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Dec 29, 2016 - Polycycloiridals with a Cyclopentane Ring from Iris tectorum. Chun-Lei Zhang,. †,‡. Zhi-You Hao,. †. Yan-Fei Liu,. †. Yan Wang,...
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Polycycloiridals with a Cyclopentane Ring from Iris tectorum Chun-Lei Zhang,†,‡ Zhi-You Hao,† Yan-Fei Liu,† Yan Wang,† Guo-Ru Shi,† Zhi-Bo Jiang,† Ruo-Yun Chen,† Zheng-Yu Cao,‡ and De-Quan Yu*,† †

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China ‡ Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Development, School of TCM, China Pharmaceutical University, Nanjing 211198, People’s Republic of China S Supporting Information *

ABSTRACT: Six new iridal-type triterpenoids containing an unprecedented cyclopentane ring, polycycloiridals E−J (1−6), were isolated from a large-scale re-extraction of Iris tectorum. A possible biosynthesis pathway is postulated. The known spirioiridotectal D (7) was also obtained in the current investigation, and its structure was unequivocally defined using X-ray diffraction data. Compound 7 suppressed LPS-activated NO production in the BV2 cell line with an IC50 value of 0.54 μM.

Iris tectorum Maxim., a widely cultivated decorative garden plant, is an abundant source of iridals with unique structures. To date, approximately 80 naturally occurring iridals have been reported.1 These molecules exhibit various biological properties including cytotoxicity,2,3 ichthyotoxicity,4 antiplasmodial effects,5 and PKC activation.6,7 These fascinating structures have promising biological activities, resulting in several synthesis efforts for this class of compounds.8−11 In continuing efforts toward the discovery of structurally intriguing iridals from this plant,12−14 trace amounts of polycycloiridals E−J (1−6), together with the known spirioiridotectal D (7), were isolated during a large-scale re-extraction of the rhizomes of I. tectorum. Compounds 1−6 possessed a unique cyclopentane ring resulting from cyclization of the homofarnesyl side chain. An X-ray diffraction analysis of compound 7 confirmed the earlier structural assignment.12 Herein, the isolation and structure elucidation of 1−6 and investigation of the inhibitory effects against lipopolysaccharide (LPS)-induced nitric oxide (NO) production are discussed.

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RESULTS AND DISCUSSION Polycycloiridal E (1), a colorless gum, had a molecular formula of C30H46O6 as determined by an HRESIMS ion at m/z 525.3201 [M + Na]+, indicative of eight indices of hydrogen deficiency. The IR (1711, 1615 cm−1) and UV (238 nm) data indicated the presence of an α,β-unsaturated formyl group.15 The 1H NMR spectrum showed signals assignable to a formyl proton (δH 10.18, Table 1), three olefinic protons (δH 6.02, 5.43, and 5.39), oxygenated methylene protons (δH 4.20 and 3.57), two oxygenated methine protons (δH 5.41 and 5.14), and © 2016 American Chemical Society and American Society of Pharmacognosy

six methyl protons (δH 1.84, 1.78, 1.27, 1.21, 1.19, and 1.12). These signals are characteristic of iridals.15 The 13C NMR spectrum revealed 30 carbon resonances that were attributed to a formyl carbonyl carbon, six olefinic carbons, and 23 aliphatic carbons including six oxygen-bearing carbons. A doubly Received: September 1, 2016 Published: December 29, 2016 156

DOI: 10.1021/acs.jnatprod.6b00796 J. Nat. Prod. 2017, 80, 156−161

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Table 1. 1H NMR Spectroscopic Data for Compounds 1−6 in CDCl3 (600 MHz) position

1

2

3

4

5

6

1 3

1.84, s 4.20, dt (12.0, 2.4) 3.57, td (12.0, 2.4) 1.76, m 2.91, m; 1.40, m 3.17, d (10.2) 3.23, brd (13.2) 2.60, td (13.2, 4.2) 1.72, m 1.64, m; 1.40, m 5.14, dd (16.2, 8.4) 5.39, d (8.4) 6.02, d (15.6) 5.43, dd (15.6, 10.2) 2.46, dd (10.2, 4.8) 1.64, m 1.78, m 1.91, m 1.21, s 10.18, s 5.41, s 1.27, s 1.78, s 1.12, s 1.19, s

10.32, s 4.21, dt (12.0, 2.4) 3.57, td (12.0, 2.4) 1.70, m 2.96, m; 1.46, m 3.81, d (9.6) 2.64, brd (13.2) 2.53, brd (13.2) 1.64, m 1.72, m; 1.68, m 5.13, dd (16.2, 8.4) 5.37, d (8.4) 6.01, d (15.6) 5.45, dd (15.0, 10.2) 2.45, dd (10.2, 4.8) 1.65, m 1.78, m 1.90, m 1.21, s 1.78, s 5.42, s 1.27, s 1.76, s 1.12, s 1.18, s

1.84, brs 4.21, dt (12.0, 2.4) 3.57, td (12.0, 2.4) 1.75, m 2.91, m; 1.40, m 3.17, d (10.2) 3.23, brd (13.2) 2.60, td (13.2, 4.2) 1.71, m 1.65, m; 1.42, m 5.14, dd (16.2, 8.4) 5.39, d (8.4) 6.02, d (15.6) 5.43, dd (15.6, 10.2) 2.46, dd (10.2, 4.8) 1.66, m 1.78, m 1.92, m 1.21, s 10.19, s 5.41, s 1.28, s 1.78, brs 1.13, s 1.18, s

1.84, d (1.8) 4.21, dt (12.6, 2.4) 3.57, td (12.6, 2.4) 1.75, m 2.91, m; 1.41, m 3.17, d (10.2) 3.23, brd (14.4) 2.60, brtd (14.4, 4.8) 1.69, m 1.62, m; 1.40, m 5.14, dd (16.2, 7.8) 5.39, d (7.8) 6.07, d (15.6) 5.45, dd (15.6, 8.4) 2.41, m 1.76, m 1.84, m 2.40, m 4.68, brs; 4.66, brs 10.19, s 5.40, s 1.27, s 1.79, s 1.15, s 1.67, s

10.33, s 4.21, dt (12.0, 2.4) 3.58, td (12.0, 2.4) 1.72, m 2.95, m; 1.47, m 3.82, d (10.2) 2.65, td (13.8, 4.8) 2.53, brd (13.8, 4.8) 1.67, m 1.70, m; 1.37, m 5.13, dd (16.8, 8.4) 5.38, d (8.4) 6.07, d (15.6) 5.45, dd (15.6, 8.4) 2.42, m 1.76, m 1.84, m 2.41, m 4.68, brs; 4.66, brs 1.78, s 5.42, s 1.27, s 1.78, s 1.15, s 1.67, s

10.33, s 4.20, dt (12.6, 2.4) 3.58, td (12.6, 2.4) 1.72, m 2.96, m; 1.47, m 3.82, d (9.6) 2.64, td (13.8, 4.8) 2.53, brd (13.8, 4.8) 1.67, m 1.70, m; 1.37, m 5.13, dd (16.8, 8.4) 5.38, d (8.4) 6.05, d (15.6) 5.45, dd (15.6, 8.4) 2.40, m 1.79, m 1.82, m 2.39, m 4.68, brs; 4.67, brs 1.78, s 5.42, s 1.26, s 1.77, s 1.15, s 1.67, s

4 5 6 8 9 12 13 14 16 17 18 20 21 22 24 25 26 27 28 29 30

oxygenated carbon signal at δC 110.02 (Table 2) was observed in the 13C NMR spectrum of 1, suggesting the presence of an acetal moiety. A detailed comparison of the spectroscopic data of 1 with those of the known polycycloiridal A showed that they shared a common 6/5/7 tricyclic ring system.14 In addition, five oxygenated carbon signals at δC 81.18, 75.13, 74.25, 72.40, and 70.32 were observed, of which three were assignable to C-13, C-10, and C-3, indicating the presence of two hydroxy groups on the homofarnesyl side chain. HMBC cross-peaks between Me-24/Me-30 and a tertiary carbon at δC 72.40, together with HMBC cross-peaks from Me-29 to a tertiary carbon at δC 81.18, placed the two hydroxy groups at C23 and C-19. The locations of the two conjugated double bonds were assigned by the interpretation of its 2D NMR data (Figure 1). These data included three rings, three double bonds, and a formyl moiety, accounting for seven indices of unsaturation and implying the presence of one more ring in 1. The presence of the cyclopentane moiety was deduced from the HMBC cross-peaks of H-18 and C-22 and of H-22 and C18. The NOESY correlations of H-25/H-8, as well as Me-1/H6, indicated an E-configured Δ2(7) double bond. The Egeometry of the Δ14(15)-double bond was assigned by the NOESY cross-peaks of H-14/H-16 (Figure 2). The Econfiguration of the Δ16(17)-double bond was confirmed by the 15.6 Hz coupling constant between H-16 and H-17. The absolute configuration of the multisubstituted cyclohexane ring system was determined by chemical degradation and X-ray analysis.16,17 On the basis of biosynthetic considerations, the absolute configurations of C-6, C-10, and C-11 were assumed to be (6R,10S,11R), which were consistent with all the iridals reported so far.18−20 The NOESY correlation between H-27 and H-14/H-26 as well as the absence of NOESY cross-peaks of H-27/H-13 suggested the (13R) and (26R) configurations (Figure 2).4 H-17 displayed a significant NOESY correlation

with H-22/Me-29, whereas there was no correlation between Me-29 and H-18. Thus, the cyclopentane ring was concluded to have the (18R*,19R*,22S*) relative configuration as shown. Polycycloiridal F (2) gave the molecular formula C30H46O6, similar to E (1), indicated by HRESIMS (m/z 525.3202 [M + Na]+). The spectroscopic data of 2 revealed a close resemblance to those of 1, except for the observation of an upfield shift (Δδ 0.59) of H-8 and a downfield shift (Δδ 0.64) of H-6 in the 1H NMR spectrum of 2, as well as an upfield shift of C-6 (Δδ 4.1) and a downfield shift of C-8 (Δδ 3.9) in the 13 C NMR spectrum of 2 compared with those of 1. The above data indicated that compounds 2 and 1 were a pair of geometrical isomers with respect to the α,β-unsaturated formyl group.21 The Z-configuration of the Δ2(7) double bond was supported by the NOESY correlations of H-1/H-6, as well as Me-25/H-8. Similar to a previous study of iridobelamal and isoiridogermanal,13 2 and 1 interchanged in acidic solution. Polycycloiridal G (3) was deduced to have the same molecular formula, C30H46O6, as 1 based on its HRESIMS ion at m/z 525.3202 [M + Na]+. Although the NMR data of compounds 3 and 1 were almost superimposable, their chromatographic data indicated two different compounds. The 13C NMR signals of the cyclopentane moiety and its adjacent carbons exhibited larger variations (C-15, δC 136.50; C-16, 134.04; C-19, 81.18; C-20, 39.81; C-29, 24.01; C-30, 28.31 for 1 vs C-15, δC 136.58; C-16, 134.13; C-19, 81.27; C20, 39.74; C-29, 24.11; C-30, 28.23 for 3) than the other carbons of the two compounds, indicating that they might possess different configurations at the cyclopentane ring. The NOESY correlation between H-17 and H-22/Me-29 and the absence of NOESY cross-peaks of Me-29/H-18 revealed that 3 and 1 had different configurations of the cyclopentane moiety from that of 1. Considering the mechanism of their biosynthesis, the reason for the presence of the different 157

DOI: 10.1021/acs.jnatprod.6b00796 J. Nat. Prod. 2017, 80, 156−161

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Table 2. 13C NMR Spectroscopic Data for Compounds 1−6 in CDCl3 (150 MHz)a position

1

2

3

4

5

6

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

10.80 129.13 70.32 32.35 31.31 49.03 165.53 20.46 39.33 74.25 60.52 43.34 75.13 129.45 136.50 134.04 132.45 56.15 81.18 39.81 24.26 55.31 72.40 29.33 190.88 110.02 27.96 13.26 24.01 28.31

190.1 129.1 70.5 31.6 32.3 44.9 165.3 24.4 39.8 74.1 60.5 43.6 75.0 129.4 136.4 134.0 132.5 56.1 81.2 39.8 24.2 55.3 72.3 29.3 10.7 110.0 28.2 13.3 24.0 28.3

10.81 129.14 70.33 32.36 31.31 49.05 165.53 20.47 39.37 74.28 60.55 43.36 75.09 129.50 136.58 134.13 132.43 56.11 81.27 39.74 24.28 55.28 72.40 29.33 190.88 110.02 28.01 13.30 24.11 28.23

10.8 129.2 70.3 32.4 31.3 49.1 165.5 20.5 39.4 74.3 60.5 43.4 75.3 129.6 136.6 136.2 128.9 58.3 81.1 39.7 27.2 50.9 146.7 110.3 190.8 110.0 28.0 13.3 24.8 19.7

190.12 129.18 70.51 31.62 32.39 45.00 165.08 24.37 38.78 74.23 60.53 43.68 75.02 129.57 136.49 136.20 128.92 58.32 81.11 39.68 27.30 50.85 146.65 110.27 10.75 110.01 28.27 13.27 24.79 19.72

190.13 129.19 70.52 31.63 32.41 45.01 165.10 24.37 38.79 74.24 60.52 43.65 75.06 129.59 136.40 136.26 128.72 58.35 81.14 39.89 27.28 50.81 146.47 110.39 10.75 110.00 28.28 13.28 24.75 19.70

Figure 2. Selected NOESY (↔) correlations for 1.

H-17/Me-29. On the basis of the similarity of key NOESY correlations and biosynthesis considerations (Scheme 1), the two compounds were assumed to have identical relative configurations of their cyclopentane moieties. The molecular formula of polycycloiridal I (5) was deduced to be C30H44O5, identical to that of 4, as indicated by the HRESIMS data (m/z 507.3086 [M + Na]+). Its NMR spectroscopic data resembled those of 4 except for small differences in the chemical shifts of C-8, C-6, H-8, and H-6. The differences between these signals for compounds 5 and 4 closely resembled those observed for 2 and 1. Therefore, compounds 5 and 4 were a pair of Z and E Δ2(7) geometrical isomers, respectively, in terms of the α,β-unsaturated formyl moiety. The interconversion of the two isomers occurred in acidic solution, further confirming this conclusion. Polycycloiridal J (6) had a molecular formula of C30H44O5, as indicated by its HRESIMS (m/z 507.3080 [M + Na]+) data. The NMR data of compound 6 were nearly superimposable with those of 5. However, their chromatographic data indicated two different compounds. Subtle differences in the 13C NMR signals for the cyclopentane ring and its adjacent carbons were observed in the 13C NMR spectrum (C-15, δC 136.49; C-16, 128.92; C-20, 39.68; C-23, 146.65; C-24, 110.27 for 5 vs C-15, δC 136.40; C-16, 128.72; C-20, 39.89; C-23, 146.47; C-24, 110.39 for 6). The 13C NMR chemical shifts of the remaining carbons in 6 were highly similar to those of 5. As in the case of 3 and 1, compounds 6 and 5 were quasi-enantiomers with respect to their cyclohexene moieties. This assumption was supported by the NOESY data. The possible biosynthesis pathways to polycycloiridals E−J were similar to those of polycycloiridals A−D.14 Their biosynthesis precursors were likely the (13R)-spirobicyclic hemiacetal (I) and its geometrical isomer (II),4,22 which underwent a series of transformations including epoxidation, nucleophilic addition, and intramolecular dehydration to form 1−6 (Scheme 1). Crystallization of 7 from MeOH/H2O (10:1) resulted in colorless crystals. The X-ray diffraction experiment unequivocally defined the absolute configuration of 7 with a Flack parameter of 0.1 (Figure 3). In a biological screen, compound 7 suppressed LPS-induced NO production in murine microglia BV2 cells, giving an IC50 value of 0.54 μM; this is 3-fold more potent than curcumin (IC50 = 2.36 μM), which served as a positive control. However, the other compounds were inactive (IC50 > 10 μM).

a

The 13C NMR data of 1, 3, 5, and 6 are listed to two decimal places for detailed comparison.

Figure 1. Selected HMBC (→) and 1H−1H COSY (bold) correlations for 1.

structures likely results from the fact that the step following epoxidation is not enzymatically controlled (Scheme 1), as in the case of polycycloiridals A and C.14 Polycycloiridal H (4) had a molecular formula of C30H44O5, 18 atomic mass units less than 1, as deduced from HRESIMS (m/z 507.3105 [M + Na]+). The spectroscopic data of 4 showed a close structural similarity with those of 1. Comparison of the NMR spectra of compounds 4 and 1 showed that the C-23 hydroxy proton in compound 1 was absent in 4 and that a terminal double bond was present in 4. Similar NOESY correlations were detected for H-17/H-22 and 158

DOI: 10.1021/acs.jnatprod.6b00796 J. Nat. Prod. 2017, 80, 156−161

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Scheme 1. Putative Biosynthesis Pathway toward Compounds 1−6

data were acquired using a Micromass Autospec-Ultima ETOF spectrometer. HPLC separations were performed using a Shimadzu instrument equipped with an SPD-20A detector, an LC-6AD pumping system, and a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). HPLC analyses were conducted on an Agilent 1260 instrument equipped with a DAD detector. TLC experiments were conducted on GF254 silica gel plates (Qingdao Haiyang Chemical Factory, Qingdao, China). Column chromatography experiments were carried out on Sephadex LH-20 (GE), ODS (50 μm, YMC, Japan), and silica gel (200−300 mesh, Qingdao Haiyang Chemical Factory, Qingdao, China). Plant Material. The rhizomes of I. tectorum were purchased from Lotus Pond Chinese Medicinal Herbs Wholesale Market in Chengdu, Sichuan Province, China, and were authenticated by Professor Lin Ma from the Institute of Materia Medica, Peking Union Medical College. A voucher specimen (ID-S-2469) was deposited at the Herbarium of the Department of Medicinal Plants, the Institute of Materia Medica, Peking Union Medical College, Beijing. Extraction and Isolation. The air-dried rhizomes of I. tectorum (100 kg) were crushed to a coarse powder and refluxed with 95% EtOH (3 × 100 L). The combined extracts were concentrated under reduced pressure to give a residue (15 kg), which was diluted with H2O (70 L) and successively partitioned with EtOAc and n-BuOH. The EtOAc extract (8 kg) was applied to a macroporous resin column eluted with 40%, 70%, and 85% EtOH in H2O, successively. After evaporation of the solvent, the 70% EtOH eluate (480 g) was dissolved in MeOH (5 L) and filtered to remove undissolved substance. After removing residual flavonoids in the filtrate by passing over Sephadex LH-20 (MeOH/CHCl3, 1:1), an iridal-enriched

Figure 3. ORTEP drawing of spirioiridotectal D.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-2000 polarimeter. The UV spectra were recorded on a JASCO V-650 spectrophotometer. The IR spectra were measured on a Nicolet 5700 FT-IR spectrometer. NMR measurements were recorded in CDCl3 on a VNS-600 spectrometer, and HRESIMS 159

DOI: 10.1021/acs.jnatprod.6b00796 J. Nat. Prod. 2017, 80, 156−161

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using the direct methods. A mixture of independent and constrained refinement was used to treat all hydrogen atoms, while the leastsquares method was used to refine anisotropically all non-hydrogen atoms. The absolute configuration was established based on the Flack parameter of 0.1(3). A supplementary publication (CCDC 1501999) containing the crystallographic data for 7 has been deposited in the Cambridge Crystallographic Data Center. These data can be accessed free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or email: [email protected]).

portion (100 g) was obtained, which was further chromatographed on an ODS RP-C18 column (80 × 6 cm) with gradient elution (50−100% MeOH in H2O) to afford six fractions (F1−F6). Fraction 3 (9 g) was submitted to a silica gel column eluted with CH2Cl2/MeOH (25:1− 15:1) to give four subfractions (F3a−F3d). F3c was fractionated by preparative TLC (CH2Cl2-/MeOH, 15:1) to produce two subfractions (F3ca and F3cb). F3ca was separated by preparative HPLC (5 mL/ min, MeOH/H2O, 71:29) to obtain 1 (tR = 58.5 min, 13 mg) and 2 (tR = 49.4 min, 21 mg). F3cb was also fractionated by preparative HPLC (5 mL/min, MeOH/H2O, 67:33), affording 3 (tR = 101.6 min, 7 mg). F4 (7 g) was fractionated over RPC18 column chromatography (MeOH/H2O, 60:40−70:30) and purified by preparative HPLC (5 mL/min, MeOH/H2O, 68:32) to afford 7 (tR = 106.1 min, 58 mg). F6 (5 g) was applied to a Sephadex LH-20 column eluted with CHCl3/ MeOH (1:1), followed by further purification via preparative HPLC (5 mL/min, MeCN/H2O, 60:40), to afford 4 (tR = 90.5 min, 3 mg), 5 (tR = 99.1 min, 3 mg), and 6 (tR = 85.2 min, 5 mg). Polycycloiridal E (1): colorless gum; [α]20D +20 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 238 (4.26); IR νmax 3391, 2926, 2868, 1711, 1655, 1615, 1467, 1377 cm−1; 1H NMR (CDCl3, 600 MHz) see Table 1; 13C NMR (CDCl3, 150 MHz) see Table 2; (+)-HRESIMS m/z 525.3201 [M + Na]+ (calcd for C30H46O6Na, 525.3187). Polycycloiridal F (2): colorless gum; [α]20D −10 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 238 (4.33), 216 (4.52), 254 (4.19), 292 (3.92) nm; IR νmax 3407, 2932, 2873, 1709, 1662, 1614, 1449, 1379 cm−1; 1H NMR (CDCl3, 600 MHz) see Table 1; 13C NMR (CDCl3, 150 MHz) see Table 2; (+)-HRESIMS m/z 525.3202 [M + Na]+ (calcd for C30H46O6Na, 525.3187). Polycycloiridal G (3): colorless gum; [α]20D +20 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 240 (4.16) nm; IR νmax 3431, 2927, 1659, 1446, 1378 cm−1; 1H NMR (CDCl3, 600 MHz) see Table 1; 13C NMR (CDCl3, 150 MHz) see Table 2; (+)-HRESIMS m/z 525.3202 [M + Na]+ (calcd for C30H46O6Na, 525.3187). Polycycloiridal H (4): colorless gum; [α]20D +36 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 241 (4.24) nm; IR νmax 3447, 2926, 1865, 1725, 1661, 1614, 1451 cm−1; 1H NMR (CDCl3, 600 MHz) see Table 1; 13C NMR (CDCl3, 150 MHz) see Table 2; (+)-HRESIMS m/z 507.3105 [M + Na]+ (calcd for C30H44O5Na, 507.3081). Polycycloiridal I (5): colorless gum; [α]20D +25 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 241 (4.37) nm; IR νmax 3437, 2942, 2870, 1722, 1661, 1614, 1449 cm−1; 1H NMR (CDCl3, 600 MHz) see Table 1; 13C NMR (CDCl3, 150 MHz) see Table 2; (+)-HRESIMS m/z 507.3086 [M + Na]+ (calcd for C30H44O5Na, 507.3081). Polycycloiridal J (6): colorless gum; [α]20D +14 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 241 (4.22) nm; IR νmax 3461, 2932, 2870, 1722, 1659, 1614, 1448 cm−1; 1H NMR (CDCl3, 600 MHz) see Table 1; 13C NMR (CDCl3, 150 MHz) see Table 2; (+)-HRESIMS m/z 507.3080 [M + Na]+ (calcd for C30H44O5Na, 507.3081). Measurement of NO Production in the BV2 Cell Line. Compounds 1−7 were tested for their inhibitory effect on LPSinduced NO production in the BV2 cell line. The LPS was purchased from Sigma-Aldrich, and the BV2 cell line was obtained from the Cell Culture Center at Peking Union Medical College, Beijing, China. The BV2 cells cultured in a 96-well plate were pretreated with the compounds for 24 h followed by an additional 24 h of LPS (0.3 μg/ mL) exposure. The Griess reaction was used to determine the nitrite content in the culture supernatant. NaNO2 served as a standard to assess the NO2− concentration. The OD values were recorded at 550 nm. Curcumin was used as the positive control. Cell viability was measured by an MTT assay.23 X-ray Crystallography Analysis of 7. The data of compound 7 were as follows: C30H46O5, M = 486.67, monoclinic, P21, analytic temperature = 100 K, a = 7.3909(6) Å, b = 9.0127(3) Å, c = 18.0475(8) Å, β = 91.162(4)°, V = 2828.16(19) Å3, Z = 4, and Dcalcd = 1.143 mg·mm−3. A total of 17 049 collected reflections, including 9024 independent reflections (I > 2σ(I), R1 = 0.0738, wR2 = 0.1391), were collected with 2θ from 3.57° to 63.36°. The experiment was conducted on an Agilent Xcalibur Eos Gemini diffractometer equipped with Cu Kα radiation (μ = 0.60 mm−1) using the multiscan technique. The structure solution was achieved with the SHELXS-97 program package

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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.6b00796. Copies of the UV, IR, 1D and 2D NMR, MS, and CD spectra for compounds 1−6 and the X-ray crystallographic data for compound 7 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (D.-Q. Yu): +86-10-63165224. Fax: +86-10-63017757. Email: [email protected]. ORCID

De-Quan Yu: 0000-0003-4774-6419 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by grants from the Postdoctoral Science Foundation of China (Nos. 2016T90530, 2015M571844), the Jiangsu Provincial Natural Science Foundation (No. BK20160754), the National Natural Science Foundation of China (No. 81603389), and the State Key Laboratory of Bioactive Substance and Function of Natural Medicines (No. GTZC201201), Institute of Materia Medica, Peking Union Medical College.

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