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Sesquiterpenoids From Cultures of The Edible Mushroom Craterellus
Sesquiterpenoids From Cultures of The Edible Mushroom Craterellus
Sesquiterpenoids From Cultures of The Edible Mushroom Craterellus
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
A R T I C L E I N F O A B S T R A C T
Keywords: Three illudin sesquiterpenoids, craterellins A–C, and one gymnomitrane sesquiterpenoids, gymnomitr-3-en-
Craterellus cornucopioides 10β,15-diol, were isolated from cultures of the basidiomycete Craterellus cornucopioides, along with four pre-
Sesquiterpenoids viously reported compounds: illudin F, illudin M, illudin T and illudalenol. Structures of new compounds were
Illudane elucidated on the basis of extensive spectroscopic analysis and their cytotoxic activities on five tumor cell lines
Gymnomitrane
were evaluated.
Cytotoxicities
⁎
Corresponding authors.
E-mail addresses: tfeng@mail.scuec.edu.cn (T. Feng), jkliu@mail.kib.ac.cn (J.-K. Liu).
http://dx.doi.org/10.1016/j.phytol.2017.06.007
Received 3 March 2017; Received in revised form 8 June 2017; Accepted 16 June 2017
1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
H. Guo et al. Phytochemistry Letters 21 (2017) 114–117
O O O 15 H OH
Fig. 1. Structures of compounds 1–8.
HO HO 12
8 1 10
9 2
14 7 OH 3 11
15
3 11 OH 1
7
9
6 5 5
4
HO 12 HO 13
13 O 14
1 2 3 4
HO O O O O
OH OH OH
HO HO OH
OH
5 6 7 8
Table 1
1
H NMR data of compounds 1–4.
No. 1a 2a 3b 4a
115
H. Guo et al. Phytochemistry Letters 21 (2017) 114–117
primary alcohol at δH 4.03 and 4.05 (each 1H, d, J = 12.3 Hz) and δC MeOH, 1:1) to give 5 (7.0 mg). Fraction F was chromatographed over a
67.2, a secondary alcohol at δH 4.16 (1H, dd, J = 10.1, 5.6 Hz) and δC silica gel column using PE–acetone (10:1 → 0:1) to produce fractions
76.1, and a atrisubstituted double bond at δC 122.1 and 142.9. These F01-F06. Compounds 2 (6.0 mg, rt = 11.2 min) and 4 (10.0 mg,
data indicate a tricyclic sesquiterpenoid with similarities to those of 3- rt = 11.9 min) were afforded from fraction F04 by preparative HPLC
gymnomitren-15-ol (Buchanan et al., 1996), except for an additional (CH3CN–H2O, 22:78, flow rate = 10 mL/min, 30 min), compound 6
hydroxyl substitution. The COSY spectrum afforded a fragment of H-8/ (6.0 mg, rt = 12.4 min) was also obtained by preparative HPLC
H-9/H-10, while the HMBC spectrum showed correlations of H-10 (δH (CH3CN–H2O, 30:70, flow rate = 10 mL/min, 30 min) from the F03
4.16) with C-9 (δC 34.7), C-10 (76.5) and C-11 (55.1). These 2D-NMR fraction. Fraction F05 was subjected to a RP-18 column (MeOH–H2O,
experiments indicated that the additional hydroxyl group was located 38:62), then purified on a silica gel column (PE-acetone, 2:1) to afford 7
at C-10. The ROESY correlation of H-10/H-15 indicated that OH-10 (4.5 mg) and 8 (6.5 mg).
should be β oriented. From the above data, the structure of this ses- D −76.0 (c 0.22, MeOH); UV
Craterellin A (1), colorless oil; [α]15
quiterpenoid was established as gymnomitr-3-en-10β,15-diol (4). (MeOH) λmax (log ε) 286 (3.3), 205 (3.3) nm; IR (KBr) νmax 3441, 3428,
All new compounds were evaluated for their cytotoxicities against 2823, 1664, 1657, 1423, 1186, 960 cm−1; 1H NMR (CDCl3, 500 MHz)
five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and and 13C NMR (CDCl3, 125 MHz) data, see Tables 1 and 2; HR-EI-MS m/z
SW-480) using the MTT method. Compound 3 exhibited moderate cy- 232.1461 (calcd for C15H20O2, 232.1463).
totoxicity against A-549 with IC50 value of 21.0 μM. D −47.5 (c 0.24, MeOH); UV
Craterellin B (2), colorless oil; [α]15
(MeOH) λmax (log ε) 240 (3.3), 195 (3.0) nm; IR (KBr) νmax 3430, 3425,
3. Experimental 2831, 1662, 1438, 1178, 978 cm−1; 1H-NMR (CDCl3, 400 MHz) and 13C
NMR (CDCl3, 100 MHz) data, see Tables 1 and 2; HR-EI-MS m/z
3.1. General experimental procedures 250.1572 (calcd for C15H22O3, 250.1569).
D −49.8 (c 0.24, MeOH); UV
Craterellin C (3), colorless oil; [α]15
Optical rotations were measured on a Jasco-P-1020 polarimeter. UV (MeOH) λmax (log ε) 247 (3.2), 207 (2.8), 194 (2.9) nm; IR (KBr) νmax
spectra were run on a Shimadzu UV-2401 PC spectrophotometer. IR 3426, 2834, 1673, 1424, 1160, 965 cm−1; 1H-NMR (DMSO-d6,
spectra were obtained using a Bruker Tensor 27 FT-IR spectrometer 600 MHz) and 13C NMR (DMSO-d6, 150 MHz) data, see Tables 1 and 2;
with KBr pellets. NMR spectra were acquired with Bruker instruments HR-EI-MS m/z 266.1521 (calcd for C15H22O4, 266.1518).
(Avance III 600, DRX-500 and Bruker AV 400). HREIMS were measured D −26.7 (c 0.17,
Gymnomitr-3-en-10β,15-diol (4), colorless oil; [α]15
on a Waters Auto Premier P776 mass spectrometer respectively. MeOH); UV (MeOH) λmax (log ε) 203 (2.9) nm; IR (KBr) νmax 3425,
Preparative HPLC was performed on an Agilent 1100 series with a DAD 2930, 2869, 1462, 1049 cm−1; 1H NMR (CDCl3, 600 MHz) and 13C
detector and a Zorbax SB-C18 (5 μm, 9.4 × 150 mm) column. NMR (CDCl3, 150 MHz) data, see Tables 1 and 2; HR-EI-MS m/z
Preparative MPLC was carried out on a Büchi apparatus equipped with 236.1776 (calcd for C15H24O2, 236.1776).
Büchi fraction collector C-660, Büchi pump module C-605 and manager
C-615. Silica gel (200–300 mesh and 80–100 mesh, Qingdao Marine 3.4. Cytotoxic assay
Chemical Inc., China), RP-18 gel (40–75 μm, Fuji Silysia Chemical Ltd.,
Japan) and Sephadex LH-20 (Amersham Biosciences, Sweden) were Five human cancer cell lines, breast cancer SK-BR-3, hepatocellular
used for column chromatography. Fractions were monitored by TLC carcinoma SMMC-7721, human myeloid leukemia HL-60, pancreatic
(Qingdao Marine Chemical Inc., China) and spots visualized by heating cancer PANC-1, and lung cancer A-549 were used. Cells were cultured
silica gel plates immersed in vanillin-H2SO4 in EtOH. in RPMI-1640 or in DMEM medium (Hyclone, USA), supplemented with
10% fetal bovine serum (Hyclone, USA) in 5% CO2 at 37 °C. The cy-
3.2. Fungal material and cultivation conditions totoxicity assay was performed according to 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) method in 96-well mi-
The fungus C. cornucopioides was collected at Ruili city in Yunnan croplates (Mosmann, 1983). Briefly, 100 μL of adherent cells were
Province, People’s Republic of China, in July 2007. The fungus was seeded into each well of 96-well cell culture plates and allowed to
identified by Prof. Mu Zang at the Kunming Institute of Botany. The adhere for 12 h before addition of test compounds, while suspended
voucher specimen was deposited at the Herbarium of Kunming Institute cells were seeded just before drug addition with initial density of
of Botany (No. HFC20120809). Culture medium: glucose (5%), pork 1 × 105 cells/mL. Each tumor cell line was exposed to the test com-
peptone (0.15%), yeast (0.5%), KH2PO4 (0.05%), MgSO4 (0.05%), The pound at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μM (dissolved in
initial pH was adjusted to 6.0, the fermentation was first carried out on DMSO) in triplicates for 48 h, and all tests were done in twice with
an erlenmeyer flask for six days till the mycelium biomass reached to cisplatin (Sigma, USA) as a positive control (IC50: SW480, 12.0 μM;
the maximum. Later it was transferred to a fermentation tank (100 L) at SMMC-7721, 10.2 μM; HL-60, 3.1 μM; MCF-7, 17.5 μM; A-549, 9.1 μM).
24 °C and 250 rpm for twenty days, ventilation was settled to 1.0 vvm After compound treatment, cell viability was detected and a cell growth
(vvm: air volume/culture volumn/min). curve was graphed. IC50 values were calculated by Reed and Muench’s
method (Reed and Muench, 1938).
3.3. Extraction and isolation
Acknowledgments
The culture broth (80 L) was evaporated to 12 L, then extracted
three times with EtOAc (3 × 10 L). The combined EtOAc extracts were
This work was financially supported by the National Natural Science
evaporated in vacuo to give a residue (80.0 g). This was subjected to
Foundation of China (81561148013, U1132607, 81373289), Natural
silica gel column chromatography (CC) with a gradient elution system
Science Fund of Liaoning province (2015020680), and Guangdong
of CHCl3-MeOH (1:0 → 0:1) to obtain ten fractions (A–J). Fraction D
Provincial Key Laboratory of Applied Botany, South China Botanical
was subjected to preparative MPLC with a reversed-phased C18 column
Garden, CAS (AB2015001).
(MeOH–H2O, 0:1 → 6:4) to obtain subfractions D01-D08. Fraction D02
was separated by silica gel CC eluted with petroleum ether (PE)-acetone
(3:1) and further purified by preparative HPLC (AcCN–H2O, 40%) to Appendix A. Supplementary data
give 1 (4.0 mg, retention time (rt) = 12.3 min) and 3 (5.0 mg,
rt = 8.2 min). Fraction D05 was chromatographed on a RP-18 column Supplementary data associated with this article can be found, in the
(MeOH–H2O, 55:45) and then purified on Sephadex LH-20 CC (CHCl3- online version, at http://dx.doi.org/10.1016/j.phytol.2017.06.007.
116
H. Guo et al. Phytochemistry Letters 21 (2017) 114–117
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