Bioactive Sesquiterpenes and Lignans from the Fruits of Xanthium

Article pubs.acs.org/jnp

Bioactive Sesquiterpenes and Lignans from the Fruits of Xanthium sibiricum Yu-Sheng Shi,† Yun-Bao Liu,† Shuang-Gang Ma,† Yong Li,† Jing Qu,† Li Li,† Shao-Peng Yuan,† Qi Hou,† Yu-Huan Li,‡ Jian-Dong Jiang,†,‡ and Shi-Shan Yu*,† †

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, and ‡Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Seven new sesquiterpenes (1, 3−8), a new sesquiterpene natural product (2), and two new lignans (9 and 10), together with 15 known compounds, were isolated from the fruits of Xanthium sibiricum. The structures of the new compounds were established by NMR spectroscopic analysis, ECD calculations, and Mo2(OAc)4-induced circular dichroism, with the structures of 1 and 4 confirmed by single-crystal X-ray diffraction. Compound 1 is the first example of a 3/5/6/5 tetracyclic eudesmane sesquiterpene lactone formed at C-6 and C-7. In turn, compound 4 is the first example of a natural xanthane tetranorsesquiterpene, while compounds 5−8 are the first xanthane trinorsesquiterpenes found to date. Compounds 8, 11−15, 17, and 24 exhibited indirect anti-inflammatory activity by suppressing the lipopolysaccharide-induced proinflammatory factors in BV2 microglial cells, with IC50 values between 1.6 and 8.5 μM. Furthermore, compounds 13 and 17 exhibited anti-inflammatory activity against ear edema in mice produced by croton oil, with inhibition rates of 46.9% and 37.7%, respectively. Compounds 8, 11, 12, 23, and 24 exhibited potent activity against influenza A virus (A/FM/1/47, H1N1) with IC50 values between 3.7 and 8.4 μM. constituents from X. sibiricum, a 95% EtOH extract of the fruits of this species was investigated. Consequently, nine new compounds (1, 3−10), a new natural product (2), and 15 known compounds (11−25; Figure S1, Supporting Information) were isolated from X. sibiricum fruits. The isolation and structure elucidation of compounds 1−10 and the bioactivities of all the isolated compounds are reported herein.

Xanthium (Asteraceae) is a relatively small genus of plants of worldwide distribution, with four species and two varieties growing in mainland China.1 Xanthium sibiricum L., an annual herb,2 has been used as a traditional Chinese medicine for treating fever, leucoderma, sinusitis, scrofula, headache, herpes, and cancer.3 Previous investigations have led to the isolation of several compounds with biological effects such as cytotoxic and hypoglycemic activities.4 A decoction of X. sibiricum is regarded as curative for rhinitis.5 Bioassays have shown that 95% EtOH and EtOAc extracts of X. sibiricum exhibited moderate antiinflammatory activity against croton oil-induced ear edema in mice, with inhibition rates of 37.5% and 43.5%, respectively, at a dose of 100 mg/mL.6 In 2008, Yoon et al. reported antiinflammatory activity of an EtOAc extract and the pure compounds xanthinosin and 4-oxobedfordia acid from X. sibiricum.7 In a previous study,6 a pair of rare racemic spirodienone sesquineolignans was isolated from an extract of this medicinal plant, but they exhibited only weak antiinflammatory activity. To search for further anti-inflammatory © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Seven new sesquiterpenes (1, 3−8), a new sesquiterpene natural product (2), and two new lignans (9 and 10) were isolated from an EtOAc-soluble fraction of the 95% EtOH extract prepared from the fruits of X. sibiricum by a combination of different chromatographic methods. Received: December 6, 2014

A

DOI: 10.1021/np500951s J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

Table 1. 1H NMR and 13C NMR Data of Compounds 1−4 1a

2b

position

δC, type

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

27.7, CH 17.1, CH2

1.51 m 0.95 (2H, m)

27.5, CH 17.7, CH2

23.4, CH 147.0, C 65.3, CH 78.7, CH

2.06 m

23.3, CH 149.8, C 70.2, CH 66.4, CH

152.5, C 196.5, C 54.2, CH2

δH (J in Hz)

2.83 d (11.4) 4.83 dd (11.4, 1.8)

2.85 d (16.2) 2.57 d (16.2)

38.9, C 132.9, C 172.8, C 9.9, CH3 20.8, CH3 110.3, CH2

δC, type

152.7, C 150.6, C 121.1, CH

3c

δH (J in Hz) 1.71 0.95 0.90 2.01

δC, type

m m m m

140.7, C 127.3, CH

3.06 d (11.4) 4.75 dd (11.4, 1.8)

24.5, 46.4, 81.3, 36.9,

6.33 s

34.5, CH 40.3, CH 179.3, C

42.0, C 125.3, C 172.9, C 2.05 0.80 5.31 5.19

d (1.8) s brs brs

9.4, CH3 23.4, CH3 109.6, CH2

CH2 CH CH CH2

10.1, CH3 17.5, CH3 45.7, CH2 2.05 0.84 5.31 5.15

d (1.8) s brs brs

172.4, C 51.6, CH3

4d

δH (J in Hz) 5.60 dd (9.0, 3.0) 1.90 m 1.96 m 4.45 ddd (12.0, 11.0, 3.0) 2.16 ddd (14.0, 11.0, 4.0) 1.74 ddd (14.0, 4.0, 3.0) 2.48 m 2.62 dq (7.2, 7.8)

1.06 0.99 3.10 3.00

d d d d

(7.8) (7.8) (15.6) (15.6)

δC, type 202.6, C 142.5, CH 135.2, CH 49.4, CH 80.6, CH 36.2, CH2 42.6, CH 41.5, CH 177.5, C 13.4, CH3 15.7, CH3

δH (J in Hz) 6.69 brd (10.0) 6.08 dd (10.0, 5.0) 3.16 m 4.20 ddd (12.0, 10.0, 3.0) 2.08 overlap 1.91 ddd (15.0, 5.0, 3.0) 3.08 m 2.67 dq (12.0, 7.0)

1.32 d (7.0) 1.08 d (6.5)

3.65 s

a

Data were recorded at 600 MHz for proton and at 150 MHz for carbon in CDCl3. bData were recorded at 600 MHz for proton and at 150 MHz for carbon in methanol-d4. cData were recorded at 600 MHz for proton and at 150 MHz for carbon in pyridine-d5. dData were recorded at 500 MHz for proton and at 125 MHz for carbon in Me2CO-d6.

Compound 1, colorless crystals (in CH3OH), gave a molecular formula of C15H16O3 based on the HRESIMS (m/ z 245.1179 [M + H]+, calcd 245.1172) and NMR data. The IR spectrum suggested the presence of carbonyl groups (1763 and 1695 cm−1). The 1H NMR data (Table 1) revealed two methyl signals, δH 2.05 (3H, d, 1.8 Hz) and 0.80 (3H, s), and resonances for two olefinic protons, δH 5.31 (1H, brs) and 5.19 (1H, brs). The 13C NMR data (Table 1) showed 15 resonances, including a conjugated carbonyl (δC 196.5), an ester carbonyl (δC 172.8), four olefinic carbons (δC 152.5, 147.0, 132.9, 110.3), and an oxygenated carbon (δC 78.7), which are characteristic signals of a eudesmane sesquiterpene lactone.8 The 1H−1H COSY and HSQC spectra supported the presence of a three-membered ring (Figure 1, ring A), while

HMBC correlations (Figure 1) from H-1 and H2-2 to C-10 indicated that C-1 and C-10 are connected directly. In turn, HMBC correlations from Me-14 to C-5/C-10 suggested a connectivity between C-5 and C-10. HMBC correlations from the terminal olefinic protons, H2-15 to C-3/C-4, indicated the presence of fragment C(5)H−C(4)[C(3)H]−C(15)H2. These results suggested that a five-membered ring (ring B) is fused to ring A at C-1 and C-3. Ring C was established by structural HMBC correlations from H-1 to C-9/C-10, from Me-14 to C9/C-10, and from H2-9 to C-7/C-8 as well as the 1H−1H COSY correlation of H-5 and H-6. Analysis of the degrees of unsaturation of 1 and the chemical shifts of H-6 (δH 4.83) and C-6 (δC 78.7), as well as the HMBC correlations from Me-13 to C-7/C-11/C-12 and from H-6 to C-7, revealed the presence B



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Figure 1. Key 1H−1H COSY and HMBC correlations of 1−10.

of an α,β-unsaturated lactone (ring D), as shown in Figure 1. However, no HMBC correlation was observed between H-6 and C-12. Compound 1 is the first example of a 3/5/6/5 tetracyclic eudesmane sesquiterpene lactone formed at C-6 and C-7. The relative configuration of 1 was deduced mainly from the NOESY spectrum. NOESY correlations were observed for Me14 with H2-2/H-6 and for H2-2 and H-6 after irradiation of Me14 (Figure 3). These correlations indicated that Me-14 and the

assignment of the absolute configurations as (1R,3S,5S,6R,10S). Thus, the structure of 1 was characterized as shown, and the compound has been given the trivial name sibirolide A. Compound 2 was isolated as a yellow, amorphous powder and was assigned a molecular formula of C15H16O3, based on the HRESIMS (m/z 245.1169 [M + H]+, calcd 245.1172) and NMR data. The 1H NMR spectrum showed two methyl groups, δH 0.84 (3H, s) and 2.05 (3H, d, 1.8 Hz), and three olefinic protons, δH 5.31 (1H, brs), 5.15 (1H, brs), and 6.33 (s) (Table 1). The 13C NMR and DEPT data exhibited the presence of an ester carbonyl carbon (δC 172.9), six olefinic carbons (δC 152.7, 150.6, 149.8, 125.3, 121.1, 109.6), an oxygenated carbon (δC 66.4), and two methyl carbons (δC 9.4, 23.4) (Table 1). The 1H and 13C NMR spectra of 2 were found to be similar to those of 1. A major difference found between these compounds was that the carbonyl carbon (C-8) and the methylene carbon (C-9) in 1 are replaced by two olefinic carbons (including one oxygenated) in 2. Another major change in the 13C NMR data was that the signal of C-6 (δC 66.4) shifted to higher field relative to 1. In addition, the signals of C-6 (δC 66.4) and C-8 (δC 150.6) in 2 resembled closely those of 6α,10α-dihydroxy-1oxoeremophila-7(11),8(9)-dien-12,8-olide (δC‑6 66.6, δC‑8 150.2) and 6β,10β-dihydroxyeremophila-7(11),8(9)-dien-12,8olide (δaddition, δC‑8 149.1).9 These results suggested that the lactone ring in 2 is formed at C-7 and C-8. The relative configuration at C-1, C-3, C-5, C-6, and C-10 was established in the same manner as described for 1, as shown in Figure 3. According to the relative configuration determined for 2, the absolute configuration of this compound could be proposed as (1S,3R,5R,6S,10R) (2a) or (1R,3S,5S,6R,10S) (2b). The calculated ECD spectrum of 2a displayed a CD curve similar to the experimental spectrum of 2 (Figure S23, Supporting Information). The structure of 2 (sibirolide B) was characterized as shown, which was obtained by chemical conversion from linderene.10 However, the NMR data and relative and absolute configurations of 2 were not reported earlier. Compound 3 was obtained as a white, amorphous powder. Its molecular formula, C14H20O4, was established based on the

Figure 2. ORTEP diagram of 1.

three-membered ring A are on the same face of ring B, while Me-14 and H-6 on the same face of ring C. The coupling constant between H-5 and H-6 (J = 11.4 Hz, antiperiplanar) revealed that both H-5 and H-6 are attached at axial bonds of the six-membered ring, indicating H-5 and H-6 to be on the opposite face of ring C, as shown in Figure 3. According to the relative configuration determined for 1, the absolute configurations of 1 could be proposed as (1R,3S,5S,6R,10S) (1a) or (1S,3R,5R,6S,10R) (1b). The calculated ECD spectrum of 1a displayed a CD curve similar to the experimental spectrum of 1 (Figure S12, Supporting Information). The X-ray crystallographic structure of 1 (Figure 2), obtained by anomalous scattering of Cu Kα radiation, allowed the unambiguous C



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Figure 3. Key NOE correlations of 1−3 and 5−8.

HRESIMS data (m/z 253.1438 [M + H]+, calcd 253.1434). The 1H and 13C NMR data showed the presence of three methyls, three methylenes, five methines, and three quaternary carbons (Table 1). The 1H−1H COSY and HSQC spectra supported the presence of the following spin system in the structure of 3: C(2)H−C(3)H2−C(4)H[C(8)H−C(10)H3]− C(5)H−C(6)H2−C(7)H−C(11)H3 (Figure 1). HMBC correlations from H2-3/Me-11 to C-1 revealed the connectivity of a seven-membered ring A. In turn, HMBC correlations from Me10 to C-4/C-8/C-9 and from H-8 to C-9, together with the chemical shift of C-5 (δC 81.3), established a five-membered ring B. The (CH3OCOCH2−) group was established by HMBC correlations from H 2-12/Me-14 to C-13. The (CH3OCOCH2−) group was located at C-1, which was confirmed by the key HMBC correlations from H2-12 to C1/C-2/C-7 (Figure 1). The relative configuration of 3 was deduced from the NOESY spectrum. NOESY correlations of H-5 with H-6a/Me-11 indicated that H-5, H-6a, and Me-11 are on the same face of the seven-membered ring, while those from H-6b to H-4/H-7 suggested that H-4, H-7, and H-6b are on the same face of the ring, opposite H-5 and Me-11. A further NOESY correlation from H-5 to Me-10 was used to infer that H-5 and Me-10 are on the same face of the lactone ring (Figure 3). According to the relative configuration determined for 3, the absolute configurations could be proposed as (4R,5S,7S,8S) (3a) or (4S,5R,7R,8R) (3b). The calculated ECD spectrum of 3a displayed a CD curve similar to the experimental spectrum of 3 (Figures S33 and S34, Supporting Information). Thus, the structure of this new dinorsesquiterpene (norxanthantolide A, 3) was proposed as shown.

Compound 4 was obtained as colorless crystals (in CH3OH) and had a molecular formula of C11H14O3 based on the HRESIMS data (m/z 217.0843 [M + Na]+, calcd 217.0835). The 1H NMR spectrum showed two methyl doublets, δH 1.32 (Me-10) and 1.08 (Me-11), and two olefinic protons, δH 6.69 (H-2) and 6.08 (H-3) (Table 1). The 13C NMR data exhibited the presence of 11 carbon resonances, including an ester carbonyl at δC 177.5, a carbonyl at δC 202.6, two olefinic carbons at δC 142.5 and 135.2, and an oxygenated carbon at δC 80.6 (Table 1). The 1H−1H COSY and HSQC spectra supported the presence of the spin system C(2)H−C(3)H2− C(4)H[C(8)H−C(10)H3]−C(5)H−C(6)H2−C(7)H−C(11)H3 (Figure 1). HMBC correlations from H-2 to C-7 and from H2-6/Me-11 to C-1 revealed the connectivity of a sevenmembered ring A. The long-range 1H−13C couplings observed in the HMBC correlations from Me-10 to C-4/C-8/C-9, in combination with the chemical shifts of C-5 and H-5 (δC 80.6; δH 4.20), were used to establish a five-membered ring B (Figure 1). Compound 4 is the first example of a natural xanthane tetranorsesquiterpene. However, no NOESY correlations were observed between H-5 and H-8/Me-11. Fortunately, a suitable single crystal was obtained for X-ray diffraction, which established the relative configurations as well as the absolute configurations of each chiral center (4R,5S,7S,8R) using the anomalous scattering of Cu Kα radiation. The resulting ORTEP drawing, with the atom-numbering scheme indicated, is shown in Figure 4. The absolute configuration of 4 also could be determined from the calculated spectrum. According to the relative configuration determined for 4 by X-ray diffraction, the absolute configurations of 4 could be proposed as D



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H3 (Figure 1). In 5 and 6, HMBC correlations from H-2 to C1/C-7 and from Me-11 to C-1/C-6/C-7 revealed the connectivity of the seven-membered ring A. HMBC correlations from Me-10 to C-4/C-8/C-9, together with the chemical shifts of C-5 (δC 83.0 in 5 and 82.9 in 6), established the fivemembered ring B. The carboxylic group (C-12) was located at C-1, which was confirmed by the key HMBC correlation from H-2 to C-12 in 5 and 6 (Figure 1). Compounds 5 and 6 are the first examples of xanthane trinorsesquiterpenes that have been discovered. For 5, NOEs were observed for Me-11 and H-6a after irradiation of H-5, which indicated that H-5, Me-11, and H-6a are on the same face of the seven-membered ring. In turn, NOE correlations of H-6b with H-4/H-7 demonstrated that H-4, H7, and H-6b are on the same face of the ring, opposite H-5 and Me-11. Further NOE correlations from H-5 to Me-10 showed that H-5 and Me-10 are on the same face of the lactone ring (Figure 3). According to the relative configuration determined for 5, alternative absolute configurations could be proposed as (4R,5S,7S,8S) (5a) or (4S,5R,7R,8R) (5b). The calculated ECD spectrum of 5a displayed CD curves similar to the experimental spectrum of 5 (Figure S55, Supporting Information). Thus, the structure of the new trinorsesquiterpene (norxanthantolide C) was established as shown. For 6, NOEs were observed for Me-11 and H-3b after irradiation of H-5, which indicated that H-5, Me-11, and H-3b are on the same face of the seven-membered ring. NOE correlations of H-3a with H-4 suggested that H-4 and H-3a are on the same face of the ring, opposite H-5 and Me-11. Further NOE correlations from H-5 to H-8 showed that H-5 and H-8 are on the same face of the lactone ring (Figure 3). Thus, compounds 6 and 5 were found to possess an identical relative configuration, except for C-8. In addition, the large coupling constant (JH‑4, H‑8 = 12.0 Hz) between H-4 and H-8 in 6 was the same as that of 4 and revealed that the relative orientation of C-8 in 6 is α, identical to that of 4. Their similar Cotton effect observed at 225 nm (Figures S44 and S56, Supporting

Figure 4. ORTEP diagram of 4.

(4R,5S,7S,8R) (4a) or (4S,5R,7R,8S) (4b). The calculated ECD spectrum of 4a displayed a CD curve similar to the experimental spectrum of 4 (Figure S43, Supporting Information). Thus, the structure of this new tetranorsesquiterpene (norxanthantolide B, 4) was established as shown. Compounds 5 and 6 were obtained as white, amorphous powders, and both give the same molecular formula (C12H16O4) from their HRESIMS data and similar 1H and 13 C NMR spectra, suggesting them to be stereoisomers. The 1H NMR and 13C NMR spectra (Table 2) of 5 and 6 showed the presence of two methyls, two methylenes, five methines (including one oxygenated and one olefinic carbon), and three quaternary carbons (one olefinic carbon, one carboxylic carbonyl, and one ester carbonyl). The 1H−1H COSY and HSQC spectra demonstrated the occurrence of the following spin system in the structures of 5 and 6: C(2)H2−C(3)H− C(4)H[C(8)H−C(10)H3]−C(5)H−C(6)H2−C(7)H−C(11)Table 2. 1H NMR and 13C NMR Data of Compounds 5−8 5a position

δC, type

1 2 3a

140.8, C 141.1, CH 25.9, CH2

3b 4 5

47.3, CH 83.0, CH

6a

37.8, CH2

6b 7 8 9 10a 10b 11 12

30.3, CH 41.7, CH 182.2, C 10.6, CH3 19.5, CH3 171.5, C

δH (J in Hz)

6b δC, type

7b

δH (J in Hz)

δC, type

7.16 dd (9.5, 3.5) 2.40 ddd (11.5, 9.5, 2.0) 2.20 overlap 2.06 m 4.61 ddd (11.0, 11.0, 3.0) 2.20 overlap

140.6, C 140.6, CH 7.21 dd (9.0, 3.0) 28.2, CH2 2.59 ddd (11.0, 9.5, 3.0) 2.25 m 51.8, CH 1.65 m 82.9, CH 4.45 ddd (12.0, 10.5, 3.0) 37.3, CH2 2.26 m

1.65 ddd (12.5, 12.5, 4.0) 3.38 m 2.66 dq (8.0, 7.2)

1.66 ddd (12.6, 12.6, 3.6) 3.45 m 2.52 dq (12.0, 7.2)

1.16 d (7.2) 1.10 d (7.2)

30.1, CH 42.9, CH 181.0, C 12.7, CH3 19.2, CH3 171.5, C

1.17 d (7.2) 1.15 d (7.2)

153.8, C 151.1, CH 29.2, CH2

51.6, CH 82.7, CH 37.2, CH2

26.8, CH 42.9, CH 180.9, C 12.7, CH3 19.2, CH3 196.4, CH

δH (J in Hz) 6.90 dd (9.0, 3.0) 2.67 ddd (11.4, 9.0, 2.4) 2.35 m 1.61 m 4.50 ddd (14.0, 11.0, 3.0) 2.22 ddd (14.0, 3.0, 3.0) 1.58 ddd (14.0, 14.0, 3.0) 3.35 overlap 2.55 dq (12.0, 7.2) 1.18 d (7.2) 1.12 d (7.2) 9.30 s

8b δC, type 150.1, C 149.8, CH 27.7, CH2

47.0, CH 81.3, CH 36.5, CH2

δH (J in Hz) 6.80 dd (9.0, 3.0) 3.00 ddd (11.4, 9.0, 3.0) 2.39 overlap 2.60 m 4.33 ddd (12.0, 10.0, 2.4) 2.39 overlap 1.80 ddd (12.6, 12.6, 4.2) 3.43 m

25.6, CH 138.9, C 169.5, C 119.3, CH2 6.21 5.43 19.3, CH3 1.07 194.4, CH 9.26

d (3.6) d (3.6) d (7.2) s

a

Data were recorded at 500 MHz for proton and at 125 MHz for carbon in methanol-d4. bData were recorded at 600 MHz for proton and at 150 MHz for carbon in methanol-d4. E



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calcd 391.1387 in both cases). The 1H and 13C NMR spectra (Table 3) of both compounds showed similar chemical shifts

Information) suggested the absolute configuration of C-5 (5S) of 6 to be identical to that of 5. Furthermore, the configurations of C-4 (4R), C-7 (7S), and C-8 (8R) were determined based on the aforementioned relative configurations. Thus, the absolute configuration of 6 was determined to be 4R,5S,7S,8R, and the structure of this new trinorsesquiterpene (norxanthantolide D, 6) was established as shown. Compound 7, obtained as a white, amorphous powder, gave a quasimolecular ion [M + H]+ at m/z 209.1177 (calcd 209.1172) by positive-ion HRESIMS, which was consistent with a molecular formula of C12H16O3. The NMR data (Table 2) revealed the presence of two methyls, two methylenes, six methines (one oxygenated carbon, one olefinic carbon, and one aldehyde group), and two quaternary carbons (one olefinic carbon and one ester carbonyl). The 1H and 13C NMR spectra of 7 were found to be similar to those of 6. A major difference between these compounds found is that the carboxylic acid group (δC 171.5, C-12) in 6 is replaced by an aldehyde group (δC 196.4, C-12) in 7, which was confirmed by HMBC correlations of H-2/C-12, H-12/C-2, and H-12/C-7 (Figure 1). NOE correlations of H-5/Me-11 and H-3b/H-5 showed that H-5, Me-11, and H-3b are on the same face of the sevenmembered ring A, while NOE correlations of H-3a with H-4 indicated them to be on the same face of ring A, opposite H-5 and Me-11. However, no NOE correlation was observed between H-5 and H-8. The relative configuration of C-8 was deduced from the coupling constant between H-4 and H-8 (JH‑4, H‑8 = 12.0 Hz), which revealed that H-4 and H-8 are on the opposite face of the lactone ring (Figure 3). According to the relative configuration apparent for 7, possible absolute configurations of 7 could be proposed as (4R,5S,7S,8R) (7a) or (4S,5R,7R,8S) (7b). The calculated ECD spectrum of 7a displayed a CD curve similar to the experimental spectrum of 7 (Figures S74 and S75, Supporting Information). Thus, the absolute configuration of 7 was determined to be the same as 7a. Accordingly, the structure of 7 (norxanthantolide E) was proposed as shown. Compound 8, obtained as a white, amorphous powder, was deduced to have a molecular formula of C12H14O3 by the HRESIMS (m/z 207.1016 [M + H]+; calcd 207.1016) and from the 13C NMR data, as well as six degrees of unsaturation. Comparison of the 1H and 13C NMR data (Table 2) of 8 with those of 7 indicated the structural similarity between these two compounds. However, 7 was found to contain a methyl carbon (δC 12.7, C-8) and a methine carbon (δC 42.9, C-10), while two extra olefinic carbons (δC 119.3 and 138.9) were observed for 8. The key HMBC correlations of H2-10 with C-4/C-8/C-9 were used to locate the double bond between C-8 and C-10 in 8 (Figure 1). NOE correlations of H-5 with Me-11/H-6a suggested that H-5, Me-11, and H-6a are on the same face of the seven-membered ring A. In turn, the NOE correlations of H-6b with H-4/H-7 demonstrated H-4 and H-7 to be on the opposite face of the ring (Figure 3). According to the established relative configuration of 8, the absolute configuration of this compound could be proposed as (4R,5S,7S) (8a) or (4S,5R,7R) (8b). The calculated ECD spectrum of 8a and the experimental spectrum of 8 were in good agreement (Figure S85, Supporting Information). Thus, the structure of this new trinorsesquiterpene (norxanthantolide F) (8) was determined as shown. Compounds 9 and 10 were obtained as amorphous powders. Their common molecular formula, C20H22O8, was established by their respective HRESIMS data (m/z 391.1397 [M + H]+,

Table 3. 1H NMR and 13C NMR Data of Compounds 9 and 10a position 1 2 3 4 5 6 7 8 9a 9b OCH3-3 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′a 9′b OCH3-3′

δC, type 130.5, 110.9, 149.5, 148.2, 116.6, 120.1, 90.8, 54.8, 64.8,

C CH C C CH CH CH CH CH2

57.1, 131.0, 114.6, 146.1, 155.1, 134.1, 120.7, 199.8, 75.9, 66.5,

CH3 C CH C C C CH C CH CH2

56.7, CH3

δH (J in Hz) 6.89 s

6.72 6.78 5.59 3.53 3.83 3.80 3.87

d (8.0) d (8.0) d (6.5) m m m s

7.52 brs

7.59 brs 5.06 3.70 3.84 3.76

t (5.5) dd (12.0, 5.5) m s

a

Data were recorded at 600 MHz for proton and at 150 MHz for carbon in methanol-d4.

and coupling constants to those of sisymbrifolin.11 Comparison of the 1H and 13C NMR data of 9 and 10 suggested that the oxygenated methine group (δC 57.7, C-7′) in sisymbrifolin is replaced by a carbonyl group (δC 199.8) in 9 and 10. This deduction was supported by HMBC correlations from H-2′/H6′/H-9′ to C-7′ (Figure 1). Based on the reversed helicity rule of the 1Lb CD band for the 7-methoxy-2,3-dihydrobenzo[b]furan chromophore,12 a positive Cotton effect at 276 nm in the ECD spectrum of 9 indicated it to have a 7S,8R configuration (Figure S86, Supporting Information), while a negative Cotton effect at 278 nm in the ECD spectrum of 10 indicated a 7R,8S configuration (Figure S96, Supporting Information). The absolute configuration of C-8′ was established using the Mo2(OAc)4-induced circular dichroism (ICD) method developed by the groups of Snatzke and Frelek.13 The negative Cotton effects observed at 337 nm in the ICD (Figure 5) permitted assignment of the 8′S absolute configurations for 9, whereas the positive Cotton effects observed at 337 nm in the ICD (Figure 6) permitted assignment of the 8′R absolute configuration for 10. Therefore, the absolute configurations of 9 and 10 were determined to be 7S,8R,8′S and 7S,8R,8′R, respectively. These compounds were assigned as (−)-7′dehydrosismbrifolin and (+)-7′-dehydrosismbrifolin. The known compounds, 2-desoxy-6-epi-parthemollin (11),14 xanthatin (12),3b xanthinosin (13),3b 11α,13-dihydro-4Hxanthalongin (14),15 11α,13-dihydroxanthatin (15),16 2deacetyl-11β,13-dihydroxyxanthinin (16),17 hydroxylindestenolide (17),18 11β-13-dihydroxanthatin (18),19 linderanlide C (19),20 dihydrophaseic acid (20),21 phaseic acid (21),22 dihydrodiconiferyl alcohol (22),23 threo-guaiacylglycerol-8′vanillic acid ether (23),24 caffeic acid ethyl ester (24),25 and F



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Figure 5. CD spectrum of 9 in DMSO containing Mo2(OAc)4 with the inherent CDs subtracted, confirming the Mo24+ complexes of 9.

Figure 6. CD spectrum of 10 in DMSO containing Mo2(OAc)4 with the inherent CDs subtracted, confirming the Mo24+ complexes of 10.

3,4-dihydroxybenzoic acid ethyl ester (25)26 (Figure S1, Supporting Information), were isolated and identified by comparing their experimental physical and spectroscopic data with literature values. Nitric oxide (NO) plays an important role in the inflammatory process, and an inhibitor of NO production may be considered as a potential anti-inflammatory agent.27 The isolated compounds 8, 11−15, 17, and 24 indirectly exhibited anti-inflammatory activity by suppressing lipopolysaccharide-induced NO production in mouse macrophages with IC50 values of 3.0, 8.5, 1.6, 5.6, 5.7, 3.7, 3.9, and 2.5 μM, respectively. Dexamethasone was used as the positive control, with an IC50 value of 2.5 × 10−2 μM. The other isolated compounds were inactive (IC50 > 10 μM) for the inhibition of NO production. The in vivo anti-inflammatory activities of compounds 8, 11, 13, 15, 16, and 17 were tested. As a result, compounds 13 and 17 were found to exhibit anti-inflammatory activity against croton oil-induced ear edema in mice with inhibition rates of 46.9% and 37.7%, respectively, at a dose of 50 mg/mL. Dexamethasone was again used as the positive control, with an inhibition rate of 70.5%, at a dose of 1 mg/mL. Compounds 8, 11, 15, and 16 gave negative data, with inhibition rates of less than 20.0%. Other isolated compounds, which were insufficient in quantity for the in vivo antiinflammatory activity assay, were not investigated in this manner. The in vivo results obtained support the use of the traditional Chinese medicine X. sibiricum to treat inflammatory disease. The isolated compounds 11, 12, and 16 exhibited potent activities against Coxsackie virus B3 (CVB3), with IC50 values of 6.4, 6.4, and 6.4 μM and selectivity index values (SI = TC50/ IC50) of 3.0, 7.5, and 12.1, respectively (Table 4). Compounds 8, 11, 12, 23, and 24 exhibited activity against influenza A virus (A/FM/1/47, H1N1) with IC50 values of 6.4, 8.6, 8.4, 8.4, and 3.7 μM and SI values of 7.5, 2.2, 6.8, 11.9, and 5.2, respectively (Table 5). To the best of our knowledge, antiviral constituents from a plant in the genus Xanthium are being reported for the first time herein. The traditional efficacy of X. sibiricum is to treat rhinitis,5 which is commonly caused by bacteria or viruses,

Table 4. Antiviral Activity against Coxsackie Virus B3 and Cytotoxicity for Compounds 8, 11, 12, 16, 19, and 25a in Vero Cellsb compound

TC50c (μM)

8 11 12 16 19 25 ribavirine pleconarile

69.3 19.2 48.1 77.6 69.3 23.1 8196 40.5

± ± ± ± ± ± ± ±

1.21 0.83 3.92 10.05 6.46 10.52 0 1.2

IC50 (μM) 19.2 6.4 6.4 6.4 19.2 11.3 1200 0.0025

± ± ± ± ± ± ± ±

5.01 6.20 3.31 5.65 7.86 2.35 10.1 2.1

SId 3.6 3.0 7.5 12.1 3.6 2.1 6.8 16 200

a

Compounds 1−7, 9, 10, 13−15, 17, 18, and 20−24 all gave IC50 values of >35 μM. bData represent the mean values for three independent determinations. cCytotoxic concentration required to inhibit Vero cell growth by 50%. dSelectivity index value is TC50/IC50. e Positive control.

Table 5. Antiviral Activity against Influenza A and Cytotoxicity for Compounds 8, 11, 12, 19, 23, and 24a in MDCK Cellsb compound

TC50c (μM)

8 11 12 19 23 24 oseltamivire

48.1 19.3 57.7 100.0 100.0 19.3 3120

± ± ± ± ± ± ±

5.32 8.21 3.18 1.03 2.32 1.71 0.00

IC50 (μM)

SId

± ± ± ± ± ± ±

7.5 2.2 6.8 3.0 11.9 5.2 1143

6.4 8.6 8.4 33.3 8.4 3.7 2.7

1.51 2.62 3.35 3.31 4.66 35.82 0.06

a Compounds 1−7, 9, 10, 13−18, 20−22, and 25 all gave IC50 values of >35 μM. bData represent the mean values for three independent determinations. cCytotoxic concentration required to inhibit MDCK cell growth by 50%. dSelectivity index value is TC50/IC50. ePositive control.

including influenza A virus.28 The five isolated compounds mentioned above that exhibited potent activity against the G



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and eluted with MeOH/H2O mixtures (30:70, 50:50, 70:30, and 100:0) to give fractions ED1 to ED15. ED10 (520 mg) was purified by RP-semipreparative HPLC (20% MeOH/H2O) to afford compounds 20 (12.0 mg) and 21 (10.0 mg). EF (120 g) was separated by flash chromatography over MCI gel (5 × 60 cm), eluted with a gradient of increasing amount of CH3OH (0−100%) in H2O, to give fractions EFA−EFD. EFA (12.0 g) was subjected to RP flash CC (10−95% MeOH in H2O) to give subfractions EFA1−EFA20. EFA-5 (500 mg) was subjected to RP-semipreparative HPLC (15% MeOH in H2O) to yield 23 (10.0 mg), 24 (10.0 mg), and 25 (12.0 mg). EFA-16 was subjected to RP-semipreparative HPLC (35% MeOH in H2O) purification to yield 22 (12.5 mg). EFA-18 (50 mg) was initially purified by RP-semipreparative HPLC (40% MeOH in H2O), and then 9 (7.0 mg) and 10 (6.0 mg) were separated from one another by chiral semipreparative HPLC (20% isopropyl alcohol in hexane). Sibirolide A (1): colorless crystals (CH3OH); mp 100−101 °C; [α]25D −61.0 (c 0.03, CH3OH); UV (MeOH) λmax (log ε) 201 (3.68), 226 (3.24) nm; ECD (MeOH) nm (Δε) 200 (+7.50), 233 (+4.11), 377(−0.85); IR νmax 3019, 2960, 2920, 1763, 1695, 1322, 1201, 1092, 988, 895, 798, 750 cm−1; 1H NMR and 13C NMR, see Table 1; HRESIMS m/z 245.1179 [M + H]+ (calcd for 245.1172, C15H17O3). Sibirolide B (2): yellow, amorphous powder; [α]25D +80.6 (c 0.05, CH3OH); UV (MeOH) λmax (log ε) 202 (3.49), 282 (3.11) nm; ECD (MeOH) nm (Δε) 207 (+1.63), 221 (−2.25), 257 (+1.39), 292 (−3.41); IR νmax 3369, 2921, 1409, 1088, 799 cm−1; 1H NMR and 13C NMR, see Table 1; HRESIMS m/z 245.1196 [M + H]+ (calcd for 245.1172, C15H17O3). Norxanthantolide A (3): white, amorphous powder; [α]25D −82.0 (c 0.11, CH3OH); UV (MeOH) λmax (log ε) 203 (3.15) nm; ECD (MeOH) nm (Δε) 214 (−0.97); IR νmax 2983, 1683, 1457, 1209, 1149, 985, 801, 725, 605 cm−1; 1H NMR and 13C NMR, see Table 1; HRESIMS m/z 253.1438 [M + H]+ (calcd for 253.1434, C14H21O4). Norxanthantolide B (4): colorless crystals (CH3OH); mp 89−90 °C; [α]25D −33.7 (c 0.06, CH3OH); UV (MeOH) λmax (log ε) 203 (3.67), 219 (3.57), 278 (2.97) nm; ECD (MeOH) nm (Δε) 201 (+0.53), 244 (−0.13), 285 (−0.08), 355 (+0.08); IR νmax 2975, 2934, 1767, 1673,1454, 1179, 1056, 1001, 913, 804 cm−1; 1H NMR and 13C NMR, see Table 1; HRESIMS m/z 217.0843 [M + Na]+ (calcd for 217.0835, C11H14O3Na). Norxanthantolide C (5): white, amorphous powder; [α]25D −101.1 (c 0.13, CH3OH); UV (MeOH) λmax (log ε) 215 (3.23) nm; ECD (MeOH) nm (Δε) 225 (−0.84); IR νmax 2972, 1774, 1678, 1416, 1260, 1207, 988, 842 cm−1; 1H NMR and 13C NMR, see Table 2; HRESIMS m/z 225.1191 [M + H]+ (calcd for 225.1121, C12H17O4). Norxanthantolide D (6): white, amorphous powder; [α]25D −68.2 (c 0.15, CH3OH); UV (MeOH) λmax (log ε) 214 (3.82) nm; ECD (MeOH) nm (Δε) 225 (−1.47); IR νmax 2974, 2929, 1778, 1691, 1418, 1258, 1207, 988, 830 cm−1; 1H NMR and 13C NMR, see Table 2; HRESIMS m/z 225.1191 [M + H]+ (calcd for 225.1121, C12H17O4). Norxanthantolide E (7): white, amorphous powder; [α]25D −25.3 (c 0.06, CH3OH); UV (MeOH) λmax (log ε) 201 (3.57), 221 (3.17) nm; ECD (MeOH) nm (Δε) 228 (−1.34), 324 (−0.09); IR νmax 3428, 2970, 2937, 1776, 1683, 1455, 1175, 981 cm−1; 1H NMR and 13 C NMR, see Table 2; HRESIMS m/z 209.1177 [M + H]+ (calcd for 209.1172, C12H17O3). Norxanthantolide F (8): white, amorphous powder; [α]25D −55.5 (c 0.06, CH3OH); UV (MeOH) λmax (log ε) 220 (3.71) nm; ECD (MeOH) nm (Δε) 210 (−11.70), 235 (+2.22), 314 (−2.25); IR νmax 2970, 2933, 1754, 1677, 1253, 1178, 1143, 977, 820, 696 cm−1; 1H NMR and 13C NMR, see Table 2; HRESIMS m/z 207.1016 [M + H]+ (calcd for 207.1016, C12H15O3). (−)-7′-Dehydrosismbrifolin (9): white, amorphous powder; [α]25D −33.0 (c 0.04, CH3OH); UV (MeOH) λmax (log ε) 203 (3.35), 289 (2.62) nm; ECD (MeOH) nm (Δε) 211 (−3.49), 248 (−3.43), 276 (+4.28), 318 (−6.45); IR νmax 3360, 2920, 2850, 1658, 1480, 1323 cm−1; 1H NMR and 13C NMR, see Table 3; HRESIMS m/z 391.1397 [M + H]+ (calcd for 391.1387, C20H23O8). (+)-7′-Dehydrosismbrifolin (10): white, amorphous powder; [α]25D +30.0 (c 0.04, CH3OH); IR νmax 3359, 2920, 2851, 1659, 1468, 1324

influenza A virus are active constituents that may have beneficial effects against rhinitis.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an XT5B micromelting point apparatus and are uncorrected. Optical rotations were measured on a JASCO P-2000 automatic digital polarimeter. UV spectra were measured on a JASCO V650 spectrophotometer. ECD spectra were recorded on a JASCO J815 spectropolarimeter. IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer. NMR data were recorded on Inova 500, SX-600, or AVANCE III 800 spectrometers for 1D and 2D spectra. ESIMS data were measured on an Agilent 1100 Series LC/MSD ion trap mass spectrometer, and HRESIMS data were recorded on an Agilent Technologies 6250 Accurate-Mass Q-TOF LC/MS spectrometer. Preparative HPLC was performed on a Shimadzu LC-6AD instrument with an SPD-10A detector using a YMC-Pack ODS-A column (250 × 20 mm, 5 μm) or a Chiralpak AD-H column (250 × 20 mm, 5 μm). Column chromatography (CC) was performed on silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Sweden), and ODS (45−70 μm, Merck). Preparative thin-layer chromatography (TLC) was performed on glass precoated with silica gel GF254 (Qingdao Marine Chemical Inc.). Plant Material. The fruits of Xanthium sibiricum (100 kg) were collected at Helen City in Heilongjiang Province, People’s Republic of China, September 2012, and identified by Prof. Lin Ma from the Institute of Materia Medica at the Chinese Academy of Medical Sciences and Peking Union Medical College. A voucher specimen (IDS-2444) was deposited at the herbarium of the Institute of Materia Medica at the Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China. Extraction and Isolation. The dried fruits of X. sibiricum (100 kg) were milled and extracted with 95% EtOH (150 L × 2 h × 3). The resulting dried extract (8.0 kg) was concentrated in vacuo, suspended in H2O (50 L), and partitioned successively with petroleum ether (60−90 °C), EtOAc, and n-BuOH. The EtOAc extract (380 g) was subjected to silica gel CC (20 × 60 cm) and eluted with CH2Cl2/ CH3OH (100:1, 50:1, 20:1, 10:1, 5:1, 4:1, 1:1, and 0:1 v/v) to obtain seven fractions (EA−EG). Fraction EA (30 g) was chromatographed over silica gel (10 × 50 cm) using petroleum ether/acetone mixtures (from 80:1 to 0:1 v/v) to produce seven fractions (EA1−EA7). Fraction EA1 (6.6 g) was applied to silica gel CC (3.0 × 40 cm) and eluted with petroleum ether/acetone (100:1 to 0:1 v/v) to yield fractions EA1-1 to EA1-8. EA1-3 (150 mg) was purified by preparative TLC (petroleum ether/acetone, 20:1, Rf = 0.2) to obtain 1 (7.1 mg). EA1-5 (200 mg) was separated by RP-semipreparative HPLC (70% MeOH/H2O) to afford compounds 17 (80.5 mg) and 18 (10.6 mg). EA1-6 (180 mg) was purified by RP-semipreparative HPLC (70% MeOH/H2O) to obtain 2 (1.1 mg). Next, EA1-8 (600 mg) was separated by RP-semipreparative HPLC (70% MeOH/H2O) to afford compounds 11 (70.5 mg), 12 (10.0 mg), and 15 (50.6 mg). Fraction EA3 (12.5 g) was subjected to silica gel CC (5 × 50 cm) and eluted with a gradient of increasing CH3OH (0−100%) in CH2Cl2 to provide fractions EA3-1−EA3-7. EA3-5 (1.8 g) was separated by RPsemipreparative HPLC (50% MeOH/H2O) to afford compounds 13 (120 mg), 14 (11.0 mg), and 16 (55.0 mg). EA3-6 (1.2 g) was separated by RP-semipreparative HPLC (60% MeOH/H2O) to furnish compound 19 (10.0 mg). Fraction EC (35 g) was subjected to silica gel CC (6 × 80 cm) and eluted with a gradient of increasing MeOH (0−100%) in CH2Cl2 to give fractions EC1−EC5. The separation of EC3 (5.0 g) using an RP-C18 silica gel column (2.5 × 30 cm) and subsequent elution with MeOH/H2O (30:70, 50:50, 70:30, and 100:0 v/v) provided fractions EC3-1 to EC3-16. EC3-3 (280 mg) was separated by RP- semipreparative HPLC (30% MeOH/H2O) to give compounds 4 (5.0 mg), 5 (10.2 mg), and 6 (5.0 mg). EC3-7 (120 mg) was separated by RP-semipreparative HPLC (30% MeOH/H2O) to produce compounds 3 (8.0 mg), 7 (15.0 mg), and 8 (60.0 mg). ED (20.8 g) was chromatographed over RP-C18 silica gel (3.5 × 40 cm) H



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cm−1; UV (MeOH) λmax (log ε) 203 (3.37), 289 (2.61) nm; ECD (MeOH) nm (Δε) 216 (+6.29), 245 (+3.81), 278 (−3.67), 317 (+6.86); 1H NMR and 13C NMR, see Table 3; HRESIMS m/z 391.1397 [M + H]+ (calcd for 391.1387, C20H23O8). Crystallographic data for 1: C15H16O3, M = 244.28, colorless tetragonal crystal (MeOH), 0.65 × 0.20 × 0.15 mm3, orthorhombic, space group P41212; a = 9.6557(17) Å, b = 9.6557(17) Å, c = 26.319(4) Å, V = 2453.8(7) Å3, T = 120(7) K, Z = 8, ρcalcd = 1.322 g/ cm−3, μ = 0.740 mm−1, F(000) = 1040, 9461 reflections in h (−11/8), k (−11/10), l (−31/30), measured in the range 9.76° ≤ θ ≤ 137.88°, completeness θmax = 99.9%, 2288 independent reflections, Rint = 0.0308, 165 parameters, 0 restraints, GOF = 1.025. Final R indices: R1 = 0.0358, wR2 = 0.0839. R indices (all data): R1 = 0.0334, wR2 = 0.0821, and largest difference peak and hole: 0.172 and −0.156 e Å−3. The absolute structure was determined using a Flack parameter of 0.2(2). Crystallographic data have been deposited at the Cambridge Crystallographic Data Centre and allocated the deposition number CCDC 1017147. The data can be obtained free of charge via www. ccdc.cam.ac.uk/products/csd/request. Crystallographic data for 4: C11H14O3, M = 194.22, colorless orthorhombic crystal (MeOH), 0.70 × 0.07 × 0.02 mm 3 , orthorhombic, space group P212121; a = 6.1491(5) Å, b = 8.0979(5) Å, c = 20.2092(14) Å, V = 1006.31(12) Å3, T = 103(0) K, Z = 4, ρcalcd = 1.282 g/cm−3, μ = 0.759 mm−1, F(000) = 416, 5435 reflections in h (−7/6), k (−9/9), l (−16/24), measured in the range 8.76° ≤ θ ≤ 44.12°, completeness θmax = 99.0%, 1931 independent reflections, Rint = 0.0357, 129 parameters, 0 restraints, GOF = 1.055. Final R indices: R1 = 0.0448, wR2 = 0.1096. R indices (all data): R1 = 0.0479, wR2 = 0.1139, and largest difference peak and hole: 0.215 and −0.241 e Å−3. The absolute structure was determined using a Flack parameter of −0.2 (3). Crystallographic data have been deposited at the Cambridge Crystallographic Data Centre and allocated the deposition number CCDC 1017148. The data can be obtained free of charge via www. ccdc.cam.ac.uk/products/csd/request. Determination of Absolute Configuration of the 8′,9′-Diol Units in 9 and 10 by Snatzke’s and Frelek’s Method. According to a previously published procedure,13,29 a 1:1.2 mixture of diol/ Mo2(OAc)4 was subjected to ECD measurements at a concentration of 0.5 mg/mL for 9 and 10. The first ECD spectrum was recorded immediately after mixing, and its evolution was monitored over time until the signal was stationary (approximately 10 min after mixing). The inherent ECD was subtracted. The observed sign of the diagnostic band at 310−340 nm in the induced ECD spectrum was correlated to the absolute configuration of the 8′,9′-diol unit. In Vitro Anti-inflammatory Activity Assays. C57BL6/J mouse macrophages were cultured in 48-well plates in RPMI1640 medium at 37 °C for 24 h. Then, the cells were divided into four groups: the blank group (RPMI1640 medium only), the LPS group (1 μg/mL LPS in RPMI1640 medium), the experimental group (1 μg/mL LPS and the test compounds in RPMI1640 medium), and the positive control group (10−6 M dexamethasone in RPMI1640 medium). The cells were then incubated at 37 °C for an additional 24 h. From each well, 100 μL of the supernatant was mixed with the same amount of Griess reagent, and the absorbance value was measured at 570 nm using a microplate reader. Sodium nitrite was used as the standard to calculate the NO2− concentration.30,31a In Vivo Anti-inflammatory Assays. The topical anti-inflammatory activity was evaluated based on the inhibition of croton oilinduced ear edema in mice. Animal experiments were performed in accordance to the Institutional Guidelines for Animal Care and Use of the Chinese Academy of Medical Sciences and Peking Union Medical College. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Chinese Academy of Medical Sciences and Peking Union Medical College (permit pumber: 002973). The croton oil (Croton tiglium L. seed oil) was obtained from SigmaAldrich (St Louis, MO, USA). In ICR male mice (18−20 g, n = 10 per group), 0.4 mg of croton oil in 1 mL of acetone was applied to the left ear topically to induce ear edema. The candidate compounds (50 mg/ mL) were administered 1 h prior to croton oil treatment. Four hours after the application of croton oil, the animals were euthanized using

sodium pentobarbital, and 8 mm (diameter) punches of ear tissues were collected for measuring the weight of the left and right ear patches. The edematous response was measured based on the weight difference between two plugs (8 mm diameter) of the treated (left) and untreated (right) ears. The anti-inflammatory activity is expressed as the percentage reduction in edema in treated mice compared with control mice. Dexamethasone was used as a positive control.31 Antiviral Assays. African green monkey kidney cells (Vero) and Madin-Darby canine kidney (MDCK) cells were all obtained from the Institute of Virology at the Chinese Academy of Preventive Medicine. Coxsackie viruses (Cox B3, Nancy strain) and influenza A (A/FM/1/ 47, H1N1) were all obtained from the American Type Culture Collection (ATCC). Cytotoxicity Assay. The cytotoxicity of the compounds for Vero or MDCK cells was assayed by a cytopathic effect (CPE) assay. Briefly, cells (2.5 × 104 cells/well) were seeded into 96-well culture plates and were incubated overnight. Then, the medium was removed and different concentrations of compounds were applied in triplicate. After a 2-day incubation, the cytotoxicity of the compounds was determined using a CPE assay. The TC50 value was defined as the concentration that inhibits 50% cellular growth in comparison with the controls and calculated by the Reed and Muench method.32 Cytopathic Effect Inhibition Assay for Anti-CVB3 Activity. The anti-CVB3 activity of compounds was assayed by the CPE inhibition method. Briefly, Vero cells (2.5 × 104 cells/well) were plated into 96-well culture plates for an incubation period of 24 h. The medium was then removed, and the cells were infected with 100 μL of CVB3 of 100 TCID50 for 2 h. Then, various concentrations of compounds were supplemented during incubation until the CPE of the control group cells reached 4+. Each experiment was tested in triplicate and performed at least three times separately. The IC50 value is defined as the minimal concentration of inhibitor required to inhibit 50% of CPE as determined by the Reed and Muench method.32 The selectivity index was calculated as the ratio of TC50/IC50. Cytopathic Effect Inhibition Assay for Anti-influenza Activity. The anti-influenza activity of compounds was assayed by a CPE inhibition method. Briefly, MDCK cells (2.5 × 104 cells/well) were plated into 96-well culture plates for incubation of 24 h. The medium was then removed, and cells were washed by PBS and infected with 100 μL of influenza A (A/FM/1/47, H1N1) of 100 TCID50 for 2 h. Then, various concentrations of compounds were supplemented during incubation until the CPE of the control group cells reached 4+. Each experiment was tested in triplicate and performed at least three times separately. The IC50 value was defined as the minimal concentration of inhibitor required to inhibit 50% of CPE as determined by the Reed and Muench method.32 The selectivity index was calculated as the ratio of TC50/IC50.

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ASSOCIATED CONTENT

S Supporting Information *

Spectroscopic data for compounds 1−10. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/np500951s.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-10-63165326. Fax: 86-10-63017757. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by grants from the Natural Science Foundation of China (No. 21132009) and the National Science and Technology Project of China (No. 2012ZX09301002-002). We are grateful to Prof. Lin Ma (from the Institute of Materia I



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(29) Bari, L. D.; Pescitelli, G.; Prarelli, C.; Pini, D.; Salvadori, P. J. Org. Chem. 2001, 66, 4819−4825. (30) Sacco, R. E.; Waters, W. R.; Rudolph, K. M.; Drew, M. L. Comp. Immunol. Microbiol. Infect. Dis. 2006, 29, 1−11. (31) (a) García-Argáez, A. N.; Ramírez Apan, T. O.; Delgado, H. P.; Velázquez, G.; Martínez-Vázquez, M. Planta Med. 2000, 66, 279−281. (b) Mencherini, T.; Cau, A.; Bianco, G.; Della Loggia, R.; Aquino, R. P.; Autore, G. J. Pharm. Pharmacol. 2006, 59, 891−897. (32) Reed, L. J.; Muench, H. Am. J. Hyg. 1938, 27, 493−497.

Medica at the Chinese Academy of Medical Sciences and Peking Union Medical College) for identifying the plant.

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REFERENCES

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