(±)-Homocrepidine A, a Pair of Anti-inflammatory Enantiomeric

Dec 28, 2015 - A pair of racemic indolizidine enantiomers, (±)-homocrepidine A (1), and a piperidine derivative, homocrepidine B (2), were isolated f...
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(±)-Homocrepidine A, a Pair of Anti-inflammatory Enantiomeric Octahydroindolizine Alkaloid Dimers from Dendrobium crepidatum Yang Hu,† Chaofeng Zhang,*,† Xin Zhao,† Yue Wang,† Deqiang Feng,‡ Mian Zhang,† and Haifeng Xie§ †

Research Department of Pharmacognosy, China Pharmacaeutical University, Nanjing, 211198, People’s Republic of China Institute of National Medicine, Yunnan University of Traditional Chinese Medicine, Kunming, 650000, People’s Republic of China § Chengdu Biopurity Phytochemicals Ltd., Chengdu, 611131, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: A pair of racemic indolizidine enantiomers, (±)-homocrepidine A (1), and a piperidine derivative, homocrepidine B (2), were isolated from Dendrobium crepidatum along with the known alkaloid crepidine (3). The racemic mixture of 1 was separated into a pair of enantiomers, (+)-1 and (−)-1, by HPLC using a chiral chromatographic substrate, which represents the first successful example of resolving indolizidine racemic mixtures. The absolute configurations of (+)-1 and (−)-1 were assigned from single-crystal X-ray diffraction data. The evaluation of anti-inflammatory activity with LPS-induced RAW 264.7 macrophages revealed that (+)-1 strongly inhibited the production of nitric oxide (IC50, 3.6 μM) and significantly decreased the expression of inducible nitric oxide synthase, while (−)-1 and (±)-1 only had moderate inhibitory effects (IC50, 22.8 and 14.7 μM). Compound 2 showed moderate antiinflammatory activity (IC50, 27.6 μM).

I

ndolizidine (octahydroindolizine) alkaloids, possessing a common 1-azabicyclo[4.3.0]nonane core, are widely distributed in bacteria, fungi, higher plants, invertebrates, and vertebrates of both terrestrial and marine sources.1,2 Three potent glycosidase inhibitors, i.e., lentiginosine, swainsonine, and castanospermine, are among the best-known plant-derived polyhydroxylated indolizidines.3−5 Owing to their promising application in the treatment of viral infections, cancer, and metabolic disorders,6−9 indolizidine alkaloids have stimulated efforts in their isolation, synthesis, structural modification, and structure−activity relationships. The plants of the Dendrobium genus (Orchidaceae), comprising more than 1,000 species, are widespread throughout Asia, Europe, and Australia. Many Dendrobium species have been used as medicinal herbs in China for a long time with a view to nourishing the stomach, promoting the production of body fluid, and clearing fever.10 Phytochemical studies revealed that the plants of this genus contained diversified components, such as alkaloids, coumarins, bibenzyls, fluorenones, phenanthrenes, and sesquiterpenoids. However, only six indolizidine alkaloids were isolated from Dendrobium plants, five from D. crepidatum Lindl. et Paxt. and one from D. primulinum Lindl.11−15 D. crepidatum is a perennial epiphytic plant distributed in southern China,16 and its stems are used as a folk medicine for the treatment of cancer, diabetes, fever, and cataracts.17,18 In the pharmacological and chemical investigation of D. crepidatum, a pair of racemic indolizidine dimers, (±)-homocrepidine A (1), and the piperidine derivative homocrepidine B (2) were isolated along with the known crepidine (3) (Figure 1) from the total alkaloid extract. The extract showed medium anti-inflammatory activity with the model of toluene-induced ear edema in mice.19 It is reported © XXXX American Chemical Society and American Society of Pharmacognosy

Figure 1. Structures of (±)-1, 2, and 3.

that the enantiomers often have different biological activities and even different metabolic fates.20 Therefore, the racemate was resolved through HPLC using a CHIRALPAK AD-H column, and the absolute configurations of the enantiomers were determined by single-crystal X-ray diffraction. The antiinflammatory effects of the isolated compounds were evaluated with lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Homocrepidine A (1) was obtained as white crystals. Its molecular formula was established as C33H44N2O3 with 13 Received: September 10, 2015

A

DOI: 10.1021/acs.jnatprod.5b00801 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. NMR Spectroscopic Data for Compounds 1 and 2 (δ in ppm, J in Hz)a 1 position

δC, type

1, 1′

30.7, CH2

2, 2′ 3, 3′

21.1, CH2 50.8, CH2

5, 6, 7, 8,

64.9, 76.6, 40.8, 35.7,

5′ 6′ 7′ 8′

9, 9′

63.6, CH

1.80−1.82, m 1.25−1.30, m 1.54, m 2.18−2.20, m 1.75−1.80, m 3.03, dd (2.9, 6.2) 1.80−1.82, 1.70−1.82, 1.15−1.20, 2.18−2.20,

m brd (12.7) brd (12.7) m

2 position

δC, type

C-2, 2′, 8, 8′, 9, 9′

1

29.9, CH3

C-1, 1′, 3, 3′ C-2, 2′, 17, 17′

2 3

C-6, 6′, 7, 7′, 11, 11′, 17, 17′

HMBC(H→C)

208.8, C 43.5, CH2

2.37, t (7.2)

C-2, 4, 5

C-8, 8′, 10, 10′

4 5 6

23.8, CH2 25.3, CH2 36.3, CH2

1.52−1.56, m 1.30−1.32, m 1.35−1.38, m

C-2, 3, 5 C-4, 6, 2′, 6′ C-5, 2′, 3′

C-1, 1′, 7, 7′, 10, 10′ C-1, 1′, 8, 8′

2′ 3′

56.7, CH 36.8, CH2

C-3′, 6′ C-6, 2′, 7′

38.7, CH 72.9, C 59.2, CH2

2.57−2.65, 1.52−1.56, 1.10−1.22, 1.86−1.97,

0.50, d (6.6)

C-6, 6′,7, 7′, 8, 8′

7.51, brd (7.7)

C-6, 6′, 14, 14′, 16, 16′

4′ 5′ 6′

13, 14, 15, 16, 17,

127.8, 124.3, 128.0, 126.4, 43.0,

7.13, 7.03, 7.21, 7.29, 1.78, 2.06,

C-14, 14′, 16, 16′ C-6, 6′, 16, 16′ C-11, 11′, 13, 13′ C-11, 11′, 15, 15′ C-5, 5′, 6, 6′, 18

7′ 8′ 9′, 13′ 10′, 12′ 11′

CH CH CH CH CH2

brd brd brd brd brd brd

(7.1) (7.5) (7.1) (7.7) (2.9, 18.6) (6.4, 18.6)

HMBC(H→C) C-2, 3

15.4, CH3 143.3, C 126.9, CH

13′ 14′ 15′ 16′ 17′

δH (J in Hz) 2.07, s

10, 10′ 11, 11′ 12, 12′

18 a1

CH C CH CH2

δH (J in Hz)

15.2, 144.5, 125.0, 128.0, 126.5,

CH3 C CH CH CH

m m m m

C-3′, 7′

2.76, d (11.7) 2.80, d (11.7) 0.57, d (6.6)

C-2′, 5′

7.38, brd (7.3) 7.26, brd (7.3) 7.15, brd (7.3)

C-5′, 8′, 11′ C-8′, 11′ C-9′, 13′

C-3′, 4′, 5′

207.3, C

H (300 MHz) and 13C (75 MHz) data in CDCl3

indices of hydrogen deficiency by the protonated molecular ion peak of [M + H]+ at m/z 517.3436 (calcd 517.3443 for C33H44N2O3) in the positive HRESIMS in conjunction with 13 C NMR data. The IR spectrum showed the presence of hydroxy (3421 cm−1), aromatic (1639, 1447, 759 cm−1), and carbonyl (1720 cm−1) groups. The 1H NMR data (Table 1) showed 10 aromatic protons of two monosubstituted benzene moieties at δH 7.51 (2H, brd, 7.7 Hz, H-12, 12′), 7.13 (2H, brd, 7.1 Hz, H-13, 13′), 7.03 (2H, m, 7.5 Hz, H-14, 14′), 7.21 (2H, brd, 7.1 Hz, H-15, 15′), and 7.29 (2H, brd, 7.7 Hz, H-16, 16′); six methines at δH 1.80−1.82 (2H, overlapped, H-7, 7′), 2.18− 1.20 (2H, overlapped, H-9, 9′), and 3.03 (2H, dd, 2.9 and 6.2 Hz, H-5, 5′); and two methyl groups at δH 0.49 (6H, d, 6.6, H310, 10′), which were unambiguously designated by the HSQC experiment. The 13C NMR (DEPT) spectrum (Table 1) exhibited 33 carbon resonances assigned to a carbonyl and two quaternary carbons, six sp3 methines, 10 methylenes, two methyls, 10 aromatic methines, and two oxygenated tertiary carbons. Taking the molecular formula and the 1H NMR spectrum into consideration, a benzyl group and two rings in subunit A (Figure 2) accounted for six of the 13 degrees of unsaturation. The HMBC correlations of H-12/H-16/6-OH to C-6, H-5 to C-6/C-7/C-11, and H-12/H-16 to C-14 established the substitution of a phenyl and a hydroxyl group on the oxygenated tertiary C-6 (δC 76.6). In addition, the HMBC correlations of H3-10 to C-6/C-7/C-8 suggested that the methyl group was located at C-7. The 1H−1H COSY data revealed the spin system of C-1/C-3 and C-7/C-9 as shown in Figure 2. From the above evidence, the structure of subunit A was established. Combined with ESIMS data and the unsaturation degree, the existence of subunit B was confirmed by the correlations of H-5 to C-11/C-17 and H2-17 to C-5/C6/C-18 in the HMBC spectrum. Therefore, two indolizidine (subunit A) moieties were connected via a carbonyl group (C-

Figure 2. 1H−1H COSY, HMBC, and NOESY correlations of 1.

18) to form a dimer. The NOESY correlations of H-5/H-7/H-9 suggested that these protons had the same orientation (Figure 2), while H-5 had the opposite orientation of H-7 and H-9 in dendrocrepine.6 Moreover, the NOESY correlations of H3-10/ 6-OH/H2-17 indicated that H3-10, 6-OH, and H2-17 were also cofacial. Thus, the 2D structure of 1 was established as shown in Figure 1. The structure and relative configuration of 1 were confirmed by a single-crystal X-ray diffraction experiment with Cu Kα radiation (Figure 3) (deposition number: CCDC 1052365). B

DOI: 10.1021/acs.jnatprod.5b00801 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. X-ray ORTEP drawing of homocrepidine A (1).

Figure 5. X-ray ORTEP drawing of (−)-homocrepidine A.

However, the crystal structure of 1 was found to exhibit centrosymmetric space group P1 ̅, suggesting that it was a racemic mixture at large, which was further supported by the small value of [α]D (−2.8). Subsequently, the racemate of (±)-1 was resolved by HPLC using a CHIRALPAK AD-H column (250 × 10 mm, 5 μm) to afford enantiomers (+)-1 and (−)-1, which were obtained in a ratio of ca. 1:1 (Figure 4). The

deshielded phenyl moiety at δH 7.38 (2H, brd, 7.3 Hz, H-9′, 13′), 7.15 (1H, t, 7.3 Hz, H-11′), and 7.26 (2H, brd, 7.3 Hz, H10′, 12′) and two high-field methyl groups at δH 0.57 (3H, d, 6.6 Hz, H-7′) and 2.07 (3H, s, H-1). The 13C NMR spectrum (Table 1) showed 18 carbon signals assigned to one phenyl group, one quaternary carbon, a carbonyl carbon, two methines, six methylenes, and two methyls, which were confirmed by DEPT and HSQC spectra. In the HMBC spectrum (Table 1), the correlations of H-9′/ H-13′/5′-OH to C-5′ and H3-7′ to C-3′/C-4′/C-5′ suggested that a phenyl moiety and a hydroxy group were located at C-5′ and a methyl group was attached at C-4′. The link of C-7′/C4′/C-3′/C-2′/C-6/C-5/C-4/C-3 was confirmed by the crosspeaks of H-2′/H2-6/H2-5/H2-4/H2-3 in the 1H−1H COSY spectrum (Figure 6). The HMBC correlations of H3-1 to C-2/

Figure 4. HPLC chromatogram of (+)-1 and (−)-1 on a chiral AD-H column.

first peak at 5.96 min (retention time) confirmed the (+)-enantiomer ([α]20D +34.6, c 0.1, MeOH), and the second peak at 7.00 min afforded the (−)-enantiomer ([α]20D −29.4, c 0.1, MeOH). Enantiomers (+)-1 and (−)-1 showed identical MS and NMR spectra, indicating the successful resolution of the enantiomers. Compound (−)-1 crystallized as colorless needles in CH2Cl2/MeOH (10:1) and gave an X-ray crystal structure with 1.5 Å resolution (deposition number: CCDC 1053880). Single-crystal X-ray diffraction with Cu Kα radiation established the absolute configuration of (−)-1 as 5R, 6S, 7R, 9S, 5′R, 6′S, 7′R, 9′S (Figure 5). Correspondingly, the absolute configuration of (+)-1 was assigned as 5S, 6R, 7S, 9R, 5′S, 6′R, 7′S, 9′R. The experimental circular dichroism (ECD) data of (+)-1 and (−)-1 (Figure 1, Supporting Information) clearly showed the enantiomeric relationship. Homocrepidine B (2) was obtained as a white, amorphous powder. Its molecular formula was established as C18H27O2N by the protonated molecular ion peak at m/z 290.2122 [M + H]+ (calcd 290.2115 for C18H27O2N) in the positive HRESIMS in conjunction with 13C NMR data. The IR spectrum exhibited absorption bands at 3425 cm −1 (hydroxy), 1710 cm −1 (carbonyl), and 1640, 1451, 766 cm−1 (phenyl moieties). The 1 H NMR data (Table 1) contained proton resonances for a

Figure 6. 1H−1H COSY, HMBC, and NOESY correlations of 2.

C-3, H2-3 to C-2/C-4/C-5, and H2-6 to C-2′/C-3′/C-5 indicated the presence of a 2-oxohexyl group. The NOESY correlations of H-4′/H-2′ suggested that H-4′ and H-2′ have the same orientation. On the basis of the above analysis, the structure of 2 was identified as 6-(5′-hydroxy-4′-methyl-5′phenylpiperidin-2′-yl)hexan-2-one (homocrepidine B). The anti-inflammatory activity of all compounds was tested with the model of LPS-induced RAW 264.7 mouse macrophages. Compounds 1−3 were nontoxic or at low toxicity for LPS-induced RAW 264.7 cells with inhibitions of cell viability < 10% at 100 μM by the MTT assay. Indices of nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression were used to evaluate their anti-inflammatory effects. The IC50 values (95% confidence interval) for NO inhibition were 3.6 (1.4−9.1) μM for (+)-1, 22.8 (20.1−25.9) μM for (−)-1, and 14.7 (10.9−19.9) μM for (±)-1, while 42.2 (32.9−54.0) μM for indomethacin, a positive control. C

DOI: 10.1021/acs.jnatprod.5b00801 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Preparative HPLC was performed on a CHIRALPAK AD-H column (10 mm × 250 mm, 5 μm). Plant Material. The stems of D. crepidatum (10 kg) were collected from Menglian, Yunnan Province, in November 2008, and authenticated by Prof. Luoshan Xu (China Pharmaceutical University). A voucher specimen (No. MG-0410) was deposited in the Research Department of Pharmacognosy, China Pharmaceutical University, Nanjing, P. R. China. Extraction and Isolation. The dried stems of D. crepidatum (10 kg) were extracted with 80% EtOH (3 × 150 L), 2.5 h each. After removal of the EtOH in vacuo, the combined extract was suspended in H2O (50 L) and partitioned successively with petroleum ether (60−90 °C), EtOAc, and n-BuOH. The EtOAc extract was subjected to silica gel column chromatography (petroleum ether/EtOAc, 98:2 → 1:1) to afford six fractions (Fr.1−Fr.6). Fr.6 was further separated with silica gel column chromatography (CHCl3/MeOH, 100:1 → 5:1) to afford six subfractions (Fr.6a−Fr.6f). Fr.6b, Fr.6c, and Fr.6e were purified on an RP-18 silica gel column to give compounds 1 (70.1 mg), 2 (20.6 mg), and 3 (9.4 mg). Enantioseparation of (±)-homocrepidine A (1) was carried out on an Agilent 1100 liquid chromatograph equipped with an Agilent VWD UV−vis spectrometric detector, using a CHIRALPAK AD-H column (10 × 250 mm) and an eluting mixture of n-hexane/2-propanol (95:5) at a flow rate of 0.8 mL/min, and the detector was set at 220 nm. (±)-Homocrepidine A (1): colorless crystals (CHCl3/MeOH, 10:1); mp 164−165 °C; [α]25D −2.8 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 203 nm; IR (KBr) νmax 3421, 1720, 1639, 1447 cm−1; 1H and 13C NMR, see Table 1; positive HRESIMS m/z 517.3436 [M + H]+ (calcd for C33H45N2O3, 517.3425). (+)-(5S,6R,7S,9R,5′S,6′R,7′S,9′R)-Homocrepidine A [(+)-1]: colorless powder; [α]25D +34.6 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 231 (−0.05), 355 (−0.2), 369 (−0.2), 379 (−0.02) nm. (−)-(5R,6S,7R,9S,5′R,6′S,7′R,9′S)-Homocrepidine A [(−)-1]: colorless crystals; [α]25D −29.4 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 231 (+0.05), 350 (+0.2), 364 (−0.2), 374 (−0.04) nm. Homocrepidine B (2): amorphous powder; [α]25D +50.76 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 203 nm; IR (KBr) νmax 3442, 1641, 1083, 699 cm−1; 1H and 13C NMR, see Table 1; positive HRESIMS m/z 290.2122 [M + H]+ (calcd for C18H28NO2, 290.2115). X-ray Crystallographic Analysis Data of Homocrepidine A (1). C33H44N2O3, M = 516.70, monoclinic, space group P1; unit cell dimensions were determined to be a = 12.1696(3) Å, b = 12.2213(3) Å, c = 23.5426(6) Å, α = 89.717(2)°, β = 89.436(2)°, γ = 63.420(2)°, V = 3131.23(14) Å3, Z = 4, Dx = 1.096 mg/cm3, F(000) = 1120, μ(Cu Kα) = 0.543 mm−1. Single crystals were measured on a Sapphire CCD with graphite-monochromated Cu Kα radiation (λ = 1.541 84 Å) at 289(2) K; 40 420 reflections were collected until θmax = 69.795°, in which 11 595 independent unique reflections were observed [Rint = 0.0196]. The structure was solved by direct methods using the SHELXS-97 program and refined by full-matrix least-squares on F2. The final refinement gave R = 0.0614, Rw = 0.1950, and S = 1.055. Crystallographic data for homocrepidine A (1) have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication No. CCDC 1052365. X-ray Crystallographic Analysis Data of (−)-(5R,6S,7R,9S,5′R,6′S,7′R,9′S)-Homocrepidine A [(−)-1]. C33H44N2O3, M = 516.70, monoclinic, space group P21; unit cell dimensions were determined to be a = 9.3519(2) Å, b = 14.7084(3) Å, c = 11.0943(2) Å, α = 90°, β = 91.709(2)°, γ = 90°, V = 1525.36(5) Å3, Z = 2, Dx = 1.125 mg/cm3, F(000) = 560, μ(Cu Kα) = 0.558 mm−1. Single crystals were measured on a Sapphire CCD with graphite-monochromated Cu Kα radiation (λ = 1.541 84 Å) at 289(2) K; 13 221 reflections were collected until θmax = 69.496°, in which 5554 independent unique reflections were observed [Rint = 0.0223]. The structure was solved by direct methods using the SHELXS-97 program and refined by full-matrix least-squares on F2. The final refinement gave R = 0.0453, Rw = 0.1294, S = 1.060, and crystallographic data for (−)-homocrepidine A have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication No. CCDC 1053880.

Compound (+)-1 showed the highest potency against NO production, and (−)-1 was less active. Unsurprisingly, the racemic mixture, (±)-1, showed a moderate inhibitory effect on the NO synthesis induced by LPS. Compound (+)-1 (5 μM) significantly inhibited the overexpression of iNOS (an enzyme catalyzing the production of NO from L-arginine) in LPSstimulated RAW 264.7 cells by the Western blotting assay (p < 0.05, Figure 7), while (±)-1 and (−)-1 showed no notable

Figure 7. Inhibition of (+)-1, (−)-1, and (±)-1 on LPS-induced iNOS overexpression in RAW 264.7 cells. Cells were pretreated with 5 μg/ mL LPS or solvent and then exposed to 5 μM compound for 24 h. Cells were harvested, and extracted proteins were resolved by SDSPAGE and analyzed by immunoblotting. Histogram represents results of densitometric analysis of iNOS normalized to β-actin. Data are presented as mean ± SD (n = 3−5). ##p < 0.01 vs control group (without LPS), *p < 0.05 vs the model group (LPS alone).

suppressing effects at the same concentration. Compound 2 showed moderate inhibition on LPS-induced NO production with an IC50 value of 27.6 μM (95% confidence interval, 22.4− 34.1 μM), but 3 showed no distinct effect at 100 μM. These results indicated that racemic indolizidine alkaloids have different biological activities, (+)-indolizidine showing higher activity than (−)−indolizidine. Similar conclusions can be found in previous studies; for example, (−)-lentiginosine was about 35 times less potent than (+)-lentiginosine,21 (+)-swainsonine was a potent specific inhibitor of L-rhamnosidase (Ki 0.45 μM), while (−)-swainsonine is less active.22 In this study, the decreased potency of known compound 3 may be partly due to its racemic nature.23−25 In conclusion, the chiral resolution of a pair of new enantiomeric indolizidine dimers, (±)-homocrepidine A (1), permitted definition of the relative and absolute configurations of the enantiomers. This is also the first time that the configurations of indolizidine enantiomers were unambiguously determined. (+)-Homocrepidine A had much stronger antiinflammatory activity than (−)- or (±)-homocrepidine A.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were obtained on an X-4 micro melting point apparatus without correction. Optical rotations were measured on a Jasco P-1030 polarimeter with a 1 cm cell at room temperature. UV spectra were recorded on a Shimadzu UV1800 spectrophotometer. ECD spectra were obtained on a Jasco J-810 spectropolarimeter at room temperature. IR spectra were recorded on a Shimadzu 8400s Plus Fourier transform infrared spectrometer using KBr pellets. HRESIMS spectra were acquired on an Agilent 6210 LC/MSD TOF mass spectrometer. NMR spectra were measured on a Bruker AV-300 spectrometer. TLC analyses were carried out using prepared silica gel GF254 plates (Qingdao Marine Chemical Inc., China). Column chromatography was performed on silica gel (200−300 mesh, Qingdao Marine Chemical Inc., China), Sephadex LH-20 (Pharmacia Fine Chemical Co. Ltd., Uppsala, Sweden), and reversed-phase C18 silica gel (Pharmacia Fine Chemical Co., Ltd., Uppsala, Sweden). All solvents used in column chromatography were purchased from Xilong Chemical Co., Ltd. D

DOI: 10.1021/acs.jnatprod.5b00801 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Measurement of Cell Viability and Nitrite.26 RAW 264.7 cells (Cell Bank of Chinese Academic of Sciences, Shanghai, China) were cultured in Dulbecco’s modified Eagle’s medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (Biological Industries, Israel), 100 units/mL penicillin, 100 μg/mL streptomycin, 2 mM Lglutamine, and 1 mM nonessential amino acids (all from Biosharp, China). Cells were plated on 96-well plates at a concentration of 4 × 105 cells/well. After incubation of cells at 37 °C with 5% CO2 for 4 h, test compounds and indomethacin (Sigma, USA) dissolved in 0.1% DMSO, with the final concentrations ranging from 0.1 to 100 μM, were added to the plates at 20 μL/well. Following incubation for 1 h, the LPS solution (10 μL/well, 5 μg/mL) was added, and the cells were incubated for a further 20 h. Cell viability was determined by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Biosharp, China) assay. Nitrite was measured by adding 50 μL of the Griess reagent (Beyotime, China) to 50 μL of cell suspension for 5 min. The absorbance was measured at 540 nm with a microplate reader (Epoch, Bio-Tek, USA). All samples were assayed in at least triplicate. Western Blot Analysis. Cells were lysed in a cold buffer containing 1% phenyl-methanesulfonyl fluoride. Total protein content was determined with a Bradford protein assay kit (Beyotime, China), and 25 μg of protein was separated on SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in Tris-buffered saline−Tween for 60 min and incubated with primary antibody against iNOS (SunShinebio, China) at 4 °C overnight. After washing with TBS−Tween, the membrane was incubated in HRP-conjugated secondary antibody for 1 h, reacted with an enhanced chemiluminescence reagent (Beyotime), and exposed to Kodak Scientific film to detect the immunoblots.27 Densitometry was performed on scanned immunoblot images using Image-Pro Insight software (Media Cybernetics, USA).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00801. UV, IR, HRESIMS, and NMR spectra, ECD data, and bioassay data of compounds 1 and 2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-25-86185140. Fax: +86-25-86185137. E-mail: [email protected] (C.-F. Zhang). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Science and Technology Project of China (2011ZX09307-002-02) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.5b00801 J. Nat. Prod. XXXX, XXX, XXX−XXX