Bioactive eudesmane and germacrane derivatives from Inula wissmanniana Hand.-Mazz.

Phytochemistry 96 (2013) 214–222

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Bioactive eudesmane and germacrane derivatives from Inula wissmanniana Hand.-Mazz. Xiang-Rong Cheng a,b, Shou-De Zhang c, Chun-Hui Wang a, Jie Ren a, Jiang-Jiang Qin a, Xue Tang b, Yun-Heng Shen d, Shi-Kai Yan a, Hui-Zi Jin a,⇑, Wei-Dong Zhang a,d,⇑ a

School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu Province, PR China School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China d School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China b c

a r t i c l e

i n f o

Article history: Received 23 January 2013 Received in revised form 28 May 2013 Available online 1 November 2013 Keywords: Inula wissmanniana Hand.-Mazz. Asteraceae Eudesmane Germacrane NO production Cytotoxicity

a b s t r a c t Phytochemical investigation of Inula wissmanniana Hand.-Mazz. afforded 21 eudesmane and germacrane derivatives, including rare 4,5-secoeudesman-12,5-olide, eudesman-12,5-olide, 3,4-secoeudesman-12oic acid, and germacra-4-en-12,6-olides. Their structures were elucidated by combinative analyses of MS, NMR, electronic circular dichroism, and X-ray crystallography data. Moreover, most of the isolates exhibited inhibition against lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages and cytotoxicity in HepG2, PC-3, and MGC-803 tumor cells. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The genus Inula (Asteraceae family) is distributed throughout Asia, Europe, and Africa, comprising a number of medicinal plants. Half of the Inula species in China, ca. 10 species, are mainly employed as expectorants, antitussives, diaphoretics, antiemetics, and bactericides (Beijing Institute of Botany, 1979; Cheng et al., 2011; Zhao et al., 2006). Sesquiterpenoids, a group of structurally diverse secondary metabolites, are major constituents of plants belong to the genus Inula, of which this remarkable biological activity has attracted much attention (Cheng et al., 2011; Cheng et al., 2012; Gökbulut et al., 2012; Khan et al., 2010; Qin et al., 2010; Qin et al., 2011b; Zhao et al., 2006). Previous pharmacological studies have demonstrated significant effects of sesquiterpenoids isolated from Inula species, such as anti-inflammatory (Cheng et al., 2011; Cheng et al., 2013; Qin et al., 2010; Qin et al., 2011b; Zhao et al., 2006), cytotoxic (Cheng et al., 2012; Cheng et al., 2013; Zhao et al., 2006), antiprotozoal (Gökbulut et al., 2012), and allelochemical properties (Khan et al., 2010).

⇑ Corresponding authors at: School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China. Tel./fax: +86 021 34205989 (W.-D. Zhang). E-mail addresses: [email protected] (H.-Z. Jin), [email protected] (W.-D. Zhang). 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.10.006

As a part of an ongoing research program for bioactive sesquiterpenoids derived from Inula species, Inula wissmanniana Hand.-Mazz., a plant endemic to Yunnan province, PR China, was examined as there were no phytochemical studies reported previously. In this contribution, twelve new and nine known eudesmane and germacrane derivatives were isolated. Their structures were assigned by spectroscopic analyses. Compound 1 is a rare 4,5-secoeudesman-12,5-olide whose absolute configuration was determined by electronic circular dichroism (ECD) analysis. Compound 2 is a rare eudesman-12,5-olide as well, while 3 is the first naturally occurring 3,4-secoeudesmane. Compounds 4–8, containing a cyclodec-4-ene system, differ from most of the natural germacran-12,6-olides which feature double bonds or epoxides at carbons 1(10) and 4(5). The absolute stereochemistries of 4, 6, 9, 12, and 18 were further assigned by X-ray crystallographic studies using Cu Ka diffraction. Moreover, all isolates were tested for their inhibition against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages and cytotoxicity in HepG2, PC-3, and MGC-803 tumor cells. 2. Results and discussion Twelve new and nine known sesquiterpenoids (Fig. 1) were isolated from the CH2Cl2 partitioned ethanolic extract of the whole plants of I. wissmanniana by using column chromatographic meth-

X.-R. Cheng et al. / Phytochemistry 96 (2013) 214–222

215

Fig. 1. Structures of compounds 1–21.

ods. The known sesquiterpenoids were identified as inulacappolide (13) (Xie et al., 2007), haageanolide (14) (Bohlmann et al., 1978), maroniolide (15) (Bohlmann and Grenz, 1979), 1b-hydroxyalantolactone (16) (Bohlmann et al., 1978), ivangustin (17) (Herz et al., 1967), 11,13-dehydroisohyposantonin (18) (Srikrishna, 1987), isohyposantonin (19) (Huang, 1948), 5a-hydroxyasperilin (20) (Bohlmann et al., 1981), and 4a,15-dihydro-5a-hydroxyasperilin (21) (Qin et al., 2010), respectively, by comparing their physical and spectroscopic data with those reported in the literature. Furthermore, the absolute stereochemistry of 18 was established as 6R,7S by X-ray crystallographic analysis. Compound 1 was obtained as a colorless oil. Its molecular formula was established to be C15H18O3 by means of HRESIMS (m/z 269.1159 [M+Na]+) and NMR spectroscopy. The IR absorptions implied the presence of carbonyls (1726 cm1) and olefinic bonds (1629 cm1). In accordance with the IR absorptions, one ester carbonyl (dC 163.5), two olefinic quaternary carbons (dC 150.6 and 138.0), three olefinic methines (dC 133.0, 122.9, and 100.4), and one olefinic methylene (dC 127.4) were observed in the 13C NMR and DEPT spectra, accounting for four degrees of unsaturation. The residual carbon resonances in the 13C NMR spectrum were also classified by DEPT and HSQC experiments to be two methyls (dC 22.0 and 22.9), three methylenes (dC 32.1, 29.3, and 35.3), one methine (dC 36.2), and two quaternary carbons (dC 107.2 and 47.8). On the basis of the assignments as above, the remaining three degrees of unsaturation suggested a tricyclic ring system in 1. In its 1H NMR spectrum, the spin couplings and splittings of H-1–H-3 (Table 1) indicated a H-1/H-2/H-3 consecutive proton system which was further confirmed by 1H–1H COSY analysis, while those of the methyls (CH3-14 and CH3-15) suggested the connections from methyls to quaternary carbons (C-4 and C-10) which were further supported by HMBC data (Fig. 2). Moreover, the proton sequence H2-6/H-7/H2-8/H2-9 deduced from extensive analysis of the 1H–1H COSY spectrum, together with the HMBC cross-peaks from H-7 (dH 2.98) to C-5, C-12, and C-13, from H2-13 (dH 6.47 and 5.59) to C-7 and C-12, from H3-14 (dH 1.11) to C-1, C-5, and C-9, and from H3-15 (dH 1.95) to C-3 (Fig. 2) estab-

lished the structure of 1 to be a 4,5-secoeudesmane with a rare sixmembered 12,5-olide moiety. Strong NOESY correlations from H-7 to H2-6, together with the coupling constant between them (3.2 Hz), established an a-oriented H-7 and a cis-fused lactone (Fig. 2), while the lack of correlations from H-7 and H2-6 to H3-14 indicated a b-oriented methyl moiety. A theoretical calculation of the electronic circular dichroism (ECD) spectrum performed using the time-dependent density functional theory (DFT) method in Gaussian 03 software (Crawford, 2006; Frisch et al., 2004; Ishida et al., 2006; Qin et al., 2011a) was applied to determine the absolute configuration of 1. The conformational diversity of 1a and 1b was however ignored, due to the rigid nature of the skeleton (Berova et al., 2007; Gao et al., 2012). 1a and 1b were optimized by using DFT at the B3LYP/6-31G⁄ level to afford putative preferred conformers. The ECD spectra of 1a and 1b were calculated at the B3LYP/6-31G⁄//B3LYP/6-31G⁄ level with the COSMO model in MeOH solution. As depicted in Fig. 3, the calculated ECD spectrum for 1a (5R,7R,10S enantiomer) matched with the experimentally determined spectrum. Hence compound 1 was assigned the structure (5R,7R,10S)-4,5-epoxy-4,5-secoeudesma1,3-dien-12,5-olide. Compound 2 was obtained as a white amorphous powder with a molecular formula C15H20O2 established by HRESIMS (m/z 255.1366 [M+Na]+). The similarity of 1H and 13C NMR spectroscopic data between 1 and 2 (Table 1), especially those for C-11, C-12, and CH2-13 indicated that 2 was also a eudesman-12,5-olide. Extensive analyses of the 1H–1H COSY and HMBC spectra supported the assignment as above and established the structure of 2. HMBC correlations from H3-15 (dH 1.74) to C-3 and C-4 indicated a ring double bond at 3(4). In the NOESY spectrum, strong correlations between H-7 and H2-6, together with no correlation between H-7 and H3-14, indicated the relative configuration of 2 was consistent with that of 1. Hence compound 2 is eudesma-3,11(13)-dien12,5b-olide. Compound 3 was obtained as a white amorphous powder with a molecular formula C15H20O4 as deduced from HRESIMS (m/z 287.1263 [M+Na]+). Its IR spectrum showed absorption bands of

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Table 1 H (400 MHz) and

1

Position

C (100 MHz) NMR spectroscopic data for compounds 1–3 and 8 (in CDCl3, d in ppm).

1

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

13

2

3

8

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

5.40 d (11.6)

133.0

31.0

6.89 d (15.8)

166.9

122.9

22.5

6.10 dd (15.8, 7.6)

129.9

4.97 d (7.6)

100.4

123.0

9.57 d (7.6)

194.1

1.34 m 0.95 m 1.68 m 1.68 m 2.30 m 2.30 m

23.1

5.67 dd (11.6, 7.6)

1.25 m 1.25 m 2.14 m 2.07 (overlap) 5.36 br s

2.45 dd (13.2, 3.6) 1.65 m 2.98 br t (3.2) 1.85 ddd (14.4, 7.2, 4.0) 1.50 m 1.61 m 1.53 m

6.47 5.59 1.11 1.95

150.6 107.2 32.1 36.2 29.3 35.3 47.8 138.0 163.5 127.4

br s br s s s

22.0 22.9

2.07 (overlap) 1.95 m 2.89 t (3.0) 1.91 m 1.59 m 1.60 m 1.35 m

6.50 5.55 1.03 1.74

br s br s s d (1.6)

135.7 86.4 33.2 35.1 29.6 32.3 36.8 138.7 166.3 127.8 22.3 17.5

2.70 dd (12.4, 2.0) 1.86 m 1.71 m 2.55 br t (11.6) 1.76 m 1.50 m 1.60 m 1.53 m

6.34 5.69 1.20 2.08

br s br s s s

209.3 58.0 29.4 38.4 26.6 39.0 39.7 145.0a 170.0a 126.0a 16.9 31.5

5.18 d (10.2) 4.59 dd (10.2, 10.0) 2.32 (overlap) 2.01 m 1.68 m 3.63 dd (11.6, 4.0) 1.87 m

6.24 5.56 1.02 1.81

d (3.0) d (3.0) d (6.8) s

18.6 35.9 141.3 125.3 81.8 45.8 32.2 77.3 30.7 139.6 170.3 118.8 17.4 16.9

Approximate data speculated from HSQC and HMBC spectra.

-1

Δε / M .cm

-1

Fig. 2. Key 1H–1H COSY, HMBC, and NOESY correlations of 1.

30 20 10 0 200 -10

250

300

350

400

λ/nm

-20 -30 Fig. 3. Stereoisomers of 1 and their calculated and experimental ECD spectra ( experiment in MeOH, 1a, 1b).

hydroxyl groups (3434 cm1), carbonyl groups (1686 cm1), and olefinic bonds (1629 cm1). Twelve clear carbon signals, including two quaternary carbons (one keto carbonyl), five methines (one aldehyde and two olefinic), three methylenes, and two methyls were observed in the 13C NMR and DEPT spectra, accounting for

C12H17O2. The 1H NMR spectrum exhibited two methyl singlets at dH 1.20 and 2.08, one pair of trans-olefinic protons at dH 6.89 (d, J = 15.8 Hz) and 6.10 (dd, J = 15.8, 7.6 Hz), and one aldehyde proton at dH 9.57 (d, J = 7.6 Hz). All protons were assigned to their corresponding carbons by an HSQC experiment. The 1H–1H COSY experiment established the following proton sequences: H-1/H-2/ H-3 and H-5/H2-6/H-7/H2-8/H2-9. The HMBC correlations from H3-14 to C-1, C-5, and C-9 and from H3-15 to C-5 further linked the proton sequences above. The residual three-carbon fragment connected to C-7 was presumed to be an a-methylene-c-carboxyl unit, as evidenced by the molecular formula determined, while olefinic protons were at dH 6.34 and 5.69 with EIMS fragments at m/z 45, 71, and 219. The coupling constants between H-5 and H2-6 (12.4, 2.0 Hz), H-7 and H2-6 and H2-8 (br t, 11.6 Hz) established that both H-5 and H-7 were vertical bonds in the chair conformation of cyclohexane. The key NOESY correlations from H-5 to H-1 further indicated a b-oriented H3-14. Hence compound 3 is 3,4-dioxo-5a,7aH-3,4-secoeudesma-1,11(13)-dien-12-oic acid. Compound 4 was isolated as colorless square crystals (CH2Cl2) with a molecular formula C22H30O6 as deduced from HRESIMS (m/ z 413.1943 [M+Na]+). Its IR spectrum exhibited absorption bands for carbonyls (1768 and 1726 cm1) and olefinic bonds (1661 cm1). All the 22 carbon signals in the 13C NMR spectrum were classified by DEPT and HSQC experiments as six quaternary carbons (three ester carbonyls and three olefinic), seven methines (three oxygenated and two olefinic), four methylenes (one olefinic), and five methyls, respectively. Seven of the double bond equivalents were accounted for by a typical a-methylene-c-lactone unit (dH 6.27 and 5.67; dC 169.8, 138.6, and 119.7) (Qin et al., 2010; Qin et al., 2011b), an angeloyl group (dH 6.09, 1.98, and 1.87; dC 167.4, 127.8, 138.5, 15.9, and 20.6) (Xie et al., 2007), an acetyl group (dH 2.06; dC 170.3 and 21.3), and a pair of ring double bond (dH 5.39; dC 138.2 and 128.0) (Table 2) and thus the molecule is monocarbocyclic. Extensive analyses of 1H–1H COSY and HMBC plots suggested the structure of 4 to be a germacranolide esterified by acetic acid and angelic acid moiety at C-2 and C-9, respectively. A NOESY experiment was applied to determine the relative configuration of 4. The strong cross-peaks from H3-15 to H-2 and H-6 indicated a b orientation, while the cross-peaks from H-7 to H-9 and H-10 indicated an a orientation. The key cross-peak from H-5 to H-7 indi-

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cated a trans-double bond at 4(5). Moreover, the coupling constant between H-6 and H-7 (10.4 Hz) is consistent with their trans-relationship (Maruyama et al., 1983). An X-ray analysis using Cu Ka radiation allowed the assignment of the absolute configuration as drawn (Fig. 4) with a Flack parameter of 0.05(14) (Flack, 1983). Thus, the structure of 4 is (2R,4E,6R,7S,9S,10R)-2-acetoxy-9-angeloyloxygermacra-4,11(13)-dien-12,6-olide. The spectroscopic data of 5 clearly established that it was the deacetyl-derivative of 4 (Table 2) and hence it was assigned the structure 4E-9b-angeloyloxy-2a-hydroxy-7a,10aH-germacra4,11(13)-dien-12,6a-olide. Detailed comparison of spectroscopic data between 6 and 5 (Table 2) indicated that 6 was the dehydroxyl-derivative of 5. The absolute stereochemistry of 6 was further determined by an X-ray analysis using Cu Ka radiation (Fig. 5). Hence compound 6 is (4E,6R,7S,9S,10R)-9-angeloyloxygermacra4,11(13)-dien-12,6-olide. Similar NMR spectroscopic data were observed between 7 and 6 (Table 2), except that the angeloyl group of 6 was found to be a methacryl group in 7. Likewise, comparing the molecular formula and NMR spectroscopic data of 8 with those of 6 (Tables 1 and 2) showed 8 to be a deangeloyl-derivative of 6. Detailed investigations of the 1H–1H COSY, HMBC, and NOESY plots of 7 and 8 further confirmed their structures to be 4E-9b-methacryloxy-7a, 10aH-germacra-4,11(13)-dien-12,6a-olide and 4E-9b-hydroxy7a,10aH-germacra-4,11(13)-dien-12,6a-olide, respectively. Compound 9 was obtained as a white amorphous powder with its molecular formula C15H20O4 deduced from HRESIMS (m/z 287.1263 [M+Na]+). Analysis of the spectroscopic data of 9 (Table 3) suggested that it was a eudesmane sesquiterpenoide with an amethylene-c-carboxylic acid moiety. The 1H–1H COSY, HMBC, and NOESY experiments further established its structure and its relative configuration. An X-ray analysis using Cu Ka radiation allowed assignment of the absolute stereochemistry as drawn Table 2 H (400 MHz) and

1

Position

1a 1b 2a 2b 3a 3b 4 5 6 7 8a 8b 9 10 11 12 13a 13b 14 15 Ac-1 2 Ang-1 2 3 4 1’ Meacr-1 2 3a 3b 1’

Fig. 4. X-ray crystal structure of 4 (ORTEP drawing).

(Fig. 6). Hence compound 9 is (1R,5S,7R,10R)-1-hydroxy-2-oxoeudesma-3,11(13)-dien-12-oic acid. Compound 10 was obtained as a white amorphous powder with a molecular formula of C15H18O5 as deduced from HRESIMS (m/z 301.1055 [M+Na]+) and 13C NMR data. The 1H and 13C NMR spectra showed typical as signals for an a-methylene-c-lactone unit [dH 6.24 (d, J = 1.0 Hz) and 5.72 (d, J = 1.0 Hz); dC 168.6, 139.2, and 122.3], a ring double bond [dH 6.52 (d, J = 10.4 Hz) and 6.01 (d, J = 10.4 Hz); dC 147.0 and 126.9], and a keto carbonyl group (dC 202.0) (Table 3). The residual two degrees of unsaturation, together with the quaternary carbon resonance at dC 48.7, indicated that 10 was a eudesmane sesquiterpene lactone. Analyses of 1H–1H COSY and HMBC plots established the structure of 10. The key HMBC cor-

13

C (100 MHz) NMR spectroscopic data for compounds 4–7 (in CDCl3, d in ppm).

4

5

6

7

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

1.73 br d (16.0) 1.50 m 5.34 m

30.1

1.71 br d (16.0) 1.41 m 4.38 m

33.1

1.50 m 1.04 m 1.68 m 1.68 m 2.30 m 2.30 m

24.6

1.52 m 1.06 m 1.69 m 1.69 m 2.32 m 2.32 m

24.5

2.85 dd (12.0, 7.8) 2.35 dd (12.0, 10.4) 5.39 br d (10.4) 4.56 dd (10.4, 10.4) 2.43 m 2.11 ddd (14.7, 3.5, 3.5) 1.47 m 4.78 dd (11.6, 2.8) 2.32 m

6.27 5.67 1.09 1.87

d (3.2) d (3.2) d (6.8) s

2.06 s

6.09 q (7.2) 1.98 br d (7.2) 1.87 br s

70.1 42.4 138.2 128.0 80.6 46.1 28.7 79.0 28.5 138.6 169.8 119.7 20.4 17.4 170.3 21.3 167.4 127.8 138.5 15.9 20.6

2.80 dd (12.1, 7.6) 2.31 m 5.34 br d (10.4) 4.54 dd (10.4, 10.2) 2.42 m 2.09 ddd (14.7, 3.4, 3.4) 1.47 m 4.74 dd (11.7, 2.9) 2.31 m

6.24 5.65 1.15 1.81

d (3.6) d (3.6) d (6.8) s

6.09 q (7.2) 1.98 dd (7.2, 1.5) 1.87 br s

68.4 46.1 139.3 127.1 81.0 46.2 28.9 79.6 28.4 138.8 170.1 119.8 21.3 17.5

167.7 128.0 138.5 16.0 20.8

5.21 br d (10.2) 4.59 dd (10.2, 10.0) 2.38 m 2.11 ddd (14.4, 3.2, 3.2) 1.63 m 4.82 dd (11.6, 3.6) 1.98 (overlap)

6.26 5.64 0.95 1.83

d (3.4) d (3.4) d (6.8) s

6.08 q (7.2) 1.98 d (7.2) 1.87 br s

18.7 35.9 141.4 125.4 81.5 45.8 28.9 78.5 30.0 139.0 170.1 119.5 17.2 17.0

5.21 d (10.4) 4.59 dd (10.4, 10.0) 2.36 m 2.09 m 1.63 m 4.78 dd (12.0, 2.8) 1.99 m

6.26 5.64 0.94 1.83

d (3.2) d (3.2) d (6.8) s

18.8 36.0 141.6 125.5 81.7 45.8 28.8 79.2 30.2 139.1 170.3 119.7 17.4 17.1

167.6 128.0 138.2 15.9 20.7

6.08 br s 5.56 t (1.6) 1.94 br s

167.2 136.6 125.7 18.4

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Fig. 6. X-ray crystal structure of 9. 1

Fig. 5. X-ray crystal structure of 6.

relation from H-6 to C-12 (dC 168.6) assigned 10 to be a eudesman12,6-olide. The strong NOESY correlations from H3-15 to H-7 and H-6 with no correlation from H3-14 to H-7 and H-6 suggested the relative configuration of 10. Thus, 10 was elucidated to be 4b,5a-dihydroxy-1-oxoeudesma-2,11(13)-dien-12,6b-olide. Compound 11 was also a eudesman-12,6-olide as evidenced by its molecular formula C15H16O3 deduced from HRESIMS (m/z 267.0994 [M+Na]+) and structural features established by 1D NMR spectra (Table 3). The presence of a 1,4-dien-3-one moiety was apparent (Table 3). The NOESY experiment also established the relative stereochemistry. The coupling constant between H-6 and H-7 (5.6 Hz) confirmed their cis-relationship (Arantes et al., 2010; Srikrishna, 1987). Thus, 11 is 3-oxoeudesma-1,4,11(13)-trien-12,6b-olide. Compound 12 was obtained as colorless square crystals with the molecular formula C15H18O2. Its 1D NMR spectrum indicated the presence of a symmetrical aromatic system and a characteristic a-methylene-c-carboxylic acid moiety (Table 3). Analyses of its

Table 3 H (400 MHz) and

1

Position

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

13

H–1H COSY and HMBC spectra established the structure of 12 to be a rearranged eudesmane with an aromatic ring. This was confirmed by an X-ray analysis using Cu Ka radiation which also established the absolute stereochemistry as drawn (Fig. 7). Thus compound 12 is (7R)-14(10 ? 1)-abeoeudesma-1,3,5(10),11(13)tetraen-12-oic acid. Sesquiterpenoids, especially those sesquiterpene lactones possessing a,b-unsaturated carbonyl moieties, have been reported as potential anti-inflammatory or antitumor agents (Cheng et al., 2011; Cheng et al., 2012; Qin et al., 2010; Qin et al., 2011b; Zhao et al., 2006). In continuation of a screening program for bioactive sesquiterpenoids, all isolates were tested for their inhibitory effects against LPS-induced NO production in RAW264.7 macrophages and cytotoxicity against HepG2, HeLa, PC-3, and MGC-803 cell lines. The IC50 values are listed in Table 4. All isolates showed no significant toxicity in RAW264.7 macrophages up to 20 lM as demonstrated by the MTT assay and cytological observation, but most exhibited strong inhibitory effects against NO production in this cell line. Compounds 1, 11, 14, and 18 gave strong NO inhibition effects with IC50 values of 0.68, 0.68, 0.97, and 0.65 lM, respectively. Eudesmanolides 16, 17, 20, and 21 displayed moderate NO inhibition, which is consistent with previously reported data (Qin et al., 2010). Germacranolides 4–6 exhibited moderate NO inhibition with IC50 values of 4.90, 2.17, and 5.09 lM, respectively, indicating that the hydroxyl group at C-2 of 5 might increase the inhibitory effects, supporting that the position of hydroxyl group in sesquiterpene lactones might be beneficial for NO inhibitory activity (Cheng et al., 2011). The appreciable cytotoxicity of compounds 13 and 16

C (100 MHz) NMR spectroscopic data for compounds 9–12 (in CDCl3, d in ppm).

9

10

dH (J in Hz)

dC

3.48 s

78.8 198.5 124.0 165.1 41.2 28.6

5.88 br s 2.86 br d (12.8) 2.11 m 1.39 m 2.62 m 1.78 m 1.61 m 2.11 m 1.34 m

6.72 6.37 0.87 1.93

br s br s s s

39.7 26.0 33.2 40.2 144.5 171.2 125.5 16.0 22.3

11

dH (J in Hz) 6.01 d (10.4) 6.52 d (10.4)

4.47 d (4.8) 3.24 m 1.89 m 1.57 m 1.99 m 1.78 m

6.24 5.72 1.48 1.57

d (1.0) d (1.0) s s

12

dC

dH (J in Hz)

dC

202.0 126.9 147.0 73.1 73.6 78.7

6.79 d (10.0) 6.28 d (10.0)

157.2 126.0 185.9 138.0 148.3 75.5

38.3 23.8

3.11 m 1.92 m 1.80 m 1.77 m 1.55 dd (14.0, 4.4)

26.8 48.7 139.2 168.6 122.3 21.3 24.7

5.41 d (5.6)

6.27 5.71 1.30 2.05

d (1.2) d (1.2) s s

40.8 24.4 33.8 38.9 140.3 169.9 121.9 25.1 10.7

dH (J in Hz) 6.94 s 6.94 s

2.99 m 2.46 dd (15.8, 10.5) 2.93 m 2.11 m 1.71 m 2.82 m 2.70 m

6.44 5.73 2.22 2.22

s s s s

dC 133.8 126.9 126.9 133.74 134.48 33.5 35.1 28.1 27.4 134.52 144.9 173.3 125.4 19.47 19.43

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conclusion, compounds 1–3 were shown to be a rare 4,5-secoeudesman-12,5-olide, a eudesman-12,5-olide, and a 3,4-secoeudesmane, respectively. Compounds 4–8 are germacranolides lacking the usual 1(10) unsaturation. The obtained IC50 values demonstrated that most of the isolated sesquiterpenoids exhibited strong inhibition against LPS-induced NO production in RAW264.7 macrophages and cytotoxicity in HepG2, PC-3, and MGC-803 tumor cells. 4. Experimental 4.1. General

Fig. 7. X-ray crystal structure of 12.

against human tumor cells has been reported previously (Xie et al., 2007; Qi et al., 2008) and was replicated in this study (Table 4). Moreover, the cytotoxicity assay in four human tumor cell lines suggested that half of the isolates, including new compounds 1, 4–8, and 11, showed strong toxicity against HepG2, PC-3, and MGC-803 cells, while most of the isolates showed weak toxicity against HeLa cells. Eudesmanes 3, 9, and 12 each possessing a-methylene-c-carboxylic acid moiety exhibited weak inhibitory effects against NO production in RAW264.7 macrophages (IC50 > 20 lM) and toxicity against the tested tumor cell lines (IC50 > 50 lM), supporting that the a-methylene-c-lactone is a structural prerequisite for the NO inhibitory and cytotoxic properties (Cheng et al., 2011; Cheng et al., 2012; Qin et al., 2010; Qin et al., 2011b).

Melting points were obtained using a RY-2 melting point apparatus (Tianjin Analytical Instrument Factory, Tianjin, China). Optical rotations were performed on a Jasco P-2000 polarimeter. IR spectra were recorded on Bruker FTIR Vector 22 spectrometer using KBr pellets. ESIMS were measured on an Agilent 1100 series mass spectrometer, whereas HRESIMS were acquired using a MAT212 magnetic sector mass spectrometer. EIMS was performed on a Autospec Premier P708. NMR experiments were recorded on a Bruker Avance III spectrometer using TMS as internal standard. Preparative HPLC was conducted on a Shimadzu LC-6AD series equipped with an SPD-20 spectrophotometer using Shimadzu PRC-ODS EV0233 column. Column chromatography (CC) separations were carried out by using silica gel (100–200 mesh and 200–300 mesh, Qingdao Haiyang Chemical Group Corporation, Qingdao, China), MCI gel CHP-20P (75–150 lm; Mitsubishi Chemical Industries Co., Ltd.), Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) as packing materials. Precoated silica GF254 plates were used for analytical TLC (Qingdao Haiyang Chemical Company, Ltd.).

3. Conclusion 4.2. Plant material Isolation, structural elucidation, and NO inhibition and cytotoxicity evaluation of twelve new and nine known eudesmane (1–3, 9–12, and 16–21) and germacrane (4–8 and 13–15) derivatives from the whole plants of I. wissmanniana are reported herein. In

Whole plants of I. wissmanniana were collected from Pingbian county, Yunnan province, PR China, in August 2010, and identified by Prof. Han-Ming Zhang (Department of Pharmacognosy, Second

Table 4 IC50 values of all isolates with regard to NO inhibition and cytotoxicity. Compounds

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Aminoguanidine Doxorubicin a b

NO inhibition (lM)a

Cytotoxicity (lM)b

RAW264.7

HepG2

HeLa

PC-3

MGC-803

0.68 ± 0.05 7.59 ± 0.37 >20 4.90 ± 0.16 2.17 ± 0.17 5.09 ± 0.31 1.09 ± 0.09 1.75 ± 0.12 >20 5.05 ± 0.41 0.68 ± 0.08 >20 10.28 ± 0.94 0.97 ± 0.05 4.93 ± 0.40 1.65 ± 0.13 5.14 ± 0.29 0.65 ± 0.08 5.78 ± 0.52 5.03 ± 0.30 2.51 ± 0.14 0.37 ± 0.06

3.46 ± 0.14 >50 >50 3.14 ± 0.12 2.68 ± 0.17 3.15 ± 0.29 14.44 ± 2.06 6.52 ± 0.45 >50 23.54 ± 2.47 3.37 ± 0.52 >50 12.82 ± 1.30 3.28 ± 0.25 7.02 ± 0.57 7.38 ± 0.64 39.09 ± 5.41 2.98 ± 0.22 >50 10.96 ± 0.93 40.10 ± 3.13

16.48 ± 2.34 >50 >50 >50 27.93 ± 2.77 47.05 ± 5.90 >50 >50 >50 >50 46.20 ± 4.71 >50 >50 >50 43.06 ± 3.47 34.87 ± 4.10 49.46 ± 5.15 5.68 ± 0.79 >50 >50 >50

7.76 ± 0.58 >50 >50 13.29 ± 1.07 17.96 ± 1.35 18.70 ± 1.63 34.44 ± 2.47 43.98 ± 3.81 >50 41.09 ± 4.03 >50 >50 47.76 ± 5.19 11.18 ± 0.92 11.96 ± 1.11 22.17 ± 1.68 44.22 ± 4.12 4.71 ± 0.33 >50 21.63 ± 0.15 38.93 ± 2.77

3.65 ± 0.18 25.78 ± 2.26 28.11 ± 2.49 3.49 ± 0.28 3.52 ± 0.31 15.03 ± 1.30 4.73 ± 0.37 17.33 ± 1.89 >50 12.79 ± 1.86 3.46 ± 0.25 >50 18.52 ± 1.38 3.30 ± 0.26 9.25 ± 0.42 3.41 ± 0.38 12.85 ± 0.97 3.37 ± 0.11 >50 9.71 ± 0.65 38.23 ± 2.14

0.17 ± 0.02

0.43 ± 0.05

0.32 ± 0.01

0.27 ± 0.01

Inhibitory effects of 1–21 against LPS-induced NO production in RAW264.7 macrophages (n = 4). Cytotoxicity of 1–21 against HepG2, HeLa, PC-3, and MGC-803 human tumor cells (n = 3).

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Military Medical University). A voucher specimen (No. DJ 20100801) has been deposited at School of Pharmacy, Shanghai Jiao Tong University, Shanghai, PR China.

4.3. Extraction and isolation Air-dried whole plants of I. wissmanniana (10.0 kg) were chopped into small pieces, these being percolated with EtOH– H2O (95:50, 5, v/v) four times (30 L, 24 h, 12 h, 12 h, 12 h, respectively) at room temperature to give a crude extract (631.4 g), which was suspended in H2O and then extracted with petroleum ether (PE, 60–90 °C) to remove the fatty components. The residual crude extract was further partitioned with CH2Cl2 to give a CH2Cl2-soluble fraction (179.6 g). An aliquot of the CH2Cl2-soluble fraction (119.2 g) was subjected to silica gel (100–200 mesh) CC (10  100 cm) eluted successively with CH2Cl2/MeOH gradient (100:0–0:1, v/v) to give eight fractions (A–H). Fraction A (17.0 g) was applied to a silica gel column (200–300 mesh, 6  70 cm) eluted with PE/EtOAc EtOAc (EtOAc) (20:1, v/v) to afford subfractions A1A6. Subfraction A2 afforded colorless square crystals (6, 415.6 mg), and the mother liquor was further purified by preparative HPLC (MeOH–H2O (70:30, v/v) as eluant) to yield compounds 1 (tR 22.3 min, 10.7 mg), 18 (tR 24.4 min, 77.7 mg), and 2 (tR 28.9 min, 1.5 mg), respectively. Subfraction A3 gave colorless square crystals (4, 3.2 mg), and the residual mother liquor was further purified by preparative HPLC ((MeOH–H2O 70:30, v/v) as eluant) to yield compound 19 (tR 26.6 min, 17.0 mg). Subfraction A4 was purified by preparative HPLC ((MeOH–H2O 70:30, v/v) as eluant) to give compounds 7 (tR 40.0 min, 2.5 mg) and 12 (tR 74.0 min, 26.0 mg). Fractions BD were applied to MCI gel column (4  40 cm) eluted with MeOH–H2O (80:20, v/v) to afford subfractions B1D1 (12.5 g, 9.1 g, and 9.3 g), respectively. Subfraction B1 was further purified over a Sephadex LH-20 column (3  100 cm) eluted with CH2Cl2/MeOH (1:1, v/v), following by preparative HPLC (MeOH– H2O, 55:45, v/v as eluant) to yield compounds 11 (tR 13.9 min, 7.0 mg), 14 (tR 21.3 min, 6.3 mg), 8 (tR 32.0 min, 25.6 mg), 13 (tR 58.2 min, 31.1 mg), and 5 (tR 75.0 min, 30.0 mg). Purification using a Sephadex LH-20 column and preparative HPLC (MeOH–H2O (1:1, v/v) as eluant) of subfraction C1 afforded compounds 10 (tR 11.0 min, 3.0 mg), 9 (tR 19.0 min, 5.0 mg), 21 (tR 34.2 min, 4.1 mg), 20 (tR 37.0 min, 3.2 mg), and 3 (tR 45.0 min, 6.4 mg) likewise. Sephadex LH-20 CC followed by preparative HPLC (MeOH– H2O (45:55, v/v) as eluant) of subfraction D1 afforded compounds 15 (tR 28.0 min, 3.0 mg), 16 (tR 35.0 min, 258.0 mg), and 17 (tR 40.0 min, 193.0 mg). The purity of all isolates (>95.0%) was determined by HPLC.

4.3.1. (5R,7R,10S)-4,5-Epoxy-4,5-secoeudesma-1,3-dien-12,5-olide (1) Colorless oil; C15H18O3; a20 D – 211.8 (c 0.10, MeOH); CD (c 2.03  104 M, MeOH) k (De) 256 (101.6); UV (MeOH) kmax (log e): 213 (2.83), 256 (2.83) nm; IR (KBr) mmax 2930, 1726, 1629, 1460, 1383, 1276, 1150, 1081, 1033 cm1; ESIMS m/z 269.1 [M+Na]+, m/z 245.1 [MH]; HRESIMS m/z 269.1159 [M+Na]+ (calcd for C15H18O3Na, 269.1154); for 1H and 13C NMR spectroscopic data, see Table 1.

4.3.2. Eudesma-3,11(13)-dien-12,5b-olide (2) White amorphous powder; C15H20O2; a20 D – 14.7 (c 0.10, CH2Cl2); UV (MeOH) kmax (log e): 211 (2.52) nm; IR (KBr) mmax 2927, 2861, 1715, 1627, 1458, 1383, 1303, 1230, 1180, 1152, 1102, 1033, 1000, 951 cm1; ESIMS m/z 255.1 [M+Na]+, m/z 231.1 [MH]; HRESIMS m/z 255.1366 [M+Na]+ (calcd for C15H20O2Na, 255.1361); for 1H and 13C NMR spectroscopic data, see Table 1.

4.3.3. 3,4-Dioxo-5a,7aH-3,4-secoeudesma-1,11(13)-dien-12-oic acid (3) White amorphous powder; C15H20O4; a20 D – 48.0 (c 0.10, MeOH); UV (MeOH) kmax (log e): 225 (3.03) nm; IR (KBr) mmax 3434, 2932, 1686 (trough from 1731 to 1648), 1629, 1565, 1414, 1383, 1109, 1081 cm1; ESIMS m/z 287.1 [M+Na]+, m/z 263.1 [MH]; HRESIMS m/z 287.1263 [M+Na]+ (calcd for C15H20O4Na, 287.1259); for 1H and 13C NMR spectroscopic data, see Table 1. 4.3.4. (2R,4E,6R,7S,9S,10R)-2-Acetoxy-9-angeloyloxygermacra4,11(13)-dien-12,6-olide (4) Colorless square crystals (CH2Cl2); C22H30O6; mp 157–160 °C; a20 D + 119.0 (c 0.10, CH2Cl2); IR (KBr) mmax 2931, 1768, 1726, 1661, 1456, 1382, 1314, 1245, 1162, 1134, 1071, 1019, 975 cm1; ESIMS m/z 413.2 [M+Na]+, m/z 389.2 [MH]; HRESIMS m/z 413.1943 [M+Na]+ (calcd for C22H30O6Na, 413.1940); for 1H and 13 C NMR spectroscopic data, see Table 2. 4.3.5. 4E-9b-Angeloyloxy-2a-hydroxy-7a,10aH-germacra-4,11(13)dien-12,6a-olide (5) White amorphous powder; C20H28O5; mp 134–142 °C; a20 D + 104.9 (c 0.10, CH2Cl2); IR (KBr) mmax 3430, 2928, 2861, 1763, 1707, 1650, 1453, 1381, 1245, 1150, 1021, 975 cm1; ESIMS m/z 371.1 [M+Na]+, m/z 347.1 [MH]; HRESIMS m/z 371.1836 [M+Na]+ (calcd for C20H28O5Na, 371.1834); for 1H and 13C NMR spectroscopic data, see Table 2. 4.3.6. (4E,6R,7S,9S,10R)-9-Angeloyloxygermacra-4,11(13)-dien-12,6olide (6) Colorless square crystals (CH2Cl2); C20H28O4; mp 115–125 °C; a20 D + 90.0 (c 0.10, CH2Cl2); IR (KBr) mmax 2937, 2923, 2856, 1766, 1705, 1643, 1450, 1379, 1234, 1153, 1144, 1037, 973 cm1; ESIMS m/z 355.2 [M+Na]+, m/z 331.2 [MH]; HRESIMS m/z 355.1856 [M+Na]+ (calcd for C20H28O4Na, 355.1855); for 1H and 13C NMR spectroscopic data, see Table 2. 4.3.7. 4E-9b-Methacryloxy-7a,10aH-germacra-4,11(13)-dien-12,6aolide (7) White amorphous powder; C22H30O6; a20 D + 77.1 (c 0.05, CH2Cl2); IR (KBr) mmax 2930, 2863, 1770, 1713, 1633, 1571, 1454, 1383, 1317, 1281, 1250, 1141, 1072, 1010, 976 cm1; ESIMS m/z 341.2 [M+Na]+, m/z 317.2 [MH]; HRESIMS: m/z 341.1733 [M+Na]+ (calcd for C19H26O4Na, 341.1729); for 1H and 13C NMR spectroscopic data, see Table 2. 4.3.8. 4E-9b-Hydroxy-7a,10aH-germacra-4,11(13)-dien-12,6a-olide (8) White amorphous powder; C15H22O3; a20 D + 69.6 (c 0.10, CH2Cl2); IR (KBr) mmax 3441, 2936, 2865, 1765, 1663, 1457, 1382, 1274, 1249, 1144, 1079, 1012, 973 cm1; ESIMS m/z 273.1 [M+Na]+, m/z 249.1 [MH]; HRESIMS: m/z 273.1466 [M+Na]+ (calcd for C15H22O3Na, 273.1467); for 1H and 13C NMR spectroscopic data, see Table 1. 4.3.9. (1R,5S,7R,10R)-1-Hydroxy-2-oxoeudesma-3,11(13)-dien-12-oic acid (9) Colorless square crystals (CH2Cl2); C15H20O4; mp 160–180 °C; a20 D + 89.1 (c 0.10, MeOH); UV (MeOH) kmax (log e): 245 (3.02), 212 (2.77) nm; IR (KBr) mmax 3430, 2941, 1695, 1658, 1435, 1381, 1268, 1185, 1152, 1048, 951 cm1; ESIMS m/z 287.1 [M+Na]+, m/z 263.1 [MH]; HR-ESI-MS m/z 287.1263 [M+Na]+ (calcd for C15H20O4Na, 287.1259); for 1H and 13C NMR spectroscopic data, see Table 3.

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4.3.10. 4b,5a-Dihydroxy-1-oxoeudesma-2,11(13)-dien-12,6b-olide (10) White amorphous powder; C15H18O5; a20 – 42.0 (c 0.025, D MeOH); UV (MeOH) kmax (loge): 217 (2.79) nm; IR (KBr) mmax 3434, 2935, 1764, 1680, 1633, 1452, 1382, 1266, 1157, 1119, 1089 cm1; ESIMS m/z 301.1 [M+Na]+, m/z 277.1 [MH]; HRESI-MS m/z 301.1055 [M+Na]+ (calcd for C15H18O5Na, 301.1052); for 1H and 13C NMR spectroscopic data, see Table 3. 4.3.11. 3-Oxoeudesma-1,4,11(13)-trien-12,6b-olide (11) White amorphous powder; C15H16O3; mp 61–75 °C; a20 D – 239.9 (c 0.10, MeOH); UV (MeOH) kmax (log e): 245 (3.33) nm; IR (KBr) mmax 2931, 2871, 1766, 1660, 1630, 1515, 1450, 1377, 1318, 1263, 1205, 1144, 1108, 1066, 1012, 965 cm1; ESIMS m/z 267.1 [M+Na]+, m/z 243.1 [M H]; HR-ESI-MS m/z 267.0994 [M+Na]+ (calcd for C15H16O3Na, 267.0997); for 1H and 13C NMR spectroscopic data, see Table 3. 4.3.12. (7R)-14(10 ? 1)-Abeoeudesma-1,3,5(10),11(13)-tetraen-12oic acid (12) Colorless square crystals (CH2Cl2); C15H18O2; mp 102–105 °C; a20 D + 42.3 (c 0.10, MeOH); UV (MeOH) kmax (loge): 220 (3.07) nm; IR (KBr) mmax 2926, 1691, 1623, 1433, 1384, 1323, 1283, 1226, 1162, 1104, 1030, 953 cm1; ESIMS m/z 253.1 [M+Na]+, m/z 229.1 [M H]; HR-ESI-MS m/z 253.1207 [M+Na]+ (calcd for C15H18O2Na, 253.1204); for 1H and 13C NMR spectroscopic data, see Table 3. 4.4. Crystallographic data of compounds 4, 6, 9, 12, and 18 4.4.1. Crystallographic data of 4 C22H30O6, CH2Cl2, M = 390.46, orthorhombic, space group P2 (1) 2(1) 2(1), a = 6.7751 (3) Å, a = 90°; b = 8.9058 (4) Å, b = 90°; c = 35.0890 (14) Å, c = 90°; V = 2117.19 (16) Å3, Z = 4, qcalcd = 1.225 mg/m3, crystal size 0.20  0.15  0.10 mm3. Cu Ka (k = 1.54178 Å), F (0 0 0) = 840, T = 133 (2) K. The final R values were R1 = 0.0317, and wR2 = 0.0873, for 22,899 observed reflections [I > 2r (I)]. The absolute structure parameter was 0.05 (15). 4.4.2. Crystallographic data of 6 C20H28O4, CH2Cl2, M = 332.42, orthorhombic, space group P2 (1) 2(1) 2(1), a = 11.9746 (5) Å, a = 90°; b = 12.3428 (5) Å, b = 90°; c = 12.4853 (5) Å, c = 90°; V = 1845.33 (13) Å3, Z = 4, qcalcd = 1.197 mg/m3, crystal size 0.20  0.15  0.10 mm3. Cu Ka (k = 1.54178 Å), F (0 0 0) = 720, T = 273 (2) K. The final R values were R1 = 0.0276, and wR2 = 0.0767, for 20,005 observed reflections [I > 2r (I)]. The absolute structure parameter was 0.02 (15). 4.4.3. Crystallographic data of 9 C15H20O4, CH2Cl2, M = 264.31, monoclinic, space group P2 (1), a = 8.0209 (10) Å, a = 90°; b = 9.4151 (10) Å, b = 93.205 (10)°; c = 8.8624 (10) Å, c = 90°; V = 668.220 (13) Å3, Z = 2, qcalcd = 1.314 mg/m3, crystal size 0.20  0.14  0.11 mm3. Cu Ka (k = 1.54178 Å), F (0 0 0) = 284, T = 133 (2) K. The final R values were R1 = 0.0312, and wR2 = 0.0863, for 4267 observed reflections [I > 2r (I)]. The absolute structure parameter was 0.01 (15). 4.4.4. Crystallographic data of 12 C15H18O2, CH2Cl2, M = 230.29, monoclinic, space group P2 (1), a = 8.3226 (2) Å, a = 90°; b = 11.6396 (3) Å, b = 97.028 (2)°; c = 25.9362 (7) Å, c = 90°; V = 2493.61 (11) Å3, Z = 8, qcalcd = 1.227 mg/m3, crystal size 0.18  0.10  0.08 mm3. Cu Ka (k = 1.54178 Å), F (0 0 0) = 992, T = 133 (2) K. The final R values were R1 = 0.0442, and wR2 = 0.1269, for 15,926 observed reflections [I > 2r (I)]. The absolute structure parameter was 0.11 (18).

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4.4.5. Crystallographic data of 18 C15H16O2, CH2Cl2, M = 228.28, orthorhombic, space group P2 (1) 2(1) 2(1), a = 5.9667 (2) Å, a = 90°; b = 7.4596 (2) Å, b = 90°; c = 27.2161 (7) Å, c = 90°; V = 1211.37 (6) Å3, Z = 4, qcalcd = 1.252 mg/m3, crystal size 0.20  0.10  0.02 mm3. Cu Ka (k = 1.54178 Å), F (0 0 0) = 488, T = 173 (2) K. The final R values were R1 = 0.0298, and wR2 = 0.0837, for 18,112 observed reflections [I > 2r (I)]. The absolute structure parameter was 0.3 (2). Crystallographic data for 4, 6, 9, 12, and 18 have been deposited at the Cambridge Crystallographic Data Centre (deposition NO. CCDC 835957, 835958, 877970, 852612, and 852611, respectively). Copies of the data could be obtained free of charge on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 1223 336033 or e-mail: data_request@ ccdc.cam.ac.uk). 4.5. Methods of computational calculations Excitation energy (in nm) and rotatory strength R (velocity form Rvel and length form Rlen in 1040 erg-esu-cm/Gauss) between different states were calculated by time-dependent DFT at B3LYP/631G⁄//B3LYP/6-31G⁄ level in MeOH solution. All calculations are performed by the Gaussian03 program package (Frisch et al., 2004). ECD spectra were then simulated by overlapping Gaussian 1 functions for each transition according to DeðEÞ ¼ 2:29710 39  PA 2 1 ½ðEE Þ=ð2 r Þ t pffiffiffiffiffiffiffi D E e , where r is the width of the band at 1/e t i 2pr height and DEi and Ri are the excitation energies and rotatory strengths for transition i, respectively, r = 0.20 eV and Rvel have been used in this work. Conformational analysis has been carried out and theoretically weighted ECD spectra have been simulated at different levels mentioned above. 4.6. Bioassays for NO inhibitory and cytotoxic activity NO inhibitory assays were conducted according to procedures previously described (Cheng et al., 2011). Briefly, RAW264.7 macrophages grown on 100 mm culture dishes were harvested and seeded in 96-well plates at 2  105 cells/well for NO production. Plates were pretreated with various concentrations of samples (20, 5, 0.5, and 0.1 lM) for 30 min, and then incubated for 24 h with or without 1 lg/mL of LPS. Aminoguanidine (Sigma–Aldrich, purity P 98.0%) was used as a positive control. The nitrite concentration in the culture supernatant was measured by the Griess reaction. Cell viability was measured using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays (Sigma– Aldrich). The cytotoxic assays were conducted according to procedures previously described (Cheng et al., 2012). Briefly, four tumor cell lines, HepG2, HeLa, PC-3, and MGC-803 were harvested and then cultivated in 96-well plates at a concentration of 0.40.6  105 cells/mL, 100 lL/well. After 12 h incubation, the medium containing different concentrations of samples was added. Doxorubicin (Wako Pure Chemical Industries Ltd.) was used as a positive control, whereas 1% DMSO was used as a solvent control. After 72 h, 10 lL CCK-8 (Cell Counting Kit-8, Dojindo Laboratories) were added into each well and incubated for another 2–4 h. Cell survival rate were determined by measuring the absorbance with an ELISA reader (Bio-Rad) at 450 nm. Acknowledgments The work was supported by program NCET Foundation, NSFC (81230090 and 81102778), partially supported by Global Research Network for Medicinal Plants (GRNMP), Shanghai Leading Academic Discipline Project (B906), FP7-PEOPLE-IRSES-2008 (TCMCANCER Project 230232), Key laboratory of drug research

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