New Apoptosis-Inducing Sesquiterpenoids from the Mycelial Culture

Dec 23, 2014 - Yongbiao Zheng†, Haiyue Pang†, Jifeng Wang‡, Guowei Shi‡, and Jianzhong Huang† .... Higher Fungi. He-Ping Chen , Ji-Kai Liu. ...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/JAFC

New Apoptosis-Inducing Sesquiterpenoids from the Mycelial Culture of Chinese Edible Fungus Pleurotus cystidiosus Yongbiao Zheng,*,† Haiyue Pang,† Jifeng Wang,‡ Guowei Shi,‡ and Jianzhong Huang† †

Engineering Research Centre of Industrial Microbiology, Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, Fujian 350117, People’s Republic of China ‡ Fudan Institute of Urology, the Fifth People’s Hospital of Shanghai, Fudan University, Shanghai 200240, People’s Republic of China S Supporting Information *

ABSTRACT: Two new bisabolane-type sesquiterpenoids, pleuroton A (1) and pleuroton B (2), and three clitocybulol derivatives, clitocybulol D (3), clitocybulol E (4), and clitocybulol F (5), were obtained from the mycelial culture of edible fungus Pleurotus cystidiosus O. K. Mill by repeated column chromatography over RP-18, Sephadex LH-20, and silica gel. Their structures were determined according to nuclear magnetic resonance data, high-resolution electron impact mass spectrometry, and circular dichroism spectra. These new sesquiterpenoids exhibited significant cytotoxicity against two human prostate cancer DU-145 and C42B cells in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The median inhibitory concentration (IC50) of compounds 1, 2, 3, 4, and 5 was 174, 28, 233, 162, and 179 nM, respectively, against the DU-145 cell and was 104, 52, 163, 120, and 119 nM, respectively, against the C42B cell. Especially, pleuroton B (2) exhibited the strongest cytotoxity among these sesquiterpenoids, which was confirmed by the colony formation assay. Furthermore, pleuroton B (2) could trigger the apoptosis of DU-145 cells through the detection of apoptosis cells using annexin V-FITC staining by flow cytometry, the observation of condensed nuclei in the apoptosis cells, and the western blot analysis for the expression of apoptosis-related proteins Bcl-2, Bak, and Bax. Analysis of structure−activity relationships of these sesquiterpenoids revealed that the unusual functional moiety of pleuroton B should contribute to its significant bioactivity. These results display the pharmacological potential of P. cystidiosus. KEYWORDS: edible fungus, Pleurotus cystidiosus, sesquiterpenoids, apoptosis inducing

■

■

INTRODUCTION Edible and medicinal fungi are a rich source of natural products with pharmacological potential; for example, davallialactone from Inonotus xaranticus could improve the aging process,1 thelephantin O from Thelephora aurantiotincta could inhibit the proliferation of cancer cells,2 etc. The genus Pleurotus is one of the major genera of edible fungi widely cultivated in China. Many different metabolites have been isolated from this genus. These include novel sesquiterpene pleurospiroketals3 A−E and new monoterpenoids with inhibitory activity against nitric oxide production from Pleurotus cornucopiae,4 diterpenoid eryngiolide A with cytotoxicity from Pleurotus eryngii,5 ubiquinone-9 with an inhibitory effect on mammalian DNA topoisomerase I from P. eryngii,6 and methane-type monoterpene pleurolactone from P. eryngii.7 Our recent experimental results indicated the ethyl acetate crude extracted from the mycelial culture of Pleurotus cystidiosus had shown significant inhibitory effects against prostate cancer DU-145 cells in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To investigate new pharmacological constituents, the strain P. cystidiosus was cultured in dishes laid with potato dextose agar media on a large scale. As a result, two new bisabolane-type sesquiterpenoids, pleuroton A (1) and pleuroton B (2), and three clitocybulol derivatives, clitocybulol D (3), clitocybulol E (4), and clitocybulol F (5), were obtained from the mycelial culture of P. cystidiosus by repeated column chromatography over RP18, Sephadex LH-20, and silica gel. © 2014 American Chemical Society

MATERIALS AND METHODS

General Procedures. Ultraviolet (UV) spectra were measured on Shimadzu UV2401PC (Tokyo, Japan). Infrared (IR) spectra were recorded on a Bruker Tensor-27 FTIR spectrophotometer (Ettlingen, Germany). Optical rotations were measured with Jasco P-1020 automatic polarimeter (Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were recorded at 500 MHz for 1H NMR and 150 MHz for 13C NMR on a Bruker AMX-500 spectrometer (Bruker BioSpin Group, Zurich, Switzerland). High-resolution electron impact mass spectrometry (HREI−MS) spectra were recorded on AutoSpec Premier P776 mass spectrometer (Milford, MA). Circular dichroism spectra were measured on a Chirascan Plus spectroscope (Applied Photophysics, Leatherhead, Surrey, U.K.). Column chromatography was performed on silica gel (Qingdao Marine Chemical Company, Qingdao, China), reverse-phase octadecyl-silica RP-18 (Merck, Darmstadt, Germany), and Sephadex LH-20 (Amersham Biosciences, Piscataway, NJ). Thin-layer chromatography was performed on the precoated silica gel GF254 plates (Qingdao Marine Chemical Company, Qingdao, China). Organic solvents used were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). MTT, 4,6-diamidino-2-phenylindole (DAPI), RNaseA, and anti-β-actin were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-Bcl-2, antiBax, and anti-Bak were purchased from Cell Signaling Technology (Danvers, MA). Annexin V-FITC Apoptosis Detection Kit was supplied by BD Biosciences, San Jose, CA. Received: Revised: Accepted: Published: 545

October 11, 2014 December 23, 2014 December 23, 2014 December 23, 2014 DOI: 10.1021/jf504931n J. Agric. Food Chem. 2015, 63, 545−551

Article

Journal of Agricultural and Food Chemistry Table 1. 1H and 13C NMR Spectral Data of Compounds 1 and 2, Recorded at 500 MHz in CD3OD δH (multiplicity, J in Hz)

δC

position

1

2

1

1 2 3 4α 4β 5α 5β 6 7 8 9 10 11 12 13 14a 14b 15

4.36 (t, J = 8.0, 1H) 3.07 (d, J = 8.0, 1H)

4.28 (t, J = 8.2, 1H) 3.47 (d, J = 8.2, 1H)

1.49 1.49 2.11 1.79 2.90

1.51 (m, 2H)

85.1 75.7 73.6 33.8

(o, 2H) (o, 2H) (m, 1H) (brd, 1H) (brs, 1H)

2.17 (o, 1H) 1.80 (brd, 1H) 3.14 (brs, 1H)

6.40 (d, J = 1.2, 1H)

1.96 2.15 5.22 5.05 1.20

1.95 2.15 5.24 5.17 1.20

(s, 3H) (s, 3H) (t, J = 2.4, 1H) (t, J = 2.4, 1H) (s, 3H)

d d s t

84.6 77.7 73.8 33.8

19.8 t 43.2 149.3 86.4 199.8 120.4 161.0 28.2 21.4 106.9

4.77 (d, J = 2.1, 1H) 6.44 (d, J = 1.1, 1H)

2

(d, J = 1.0, 3H) (d, J = 1.0, 3H) (d, J = 3.1, 1H) (t, J = 3.1, 1H) (s, 3H)

d d s t

19.9 t

d s d s d s q q t

44.3 152.0 105.8 196.8 119.7 161.6 28.3 21.4 110.4

26.9 q

d s s s d s q q t

27.0 q

Table 2. 1H and 13C NMR Spectral Data of Compounds 3−5, Recorded at 500 MHz in CD3OD δH (multiplicity, J in Hz) position 1α 1β 2 3 4 5α 5β 6 7α 7β 8 9 10α 10β 11 12a 12b 13 14 15

3 4.02 (s, 1H)

δC

4

5

2.25 (dd, J = 16.2, 1.0, 1H) 2.14 (dd, J = 16.2, 1.0, 1H)

1.86 (o, 1H) 2.49 (d, J = 16.15, 1H)

4.34 (dt, J = 2.1, 10.1, 1H) 4.47 (dt, J = 2.1, 10.1, 1H)

4.26 (dt, J = 11.0, 2.1, 1H) 4.47 (dt, J = 11.0, 2.1, 1H)

4.24 (dt, J = 12.9, 2.45, 1H) 4.41 (dt, J = 12.9, 2,0, 1H)

3.66 (d, J = 2.4, 1H)

3.63 (d, J = 2.8, 1H)

2.51 (m, 1H)

2.36 (m, 1H)

2.06 (dd, 14.0, 7.0, 1H) 1.82 (o, 1H) 2.26 (o, 1H)

2.24 (dt, J = 1.5, 14.0, 1H) 2.18 (dt, J = 1.5, 14.0, 1H)

2.49 (m, 1H) 2.06 (dd, J = 15.7, 1.2, 1H)

2.28 (o, 1H) 2.15 (d, J = 15.4, 1H)

5.27 5.13 1.25 1.08 0.97

(t, J = 2.1, 1H) (t, J = 2.1, 1H) (d, J = 2.4, 3H) (s, 3H) (s, 3H)

5.27 5.13 1.11 1.04 3.40

(t, J = 2.1, 1H) (t, J = 2.1, 1H) (d, J = 7.5, 3H) (s, 3H) (o, 2H)

5.25 5.12 1.00 1.10 3.29

Fungus Material and Fermentation. The strain was isolated from the basidiocarp of P. cystidiosus, which grew in the Qingyun mountains in Yongtai, Fujian, China. Inferred from the comparison of internal transcribed spacer (ITS) DNA sequences (GenBank accession number KP164598; see Figure S1 of the Supporting Information), the strain was identified as P. cystidiosus O. K. Mill8 and has been deposited in the China Center for Type Culture Collection (CCTCC number 2014256). The strain P. cystidiosus was cultured in 750 dishes each containing ca. 20 mL of potato dextose agar media with the total volume of 15 L. These dishes were incubated at 28 °C for 43 days. Extraction and Isolation. The fermented mycelia, some of which have formed the synnematous anamorph in black (see Figure S2 of the Supporting Information), together with the cultured substrate in these dishes, were merged and extracted with AcOEt/MeOH/AcOH (80:15:5, v/v/v). The organic crude extract was partitioned with AcOEt and double-distilled water. The AcOEt phase was dried over sodium sulfate (anhydrous) and concentrated under reduced pressure

(t, J = 2.4, 1H) (t, J = 2.4, 1H) (o, 3H) (o, 3H) (o, 2H)

3

4

5

88.5 d

45.9 t

45.9 t

136.1 77.7 150.7 68.4

s s s t

131.5 77.7 151.0 68.4

s s s t

132.1 77.8 151.3 67.9

s s s t

104.7 s 76.9 d

104.6 s 76.0 d

105.2 s 37.3 t

39.7 d 141.5 s 43.1 t

39.5 d 140.4 s 41.7 t

31.5 d 143.0 s 41.7 t

43.3 s 107.9 t

43.6 s 107.8 t

43.5 s 107.5 t

18.8 q 28.4 q 23.1 q

17.4 q 25.6 q 71.3 t

19.6 q 25.5 q 71.3 t

to afford 4.41 g of a crude organic extract. The crude extract was subjected to medium-pressure liquid chromatography (MPLC) over RP-18 silica gel (170 g) using a stepwise gradient of 30, 50, 70, and 100% (v/v) MeOH in water and to afford fraction 1 (154.8 mg) obtained from 30% MeOH in water and fraction 2 (495.7 mg) obtained from 50% MeOH in water. Fraction 1 was then subjected to a Sephadex LH-20 column eluted with MeOH to afford fraction 11 (48.4 mg). Fraction 11 was separated to fraction 111 (4.6 mg) and fraction 112 (15.4 mg) by MPLC over RP-18 silica gel (30 g) (from 25% MeOH in water). Fraction 111 was subjected to silica gel (1.0 g) chromatography using a CHCl3/MeOH solvent gradient to yield compound 3 (1.7 mg). Fraction 112 was further subjected to a Sephadex LH-20 column (100 g) eluted with MeOH to afford fraction 1121 (3.0 mg). Fraction 1121 was subjected to silica gel (1.0 g) chromatography using a CHCl3/MeOH solvent gradient to yield compound 5 (2.3 mg). Fraction 2 was divided to fraction 21 (222.9 mg) (from 35% MeOH in water) and fraction 22 (134.1 mg) (from 546

DOI: 10.1021/jf504931n J. Agric. Food Chem. 2015, 63, 545−551

Article

Journal of Agricultural and Food Chemistry

fluorescence microscopy at 340 nm (excitation) and 488 nm (emission). Western Blot Analysis. After treatment with or without a compound for different time courses, cells were harvested and lysed in ice-cold lysis buffer [20 mM Tris−HCl at pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM sodium orthovanadate, 1 mg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride]. The lysate was mixed with an equal volume of 2× loading buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, and 0.2 mg/mL bromophenol blue in 0.1 M Tris−HCl at pH 6.8) and boiled for 10 min immediately. The boiled lysates were subjected to 8−12% SDS−polyacrylamide gels, developed under 100 V, and then transferred to Immobilon-P membranes (Millipore). After blocking with 5% skim milk in phosphate-buffered saline with 0.1% Tween-20 for 1 h and incubation overnight with the corresponding primary antibodies in the blocking solution at 4 °C, primary antibodies were detected using either a peroxidase-conjugated immunopure goat anti-rabbit IgG (H + L) or peroxidase-conjugated immunopure goat anti-mouse IgG (H + L) secondary antibody and enhanced chemiluminescence.

45% MeOH in water) by MPLC over RP-18 silica gel (30 g). Fraction 21 was continuously subjected to a Sephadex LH-20 (120 g) column eluted with MeOH and to silica gel (2.0 g) chromatography using a CHCl3/MeOH solvent gradient to yield compound 1 (6.1 mg). Fraction 22 was further subjected to a Sephadex LH-20 (120 g) column eluted with MeOH and to afford fraction 221 (16.6 mg) and fraction 222 (49.0 mg). Fraction 221 was subjected to silica gel (1.0 g) chromatography using a CHCl3/MeOH solvent gradient (200:1) to yield compound 2 (3.0 mg). Fraction 222 was also subjected to silica gel (1.0 g) chromatography using a CHCl3/MeOH solvent gradient (50:1) to yield compound 4 (23.5 mg). The physical properties and spectroscopic data of five new sesquiterpenoids are as follows: Compound 1: colorless oil. [α]25 D : +51.7 (c 0.23, methanol). UV (methanol) λmax, nm: 216. HREI−MS, m/z: 266.1521 (calcd for C15H22O4, 266.1518). IR (KBr) νmax: 3441, 1749, 1623 cm−1. 1H and 13 C NMR spectral data are shown in Table 1. Compound 2: colorless oil. [α]25 D : +79.7 (c 0.32, methanol). UV (methanol) λmax, nm: 247. HREI−MS, m/z: 282.1461 (calcd for C15H22O5, 282.1467). IR (KBr) νmax: 3443, 1758, 1684, 1622 cm−1. 1H and 13C NMR spectral data are shown in Table 1. Compound 3: colorless oil. [α]25 D : −50.0 (c 0.17, methanol). UV (methanol) λmax, nm: 209. HREI−MS, m/z: 282.1465 (calcd for C15H22O5, 282.1467). IR (KBr) νmax: 3426, 1630 cm−1. 1H and 13C NMR spectral data are shown in Table 2. Compound 4: colorless oil. [α]25 D : −45.8 (c 0.20, methanol). UV (methanol) λmax, nm: 207. HREI−MS, m/z: 282.1466 (calcd for C15H22O5, 282.1467). IR (KBr) νmax: 3423, 1631 cm−1. 1H and 13C NMR spectral data are shown in Table 2. Compound 5: colorless oil. [α]25 D : −51.3 (c 0.07, methanol). UV (methanol) λmax, nm: 207. HREI−MS, m/z: 266.1517 (calcd for C15H22O4, 266.1518). IR (KBr) νmax: 3418, 1631 cm−1. 1H and 13C NMR spectral data are shown in Table 2. MTT Cell Viability Assay. MTT cell viability assay was used to evaluate the inhibitory effect of these compounds on two human prostate cancer DU-145 and C42B cells. DU-145 and C42B cells were cultured in Dulbecco’s modified Eagle’s media (DMEM) complemented with 10% fetal bovine serum and 2 mM L-glutamine. The cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. DU-145 and C42B cells (1 × 105/well) were cultured for 72 h in 96-well plates (Falcon, CA) in the presence of various concentrations of compounds. All wells (100 μL each) were incubated with 20 μL of 5 mg/mL MTT solution for 3 h at 37 °C. Then, 100 μL of “triplex solution” [10% sodium dodecyl sulfate (SDS), 5% isobutanol, and 12 mM hydrochloric acid (HCl)] was added to each well and incubated overnight at 37 °C. Absorbance was read at 595 nm on a microplate reader (M-3350, Bio-Rad) with 655 nm as a reference. Colony Formation Assay. DU-145 cells were cultured in 6 well plates (1000/well) overnight and with new medium replaced in the presence of compound 2, and the plates continued to incubate at 37 °C with 5% CO2 for 10 days. On the last day, the medium was removed, and after washing with phosphate-buffered saline (PBS) and fixing with methanol, the colonies were stained with crystal violet solution for 3 h at room temperature. The images were acquired with a scanner, and visible colony numbers were counted after washing and air drying. Assessing Apoptosis by Annexin-V/Propidium Iodide (PI) Staining. Cells were seeded in a 6 cm dish 1 day before compound treatment. After treatment with compound 2 at 35 nM for 24 h, DU145 cells were stained with annexin V and PI following the protocol of the manufacturer. Subsequently, cells were analyzed by a FACSCalibur flow cytometer and BD CellQuest Pro software under the examination of FL1 channel for FITC and FL3 detector for PI, as described elsewhere. Assessing Apoptosis Using DAPI Staining. After treatment with compound 2, cells were collected and washed once with 2 mL of ice-cold PBS, fixed with 1 mL of 4% paraformaldehyde for 20 min, and washed once again with 2 mL of ice-cold PBS. The cells were incubated in 1 mL of DAPI at 50 μg/mL containing 100 μg/mL RNaseA. This mixture was incubated for 30 min at 37 °C. After washing with 2 mL of PBS 3 times, the cells were observed using

■

RESULTS AND DISCUSSION Identification of New Sesquiterpenoids. Compound 1 was obtained as a colorless oil. The molecular formula of compound 1 was found to be C15H22O4 (five degrees of unsaturation) based on the HREI−MS peak at m/z 266.1521 [M]+ and 1H and 13C NMR data (Table 1 and Figure S3 of the Supporting Information). In the IR spectra, the prominent absorptions indicated the presence of hydroxyl (3441 cm−1), carbonyl (1749 cm−1), and unsaturated double bond (1623 cm−1) groups. NMR data [1H, 13C, and distortionless enhancement by polarization transfer (DEPT)] revealed resonances for three methyls, three methylenes, including one terminal sp2 methylene (δH 5.22 and 5.05, δC 106.9), five methines, including three oxygenated methines (δH 4.36, δC 85.1; δH 3.07, δC 75.7; and δH 4.77, δC 86.4) and one sp2 methine (δH 6.44, δC 120.4), and four quaternary carbons, including one oxygenated carbon (δC 73.6), one carbonyl carbon (δC 199.8), and two sp2 quaternary carbons (δC 149.3 and 161.0). One sp2 methylene, one sp2 methine, one carbonyl carbon, and two sp2 quaternary carbons represented three double bond equivalents. Therefore, compound 1 must be bicyclic to account for five double bond equivalents required by the molecular formula. The cross-peaks of H-1 and H-2, H-1 and H-6, H-5α/β and H-4α/β, and H-5α/β and H-6 in the 1 H−1H correlation spectroscopy (COSY) spectrum showed the spin system of H-1, H-2, H-4α/β, H-5α/β, and H-6. Further, the heteronuclear multiple-bond correlations (HMBCs) from H3-14 to C-2, C-3, and C-4, from H2-14 to C-6 and C-8, from H-1 to C-2, C-7, and C-8, from H-6 to C-7, and from H-8 to C7 could be observed and allowed for the establishment of the octahydrobenzofuran moiety of compound 1, together with the oxygenated nature of C-1, C-2, C-3, and C-8. HMBC correlations from H3-12/13 to C-11, C-10, and C-13/12 and from H-10 to C-9 and C-12/13, together with the chemical shift of C-9 (δC 199.8 s), allowed for the establishment of another moiety of the carbonyl-substituted isoprene in compound 1, which was connected to C-8 according to the key HMBC from H-8 to C-9. On the basis of the above data, the planar structure of compound 1 was formed. The relative configuration of compound 1 was established by nuclear Overhauser effect spectrometry (NOESY) experiments. The presence of NOESY correlations of H-1 with H-6 and H-6 with 547

DOI: 10.1021/jf504931n J. Agric. Food Chem. 2015, 63, 545−551

Article

Journal of Agricultural and Food Chemistry H-5α indicated the α orientation of these protons. Further, the NOESY cross-peaks between H-2 and H3-15 and H-2 and H-8 established the relative configuration at C-2, C-3, and C-8 and the β orientation of these protons (Figure 1). Therefore, the structure of compound 1 was established as a derivative of bisabolane-type sesquiterpenoid cheimonophyllons9,10 and named pleuroton A.

4.02, δC 88.5; δH 3.66, δC 76.9), and six quaternary carbons, including two oxygenated carbon (δC 77.7, 104.7 s) and three sp2 quaternary carbons (δC 136.1, 150.7, and 141.5), indicating a tricyclic sesquiterpene backbone. HMBC correlations from the protons of two methyl groups at 0.97 (s, H-15) and 1.08 (s, H-14) to the corresponding carbons (C-1, C-11, and C-10) indicated the presence of gem-dimethyl groups and deduced the one 5-carbon moiety, including C-1, C-11, C-10, C-14, and C15. According to HMBCs from H3-13 to C-7, C-8, and C-9, from H-8 to C-6, C-7, C-9, and C-13, and from H-7 to C-3, C6, C-8, C-9, and C-13, together with the 1H−1H COSYs of H-8 and H-7 and H-8 and H-13, and the consideration of the chemical shift of C-6 (δC 104.7 s), C-2 (δC 136.1 s), and C-9 (δC 141.5 s), a moiety of three hydroxy-substituted cyclohexenes could be recognized in compound 3. Additionally, a furan moiety fusing with terminal alkene was assigned by analyzing the HMBC from H2-12 to C-3, C-4, and C-5 and from H2-5 to C-3, C-4, C-6, and C-12. Therefore, the planar structure of compound 3 was established (Figure 1). Following a similar strategy, compounds 4 and 5 were recognized. Compound 4 has the molecular formula of C15H22O5 based on the HREI−MS peak at m/z 282.1466 [M]+ and NMR data (Table 2 and Figure S6 of the Supporting Information), requiring five degrees of unsaturation. The IR spectrum indicated the existence of hydroxyl (3423 cm−1) and unsaturated double bond (1631 cm−1) groups. Analysis of NMR data of compound 4 revealed 15 carbon signals attributed to two methyls, five methylenes, including one terminal sp2 methylene (δH 5.27 and 5.13, δC 107.8) and two oxygenated methylene (δH 4.47 and 4.26, δC 68.4; δH 3.40, δC 71.3), two methines, including one oxygenated methine (δH 3.63, δC 76.0), and six quaternary carbons, including two oxygenated carbon (δC 77.7 and 104.6) and three sp2 quaternary carbons (δC 131.5, 151.0, and 140.4). Compound 5 has the molecular formula of C15H22O4 based on the HREI−MS peak at m/z 266.1517 [M]+ and NMR data (Table 2 and Figure S7 of the Supporting Information), requiring five degrees of unsaturation. The IR spectrum indicated the existence of hydroxyl (3418 cm−1) and unsaturated double bond (1631 cm−1) groups. Analysis of NMR data of compound 5 revealed 15 carbon signals attributed to two methyls, six methylenes, including one terminal sp2 methylene (δH 5.25 and 5.12, δC 107.5) and two oxygenated methylenes (δH 4.41 and 4.24, δC 67.9; δH 3.29, δC 71.3), one methine, and six quaternary carbons, including two oxygenated carbon (δC 77.8 and 105.2) and three sp 2 quaternary carbons (δC 132.1, 151.3, and 143.0). Detailed comparisons of NMR data (Table 2) of compounds 3−5 revealed that these compounds had a carbon skeleton similar to clitocybulols A and C.11 However, the CHOH group at C-1 and the methyl group at C-14 in compound 3 were replaced by CH2 at C-1 and a hydroxymethyl at C-14 in compound 4 (Figure 1). In addition, the CHOH group at C-7 in compound 4 was substituted for CH2 at C-7 in compound 5 (Figure 1). The stereochemistry of clitocybulols A and C was determined by applying the octant rule12 and based on the circular dichroic spectra of these compounds.11 The negative Cotton effects at 212 nm for compound 3, 216 nm for compound 4, and 216 nm for compound 5 (see Figure S8 of the Supporting Information) suggest that three compounds have the same configuration as clitocybulols A and C at C-3 and C-6. This configuration was further supported by the same 25 sinistral optical rotations ([α]25 D , −50.0 for compound 3; [α]D , 25 −45.8 for compound 4; [α]D , −51.3 for compound 5; [α]25 D,

Figure 1. New sesquiterpenoids of P. cystidiosus.

Compound 2 was obtained as a colorless oil. The molecular formula of compound 2 was determined to be C15H22O5 (five degrees of unsaturation) based on the HREI−MS peak at m/z 282.1461 [M]+ and 1H and 13C NMR data (Table 1 and Figure S4 of the Supporting Information). NMR data (1H, 13C, and DEPT) displayed three methyls, three methylenes, including one terminal sp2 methylene (δH 5.24 and 5.17, δC 110.4), four methines, including two oxygenated methines (δH 4.28, δC 84.6; δH 3.47, δC 77.7) and one sp2 methine (δH 6.40, δC 119.7), and five quaternary carbons, including two oxygenated carbon (δC 73.8, 105.8), one carbonyl carbon (δC 196.8), and two sp2 quaternary carbons (δC 152.0; δC 161.6). One sp2 methylene, one sp2 methine, one carbonyl carbon, and two sp2 quaternary carbons represented three degrees of unsaturation. Thus, compound 2 must be bicyclic to account for five degrees of unsaturation required by the molecular formula. Comprehensive inspection of the NMR data [1H, 13C, heteronuclear single-quantum coherence (HSQC), HMBC, and 1H−1H COSY] revealed that compound 2 was similar to compound 1, except that the oxygenated methine (δC 84.6) at C-8 in compound 1 was replaced by the dioxygenated quaternary carbon (δC 105.8) at the same position in compound 2 (Figure 1). The similar NOESY correlations of H-1 with H-6, H-6 with H-5α, H-2 with H3-15, and H-2 with H-4β, established the relative configuration of compound 2. Therefore, compound 2 was determined as an analogue of cheimonophyllons and named pleuroton B (2). The molecular formula of compound 3 was assigned to C15H22O5 based on the HREI−MS peak at m/z 282.1465 [M]+ and NMR data (Table 2 and Figure S5 of the Supporting Information), requiring five degrees of unsaturation. The IR spectrum indicated the existence of hydroxyl (3426 cm−1) and unsaturated double bond (1630 cm−1) groups. Analysis of NMR data (1H, 13C, and DEPT) of compound 3 revealed 15 carbon signals attributed to three methyls, three methylenes, including one terminal sp2 methylene (δH 5.27 and 5.13, δC 107.9) and one oxygenated methylene (δH 4.34 and 4.47, δC 68.4), three methines, including two oxygenated methines (δH 548

DOI: 10.1021/jf504931n J. Agric. Food Chem. 2015, 63, 545−551

Article

Journal of Agricultural and Food Chemistry −80.0 for clitocybulol A; and [α]25 D , −57.7 for clitocybulol C) and NOESY experiments. NOSEY correlations between H-7 and H3-13, indicating that they are directed on the same side of compound 3, and between H-1 and H-8, which are pointed at the other side, were observed and deduced the stereochemistry of compound 3 (Figure 1). The presence of NOESY correlations between H-8 and H2-15, between H2-15 and H10α, and between H-8 and H-1α indicated the α orientation of these protons in compound 4. Further, the NOESY cross-peak between H-7 and H3-13 revealed the β orientation of these protons in compound 4 (Figure 1). NOESY correlations between H-8 and H-1α and H-7α and between H2-15 and H5α, which showed the α orientation of these protons, and between H-1β and H-7β, between H-10β and H3-13, and between H-10β and H3-14, which showed the β orientation, were observed in compound 5 and supported its stereochemistry (Figure 1). Compounds 3, 4, and 5 were named clitocybulols D, E, and F, respectively. Bioactivity Evaluation. The experimental results of the MTT cell viability assay indicated that these new sesquiterpenoids exhibit significant cytotoxicity against two human prostate cancer DU-145 and C42B cells. The median inhibitory concentration (IC50) of compounds 1−5 against DU-145 and C42B cells are shown in Table 3. Consistently, the colony Table 3. Growth Inhibition against DU-145 and C42B Cells of Compounds 1−5 growth inhibition (IC50, nM) compound

DU-145

C42B

1 2 3 4 5

174 28 233 162 179

104 52 163 120 119

Figure 3. Analysis of apoptosis induced by compound 2. (A) Detection of apoptosis using annexin V-FITC staining. After treatment with compound 2 at 35 nM for 24 h, DU-145 cells were analyzed by a FACSCalibur flow cytometer. The horizontal axis represents annexin V intensity, and the vertical axis shows PI staining. The lines divide each plot into four quadrants: lower left quadrant, living cells; lower right quadrant, early apoptotic cells; upper left quadrant, necrotic cells; and upper right quadrant, late apoptotic cells. (B) Microscopy of DU145 cells treated with compound 2. DU-145 cells were treated with compound 2 at 35 nM for 24 h. The images were captured using fluorescence (at 600×) for nuclear staining with compound 2 at 0 nM (vehicle) or 35 nM. Apoptotic cells with condensed nuclei were observed using nuclear staining, as indicated with red arrows. (C) Western blot analysis for the expression of apoptosis-related proteins in the DU-145 cells. The expression levels of Bcl-2, Bak, and Bax were determined by western blotting in the DU-145 cells after treatment for the indicated time with compound 2 at 35 nM. β-Actin was used for normalization and verification of protein loading.

formation assay showed that the number of colonies formed in the cells treated with compound 2 significantly decreased in comparison to that of the control group (Figure 2). Furtherly,

Figure 2. Colony formation assay (A, DU-145 cells were cultured in the medium not containing compound 2; B, DU-145 cells were cultured in the medium containing compound 2 at 17.5 nM; and C, DU-145 cells were cultured in the medium containing compound 2 at 35 nM).

controls, but the anti-apoptotic protein Bcl-2 level was decreased (Figure 3C). Taken together, these results indicated that compound 2 triggered the apoptosis of the DU-145 cells. Sesquiterpenoids 1 and 2 possess a bisabolane-type skeleton, whereas sesquiterpenoids 3−5 are clitocybulol derivatives. In the previous reports, cheimonophyllons A−E and cheimonophyllal, analogues of compounds 1 and 2, exhibited nematicidal, cytotoxic, and antimicrobial activities,9,10 but no activities were observed about clitocybulols.11 In the present work, pleuroton B (2) exhibited significant cytotoxicity at a higher level than other sesquiterpenoids, pleuroton A (1), and clitocybulols D−F (3−5). In particular, although compound 1 possesses one α,β-

flow cytometric analysis revealed that the proportion of apoptotic cells after treatment with compound 2 was 13.36%, which was dramatically increased in comparison to 0.29% of the control cells (Figure 3A). Chromatin condensations induced by the treatment with compound 2 for 24 h could be observed by DAPI staining in DU-145 cells (Figure 3B). We observed that the expression levels of pro-apoptotic protein Bak and Bax increased significantly in the treated cells compared to the 549

DOI: 10.1021/jf504931n J. Agric. Food Chem. 2015, 63, 545−551

Journal of Agricultural and Food Chemistry

■

unsaturated carbonyl moiety and one exomethylene as compound 2, compound 2 has one additional hydroxyl at C8 compared to compound 1 and has ca. 2−6 times stronger cytotoxicity against the two prostate cancer cells than compound 1. Sesquiterpenoids are the major secondary metabolites of fungi, plant, algae, etc. The diversity of their structures is often beyond people’s imagination. In recent years, many new sesquiterpenoids were discovered and possessed a wide variety of bioactivities, such as antidiabetic and anti-inflammatory,13 anti-aging,14 antimelanogenesis,15 antineuroinflammatory,16 antivirus,17 antimicrobial,18 acetylcholinesterase inhibitory activity,19,20 protein tyrosine phosphatase,21 antifouling,22 etc. Moreover, the relationships between the structure and bioactivities of sesquiterpenoids have been summarized in several reviews23−25 and revealed that the α,β-unsaturated carbonyl structural groups, including α-methylene-γ-lactones, α,β-unsaturated cyclepentanone, etc., are the active functional groups, which could react as Michael acceptors with nucleophiles by Michael addition and display promising cytotoxity, such as bigelovin,26 ergolide,26 kaunial,27 germacranolide,28 eudesmane derivative,29 etc. Additionally, several studies in bisabolane-type sesquiterpenoids had revealed more interesting results about structure−activity relationships. The bisabolane-type sesquiterpenoid dihydro-ar-turmerone, which contains an oxidation functional group and the single bond moiety in its skeleton, has a higher acetylcholinesterase inhibitory activity than other analogues.30 3,6-Epidioxy-1,10bisaboladiene (EDBD), the bisabolane sesquiterpenoid endoperoxide, showed stronger antitumor activity than α-curcumene, a bisabolane sesquiterpene that lacks the endoperoxide moiety of EDBD.31 The additional hydroxyl at C-8 in compound 2 could act as an electron-withdrawing group and polarize the exomethylene, resulting in the susceptibility to nucleophilic attack and forming a bifunctional center, which plays a key role in the cytotoxity against the prostate cancer cells. Structure−activity relationship studies will contribute to the modification of new lead compounds.

■

REFERENCES

(1) Yang, T. K.; Lee, Y. H.; Paudel, U.; Bhattarai, G.; Yun, B. S.; Hwang, P. H.; Yi, H. K. Davallialactone from mushroom reduced premature senescence and inflammation on glucose oxidative stress in human diploid fibroblast cells. J. Agric. Food Chem. 2013, 61, 7089− 7095. (2) Norikura, T.; Fujiwara, K.; Yanai, T.; Sano, Y.; Sato, T.; Tsunoda, T.; Kushibe, K.; Todate, A.; Morinaga, Y.; Iwai, K.; Matsue, H. pTerphenyl derivatives from the mushroom Thelephora aurantiotincta suppress the proliferation of human hepatocellular carcinoma cells via iron chelation. J. Agric. Food Chem. 2013, 61, 1258−1264. (3) Wang, S. J.; Bao, L.; Han, J. J.; Wang, Q. X.; Yang, X. L.; Wen, H. A.; Guo, L. D.; Li, S. J.; Zhao, F.; Liu, H. W.; Pleurospiroketals, A.-E. Perhydrobenzannulated 5,5-spiroketal sesquiterpenes from the edible mushroom Pleurotus cornucopiae. J. Nat. Prod. 2013, 76, 45−50. (4) Wang, S. J.; Bao, L.; Zhao, F.; Wang, Q. X.; Li, S. J.; Ren, J. W.; Li, L.; Wen, H.; Guo, L. D.; Liu, H. W. Isolation, identification, and bioactivity of monoterpenoids and sesquiterpenoids from the mycelia of edible mushroom Pleurotus cornucopiae. J. Agric. Food Chem. 2013, 61, 5122−5129. (5) Wang, S. J.; Li, Y. X.; Bao, L.; Han, J. J.; Yang, X. J.; Li, H. R.; Wang, Y. Q.; Li, S. J.; Liu, H. W.; Eryngiolide, A. a cytotoxic macrocyclic diterpenoid with an unusual cyclododecane core skeleton produced by the edible mushroom Pleurotus eryngii. Org. Lett. 2012, 14, 3672−3675. (6) Bae, J. S.; Park, J. W.; Park, S. H.; Park, J. B.; Rho, Y. H.; Ryu, Y. B.; Lee, K. S.; Park, K. H.; Bae, Y. S. Apoptotic cell death of human leukaemia U937 cells by ubiquinone-9 purified from Pleurotus eryngii. Nat. Prod. Res. 2009, 23, 1112−1119. (7) Liu, L. Y.; Li, Z. H.; Liu, J. K. A new menthane-type monoterpene from Pleurotus eryngii. Chin. J. Nat. Med. 2013, 11, 71−73. (8) Zervakis, G. I.; Moncalvo, J. M.; Vilgalys, R. Molecular phylogeny, biogeography and speciation of the mushroom species Pleurotus cystidiosus and allied taxa. Microbiology 2004, 150, 715−726. (9) Stadler, M.; Anke, H. New nematicidal and antimicrobial compounds from the basidiomycete Cheimonophyllum candidissimum (Berk & Curt.) sing. I. Producing organism, fermentation, isolation, and biological activities. J. Antibiot. 1994, 47, 1284−1289. (10) Stadler, M.; Anke, H.; Stener, O. Six new antimicrobial and nematicidal bisabolanes from the basidiomycete Cheimonophyllum candidissimum. Tetrahedron 1994, 50, 12649−12654. (11) Ayer, W. A.; Shan, R.; Trifonov, L. S.; Hutchison, L. J. Sesquiterpenes from the nematicidal fungus Clitocybula oculus in honour of professor GH Neil Towers 75th birthday. Phytochemistry 1998, 49 (2), 589−592. (12) Scott, A.; Wrixon, A. A symmetry rule for chiral olefins. Tetrahedron 1970, 26, 3695−3715. (13) Chung, Y. M.; Wei, C. K.; Chuang, D. W.; El-Shazly, M.; Hsieh, C. T.; Asai, T.; Oshima, Y.; Hsieh, T. J.; Hwang, T. L.; Wu, Y. C. An epigenetic modifier enhances the production of anti-diabetic and antiinflammatory sesquiterpenoids from Aspergillus sydowii. Bioorg. Med. Chem. 2013, 21, 3866−3872. (14) Park, J. H.; Jung, Y. J.; Antar Aziz Mohamed, M.; Hoon Lee, T.; Lee, C. H.; Han, D.; Song, M. C.; Kim, J.; Baek, N. I. New bisabolane sesquiterpenes from the rhizomes of Curcuma xanthorrhiza Roxb. and their inhibitory effects on UVB-induced MMP-1 expression in human keratinocytes. Helv. Chim. Acta 2014, 97, 438−446. (15) Lee, J. W.; Yun, C. Y.; Roh, E.; Lee, C.; Jin, Q.; Bang, K. K.; Jung, S. H.; Lee, D.; Lee, M. K.; Kim, Y. Melanogenesis inhibitory bisabolane-type sesquiterpenoids from the roots of Angelica koreana. Bioorg. Med. Chem. Lett. 2012, 22, 2927−2931. (16) Wang, L. J.; Xiong, J.; Liu, S. T.; Liu, X. H.; Hu, J. F. Sesquiterpenoids from Chloranthus henryi and their anti-neuroinflammatory activities. Chem. Biodiversity 2014, 11, 919−928. (17) Lv, J. J.; Yu, S.; Wang, Y. F.; Wang, D.; Zhu, H. T.; Cheng, R. R.; Yang, C. R.; Xu, M.; Zhang, Y. J. Anti-hepatitis B virus norbisabolane sesquiterpenoids from Phyllanthus acidus and the establishment of their absolute configurations using theoretical calculations. J. Org. Chem. 2014, 79, 5432−5447.

ASSOCIATED CONTENT

S Supporting Information *

ITS DNA sequences of P. cystidiosus (Figure S1), mycelia of P. cystidiosus cultured in a dish (Figure S2), 1H and 13C NMR spectra of compounds 1−5 (Figures S3−S7) and circular dichroic spectra of compounds 3−5 (Figure S8). This material is available free of charge via the Internet at http://pubs.acs.org.

■

Article

AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: +86-591-22868193. E-mail: yongbiaozheng@ fjnu.edu.cn. Funding

This work was financially supported by the Natural Science Foundation of Fujian Province of China (2013J01122), the Science Foundation of Fuzhou City (2013G101), and the Science Fund of the National Health and Family Planning Commission of China (WKJ-FJ-20). Notes

The authors declare no competing financial interest. 550

DOI: 10.1021/jf504931n J. Agric. Food Chem. 2015, 63, 545−551

Article

Journal of Agricultural and Food Chemistry (18) Ma, K.; Bao, L.; Han, J.; Jin, T.; Yang, X.; Zhao, F.; Li, S.; Song, F.; Liu, M.; Liu, H. New benzoate derivatives and hirsutane type sesquiterpenoids with antimicrobial activity and cytotoxicity from the solid-state fermented rice by the medicinal mushroom Stereum hirsutum. Food Chem. 2014, 143, 239−245. (19) Chen, P.; Wang, P. P.; Jiao, Z. Z.; Xiang, L. Sesquiterpenoids from the fruits of Alpinia oxyphylla and their anti-acetylcholinesterase activity. Helv. Chim. Acta 2014, 97, 388−397. (20) Zhang, J.; Fan, P.; Zhu, R.; Li, R.; Lin, Z.; Sun, B.; Zhang, C.; Zhou, J.; Lou, H. Marsupellins A−F ent-longipinane-type sesquiterpenoids from the Chinese liverwort Marsupella alpine with acetylcholinesterase inhibitory activity. J. Nat. Prod. 2014, 77, 1031−1036. (21) Fan, Q. Y.; Dong, Z. J.; Li, Z. H.; Yin, X.; Yang, X. Y.; Feng, T.; Wei, K.; Liu, J. K.; Zhao, B. H. Two new ylangene-type sesquiterpenoids from cultures of the fungus Postia sp. J. Asian Nat. Prod. Res. 2014, 16, 254−258. (22) Chen, D.; Chen, W.; Liu, D.; van Ofwegen, L.; Proksch, P.; Lin, W. Asteriscane-type sesquiterpenoids from the soft coral Sinularia capillosa. J. Nat. Prod. 2013, 76, 1753−1763. (23) Wang, G. W.; Qin, J. J.; Cheng, X. R.; Shen, Y. H.; Shan, L.; Jin, H. Z.; Zhang, W. D. Inula sesquiterpenoids: Structural diversity, cytotoxicity and anti-tumor activity. Expert Opin. Invest. Drugs 2014, 23, 317−345. (24) Vasas, A.; Hohmann, J. Xanthane sesquiterpenoids: Structure, synthesis and biological activity. Nat. Prod. Rep. 2011, 28, 824−842. (25) Ghantous, A.; Gali-Muhtasib, H.; Vuorela, H.; Saliba, N. A.; Darwiche, N. What made sesquiterpene lactones reach cancer clinical trials? Drug Discovery Today 2010, 15, 668−678. (26) Wang, Q.; Zhou, B. N.; Zhang, R. W.; Lin, Y. Y.; Lin, L. Z.; Gil, R. R.; Cordell, G. A. Cytotoxicity and NMR spectral assigments of ergolide and bigelovin. Planta Med. 1996, 62, 166−168. (27) Maldonado, E. M.; Svensson, D.; Oredsson, S. M.; Sterner, O. A novel cytotoxic terpenoid from the flowers of Kaunia lasiophthalma Griseb. Phytochem. Lett. 2014, 8, 105−108. (28) Harvala, E.; Aligiannis, N.; Skaltsounis, A. L.; Pratsinis, H.; Lambrinidis, G.; Harvala, C.; Chinou, I. Cytotoxic germacranolides from Inula verbascifolia subsp. methanea. J. Nat. Prod. 2002, 65, 1045− 1048. (29) Maldonado, E. M.; Svensson, D.; Oredsson, S. M.; Sterner, O. Cytotoxic sesquiterpene lactones from Kauna lasiophthalma Griseb. Sci. Pharm. 2014, 82, 147−160. (30) Fujiwara, M.; Yagi, N.; Miyazawa, M. Acetylcholinesterase inhibitory activity of volatile oil from Peltophorum dasyrachis Kurz ex Bakar (yellow batai) and bisabolane-type sesquiterpenoids. J. Agri. Food Chem. 2010, 58, 2824−2829. (31) Nishikawa, K.; Aburai, N.; Yamada, K.; Koshino, H.; Tsuchiya, E.; Kimura, K. The bisabolane sesquiterpenoid endoperoxide, 3,6epidioxy-1,10-bisaboladiene, isolated from Cacalia delphiniifolia inhibits the growth of human cancer cells and induces apoptosis. Biosci., Biotechnol., Biochem. 2008, 72, 2463−2466.

551

DOI: 10.1021/jf504931n J. Agric. Food Chem. 2015, 63, 545−551