the normal cell lines (QSG7701) were obtained from Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C, 5% CO2. Cell Viability Assay. Cell viability was determined by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.28 Cells were plated on a 96-well plate at 6 × 103 cells/well and exposed to the test compounds (0, 1, 5, 10, 25, 50 μM) for 24 h. Cultures were also treated with 0.1% DMSO as the vehicle control. After 24 h of treatment, 10 μL of MTT solution (5 mg/mL) was added to each well and the plates were incubated for 2−4 h at 37 °C. The supernatant was then removed from formazan crystals, and 100 μL of DMSO was added to each well. The absorbance at 570 nm was read using an OPTImax microplate reader. The cell viability was calculated by dividing the mean optical density (OD) of compoundcontaining wells by that of 0.1% DMSO-control wells. Cell Cycle Assay. The DNA content of cells in the G0/G1, S, and G2/M phases can be determined by flow cytometry.29 Briefly, Huh7 cells were incubated with the test compound (5, 10, and 20 μM) for 24 h. After 24 h of treatment, the cells were collected and washed with PBS containing 2% FBS. A total of 3 × 105 cells/mL were fixed with cold absolute EtOH overnight at 4 °C in a 15 mL polypropylene and V-bottomed tube. After washing with PBS twice, the cells were incubated with 1.0 mL of propidium iodide (PI) staining solution (3.8 mM sodium citrate, 20 μg/mL PI in PBS). Then 50 μL of RNase A solution (10 μg/mL RNase A) was added, and the cells were incubated for 30 min at room temperature in the dark. The DNA contents were analyzed using flow cytometry. Cell Apoptosis Assay. The apoptotic cells were measured by flow cytometry using the annexin V-FITC and PI double staining kit.30 Briefly, Huh7 cells were treated with the test compound (5, 10, and 20 μM) for 24 h. After 24 h of treatment, the cells were collected, washed with PBS, and resuspended in 200 μL of binding buffer containing 5 μL of annexin V-FITC (10 μg/mL) for 10 min in the dark. The cells were incubated with 10 μL of PI (20 μg/mL), and the samples were immediately analyzed by flow cytometry. Intracellular ROS Production Measurement. ROS levels were detected using a flow cytometer and a microplate spectrophotometer. Briefly, Huh7 cells were treated with the test compound (5, 10, and 20 μM) for 24 h. After 24 h of treatment, cells were harvested and washed with PBS and suspended in DMEM containing 10 μM 5(6)-carboxy2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA; Invitrogen) at 37 °C for 30 min. The cells were washed again, and flow cytometry analysis was performed.31 Statistical Analysis. The data are expressed as the means ± SEM. Data were analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett’s test. A value of p < 0.05 was considered significant.

Article

ASSOCIATED CONTENT

S Supporting Information *

HRESIMS and 1D and 2D NMR spectra of the new triterpenoids abiespirones A−H are presented. The HPLC plots of abiespirones A and B are also presented. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(Y. H. Shen) E-mail: [email protected]. Tel: +8621-81871244. *(W. D. Zhang) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by the NCET Foundation, NSFC (81230090), Shanghai Leading Academic Discipline Project (B906), Key Laboratory of Drug Research for Special Environments, PLA, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300), the Scientific Foundation of Shanghai China (12401900801, 13401900101), National Major Project of China (2011ZX09307-002-03), and the National Key Technology R&D Program of China (2012BAI29B06).

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