Highly Oxidized Guaianolide Sesquiterpenoids ... - ACS Publications

Feb 5, 2018 - Among them, Chrysanthemum indicum is used in traditional Chinese medicine with heat-clearing and detoxifying functions and employed to ...
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Article Cite This: J. Nat. Prod. 2018, 81, 378−386

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Highly Oxidized Guaianolide Sesquiterpenoids with Potential Antiinflammatory Activity from Chrysanthemum indicum Gui-Min Xue,† Xiao-Qing Li,† Chen Chen,† Kang Chen,† Xiao-Bing Wang,† Yu-Cheng Gu,‡ Jian-Guang Luo,*,† and Ling-Yi Kong*,† †

Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, People’s Republic of China ‡ Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom S Supporting Information *

ABSTRACT: Ten new highly oxidized monomeric (1−8) and dimeric guaianolides (9 and 10), along with two known guaianolide derivatives (11 and 12), were isolated from the aerial parts of Chrysanthemum indicum using a bioassay-guided fractionation procedure. The new compounds were characterized by the basic analysis of the spectroscopic data obtained, and the absolute configurations were determined by both empirical approaches and ECD calculations. Inhibitory effects of 1−12 on nitric oxide production were investigated in lipopolysaccaride (LPS)-mediated RAW 264.7 cells, and most of them (1−8 and 11) displayed IC50 values in the range 1.4−9.7 μM. Moreover, a mechanistic study revealed that the potential anti-inflammatory activity of compound 1 appears to be mediated via suppression of an LPS-induced NF-κB pathway and downregulation of MAPK activation.

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esquiterpenoids, a class of metabolites with their C15 cores derived biosynthetically from isoprenoid precursors, are distributed widely in some higher plant families, including the Asteraceae, Cornaceae, Magnoliaceae, and Rutaceae.1 Extensive research focused on sesquiterpenoids with different skeletons and diverse activities has been carried out.1−3 The genus Chrysanthemum, belonging to the family Asteraceae, comprises approximately 30 species. Among them, Chrysanthemum indicum is used in traditional Chinese medicine with heat-clearing and detoxifying functions and employed to treat diseases associated with inflammation (colitis, stomatitis),4 infectious diseases (pertussis, pneumonia),5 and some other conditions including cancer, fever, hypertension, and vertigo.4,6 Phytochemical work on this species has revealed the presence of a number of monomeric, dimeric, and trimeric sesquiterpenoids.4,7 Cellular studies have demonstrated that certain sesquiterpenes from plants have exhibited promising potential anti-inflammatory activities. For example, helenalin directly modulates NF-κB,8 while parthenolide exerts an effect via inhibition of IκB kinase.9 Previously, a report on an extract of C. indicum indicated that sesquiterpenes might be the constituents responsible for its putative anti-inflammatory effects.10 Motivated by this finding and considering the structural diversity of sesquiterpenes in Asteraceae plants, the chemical constituents of C. indicum were studied via a bioassay-guided fractionation procedure. As a result, 12 highly oxygenated guaianolides (1−8, 11, and 12) and guaianolide dimers (9 and 10) were obtained. In this report, their isolation, structural characterization, and potential anti-inflammatory activities are described. © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Chrysanthemulide A (1), a white powder, afforded a molecular formula of C20H24O7 based on the (+)-HRESIMS ion peak at m/ z 399.1412 [M + Na]+ (calcd for 399.1414), corresponding to nine degrees of hydrogen deficiency. The characteristic IR absorptions demonstrated the presence of hydroxy (3484 cm−1) and ester carbonyl (1769 and 1716 cm−1) groups. Its 1H NMR spectrum exhibited typical signals for two methyl groups (δH 1.09, s, 3H; 1.56, s, 3H), four oxymethine protons (δH 3.33, br s; Received: October 17, 2017 Published: February 5, 2018 378

DOI: 10.1021/acs.jnatprod.7b00867 J. Nat. Prod. 2018, 81, 378−386

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Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) Spectroscopic Data for Compounds 1−5 1a position

δC

1 2 3 4 5 6 7 8

75.9 56.6 57.4 71.2 42.5 82.3 40.9 31.2

9 10 11 12 13

73.8 72.7 138.7 170.1 119.1

14 15 1′ 2′ 3′ 4′ 5′

24.9 20.8 166.4 127.2 140.1 20.8 16.0

a

δH (J in Hz) 3.60, br s 3.33, br s 2.94, d (10.5) 4.04, t (10.5) 3.52, m (10.2) 2.18, ddd (16.3, 10.2, 6.0) 2.08, d (16.3) 4.93, dd (6.0, 1.3)

6.19, d (3.3) 5.39, d (3.3) 1.09, s 1.56, s

6.16, qq (1.3, 7.2) 2.00, dq (1.3, 7.2) 1.93, dq (1.3, 1.3)

2a δC 76.0 56.7 57.4 71.2 42.5 82.2 41.0 31.1

74.0 73.0 138.8 170.2 119.2 24,9 19.6 166.5 128.4 138.7 14.7 12.5

δH (J in Hz) 3.60, br s 3.34, br s 2.95, d (10.5) 4.05, t (10.5) 3.52, m 2.17, m 2.06, d 4.92, dd (6.0, 1.5)

6.20, d (3.3) 5.39, d (3.3) 1.08, s 1.57, s

6.90, q (7.0) 1.85, d (7.0) 1.88, s

3a δC 76.0 56.6 57.4 71.3 42.4 82.4 40.9

72.7 72.7 138.7 170.2 119.2 24.9 19.6 164.9 115.4 160.3 27.6 20.5

δH (J in Hz) 3.59, br s 3.33, br s 2.95, d (10.5) 4.63, t (10.5) 3.51, m 2.15, m 2.06, d (15.9) 4.88, d (6.0)

6.19, s 5.40, s 1.06, s 1.56, s 5.74, s 2.21, s 1.94, s

4a δC 78.1 54.5 59.2 75.8 51.3 78.3 44.6 24.4

77.5 71.8 137.7 168.7 120.2 24.7 19.6 166.5 127 141.4 16.1 21.0

δH (J in Hz) 3.37, br s 3.34, br s 2.59, d (12.5) 4.27, dd (12.5, 9.8) 2.96, m 2.53, m 1,79, m 5.16, d (4.5)

6.23, d (3.3) 5.16, d (3.3) 1.38, s 1.55, s

6.18, qq (1.1, 7.2) 2.04, dq (1.1, 7.2) 1.95, dq (1.1, 1.1)

5a δC 74.0 54.5 59.3 75.9 51.5 78.3 44.6 27.4

78.1 71.8 137.7 168.8 120.2 24.6 19.6 167.0 128.1 139.6 14.9 12.4

δH (J in Hz) 3.40, br s 3.40, br s 2.63, d (12.5) 4.27, dd (12.5, 9.8) 2.95, m 2.56, m 1.79, m 5.12, d (4.5)

6.23, d (3.3) 5.50, d (3.3) 1.38, s 1.60, s

6.99, q (7.0) 1.83, d (7.0) 1.87, s

Data were recorded in CDCl3.

3.60, br s; 4.04, t, J = 10.5 Hz; 4.93, dd, J = 5.9, 1.3 Hz), a pair of exocyclic methylene protons (δH 5.39, 6.19, each, d, J = 3.3 Hz), and an angeloyloxy group (Table 1).11 In the 13C NMR spectrum, apart from five characteristic carbon signals for an angeloyloxy group, 15 carbon resonances were observed and ascribed to three sp2 carbons (one lactone carbonyl and two olefinic carbons) and 12 sp3 carbons (including seven oxygenbearing carbons), with the aid of an HSQC experiment. These characteristic signals, in combination with the HRESIMS data, implied that 1 is a highly oxidized sesquiterpenoid possessing an angeloyloxy group substitution.12,13 In the HMBC spectrum, the cross-peaks from H-13 to C-7 and C-12 and from H-7 to C-12 and C-13 suggested the existence of an α-methylene-γ-lactone moiety.11 The observable HMBC correlations of H-5/C-2, C-3, C-7, and C-10; H-6/C-1 and C-8; H-9/C-1 and C-7; H-14/C-1, C-2, C-9, and C-10; and H-15/C3, C-4, and C-5 (Figure 1A) were used to establish the molecular skeleton of 1 as being the same as that of a typical guaianolide sesquiterpene.11−13 An observed chemical shift (δC 72.7, C-10), along with HMBC corrections from H-5, H-8, and H-14 to C-10, indicated the location of a tertiary hydroxy group at C-10. The esterification of the hydroxy unit at C-9 with angelic acid was elucidated from the HMBC correlation of H-9/C-1′ (δC 166.4). Thus, the molecule required two epoxy rings to satisfy the remaining two degrees of unsaturation, suggesting that 1 possesses 1,2:3,4-diepoxy groups attached to the cyclopentane ring. These were supported by typical upfield oxygen-bearing 13C NMR spectroscopic chemical shifts (δC 56.6, C-2; 57.4, C-3) and the observed HMBC correlations from H-15 to C-3 and C-5 and H-14 to C-1 and C-2. Thus, the planar structure of 1 was assigned as being that of a highly oxidized guaianolide. The relative configuration of 1 was determined from its ROESY spectrum and J-based configurational analysis. The magnitude of JH‑5,H‑6 = 10.5 Hz was indicative of them being in an

Figure 1. Key HMBC (A) and ROESY (B) correlations and experimental and calculated ECD spectra (C) of compound 1.

axial position on opposite sides of the molecule; thus H-5 and H6 were deduced to have an α- and a β-orientation, respectively. The observable ROESY correlation of H-5/H-7 supported these protons as having cofacial orientations, and they are assigned as α-oriented, while H-9 and CH3-14 were defined as β-oriented owing to the ROESY cross-peaks of H-6/H-8β, H-8β/H-9, and H-9/H-14 (Figure 1B). Comparison of the NMR data of 1 was made with those of canin (1α,2α:3α,4α-diepoxide) and artecanin (1β,2β:3β,4β-diepoxide),12 with both epoxy groups in 1 being assigned as β-oriented according to the characteristic chemical shifts of H-5 (δH 2.94, d, J = 10.5 Hz) and C-5 (δC 42.5)12 and were confirmed by the ROESY correlations of H-3/H-2, H-5/H15, and H-15/H-3 (Figure 1B). 379

DOI: 10.1021/acs.jnatprod.7b00867 J. Nat. Prod. 2018, 81, 378−386

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Figure 2. Key ROESY correlations of compound 4, 6, 7, and 8.

Table 2. 1H NMR (500 MHz) and 13C NMR (125 MHz) Spectroscopic Data for Compounds 6−10 6a position

a

δC

1 2 3 4 5 6 7 8

73.5 63.1 77.6 87.5 56.6 78.6 42.3 28.3

9

78.2

10 11 12 13

71.9 137.9 169.2 120.6

14 15 1′ 2′ 3′

23.2 23.4 166.6 127.1 141.3

4′ 5′ 6′ 7′ 8′

16.2 21.0

δH (J in Hz) 3.81, d (3.1) 3.78, br s 2.32, d (11.5) 4.54, dd (11.5, 10.0) 3.03, m 2.55, ddd (15.2, 5.1, 3.1) 1.80, m 5.17, dd, (5.0, 1.4)

6.25, d (3.3) 5.52, d (3.3) 1.40, s 1.49, s

6.21, qq (1.3, 7.3) 2.06, dq (1.3, 7.3) 1.98, dq (1.3, 1.3)

7a δC 69.0 62.5 64.3 86.4 57.8 79.5 42.0 28.6 78.2 72.5 137.1 168.4 121.2 22.8 20.5 166.2 135.6 127.4 18.2

8a

δH (J in Hz) 3.57, s 4.13, s 2.59, d (11.5) 4.41, dd (11.5, 10.0) 3.14, m 2.58, m 1.82, m 5.12, dd (5.0, 2.4)

6.29, d (3.3) 5.57, d (3.3) 1.38, s 1.52, s

5.57, br s 6.30, br s 2.00, s

δC 98.6 133.6 137.7 93.8 69.7 78.7 41.8 70.7 30.5 74.2 139.3 169.7 120.1 21.8 13.8 165.7 115.1 159.9 27.8 20.6

δH (J in Hz) 6.44, d (5.6) 6.35, d (5.6) 2.65, d (10.0) 3.79, t (10.0) 3.44, m 5.07, t (8.4) 2.35, m 1.97, m

6.17, d (3.5) 5.43, d (3.5) 1.36, s 1.71, s 5.77, s

2.20, s 1.94, s

9a δC 74.6 65.4 78.3 86.4 56.6 76.8 48.8 24.5 38.6 70.1 56.6 178.1 35.9 15.3 24.9 64.0 133.7 141.5 58.0 66.6 79.7 43.6 23.9

9′ 10′ 11′ 12′ 13′

35.1 73.2 140.9 170.8 119.0

14′ 15′

30.1 15.3

10a

δH (J in Hz)

δC

3.85, d (3.0) 5.83, d (3.0) 2.26, d (10.6) 4.47, d (10.6) 2.57, dt (9.0, 4.1) 1.99, m 1.71, m 1.90, m 1.87, m

2.47, d (11.7) 1.42, d (11.7) 1.31, s 1.43, s 5.91, d (5.5) 6.30, d (5.5)

2.27, d (9.7) 4.17, t (9.7) 3.24, m 2.22, m 1.51, m 1.88, m

6.11, d (3.3) 5.37, d (3.3) 1.34, s 1.39, s

73.5 65.0 77.7 87.6 57.2 76.6 22.2 36.8 71.8 56.9 178.4 35.9 27.4 24.1 64.1 133.8 141.4 57.6 66.7 79.9 43.7 23.6 35.0 73.1 141.0 170.9 119.0 29.9 15.3

δH (J in Hz) 3.95, d (3.0) 3.83, d (3.0) 2.17, d (10.6) 4.78, t (10.6) 2.43, m 2.08, m 1.78, m 1.92, m 1.85, m

2.54, d (11.7) 1.37, d (11.7) 1.16, s 1.37, s 5.90, d (5.5) 6.30, d (5.5)

2.33, d (9.7) 4.17, d (9.7) 3.22, m 2.24, m 1.48, m 1.86, m

6.11, d (3.3) 5.38, d (3.3) 1.32, s 1.40, s

Data were recorded in CDCl3.

determined from the similarities of the experimental and the calculated ECD curves. Therefore, the structure of 1 was elucidated and has been named chrysanthemulide A. The same molecular formula, C20H24O7, for 2 and 3 (chrysanthemulides B and C) was established based on their sodium adduct ions in the HRESIMS at m/z 399.1411 and 399.1416, respectively. Analysis of their NMR spectroscopic data suggests that both 2 and 3 share the same guaiane-type sesquiterpene skeleton as 1, except for a difference in their respective side chain moiety. Their respective 1H NMR spectra

The configuration of C-7 in guaianolide sesquiterpene lactones possessing an α-methylene-γ-lactone group can be determined following the Geissman rule.14,15 In the electronic circular dichroism (ECD) spectrum, the negative Cotton effect at 250 nm (Δε = −2.1) for a C-6, C-7 trans-fused α-methylene-γlactone chromophore in the ECD curve was observed (Figure 1C), which indicated a 7S configuration. Moreover, the ECD quantum chemical calculation was employed for determination of its absolute configuration.5 As shown in Figure 1C, the 1S,2R,3S,4R,5S,6S,7S,9R,10R absolute configuration for 1 was 380

DOI: 10.1021/acs.jnatprod.7b00867 J. Nat. Prod. 2018, 81, 378−386

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Figure 3. Key HMBC and ROESY correlations of compound 9.

displayed a similar negative Cotton effect at 250 nm, which was attributed to a C-6, C-7 trans-fused α-methylene-γ-lactone chromophore, which defined a 7S stereochemistry. Thus, the structure of 6 was assigned as shown. A cluster of sodium adduct ion peaks at m/z 421.1022/ 423.0998 with a ratio of 3:1 was observed in the HRESIMS of 7, suggesting that it possesses a monochlorinated structure with the molecular formula of C20H23O7Cl. Comparison of 1H and 13C NMR spectra between 7 and 6 indicated that the OH-3 in 6 is replaced by a Cl atom in 7, which was confirmed by the upfield chemical shift at C-3 (δC 64.3) and HMBC correlation between H-15 and C-3.15 In addition, the resonances for a methacrylate group (Table 2) were observed in 7, instead of signals of an angeloyloxy unit in 6. The methacrylate unit was situated at C-9, as determined from the HMBC cross-peak of H-9/C-1′. The H-9 proton was defined to be β-oriented according to the ROESY correlations from H-14 to H-6 and H-9 (Figure 2). The Cl-3β configuration was deduced by the ROESY correlation of H-3/H5 (Figure 2). The similarity of the Cotton effect at 250 nm (Figure S7-1, Supporting Information) of 7 to that of 6 suggested a 7S configuration in both compounds. Furthermore, its (1R,1R,3S,4S,5S,6S,7S,9R,10R) absolute configuration was also defined by the calculated ECD data (Figure S2, Supporting Information). Therefore, the structure 7 was deduced for chrysanthemulide G. To date, more than 60 chlorine-containing guaianolide sesquiterpenoids have been isolated from the Asteraceae species,18−20 and no chlorine-containing solvents were used during the process of extraction and isolation. Therefore, it is believed that compound 7 is a naturally occurring substance. The molecular formula of chrysanthemulide H (8) was assigned as C20H24O7 by the analysis of its HRESIMS data. The typical 1H NMR signals for an endo-double bond (δH 6.35, 6.44, each, d, J = 5.6 Hz), a senecioyl group (δH 1.94, br s; 2.20, s; 5.77, br s), an exomethylene group (δH 5.43, 6.17, each, d, J = 3.5 Hz), two methyls (δH 1.36, s; 1.71, s), and two oxygen-bearing carbon protons (3.97, t, J = 10.0 Hz; 5.07, t, J = 8.4 Hz) were observed (Table 2). In the 13C NMR spectrum, 15 carbon resonances occurred aside from the signals for a senecioyl group, suggesting that compound 8 is also a sesquiterpenoid. Comparison of the NMR data with those of 8-tigloyldesacetylezomontanin (11)21 indicated that the tigloyl unit at C-8 in 11 is replaced by a senecioyl group in 8. This deduction was supported by the HMBC correlation of H-8/C-1′. The ROESY correlations (Figure 2) between H-6/H-8, H-8/H-2, and H-2/H-14 suggested that the H-6, H-8, and H-14 protons are β-oriented,

exhibited resonances for a tigloyloxy moiety (Table 1) in 2 and a senecioyloxy unit (Table 1) in 3.16 The HMBC (H-9/C-1′) and ROESY (H-6/H-8β, H-9/H-8β and H-14) correlations enabled the determination of the α-orientation of the C-9 substituent in both 2 and 3. The ECD spectra of 2 and 3 were similar to that of 1 (Figures S2-1 and S3-1, Supporting Information), and thus their 7S configurations were also determined by the Geissman rule.14,15 The structures of compounds 2 and 3 were proposed as shown. The HRESIMS of chrysanthemulide D (4) displayed a positive-ion peak at m/z 399.1416 [M + Na]+, suggesting that this compound shares the same molecular formula (C20H24O7) as those of 1−3. The resonances in its NMR data (Figures S4-4− S4-7, Supporting Information) demonstrated the planar structure of 4 was identical to that of 1. However, the ROESY correlations (Figure 2) of H-15/H-6 and H-3; and H-14/H-6 and H-2 were indicative of a 1β,2β:3β,4β-diepoxy moiety in 4, which was opposite those of 1−3, and further supported by the upfield-shifted resonance of H-5 at δH 2.59 (d, J = 12.5 Hz), due to the shielding effects of the two epoxy groups. The ECD spectrum of 4 was similar to those of 1−3 (Figure S4-1, Supporting Information), suggesting a 7S absolute configuration of 4. The molecular formula of 5 was consistent with that of 4 based on the HRESIMS data. The NMR data suggest they share the same carbon skeleton with a 1β,2β:3β,4β-diepoxy unit in the cyclopentane ring. On comparison of 5 with 4, the difference was the replacement of the angeloyloxy group in 4 by a tigloyloxy substituent in 5 (Table 1), which was in agreement with the HMBC and ROESY correlations observed (Figure 2). Similarly, the 7S configuration of chrysanthemulide E (5) was deduced by the analysis of the ECD spectrum (Figure S5-1, Supporting Information), according to the Geissman rule.14,15 Chrysanthemulide F (6) possessed a molecular formula of C20H26O8 according to the HRESIMS data (m/z 417.1517 [M + Na]+), with 18 mass units more than that of 4. An analysis of the NMR data of 6 suggested a similar structure to 4, with the main differences occurring in the cyclopentane ring. The downfieldshifted resonances at C-3 (ΔδC +18.3) and C-4 (ΔδC +11.6), coupled with HMBC cross-peaks from H-15 to C-3 (δC 77.6) and C-4 (δC 87.5), suggested the opening ring of the 3,4-epoxy group as in 4,12,17 which was also consistent with the rest of the indices of unsaturation and the molecular weight. The ROESY correlations of H-3/H-15, H-15/H-6, and H-3/H-6 suggested both protons H-3 and H-15 to be β-oriented (Figure 2). The ECD spectrum (Figure S6-1, Supporting Information) of 6 381

DOI: 10.1021/acs.jnatprod.7b00867 J. Nat. Prod. 2018, 81, 378−386

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and thus the 1,4-peroxide bridge and senecioyl group are αoriented, while the correlations of H-5/H-7 confirmed the αorientation of these protons. A negative Cotton effect at 226 nm for the n−π* transition for the C-8 substituent was observed, indicative of an 8S configuration of 8 according to the Geissman rule.22 In addition, calculated ECD data (Figure S2, Supporting Information) facilitated the determination of the 1S,4R,5S,6S,7S,8S,10R absolute configuration of 8. The HRESIMS of chrysanthemulide I (9) displayed a sodium adduct ion peak at m/z 565.2404 ([M + Na]+, calcd for 565.2408), suggesting its molecular formula of C30H38O9. Its 1H NMR data revealed the signals for four methyls (δH 1.31, s; 1.34, s; 1.39, s; 1.43, s), an endo-double bond (δH 5.91, 6.30, each, d, J = 5.5 Hz), and an exomethylene group (δH 5.37, 6.11, each, d, J = 3.5 Hz) (Table 2). The 13C NMR spectrum exhibited the presence of 30 carbons, including four methyl carbons, six methylene carbons (including one olefinic), 10 methine carbons (including four oxygenated and two olefinic), and 10 quaternary carbons (including two ester carbonyls, four oxygenated, and one olefinic) (Table 2). These assignments, coupled with the HRESIMS data, implied that 9 is a sesquiterpene lactone dimer.23 The structure of 9 (chrysanthemulide I) was deduced mainly by analysis of its HMBC data. Starting from the characteristic signal of H-6, the HMBC correlations of H-6/C-1, C-8, C-11, and C-12; H-7/C-5, C-9, C-12, and C-13; H-13/C-7 and C-12; C-14/C-1, C-2, and C-9; and H-14/C-2 and C-5 (Figure 3) suggested that a part of the molecule of 9 is a highly oxidized guaianolide sesquiterpenoid with the same planar structure as 10epiajafinin (12) except for the signals of a Δ11,13 exomethylene group.24 A second unit found is also a guaianolide moiety that was established by a similar HMBC correlation analysis. An αmethylene-γ-lactone and a Δ2′,3′ endo-double bond moiety were present in this second unit as supported by the HMBC crosspeaks of H-13′/C-11′ and C-12′, H-14′/C-2′, and H-15′/C-3′. The linkage of the two units of 9 was confirmed by the HMBC correlations of H-13/C-7 and C-10', H-15′/C-11 and C-5', and H-3′/C-11 (Figure 3), indicating their direct carbon−carbon formations via C-11/C-4′ and C-13/C-1′. Consequently, the planar structure of 9 could be completely established. The relative configuration of 9 was resolved by its coupling constants and ROESY correlations. The large coupling constants of H-5/H-6 (JH‑5/H‑6 = 10.6 Hz) and H-5′/H-6′ (JH‑5′/H‑6′ = 9.7 Hz) indicated their trans spatial relationship. The successive cross-peaks of H-5′/H-7′, H-5′/H-13β, H-13β/H-6, H-6/H-14, H-6/H-3, and H-14/H-2 confirmed that these protons are cofacial with a β-orientation, as shown in Figure 3. The αoriented H-5, H-7 H-6′, and H-14′ and the C-2′−C-3′ bridge were determined by ROESY correlations between H-6′/H-2′, H6′/H-3′, H-2′/H-14′, H-15′/H-7, and H-7/H-5 (Figure 3). Accordingly, compound 9 was confirmed as shown in Figure 3. Chrysanthemulide J (10) was found to have the same molecular formula as 9 based on their HRESIMS data. Analysis of the 2D NMR data of 10 enabled an identical planar structure as 9 to be proposed. However, the configuration of 10 at C-4 was opposite that of 9, and CH3-15 was defined as β-oriented owing to the ROESY correlations between H-15/H-6 and H-3.12,13 The ECD spectra of 9 and 10 were very similar, and the experimental ECD spectrum for 9 was nearly consistent with the calculated one of 1R,2S,3R,4R,5S,6S,7S,10R,11R,1′R,4′R,5′S,6′S,7′S,10′R (Figure S2, Supporting Information), suggesting the absolute configuration for 9 as shown in Figure 3. Furthermore, the similar negative Cotton effect at 258 nm between 9 and 10 (Figures S9-1

and S10-1, Supporting Information), together with a consideration of the Geissman rule, 25 indicated the absolute configurations of 9 and 10 to be as shown. Two known compounds were identified as 8-tigloyldesacetylezomontanin (11)21 and 10-epiajafinin (12),24 based on their obtained spectroscopic data. A bioassay-guided separation of C. indicum led to the isolation of compounds 1−12. All the isolates were tested for their effects on nitric oxide (NO) production inhibition in lipopolysaccharide (LPS)-activated RAW 264.7 cells. In order to exclude the inhibition of NO production caused by cytotoxicity, cell viability was evaluated by the MTT method. Results revealed that no obvious cytotoxicity (over 90% cell survival) for most of compounds at concentrations up to 15 μM was observed. As all isolates inhibited NO release, as shown in Table 3, compared Table 3. Nitric Oxide Inhibitory Activity of Compounds 1−12 in LPS-Induced RAW264.7 Macrophages compound

IC50 (μM)

compound

IC50 (μM)

1 2 3 4 5 6 a L-NMMA

1.4 ± 0.12 3.9 ± 0.32 4.8 ± 0.76 4.0 ± 0.59 6.3 ± 0.35 7.1 ± 1.01 25.8 ± 2.01

7 8 9 10 11 12

9.7 ± 1.42 7.2 ± 0.48 >50 >50 3.4 ± 0.31 21.5 ± 2.11

a L-NMMA was the positive control. Each value represents the mean ± SD of three independent experiments.

with the positive control NG-monomethyl-L-arginine (L-NMMA, IC50 25.8 ± 2.01 μM), most of the isolated compounds (1−12) that displayed NO inhibitory activity possess an α-methylene-γlactone moiety, which suggests that this functionality, usually as a Michael acceptor, may play a significant role in mediating the activities.25 The structure differences of the highly oxidized guaianolide sesquiterpenoids 1−8 and 12 reside mainly in the cyclopentane ring and the substituent at C-9. The enhanced NO inhibitory activity of compounds 1−8 (IC50 1.4−9.7 μM) versus 12 (IC50 21.5 μM) suggested the important role of α,βunsaturated acyl substitution at C-9. The type of substituent at C9 also influenced compound activity, and the following trend in activity for the ester groups was observed: angeloyloxy > tigloyloxy > senecioyloxy (1 vs 2 and 3; 4 vs 5; 8 vs 11). In addition, compounds 1−5 were more active than 6, 7, and 12, indicating the necessity of a diepoxide functionality in the cyclopentane ring. Since compound 1 displayed the strongest inhibition on NO release, it was selected for a mechanistic investigation. The proinflammatory mediator NO, a key factor in inflammation-related diseases,26,27 was evaluated in LPS-induced RAW 264.7 macrophages. Compound 1 displayed no cytotoxicity at concentrations up to 6.25 μM (Figure 4A); thus, the NO levels were detected under this concentration in subsequent experiments. As shown in Figure 4B, the significant NO production inhibitory effect of 1 with an IC50 of 1.4 ± 0.12 μM was observed. As is well known, the production of NO is closely related to the key proteins iNOS and COX-2.28 Therefore, the expression levels on these proteins were detected by Western blotting. As shown in Figure 4C, a slight down-regulation of COX-2 expression was observed, but the levels of iNOS were completely inhibited in the presence of 1 at 2.5 μM. Based on these observations, it was concluded that 1 exerted inhibitory effects on 382

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Figure 4. Effects of 1 on LPS-induced iNOS and COX-2 protein levels. Effects of compound 1 on cell viability (1.5625−50 μM) (A) and NO inhibitory activity (0.1953−6.25 μM) in RAW264.7 cells (B). A Western blot assay was used to detect iNOS and COX-2 protein levels (C). ###p < 0.001, compared to the control group; ***p < 0.001, compared to the LPS-treated group.

Figure 5. Effects of 1 on nuclear translocation of NF-κB (p65) (A) and phosphorylation of STAT3 (B) in RAW264.7 cells by Western blotting (###p < 0.001, compared to the control group; *p < 0.05, **p < 0.01, ***p < 0.001, compared to the LPS-treated group).

Western blotting. Notably, the levels of p-STAT3 were downregulated upon the treatment of 1, and also the total NF-κB was suppressed (Figure 5B). Thus, these data demonstrated that compound 1 inhibited the NF-κB signaling pathway partially via suppression of STAT3 to exert anti-inflammatory effects. The MAPKs pathway is also well known to mediate the inflammation process.28 It has been demonstrated that the inhibition of MAPKs (ERK, JNK, and P38) by specific pharmacological antagonists can inhibit LPS-stimulated overexpression of iNOS and COX-2, suggesting that all three kinases participate in the inflammation-mediated response.28 Therefore, the effect of 1 on MAPKs was investigated, and as a result, compound 1 decreased the expression of these three kinases

NO production mainly through suppression of iNOS in LPSinduced RAW264.7 macrophages. NF-κB occurs as a key transcriptional regulator in inflammatory mediators.28 The translocation of activated NF-κB from the cytoplasm to nuclear binding with DNA can up-regulate iNOS and COX-2 levels.29 Therefore, the effect of 1 on the activated NF-κB (p65) was analyzed using Western blotting. The results showed that the nuclear NF-κB levels were down-regulated (Figure 5A), indicating that its nuclear translocation procedure was suppressed by 1. In addition, previous investigations have demonstrated that phosphorylation of STAT3 (p-STAT3) regulates iNOS levels via the NF-κB pathway.30 Therefore, total NF-κB and p-STAT3 in protein levels was determined by 383

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Figure 6. Effects of 1 on phosphorylation of MAPKs in LPS-induced RAW264.7 cells. Cells were pretreated in the presence or absence of 1 (1.25, 2.5, 5.0 μM) for 1 h and then incubated with LPS (0.1 μg/mL) for 4 h. Protein levels were analyzed by using Western blotting. ###p < 0.001, ##p < 0.01 compared to the control group; *p < 0.05, **p < 0.01, ***p < 0.001, compared to the LPS-treated group. subfrations (Fr.A−Fr.E), and Fr.D revealed the most potent inhibitory activity against NO production (IC50 1.8 μg/mL, Table S1). Thus, Fr. D (40 g) was loaded onto Sephadex LH-20 to yield five fractions (Fr.D1− Fr.D5), and Fr. D4 was selected for further studies based on its best NO inhibitory effects (Table S1). Fr. D4 (6.2 g) was chromatographed on MPLC (100 g, 20−100% MeOH/H2O, v/v) to afford five fractions (Fr.D4-1−Fr.D4-6). Fr.D4-2 was purified subsequently by preparative HPLC using 45% MeOH/H2O (v/v) to yield 1 (33.1 mg), 3 (13.8 mg), 4 (5.5 mg), and 5 (5.1 mg). Fr.D4-3 was chromatographed by RP-C18 CC to afford 6 (15 mg) and Fr.D4-3a, while Fr.D4-3a was separated by preparative HPLC eluted with 48% MeOH to produce 2 (4.4 mg), 7 (2.2 mg), and 12 (20.0 mg). Fr.D4-4 was subjected to RP-C18 CC to afford five fractions (Fr.D4-4a−Fr.D4-4e). Then, Fr.D4-4c and Fr.D44d were purified by preparative HPLC with 55% MeOH to yield 8 (3.1 mg), 9 (3.5 mg), and 11 (6.1 mg), and with 50% CH3CN−H2O (v/v) to yield 9 (4.8 mg) and 10 (3.8 mg). Chrysanthemulide A (1): white powder; [α]25 D +11.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (3.89) nm; ECD (MeOH) λmax (Δε) 205 (−3.2), 222 (+12.7), 250 (−2.1) nm; IR (KBr) νmax 3484, 1769, 1716, 1459, 1387, 1233, 1159, 1040, 1014, 953, 848, 726 cm−1; 1H and 13C NMR data (Table 1); HRESIMS m/z 399.1412 [M + Na]+ (calcd for C20H24O7Na, 399.1414). Chrysanthemulide B (2): white powder; [α]25 D −1.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (3.63) nm; ECD (MeOH) λmax (Δε) 204 (−23.2), 223 (+45.6), 263 (−2.4) nm; IR (KBr) νmax 3450, 1768, 1710, 1641, 1384, 1269, 1151, 1014, 816 cm−1; 1H and 13C NMR data (Table 1); HRESIMS m/z 399.1411 [M + Na]+ (calcd for C20H24O7Na, 399.1414). Chrysanthemulide C (3): white powder; [α]25 D −5.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (3.83) nm; ECD (MeOH) λmax (Δε) 207 (−7.5), 224 (+6.4), 259 (−1.3) nm; IR (KBr) νmax 3459, 1769, 1711, 1646, 1443, 1384, 1229, 1143, 1077, 1012, 951, 851 cm−1; 1H and 13C NMR data (Table 1); HRESIMS m/z 399.1416 [M + Na]+ (calcd for C20H24O7Na, 399.1414).

(Figure 6). Thus, this demonstrated that the inhibitory effects on LPS-induced iNOS overexpression by 1 were mainly via the suppression of the MAPK signaling pathway.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO P-1020 spectropolarimeter in MeOH. The UV spectra were determined by a UV-2450 visible spectrophotometer (Shimadzu, Tokyo, Japan). A JASCO 810 spectropolarimeter (JASCO, Tokyo, Japan) was used to measure ECD spectra. Infrared spectra were collected using KBr disks on a Tensor 27 infrared spectrometer (Bruker). Bruker AV-500 and AV-600 NMR spectrometers (Bruker, Karlsruhe, Germany) were used to record NMR data in CDCl3, with tetramethylsilane as the internal standard. High-resolution electrospray ionization mass spectra (HRESIMS) were acquired on an Agilent 6520B Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). An Agilent 1260 Series instrument with an RP-C18 column (20 × 200 mm i.d., Shim-pack, Shimadzu, Tokyo, Japan) and an Agilent 1200 Series (Agilent Technologies) were used for analysis. Semipreparative HPLC was run on a Shimadzu LC-6A system (Shimadzu) coupled with a Shim-pack RP-C18 column (200 × 20 mm, 10 μm). Column chromatography (CC) was performed on MCI (Mitsubishi, Japan), Sephadex LH-20 (Uppsala, Sweden), and RP-C18 silica (40−63 μm, FuJi, Japan). Plant Material. The aerial parts of C. indicum were collected in Kunming, Yunnan Province, People’s Republic of China, in September 2016. A voucher specimen (No. 160920) was deposited at the Department of Natural Medicinal Chemistry, China Pharmaceutical University. Extraction and Isolation. The aerial parts of C. indicum (2.0 kg) were extracted using 95% EtOH (3 × 10 L) under reflux. The extract was concentrated in vacuo at 40 °C. The residue (100 g) was first fractionated by an MCI gel chromatographic column, eluted with a step gradient system of MeOH/H2O (v/v, 30−100%), to afford five 384

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Chrysanthemulide D (4): white powder; [α]25 D −5.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 213 (3.73) nm; ECD (MeOH) λmax (Δε) 208 (−0.3), 221 (+5.2), 251 (−2.4) nm; IR (KBr) νmax 3497, 1767, 1716, 1646, 1457, 1383, 1294, 1233, 1157, 1011, 999, 941, 848, 763 cm−1; 1H and 13C NMR data (Table 1); HRESIMS m/z 399.1416 [M + Na]+ (calcd for C20H24O7Na, 399.1414). Chrysanthemulide E (5): white powder; [α]25 D −1.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (3.66) nm; ECD (MeOH) λmax (Δε) 204 (−0.1), 221 (+0.8), 253 (−0.2) nm; IR (KBr) νmax 3465, 1769, 1708, 1654, 1384, 1262, 1156 cm−1; 1H and 13C NMR data (Table 1); HRESIMS m/z 399.1412 [M + Na]+ (calcd for C20H24O7Na, 399.1414). Chrysanthemulide F (6): white powder; [α]25 D −45.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (4.09) nm; ECD (MeOH) λmax (Δε) 208 (−0.7), 222 (+2.5), 252 (−1.2) nm; IR (KBr) νmax 3472, 1769, 1717, 1640, 1459, 1384, 1293, 1157, 1043, 998, 926, 802 cm−1; 1H and 13C NMR data (Table 2); HRESIMS m/z 417.1517 [M + Na]+ (calcd for C20H26O8Na, 417.1520). Chrysanthemulide G (7): white powder; [α]25 D −20.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (3.64) nm; ECD (MeOH) λmax (Δε) 208 (−0.7), 222 (+2.5), 252 (−1.2) nm; IR (KBr) νmax 3856, 1770, 1718, 1637, 1384, 1294, 1264, 1230, 1037, 954, 813 cm−1; 1H and 13C NMR data (Table 2); HRESIMS m/z 421.1022 [M + Na]+ (calcd for C20H23O7Cl, 421.1025). Chrysanthemulide H (8): white powder; [α]25 D +6.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (4.01) nm; ECD (MeOH) λmax (Δε) 200 (+7.0), 224 (−2.6), 256 (−1.7) nm; IR (KBr) νmax 3450, 1772, 1714, 1646, 1446, 1383, 1349, 1228, 1145, 1078, 1005, 942, 815 cm−1; 1H and 13 C NMR data (Table 2); HRESIMS m/z 399.1412 [M + Na]+ (calcd for C20H24O7Na, 399.1414). Chrysanthemulide I (9): white powder; [α]25 D −25.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.81) nm; ECD (MeOH) λmax (Δε) 200 (+6.5), 230 (−2.1), 256 (−1.6) nm; IR (KBr) νmax 3447, 1742, 1638, 1383, 1161, 1059, 819 cm−1; 1H and 13C NMR data (Table 3); HRESIMS m/z 565.2404 [M + Na]+ (calcd for C30H38O9Na, 565.2408). Chrysanthemulide J (10): white powder; [α]25 D −29.4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (3.85) nm; ECD (MeOH) λmax (Δε) 200 (+5.6), 228 (−2.1), 252 (−1.3) nm; IR (KBr) νmax 3432, 1751, 1638, 1458, 1383, 1265, 1138, 1061, 991, 905 cm−1; 1H and 13C NMR data (Table 3); HRESIMS m/z 565.2407 [M + Na]+ (calcd for C30H38O9Na, 565.2408). Quantum Chemical ECD Calculations. The relative configurations of 1, 7, 8, and 9 were established initially according to their ROESY NMR spectra. Their conformers were first identified using the TD-SCF method at the B3LYP/6-311+G(d,2p) level. Then, on the basis of Boltzmann distribution, the selected conformers were further optimized at the B3LYP/6-311G(d, 2p) level in MeOH, and TDDFT ECD calculations were measured with the Gaussian 09 program package. The ECD curves of different conformers were simulated by SpecDis and weighted according to the Boltzmann distribution after UV correction. Cell Culture. The murine RAW 264.7 macrophage cells were obtained from Cell Bank of the Shanghai Institute of Cell Biology and Biochemistry, Chinese Academy of Sciences (Shanghai, People’s Republic of China). The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and penicillin− streptomycin (100 U/mL) at 37 °C with 5% CO2. Cell Viability Assay. An MTT assay was used to evaluate RAW264.7 cell viability as previously described.28 Briefly, cells were plated in 96-well plates (5 × 103 cells/well) for 18 h and then incubated with compounds 1−12 (purity ≥95%) in various concentrations with or without LPS (1.0 μg/mL). Eighteen hours later, the prepared MTT solution (20 μL, 5 mg/mL) was added, and the cells were incubated for another 4 h. After the formazan that formed was fully dissolved in DMSO (150 μL/well), the absorbance was read at 570 nm on a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The viability of RAW264.7 cells for the control group (with DMSO only) is defined as 100%.

Measurement of NO Release. The accumulated nitrite in the supernatant was evaluated using the Griess reagent29 (Beyotime, Nanjing, People’s Republic of China). RAW264.7 macrophage cells were seeded in 96-well culture plates (6 × 104 cells/well) for 18 h. Then, various concentrations of test compounds were added, with the presence of 1 μg/mL LPS. Eighteen hours later, Griess reagent and culture supernatant were mixed and then incubated for 10 min. The optical density of the mixture was read at 540 nm using an automated microplate reader. The NO inhibitory rate was measured in relation to the control group (cells were treated with DMSO only). NGMonomethyl-L-arginine (L-NMMA; purity ≥99%, Beyotime, Shanghai, People’s Republic of China) served as a positive control.31 Western Blot Analysis. Cells were initially treated with 1 with LPS (1 μg/mL) stimulation. The total proteins were extracted and immunoblotted as previously described.29 Briefly, the harvested cells were lysed by 1% RIPA (radio-immunoprecipitation assay) (Amresco, Solon, OH, USA) to achieve the cellular lysates. Then, the lysates were centrifuged to obtain the total protein, and the protein concentration was measured by the BCA protein assay. Total proteins were electrophoresed on SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were washed with TBST buffer, treated with 5% skimmed milk for 2 h at 25 °C, and then treated with primary antibodies for 12 h at 4 °C. After being washed with TBST, the membranes were probed with secondary antibody at room temperature. Lastly, the protein blots were recorded on a ChemiDOC XRS+ system (Bio-Rad Laboratories).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00867. 1D and 2D NMR, HRESIMS, UV, IR, and ECD spectra of compounds 1−10 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.-G. Luo). *Tel/Fax: +86-25-83271405. E-mail: [email protected] (L.-Y. Kong). ORCID

Ling-Yi Kong: 0000-0001-9712-2618 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was funded by the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and a Syngenta Ph.D. Fellowship awarded to G.M.X.

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