全 文 ： 421 Journal of Chinese Pharmaceutical Sciences http://www.jcps.ac.cn
Two terpenoids and a polyketide from the endophytic fungus Trichoderma
sp. Xy24 isolated from mangrove plant Xylocarpus granatum
Min Zhang1,2, Ning Li1,2, Ridao Chen2, Jianhua Zou2, Chunmei Wang1, Jungui Dai1,2*
1. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China
2. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy
of Medical Sciences & Peking Union Medical College, Beijing 100050, China 
Abstract: Three known compounds were isolated from the endophytic fungus Trichoderma sp. Xy24 from a mangrove plant
Xylocarpus granatum by using various column chromatography techniques. Their structures were identified as harzianone (1),
trichoacorenol (2), and trichodimerol (3) by extensive spectroscopic analysis. Among them, 1 was a harziane diterpene, 2 was
a sesquiterpene alcohol, and 3 was a polyketide with a completely symmetric configuration. Compound 3 exhibited medium
inhibitory activity with an IC50 value of 74.6 μM using a NA (H7N9)/MUNANA model.
Keywords: Endophytic fungus, Trichoderma sp. Xy24, Terpenoid, Polyketide, Xylocarpus granatum
CLC number: R284 Document code: A Article ID: 1003–1057(2014)6–421–04
Received: 2014-04-07, Revised: 2014-05-05, Accepted: 2014-05-20.
Foundation items: Science & Technology Project of Guangdong
Province (Grant No. 2011A080403020) and the Fundamental Research
Funds for the Central Universities (Grant No. 2012N06).
*Corresponding author. Tel.: 86-10-63165195,
E-mail: jgdai@imm.ac.cn 
http://dx.doi.org/10.5246/jcps.2014.06.056
1. Introduction
“Mangrove” designates an intertidal wetland ecosystem
formed by a very special association of animals and
plants which proliferate luxuriantly in the coastal areas
and river estuaries throughout the low lying tropical
and sub-tropical latitudes[1]. It includes as many as
50 tree species in about 13 genera and 8 families[2]. As
for their special living environment, mangroves have
been important habitats for varieties of endophytic
fungi. Recently, endophytic fungi have drawn much
more worldwide attention. Many secondary metabolites
with good biological activities have been found from
the endophytic fungi[3].
Trichoderma is widely used as a biocontrol microbe
against plant diseases in the world, which is attributed
to the biosynthesis of a wide array of secondary
metabolites, transformation of a great variety of natural
and xenobiotic compounds and production of various
degradative enzymes[4]. In recent years, many secondary
metabolites exhibiting a variety of biological activities,
such as antibacterial activities[5] and anti-influenza virus






activities[6], have been isolated from the Trichoderma
species. In this study, the endophytic fungus Trichoderma
sp. Xy24 was isolated from the leaves, stems and peels
of a mangrove plant Xylocarpus granatum collected
in Sanya, Hainan province, China[7]. As part of our
ongoing search for structurally novel metabolites with
interesting biological activities from endophytic
fungi[8,9], Trichoderma sp. Xy24 was scaled up to 10 L
level, and three compounds (Fig. 1) were isolated
and identified as harzianone (1), trichoacorenol (2),
and trichodimerol (3) by various spectroscopic data
analyses. Among them, 1 was a harziane diterpene,
2 was a sesquiterpene alcohol, and 3 was a polyketide
with a completely symmetric configuration. Their
antiviral activity was tested and compound 3 exhibited
medium inhibition with an IC50 value of 74.6 μM.
Herein, we report their detailed isolation, structural
characterization, and antiviral activity.
2. Experimental
2.1. General procedures
Optical rotations were measured on a PerkinElmer
Model 341 LC polarimeter. The NMR spectra were
collected using a Varian MP-400 spectrometer. Chemical
shifts (δ) are given in parts per million (ppm), coupling
constants (J) are given in hertz (Hz). HR-ESI-MS were
422 Zhang, M. et al. / J. Chin. Pharm. Sci. 2014, 23 (6), 421–424
measured on a Q-trap ESI mass spectrometer (Applied
Biosystems/MDS Sciex, Carlsbad, CA, USA). ESI-MS
data were measured on a LCQ Fleet mass spectrometer
(Thermo, USA). Column chromatography (CC) was
carried out with silica gel (200−300 mesh, Qingdao
Marine Chemical Inc. Qingdao, China). Semi-preparative
HPLC was performed on a Shimadzu HPLC instrument
equipped with a Shimadzu RID-10A detector and a Grace
Allsphere silica column (250 mm×10 mm, 5 μm) by
eluting with mixtures of n-hexane and ethyl acetate
(EtOAc) or a Grace Adsorbosphere C18 column
(250 mm×10 mm, 5 μm) by eluting with mixtures of
CH3CN and H2O. Analytical TLC was carried out on
pre-coated silica gel GF254 plates (Qingdao Marine
Chemical Industry, Qingdao, China), and spots were
visualized under UV light and by spraying with 5%
H2SO4 in 95% EtOH followed by heating at 120 °C.
All solvents used for CC were of analytical grade
(Beijing Chemical Works, Beijing, China).
2.2. Fungal material
The fungal strain Trichoderma sp. Xy24 was isolated
from the leaves, stems and peels of mangrove plant
Xylocarpus granatum collected in Sanya district, Hainan
Province of China. It was identified as Trichoderma
species according to the morphological and molecular
(ITS1-5.8S-ITS2 rDNA sequence) analyses by our
research group. The strain was deposited at the
Institute of Materia Medica, Chinese Academy of
Medical Sciences.
2.3. Fermentation, extraction and isolation
The fungal strain was maintained on slants of
modified potato dextrose agar (PDA) medium (potato
200 g, glucose 20 g, distilled water 1 L, KH2PO4 3 g,
MgSO4 0.73 g, vitamin B1 10 mg, agar 8.0 g, pH 6.0;
the media were autoclaved at 121 °C for 30 min) at
4 °C. Seed cultures were performed in Erlenmeyer
flasks (250 mL) containing 100 mL of PDA liquid
medium on a shaker at 150 r/min at 25 °C for 2 d, after
that 5 mL seed cultures were inoculated into each
1000 mL flask with 250 mL medium and cultivated for
10 d. Cultures (10 L) were filtered under reduced
pressure to afford the filtrate and mycelia. The filtrate
was partitioned with EtOAc and the EtOAc extract was
evaporated under reduced pressure to yield 3 g of
residue. The dried mycelia were extracted with methanol
by the ultrasonic extraction method to afford 10 g of
residue. The residues of the two parts were combined
on the basis of their TLC analysis in which they
showed the similar TLC pattern. The combined extract
was subjected to silica gel CC eluting with a petroleum
ether EtOAc gradient (100:0–0:100, v/v) to give nine
fractions based on TLC analysis.
Fraction 1 (1.2 g) was initially separated via normal-
phase semi-preparative HPLC using n-hexane–EtOAC
(25:1, v/v) at 4 mL/min to obtain three fractions
(Fr1.1–Fr1.3), Fr1.1 (65.0 mg) was further separated
by reversed-phase semi-preparative HPLC eluting
with CH3CN–H2O (85:15, v/v) at 3 mL/min to obtain
three fractions (Fr1.1.1–Fr1.1.3), and Fr1.1.3 (15.5 mg,
tR 19.6 min) was successively subjected to Sephadex
LH-20 column chromatography to afford compound 1
(11.1 mg). The Fr1.3 (20.3 mg) was further isolated
by reversed-phase semi-preparative HPLC eluting
with CH3CN–H2O (45:55, v/v) at 3 mL/min to yield
compound 2 (15.0 mg, tR 16.6 min). Fraction 3 (269 mg)
was purified by normal-phase semi-preparative
HPLC using a mobile phase of n-hexane–EtOAC
(4:1, v/v) at 4 mL/min to give compound 3 (22.1 mg,
tR 22.4 min).
Figure 1. Chemical structures of compounds 1–3.
3
2
14
6
5
8
12
11
O
2019
H
18
117
16
H
1
(S)
5
(R)
7
9
(S) 1
3
(S)
12
OH
1514
2
1
6
4
3
O O
HO OH
OH
13
HO
14
8
12
O
O
3
423 Zhang, M. et al. / J. Chin. Pharm. Sci. 2014, 23 (6), 421–424
3.1. Harzianone (1)
Colorless oil; +42.9° (c 0.14, MeOH); positive
ESI-MS m/z 287.12 [M+H]+, 309.09 [M+Na]+; 1H NMR
(400 MHz, CDCl3) δ: 2.51 (1H, d, J 16.2 Hz, H-12b),
2.41 (1H, m, H-5), 2.36 (1H, d, J 16.2 Hz, H-12a),
2.14 (1H, dd, J1 11.4 Hz, J2 8.9 Hz, H-14), 2.07 (1H,
s, H-20), 2.02 (1H, m, H-4b), 1.93 (1H, m, H-3b),
1.90 (1H, m, H-8b), 1.85 (1H, m, H-15b), 1.80 (1H, m,
H-7b), 1.64 (1H, m, H-2), 1.46 (3H, s, H-19), 1.35
(1H, dd, J1 13.3 Hz, J2 8.9 Hz, H-15a), 1.30 (1H, m,
H-3a), 1.24 (3H, m, H-4a, H-7a, and H-8a), 1.01 (3H,
s, H-17), 1.01 (3H, d, J 7.3 Hz, H-18), 0.83 (3H, s,
H-16); 13C NMR (100 MHz, CDCl3) δ: 199.5 (C-11),
150.2 (C-10), 146.6 (C-9), 60.1 (C-12), 52.4 (C-14),
51.0 (C-6), 46.4 (C-1), 43.0 (C-2), 41.0 (C-13), 30.4
(C-7), 29.5 (C-8), 29.4 (C-5), 27.7 (C-15), 26.2 (C-16),
26.0 (C-3), 25.5 (C-4), 22.8 (C-20), 22.7 (C-17), 21.8
(C-19), 20.9 (C-18). All these data were in good
agreement with those of harzianone[10].
3.2. Trichoacorenol (2)
White powder; –16.6° (c 0.12, CHCl3); EI-MS
m/z 222 [M]+; 1H NMR (400 MHz, CDCl3) δ: 5.45 (1H,
m, H-9), 4.25 (1H, br s, H-7), 2.10 (1H, m, H-10b),
1.79 (1H, m, H-10a), 1.76 (5H, br s, H3-15, H-2b, and
H-6b), 1.52-1.73 (3H, m, H-4, H-3, and H-11), 1.41
(1H, m, H-2), 1.09−1.28 (3H, m, H-3, H-1, and H-6),
0.89 (3H, d, J 6.5 Hz, H-13), 0.83 (3H, d, J 6.5 Hz,
H-12), 0.82 (3H, d, J 6.8 Hz, H-14); 13C NMR (100
MHz, CDCl3) δ: 132.4 (C-6), 125.2 (C-9), 68.8 (C-7),
59.9 (C-1), 46.7 (C-4), 45.1 (C-5), 35.6 (C-8), 35.4
(C-10), 30.3 (C-11), 28.9 (C-3), 26.5 (C-2), 23.4 (C-13),
23.0 (C-12), 19.0 (C-15), 14.3 (C-14). By comparing all
the above data with those published in the literature[11],
compound 2 was established to be trichoacorenol.
3.3. Trichodimerol (3)
Yellow powder; positive HR-ESI-MS m/z 497.2142
[M+H]+ (calcd. for C28H33O8, 497.2170); 1H NMR
(400 MHz, CDCl3) δ: 7.32 (2H, dd, J1 10.4 Hz, J2 14.4 Hz,
H-9 and H-9), 6.27 (2H, m, H-10 and H-10), 6.21
(2H, m, H-11 and H-11), 6.14 (2H, m, H-8 and H-8),
2.99 (2H, s, H-1 and H-1), 1.89 (6H, d, J 6.4 Hz, H-12
and H-12), 1.45 (6H, s, H-14 and H-14), 1.42 (6H,
s, H-13 and H-13); 13C NMR (100 MHz, CDCl3) δ:
197.9 (C-5 and C-5), 175.9 (C-7 and C-7), 143.6 (C-9
and C-9), 140.4 (C-11 and C-11), 130.9 (C-10 and
C-10), 118.5 (C-8 and C-8), 104.0 (C-3 and C-3), 102.7
(C-6 and C-6), 78.7 (C-2 and C-2), 58.8 (C-4 and C-4),
57.5 (C-1 and C-1), 21.2 (C-13 and C-13), 18.9 (C-14
and C-14), 18.7 (C-12 and C-12). These data were in
good accordance with those of trichodimerol[12,13].
4. Antiviral activity
Compounds 1−3 were tested for their antiviral
activity using a NA (H7N9)/MUNANA model as well
as zanamivir as a positive control[14,15]. Compound 3
exhibited good inhibitory activity with an IC50 value of
74.6 μM and the inhibition rate was 31.7% at 10–5 mol/
L, whereas the other two showed weaker activity.
Acknowledgements
This work was financially supported by the Science &
Technology Project of Guangdong Province (Grant No.
2011A080403020) and the Fundamental Research Funds
for the Central Universities (Grant No. 2012N06).
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红树林木果楝属植物内生真菌Trichoderma sp. Xy24的
两个萜类和一个聚酮类化合物
张敏1,2, 李宁1,2, 陈日道2, 邹建华2, 王春梅1, 戴均贵1,2*
1. 北京中医药大学 中药学院, 北京 100102
2. 中国医学科学院 北京协和医学院药物研究所 天然药物活性物质与功能国家重点实验室, 北京 100050  

摘要: 从一株红树林木果楝属植物内生真菌Trichoderma sp. Xy24中分离得到3个化合物,包括一个Harziane二萜
harzianone (1), 一个菖蒲烷型倍半萜醇trichoacorenol (2)以及一个具有完全对称结构的聚酮类化合物trichodimerol (3)。
其化学结构通过旋光、质谱以及核磁共振波谱等波谱方法确定。化合物3具有中等程度的抗病毒活性。
关键词: 内生真菌; Trichoderma sp. Xy24; 萜类化合物; 聚酮类化合物; 木果楝