Bioactive α-Pyrone Derivatives from the Endolichenic Fungus

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Bioactive α‑Pyrone Derivatives from the Endolichenic Fungus Dothideomycetes sp. EL003334 Gil Soo Kim,†,‡,∥ Wonmin Ko,§,∥ Jong Won Kim,† Min-Hye Jeong,⊥ Sung-Kyun Ko,†,‡ Jae-Seoun Hur,⊥ Hyuncheol Oh,§ Jae-Hyuk Jang,*,†,‡ and Jong Seog Ahn*,†,‡ †

Anticancer Agent Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Korea Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon 34141, Korea § College of Pharmacy, Wonkwang University, Iksan 54538, Korea ⊥ Korean Lichen Research Institute, Sunchon National University, Suncheon 57922, Korea ‡

S Supporting Information *

ABSTRACT: Two new α-pyrones, dothideopyrones E (1) and F (2), were isolated from a culture of the endolichenic fungus Dothideomycetes sp. EL003334. Their structures were elucidated by spectroscopic data analysis. Their absolute configurations were established by the modified Mosher’s method. Compound 2 inhibited nitric oxide (NO) production with IC50 values of 15.0 ± 2.8 μM in lipopolysaccharide (LPS)-induced BV2 cells. Compound 2 diminished the protein expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Additionally, 2 decreased the mRNA expression levels of pro-inflammatory cytokines, such as tumor necrosis factorα (TNF-α), interleukin (IL)-1β, and IL-6.

E

ndolichenic fungi are a group of fungi that inhabit the thalli of lichens without causing disease symptoms. They were first reported a decade ago, and new metabolites have been found in them.1 These metabolites possess various bioactivities, including cytotoxic,2 antibacterial,3 and anti-Aβ42 aggregation.4 The bioactive compounds from endolichenic fungi are from various classes, including alkaloids,5 steroids,6 peptides,7 and pyrones.8 We now report bioactive secondary metabolites from the endolichenic fungus Dothideomycetes sp. EL003334 isolated from the lichen Stereocaulon tomentosum. The strain was provided by the Korean Lichen & Allied Bioresource Center (KoLABIC) at Sunchon National University, Republic of Korea. The HPLC-PDA profile of the EtOAc extract showed several pyrone derivatives, which exhibited a characteristic UV maximum absorption at approximately 300 nm. The LC-MSUV data of the culture extract suggested the presence of unusual pyrone derivatives. The fungus Dothideomycetes sp. EL003334 was grown in potato dextrose broth, and culture extracts were fractionated using reversed-phase C18 vacuum liquid column chromatography and HPLC with a Cholester packed column; this provided two new α-pyrone derivatives (1 and 2). Herein, we report the isolation, structure elucidation, and biological activities of dothideopyrones E (1) and F (2). Compound 1 was isolated as a colorless oil, and its molecular formula was determined as C16H26O6 by the HRESIMS and NMR data. The UV maximum absorption (approximately 300 nm) suggested the presence of a pyrone unit (Figure S3, © XXXX American Chemical Society and American Society of Pharmacognosy

Supporting Information).9 The 1H NMR spectrum showed an olefinic proton (δH 6.56), three exchangeable protons (δH 5.70, 4.54, and 4.28), two oxygenated methines (δH 4.29 and 3.55), an oxygenated methylene (δH 4.20), a methoxy proton (δH 3.91), a methyl proton (δH 1.02), and 6 methylene groups (δH 1.67−1.20) indicating an alkyl chain. The 1H, 13C, and DEPT NMR data in conjunction with the HSQC of 1 suggested the presence of 16 carbons, containing a carbonyl carbon (δC 164.0), three nonprotonated carbons (δC 169.1, 168.2, and 104.1), an olefinic methine carbon (δC 93.5), two oxymethine carbons (δC 69.8 and 66.2), a methoxy carbon (δC 57.3), seven methylene carbons (δC 52.6, 39.5, 35.3, 29.6, 29.4, 25.8, and Received: December 5, 2017

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DOI: 10.1021/acs.jnatprod.7b01022 J. Nat. Prod. XXXX, XXX, XXX−XXX

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and H-9′ (δH 0.86). The structure of 2 was determined as shown in Figure 1 and designated as dothideopyrone F (2). To determine the absolute configurations of 1 and 2, the modified Mosher’s method was applied.10 All hydroxy groups, a primary alcohol and two secondary alcohols, in 1 were derivatized with R-(−)- and S-(+)-α-methoxy-α(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) to yield the S- and R-MTPA esters. Calculating the Δδ values suggested the 1′S, 8′R configuration in 1 (Figure 2). The absolute

25.1), and one methyl carbon (δC 24.2) (Table 1). Crowded methylene carbons around 30 ppm also suggested the presence Table 1. 1H and 13C NMR Spectroscopic Data for 1 and 2 in DMSO-d6 1

a

position

δC,a type

2 3 4 5 6 1′ 2′

164.0, C 104.1, C 168.2, C 93.5, CH 169.1, C 69.8, CH 35.3, CH2

3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 1′-OH 8′-OH 1″-OH 4-OMe

25.1, CH2 25.8, CH2 29.4, CH2 29.6, CH2 39.5, CH2 66.2, CH 24.2, CH3 52.6, CH2

57.3, CH3

2

δH (J in Hz)b

δC,a type

δH (J in Hz)b

6.56, s

164.0, C 104.1, C 168.2, C 93.5, CH 169.1, C 69.8, CH 35.3, CH2

6.56, s

4.29, ovlc 1.67, m 1.52, m 1.20−1.40, ovl 1.20−1.40, ovl 1.20−1.40, ovl 1.20−1.40, ovl 1.20−1.40, ovl 3.55, m 1.02, d (6.2) 4.20, d (5.5) 5.70, d (5.3) 4.28, ovl 4.54, t (5.6) 3.91, s

25.1, 29.1, 29.3, 29.4, 31.7, 22.6, 14.4, 52.6,

CH2 CH2 CH2 CH2 CH2 CH2 CH3 CH2

57.3, CH3

4.29, m 1.68, m 1.57, m 1.20−1.40, ovl 1.20−1.40, ovl 1.20−1.40, ovl 1.20−1.40, ovl 1.20−1.40, ovl 1.20−1.40, ovl 0.86, t (7.1) 4.20, d (5.5) 5.70, d (4.2)

Figure 2. ΔδS−R values in ppm of the MTPA ester of 1 and 2.

configuration of 2 was established by the same method as that of 1. The results indicate the S-configuration at C-1′ in 2 (Figure 2). The absolute configuration of 2 was confirmed by comparison of the specific rotation value with dothideopyrone A. Specific rotation values of 2 were [α]21D −118.72 (c 0.05, MeOH), which were in agreement with an S-configuration at C-1′ of dothideopyrone A ([α]25D −77 (c 0.22, CHCl3)).11 The effects of compounds 1 and 2 on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced BV2 cells were investigated. Compound 2 inhibited NO production with IC50 values of 15.0 ± 2.8 μM, whereas compound 1 was inactive (Table S1, Supporting Information and Figure 3A). Prior to further examining the inhibitory effects of compound 2 on LPSstimulated BV2 cells, the cytotoxic effects were assessed in order to exclude the possibility of a direct effect on BV2 cells; BV2 cell viability was not altered by compound 2 at concentrations of up to 40 μM for 24 h, but was decreased by exposure to compound 2 at a concentration of 80 μM for 24 h (Figure S26). Thus, in subsequent experiments using BV2 cells, the maximum concentration of compound 2 was limited to 40 μM. Compound 2 decreased the prostaglandin E2 (PGE2) production in a dose-dependent manner in LPS-induced BV2 cells, measured by enzyme immunoassay (Figure 3B). Furthermore, protein expression levels of inducible nitric oxide synthesis (iNOS) and cyclooxygenase-2 (COX-2) in the BV2 cells were significantly up-regulated in response to LPS, while compound 2 suppressed the iNOS and COX-2 protein expression in a dose-dependent manner (Figure 3C,D). To further examine the anti-inflammatory effects of compound 2 in the LPS-stimulated BV2 cells, the mRNA expression levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were estimated by RT-qPCR analysis in cells stimulated with LPS (1 μg/mL) for 6 h. The transcript levels of IL-1β, IL-6, and TNFα were decreased in a dose-dependent manner in BV2 cells stimulated with LPS (Figure 4A−C). Dothideopyrones E (1) and F (2) shared structural similarities with known α-pyrone derivatives, dothideopyrones A−D isolated from the endophytic fungus Dothideomycetes sp. LRUB20, which was isolated from a Thai medicinal plant, Leea rubra Blume ex Spreng. (family Leeaceae). Previous reports have demonstrated dothideopyrones A−C were inactive, and dothideopyrone D showed weak activity against cancer cell lines.11 Compound 2 showed moderate anti-inflammatory effects in LPS-stimulated BV2 microglial cell. Activated

4.54, t (5.6) 3.92, s

Recorded at 175 MHz. bRecorded at 700 MHz. cOverlapped signals.

of an aliphatic chain. The planar structure of 1 was elucidated by analyzing the 2D NMR data, including the COSY, HSQC, and HMBC data (Figure 1). The HMBC correlations of OMe-

Figure 1. 1H−1H COSY and HMBC correlations of compounds 1 and 2.

4 to C-4 and C-5, H-1″ to C-2 and C-3, and H-5 to C-3 suggested the presence of a pyrone core structure. A primary alcohol was assigned by HMBC and COSY correlations from OH-1″ to H-1″. The HMBC correlations from OH-1′ to C-2′ and from OH-8′ to C-7′/C-9′ established secondary alcohols on the alkyl chain. In addition, the COSY correlations from OH-1′ to H-2′ and from OH-8′ to H-9′ supported the position of secondary alcohols. The H-2′ to C-6 correlation of HMBC implied that the aliphatic chain was connected to the α-pyrone core. The planar structure of 1 was elucidated as depicted. Compound 2 was obtained as a yellow oil, and its molecular formula established as C16H26O5 by HRESIMS and NMR. The UV maximum absorption and 1D NMR data were very similar to those of 1 (Table 1 and Figure S16, Supporting Information), suggesting the same carbon skeleton. However, the exchangeable signal of OH-8′ (δH 4.28) in 1 was not present in the 1H NMR data of 2. The oxymethine signal of 1 at the C-8′ (δC 66.2) position was replaced by a methylene (δC 22.6) in the 13C NMR spectrum of 2. The dehydroxylation of C-8′ was supported by the shielded signals of C-9′ (δC 14.4) B

DOI: 10.1021/acs.jnatprod.7b01022 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. Effects of 2 on the nitrite content (A), PGE2 production (B), and protein expression levels of iNOS (C) and COX-2 (D) in BV2 stimulated with LPS. The cells were pretreated for 3 h with the indicated concentrations of 2 and stimulated for 24 h with LPS (1 μg/mL). The measurement of the nitrite concentration, PGE2 assay, and Western blot analysis were performed as described in the Experimental Section. *p < 0.05 compared with the group treated with LPS.

Figure 4. Effects of 2 on IL-1β (Il1b) (A), IL-6 (Il6) (B), and TNF-α (Tnf) (C) mRNA expression levels in BV2 cells. Cells were incubated with the indicated concentrations of 2 for 3 h and treated for 6 h with LPS (1 μg/mL). RNA quantification of Il1b, Il6, and Tnf expression was performed as described in the Experimental Section. The data shown represent the mean values of three independent experiments. *p < 0.05 compared with the group treated with LPS. T3 column (Waters, 2.5 μm, 2.1 × 150 mm). Open column chromatography was performed with an ODS (Cosmosil, 75 μm). Analytical C18 (YMC, 5 μm, 4.6 × 150 mm), Cholester packed column (Cosmosil, 5 μm, 10 × 250 mm), and semipreparative C 18 (Optimapak, 10 μm, 10 × 250 mm) columns were used for HPLC on a YL9100 HPLC system equipped with a photodiode array detector (YL9160) that uses HPLC grade solvents (Burdick & Jackson). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco BRL Co. All other chemicals were obtained from Sigma-Aldrich Co. Primary antibodies (COX-2: sc-1745; iNOS: sc-650; Actin: sc-1616, Cell Signaling Technology) and the following secondary antibodies were used: goat, ap106p, and rabbit, ap132p (Millipore). Enzymelinked immunosorbent assay (ELISA) kits for PGE2 were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Fungus Strain. Endolichenic fungus Dothideomycetes sp. EL003334 was obtained from KoLABIC at Sunchon National University, Korea. EL003334 was isolated from the lichen Stereocaulon tomentosum (CL130173) collected from Chile in January 2013 during a field trip in the National Park of Torres Del Paine, Patagonia, organized by Dr. Pereira at Talca University, Talca, Chile. The permit to collect the lichen specimens from the location was issued by the Administration of the National Forestry Corporation of Punta Arenas and by the Administration of the National Park Torres del Paine, Magallanes region and Chilean Antarctic, which is part of the National System of Protected Wild Areas of the State of Chile. Fungus ITS Sequencing. The endolichenic fungi (ELF) were grown and maintained on potato dextrose agar (PDA) medium (BD

microglia cells produce neuroinflammatory factors, including NO, PGE2, and TNF-α, that respond to danger in the central nervous system (CNS).12,13 However, uncontrolled neuroinflammatory factors contribute to neurodegeneration, leading to changes in the CNS, and contribute to diseases such as Alzheimer’s and Parkinson’s disease; the control of neuroinflammation could be a pharmacologic target for neurodegenerative disease.14,15 It is possible that 2 might be a promising therapeutic lead-agent to prevent neurodegenerative diseases.

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EXPERIMENTAL SECTION

General Experimental Procedures. The specific rotations were measured on a JASCO P-1020 polarimeter that uses a 100 mm glass microcell. UV spectra were obtained on an Optizen 2120 UV spectrophotometer. IR spectra were recorded on a Bruker VERTEX 80 V FT-IR spectrometer. The NMR spectra were recorded on Bruker AVANCE HD 700, 800, and 900 NMR spectrometers at the Korea Basic Science Institute (KBSI) in Ochang, South Korea. Chemical shifts were referenced to a residual solvent signal (DMSO-d6 1H δH 2.50, 13C δC 39.51). High-resolution electrospray ionization mass spectrometry (HRESIMS) data were acquired with a Q-TOF mass spectrometer on a SYNAPT G2 (Waters) at KBSI in Ochang, South Korea. Liquid chromatography−mass spectrometry (LC-MS) was performed with a Thermo LTQ XL linear ion trap attached to an ESI source that was connected to a Thermo Scientific Dionex Ultimate 3000 Rapid Separation LC system (ESI-LC-MS) using a Waters HSS C

DOI: 10.1021/acs.jnatprod.7b01022 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Difco, Sparks, MD, USA) at 25 °C. The total DNA of ELF was extracted using a DNeasy plant mini kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS) region of the rDNA gene was amplified with the universal primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) 16 and ITS4A (5′-ATTTGAGCTCTTCCCGCTTCA-3′).17 The PCR reaction mix compositions were standard, and the cycling conditions were as follows: denaturation at 94 °C for 5 min, followed by 30 cycles at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s; after the 30th cycle finished, a final extension step at 72 °C for 10 min was done. The PCR product was concentrated and purified using a PCR quick-spin PCR product purification kit (INTRON Biotechnology, Seongnam, Korea), after which it was sequenced using the same primers. The sequence of EL003334 (NCBI accession no. MF924729) was registered at the NCBI. Extraction and Isolation. The fungus Dothideomycetes sp. EL003334 was grown in potato dextrose broth (PDB, 1 L) for 28 days at 28 °C on a rotary shaker operating at 140 rpm. The culture broth was extracted with an equal volume of acetone followed by filtration and extraction three times with an equal volume of ethyl acetate. The crude extract (140.7 mg) was fractionated by reversedphase C18 vacuum liquid column chromatography (3.5 × 13.5 cm), eluted with a stepwise gradient of MeOH in water (20%, 40%, 60%, 80%, and 100%; v/v) to yield 10 fractions. Fraction 5 (60% MeOH) was purified by HPLC with a Cholester-packed column (Cosmosil, 5 μm, 10 × 250 mm) that used an isocratic condition with MeCN−H2O (2:8, 2 mL/min) to yield compound 1 (7.0 mg). Fraction 7 (80% MeOH) was subjected to HPLC using 42% MeCN isocratic elution with a Cholester-packed column to yield compound 2 (10.4 mg). Dothideopyrone E (1): colorless oil; [α]21D −92.68 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 215 (3.79), 300 (3.93) nm; IR (ATR) νmax 3345, 3259, 2920, 2852, 1678, 1639, 1556, 1467, 1376, 1276 cm−1; 1H and 13C NMR spectroscopic data, Table 1; HRESIMS m/z 337.1612 [M + Na]+ (calcd for C16H26O6Na, 337.1627). Dothideopyrone F (2): yellow oil; [α]21D −118.72 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 215 (3.74), 300 (3.78) nm; IR (ATR) νmax 3310, 2920, 2854, 1672, 1633, 1554, 1461, 1385, 1253 cm−1; 1H and 13 C NMR spectroscopic data, Table 1; HRESIMS m/z 321.1674 [M + Na]+ (calcd for C16H26O5Na, 321.1678). MTPA Esterification. (R)-MTPA-Cl (20 μL) and anhydrous pyridine (16 μL) were added to compound 1 (0.7 mg), which was dissolved in anhydrous CH2Cl2 (500 μL). The mixture was stirred at 800 rpm for 2 h at room temperature. The reaction mixture was partitioned by water (1 mL) with diethyl ether (3 mL). After confirming the successful product formation by LC/MS (ESIMS m/z 985 [M + Na]+), the solvent layer was purified by HPLC (Optimapak, C18, 10 μm, 10 × 250 mm; flow rate 2 mL/min; 85% MeCN) to yield (S)-MTPA-ester 1a (0.3 mg). Treatment of 1 (0.7 mg) with (S)MTPA-Cl (20 μL) as described above yielded the corresponding (R)MTPA ester 1b (0.3 mg). Compound 2 was also reacted with (R)- and (S)-MTPA-Cl to yield the respective Mosher esters. The product formation was confirmed by LC-MS (ESIMS m/z 753 [M + Na]+). The 1H chemical shifts around the stereogenic centers of the MTPA esters were assigned by the 1H NMR and COSY spectra of the derivatives. S-MTPA ester of compound 1 (1a): 1H NMR (DMSO-d6, 900 MHz) δH 7.51−7.42 (15H, m, aromatic), 6.88 (1H, s, H-5), 5.77 (1H, dd, J = 8.4, 5.3 Hz, H-1′), 5.13 (2H, m, H-1″), 5.08 (1H, qd, J = 12.5, 6.3 Hz, H-8′), 3.95 (3H, s, OMe), 3.49 (3H, s, OMe), 3.48 (3H, s, OMe), 3.45 (3H, s, OMe), 1.98 (1H, br s), 1.95 (1H, br s), 1.85 (1H, m), 1.83 (1H, m, H-2′), 1.55−1.41 (4H, m), 1.29 (3H, d, J = 6.3 Hz, H-9′) 1.16−1.02 (8H, m). R-MTPA ester of compound 1 (1b): 1H NMR (DMSO-d6, 800 MHz) δH 7.49−7.38 (15H, m, aromatic), 6.68 (1H, s, H-5), 5.77 (1H, dd, J = 8.4, 5.3 Hz, H-1′), 5.11 (2H, m, H-1″), 5.07 (1H, qd, J = 12.5, 6.3 Hz, H-8), 3.86 (3H, s, OMe), 3.50 (3H, s, OMe), 3.46 (3H, s, OMe), 3.45 (3H, s, OMe), 1.98 (1H, br s), 1.94 (1H, br s), 1.92 (1H, m), 1.89 (1H, m, H-2′), 1.60 (1H, m), 1.55 (1H, m, H-7′), 1.31−1.22 (12H, m), 1.19 (3H, d, J = 6.2 Hz, H-9′).

S-MTPA ester of compound 2 (2a): 1H NMR (DMSO-d6, 800 MHz) δH 7.52−7.41 (10H, m, aromatic), 6.89 (1H, s, H-5), 5.79 (1H, dd, J = 8.4, 5.3 Hz, H-1′), 5.13 (2H, m, H-1″), 3.95 (3H, s, OMe), 3.48 (3H, s, OMe), 3.45 (3H, s, OMe), 1.87 (1H, m), 1.83 (1H, m, H2′) 1.29−1.12 (14H, m), 0.85 (3H, t, J = 7.2 Hz, H-9′). R-MTPA ester of compound 2 (2b): 1H NMR (DMSO-d6, 700 MHz) δH 7.49−7.39 (10H, m, aromatic), 6.69 (1H, s, H-5), 5.77 (1H, dd, J = 7.8, 5.9 Hz, H-1′), 5.11 (2H, m, H-1″), 3.87 (3H, s, OMe), 3.51 (3H, s, OMe), 3.46 (3H, s, OMe), 1.93 (1H, m), 1.89 (1H, m, H2′), 1.37−1.18 (14H, m), 0.85 (3H, t, J = 7.0 Hz, H-9′). Cell Culture and Cytotoxic Assay. BV2 cells were maintained at a density of 5 × 105 cells/mL in DMEM medium supplemented with 10% heat-inactivated FBS, penicillin G (100 units/mL), streptomycin (100 mg/mL), and L-glutamine (2 mM) and were incubated at 37 °C in a humidified atmosphere containing 5% CO2. To determine the cell viability, cells (1 × 105 cells/well in 96-well plates) were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at a final concentration of 0.5 mg/mL for 3 h, and the formazan formed was dissolved in acidic 2-propanol. The optical density was measured at 540 nm with a microplate reader (BioRad, Hercules, CA, USA). The optical density of the formazan formed in control (untreated) cells was considered to represent 100% viability. Determination of Nitrite and PGE2.13 As an indicator of NO production in BV2 cells, production of nitrite, a stable end-product of NO oxidation, was estimated. The concentration of nitrite in the conditioned media was determined by a method based on the Griess reaction.18 An aliquot of each supernatant (100 mL) was mixed with the same volume of Griess reagent [0.1% (w/v) N-(1-naphathyl)ethylenediamine and 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid] for 10 min at room temperature. The absorbance of the final product was measured spectrophotometrically at 540 nm using an ELISA plate reader. The nitrite concentration in the samples was determined from a standard curve of sodium nitrite prepared in phenol red-free DMEM. The concentration of PGE2 in the culture medium was determined using ELISA kits (R&D Systems) according to the manufacturer’s instructions. Quantitative Real-Time Polymerase Chain Reaction (qRTPCR). Total RNA was isolated from BV2 cells using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and quantified spectrophotometrically at 260 nm. Total RNA (1 μg) was reverse-transcribed using the High Capacity RNA-tocDNA kit from Applied Biosystems (Carlsbad, CA, USA). The cDNA was amplified using the SYBR Premix Ex Taq kit from TaKaRa Bio Inc. (Shiga, Japan) and the StepOnePlus Real-Time PCR system from Applied Biosystems. qRT-PCR was performed in a 20 μL total volume of 0.8 μM of each primer, 2.5 μL of cDNA sample, diethyl pyrocarbonate-treated water, and 10 μL of SYBR Green PCR Master Mix. The primer sequences were designed using Primer Quest software from Integrated DNA Technologies (Cambridge, MA, USA). The primer sequences used in this study were provided previously. The optimal conditions for the PCR amplification of the cDNA were established using the manufacturer’s instructions. In addition, the data were analyzed using the StepOne software from Applied Biosystems (Carlsbad, CA, USA). The cycle numbers at the linear amplification threshold (Ct) values for the endogenous control GAPDH and the target gene were recorded. Relative gene expression (target gene expression normalized to the expression of the endogenous control gene) was calculated using the comparative Ct method (2−ΔΔCt). Western Blot Analysis. The proteins of iNOS and COX-2 were measured by Western blot analysis. The details of the procedures for the Western blot analysis were described previously.13 The cells were lysed with 20 mM Tris HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM phenylmethylsulfonyl fluoride, 5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 1 mg/mL chymostatin). The protein concentration was determined using a Lowry protein assay kit (P5626; Sigma-Aldrich, St. Louis, MO, USA). An equal amount of protein for each sample was resolved by performing 7.5% and 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The proteins were electrophoretically transferred onto Hybond enhanced chemiluminescence (ECL) nitrocellulose membranes (Bio-Rad), D

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which were blocked with 5% skim milk and sequentially incubated with particular primary antibodies (Santa Cruz Biotechnology) and horseradish peroxidase-conjugated secondary antibodies, followed by ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Statistical Analysis. Data were presented as the mean ± standard deviation of at least three independent experiments. One-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison tests, was used to compare three or more groups. Statistical analysis was performed using the GraphPad Prism software, version 4.00 (GraphPad Software Inc., San Diego, CA, USA).

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(11) Chomcheon, P.; Wiyakrutta, S.; Sriubolmas, N.; Ngamrojanavanich, N.; Mahidol, C.; Ruchirawat, S.; Kittakoop, P. Phytochemistry 2009, 70, 121−127. (12) Pišlar, A.; Božić, B.; Zidar, N.; Kos, J. Neuropharmacology 2017, 114, 88−100. (13) Ko, W.; Sohn, J. H.; Jang, J. H.; Ahn, J. S.; Kang, D. G.; Lee, H. S.; Kim, J. S.; Kim, Y. C.; Oh, H. Chem. Biol. Interact. 2016, 244, 16− 26. (14) Gao, H. M.; Hong, J. S. Trends Immunol. 2008, 29, 357−365. (15) Ransohoff, R. M. Science 2016, 353, 777−783. (16) Gardes, M.; Bruns, T. D. Mol. Ecol. 1993, 2, 113−118. (17) Innis, M.; Gelfand, D.; Sninsky, J.; White, T. PCR Protocols: a Guide to Methods and Applications; Academic Press: San Diego, 1990; pp 315−321. (18) Titheradge, M. A. Methods Mol. Biol. 1998, 100, 83−91.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b01022. NMR and HRESIMS spectroscopic data of compounds 1 and 2 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(J.-H. Jang) Tel: +82-43-240-6164. Fax: +82-43-240-6169. Email: [email protected]. *(J. S. Ahn) Tel: +82-43-240-6160. Fax: +82-43-240-6169. Email: [email protected]. ORCID

Jae-Hyuk Jang: 0000-0002-4363-4252 Author Contributions ∥

G. S. Kim and W. Ko contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the International Joint Research Project (ASIA-16-011) of the NST (National Research Council of Science & Technology), Young Researcher Program (NRF2017R1C1B2002602) of the NRF (National Research Foundation of Korea), and KRIBB Research Initiative Program funded by the Ministry of Science ICT (MSIT) of the Republic of Korea. We thank the Korea Basic Science Institute, Ochang, Korea, for providing the NMR (700, 800, and 900 MHz) and HRESIMS data.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.7b01022 J. Nat. Prod. XXXX, XXX, XXX−XXX