Inflammatory Inhibitory Activity of Sesquiterpenoids from Atractylodes macrocephala Rhizomes

Abstract

Three new sesquiterpenoids, 13-hydroxyl-atractylenolide II (1), 4-ketone-atractylenolide III (2), and eudesm-4(15)-ene-7β,11-diol (3), along with eleven known compounds (4–14), were isolated from the rhizomes of Atractylodes macrocephala. The structures and relative configurations of 1–3 were determined by analysis of the spectroscopic data, and the absolute configurations of 1 and 2 were assigned by circular dichroism technique. The anti-inflammatory activities of these isolates were evaluated against lipopolysaccharide-induced nitric oxide production in macrophage RAW264.7 cells; compounds 4, 7, and 8 exhibited moderate efficacy with IC₅₀ values of 48.6±0.5, 46.4±3.2, and 32.3±2.9 µM, respectively.

The genus Atractylodes DC, which consists of seven species in the world that are distributed widely in East Asia, has been taxonomically placed in the Compositae family.¹⁾ One of the noteworthy species is Atractylodes macrocephala KOIDZ. Although it has been used as an important herbal medicine for the treatment of hypofunction of the spleen with the loss of appetite, abdominal distension, diarrhea, dizziness, and palpitation due to the retention of phlegm and fluid, edema, spontaneous sweating, and threatened abortion for thousands of years,²⁾ its active compounds have not been characterized fully. Previous phytochemical investigations of A. macrocephala revealed the presence of polyacetylenes,³⁾ polysaccharides,⁴⁾ sesquiterpenoids,⁵⁾ phenylpropanoids, and coumarins⁶⁾ with several pharmacological functions, such as the anti-inflammatory⁷⁾ and inhibitory effects on the proliferation of cultured human tumor cells,⁸⁾ aromatase activity⁹⁾ and modulatory activity on the γ-aminobutyric acid type A receptor.¹⁰⁾ The atractylodes rhizome is one of the main active components of some Korean folk prescriptions, such as “Guibitang” and “Bojungikgitang,” which have been used to enhance of the deficiency of vital energy and synergistic effects.¹¹⁾

This study examines whether new metabolites also contribute to the biological activity of A. macrocephala. Three new eudesmane-type sesquiterpenoids, i.e., 13-hydroxyl-atractylenolide II (1), 4-ketone-atractylenolide III (2) and eudesm-4(15)-ene-7β,11-diol (3), together with eleven known compounds (4–14) were isolated. Their structures were elucidated by combination analysis based on their NMR and mass spectra. The ¹H- and ¹³C-NMR spectra were assigned based on heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC) and nuclear Overhauser effect spectroscopy (NOESY) experiments. This paper reports the isolation and structure determination of the isolated compounds as well as their nitric oxide (NO) production inhibition.

Results and Discussion

The dried rhizome of A. macrocephala was extracted in methanol, and the obtained extracts were partitioned successively into n-hexane, chloroform (CHCl₃), ethyl acetate (EtOAc), butanol (n-BuOH), and water. Repeated silica gel and reverse phase column chromatography of the CHCl₃ fraction afforded three new sesquiterpenoids (1–3) and eleven known ones (4–14). The chemical structures of the known compounds were determined to be atractylenolide II (4),¹²⁾ atractylenolide III (5),¹³⁾ asterolid (6),¹⁴⁾ 8β-methoxyatractylenolide (7),¹⁵⁾ atractylenolide V (8),¹⁶⁾ atractylenolide I (9),¹⁷⁾ atractylenolactam (10),⁵⁾ Z-methyl caffeate (11),¹⁸⁾ ferulic acid (12),¹⁹⁾ Z-5-hydroxy ferulic acid (13),²⁰⁾ and 2-hydroxy ferulic acid (14)²¹⁾ based on an analysis of the ¹H- and ¹³C-NMR spectra and confirmed by a comparison with those reported in the literature (Fig. 1).

Fig. 1. Structure of Isolated Compounds 1–14

Compound 1 was obtained as a yellow powder. The molecular formula C₁₅H₂₀O₃ was deduced from its high resolution-electron ionization (HR-EI)-MS (m/z 248.1412 [M]⁺, Calcd for C₁₅H₂₀O₃ 248.1412). The UV-visible spectrum absorption maximum was observed at 221 nm. The Fourier transform-infrared (FT-IR) absorption spectras revealed the presence of C=O functionality (1766 cm⁻¹). The ¹H-NMR spectrum showed signals of one methyl group at δ_H 0.90 (3H, s, H-14), two exocyclic methylene protons at δ_H 4.58 (1H, s, H-15a) and 4.88 (1H, s, H-15b), and one hydroxymethyl group δ_H 4.40 (2H, s, H-13). The ¹³C-NMR spectrum and distortionless enhancement by polarization transfer (DEPT) spectrum of 1 displayed 15 carbon signals, including one methyl, five methylenes, one exocyclic methylene, one hydroxymethyl, one aliphatic methine, one oxygenated methine, one aliphatic quaternary, three olefinics and one carbonyl carbon (Table 1). The above spectra indicated that 1 is a eudesmanolide derivative, which possesses an exocyclic group at C-4 of the decalin ring.¹⁷⁾ The HMBC spectrum of 1 revealed the cross-peaks of the methyl signal at δ_H 0.90 (3H, s) with C-1 (δ_C 40.9), C-5 (δ_C 50.3), C-9 (δ_C 47.5), and C-10 (δ_C 36.9), and which was assigned the methyl group at C-10. Furthermore, the HMBC correlations among the hydroxymethyl signal at δ_H 4.40 (2H, s) and oxygenated methine signal at δ_H 4.91 (1H, dd, J=12.0, 8.0 Hz) with C-7 (δ_C 165.5), C-11 (δ_C 123.1) and C-12 (δ_C 174.2) assigned the location of the hydroxymethyl moiety at C-11 and revealed the presence of an α,β-unsaturated γ-lactone ring at C-11 (α), C-7 (β), C-8 (γ) and C-12 (C=O)¹⁵⁾ (Fig. 2). On the basis of above discussion, all of the ¹H and ¹³C chemical shifts were assigned, and its optical rotation value [α]_D²⁵ +102.2 (c=0.10, MeOH) led us to speculate that three contiguous stereocenters of 1 were the same as 4 ([α]_D²⁵ +266.1 (c=5, MeOH)¹²⁾). Additionally, observations of nuclear Overhauser effect (NOE) interactions between Me-14/H-2β and H-6β but no NOE interactions for H-14/H-5 were detected in the NOESY spectrum indicating the trans-fused of this ring junction and α,β-orientation of H-5 and Me-14, respectively (Fig. 3). Furthermore, the stereochemistry of 1 was also confirmed using the empirical rule for the α,β-unsaturated γ-lactone ring in circular dichroism (CD) spectrum.²²⁾ The positive Cotton effect CD, observed at 219 nm (Δε_max +7.5 (c=0.03, MeOH), substantiated the absolute configuration of C-8 to be S-form.²³⁾ Accordingly, 1 was assigned to 13-hydroxyl-eudesm-4(15),7(11)-dien-8α,12-olide, trivially called 13-hydroxyl-atractylenolide II.

Table 1. ¹H (400 MHz) and ¹³C (100 MHz) Spectroscopic Data for Compounds 1–3 (δ in ppm, J in Hz)

	1a)		2b)		3c)
	δH	δC	δH	δC	δH	δC
1α	1.27 (1H, m)	40.9	2.42 (1H, m)	41.4	1.34 (1H, m)	42.2
1β	1.58 (1H, m)		2.25 (1H, m)		1.65 (1H, m)
2α	1.62 (2H, m)	22.4	1.95 (2H, m)	22.9	1.26 (1H, m)	24.2
2β					1.41 (1H, m)
3α	2.10 (1H, m)	36.4	1.65 (2H, m)	40.7	2.31 (1H, dd, 11.2, 13.2)	37.6
3β	2.31 (1H, m)				2.05 (1H, m)
4		148.2		212.4		152.1
5	1.95 (1H, m)	50.3	2.45 (1H, m)	58.9	2.63 (br d, 11.6)	45.0
6α	2.87 (1H, dd, 4.0, 16.0)	25.8	2.70 (1H, dd, 2.8, 13.2)	22.2	1.47 (1H, m)	30.3
6β	2.12 (1H, m)		2.31 (1H, m)		1.55 (1H, m)
7		165.5		161.9		76.3
8	4.91 (1H, dd, 8.0, 12.0)	78.7		104.9	1.54 (2H, m)	27.3
9α	1.55 (1H, m)*	47.5	2.23 (1H, d, 4)	51.6	1.53 (1H, m)	37.4
9β	2.25 (1H, m)*		1.64 (1H, 4)		1.74 (1H, m)
10		36.9		41.2		36.2
11		123.1		123.3		75.1
12		174.2		174.1	1.54 (3H, s)	25.7
13	4.40 (2H, s)	54.9	1.80 (3H, s)	8.1	1.54 (3H, s)	25.6
14	0.90 (3H, s)	16.4	1.08 (3H, s)	17.7	0.86 (3H, s)	16.0
15a	4.58 (1H, s)	107.2			4.59 (1H, d, 1.6)	105.8
15b	4.88 (1H, s)				4.79 (1H, d, 1.6)

a) Recorded in CDCl₃. b) Recorded in CD₃OD. c) Recorded in pyridine-d₅. * Signals overlapped are labeled with multiplicity.

Fig. 2. Selected HMQC, HMBC and COSY Correlations of 1–3

Fig. 3. Key NOEs Correlations of 1–3

Compound 2 was isolated as a white amorphous powder with a molecular formula of C₁₄H₁₈O₄, accounting for six degrees of unsaturation, as determined based on a molecular ion peak at m/z 250.1204 [M]⁺ (Calcd for C₁₄H₁₈O₄ 250.1205) in HR-EI-MS. The optical rotation of 2 was determined to be [α]_D²⁵ +267.6 (c=0.10, MeOH). The ¹H-NMR spectrum of 2 exhibited signals for two methyl groups at δ_H 1.80 (3H, d, J=1.2 Hz, H-13) and 1.08 (3H, s, H-14), which were typical of Me-13 and Me-14 of eudesmanolide.²⁴⁾ The ¹³C-NMR spectrum of 2 revealed 14 carbon resonances that were assigned via the DEPT spectrum. The close similarity of the ¹H- and ¹³C-NMR data of 1 and 2 suggested that 2 is an analogue of 1. On the other hand, the differences between 1 and 2 were the presence of the ketone group at C-4 (δ_C 212.4), the downfield quaternary carbon signal at C-8 (δ_C104.9) as well as one methyl group at C-13 (δ_C 8.1) of 2 instead of the signals for the exocyclic methylene protons at C-15, methine proton at C-8 and hydroxymethyl protons at C-13 of 1. These differences were confirmed by the cross-peaks of δ_H 1.65 (2H, m, H-3) and 2.45 (1H, m, H-5) with δ_C 212.4 (C-4) in the HMBC spectrum of 2, which placed the ketone at C-4, and the other HMBC correlations between δ_H 1.80 (3H, s, H-13) with δ_C 161.9 (C-7), 123.3 (C-11) and 174.1 (C-12) placed the methyl group at C-13 (Fig. 2). The absolute configuration of 2 was also determined by its CD spectrum, in which a negative Cotton effect for n→π* was observed at 317 nm [Δε_max +0.06 (MeOH, c=0.02)] and a positive Cotton effect for π→π* 215 nm [Δε_max +7.1 (MeOH, c=0.02)]. CD of the cyclic saturated ketone (the octant rule²⁵⁾) and α,β-unsaturated γ-lactone²²⁾ to 2 indicated that the absolute configurations at C-5/C-8/C-10 of 2 were similar to 8-epia-tractylenolide III,²⁾ and tractylenolide III (5)¹³⁾ as 5R/8S/10S, respectively.²⁶⁾ Finally, the structure of 2 and its relative stereochemistry were confirmed by the NOESY experiment (Fig. 3). Based on the above information, the structure of 2 was determined to be 8β-hydroxyl-4-oxoeudesm-7(11)-en-8α,12-olide, called 4-oxo-atractylenolide III.

Compound 3 gave a quasi-molecular ion at 261.1828 m/z [M+Na]⁺ (Calcd for C₁₅H₂₆O₂Na 261.1830) in the positive-ion HR-ESI-MS, which was consistent with a molecular formula possessing three degrees of unsaturation. The ¹H-NMR spectrum revealed the presence of three methyl groups at δ_H 1.54 (6H, s, H-12, H-13) and 0.86 (3H, s, H-14), and two exocyclic methylene protons at δ_H 4.59 (1H, d, J=1.6 Hz, H-15a) and 4.79 (1H, d, J=1.6 Hz, H-15b). In accordance with the molecular formula, the ¹³C-NMR spectrum displayed 15 carbon resonances that could be attributed to three methyl groups at δ_C 25.7 (C-12), 25.6 (C-13) and 16.0 (C-14), six aliphatic methylene groups, one exocyclic methylene (δ_C 105.8, C-15), one aliphatic methine (δ_C 45.0, C-5), one olefinic quaternary (δ_C 152.1, C-4), one aliphatic quaternary (δ_C 36.2, C-10), and two hydroxyl groups (δ_C 76.3, C-7) and (δ_C 75.1, C-11) (Table 1), which are typical of an eudesmene skeleton²⁷⁾ with two hydroxyl groups, and an exocyclic double bond. These features were confirmed by the HMBC correlations between the two methyl signals at δ_H 1.54 with δ_C 76.3 (C-7) and 75.1 (C-11), and from the exocyclic protons at δ_H 4.59 and 4.79 to δ_C 37.6 (C-3), 152.1 (C-4) and 45.0 (C-5) (Fig. 2). The NMR spectrum of 3 was similar to eudesm-4(15)-ene-7α,11-diol¹⁾ except for the optical rotation values: 3 possessed a positive optical rotation [α]_D²⁵ +27.0 (c=0.10, MeOH), whereas eudesma-4(15)-ene-7α,11-diol possessed a negative optical rotation [α]_D²⁵ −26.0 (c=1.0, MeOH).²⁸⁾ The relative configuration of 3 was also confirmed by NOE experiments. The NOEs correlations between H-5 and H-12 (H-13) suggested that the junction of the eudesmane ring should be β-oriented of the hydroxyl group at C-7²⁸⁾ (Fig. 3). Therefore, the structure of 3 was elucidated unambiguously as eudesm-4(15)-ene-7β,11-diol.

NO is one of the inflammatory mediators causing inflammation in many organs. An inhibitor of NO production may have therapeutic potential for the treatment of inflammation accompanying the overproduction of NO.²⁹⁾ In murine macrophage RAW264.7 cells, lipopolysaccharide (LPS) stimulation alone was demonstrated to increase NO production. This cell system is one of the frequently-used models for an evaluation of anti-inflammatory agents.³⁰⁾ Considering the traditional use of A. macrocephala, this cell model was used as an in vitro system for the activity-guided inhibitory activity of NO production. On the other hand, studies of the anti-inflammatory activity of this plant started quite early.³¹⁾ Atractylenolide I and atractylenolide III was first studied for this activity because of their relatively high content in this medicinal plant and atractylenolide I was found to be the most active.³²⁾ In these experiments, fourteen compounds (1–14) were isolated and identified. Based on the molecular skeleton, they were classified into two groups, eudesmane (1–10) and phenol (11–14). To determine if the inhibition of NO production was due to the cytotoxicity of the compounds tested, the cell viability of RAW264.7 was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.³³⁾ The viability by the isolated compounds at the concentrations of 3–100 µM for 24 h of incubation, in which the viability of the cells were greater than 95%, the final concentration of dimethyl sulfoxide (DMSO), which was used as a solvent of the samples did not exceed 1.2%, and it did not affect the cell viability (data not shown). To assess the effects of 1–14 on LPS-induced NO production in RAW264.7 cells, the cell culture medium was harvested and NO production was measured using the Griess reaction. During the incubation time of 24 h, RAW264.7 macrophages produced very low levels of NO in the resting state. After LPS (1 µg/mL) stimulation, NO production increased dramatically. On the other hand, compounds 1–14 reduced the level of NO production 24 h after LPS stimulation in a dose-dependent manner. Among them, atractylenolide V (8) exhibited the most potent inhibition of NO production with an IC₅₀ value of 32.3±2.9 µM, followed by 8β-methoxyatractylenolide (7) (IC₅₀=46.4±3.2 µM), and atractylenolide II (4) (IC₅₀=48.6±0.5 µM). The others exhibited weak inhibition of NO production with IC₅₀ values greater than 50 µM.

Table 2. NO Production Inhibition of 1–14

Compounds	NO inhibition, IC50 (µM)a)
1	98.1±3.4
2	98.0±1.7
3	95.3±5.8
4	48.6±0.5
5	60.3±2.1
6	54.4±4.2
7	46.4±3.2
8	32.3±2.9
9	70.7±6.9
10	69.2±3.1
11	97.8±4.5
12	69.6±3.8
13	88.8±4.2
14	75.0±6.3
Celastrolb)	1.0±0.1

a) The inhibitory effects are represented as the molar concentration (µM) giving 50% inhibition (IC₅₀) relative to the vehicle control. These data represent the average values of three repeated experiments. b) Positive control for NO production.

Experimental

General Experimental Procedure

Solvents were purchased from Samchun Chemicals Company (Korea). The cancer cell lines HL-60, MCF-7, and HeLa were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), penicillin, and streptomycin were bought from GIBCO, Inc. (NY, U.S.A.). Optical rotations were measured with a JASCO DIP-370 polarimeter. UV spectra were measured with a Thermo 9423AQA2200E UV spectrometer. IR spectra were measured with a Bruker Equinox 55 FT-IR spectrometer. High pressure liquid chromatography (HPLC, Waters, U.S.A.) was used for purification and isolation with an YMC-Pack ODS-A HPLC column. NMR spectra were recorded on Varian Unity Inova 400 (¹H, 400 MHz; ¹³C, 100 MHz) spectrometers. Conventional pulse sequences were used for correlation spectroscopy (COSY), HMBC and HMQC spectroscopy. All chemical shifts are given in ppm units with reference to tetramethylsilane (TMS) as an internal standard and the coupling constants (J) are in Hz. HR-EI-MS was measured on a JMS-700 MStation mass spectrometer. TLC was carried out on precoated silica gel 60 F254 (Merck). Chromatography suppliers were used for isolation: silica-gel (Kieselgel 60, 63–200 mesh, Merck) or reverse phase silica-gel (LiChroprep^® RP-18, 40–63 mm, Merck). Optical density (OD) values in the cytotoxic activity evaluation by MTT assays were read on a TECAN-ELISA Microplate Reader.

Plant Material

The dried rhizomes of A. macrocephala were purchased from the oriental market in Ulsan-si, Korea, and identified by Professor Byung-Sun Min, Catholic University of Daegu, Korea. A voucher specimen (CUD-3064) is deposited at the Herbarium of the College of Pharmacy, Catholic University of Daegu, Korea.

Extraction and Separation

The air-dried powder of the plant material (9 kg) was extracted five times with MeOH (30 L×3) to give a crude extract, which was suspended in H₂O and partitioned successively with n-hexane, CHCl₃, n-BuOH and H₂O. The CHCl₃-soluble fraction (25.58 g) was subjected to a silica gel column running with the gradient of n-hexane–EtOAc (9 : 1–1 : 1) followed by CHCl₃–acetone (9 : 1–1 : 1) to give 10 fractions 1–10. Fraction 1 (450.2 mg) was separated on a silica gel column (n-hexan–EtOAc, from 10 : 1 to 1 : 1) to yield 4 fractions, A–D. Fraction 1B was first subjected to an RP-18 column eluted with MeOH–H₂O (1 : 1) to obtain 2 fractions 1Ba and b. The fraction 1Ba was then purified by HPLC using a solvent system of MeOH–H₂O (from 50 : 50 to 60 : 40, a flow rate of 5 mL/min) to yield compounds 8 (t_R=36.5 min, 54.23 mg) and 3 (t_R=38.3 min, 10.94 mg). Fraction 2 (230 mg) was separated on a silica gel column (n-hexane–acetone, from 5 : 1 to 1 : 1) to yield A–E. Fraction 2C (540.3 mg) was applied to a silica gel column eluted in a step gradient manner with CHCl₃–EtOAc (from 5 : 1 to 1 : 1) to afford 10 fractions 2C1–10. From 2C5 (120.4 mg), compounds 4 (12. 5 mg), 5 (6.7 mg) and 6 (23.6 mg) were obtained by repeated column chromatography with CH₂Cl₂–EtOAc (from 10 : 1 to 2 : 1). Fraction 4 (1.1 g) was subjected to RP-C18 column (MeOH–H₂O, 1 : 1) to provide 5 fractions 4A–E. Compound 7 (4.7 mg) were obtained from fraction 4D by repeated purifications of silica gel column chromatography using CH₂Cl₂–EtOAC (1 : 1). Compound 2 (t_R=45.6 min, 25.8 mg) was obtained by HPLC using solvent system of MeOH–H₂O (from 50 : 50 to 60 : 40, flow rate 5.0 mL/min). Fraction 5 (560.9 mg) was separated by medium pressure liquid chromatography (MPLC, octadecyl silica (ODS), 11 mm×300 mm, 2.0 mL/min) with the mobile phase MeOH–H₂O (1 : 1) to give six sub-fractions 5A–F. From fraction 5B, compounds 9 (3.4 mg) and 11 (1.5 mg) were separated by MPLC (IS, 11 mm×300 mm, flow rate 2.0 mL/min) using CH₂Cl₂–acetone (95 : 5). Fraction 7 (790.5 mg) was re-chromatographed over a silica gel column using a gradient solvent system of CH₂Cl₂–EtOAc (from 5 : 1 to 1 : 1) as mobile phase to yield four fractions 7A–D. Compound 1 (2.6 mg) was separated by HPLC using a solvent system of MeOH–H₂O (from 40 : 60 to 50 : 50, flow rate 5.0 mL/min). Fraction 8 (250.7 mg) was subjected to an RP-C18 column eluting with MeOH–H₂O (1 : 1) to obtain 2 fractions 8A and B. Fraction 8A was chromatographed by MPLC (ODS, 11 mm×300 mm, 2.0 mL/min) with the mobile phase (MeOH–H₂O, 1 : 1) to give 5 sub-fractions 8A1–5. From 8A4, compounds 10 (4.5 mg) and 12 were separated by MPLC (IS, 11 mm×300 mm, flow rate 2.0 mL/min) by using CH₂Cl₂–EtOAc (1 : 1). Fraction 8B was chromatographed by MPLC (IS, 11 mm×300 mm, 2 mL/min) with the mobile phase CH₂Cl₂–MeOH (1 : 1) to afford compounds 13 (1.3 mg) and 14 (6.5 mg).

13-Hydroxy-tractylenolide II (1)

Yellow powder: [α]_D²⁵ +102.2 (c=0.10, MeOH); UV (MeOH) λ_max: 221 nm; IR (KBr) ν_max 3241, 1766, 1683, 1647, 1440, 1387, 1326, 1118, 1038, 1004, 958, 890, 722 cm⁻¹; HR-EI-MS m/z: 248.1412 (Calcd for C₁₅H₂₀O₃: 248.1412); ¹H- and ¹³C-NMR (CDCl₃, 400 MHz/100 MHz) spectroscopic data, see Table 1.

4-Oxo-tractylenolide III (2)

White amorphous powder: [α]_D²⁵ +267.6 (c=0.10, MeOH); UV (MeOH) λ_max: 217 nm; IR (KBr) ν_max 3233, 1742, 1387, 1320, 1109, 1034, 1012, 945, 885, 718 cm⁻¹; HR-EI-MS m/z: 250.1204 (Calcd for C₁₄H₁₈O₄: 250.1205); ¹H- and ¹³C-NMR (CD₃OD, 400 MHz/100 MHz) spectroscopic data, see Table 1.

Eudesm-4(15)-ene-7β,11-diol (3)

White amorphous powder: [α]_D²⁵ +27 (c=0.10, MeOH); UV (MeOH) λ_max: 226 nm; IR (KBr) ν_max 3423, 2946, 1380 cm⁻¹; HR-ESI-MS (positive-ion mode) m/z: 261.1828 [M+Na]⁺ (Calcd for C₁₅H₂₆O₂Na: 261.1830); ¹H- and ¹³C-NMR (pyridine-d₅, 400 MHz/100 MHz) spectroscopic data, see Table 1.

Cell Culture

The murine macrophage cell line (RAW264.7) was purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). These cells were cultured in DMEM (GIBCO, Inc., NY, U.S.A.) supplemented with 100 U/mL of penicillin, 100 U/mL of streptomycin and 10% FBS (GIBCO, Inc.). The cells were incubated in an atmosphere of 5% CO₂ at 37°C and were sub-cultured every 3 d.

Determination of NO Production

The level of NO production was determined by measuring the amount of nitrite present in cell culture supernatants as described previously.³⁰⁾ Briefly, the RAW264.7 cells (1×10⁵ cells/well) were stimulated with or without 1 µg/mL LPS (Sigma Chemical Co., St. Louis, MO, U.S.A.) for 24 h in the presence or absence of the test compounds (3–100 µM). The cell culture supernatant (100 µL) was then reacted with 100 µL of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphtylethylenediamine dihydrochloride in distilled H₂O). The absorbance at 540 nm was determined with a micro plate reader (Emax; Molecular Devices, Sunnyvale, CA, U.S.A.), and the absorption coefficient was calibrated by using a sodium nitrite (NaNO₂) solution standard. Cell viability was measured with an MTT-based colorimetric assay. For this experiment, celastrol was used as a positive control.

Acknowledgment

This research was supported by Research Grants from Catholic University of Daegu in 2013.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials as 1D- and 2D-NMR spectra data of the new compounds 1–3.

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