Phytochemistry Letters 21 (2017) 114–117

Contents lists available at ScienceDirect

Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol

Sesquiterpenoids from cultures of the edible mushroom Craterellus MARK

cornucopioides
⁎
Hua Guoa,b, Quan-Ping Diaoa, Dong-Yan Houa, Zheng-Hui Lib, Zhong-Yu Zhouc, Tao Fengb, ,
⁎
Ji-Kai Liub,
a
School of Chemistry and Life Science, Anshan Normal University, Anshan 114005, China
b
School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan 430074, China
c
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China

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

1. Introduction formula was determined to be C15H20O2 by HREIMS, with a molecular

Illudin-type sesquiterpenoids have been widely reported as anti-
bacterial and antitumor agents (Anchel et al., 1950; McMorris and
Anchel, 1965; Murgo et al., 1999). Some basidiomycetes have been
reported to produce different illudins and related compounds (Gonzalez
del Val et al., 2003; Liu et al., 2011; Nakanishi et al., 1963; Reina et al.,
2004; Wang et al., 2011; Zhu et al., 2010). Craterellus cornucopioides (L.
Fr.) Pers. (Cantharellaceae) is an edible fungus with a wide distribution
in most parts of China, especially in the Southwestern. In our previous
work, the fungus has been reported to produce a series of keto esters
(Liu et al., 2010). In the course of our continuous searching for more
biologically active compounds, we increased the fermentation scale
(from 20 L to 80 L) of C. cornucopioides, and isolated three new illudin
and one new gymnomitrane sesquiterpenoids, named as craterellins
A–C (1–3) and gymnomitr-3-en-10β,15-diol (4), along with four pre-
viously reported sesquiterpenoids: illudin F (5) (Burgess et al., 1999),
illudin M (6) (Anchel et al., 1950), illudin T (7) (Wang et al., 2011) and
illudalenol (8) (Arnone et al., 1991) (Fig. 1). Compounds 1–4 were
evaluated for their cytotoxic activities on five human cancer cell lines.
Herein, we report the isolation, structural elucidation, and cytotoxic
activities of these compounds.
2. Results and discussion
Craterellin A (1) was obtained as a colorless oil. Its molecular
ion at m/z 232.1461 [M]+, which indicated six degrees of unsaturation.
The IR spectrum showed absorption bands for hydroxy group
(3441 cm−1) and double bond (1657 cm−1). Inspection of the 1H- and
13
C NMR spectra revealed the existence of three methyl groups, four
methylene units (including one olefinic methylene group), and two
methine units (including one oxymethane group). Additionally, six
quaternary carbons (one belonging to a ketone group and three to
double bonds) were identified from both the 13C NMR and HMBC
spectra. The proton and carbon signals (Tables 1 and 2) were assigned
on the basis of 2D-NMR data (COSY, HSQC, and HMBC). These data
revealed that compound 1 was an illudin-type sesquiterpenoid closely
related to illudin F (5) (Burgess et al., 1999) except for the absence of
two hydroxy groups at C-2 and C-8 in 5. A COSY correlation of H-2 with
H-10 and the HMBC cross-peaks of H-2 (δH 1.79) with C-1 (δC 201.8), C-
3 (27.2) and C-10 (16.2) suggested that C-2 is now a methine. Similarly,
correlations of H-8 (δH 2.30 and 2.56) with C-5 (δC 155.7), C-9 (136.4)
and C-1 (201.8) indicated that C-8 is a methylene. In the light of the
evidences mentioned above and the key 1H–1H COSY and HMBC cor-
relations (Fig. 2), the planar structure of 1 was therefore elucidated as
shown in Fig. 1. The relative configuration of 1 was established by an
ROESY experiment, which displayed correlations related to those of
illudin F (Burgess et al., 1999). As shown in Fig. 2, ROESY correlations
of H-2/H-11a and H-12a/H-13b suggested that H-2 and CH2-11 were α
oriented, CH2-12 and H-13b should be β oriented, while a correlation of
H-13a with H-6 indicated an α-orientation for this last proton. In

⁎
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

1a 2.01 dd (11.2, 4.7)

1b 1.51 d (11.0)
2 1.79 m 2.77 q (7.0) 1.90 d (4.5)
4 5.53 br s
5a 2.25 d (18.0)
5b 1.96 d (18.0) Fig. 2. Key 1H–1H COSY, HMBC and ROESY correlations of 1.
6a 4.58 s 2.44 br s 2.26 dd (18.0, 18.0)
6b
8a 2.56 d (17.0) 4.61 s 2.17, d (15.7) 1.57 m addition, the resonances of Me-14 and Me-15 were assigned considering
8b 2.30 d (17.0) 2.05, d (15.7) 1.02 m the H-6/H-14 correlation observed in the ROESY experiment (Fig. 2).
9a 1.91 m
All these data indicated that craterellin A should have the structure 1.
9b 1.67 m
10 1.08 d (7.3) 0.90 d (7.0) 1.11 s 4.16 dd (10.1, 5.6)
The molecular formula of compound 2 was determined as C15H22O3
11a 1.09 m 0.56 m 1.66 m considering the molecular ion at m/z 250.1572 [M]+ observed in its
11b 0.51 m 0.52 m 1.53 m HREIMS, corresponding to five degrees of unsaturation. The 1D- and
12a 0.69 m 0.68 m 3.67 dd (15.6, 8.3) 0.97 s 2D-NMR data suggested that the structure of 2 was related to that of 1.
12b 0.47 m 0.37 m 3.29 overlap
One difference was that the C-4,C-13 double bond in 1 was replaced in
13a 5.48 s 1.47 s 1.21 s 0.91 s
13b 5.12 s 2 with a C-13 methyl and an oxygenated quaternary carbon (C-4).
14 1.11 s 1.06 s 0.96 s 0.89 s Which was inferred from the HMBC correlations observed between H-
15 1.12 s 1.14 s 0.91 s 4.03 d (12.3) 13(δH 1.47) and C-3 (δC 31.5), C-4 (70.7) and C-5 (168.1). Additionally,
4.05 d (12.3) the OH-6 in 1 was located at C-8 in 2, taking in consideration the
OH-2 5.29 s
OH-3 4.70 s
correlations showed between H-8 (δH 4.61) and C-1 (δC 200.1), C-9
(136.1) and C-5 (168.1). In the ROESY spectrum, the correlation be-
a
Measured in CDCl3. tween H-2 and Me-13 suggested that Me-13 should be α oriented.
b
Measured in DMSO-d6. However, the ROESY spectrum could not establish the configuration of
C-8. Due to the limited amount available of 2, the configuration of C-8
could not be determined using any further method. Therefore, the
Table 2 structure of compound 2 was elucidated as shown in Fig. 1, and named
13
C NMR data of compounds 1–4. craterellin B.
No. 1a 2a 3b 4a
The molecular formula of craterellin C (3) was inferred to be
C15H22O4 on the basis of the [M]+ at m/z 266.1521 observed in
1 201.8 200.1 195.3 42.9 HREIMS. All the data suggested that the structure of 3 was closely re-
2 51.6 45.0 77.3 46.3 lated to that of illudalenol (8) (Arnone et al., 1991). However, the 2,3-
3 27.2 31.5 83.3 142.9
double bond was substituted by two hydroxyl groups at C-2 (δC 77.3)
4 141.6 70.7 83.2 122.1
5 155.7 168.1 164.3 40.2 and C-3 (83.3), which were assigned considering the HMBC correlations
6 83.4 44.9 46.6 43.4 of OH-2 (δH 5.29) with C-1 (δC 195.3), C-2 (77.3), C-3 (83.3), C-4 (83.2)
7 42.0 42.2 37.5 60.0 and C-11(36.9) (Fig. 2). In addition to four degrees of unsaturation
8 41.9 81.4 44.1 31.1
occupied by two rings and two double bonds, the remaining degree of
9 136.4 136.1 133.5 34.7
10 16.2 9.0 18.3 76.5 unsaturation required that compound 3 should have a 4,12-ether
11 17.6 4.8 36.9 55.1 moiety, which was in agreement with the HMBC correlation of H-12 (δH
12 7.9 2.8 63.8 18.9 3.67) with C-4 (δC 83.2). The relative configuration of 3 was deduced
13 112.4 23.3 19.3 23.9 from the ROESY correlations of H3-10/H2-11, H3-13/OH-2, and H3-13/
14 22.1 22.1 28.9 24.4
OH-3. Consequently, the structure of craterellin C was determined as 3.
15 28.0 27.6 29.4 67.2
The molecular formula of 4 was determined to be C15H24O2 on the
a
Measured in CDCl3. basis of HREIMS ([M]+ at m/z 236.1776), corresponding to four de-
b
Measured in DMSO-d6. grees of unsaturation. The IR spectrum showed absorption bands for
hydroxy group (3425 cm−1) and double bond (1462 cm−1). The 1H-
and 13C NMR spectra showed signals of three tertiary methyls, a

115
H. Guo et al.
primary alcohol at δH 4.03 and 4.05 (each 1H, d, J = 12.3 Hz) and δC
67.2, a secondary alcohol at δH 4.16 (1H, dd, J = 10.1, 5.6 Hz) and δC
76.1, and a atrisubstituted double bond at δC 122.1 and 142.9. These
data indicate a tricyclic sesquiterpenoid with similarities to those of 3-
gymnomitren-15-ol (Buchanan et al., 1996), except for an additional
hydroxyl substitution. The COSY spectrum afforded a fragment of H-8/
H-9/H-10, while the HMBC spectrum showed correlations of H-10 (δH
4.16) with C-9 (δC 34.7), C-10 (76.5) and C-11 (55.1). These 2D-NMR
experiments indicated that the additional hydroxyl group was located
at C-10. The ROESY correlation of H-10/H-15 indicated that OH-10
should be β oriented. From the above data, the structure of this ses-
quiterpenoid was established as gymnomitr-3-en-10β,15-diol (4).
All new compounds were evaluated for their cytotoxicities against
five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and
SW-480) using the MTT method. Compound 3 exhibited moderate cy-
totoxicity against A-549 with IC50 value of 21.0 μM.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a Jasco-P-1020 polarimeter. UV
spectra were run on a Shimadzu UV-2401 PC spectrophotometer. IR
spectra were obtained using a Bruker Tensor 27 FT-IR spectrometer
with KBr pellets. NMR spectra were acquired with Bruker instruments
(Avance III 600, DRX-500 and Bruker AV 400). HREIMS were measured
on a Waters Auto Premier P776 mass spectrometer respectively.
Preparative HPLC was performed on an Agilent 1100 series with a DAD
detector and a Zorbax SB-C18 (5 μm, 9.4 × 150 mm) column.
Preparative MPLC was carried out on a Büchi apparatus equipped with
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
Chemical Inc., China), RP-18 gel (40–75 μm, Fuji Silysia Chemical Ltd.,
Japan) and Sephadex LH-20 (Amersham Biosciences, Sweden) were
used for column chromatography. Fractions were monitored by TLC
(Qingdao Marine Chemical Inc., China) and spots visualized by heating
silica gel plates immersed in vanillin-H2SO4 in EtOH.
3.2. Fungal material and cultivation conditions
The fungus C. cornucopioides was collected at Ruili city in Yunnan
Province, People’s Republic of China, in July 2007. The fungus was
identified by Prof. Mu Zang at the Kunming Institute of Botany. The
voucher specimen was deposited at the Herbarium of Kunming Institute
of Botany (No. HFC20120809). Culture medium: glucose (5%), pork
peptone (0.15%), yeast (0.5%), KH2PO4 (0.05%), MgSO4 (0.05%), The
initial pH was adjusted to 6.0, the fermentation was first carried out on
an erlenmeyer flask for six days till the mycelium biomass reached to
the maximum. Later it was transferred to a fermentation tank (100 L) at
24 °C and 250 rpm for twenty days, ventilation was settled to 1.0 vvm
(vvm: air volume/culture volumn/min).
3.3. Extraction and isolation
The culture broth (80 L) was evaporated to 12 L, then extracted
three times with EtOAc (3 × 10 L). The combined EtOAc extracts were
evaporated in vacuo to give a residue (80.0 g). This was subjected to
silica gel column chromatography (CC) with a gradient elution system
of CHCl3-MeOH (1:0 → 0:1) to obtain ten fractions (A–J). Fraction D
was subjected to preparative MPLC with a reversed-phased C18 column
(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
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
(MeOH–H2O, 55:45) and then purified on Sephadex LH-20 CC (CHCl3-
116
Phytochemistry Letters 21 (2017) 114–117
MeOH, 1:1) to give 5 (7.0 mg). Fraction F was chromatographed over a
silica gel column using PE–acetone (10:1 → 0:1) to produce fractions
F01-F06. Compounds 2 (6.0 mg, rt = 11.2 min) and 4 (10.0 mg,
rt = 11.9 min) were afforded from fraction F04 by preparative HPLC
(CH3CN–H2O, 22:78, flow rate = 10 mL/min, 30 min), compound 6
(6.0 mg, rt = 12.4 min) was also obtained by preparative HPLC
(CH3CN–H2O, 30:70, flow rate = 10 mL/min, 30 min) from the F03
fraction. Fraction F05 was subjected to a RP-18 column (MeOH–H2O,
38:62), then purified on a silica gel column (PE-acetone, 2:1) to afford 7
(4.5 mg) and 8 (6.5 mg).
D −76.0 (c 0.22, MeOH); UV
Craterellin A (1), colorless oil; [α]15
(MeOH) λmax (log ε) 286 (3.3), 205 (3.3) nm; IR (KBr) νmax 3441, 3428,
2823, 1664, 1657, 1423, 1186, 960 cm−1; 1H NMR (CDCl3, 500 MHz)
and 13C NMR (CDCl3, 125 MHz) data, see Tables 1 and 2; HR-EI-MS m/z
232.1461 (calcd for C15H20O2, 232.1463).
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,
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
250.1572 (calcd for C15H22O3, 250.1569).
D −49.8 (c 0.24, MeOH); UV
Craterellin C (3), colorless oil; [α]15
(MeOH) λmax (log ε) 247 (3.2), 207 (2.8), 194 (2.9) nm; IR (KBr) νmax
3426, 2834, 1673, 1424, 1160, 965 cm−1; 1H-NMR (DMSO-d6,
600 MHz) and 13C NMR (DMSO-d6, 150 MHz) data, see Tables 1 and 2;
HR-EI-MS m/z 266.1521 (calcd for C15H22O4, 266.1518).
D −26.7 (c 0.17,
Gymnomitr-3-en-10β,15-diol (4), colorless oil; [α]15
MeOH); UV (MeOH) λmax (log ε) 203 (2.9) nm; IR (KBr) νmax 3425,
2930, 2869, 1462, 1049 cm−1; 1H NMR (CDCl3, 600 MHz) and 13C
NMR (CDCl3, 150 MHz) data, see Tables 1 and 2; HR-EI-MS m/z
236.1776 (calcd for C15H24O2, 236.1776).
3.4. Cytotoxic assay
Five human cancer cell lines, breast cancer SK-BR-3, hepatocellular
carcinoma SMMC-7721, human myeloid leukemia HL-60, pancreatic
cancer PANC-1, and lung cancer A-549 were used. Cells were cultured
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-
totoxicity assay was performed according to 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) method in 96-well mi-
croplates (Mosmann, 1983). Briefly, 100 μL of adherent cells were
seeded into each well of 96-well cell culture plates and allowed to
adhere for 12 h before addition of test compounds, while suspended
cells were seeded just before drug addition with initial density of
1 × 105 cells/mL. Each tumor cell line was exposed to the test com-
pound at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μM (dissolved in
DMSO) in triplicates for 48 h, and all tests were done in twice with
cisplatin (Sigma, USA) as a positive control (IC50: SW480, 12.0 μM;
SMMC-7721, 10.2 μM; HL-60, 3.1 μM; MCF-7, 17.5 μM; A-549, 9.1 μM).
After compound treatment, cell viability was detected and a cell growth
curve was graphed. IC50 values were calculated by Reed and Muench’s
method (Reed and Muench, 1938).
Acknowledgments
This work was financially supported by the National Natural Science
Foundation of China (81561148013, U1132607, 81373289), Natural
Science Fund of Liaoning province (2015020680), and Guangdong
Provincial Key Laboratory of Applied Botany, South China Botanical
Garden, CAS (AB2015001).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.phytol.2017.06.007.
H. Guo et al.
References
Anchel, M., Hervey, A., Robbins, W., 1950. Antibiotic substances from basidiomycetes
Clitocybe illudins. Proc. Natl. Acad. Sci. U. S. A. 36, 300–305.
Arnone, A., Cardillo, R., Nasini, G., Vajna de Pava, O., 1991. Secondary mold metabolites
Part 31. Isolation and structure elucidation of illudins A and B, and illudalenol, new
sesquiterpenoids from Clitocybe illudens. J. Chem. Soc. Perkin Trans. 1, 733–737.
Buchanan, M.S., Connolly, J.D., Kadir, A.A., Rycroft, D.S., 1996. Sesquiterpenoids and
diterpenoids from the liverwort Jungermannia truncata. Phytochemistry 42,
1641–1646.
Burgess, M.L., Zhang, Y.L., Barrow, K.D., 1999. Characterization of new illudanes, illudins
F, G, and H from the basidiomycete Omphalotus nidiformis. J. Nat. Prod. 62,
1542–1544.
Gonzalez del Val, A., Platas, G., Arenal, F., Orihuela, J.C., Garcia, M., Hernández, P.,
Royo, I., De Pedro, N., Silver, L.L., Young, K., Vicente, M.F., Pelaez, F., 2003. Novel
illudins from Coprinopsis episcopalis (syn. Coprinus episcopalis): and the distribution of
illudin-like compounds among filamentous fungi. Mycol. Res. 107, 1201–1209.
Liu, R., Zhou, Z.Y., Liu, J.K., 2010. Three new keto esters from cultures of the basidio-
mycete Craterellus cornucopioides. Chin. J. Nat. Med. 8, 88–90.
Liu, L.Y., Zhang, L., Feng, T., Li, Z.H., Dong, Z.J., Liu, J.K., 2011. Unusual illudin-type
sesquiterpenoids from cultures of Agrocybe salicacola. Nat. Prod. Bioprospect. 1,
117
Phytochemistry Letters 21 (2017) 114–117
87–92.
McMorris, T.C., Anchel, M., 1965. Fungal: metabolites: The structures of the novel ses-
quiterpenoids illudin-S and -M. J. Am. Chem. Soc. 87, 1594–1600.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63.
Murgo, A., Cannon, D.J., Blatner, G., Cheson, B.D., 1999. A derivative of illudin was
effective in clinical trials. Oncology 13, 233–238.
Nakanishi, K., Tada, M., Yamada, Y., Ohashi, M., Komatsu, N., Terakawa, H., 1963.
Isolation of lampterol, an antitumor substance from Lampteromyces japonicus. Nature
197, 292.
Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent endpoints. Am.
J. Hygiene 27, 493–497.
Reina, M., Orihuela, J.C., Gonzalez-Coloma, A., de Ines, C., de la Cruz, M., Gonzalez del
Val, A., Torno, J.R., Fraga, B.M., 2004. Four illudane sesquiterpenes from Coprinopsis
episcopalis. Phytochemistry 65, 381–385.
Wang, G., Yu, L.L., Zhu, Y.C., Liu, J.K., 2011. Illudin T, a new sesquiterpenoid from ba-
sidiomycete Agrocybe salicacola. J. Asian Nat. Prod. Res. 13, 430–433.
Zhu, Y.C., Wang, G., Yang, X.L., Luo, D.Q., Zhu, Q.C., Peng, T., Liu, J.K., 2010.
Agrocybone, a novel bis-sesquiterpene with a spirodienone structure from basidio-
mycete Agrocybe salicacola. Tetrahedron. Lett. 51, 3443–3445.