Bioactive Constituents from the Termite Nest-Derived Medicinal

Jan 5, 2017 - Bioactive Constituents from the Termite Nest-Derived Medicinal Fungus ...... (21) Briefly, cells were cultured in Dulbecco's modified Ea...
0 downloads 0 Views 610KB Size
Download PDF
Article pubs.acs.org/jnp

Bioactive Constituents from the Termite Nest-Derived Medicinal Fungus Xylaria nigripes Jung-Chun Chang,†,# George Hsiao,‡,# Ruo-Kai Lin,† Yueh-Hsiung Kuo,§,⊥ Yu-Min Ju,∥ and Tzong-Huei Lee*,¶ †

Graduate Institute of Pharmacognosy and ‡Graduate Institute of Medical Sciences and Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei, Taiwan 110 § Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung, Taiwan 404 ⊥ Department of Biotechnology, Asia University, Taichung, Taiwan 413 ∥ Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan 115 ¶ Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan 106 S Supporting Information *

ABSTRACT: Six new eremophilane-type sesquiterpenes, namely, nigriterpenes A−F (1−6), and one new phenolic compound, named 2-hydroxymethyl-3-pentylphenol (7), along with fomannoxin alcohol, 3-butyl-7-hydroxyphthalide, scytalone, and fomannoxin were isolated from the ethyl acetate extracts of the fermented broths of termite nest-derived Xylaria nigripes, which has long been used as a traditional Chinese medicine for treating insomnia and depression. Their structures were elucidated on the basis of spectroscopic data analysis and compared with the literature. All the pure isolates were evaluated against lipopolysaccharide-induced inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) expression, and NO production in murine brain microglial BV-2 cells. Of the compounds tested, nigriterpene C (3) and fomannoxin alcohol exerted significant inhibitory effects on two induced enzymes and NO production without any significant cellular toxicity. The most potent compound, 3, exhibited concentration-dependent inhibition on NO production and iNOS and COX-2 expression with IC50 values of 21.7 ± 4.9, 8.1 ± 2.3, and 16.6 ± 5.5 μM, respectively. These results indicated that the potential anti-inflammatory effects of nigriterpene C (3) and fomannoxin alcohol on murine brain microglial BV-2 cells may provide a rationale for the traditional medical uses of X. nigripes for treating insomnia and depression.

T

antidepressant.9 A commercial fermented mycelium capsule product of X. nigripes has been used clinically for improving sleep and cognitive disorders in mainland China thus far.10 Recent pharmacological studies also revealed that crude extracts of X. nigripes can prevent chemical hepatic injury11 and exert hypoglycemic, insulin-sensitivity enhancing,12 and cardiovascular protective effects.13 In addition, the ethyl acetate extracts of the fermented broths of X. nigripes YMJ653 exhibited significant cytotoxicity against adenocarcinomic human alveolar basal epithelial A549 cells with a GI50 of 23.7 μg/mL in our preliminary biological evaluation, indicating cytotoxic constituents in X. nigripes. All the evidence suggests that X. nigripesYMJ653 is a potential source for exploring medicinally useful compounds. That prompted us to investigate the active principles of this fungal strain, which led to the isolation and characterization of seven new compounds, 1−7 (Figure 1), and four known compounds. This report herein

he best-known fungi that macrotermitine termites cultivate are the mushroom species of the genus Termitomyces R. Heim, fruiting bodies of which emerge directly from underground fungus combs or fungus gardens built in the chambers of termite colonies.1 In Taiwan, the only macrotermitine termite, Odontotermes formosanus, cultivates basidiomycetous fungi of the Termitomyces species T. eurrhizus (Berk.) R. Heim and forms a mutualistically symbiotic association in the underground colonies.2,3 Besides Termitomyces, a fungus comb also contains many other microbes, especially Xylaria spp. (Xylariaceae),2,4 whose growth and metabolic activity seemed to play crucial roles in modifying the metabolism and development of Termitomyces. In particular, it was found that T. eurrhizus and Xylaria spp. were the dominant fungal species during the period before and after the termites abandoned their nest, respectively.5 The fruiting bodies of X. nigripes, also known as Wu-LingShen, have long been used in traditional Chinese medicine for enhancing memory,6 immunity, and hematopoiesis,7 treating insomnia and trauma, and as a diuretic, nerve tonic,8 and © 2017 American Chemical Society and American Society of Pharmacognosy

Received: March 19, 2016 Published: January 5, 2017 38

DOI: 10.1021/acs.jnatprod.6b00249 J. Nat. Prod. 2017, 80, 38−44

Journal of Natural Products

Article

Figure 1. Chemical structures of compounds 1−7 identified in this report.

H3-15, H3-14/H-6, and H-6/H3-13 and molecular modeling (Figure 2). For the determination of the absolute configuration the pure compound 1 was reacted with (S)-(+)- and (R)(−)-MTPACl to afford the (R)- and (S)-MTPA esters (1a and 1b), respectively. The resolving 1H chemical shift differences of 1a and 1b were shown in Figure 3, and the Δδ values of H3-13, -14, and -15 were +0.004, +0.020, and +0.080, respectively, while those of H-8 and H-9 were −0.046 and −0.026, respectively. Further supported by the molecular modelingaided (ChemBio3D Ultra 12.0, ChemBioOffice, PerkinElmer) interpretation, the configurations of C-4, -5, -6, and -8 were unambiguously assigned to be S, R, R, and R, respectively. Thus, the structure of 1 was deduced as shown in Figure 1 and was named nigriterpene A. The molecular formula for 2, C15H18O3, determined by 13C NMR and HRESIMS data was 2 Da less than that of 1. Its IR absorption bands at 3419, 1739, and 1633 cm−1 indicated the presence of a hydroxy, a conjugated γ-lactone carbonyl, and an olefinic group, respectively. When the 1H and 13C NMR data of 2 were compared with those of 1, the marked downfield shifts of H-1, H-2, C-1, and C-2 demonstrated there was a structural difference (Table 1). These assignments coupled with the interpretations of COSY and HMBC spectra of 2 indicated that two methylenes, H2-1 and H2-2, in 1 were changed to an unsaturated double bond [(δH 5.98, dd, J = 2.6, 9.8 Hz, H-1; δC 128.2, C-1) and (δH 5.81, m, H-2; δC 130.6, C-2)] in 2. Thus, the structure of 2 was determined to be as shown in Figure 1 and was named nigriterpene B. Compound 3 was obtained as a light yellow oil, and the quasi-molecular ion [M + H]+ at m/z 235.1705 in the HRESIMS established its molecular formula to be C15H22O2. The absorption bands at 3392 and 1662 cm−1 in the IR spectrum of 3 indicated the presence of a hydroxy and an olefinic moiety, respectively. The 1H NMR data of 3 were in accordance with those of 1 except that the methylene group at δH 1.48 and 1.96 (H2-6) in 3 replaced the carbinol in 1, the exomethylene moiety at δH 5.18 and 5.22 (H2-13) in 3 substituted the methyl in 1, and an additional oxymethylene at δH 4.14 and 4.32 (H2-12) instead of a carbonyl group was observed (Table 1). All the assignments were further supported by key cross-peaks of δH 1.14 (H3-14)/δC 47.5 (C-6) and δC 147.8 (C-10), δH 1.84, 1.96 (H2-6)/δC 147.8 (C-10), and δH 5.18, 5.22 (H2-13)/C-7 (δC 75.3) and C-12 (δC 65.2) in the HMBC spectrum of 3 (Figure 4). The configurations of OH-7, H-8, H3-14, and H3-15 of 3 were deduced to be β-, α-, β-, and β-oriented on the basis of the key NOESY correlations of Hpseudoequatorial-6 (δH 1.96)/H3-14 and H3-15, Hpseudoaxial-6 (δH 1.48)/H-8 and Ha-13 (δH 5.18), and H-8/Ha-13 (δH 5.18) by

described the isolation and characterization of previously unreported compounds together with their bioactivities.

■

RESULTS AND DISCUSSION From the ethyl acetate extracts of the fermented broths of X. nigripes YMJ653, 11 compounds including seven new compounds (1−7) and four known compounds, fomannoxin alcohol, fomannoxin, 3-butyl-7-hydroxyphthalide, and scytalone, were isolated and purified by sequential Sephadex LH20 open-column and reversed-phase HPLC. Of these, fomannoxin alcohol was afforded as a light yellow oil whose spectroscopic data were consistent with those of the previously reported compound isolated from a wood-rotting fungus, Fomes annosus.14 3-Butyl-7-hydroxyphthalide and scytalone were deduced to be compatible with published data.15,16 1H and 13 C NMR, IR, optical rotation, and MS of fomannoxin coincided well with those reported.14,17 Compound 1 was obtained as a colorless crystal with a molecular formula of C15H20O3, which was determined by negative mode HRESIMS and supported by 13C NMR assignments. Its IR spectrum exhibited absorption bands at 3448, 1731, and 1645 cm−1, indicating the presence of a hydroxy, a conjugated γ-lactone carbonyl, and an olefinic functionality, respectively. The 1H NMR spectrum shows distinctive resonances for a three-proton doublet at δH 0.93 (J = 6.8 Hz, H3-15), a three-proton singlet at δH 0.82 (H3-14), and a three-proton singlet at δH 1.84 (H3-13) attached to an olefinic carbon (Table 1). The 13C NMR in combination with the HSQC spectrum of 1 showed 15 carbon signals for three methyls at δC 9.0 (C-13), 15.2 (C-15), and 17.3 (C-14), three methylenes at δC 26.5 (C-2), 30.4 (C-3), and 32.8 (C-1), four methines at δC 34.4 (C-4), 69.8 (C-6), 77.2 (C-8), and 116.6 (C-9), and five other carbons at δC 48.5 (C-5), 121.5 (C-11), 147.2 (C-10), 160.3 (C-7), and 174.4 (C-12) (Table 1). The NMR data suggested 1 had a similar structural feature to that of arecolactone, an eremophilane-type sesquiterpene, isolated previously from Arecophila saccharicola,18 except that the exomethylene H2-13 in arecolactone was substituted by a methyl group at δH 1.84 (H3-13) and different positions of the carbinol and double bonds. The locations of the carbinol and two double bonds were further corroborated to be at C-6, Δ7(11), and Δ9, respectively, by key COSY cross-peaks of δH 5.42 (H-8)/δH 5.53 (H-9) and key HMBC cross-peaks of δH 0.82 (H3-14)/δC 69.8 (C-6), 147.2 (C-10), δH 4.60 (H-6)/ δC147.2 (C-10), and δH 1.84 (H3-13)/δC 160.3 (C-7) and 174.4 (C-12) (Figure 2). The relative configurations of OH-6, H-8, H3-14, and H3-15 of 1 were deduced to be α-, α-, β-, and β-oriented on the basis of the NOESY correlations of H3-14/ 39

DOI: 10.1021/acs.jnatprod.6b00249 J. Nat. Prod. 2017, 80, 38−44

40

26.5 t

30.4 t

34.4 d 48.5 s 69.8 d

160.3 s 77.2 d

116.6 d

147.2 s

121.5 s 174.4 s

9.0 q

17.3 q 15.2 q

2

3

4 5 6

7 8

9

10

11 12

13

14 15 1′ 2′ 3′ 4′ 5′ 1″

H

1

m m m m m

0.82 s 0.93 d (6.8)

1.84 d (1.6)

5.53 d (1.6)

5.42 d (1.6)

4.60 br s

1.36 1.74 1.36 1.59 2.11

2.14 m

1

16.9 q 14.3 q

9.1 q

122.6 s 174.1 s

143.6 s

118.6 d

159.5 s 77.8 d

31.4 d 46.1 s 69.6 d

32.2 t

130.6 d

128.2 d

C

13 a

H

1

0.75 s 1.01 d (6.5)

1.88 d (1.7)

5.63 d (2.6)

5.50 s

4.68 s

1.93 m 2.20 m 2.21 m

5.81 m

5.98 dd (2.6, 9.8)

2

19.1 q 15.5 q

114.4 t

153.4 s 65.2 t

147.8 s

119.2 d

75.3 s 72.1 d

45.5 d 38.7 s 47.5 t

30.7 t

28.1 t

32.3 t

C

13 a

m m m m m

H

1

4.14 4.32 5.18 5.22 1.14 0.80

d (12.4) d (12.4) br s br s s d (6.8)

5.16 s

4.29 s

1.48 d (14.4) 1.96 d (14.4)

1.25 m

2.04 2.18 1.30 1.77 1.40

3

Multiplicities were determined by phase-sensitive HSQC and 1H NMR experiments.

32.8 t

1

a

C

no.

13 a

17.2 q 16.3 q

117.8 t

138.6 s 65.3 t

169.5 s

124.1 d

146.9 s 186.8 s

41.8 d 44.1 s 154.2 d

30.2 t

28.0 t

33.0 t

C

13 a

H

1

5.22 5.28 1.15 1.07

d (1.7) d (1.7) s d (6.1)

4.19 s

6.09 s

7.02 s

1.52 m

1.39 m 1.99 m 1.56 m

2.38 m

4

Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1−7 [δ in ppm, mult (J in Hz)]

17.4 q 15.2 q

134.6 t

152.5 s 195.4 d

146.8 s

123.2 d

37.9 d 71.8 d

44.0 d 39.1 s 41.9 t

30.8 t

27.5 t

32.1 t

C

13 a

t (12.9) dd (2.5, 12.9) ddd (2.5, 9.7, 12.9) d (9.7)

m m m m m m m

br s br s s d (6.8)

9.59 s 6.12 6.35 1.02 0.78

H

1

5.29 t (1.7)

1.36 1.63 2.73 4.09

2.02 2.13 1.28 1.74 1.35 1.43 1.24

5

15.9 q 15.1 q

135.9 t

148.5 s 193.5 d

170.4 s

123.7 d

42.5 d 197.2 s

43.5 d 39.8 s 41.7 t

30.4 t

26.3 t

33.0 t

C

13 a

m m m m m m m

H

1

6.20 6.31 1.17 0.86

br s br s s d (6.3)

9.55 s

5.76 d (1.7)

1.89 t (13.5) 1.95 dd (5.0, 13.5) 3.63 dd (5.0, 13.5)

2.27 2.32 1.45 1.85 1.45 1.53 1.45

6

33.2 31.4 31.7 22.5 14.0 60.0

t t t t q t

121.5 d 128.8 d 114.4 d

141.2 s

122.7 s

156.6 s

C

13 a

H

1

2.54 1.48 1.30 1.31 0.87 4.90

t (7.9) m m m t (7.0) s

6.68 d (7.9) 7.07 t (7.9) 6.71 d (7.9)

7

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.6b00249 J. Nat. Prod. 2017, 80, 38−44

Journal of Natural Products

Article

and 13C NMR of 5 (Table 1). Upon further interpretion of the COSY and HMBC spectra of 5, key cross-peaks of H2-6/H-7, H-7/H-8, H2-6/C-4, -5, -8, -10, and -14, H-12/C-7 and -11, and H2-13/C-7, -11, and -12 were observed (Figure 5). Examination

Figure 2. Key correlation spectroscopy (COSY), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser effect spectroscopy (NOESY) of compound 1.

Figure 5. Key COSY, HMBC, and NOESY of compound 5.

of the NOESY spectrum of 5 suggested the orientations of H-7 and H-8 to be β- and α-oriented, respectively, as deduced from the key cross-peaks of H-7/H3-14 and H-8/Hb-13 (δH 6.35) (Figure 5) and the large coupling constant (J = 9.7 Hz) of mutually coupled Hquasi‑axial-7 and Hquasi‑axial-8. Therefore, 5 was concluded to possess the structure shown in Figure 1 and was named nigriterpene E. The spectroscopic data of 5 and 6 were similar and showed that the carbinol at C-8 in 5 was oxidized to be a carbonyl group in 6, this being confirmed by a conspicuous ketone signal at δC 197.2 (C-8) together with a pseudomolecular ion [M + Na]+ at m/z 255.1352 for 6, 2 Da less than that of 5, in the HRESIMS. The spectral data were fully assigned, and the structure of 6 was deduced to be as shown in Figure 1; it was named nigriterpene F. Compound 7, isolated as a yellow oil, was assigned a molecular formula of C12H18O2 by HRESIMS. Analysis of the IR spectrum of 7 suggested that it contained a benzene ring (1589 and 1467 cm−1) bearing a hydroxy functionality (3306 cm−1). In the 1H NMR data of 7, three mutually coupled aromatic proton signals at δH 6.68 (d, J = 7.9 Hz, H-4), 6.71 (d, J = 7.9 Hz, H-6), and 7.07 (t, J = 7.9 Hz, H-5) indicating a 1,2,3-trisubtituted aromatic ring in 7 were present. Upon further analysis of its two-dimensional NMR data, including key cross-peaks of H-4/H-5, H-5/H-6, H2-1′/H2-2′, H2-2′/H2-3′, H2-3′/H2-4′, and H2-4′/H2-5′ in the COSY spectrum and H21′/C-2 and C-4, H2-1″/C-1, -2, and -3, and H-5/C-1 and -3 in the HMBC spectrum (Figure 6), the locations of all the

Figure 3. Chemical shift differences between the (R)-MTPA and (S)MTPA esters of 1.

Figure 4. Key HMBC and NOESY of compound 3.

the aid of molecular modeling (Figure 4). Accordingly, 3 was assigned to be as shown in Figure 1 and was named nigriterpene C. The molecular formula of 4, C15H20O2, deduced by HRESIMS of a sodium adduct, indicated the double-bond equivalence was six including three double bonds at δC 117.8 (C-13), 124.1 (C-9), 138.6 (C-11), 146.9 (C-7), 154.2 (C-6), and 169.5 (C-10) as well as one conjugated ketone at δC 186.8 (C-8). Thus, there would be only two rings remaining in 4. The IR absorption at 1659 cm−1 coupled with 13C NMR at δC 186.8 (C-8) revealed the presence of a set of two cross-conjugated double bonds. Further comparison of the 13C NMR data of 4 with those of 3 indicated that the C-5−C-10 in 3 would covert to an α,β-unsaturated cyclohexanone in 4, and the furan ring in 3 passed through an oxidative cleavage to give a primary alcohol (δH 4.19, H2-12; δC 65.3, C-12) in 4 (Table 1). In the HMBC spectrum of 4, key cross-peaks of δH 7.02 (H-6)/δC 41.8 (C-4), 169.5 (C-10), and 186.8 (C-8) also confirmed the assignments. 4 was determined to be as shown in Figure 1 and was named nigriterpene D. Compound 5 had the same molecular formula as that of 4, as judged by HRESMIS of the sodium adduct. The IR absorption bands at 3401 and 1693 cm−1 were attributed to the presence of a hydroxy and a conjugated aldehyde carbonyl, respectively, which corresponded to signals of δH 4.09 (d, J = 9.7 Hz, H-8); δC 71.8 (C-8) and δH 9.59 (s, H-12); δC 195.4 (C-12) in the 1H

Figure 6. Key COSY and HMBC of compound 7.

functionalities borne by the benzene ring were thus determined. The structure of 7 was identified to be as shown in Figure 1 and was named 2-hydroxymethyl-3-phentylphenol. Neuroinflammation is a precise pathological process in neurodegenerative diseases.19 Among the neuroinflammatory cascades, dysregulation of microglial nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression is an important factor in causing neuronal injuries.20 As X. nigripes 41

DOI: 10.1021/acs.jnatprod.6b00249 J. Nat. Prod. 2017, 80, 38−44

Journal of Natural Products

Article

Taken together, these data show that 3 and FA may be promising candidates to develop antineuroinflammatory drugs.

shows CNS effects, the activity of the isolated metabolites on lipopolysaccharide (LPS)-induced iNOS and COX-2 expression on murine brain microglial BV-2 cells were evaluated. It was found that compound 3 and fomannoxin alcohol (FA) exhibited more potent inhibitory effects than other isolated compounds on LPS-induced iNOS and COX-2 expression (Table 2). The cellular 24 h MTT assay showed that 1, 3, and

■

General Experimental Procedures. Optical rotations were measured on a JASCO P-2000 polarimeter (Tokyo, Japan). 1H and 13 C NMR were acquired on a Bruker Avance DRX-500 and AVIII-500 spectrometer (Ettlingen, Germany). Low- and high-resolution mass spectra were obtained using an API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA) and Synapt high-definition mass spectrometry system with an ESI interface and a TOF analyzer (Waters Corp., Manchester, UK), respectively. IR spectra were recorded on a JASCO FT/IR 4100 spectrometer (Tokyo, Japan). UV spectra were measured on a Thermo UV−Vis Heλios α spectrophotometer (Thermo Scientific, Waltham, MA, USA). Sephadex LH-20 (GE Healthcare Life Sciences, Pittsburgh, PA, USA) was used for open-column chromatography. TLC was performed using silica gel 60 F254 plates (200 μm, Merck). A reflective index detector (Bischoff, Leonberg, Germany) was used for HPLC purification. Fermentation of Xylaria nigripes YMJ653. X. nigripes YMJ653 was isolated and identified by one of us (Y.M.J.) and was deposited with the accession number BCRC34219 at the Bioresource Collection and Research Center, Hsin-chu, Taiwan. ITS sequences of nuclear rDNA of this fungal strain were submitted to GenBank, and the accession number was EU179868. The mycelium of this strain was inoculated into 5 L serum bottles, each containing 60 g of Bacto malt extract (Becton, Dickinson and Company, Sparks, MD, USA) and 3 L of deionized water. The fermentation was conducted with aeration at 25−30 °C for 30 days. Extraction and Isolation. The filtered fermented broth (30 L) of X. nigripes YMJ653 was partitioned two times with equal volumes of ethyl acetate, then concentrated in vacuum to dryness (4.0 g). The crude extract was redissolved in 20 mL of MeOH, then applied onto a Sephadex LH-20 column (2.5 cm i.d. × 66.0 cm) and eluted by MeOH with a flow rate of 1.8 mL/min. Each fraction (24 mL) collected was checked for its compositions by TLC using n-hexane−ethyl acetate (1:1, v/v) for development, and dipping in vanillin−sulfuric acid was used to detect compounds with similar chromophores. All the fractions were combined into eight portions (I−VIII). Portion III (fr. 11) was further separated by HPLC on a semipreparative Phenomenex Luna PFP column (5 μm, 10 × 250 mm, Torrance, CA, USA) eluted by 35% MeCN, 2 mL/min, to afford 1 (215.2 mg, tR = 23.80 min) and 2 (87.8 mg, tR = 21.80 min). The same fraction was chromatographed by HPLC on the same column with 40% MeCN to give 3 (32.2 mg, tR = 18.50 min), 5 (14.7 mg, tR = 47.49 min), 6 (9.3 mg, tR = 50.49 min), fomannoxin alcohol (114.2 mg, tR = 24.82 min), and 3-butyl-7hydroxyphthalide (50.8 mg, tR = 36.17 min). Portion II (fr. 10) was purified by HPLC on the same column eluted by 60% MeOH, 2 mL/ min, to obtain 4 (14.9 mg, tR = 30.56 min) and 7 (27.4 mg, tR = 35.44 min). Portion IV (fr. 12) was chromatographed by HPLC on a semipreparative Thermo Hypersil-Keyston BDS Hypersil C18 column (5 μm, 10 × 250 mm, Bellefonte, PA, USA) eluted with 45% MeCN to give fomannoxin (112.6 mg, tR = 28.53 min). Portion VII (fr. 16 and 17) was purified by HPLC on the same C18 column with 20% MeCN as eluent, 2 mL/min, to afford scytalone (2.7 mg, tR = 12.68 min). Nigriterpene A (1): colorless crystal; [α]26D −125.9 (c 0.64, MeOH); IR (KBr) νmax 3448, 2947, 2853, 1731, 1691, 1645, 1460, 1439, 1382, 1306, 1180, 1102, 1053, 1027, 947, 924, 867 cm−1; UV λmax (log ε, MeCN) 202 (4.3), 223 (4.2); 1H and 13C NMR data, see Table 1; ESIMS [M − H]− m/z 247; HREIMS [M − H]− m/z 247.1324 (calcd for C15H19O3, 247.1334). Nigriterpene B (2): colorless crystal; [α]26D −130.5 (c 0.61, MeOH); IR (KBr) νmax 3419, 2972, 2853, 1739, 1633, 1383, 1301, 1030, 980, 923, 641 cm−1; UV λmax (log ε, MeOH) 229 (4.4); 1H and 13 C NMR data, see Table 1; ESIMS [M − H]− m/z 245; HREIMS [M − H]− m/z 245.1173 (calcd for C15H17O3, 245.1178). Nigriterpene C (3): light yellow oil; [α]26D −4.1 (c 0.55, MeOH); IR (KBr) νmax 3392, 2929, 2855, 1662, 1461, 1439, 1381, 1258, 1124,

Table 2. Inhibitory Effects of Compounds 1−7, Fomannoxin Alcohol, 3-Butyl-7-hydroxyphthalide, Scytalone, and Fomannoxin on LPS-Induced iNOS and COX-2 Expression in Microglial BV-2 Cells (folds)a,b iNOS resting vehicle (+LPS) compound (20 μM)

1 2 3 4 7

fomannoxin alcohol 3-butyl-7-hydroxyphthalide scytalone fomannoxin

1.0 5.7 3.2 4.5 1.8 3.6 3.4 1.9 4.3 2.9 3.0

± ± ± ± ± ± ± ± ± ± ±

COX-2

0.0 1.0c 0.6 0.5 0.3e 1.1 1.2 1.2e 1.9 1.2 1.5

1.0 6.8 6.4 4.9 2.1 3.7 5.6 2.4 6.5 4.9 4.2

± ± ± ± ± ± ± ± ± ± ±

0.0 0.5c 3.2 0.6 0.3d 0.7 2.0 0.5d 2.1 3.0 1.5

Values represent mean ± SD based on three individual experiments. Compounds 5 and 6, giving strong cytotoxicity and no available proteins for WB, were not listed. cp < 0.01. dp < 0.05. ep < 0.01.

a b

FA had no significant effect on the viability of BV-2 cells, while 5 and 6 exerted more significant inhibitory effects on the cellular viability. These results indicated 5 and 6 exhibited potent cytotoxicity (Table 3). As shown in Figure S15, Table 3. Effects of Compounds 1−6 and Fomannoxin Alcohol on Microglial BV-2 Viabilitya compound (20 μM)

viability (%)

1 2 3 4 5 6 fomannoxin alcohol

90.2 79.3 87.7 73.8 44.6 46.1 85.9

± ± ± ± ± ± ±

EXPERIMENTAL SECTION

6.9 4.9b 5.2 4.9b 4.0c 7.1c 7.9

Values are mean ± SD of three individual experiments. bp < 0.01. cp < 0.001.

a

compound 3 significantly abrogated microglial iNOS and COX-2 expression in a concentration-dependent manner (2, 5, 10, and 20 μM), and the IC50 values were calculated to be 8.1 ± 2.3 and 16.6 ± 5.5 μM, respectively. FA markedly inhibited iNOS and COX-2 expression in a concentration-dependent manner (2, 5, 10, and 20 μM) (Figure S16), and the IC50 values of iNOS and COX-2 were found to be 11.6 ± 5.9 and 12.7 ± 5.1 μM, respectively. Consistently, compound 3 significantly attenuated LPS-induced microglial NO production in a concentration-dependent manner (2, 10, 20, and 40 μM), and its IC50 was calculated to be 21.7 ± 4.9 μM (Figure S17A). The IC50 of FA on microglial NO production was calculated to be 33.8 ± 6.4 μM (Figure S17B). The positive control curcumin (Sigma, lot no. SLBH2403 V, purity ≥65%) exerted NO production inhibitory activity with an IC50 of 4.5 ± 1.5 μM. 42

DOI: 10.1021/acs.jnatprod.6b00249 J. Nat. Prod. 2017, 80, 38−44

Journal of Natural Products

■

1061, 1033, 914, 869 cm−1; 1H and 13C NMR data, see Table 1; ESIMS [M + H]+ m/z 235; HREIMS [M + H]+ m/z 235.1705 (calcd for C15H23O2, 235.1698). Nigriterpene D (4): yellow oil; [α]27D −27.2 (c 0.50, MeOH); IR (KBr) νmax 3401, 2935, 2860, 1659, 1615, 1463, 1441, 1382, 1205, 1028, 913 cm−1; UV λmax (log ε, MeOH) 247 (4.4); 1H and 13C NMR data, see Table 1; ESIMS [M + Na]+ m/z 255; HREIMS [M + H]+ m/ z 255.1367 (calcd for C15H20O2Na, 255.1361). Nigriterpene E (5): light yellow oil; [α]24D −42.6 (c 0.65, MeOH); IR (KBr) νmax 3401, 2930, 2856, 1693, 1621, 1441, 1371, 1076, 1033, 968 cm−1; 1H and 13C NMR data, see Table 1; ESIMS [M + Na]+ m/z 257; HREIMS [M + Na]+ m/z 257.1521 (calcd for C15H22O2Na, 257.1517). Nigriterpene F (6): light yellow oil; [α]24D −55.0 (c 0.63, MeOH); IR (KBr) νmax 3435, 2933, 2860, 1694, 1674, 1621, 1442, 1373, 1318, 1238, 1197 cm−1; UV λmax (log ε, MeOH) 241 (4.0); 1H and 13C NMR data, see Table 1; ESIMS [M + Na]+ m/z 255; HREIMS [M + Na]+ m/z 255.1353 (calcd for C15H20O2Na, 255.1361). 2-Hydroxymethyl-3-pentylphenol (7): light yellow oil; IR (KBr) νmax 3307, 2957, 2931, 2859, 1589, 1467,1378, 1270, 1188, 1107, 994, 752 cm−1; UV λmax (log ε, MeOH) 284 (3.5); 1H and 13C NMR data, see Table 1; ESIMS [M − H]− m/z 193; HREIMS [M − H]− m/z 193.1231 (calcd for C12H17O2, 193.1229). Cell Culture. The cell culture of the mouse BV-2 microglia cell line was performed as described in our previous report.21 Briefly, cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with L-glutamine, penicillin, streptomycin, HEPES, NaHCO3, and 10% heat-inactivated fetal bovine serum (FBS) at 37 °C under 5% CO2 with humidified air. Before experiments, a confluence of 85% of cells was changed to lower serum (0.5% FBS) media. Thereafter, cells were treated with vehicle, indicated compounds, or their different concentrations for 15 min and then stimulated with LPS (150 ng/mL) for 24 h. Cellular Viability. The viability of BV-2 cells with 24 h treatment of the isolated compound was measured by a colorimetric assay of MTT reduction as described previously.22 Western Blot Analysis. Western blot analysis was performed as previously described.23 Briefly, BV-2 cells were cultured as 80% confluence and changed to serum-free medium for 24 h. Thereafter, cells were treated with DMSO or the isolated compound, then stimulated with LPS (150 ng/mL) for 24 h. The quantitative supernatants from cellular lysates were subjected to SDS-PAGE and electrophoretically transferred onto a polyvinylidene fluoride membrane. After soak in the blocking buffer with 5% dry skim milk overnight, membranes were washed three times and sequentially incubated with primary antibodies (anti-COX-2 and -iNOS) and HRPconjugated secondary antibodies, followed by enhanced chemiluminescence detection. Data of specific protein levels are presented as the relative multiples in relation to the control groups. Measurement of Nitric Oxide Production. Production of nitric oxide (NO) was evaluated by measuring the levels of nitrite in culture medium as previously described with some modification.24 Briefly, BV2 cells were seeded in 24-well plates at 5 × 105 cells per well for 16 h. Then, the medium was changed to fresh DMEM without phenol red. The cells were pretreated with vehicle or test compounds for 30 min and stimulated with LPS (150 ng/mL) for 24 h. The culture supernatants were reacted with reconstituted cofactors solution and reconstituted nitrate reductase solution for 1 h at room temperature in the dark according to the instructions of the nitrate/nitrite colorimetric assay kit (Cayman, MI, USA). Griess reagents A and B were sequentially added and incubated for 20 min. Absorptions were measured at 550 nm using a microplate reader (MRX). Nitrite concentrations were calculated by regression from standard solutions of sodium nitrite.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00249. 1 H and 13C NMR spectra of the new compounds 1−7 (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Tel: 886-2-33661828. E-mail: [email protected]. ORCID

Tzong-Huei Lee: 0000-0001-8036-7563 Author Contributions #

J.-C. Chang and G. Hsiao contributed equally to this article.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by a grant from the Ministry of Science and Technology of the Republic of China. We thank Ms. S.-H. Wang of the Instrumentation Center of Taipei Medical University and Ms. S.-L. Huang of the Instrumentation Center of the College of Science, National Taiwan University, for the NMR data acquisition and Ms. Y.-C. Wu of the Small Molecule Metabolomics Core Facility, the Institute of Plant and Microbial Biology and Academia Sinica Scientific Instrument Center, Academia Sinica, for the MS data acquisition.

■

REFERENCES

(1) Mueller, U. G.; Gerardo, N. M.; Aanen, D. K. Annu. Rev. Ecol. Evol. Syst. 2005, 36, 563−595. (2) Ju, Y. M.; Hsieh, H. M. Mycologia 2007, 99, 936−957. (3) Mathew, G. M.; Ju, Y. M.; Lai, C. Y.; Mathew, D. C.; Huang, C. C. FEMS Microbiol. Ecol. 2012, 79, 504−517. (4) Visser, A. A.; Ros, V. I.; De Beer, Z. W.; Debets, A. J.; Hartog, E.; Kuyper, T. W.; Laessøe, T.; Slippers, B.; Anaen, D. K. Mol. Ecol. 2009, 18, 553−567. (5) Qian, Q.; Li, S.; Wen, H. Mycosystema 2011, 30, 12−17. (6) Liang, W. L.; Hsiao, C. J.; Ju, Y. M.; Lee, L. H.; Lee, T. H. Chem. Biodiversity 2011, 8, 2285−2290. (7) Ko, H. J.; Song, A.; Lai, M. N.; Ng, L. T. J. Ethnopharmacol. 2011, 138, 762−768. (8) Ma, Z. Z.; Zuo, P. P.; Chen, W. R.; Tang, L. J.; Shen, Q.; Sun, X. H.; Shen, Y. Chin. Pharma. J. 1999, 34, 374−377. (9) Zhao, Z.; Li, Y.; Huang, L.; Zhao, F.; Yu, Q.; Xiang, Z.; Zhao, Z.; Chen, H. Int. J. Clin. Exp. Med. 2014, 7, 356−362. (10) Li, Y.; Wang, X.-Y.; Ye, R.; Hu, W.-H.; Sun, S.-C.; Jiao, H.-J.; Song, X.-H.; Yuan, Z.-Z.; Zheng, G.-Q.; He, J.-C. J. Ethnopharmacol. 2013, 145, 320−327. (11) Song, A.; Ko, H.-J.; Lai, M.-N.; Ng, L.-T. Immunopharmacol. Immunotoxicol. 2011, 33, 454−460. (12) Chen, Y.-I.; Tzeng, C.-Y.; Cheng, Y.-W.; Hsu, T.-H.; Ho, W.-J.; Liang, Z.-C.; Hsieh, C.-W.; Tzeng, T.-C.; Chang, S.-L. Phytother. Res. 2015, 29, 770−776. (13) Bai, Y.; Wang, G.; Wang, H.; Chin. J. Mod. Appl. Pharm. 2014, 31, 671−674. (14) Hirotani, M.; O’Reilly, J.; Doeenlly, D. M. X. Tetrahedron Lett. 1977, 7, 651−652. (15) Makino, M.; Endoh, T.; Ogawa, Y.; Watanabe, K.; Fujimoto, Y. Heterocycles 1998, 48, 1931−1934. (16) Fabrice, V.; Michel, G. Tetrahedron 1990, 46, 2827−2834. (17) Hansson, D.; Menkis, A.; Olson, K.; Stenlid, J.; Broberg, A.; Karlsson, M. Phytochemistry 2012, 84, 31−39.

43

DOI: 10.1021/acs.jnatprod.6b00249 J. Nat. Prod. 2017, 80, 38−44

Journal of Natural Products

Article

(18) Lee, L. W.; Wang, G. J.; Lin, M. H.; Ju, Y. M.; Lin, Y. W.; Chen, F. Y.; Lee, T. H. Phytochemistry 2013, 85, 129−136. (19) Rojo, L. E.; Fernandez, J. A.; Maccioni, A. A.; Jimenez, J. M.; Maccioni, R. B. Arch. Med. Res. 2008, 39, 1−16. (20) Okorji, U. P.; Velagapudi, R.; El-Bakoush, A.; Fiebich, B. L.; Olajide, O. A. Mol. Neurobiol. 2016, 53, 6426−6443. (21) Jung, K. K.; Lee, H. S.; Cho, J. Y.; Shin, W. C.; Rhee, M. H.; Kim, T. G.; Kang, J. H.; Kim, S. H.; Kang, S. Y. Life Sci. 2006, 79, 2022−2031. (22) Wang, C. H.; Hsiao, C. J.; Lin, Y. N.; Wu, J. W.; Kuo, Y. C.; Lee, C. K.; Hsiao, G. Pharm. Biol. 2014, 52, 1451−1459. (23) Chou, Y. C.; Sheu, J. R.; Chung, C. L.; Chen, C. Y.; Lin, F. L.; Hsu, M. J.; Kuo, Y. H.; Hsiao, G. Chem.-Biol. Interact. 2010, 184, 403− 412. (24) Hsiao, G.; Fong, T. H.; Tzu, N. H.; Lin, K. H.; Chou, D. S.; Sheu, J. R. In Vivo 2004, 18, 351−356.

44

DOI: 10.1021/acs.jnatprod.6b00249 J. Nat. Prod. 2017, 80, 38−44