Nocapyrones H–J, 3,6-Disubstituted α-Pyrones from the Marine Actinomycete Nocardiopsis sp. KMF-001

Abstract

Three new 3,6-disubstituted α-pyrones, nocapyrones H–J (1–3), were isolated from the marine actinomycete Nocardiopsis sp. KMF-001. Their structures were assigned to be 3-alkylated 6-(1-methyl-1-propenyl)-2H-pyran-2-ones on the basis of UV, MS, NMR, and high resolution (HR)-FAB-MS analyses. Nocapyrone H (1) reduced the pro-inflammatory factor such as nitric oxide (NO), prostaglandin E₂ (PGE₂) and interleukin-1β (IL-1β). Moreover, nocapyrone H showed 5.82% stronger inhibitory effect on NO production than chrysin at a concentration of 10 µm in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells.

In recent decades, marine natural products have been used in the production of new pharmaceuticals due to their great chemical diversity and potent biological activity in a variety of indications.^1,2) Marine microorganisms have been considered fascinating sources for the discovery of new pharmacophores.^3,4) Especially, the largest number of new biologically active metabolites, especially in antibiotic and antitumor components, have originated from actinomycetes.^5,6) The Nocardiopsis is an actinobacterial genus consisting of former members of the genus Actinomadura that Meyer reclassified in 1976.⁷⁾ Although Nocardiopsis does not represent a major group within actinomycetes, a ubiquitous distribution of Nocardiopsis strains has been reported in various environments, such as saline soil, marine sediment, salterns, honeybee guts, and marine sponges.^8–13) Moreover, many secondary metabolites have been isolated from this genus, as for example, nocapyrones A–D,¹³⁾ pendolmycin,¹⁴⁾ lucentamycins A–D,¹⁵⁾ 2-pyranone derivatives,¹⁶⁾ nocardiopsins A and B,¹⁷⁾ fijiolides A and B,¹⁸⁾ nocazins A–C, and nocazoline A.¹⁹⁾ In addition, Nocardiopsis has also been reported to possess various biologically activities, such as cytotoxicity against HeLa cells,¹⁶⁾ tumor necrosis factor-α (TNF-α) inhibition,²⁰⁾ antimicrobial activity,²⁰⁾ protein kinase C inhibition,²¹⁾ and staurosporine-like inhibition of cyclic AMP-dependent protein kinase.²¹⁾

We are screening the neuroprotective actinobacterial metabolites related to prevent of brain inflammation through inhibition of microglial activation. Although a low degree of brain inflammation can contribute to the recovery of damaged tissue from brain injury,^22–24) excessive brain inflammation leads to neuronal cell death and is a cause of Parkinson’s disease, Alzheimer’s disease or cerebral ischemia.^25–27) Inflammatory responses in the brain primarily result from the activation of astrocytes and microglia, which are the major immune effector cells in the central nervous system (CNS).^28,29) Under normal conditions, microglia play a primary role in immune surveillance, and astrocytes serve to sustain the survival of neurons through the release of nerve growth factors and by buffering the activity of neurotransmitters.^28,30) However, activated microglia release a variety of pro-inflammatory and cytotoxic factors, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), TNF-α, nitric oxide (NO), reactive oxygen species (ROS) and arachidonic acid metabolites.³¹⁾ The accumulation of pro-inflammatory and cytotoxic factors is injurious to neurons in vitro, and these factors are thought to actively contribute to the progression of neurodegenerative diseases in vivo.^28,32–36)

In order to search for bioactive secondary metabolites from marine sources, we collected marine sediment in Ulleng Basin, the eastern sea of Korea. The actinomycete Nocardiopsis sp. KMF-001, was isolated from one sediment sample, and selected for further investigations due to anti-inflammatory activity of the organic extract. The liquid culture broth of bacterial strain KMF-001 was extracted with ethyl acetate. The ethyl acetate soluble fraction was subjected to vacuum column chromatography and repeated preparative reverse phase (RP)-HPLC to yield three new α-pyrone compounds. Their structures were established by extensive 2D-NMR experiments and high resolution (HR)-FAB-MS. These structures are characterized by 3-alkylated 6-(1-methyl-1-propenyl)-2H-pyran-2-ones, which were designated as nocapyrone H (1), I (2), and J (3). Compound 1 demonstrated neuroprotective effects in inflammation-related brain damage induced by microglial cell activation. Previously reported nocapyrones A–D are the first γ-pyrones reported from Nocardiopsis sp.,¹³⁾ while nocapyrones E–G isolated from Nocardiopsis dassonvillei HR10-5 are composed of 3,6-disubstituted α-pyrones moiety.¹⁹⁾ However, the structures of nocapyrones H (1), I (2), and J (3) are distinguished from the nocapyrones E–G by the functionality at C-6 position of the α-pyrones, which are 2-butenyl group in 1, 2, and 3, while 2-pentenyl group in the nocapyrones E–G.

Fig. 1. The Structure of Compounds 1–3

Results and Discussion

Nocapyrone H (1) was isolated as a pale yellow oil and its molecular formula was assigned as C₁₃H₁₈O₂ based on the HR-FAB-MS ([M+H]⁺ m/z 207.1389) and ¹³C-NMR data (Table 1). Its IR absorption band at 1713 cm⁻¹ and UV absorption bands at λ_max 234 and 332 nm are consistent with an extended α-pyrone chromophore.³⁷⁾ The ¹H-NMR spectrum of 1 displayed signals from two ortho-coupled protons at δ_H 7.04 (1H, d br q, J=7.0, 1.0 Hz, H-4) and 6.05 (1H, d, J=7.0 Hz, H-5), one olefinic proton at δ_H 6.61 (1H, q br q, J=7.5, 1.0 Hz, H-8), one methylene at δ_H 2.29 (2H, d, J=7.0 Hz, H₂-11), one methine at δ_H 1.95 (1H, tq, J=7.0, 7.0 Hz, H-12), two magnetically equivalent methyl doublet groups at δ_H 0.90 (6H, d, J=7.0 Hz, H₃-13 and H₃-14), and two other methyl groups at δ_H 1.81 (3H, d br t, J=7.5, 1.0 Hz, H₃-9) and 1.84 (3H, br q, J=1.0 Hz, H-10). The heteronuclear single quantum coherence (HSQC) spectrum showed correlation of all proton signals with the corresponding ¹³C-NMR signals. The ¹³C-NMR spectrum also displayed signals for one carbonyl carbon (δ_C 163.0, C-2) and two quaternary sp² carbons (δ_C 159.9 for C-6 and δ_C 126.0 for C-3); these signals, together with two proton signals at δ_H 7.04 and 6.05 in the ¹H-NMR spectrum, suggested the presence of a 3,6-disubstituted α-pyrone system. The structure of an isobutyl group was assigned by sequential ¹H–¹H correlation spectroscopy (COSY) correlation between two magnetically equivalent methyl (δ_H 0.90, H₃-13 and 14) signals, a methine (δ_H 1.95, H-12) signal, and a methylene (δ_H 2.29, H₂-11) signal. In addition, the isobutyl group was connected through the C-3 position of the pyrone, which was determined by the heteronuclear multiple bond connectivity (HMBC) correlations from H-11 to C-2 (δ_C 163.0), C-3 (δ_C 126.0), and C-4 (δ_C 140.3). The ¹H–¹H COSY spectrum also showed correlations between an olefinic proton (δ_H 6.61, H-8) and two methyl signals (δ_H 1.81, H₃-9 and δ_H 1.84, H₃-10), which indicated the presence of a 2-butenyl side chain. This 2-butenyl functional group was connected through position 6 of the α-pyrone system based on the HMBC correlation from H-8 and H₃-10 to C-6. The 2 dimensional nuclear Overhauser effect spectroscopy (2D NOESY) spectroscopic data could not be clear to assign the geometric configuration of the 2-butenyl chain. However, previously reported NMR data of gibepyrone A and fusalanipyrone proposed differences of ¹³C chemical shifts between E- and Z-butenyl group (E-form: δ_C-9 14.10/δ_C-10 12.05, Z-form δ_C-9 16.50/δ_C-10 15.70).³⁸⁾ Consequently, the geometric configuration of the 2-butenyl group in nocapyrone H (1) was consistent with the E-configuration. Thus, the structure of 1 was determined to be (E)-6-(but-2-en-2-yl)-3-isobutyl-2H-pyran-2-one.

Table 1. NMR Spectroscopic Data for Nocapyrones 1–3

Position	1		2		3
	δH, mult (J in Hz)a)	δCb)	δH, mult (J in Hz)a)	δCb)	δH, mult (J in Hz)a)	δCb)	δCc)
2		163.0, C		163.0, C		163.0, C	165.1, C
3		126.0, C		126.8, C		126.8, C	128.5, C
4	7.04, d br q (7.0, 1.0)	140.3, CH	7.08, d br q (7.0, 1.0)	139.3, CH	7.08, d br q (7.0, 1.0)	139.1, CH	142.1, CH
5	6.05, d (7.0)	100.4, CH	6.06, d (7.0)	100.5, CH	6.06, d (7.0)	100.5, CH	102.5, CH
6		159.9, C		159.7, C		159.7, C	161.0, C
7		126.8, C		127.0, C		127.2, C	128.0, C
8	6.61, q br q (7.5, 1.0)	128.2, CH	6.63, q br q (7.0, 1.0)	128.2, CH	6.62, q br q (7.0, 1.0)	128.2, CH	129.0, CH
9	1.81, d br t (7.5, 1.0)	14.1, CH3	1.83, d br t (7.0, 1.0)	14.1, CH3	1.83, d br t (7.0, 1.0)	14.1, CH3	14.2, CH3
10	1.84, br qd) (1.0)	12.0, CH3	1.85, br qd) (1.0)	12.0, CH3	1.86, br qd) (1.0)	12.0, CH3	12.1, CH3
11	2.29, d (7.0)	39.8, CH2	2.42, t (7.0)	32.4, CH2	2.44, t (7.0)	30.1, CH2	30.8, CH2
12	1.95, tq (7.0, 7.0)	27.0, CH	1.60, tq (7.5, 7.5)	21.2, CH2	1.56, me)	30.1, CH2	31.4, CH2
13	0.90, d (7.0)	22.3, CH3	0.95, t (7.0)	13.7, CH3	1.37, tq (7.5, 7.5)	22.4, CH2	23.4, CH2
14	0.90, d (7.0)	22.3, CH3			0.92, t (7.0)	13.9, CH3	14.2, CH3

a) 500 MHz, CDCl₃. b) 125 MHz, CDCl₃. c) 125 MHz, CD₃OD. d) Quintet. e) Overlapping signals with the H₂O peak. Chemical shifts (δ) are given in ppm.

Nocapyrone I (2) was obtained as a pale yellow oil, and the molecular formula assigned was C₁₂H₁₆O₂, which was based on the HR-FAB-MS ([M+H]⁺ m/z 193.1223) and ¹³C-NMR data (Table 1). The 1D and 2D NMR spectroscopic data for 2 were similar to those of 1. However, the NMR data of 2 displayed signals attributable to the linear propane chain connected to the α-pyrone moiety. The ¹H-NMR spectrum showed two methylene signals at δ_H 2.42 (t, J=7.0 Hz, H-11)/δ_H 1.60 (tq, J=7.5, 7.5 Hz, H-12) and one methyl signal at δ_H 0.95 (t, J=7.0 Hz, H₃-13). The ¹H–¹H COSY correlations between these signals also supported the presence of a propyl group in the structure of 2. The HMBC correlations indicated that the propyl unit was connected through the C-3 position of the α-pyrone moiety. The structure of 2 was accordingly assigned as (E)-6-(but-2-en-2-yl)-3-propyl-2H-pyran-2-one.

Finally, nocapyrone J (3) was isolated as a pale yellow oil, and the molecular formula assigned as C₁₃H₁₈O₂, by interpretation of the HR-FAB-MS ([M+H]⁺ m/z 207.1383) and ¹³C-NMR data (Table 1). Overall, the ¹H- and ¹³C-NMR spectroscopic of 3 were very similar to those of nocapyrone I (2) except for an additional methylene signal at δ_H 1.37 (2H, dq, J=7.5, 7.5 Hz, H-13), which indicated the presence of a butyl functionality in the structure of 3. The structure and position of the butyl group was assigned on the basis of a ¹H–¹H COSY correlation between the methylene signal (H₂-13) and a methyl signal at δ_H 0.92, and an HMBC correlation from the H₂-12 signal to C-3 signal. Although the HSQC spectrum showed a clear correlation between H₂-13 and the carbon signal at δ_C 22.4, the other two methylene carbon signals (C-11 and C-12) could not be clearly identified. These C-11 and C-12 methylene signals observed at the same chemical shift (δ_C 30.1) in the ¹³C-NMR spectrum of 3 using CDCl₃. However, these carbon signals were clearly distinguishable in the ¹³C-NMR spectrum using CD₃OD. H₂-11 and H₂-12 signals were observed at δ_H 2.46 and 1.58 in the ¹H-NMR spectrum using CD₃OD, while the chemical shifts of the C-11 and C-12 signals were δ_C 30.8 and 31.4, respectively, in the ¹³C-NMR spectrum using CD₃OD (Table 1). Therefore, the structure of compound 3 was determined to be (E)-6-(but-2-en-2-yl)-3-butyl-2H-pyran-2-one.

We investigated the effects of nocapyrones H (1), I (2), and J (3) on the inhibition of NO production in BV-2 microglial cells. Among these compounds, 1 showed significant inhibition on NO production in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells, while 2 and 3 possessed week effect on the NO production (Fig. 2A). We also compared the effect of 1, 2, and 3 with that of chrysin, which is known to reduce NO production.³⁹⁾ As the results, 1 exhibited 5.82% stronger inhibitory effect on NO production than chrysin at a concentration of 10 µm in LPS-stimulated BV-2 microglial cells (Fig. 2A). In addition, 1 increased cell viability in a dose-dependent manner (Fig. 2B). Treatment of BV-2 cells with 1 led to a modest decrease in iNOS protein levels at 10 µm (Fig. S25). The ability of 1 to decrease the production of prostaglandin E₂ (PGE₂) was investigated using BV-2 microglial cells treated with LPS (100 ng/mL). Pretreatment of primary microglial cells with 1 significantly decreased LPS-induced PGE₂ production in a dose-dependent manner (Fig. 3A). We also measured the effects of 1 on cytokines, such as TNF-α and IL-1β, which are related to the LPS-induced inflammatory response in BV-2 microglial cells. 1 suppressed IL-1β secretion in BV-2 cells (Fig. 3B), while 1 increased TNF-α secretion in a dose-dependent manner (Fig. 3C). 1 also showed neuro-protective effects on LPS-induced, microglia-mediated neurotoxicity in N2a cells (Fig. S26-A). Caspase-3 activation was blocked by 1 in a dose-dependent manner, while treatment of 1 did not change expression of the anti- and pro-apoptotic proteins Bcl-2 and Bax, respectively, induced by activated microglia in N2a cells (Fig. S26-B). Even though 1, 2, and 3 have nearly the same structures except alkyl functionalities attached at C-3 position of the α-pyrone, 1 possesses much stronger inhibitory activity on NO production in LPS-stimulated BV-2 cells than 2 and 3. Previously reported literatures demonstrated that the form of alkyl functionalities (e.g., chain length, branch, and attached position) conjugated with aromatic compounds could cause remarkable differences of biological activities.^40–43) In this case, nocapyrone H, which has a branched alkyl chain, possesses significant neuro-protective effect.

Fig. 2. The Effect of Nocapyrones H (1), I (2), and J (3) on NO Production in BV-2 Microglial Cells

BV-2 microglial cells were pretreated with 1, 5 and 10 µm nocapyrones 1–3 for 30 min and stimulated with 100 ng/mL of LPS for 24 h. Nocapyrone H (1) inhibited LPS-induced production of NO (A). Nocapyrone H (1) increased cell viability (B). All data are presented as the mean±S.E.M. of three independent experiments. * p<0.05 indicates a significant difference compared with treatment with LPS alone.

Fig. 3. The Effects of Nocapyrone H (1) on the Secretion of PGE₂ (A), IL-1β (B) and TNF-α (C) in BV-2 Microglial Cells

BV-2 cells were pretreated with nocapyrone H (1) for 30 min and then stimulated with LPS (100 ng/mL) for 24 h. All data are presented as the mean±S.E.M. of three independent experiments. * p<0.05 indicates a significant difference compared with treatment with LPS alone.

To summarize, we isolated of three new 3,6-disubstitute α-pyrone, nocapyrones H–J (1–3), from marine actinomycete Nocardiopsis sp. KMF-001. In addition, nocapyrone H (1) showed stronger inhibitory effect on NO production at a concentration of 10 µm in LPS-stimulated BV-2 microglial cells. Moreover, nocapyrone (1) reduced the pro-inflammatory factor such as PGE₂ and IL-1β in LPS-stimulated BV-2 microglial cells. We suggest that nocapyrone H (1) has neuro-protective effects in the setting of inflammation-related brain damage induced by microglial cell activation.

Experimental

General Experimental Procedures

UV spectra were obtained with a PerkinElmer Lambda 35 UV/Vis spectrophotometer. FT-IR spectra were recorded on a Thermo Scientific Nicolet iS10 spectrometer. ¹H- and ¹³C-NMR data were obtained using a Varian 500 MHz spectrometer with a carbon-enhanced cold probe in CDCl₃ and CD₃OD. The chemical shift values are reported in ppm and the coupling constants in Hz. Low-resolution electrospray ionization (ESI)-MS was measured on an Agilent Technologies VS/Agilent 1100 system. HR-FAB-MS data were obtained with the aid of a JEOL/JMS-AX505WA instrument. Lichroprep RP-18 (Merck, 40–63 µm) was used for vacuum flash chromatography. A Gilson 321 HPLC system, Luna 10 µm C18 column (250×21.20 mm, 10 µm, Phenomenex®) and Luna 10 µm C18 column (250×10.00 mm, 10 µm, Phenomenex®) with a Gilson UV/Vis-151 detector were used for the HPLC separation of compounds. A Waters 1525 HPLC-PDA system and Luna 5 µm C18 column (150×4.6 mm, 5 µm, Phenomenex®) were used to perform the HPLC analysis.

Identification and Cultivation of Strain KMF-001

Strain KMF-001 was isolated from marine sediment collected at a depth of 1198 m in the Ulleung Basin, the eastern sea of Korea. Nearly complete 16S ribosomal RNA (rRNA) gene sequencing of the KMF-001 strain revealed a 99.9% sequence identity with Nocardiopsis dassonvillei. The cultured Nocardiopsis strain KMF-001 was deposited with the Korean Culture Center of Microorganisms (KFCC11531P). The bacterial strain KMF-001 was cultured in 16 replicates at a volume of 500 mL using an A1 liquid culture medium (10 g of starch, 4 g of peptone, and 2 g of yeast extract in 1 L of sea water) for 15 d at 27°C while shaking at 200 rpm. After 15 d, the whole culture broth were extracted with ethyl acetate (16 L overall). The ethyl acetate extract was concentrated to yield 1.6 g of crude extract.

Isolation of Compounds 1–3

The crude extract was fractionated by Lichroprep RP-18 (40–63 µm, Merck, NJ, U.S.A.) flash column chromatography using step gradient elution with H₂O and CH₃OH (20%, 40%, 60%, 80%, and 100%) to give five subfractions. The 100% methanol fraction containing nocapyrones H (1) and J (3) was dried under a vacuum and subjected to reversed phase Prep HPLC separation [Gilson 321; Phenomenex Luna C18 (2) 10 µm column (21.2×250 mm); 10 mL/min; UV detection at 330 nm] using gradient elution with 10% to 100% MeCN/H₂O in 0.02% trifluoroacetic acid (TFA) for 120 min. Nocapyrone I (2) was isolated from the 80% methanol fraction by reversed phase Prep HPLC [Gilson 321; Phenomenex Luna C18 (2) 10 µm column (21.2×250 mm); 10 mL/min; UV detection at 330 nm] using gradient elution with 10% to 100% MeCN/H₂O in 0.02% TFA for 120 min. 1 (40 mg, t_R 98 min), 2 (1.4 mg, t_R 90 min), and 3 (0.8 mg, t_R 105 min) were purified by reversed phase HPLC [Gilson 321; Phenomenex Luna C18 (2) 10 µm column (10×250 mm), 4 mL/min; UV detection at 330 nm] with an MeCN/H₂O (in 0.02% TFA) gradient elution system from 50% to 100% for 60 min.

Nocapyrone H (1)

pale yellow oil; ¹H-NMR (CDCl₃, 500 MHz) and ¹³C-NMR (CDCl₃, 125 MHz) data: see Table 1; IR (film) ν_max 2955, 2926, 2866, 1713, 1644, 1558, 1463, 1382, 1087, 805 cm⁻¹; UV (MeCN) λ_max (log ε) 203 (3.91), 234 (3.99), 332 (4.00) nm; HR-FAB-MS [M+H]⁺ m/z 207.1389 (calcd for C₁₃H₁₉O₂, 207.1385)

Nocapyrone I (2)

pale yellow oil; ¹H-NMR (CDCl₃, 500 MHz) and ¹³C-NMR (CDCl₃, 125 MHz) data: see Table 1; IR (film) ν_max 2951, 2926, 2872, 1713, 1641, 1555, 1454, 1378, 1128, 1112, 811 cm⁻¹; UV (MeCN) λ_max (log ε) 200 (3.17), 234 (4.03), 328 (3.96) nm; HR-FAB-MS [M+H]⁺ m/z 193.1223 (calcd for C₁₂H₁₇O₂, 193.1229)

Nocapyrone J (3)

pale yellow oil; ¹H-NMR (CDCl₃, 500 MHz) and ¹³C-NMR (CDCl₃ or CD₃OD, 125 MHz) data: see Table 1; IR (film) ν_max 2954, 2929, 2870, 1713, 1641, 1562, 1376, 1257, 1130, 1082, 1022, 800 cm⁻¹; UV (MeCN) λ_max (log ε) 201 (3.94), 234 (3.97), 327 (3.95) nm; HR-FAB-MS [M+H]⁺ m/z 207.1383 (calcd for C₁₃H₁₉O₂, 207.1385)

Cell Culture³⁹⁾

The BV-2 cell line, derived from murine microglia, was originally developed by Dr. V. Bocchini at the University of Perugia (Perugia, Italy). BV-2 cells have both the phenotypic and functional characteristics of reactive microglia cells. BV-2 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS). N2a cells were maintained in DMEM supplemented with 10% FBS and 1% PS. The cells were cultured in a humidified atmosphere of 5% CO₂ at 37°C.

Measurement of NO Production³⁹⁾

To measure NO production, BV-2 cells were plated onto 96-well plates (3×10⁴ cells/well) and treated with 100 ng/mL of LPS in the presence or absence of compound 1 (1, 5, and 10 µm) for 24 h. The release of nitrite, a soluble oxidation product of NO, into the culture medium was determined using the Griess reaction. The supernatant (50 µL) was harvested and mixed with an equal volume of the Griess reagent (1% sulfanilamide and 0.1% N-1-napthylethylenediamine dihydrochloride in 5% phosphoric acid). After 10 min, the absorbance at 570 nm was measured using a microplate reader. Sodium nitrite was used as a standard to calculate the NO₂ concentration. Cell viability was measured using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay.

Determining Neuronal Cell Viability

To determine neuronal cell viability, LPS-challenged BV-2 microglial cells were treated with compound 1, 2, or 3. After 24 h, conditioned medium was collected and used to treat N2a cells. Cell viability was measured using a MTT assay

Measurement of IL-1β, TNF-α, and PGE₂^44–46)

To measure IL-1β, PGE₂, and TNF-α, cells were plated in a six-well plate at a density of 15×10⁵ cells/well in DMEM and incubated for 24 h before sample treatment. Medium was collected and centrifuged 24 h after treatment with LPS (100 ng/mL) in the presence or absence of compound 1. PGE₂, IL-1β, and TNF-α were measured by a competitive enzyme immunoassay kit (specific enzyme-linked immunosorbent assay (ELISA) kit for PGE₂, IL-1β, and TNF-α, R&D Systems, Minneapolis, MN, U.S.A.) according to the manufacturer’s protocol.

Western Blot Analysis^39,47,48)

For the Western blot analysis, cell lysates were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After transfer, the membranes were blocked with 5% skim milk and incubated for 12 h with primary antibodies against iNOS, cyclooxygenase-2, Bax, Bcl-2, or cleaved caspase-3 at 4°C, followed by incubation for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature. Blots were developed using ECL Western blot detection reagents (Amersham Pharmacia Biotech).

Statistical Analysis

All data are expressed as the mean±S.E.M. Statistical comparisons between the different treatment groups were performed using a one-way ANOVA with Tukey’s multiple comparison post-test. *p-Values <0.05 were considered statistically significant.

Supplemental Information Available

The spectroscopic data of nocapyrones H, I and J, as well as the biological activity data of nocapyrone H, are available.

Acknowledgment

This study was supported by the Korea Institute of Science and Technology institutional program, Grant number 2Z03840, and the Global Leading Technology Program of the Office of Strategic R&D Planning (10039303) funded by the Ministry of Knowledge Economy, Republic of Korea.

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