Article pubs.acs.org/jnp
Diterpenoids from the Roots of Euphorbia f ischeriana with Inhibitory Effects on Nitric Oxide Production Jin Woo Lee,† Chul Lee,† Qinghao Jin,† Hari Jang,† Dongho Lee,‡ Ha-Jin Lee,§ Jong Won Shin,⊥ Sang Bae Han,† Jin Tae Hong,† Youngsoo Kim,† Mi Kyeong Lee,† and Bang Yeon Hwang*,† †
College of Pharmacy, Chungbuk National University, Cheongju 28644, Korea Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea § Western Seoul Center, Korea Basic Science Institute, Seoul 03759, Korea ⊥ Beamline Department, Pohang Accelerator Laboratory/POSTECH, Pohang 37673, Korea ‡
S Supporting Information *
ABSTRACT: Bioactivity-guided isolation of a methanolic extract of Euphorbia f ischeriana led to the isolation of four new abietane-type diterpenoids, fischeriolides A−D (1−4), together with 11 known diterpenoids. Their structures were elucidated based on the interpretation of 1D and 2D NMR spectroscopic and HRESIMS data. The absolute configuration of compound 3 was determined by single-crystal X-ray diffraction analysis and electronic circular dichroism methods. Compounds 5−9 exhibited inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages with IC50 values in the range 4.9−12.6 μM.
T
12), and 4.35 (d, J = 2.0 Hz, H-11), a vinylic methyl group at δH 2.02 (d, J = 2.0 Hz, CH3-17), and three tertiary methyl groups at δH 0.93 (s, CH3-18), 0.91 (s, CH3-19), and 0.88 (s, CH3-20). The 13C NMR and HSQC spectra of 1 revealed 20 carbon signals corresponding to four methyls, four methylenes, six methines (including one olefinic carbon at δC 129.6 and three oxygenated carbons at δC 65.7, 81.8, and 69.5), and six quaternary carbons (one carbonyl at δC 174.9 and three olefinic carbons at δC 132.7, 159.5, and 126.9). The above-mentioned NMR data were found to be similar to those of 11β-hydroxy8,14-epoxy-ent-abieta-13(15)-en-16,12α-olide (5), except for an olefinic and a hydroxy group rather than an epoxy group at C-8 and C-14.5 The HMBC correlations between the vinylic methyl proton (δH 2.02) and C-13 (δC 159.5), C-15 (δC 126.9), and C16 (δC 174.9) revealed the presence of an α,β-unsaturated-γlactone moiety. Further HMBC correlations from H-11 to C-8, C-9, C-10, C-12, and C-13 and from H-14 to C-8, C-9, C-12, C-13, and C-15 indicated that two hydroxy groups are attached to C-11 and C-14, respectively (Figure 1). The COSY correlation between H-6 and H-7 indicated that a double bond is located at C-7 and C-8. The relative configuration of 1
he dried roots of Euphorbia f ischeriana Steud. (Euphorbiaceae) are a well-known traditional folk medicine in Korea used for the treatment of skin diseases, intestinal parasites, psoriasis, and cancer. Plants in the genus Euphorbia are reported to be rich sources of a variety of diterpenoids.1−8 As part of an ongoing study for the discovery of plant-derived anti-inflammatory agents, four new abietane-type diterpenoids, fischeriolides A−D (1−4), together with 11 known diterpenoids, were isolated from the n-hexane and CH2Cl2-soluble fractions of the dried roots of E. f ischeriana. Herein, we describe the isolation and structure determination of compounds 1−4 and the inhibitory effects of all isolated compounds on NO production in LPS-stimulated RAW 264.7 cells.
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RESULTS AND DISCUSSION Compound 1 was isolated as a yellow, amorphous powder. Its molecular formula of C20H28O4 was determined from the 13C NMR (Table 1) and HRESIMS data (m/z 333.2058 [M + H]+; calcd 333.2060), indicating seven indices of hydrogen deficiency. The IR spectrum showed the presence of hydroxy (3398 cm−1) and α,β-unsaturated-γ-lactone (1740 and 1694 cm−1) groups. The 1H NMR spectrum of 1 exhibited signals for an olefinic group at δH 6.00 (t, J = 2.5 Hz, H-7), three oxymethine groups at δH 4.93 (s, H-14), 4.63 (t, J = 2.0 Hz, H© 2015 American Chemical Society and American Society of Pharmacognosy
Received: September 3, 2015 Published: December 24, 2015 126
DOI: 10.1021/acs.jnatprod.5b00789 J. Nat. Prod. 2016, 79, 126−131
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Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) Data for Fischeriolides A (1) and B (2) in CDCl3a 1
2
no.
δH (J in Hz)
δC
1 2 3 4 5 6 7
2.01 (m), 1.27 (m) 1.60 (m), 1.57 (m) 1.51 (m), 1.26 (m)
38.4 18.4 42.0 33.0 49.3 23.5 129.6
8 9 10 11 12 13 14 15 16 17 18 19 20
1.36 (m) 2.20 (m), 1.95 (m) 6.00 (t, 2.5)
2.49 (m) 4.35 (d, 2.0) 4.63 (t, 2.0) 4.93 (s)
2.02 0.93 0.91 0.88
(d, 2.0) (s) (s) (s)
132.7 54.4 35.2 65.7 81.8 159.5 69.5 126.9 174.9 9.1 21.8 32.8 14.1
δH (J in Hz) 1.90 (m), 1.64 (m) 1.70 (m), 1.62 (m) 1.49 (m), 1.23 (m) 1.27 1.92 2.52 2.38
(m) (m), 1.45 (m) (dd, 19.0, 6.0) (ddd, 19.0, 11.5, 6.0)
4.84 (m) 5.00 (m)
2.22 0.92 0.96 1.14
(d, 2.0) (s) (s) (s)
δC 35.0 18.5 41.1 33.5 50.4 17.5 24.6 136.7 160.7 39.7 63.8 80.6 146.1 185.1 133.6 173.7 9.8 21.6 33.0 20.3
correlations from H-7 and H-12 to C-14 (Figure 2). The position of the double bond was assigned as C-8 and C-9 by the absence of any olefinic proton signal in the 1H NMR spectrum. The relative configuration of 2 was established by the NOESY spectrum, in which the cross-peaks of H-11/H-12 and CH3-20 were observed to confirm the β-orientation of the C-11 hydroxy group (Figure 2). Therefore, the structure of 2 (fischeriolide B) was determined as 11β-hydroxy-14-oxo-ent-abieta-8(9),13(15)dien-16,12α-olide. Compound 3, colorless needle crystals, showed a molecular formula of C20H28O4 as determined by its 13C NMR (Table 2) and HRESIMS data (m/z 333.2064 [M + H]+; calcd 333.2060). The IR spectrum exhibited the presence of hydroxy (3385 cm−1) and α,β-unsaturated-γ-lactone (1734 and 1651 cm−1) groups. The 1H and 13C NMR spectra of 3 were similar to those of 1, with the only difference being the position of both a hydroxy group and a double bond. In the HMBC spectrum of 3, the correlations from H-7 to C-5, C-8, C-9, and C-14 and from H-11 to C-8, C-9, C-10, C-12, and C-13 indicated the presence of two hydroxy groups at C-7 and C-11, respectively. Furthermore, the location of a Δ8,14 double bond was corroborated by the HMBC correlations from H-14 to C-7, C-8, C-9, C-12, C-13, and C-15 (Figure 3). The NOESY correlations of H-11/H-12 and CH3-20, H-5/H-9, and H-7/H14 and CH3-20 revealed that the hydroxy groups at both C-7
a
Assignments were supported with COSY, HSQC, and HMBC experiments.
was defined by a NOESY experiment. The NOE correlations of H-11/H-12 and CH3-20, H-5/H-9, and H-14/H-7 and CH3-17 indicated a β-orientation for both hydroxy groups at C-11 and C-14 (Figure 1). Therefore, the structure of 1 was determined as 11β,14β-dihydroxy-ent-abieta-7(8),13(15)-dien-16,12α-olide, and this compound has been named fischeriolide A. Compound 2 was obtained as a yellow, amorphous powder. Its molecular formula was determined to be C20H26O4 from the 13 C NMR (Table 1) and HRESIMS data (m/z 331.1901 [M + H]+; calcd 331.1904), indicating the presence of eight indices of hydrogen deficiency. The IR spectrum showed the presence of hydroxy (3402 cm−1) and α,β-unsaturated-γ-lactone (1747 and 1644 cm−1) groups. The 1H and 13C NMR spectra of 2 were very similar to those of 1, except for the presence of an additional carbonyl group instead of a hydroxy group at C-14. The carbonyl group was placed at C-14 by the HMBC
Figure 1. Key HMBC and NOESY correlations of compound 1. 127
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Figure 2. Key HMBC and NOESY correlations of compound 2.
and C-11 were assigned in a β-orientation (Figure 3). The absolute configuration of 3 was determined by single-crystal Xray crystallographic diffraction analysis. The Flack parameter x of 0.05(8) and the Hooft parameter y of 0.07(8) indicated that the absolute configuration of 3 was assigned correctly (Figure 4 and Experimental Section). This conclusion was supported by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra (Figure 5). Therefore, the structure of 3 (fischeriolide C) was determined as 7β,11βdihydroxy-ent-abieta-8(14),13(15)-dien-16,12α-olide. Compound 4, a yellow, amorphous powder, gave the molecular formula C20H28O4, based on the 13C NMR (Table 2) and HRESIMS data (m/z 355.1879 [M + Na]+; calcd 355.1880). The IR spectrum showed the presence of hydroxy (3359 cm−1) and α,β-unsaturated-γ-lactone (1742 and 1648 cm−1) groups. The 1H and 13C NMR data of 4 were similar to those of 2, except for the presence of a hydroxy group instead of a carbonyl group at C-14. The hydroxy group located at C14 was confirmed by the key HMBC correlations from H-14 to C-8, C-9, C-12, C-13, and C-15. Further HMBC correlations from H-7 and H-11 to C-8 and C-9 indicated a double bond to be located at C-8 and C-9 (Figure 6). The NOESY correlations of H-14/H-7α, H-12, and CH3-17 and H-11/H-12 and CH3-20 indicated that the hydroxy groups at both C-11 and C-14 are arranged in a β-orientation (Figure 6). Therefore, the structure of 4 (fischeriolide D) was determined as 11β,14β-dihydroxyent-abieta-8(9),13(15)-dien-16,12α-olide.
Table 2. 1H NMR (500 MHz) and 13C NMR (125 MHz) Data for Fischeriolides C (3) and D (4) in CDCl3a 3
4
no.
δH (J in Hz)
δC
δH (J in Hz)
δC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.92 (m), 1.41 (m) 1.60 (m), 1.59 (m) 1.50 (m), 1.31 (m)
39.3 19.0 41.7 33.0 47.4 30.5 71.8 149.2 55.7 40.5 64.2 79.6 151.9 115.1 120.6 175.0 8.6 21.6 33.6 16.6
1.80 (m), 1.46 (m) 1.58 (m), 1.57 (m) 1.47 (m), 1.21 (m)
35.7 18.6 41.4 33.0 51.3 18.1 29.0 134.8 156.5 38.1 64.4 80.5 141.2 69.7 125.0 175.5 9.2 21.5 33.2 20.4
1.80 (m) 1.97 (m), 1.58 (m) 4.46 (t, 3.5) 2.71 (br s) 4.49 (d, 4.0) 4.90 (m) 6.47 (s)
1.91 0.87 0.94 0.91
(d, 2.0) (s) (s) (s)
1.24 (m) 1.85 (m), 1.50 (m) 2.60 (m), 2.10 (m)
4.99 (d, 4.0) 4.63 (m) 4.85 (br s)
2.07 0.88 0.94 1.02
(d, 2.0) (s) (s) (s)
a
Assignments were supported with COSY, HSQC, and HMBC experiments.
Figure 3. Key HMBC and NOESY correlations of compound 3. 128
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Figure 4. X-ray ORTEP plot for the molecular structure of compound 3 drawn with 50% probability displacement ellipsoids.
lactone and 8,14-epoxide or hydroxy moieties, exhibited inhibitory effects against NO production with IC50 values ranging from 4.9 to 12.6 μM. Comparison of the inhibitory effects of compound 7 (IC50 5.6 μM) and yuexiandajisu E (IC50 > 20 μM), with the only difference being the stereochemistry of the hydroxy groups at C-8 and C-14, indicated that the configuration of ring C substituents is significant for the enhancement of NO inhibition. Even though they possess the same rosane skeleton, ebractenoid F (8) (IC50 7.4 μM) showed better activity than ebractenoid H (IC50 > 20 μM), suggesting that a catechol moiety plays an important role in the inhibition of NO production.11 Jolkinol A (9), a lathyrane-type diterpenoid, also showed an inhibitory effect, with an IC50 value of 9.4 μM. Recently, several diterpenoids from the genus Euphorbia having rosane, jatrophane, ent-abietane, myrsinol, paraliane, and pepluane skeletons were also found to have inhibitory effects on NO production.11,15−17 Jolkinolide B and 17-hydroxyjolkinolide B, ent-abietane diterpenoids from the roots of E. f ischeriana, showed inhibitory effects on LPSinduced NO, PGE2, IL-6, and TNF-α, by down-regulation of iNOS, COX-2, IL-6, and TNF-α gene expression through the suppression of MAPK phosphorylation and NF-κB activation.18 Taken together, ent-abietane (5−7), rosane (8), and lathyrane-type (9) diterpenoids from the roots of E. fischeriana exhibited promising inhibitory effects on NO production in LPS-induced RAW 264.7 macrophages. These compounds may be worthy of further investigation for the treatment of inflammatory diseases associated with enhanced production of NO.
Figure 5. Calculated and experimental ECD spectra of compound 3.
The 11 known compounds were identified as ent-11αhydroxyabieta-8(14),13(15)-dien-16,12α-olide,5 11β-hydroxy8,14-epoxy-ent-abieta-13(15)-en-16,12α-olide (5),5 jolkinolide B (6),7,9 yuexiandajisu D (7),10 yuexiandajisu E,10 ebractenoid B,11 ebractenoid F (8),11 ebractenoid H,11 ingenol,12 jolkinol A (9),13 and antiquorin,14 respectively, by comparing their physicochemical and spectroscopic data with those of published values. All compounds were evaluated for their inhibitory effects on LPS-induced NO production in RAW 264.7 macrophage cells using the Griess reagent. Aminoguanidine and indomethacin were used as positive controls (IC50 18.7 and 12.5 μM, respectively) (Table 3). Cell viability was evaluated using an MTT assay, indicating that none of the test compounds showed any significant cytotoxicity at their effective concentration for the inhibition of NO production (data not shown). entAbietane derivatives 5−7, which contain α,β-unsaturated-γ-
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO DIP-1000 polarimeter. UV spectra were recorded on a JASCO UV-550 spectrophotometer, and IR spectra were measured on a JASCO FT-IR 4100 spectrometer. ECD spectra were obtained on a JASCO J-715 spectrometer. NMR spectra were recorded on a Bruker AVANCE 500 MHz spectrometer using CDCl3 as solvent. ESIMS and HRESIMS were obtained with LCQ Fleet and maXis 4G mass spectrometers, respectively. Column chromatography 129
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Figure 6. Key HMBC and NOESY correlations of compound 4. yield compounds 3 (tR = 18.8 min, 11 mg), 7 (tR = 20.5 min, 8 mg), and yuexiandajisu E (tR = 23.8 min, 6 mg). EFH7-8 (360 mg) was further purified by preparative HPLC (MeCN−H2O, 45:55 to 80:20) to yield compounds 1 (tR = 20.3 min, 12 mg), 2 (tR = 23.8 min, 11 mg), 4 (tR = 17.7 min, 6 mg), 5 (tR = 22.0 min, 22 mg), and ebractenoid H (tR = 18.2 min, 16 mg). EFH7-9 (170 mg) was separated by preparative HPLC (MeCN−H2O, 50:50 to 90:10) to yield ent-11α-hydroxyabieta-8(14),13(15)-dien-16,12α-olide (tR = 25.0 min, 10 mg). The CH2Cl2-soluble fraction (64 g) was chromatographed on a silica gel column and eluted with a CH2Cl2−MeOH gradient system (90:1 to 0:1) to give four fractions (EFC1−EFC4). EFC2 (12.0 g) was separated by MPLC with a Lichroprep RP-18 column and eluted with a MeOH−H2O gradient system (20:80 to 100:0) to give 12 fractions (EFC2-1−EFC2-12). EFC2-7 (680 mg) was further purified by preparative HPLC (MeCN−H2O, 40:60 to 80:20) to yield ebractenoid B (tR = 25.8 min, 8 mg) and 9 (tR = 23.1 min, 15 mg). Compound 6 (54 mg) was isolated from fraction EFC211 (870 mg) by recrystallization in MeOH. EFC2-12 (530 mg) was further purified by preparative HPLC (MeCN−H2O, 70:30 to 100:0) to afford compound 8 (tR = 15.9 min, 13 mg). Fischeriolide A (1): yellow, amorphous powder; [α]25D −35.3 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 223 (3.85) nm; CD (MeOH) λmax (Δε) 225 (−7.2), 275 (+15.0) nm; IR νmax (film) 3398, 2956, 1740, 1694, 1525, 1268, 1052 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Table 1; ESIMS m/z 333 [M + H]+; HRESIMS m/z 333.2058 [M + H]+ (calcd for C20H29O4, 333.2060). Fischeriolide B (2): yellow, amorphous powder; [α]25D −40.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 282 (3.77) nm; CD (MeOH) λmax (Δε) 220 (−2.2), 240 (−5.6) nm; IR νmax (film) 3402, 1747, 1644, 1556, 1265, 1016 cm−1; 1H NMR (500 MHz, CDCl3) and 13 C NMR (125 MHz, CDCl3), see Table 1; ESIMS m/z 331 [M + H]+; HRESIMS m/z 331.1901 [M + H]+ (calcd for C20H27O4, 331.1904). Fischeriolide C (3): colorless needle crystals; [α]25D +114.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 274 (4.65) nm; CD (MeOH) λmax (Δε) 240 (−8.7), 275 (+18.6) nm; IR νmax (film) 3385, 2926, 1734, 1651, 1540, 1267, 1031 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Table 2; ESIMS m/z 333 [M + H]+; HRESIMS m/z 333.2064 [M + H]+ (calcd for C20H29O4, 333.2060). Fischeriolide D (4): yellow, amorphous powder; [α]25D −75.9 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 223 (4.47) nm; CD (MeOH) λmax (Δε) 210 (−12.4), 235 (+0.3), 283 (−4.4) nm; IR νmax (film) 3359, 2934, 1742, 1648, 1540, 1265, 1065 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Table 2; ESIMS m/z 355 [M + Na]+; HRESIMS m/z 355.1879 [M + Na]+ (calcd for C20H28NaO4, 355.1880). ECD Computational Calculations for Compound 3. A conformational search was carried out by employing the procedure implemented in Spartan ’14 software under the MMFF force field.19
Table 3. Inhibitory Effects of Compounds 1−15 on LPSInduced NO Production in Macrophage RAW 264.7 Cellsa compound fischeriolide A (1) fischeriolide B (2) fischeriolide C (3) fischeriolide D (4) 11β-hydroxy-8,14-epoxy-ent-abieta-13(15)-en-16,12α-olide (5) jolkinolide B (6) yuexiandajisu D (7) ebractenoid F (8) jolkinol A (9) aminoguanidinec indomethacinc
IC50 (μM)b >20 >20 >20 >20 12.6 ± 0.7 4.9 5.6 7.4 9.4 18.7 12.5
± ± ± ± ± ±
0.2 0.4 0.5 0.6 1.1 0.9
a Compounds 10−15 were inactive (IC50 values >20 μM). bResults are expressed as means ± SD from triplicate experiments. cPositive control.
was performed on silica gel (Merck, 70−230 mesh) and Lichroprep RP-18 (Merck, 40−63 μm). MPLC was performed on a Biotage Isolera Prime chromatography system. Preparative HPLC was performed using a Waters HPLC system equipped with two Waters 515 pumps with a 2996 photodiode-array detector using a YMC J’sphere ODS-H80 column (4 μm, 150 × 20 mm, i.d., flow rate 6 mL/ min). TLC was performed using precoated silica gel 60 F254 (0.25 mm, Merck) plates, and spots were detected by a 10% vanillin−H2SO4 in water spray reagent. Plant Material. The dried roots of E. f ischeriana were purchased from Kyungdong herbal market in Seoul, Korea, in May 2014. A voucher specimen (CBNU-2014-01-EF) was authenticated by B.Y.H. and deposited at the Herbarium of the College of Pharmacy, Chungbuk National University, Korea. Extraction and Isolation. The dried and powdered roots of E. f ischeriana (6.0 kg) were extracted with MeOH (3 × 18 L) at room temperature for 3 days. The extract was evaporated under reduced pressure, and the residue (450 g) was suspended in water and partitioned successively with n-hexane (2 × 2 L), CH2Cl2 (2 × 2 L), and EtOAc (2 × 2 L). The n-hexane-soluble fraction (86 g) was chromatographed on a silica gel column and eluted with a CH2Cl2− MeOH gradient system (90:1 to 0:1) to give seven fractions (EFH1− EFH7). EFH7 (6.0 g) was subjected to RP-18 CC and eluted with MeCN−H2O (20:80 to 100:0) to obtain 10 fractions (EFH7-1− EFH7-10). EFH7-5 (230 mg) was further purified by preparative HPLC (MeCN−H2O, 30:70 to 70:30) to yield ingenol (tR = 12.5 min, 16 mg) and antiquorin (tR = 22.4 min, 14 mg). EFH7-7 (320 mg) was separated by preparative HPLC (MeCN−H2O, 40:60 to 70:30) to 130
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Selected conformers were optimized with DFT calculations at the B3LYP/6-31+G(d,p) level using the Gaussian 09W package.20 TDDFT CD calculations for the optimized conformers were performed at the CAM-B3LYP/TZVP level with a CPCM solvent model in MeOH. The calculated CD spectra were simulated and generated with a half-bandwidth of 0.3 eV by SpecDis 1.64 software.21 The CD spectra were weighted by a Boltzmann distribution after UV correction. X-ray Crystallographic Analysis of Compound 3. A single crystal of compound 3 was mounted on the tip of a cryoloop using Paratone oil and mounted on a Bruker D8 Venture diffractometer equipped with a monochromatic fine-focus Cu Kα (λ = 1.541 78 Å) radiation source. Data collection was carried out using a PHOTON 100 CMOS detector at 193 K with APEX2 software.22 Semiempirical absorption corrections based on equivalent reflections were applied by Bruker SADABS.22 The crystal structures were solved by direct methods and refined by full-matrix least-squares refinement using the SHELXL-2014 computer program.23 The positions of all nonhydrogen atoms were refined with anisotropic displacement factors. All hydrogen atoms were placed using a riding model, and their positions were constrained relative to their parent atoms using the appropriate HFIX command in SHELXL-2014.23 The Hooft parameter y24 was 0.07(8) and was calculated using PLATON.25 Molecular graphics were computed with ORTEP-3. Crystallographic data for 3 have been deposited at the Cambridge Crystallographic Data Centre (deposition number: CCDC 1421843). Copies of these data can be obtained free of charge from the CCDC via www.ccdc. cam.ac.uk. Crystal Data of Fischeriolide C (3): C20H28O4, H2O Mr = 350.44, size 0.24 × 0.20 × 0.10 mm3, monoclinic, a = 15.8467(8) Å, b = 7.5277(4) Å, c = 17.3310(12) Å, α = 90.00°, β = 113.152(2)°, γ = 90.00°, V = 1900.90(19) Å3, T = 193(2) K, space group C2, Z = 4, μ = 0.702 mm −1; 13 224 collected reflections, 3402 independent reflections (Rint = 0.0558), R1 (all data) = 0.0623, wR2 (all data) = 0.1749, Flack parameter x = 0.05(8), Hooft parameter y = 0.07(8). Measurement of LPS-Induced NO Production and Cell Viability. RAW 264.7 cells were seeded into 96-well tissue culture plates at 2 × 106 cells/mL and stimulated with 1 μg/mL of LPS in the presence or absence of compounds. Aminoguanidine and indomethacin were used as positive controls. After incubation at 37 °C for 24 h, 100 μL of cell-free supernatant was mixed with 100 μL of Griess reagent containing equal volumes of 2% (w/v) sulfanilamide in 5% (w/v) phosphoric acid and 0.2% (w/v) N-(1-naphthyl)ethylenediamine solution to determine nitrite production. Absorbance was measured at 550 nm against a calibration curve with sodium nitrite standards. Cell viability of the remaining cells was determined by an MTT (Sigma Chemical Co., St. Louis, MO, USA)-based colorimetric assay.
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Research Foundation of Korea. The authors acknowledge the Korea Basic Science Institute for the NMR spectroscopic measurements.
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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.5b00789. 1D NMR, 2D NMR, and HRESIMS of compounds 1−4 (PDF)
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*Tel (B. Y. Hwang): +82-43-261-2814. Fax: +82-43-268-2732. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by a Medical Research Center Program (MRC, 2008-0062275) through the National 131
DOI: 10.1021/acs.jnatprod.5b00789 J. Nat. Prod. 2016, 79, 126−131
