Journal of Asian Natural Products Research

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Two new sesquiterpenes produced by the

endophytic fungus Aspergillus fumigatus from
Ligusticum wallichii

Sha Li, Jin-Feng Chen, Ling-Ling Qin, Xiao-Hua Li, Zhi-Xing Cao, Yu-Cheng Gu,
Da-Le Guo & Yun Deng

To cite this article: Sha Li, Jin-Feng Chen, Ling-Ling Qin, Xiao-Hua Li, Zhi-Xing Cao, Yu-Cheng
Gu, Da-Le Guo & Yun Deng (2018): Two new sesquiterpenes produced by the endophytic fungus
Aspergillus�fumigatus from Ligusticum�wallichii, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2018.1540606

To link to this article: https://doi.org/10.1080/10286020.2018.1540606

Published online: 17 Nov 2018.

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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2018.1540606

Two new sesquiterpenes produced by the endophytic

fungus Aspergillus fumigatus from Ligusticum wallichii
Sha Lia, Jin-Feng Chena, Ling-Ling Qina, Xiao-Hua Lia, Zhi-Xing Caoa, Yu-Cheng
Gub, Da-Le Guoa and Yun Denga
a
The Ministry of Education Key Laboratory of Standardization of Chinese Herbal Medicine, State Key
Laboratory, Breeding Base of Systematic Research Development and Utilization of Chinese Medicine
Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu
611137, China; bSyngenta Jealott’s Hill International Research Centre, Berkshire, UK

ABSTRACT ARTICLE HISTORY

Two previously undescribed sesquiterpenes along with nine Received 7 September 2018
known compounds were isolated from the fermentation broth of Revised 19 October 2018
Aspergillus fumigatus, an endophyte of Ligusticum wallichii. Their Accepted 19 October 2018
structures were elucidated through extensive spectroscopic ana-
KEYWORDS
lysis combined with quantum chemical ECD calculations. Two Sesquiterpene; Aspergillus
new compounds exhibited moderate growth inhibition against fumigatus; Ligusticum
MV4-11 and MDA-MB-231 cell lines. wallichii; ECD calculations;
cytotoxicity

1. Introduction
Natural products have played an important role in the discovery and development of
medicinal agents and endophytic fungi which live asymptomatically within the tissues
of higher plants are valuable resources for bio-activities compounds [1]. Various of
secondary metabolites such as peptides [2], polyketides [3], terpenes [4] and nitro-
gen-containing heterocycles [5] with obvious cytotoxicity had been isolated from
endophytic fungi. With the purpose of searching novel compounds with cytotoxicity
from endophytes of traditional Chinese medicine (TCM), Aspergillus fumigatus has
been isolated from the root of Ligusticum wallichii. The chemical investigation of its
fermentation broth led to the isolation of two previously undescribed sesquiterpenes,

CONTACT Dale Guo guodale@cdutcm.edu.cn; Yun Deng dengyun2000@hotmail.com The Ministry of

Education Key Laboratory of Standardization of Chinese Herbal Medicine, State Key Laboratory, Breeding Base of
Systematic Research Development and Utilization of Chinese Medicine Resources, School of Pharmacy, Chengdu
University of Traditional Chinese Medicine, School of Pharmacy, Chengdu, China.
Supplemental data for this article can be accessed at https://doi.org/10.1080/10286020.2018.1540606.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group
2 S. LI ET AL.

Figure 1. The structures of compounds 1–2 isolated from A. fumigatus.

Figure 2. The 1H-1H COSY and key HMBC correlations of 1.

fumagillene A (1) and B (2) Figure 1, along with nine known compounds (3–11).
Details of the isolation, structure elucidation, and cytotoxicity of new compounds are
discussed below.

2. Results and discussion

Compound 1 was obtained as a yellow gum, and HRESIMS ion at m/z 259.1678
[M þ Na]þ revealed its molecular formula to be C15H24O2 (calculated for
C15H24O2Naþ, 259.1669). The IR spectrum of 1 showed the presence of hydroxyl
(3420.6 cm1), methylene (2927.7 cm1), and olefinic (1623.0 cm1) groups. The 1H
NMR spectrum displayed an olefinic proton signal at d 5.04–5.12 (1H, m, H-50 ); a
pair of terminal olefinic proton signals at d 4.82 and 4.93 (each 1H, s, H-7); three
methyl signals at d 1.73 (3H, s, H-10 ), 1.71 (3H, s, H-70 ), and 1.64 (3H, s, H-80 ). The
13
C NMR and HSQC spectra of compound 1 exhibited 15 carbon signals, including
six olefinic carbons at d 150.3 (C-2), 134.6 (C-60 ), 131.3 (C-20 ), 131.2 (C-5), 120.4 (C-
50 ), 108.1 (C-7); two oxymethine carbon signals at d 73.4 (C-1) and 70.0 (C-30 ), four
methylene carbons at d 38.7 (C-6), 33.8 (C-40 ), 32.5 (C-3), 31.7 (C-4) and three
methyl signals at d 26.0 (C-70 ), 18.1 (C-80 ), 12.0 (C-10 ). The 1H-1H COSY correlations
of H-1/H-6, H-3/H-4, H-30 /H-40 , H-40 /H-50 , H-50 /H-70 , and H-50 /H-80 as well as the
HMBC correlations of H-7/C-1, H-7/C-3, H-1/C-5, H-3/C-5, H-4/C-5, H-6/C-5, H-
30 /C-10 , H-30 -C-20 , H-30 /C-5 and H-10 /C-30 indicated the primary structure of com-
pound 1 should be 5-(3-hydroxy-6-methylhept-5-en-2-ylidene)-2-methylenecyclo-
hexan-1-ol as shown in Figure 2. The NOESY correlations between H-10 (d 1.73) and
H-4 (d 2.09–2.20 and 2.27–2.37) indicated they were on the same side of the double
bond between C-10 and C-5 (Figure 3). Due to the amount of compound 1 (only
3.1 mg obtained), its absolute configuration was elucidated by quantum chemical
ECD calculations. The results showed that the calculated ECD curve of (1R,30 R)-1
was similar to the experimental ECD spectrum of 1 as shown in Figure 4. Therefore,
 JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH 3

Figure 3. The key NOESY correlations of 1 and 2.

Figure 4. Calculated ECD spectra of 1 and 2 and their experimental curves.

the absolute configuration of 1 was established as 1R,30 R. It was given a trivial name
as fumagillene A.
Compound 2 had the same molecular formula as 1 (C15H24O2), which was
deduced from HRESIMS. On comparing the 1D- and 2D-NMR spectra of 2 with
those of 1, it appeared that they might be a pair of cis-trans isomer. The NOESY cor-
relation between H-10 (d 1.74) and H-6 (d 2.59–2.67) was observed and indicated the
“E” configuration of the double bond between C-10 and C-5 (Figure 3). The experi-
mental ECD spectrum of 2 was in accordance with the calculated ECD spectrum for
(1R,30 R)-2 (Figure 4), establishing the assignment of the absolute configuration of 2
as (R,E)-5-((R)-3-hydroxy-6-methylhept-5-en-2-ylidene)-2-methylenecyclohexan-1-ol
and compound 2 was named fumagillene B.
The nine known compounds were identified as gliotoxin (3) [6], 5a,6-dihydrobis-
dethio-3,10a-bis(methylthio)gliotoxin (4) [7], verruculogen (5) [8], 12,13-dihydroxyfu-
mitremorgin C (6) [8], fumiquinazoline A (7) [9], cyclo-((S)-Pro-(R)-Val) (8) [10],
pseurotin A (9) [11], cyclo-((S)-Pro-(R)-Ile) (10) [12], and cephalimysin A (11) [13]
on the basis of the NMR and MS spectroscopic comparison with those reported in
the literatures.
MTT method was applied to evaluate the cytotoxicity of novel compounds against
MDA-ME-231 and MV4-11 cancer cell lines. Compound 1 showed moderate growth
inhibition against MV4-11 and MDA-ME-231 with IC50 values of 8.4 ± 2.9 lg/ml and
14.3 ± 5.8 lg/ml. Compound 2 showed moderate growth inhibition against MV4-11
and MDA-ME-231 with IC50 values of 11.2 ± 3.6 lg/ml and 17.3 ± 6.4 lg/ml.
4 S. LI ET AL.

3. Experimental
3.1. General experimental procedures
Optical rotations were determined on a Perkin-Elmer-241 polarimeter (Perkin Elmer,
Inc., Waltham, MA) at room temperature. UV spectra were recorded on a Perkin-
Elmer Lambda 35 UV-VIS spectrophotometer (Perkin Elmer, Inc.). IR spectra were
measured by Perkin-Elmer one FT-IR spectrometer (KBr) (Perkin Elmer, Inc.). CD
spectra were recorded on a Chirascan circular dichroism spectrometer (Applied
Photophysics Ltd, Leatherhead, UK). 1D and 2D NMR were carried out on a Bruker-
Ascend-400 MHz instrument (Bruker, Bremen, Germany) at 300 K, with TMS as
internal standard. HRESIMS were measured using a Synapt G2 HDMS instrument
(Waters Corporation Milford, MA). Preparative HPLC was performed on a NP7000
serials pump (Hanbon Sci. & Tech., Jiangsu, China) equipped with a Kromasil RP-
C18 column (10  250 mm, 5 lm, Akzo Nobel Pulp and Performance Chemicals AB,
Bohus, Sweden) using a NU3000 serials UV detector (Hanbon Sci. & Tech). Column
chromatography (CC) was performed with silica gel (200–300 mesh, Qingdao
Haiyang Chemical Co., Qingdao, China) and Sephadex LH-20 (GE-Healthcare Bio-
Sciences AB, Uppsala, Sweden). All the solvent used were of analytical grade.

3.2. Fungal material and culture

Ligusticum wallichii was collected from the Dujiangyan city, suburb of Chengdu,
China. A. fumigatus was isolated from its root, and further identified by morpho-
logical observation and sequence analyses of the ITS region of rDNA. The strain
(GenBank accession No. MH773172) was deposited at Chengdu University of TCM.
This fungus was cultivated on a 36 L scale using 1L Erlenmeyer flasks containing
400 ml of the seed PDA medium for 7 days and fermentation medium (soluble starch
80 g/L, peptone 5 g/L, NaCl 2 g/L, CaCO3 2 g/L, MgSO47 H2O 0.5 g/L, K2HPO4
0.5 g/L) for 14 days at 28  C on a rotary shaker (250 rpm).

3.3. Extraction and isolation

The fermentation broth (36 L) of A. fumigatus was filtered. The filtrate was firstly
extracted with petroleum ether and followed by EtOAc. The EtOAc solution was
dried under vacuum and yielded 10.8 g extract. The extract was separated into 11
fractions by CC on silica gel (300–400 mesh), eluting stepwise with a petroleum
ether/acetone gradient (100:0; 98:2; 96:4; 94:6; 92:8; 90:10; 85:15; 80:20; 70:30; 50:50;
0:100). The fractions eluted with 94:6 were combined (1.09g) and further subjected to
Sephadex LH-20 column chromatography (4  180 cm; CHCl3-MeOH, 1:1) afforded 3
subfractions (Fr.1–Fr.3). Fr.3 (0.31 g) was purified by a preparative HPLC using a
reversed-phase column to afford 1 (3.1 mg, wavelength: 220 nm, MeOH-H2O, 65:35,
3 ml/min, tR: 17.2 min) and 2 (2.6 mg, MeOH-H2O, wavelength: 220 nm, 65:35, 3 ml/
min, tR: 19.3 min). The fractions eluted with 92:8 were combined (1.73 g) and further
subjected to Sephadex LH-20 column chromatography (4  180 cm; CHCl3-MeOH,
1:1) afforded three subfractions (Fr.1–Fr.3). Fr.1 (0.68g) was purified by a preparative
HPLC using a reversed-phase column to afford 3 (7.2mg, wavelength: 220 nm,
 JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH 5

Table 1. 1H and 13
C NMR spectral data of 1 and 2.a
1 2
Position dH (J in Hz) dC dH (J in Hz) dC
1 4.14, dd (6.6, 4.0) 73.4 4.15, dd (8.1, 4.1) 72.8
2 – 150.3 – 150.4
3 2.07–2.16, m; 2.36–2.46, m 32.5 2.07–2.19, m; 2.31–2.43, m 33.0
4 2.09–2.20, m; 2.27–2.37, m 31.7 2.10–2.22, m; 2.33–2.43, m 34.6
5 – 131.2 – 130.6
6 2.39–2.48, m; 2.49–2.57, m 38.7 2.24–2.32, m; 2.59–2.67, m 40.4
7 4.82, s; 4.93, s 108.1 4.80, s; 4.92, s 106.9
10 1.73, s 12.0 1.74, s 12.0
20 – 131.3 – 131.2
30 4.64, dd (7.6, 6.7) 70.0 4.70, t (7.2) 70.7
40 2.10–2.20, m; 2.33–2.42, m 33.8 2.10–2.24, m; 2.31–2.41, m 30.3
50 5.04–5.12, m 120.4 5.05–5.12, m 120.1
60 – 134.6 – 135.2
70 1.71, s 26.0 1.71, s 26.1
80 1.64, s 18.1 1.64, s 18.1
a
400 MHz for 1H and 100 MHz for 13
C in CDCl3.

MeOH-H2O, 60:40, 3 ml/min, tR: 25.2 min) and 4 (5.7mg, wavelength: 220 nm, MeOH-
H2O, 55:45, 3 ml/min, tR: 37.8 min), Fr.2 (0.47g) was purified by a preparative HPLC
using a reversed-phase column to afford 7 (4.2 mg, wavelength: 220 nm, MeOH-H2O,
40:60, 3 ml/min, tR: 25.3 min). The fractions eluted with 90:10 were combined (2.52g)
and further subjected to Sephadex LH-20 column chromatography (4  180 cm;
CHCl3-MeOH, 1:1) afforded three subfractions (Fr.1–Fr.3). Fr.1 (1.45g) was purified
by a preparative HPLC using a reversed-phase column to afford 5 (6.2 mg, wavelength:
220 nm, MeOH-H2O, 65:35, 3 ml/min, tR: 28.3 min) and 6 (7.3 mg, wavelength:
220 nm, MeOH-H2O, 60:40, 3 ml/min, tR: 25.2 min), Fr.2 (0.53g) was purified by a pre-
parative HPLC using a reversed-phase column to afford 9 (5.2 mg, wavelength:
220 nm, MeOH-H2O, 65:35, 3 ml/min, tR: 22.3 min). The fractions eluted with 85:15
were combined (1.07g) and further subjected to Sephadex LH-20 column chromatog-
raphy (4  180 cm; CHCl3-MeOH, 1:1) afforded three subfractions (Fr.1–Fr.3). Fr.1
(0.60 g) was purified by a preparative HPLC using a reversed-phase column to afford
10 (23.1 mg, wavelength: 210 nm, MeOH-H2O, 30:70, 3 ml/min, tR: 15.3 min) and 11
(4.2 mg, wavelength: 220 nm, MeOH-H2O, 65:35, 3 ml/min, tR: 23.5 min), Fr.3 (0.33g)
was purified by a preparative HPLC using a reversed-phase column to afford 8
(20.7 mg, wavelength: 210 nm, MeOH-H2O, 30:70, 3 ml/min, tR: 15.5 min).

3.3.1. Fumagillene A (1)

Yellow gum, [a]20Dþ9.95 (c 0.38, MeOH); IR (KBr)  max 3420.6, 2927.7, 1623.0,
1384.7, 1094.8 cm1; UV kmax 208.0 (3.78), 238.4 (2.79) nm; ECD (c 1.61  103 M,
MeOH) De196 nm þ16.44, De200 nm –5.12, De222 nm 2.11; 1H-NMR and 13C-NMR
spectral data see Table 1; HRESIMS: m/z 259.1678 [M þ Na]þ (calcd for
C15H24O2Na, 259.1669).

3.3.2. Fumagillene B (2)

D–13.2 (c 0.11, MeOH); IR (KBr)  max 3524.8, 2926.2, 1623.1, 1384.6,
Yellow gum, [a]20
1087.8 cm1; UV kmax 204.4 (3.75), 239.3 (2.78) nm; ECD (c 9.40  10–4 M, MeOH)
De196 nm 18.65, De208 nm –6.23; 1H-NMR and 13C-NMR spectral data see Table 1;
HRESIMS: m/z 259.1675 [M þ Na]þ (calcd for C15H24O2Na, 259.1669).
6 S. LI ET AL.

3.4. MTT assay

MDA-ME-231 and MV4-11 cells were cultured in 96-well plates. The cells were
treated with the compounds at the specified concentrations for 72 h, followed by
standard MTT assays with taxol (antiproliferative activity against the MDA-ME-231
and MV4-11 cell lines with IC50 values of 0.014 ± 0.005 lg/ml and 0.039 ± 0.016 lg/ml
respectively) as positive control. The detailed process is described previously [14].

Disclosure statement
No potential conflict of interest was reported by the authors.

Acknowledgments
The authors wish to thank Dr. Jing Yang (Kunming Institute of Botany, Chinese Academy of
Sciences) for the help in ECD calculation.

Funding
This research was funded by Sichuan Provincial Youth Science and Technology Innovation
Team (2016TD0006, 2015TD0028); China Postdoctoral Science Foundation (2017M622985).

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