Fusaroside, a unique glycolipid from Fusarium sp., an endophytic fungus isolated from Melia azedarach

Article abstract of DOI:10.1039/c1ob06426f

Fusaroside (1), a unique trehalose-containing glycolipid composed of the 4-hydroxyl group of a trehalose unit attached to the carboxylic carbon of a long-chain fatty acid, was isolated from the organic extract of fermentation broths of an endophytic fungus, Fusarium sp. LN-11 isolated from the leaves of Melia azedarach. Six known compounds, phalluside (2), (9R*,10R*,7E)- 6, 9,10-trihydroxyoctadec-7-enoic acid (3), porrigenic acid (4), (9Z)-2,3-dihydroxypropyl octadeca-9-enoate (5), cerevisterol (6) and ergokonin B (7), were also isolated from this fungus. The glycolipid contains a rare branched long-chain fatty acid (C_20:4) with a conjugated diene moiety and a conjugated ketone moiety. The structure of the new compound 1 was elucidated by spectroscopic methods (1D and 2D NMR experiments, MS) and chemical degradations. The metabolites 1-5 were shown to have moderate to weak active against the brine shrimp larvae. To our knowledge, this is the first report of isolation of the first representative of a new family of glycolipids from natural sources.

Full text of DOI:10.1039/c1ob06426f

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PAPER
Fusaroside, a unique glycolipid from Fusarium sp., an endophytic fungus
isolated from Melia azedarach†
Sheng-Xiang Yang,a,b,c Hong-Peng Wang,c Jin-Ming Gao,*a Qiang Zhang,a Hartmut Laatsch*c and Yi Kuangd
Received 21st August 2011, Accepted 6th October 2011
DOI: 10.1039/c1ob06426f
Fusaroside (1), a unique trehalose-containing glycolipid composed of the 4-hydroxyl group of a
trehalose unit attached to the carboxylic carbon of a long-chain fatty acid, was isolated
from the organic extract of fermentation broths of an endophytic fungus, Fusarium sp. LN-11 isolated
from the leaves of Melia azedarach. Six known compounds, phalluside (2), (9R*,10R*,7E)-6,
9,10-trihydroxyoctadec-7-enoic acid (3), porrigenic acid (4), (9Z)-2,3-dihydroxypropyl octadeca-
9-enoate (5), cerevisterol (6) and ergokonin B (7), were also isolated from this fungus. The glycolipid
contains a rare branched long-chain fatty acid (C20 : 4) with a conjugated diene moiety and a conjugated
ketone moiety. The structure of the new compound 1 was elucidated by spectroscopic methods (1D and
2D NMR experiments, MS) and chemical degradations. The metabolites 1–5 were shown to have
moderate to weak active against the brine shrimp larvae. To our knowledge, this is the first report of
isolation of the first representative of a new family of glycolipids from natural sources.
In our continuing search for new biologically active secondary
metabolites from plant-derived fungi, we investigated chemical
Introduction
Endophytic fungi are microorganisms that live in the inter- and
constituents from an endophyte Fusarium sp. LN-11 and isolated
intracellular spaces of the tissues of apparently healthy host plants
seven compounds including a novel glycolipid, named fusaroside
and do so in a variety of relationships, ranging from symbiotic
to pathogenic.1 Recently, statistical analyses showed that 51% of
the biologically active metabolites obtained from endophytes are
previously unknown, compared with only 38% of novel substances
from soil microflora. During the last decade, endophytes have
(1), and six known compounds, 2–7. Herein, we describe the isola-
tion and structure characterization of the new natural glycolipid,
as well as toxicity to the brine shrimp larvae of five lipids 1–5.
Results and discussion
been recognized as important sources of a variety of structurally
novel active secondary metabolites with anticancer, antimicrobial
and other biological activities.1,2 As a result, the study of fungal
endophytes is currently considered a reasonable approach to the
discovery of novel, bioactive natural products.3,4
Melia azedarach Linn (Meliaceae), also known as Chinaberry or
Persian lilac tree, is a deciduous tree and has long been recognized
for its insecticidal properties.5 This plant produces a class of
modified oxygenated triterpenoids, also known as limonoids.6,7
In previous papers, we reported the isolation and structural eluci-
dation of some new metabolites from the endophytes harboured
inside the healthy tissues of M. azedarach.8,9
The fungus Fusarium sp. LN-11, originally obtained from the
leaves of M. azedarach L., was grown in liquid culture at 28 C.
◦
After 5 days, the culture broths obtained were filtered to give
the mycelium. The mycelium was then dried and extracted
ultrasonically by ethyl acetate and acetone successively to provide
a crude extract. The extract was repeatedly subjected to column
chromatography on silica gel, Sephadex LH-20, RP-18, and
preparative TLC to yield five lipids (1–5), including a new
glycolipid (1), and two sterols (6 and 7).
Fusaroside (1) was obtained as a white amorphous solid. Its
molecular formula was established as C34H54O13 by a pseudomolec-
ular ion peak at m/z 693.3457 [M+Na]+ (calcd. for C34H54O13Na:
693.3456) in the HRESIMS, with 8 degrees of unsaturation. The
1H NMR spectrum recorded for 1 in C5D5N (Table 1) showed
resonances for a series of deshielded methine groups between dH
4.1 and 5.9, characteristic of protons on sp3 carbons attached
to oxygen atoms, and a series of aliphatic methylene and methyl
protons between dH 1.2 and 2.1, indicative of a branched aliphatic
hydrocarbon fragment. These proton NMR features suggested
that fusaroside (1) was a glycolipid.10 Its positive ESI-MS/MS data
generated prominent fragment ion peaks at m/z 693 [M+Na]+,
aChemical Biological Research Institute, College of Science, Northwest
A&F University, Yangling, 712100, Shaanxi, China. E-mail: jinminggao@
nwsuaf.edu.cn; Fax: +86 29 87092335; Tel: +86 29 87092335
bDepartment of Chemical Engineering, Huainan Union University, Huainan,
232038, Anhui, China
cDepartment of Orgnic Chemistry, University of Goettingen, Tam-
mannstrasse 2, D-37077, Goettingen, Germany. E-mail: hlaatsc@gwdg.de
dDepartment of Crop Sciences, Georg-August-Universita¨t Go¨ttingen, Grise-
bachstr.6, D-37077, Go¨ttingen, Germany
† Electronic supplementary information (ESI) available: NMR spectra for
new compound 1. See DOI: 10.1039/c1ob06426f
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Table 1 1H- (500 MHz) and 13C- (125 MHz) NMR Data (ppm) of
compound 1 and trehalose in C5D5N a
(Fig. 2 and 3). The HMBC spectrum of 1 displayed correlations
observed between H-4 and C-2, C-3, C-6, between H-11 and C-9,
C-13, H-13 and C-11, C-15, between H-18 and C-16, C-20, and
between H-19 and C-17, C-20, indicating these double bonds to
be located at C-4, C-11, C-13 and C-18 (Fig. 2). This deduction
was further supported by fragmentations occurring along the fatty
acid chain observed in the EI-MS spectrum of 1, showing a set
of diagnostic fragment ions of m/z 231, 206, 137, 110, 85 and
1
trehalose
No.
dH (mult., J in Hz)
dC
HMBC
dC
1
2
173.7 s
55.1 s
3
4
5
6
196.6 s
126.1 d
148.8 d
32.4 t
1
55 (Fig. 3). Moreover, analysis of the H–1H COSY data led to
6.78 (dt, 15.0, 1.8, 1H)
7.15 (dt, 15.0, 7.2, 1H)
1.91 (m, 2H)
2, 3, 6
3, 7
4, 5, 8
the identification of two proton spin systems shown in Fig. 2,
locating the two double bonds at C-4 and C-18 of structure 1,
respectively. Likewise, these above HMBC and COSY correlations
also reinforced the presence of three features present in the fatty
acid moiety, namely a conjugated enone and a conjugated diene,
as well as of an allylic group, which were revealed by three sets of
their respective proton resonances at dH 6.78 (1H, dt, J = 15.0, 1.8
Hz, H-4), dH 7.15 (1H, dt, J = 15.0, 1.8 Hz, H-5)], and at dH 5.49
(each H, m, H-11 and H-14), 5.40 (each H, m, H-12 and H-13),
as well as at dH 5.42 (1H, dt, J = 15.0, 8.0 Hz, H-18), 5.44 (1H,
7
8
9
1.35 (m, 2H)
27.8 t
29.4 t
5, 8, 9
6, 9, 10
7, 11
8, 11, 12
9, 13
10, 14
11, 15
12, 16
13, 14, 17
14, 18
15, 19
1.27 (m, 2H)
1.92 (m, 2H) b
32.5 t b
33.0 t b
130.4 d c
130.3 d c
130.4 d c
130.6 d c
32.9 t b
32.9 t b
33.0 t b
131.3 t
125.2 t
18.0 q
22.2 q
22.3 q
95.4 d
73.4 d
72.5 d
73.5 d
71.9 d
62.0 t
10
11
12
13
14
15
16
17
18
19
20
21
22
1¢
2¢
3¢
4¢
5¢
6¢
2.09 (m, 2H) b
5.49 (m, 1H) c
5.40 (m, 1H) c
5.40 (m, 1H) c
5.49 (m, 1H) c
2.09 (m, 2H) b
1.92 (m, 2H) b
2.09 (m, 2H) b
1
5.42 (dt, 15.0, 8.0, 1H)
5.44 (m, 1H)
1.61 (d, 4.8, 3H)
1.51 (s, 3H)
16, 20
17, 20
18, 19
m, H-19), and 1.61 (3H, d, J = 4.8 Hz, H-20) in the H NMR
spectrum of 1.
1, 2, 3, 22
1, 2, 3, 21
1¢¢, 3¢, 5¢
1¢, 4¢
1¢, 4¢, 5¢
1, 2¢, 3¢, 6¢
1¢, 3¢, 6¢
4¢, 5¢
1.55 (s, 3H)
5.87 (d, 3.6, 1H)
4.21 (dd, 9.8, 3.6, 1H)
4.69 (m, 1H)
5.77 (m, 1H)
4.91 (m, 1H)
4.09 (dd, 12.0, 4.8, 1H)
4.19 (m, 1H)
95.3
73.3
74.7
72.1
74.1
62.6
1¢¢
2¢¢
3¢¢
4¢¢
5¢¢
6¢¢
5.85 (d, 4.2, 1H)
4.19 (dd, 9.8, 3.8, 1H)
4.72 (m, 1H)
4.25 (m, 1H)
4.89 (m, 1H)
4.36 (dd, 11.4, 4.8, 1H)
4.44 (dd, 12.0, 3.0, 1H)
95.9 d
73.7 d
74.9 d
72.3 d
74.4 d
62.8 t
1¢, 3¢¢, 5¢¢
1¢¢, 4¢¢
1¢¢, 4¢¢, 5¢¢
2¢¢, 3¢¢, 6¢¢
1¢¢, 3¢¢, 6¢¢
4¢¢, 5¢¢
95.3
73.3
74.7
72.1
74.1
62.6
a
Assigned by DEPT, COSY, HSQC, and HMBC experiments.b,c Assign-
ments may be interchanged.
531 [M+Na-hexose]+, 513 [M+Na-hexose-H2O]+, and 347 [M+H-
2 ¥ hexose]+, suggesting 1 to contain two hexose residues.
The 1H NMR data of fusaroside (1) (Table 1) revealed the
presence of a terminal allylic methyl at dH 1.61 (3H, d, J = 4.8 Hz),
two methyl groups H-21 (dH 1.51) and H-22 (dH 1.55) at a tertiary
carbon, methylene protons as multiplets between dH 1.27 and 2.09,
and eight olefinic protons between dH 5.40 and 7.15. Furthermore,
the 13C NMR and DEPT spectra of 1 with the aid of HSQC data
(Table 1) showed the presence of 34 carbon signals, 22 of which
are recognized as three methyls, eight aliphatic methylenes, eight
olefinic methines, and three quaternary carbons. Among them,
three quaternary carbons including an ester carbonyl at dC 173.7
(C-1), a ketone carbon at dC 196.6 (C-3) and an sp3 carbon at dC
55.1 (C-2), eight olefinic carbons between dC 125.2 and 148.8, and
two methyl groups C-21 (dC 22.2) and C-22 (dC 22.3) at a tertiary
carbon, together with the terminal methyl at dC 18.0 (C-20) were
observed. The above evidence indicated that 1 contains a long-
chain fatty acid moiety with four double bonds and a geminal-
dimethyl group.
The positions of the four double bonds in the fatty acyl
chain were determined by HMBC, COSY and EI-MS spectra
Fig. 1 Structures of metabolites 1–7.
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Fig. 2 Key COSY and HMBC correlations in 1.
Scheme 1 Microscale chemical degradation procedure of 1.
carbon resonances at dC 95.4 of glucose-1 (C-1¢) and 95.9 of
glucose-2 (C-1¢¢), in addition to a number of oxymethines and
oxymethylenes between dH 4.09 and 5.77, and between dC 62.0
and 74.9. 2D NMR (1H–1H COSY, TOCSY, HSQC, and HMBC)
experiments (Fig. 2 and Table 1) allowed the assignment of the
proton and carbon shifts for each of the two monosaccharides in
the molecule. Starting from the anomeric protons H-1¢ and H-1¢¢,
interpretation of COSY and TOCSY spectra revealed two sugar
spin systems corresponding to two hexoses. HMBC correlations
observed between H-1¢ and C-5¢ (dC 71.9) of the glucose-1, and
between H-1¢¢ and C-5¢¢ (dC 74.4) of the glucose-2, indicated that
the hexoses existed in the pyranose forms. The lack of dispersion in
the methine proton resonances between dH 4.1 and 5.9 regions of
the sugar moiety could not resolve vicinal coupling constants for
the H-3 to H-5 regions in both systems, but only vicinal coupling
Fig. 3 Proposed major EI-MS fragmentation pathway of 1.
On theother hand, the location ofonecarbonylgroupatC-3 and
two additional tertiary methyl branches at C-2 on the alkene chain
were ascertained from the HMBC spectrum, showing correlations
of H-21 with C-22, C-1, C-2, C-3, of H-22 with C-21, C-1, C-2,
C-3, of H-4 with C-3, C-2, and of H-5 with C-3 (Fig. 2). This was
further confirmed from characteristic fragment ions at m/z 231,
259, and 301 of the EI-MS spectrum of 1 (Fig. 3).
The limited amounts of 1 prevented the use of NOE spec-
troscopy to determine the configuration of the double bonds. Thus,
the geometry of the double bonds of the long-chain alkene moiety
in structure 1 can be determined from the 13C NMR chemical shifts
of the methylene carbon next to the olefinic carbon, namely, the
carbon signals observed between dC 27–28 in cis type and between
dC 32–33 in trans type.11,12 The trans configuration of these double
bonds was deduced from the chemical shifts of C-6 (dC 32.4),
C-10 (dC 33.0), C-15 (dC 32.9) and C-17 (dC 33.0). In addition,
the 4E,18E configurations of 1 were also in agreement with the
constants (J
= 3.6, JH-1¢¢-H-2¢¢ = 4.2 Hz) for the H-1 to H-2
H-1¢-H-2¢
regions observed are all small, consistent with all equatorial–
axial relationships of these protons (Table 1). This discloses that
both monosaccharides were glucose residues having a anomeric
configurations.
In order to identify the absolute configuration of the sugar, the
GC-MS retention times of the resulting silylated sugar 1b were
compared with those of silylated authentic sugars using the reac-
tion conditions described above for transformation of the natural
product.13 Analysis without further purification of 1b showed three
peaks attributable to isomeric a/b-pyranone/furanone derivatives
(Fig. S11, ESI†). These were identical, within error limits, to peaks
observed for TMS-glucose. All other investigated sugars were
clearly different. In addition, the configuration of natural glucose
is essentially D. Therefore, the results confirmed the presence of
the glucoses in 1 and showed that the absolute configurations of
both glucose residues were determined as D.
HMBC data provided evidence for the nature of the linkages
between the two monosaccharides. The anomeric proton of the
glucose-1 (H-1¢) of 1 showed cross-peaks with C-3¢ (dC 72.5),
C-5¢ (dC 71.9) and C-1¢¢, and the other anomeric proton of the
glucose-2 (H-1¢¢) with C-3¢¢ (dC 74.9), C-5¢¢ (dC 74.4) and C-1¢, in
comparison to the 13C NMR data of an authentic sample of sugar
a,a-trehalose in C5D5N, which established a 1,1-glycosidic linkage
between the two glucose residues (Fig. 2 and Table 1). These data
confirmed the disaccharide chain to be a-D-glucopyranosyl-(1-1)-
a-D-glucopyranosyl residue, also known as a,a-trehalose residue.
3
large vicinal coupling constants (3JH-4-H-5 and JH-18-H-19 values of
ca. 15 Hz) (Table 1). Therefore, it is clear that 1 possesses a fatty
acyl chain with the all-E geometry of the double bond moiety.
Determination of the length of the long-chain fatty acyl moiety
of compound 1 was carried out by chemical degradation. A
very small amount of 1 was subjected to methanolysis with
HCl in MeOH, followed by silylation with MSTFA (N-methyl-
N-ethyldimethylsilyltrifluoroacetamide) (Scheme 1),13 leading to
products, silylated fatty acid (1a) and silylated glucose (1b).
The silylated fatty acid 1a was subjected to GC-MS analysis,
allowing us to identify 1a as the sole product of the reaction
due to typical fragment ions at m/z 419 [C22H33O3SiMe3+H]+.
The structure of the fatty acid moiety in 1 was accordingly de-
termined to be (4E,11E,13E,18E)-2,2-dimethyl-3-oxo-4,11,13,18-
tetra-eneicosanoic acid, as indicated in structure 1. This rare
branched fatty acid is first to be discovered from natural sources.
1
The H and 13C NMR and HSQC NMR data revealed two
anomeric proton resonances at dH 5.87 of glucose-1 (H-1¢) and
5.85 of glucose-2 (H-1¢¢), attached to the corresponding anomeric
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Table 2 Toxicity of metabolites 1–5 with mortality rates (%)
of glycolipids is previously unreported. In addition, trehalose is a
disaccharide widely present in bacteria, yeast, and fungi, as well
as some plants.22 Recent studies have shown that trehalose does
not only primarily function as a reserve carbohydrate, but also as
a highly efficient protectant, enhancing the resistance of cellular
components against adverse conditions such as high temperature,
freezing, and low dehydration.21 The fungal metabolites that may
be formed in fungi constitute their chemical defense system against
parasites, as well as various predators such as bacteria, fungi,
animals, and insects.23 This newly discovered fungal glycolipid 1,
in combination with other lipids, might be presumed to act as
phytoalexins occurring in endophytic fungi. It should be pointed
out that the biological functions of the new glycolipid and other
substances present in the endophytic Fusarium sp. remains unclear,
and that further investigation needs to be performed.
Sample
1
2
3
4
5
mortality
47.6
64.8
26.2
20.9
18.7
Finally, the relatively downfield chemical shift of H-4¢ (dH 5.77)
suggested that the trehalose is esterified at C-4¢, as confirmed by a
cross-peak between the proton H-4¢ of the glucose-1 and the ester
carbonyl carbon C-1 of the fatty acyl group in the HMBC experi-
ment. This indicated the fatty acyl chain to be linked to C-4¢ of the
sugar chain via an ester linkage. The structure of 1 was thus un-
ambiguously established as 4¢-(4E,11E,13E,18E)-2,2-dimethyl-3-
oxo-4,11,13,18-tetraeneeicosanoyl a-D-glucopyranosyl-(1–1)-a-
D-glucopyranoside, named fusaroside, as shown in Fig. 1.
The structures of compounds 2–7 were determined on the
basis of ESI-MS and H, 13C, and 2D NMR data, as well as by
1
Conclusions
comparison with data reported in the literature, as phalluside-1
(2),14 (9R*,10R*,7E)-6,9,10-trihydroxyoctadec-7-enoic acid (3),15
porrigenic acid (4),16 (9Z)-2,3-dihydroxypropyl octadeca-9-enoate
(5),17 cerevisterol (6)18 and ergokonin B (7).19 Compounds 2–7
(except 6) are reported from endophytic fungi for the first time.
The isolated metabolites 1–5 were tested for in vitro toxicity
toward brine shrimp (Artemia salina) using the reported assay in
which fusariumin, an isocoumarin obtained from the same fungal
strain, was used as a positive control (mortality rate: 78.2%).8 They
were found to possess weak to moderate growth inhibitory effects
on brine shrimp larvae at concentrations of 10 mg ml-1, as given in
Table 2. The glycolipids 1 and 2 exhibited moderate toxicity, with
mortality rates of 47.6% and 64.8%, respectively.
It is noteworthy that phalluside-1 (2) is a glucosphingolipid that
was isolated in 1998 from the marine ascidian Phallusia fumigata;14
oxylipin 3 is a trihydroxylated fatty acid that was reported
from a traditional herb, Dracontium loretense, with toxicity to
human peripheral blood mononuclear cells;15 porrigenic acid
(4), a conjugated ketonic fatty acid, was isolated as a cytotoxic
constituent of the higher mushroom Pleurocybella porrigens;16 and
fatty acid ester 5 is a monoglyceride present in a medicinal plant
Rhazya stricta.17 Furthermore, with respect to polyoxygenated
sterols cerevisterol (6) and ergokonin B (7), the former is commonly
found in fungi, which has been previously reported as a cytotoxic
agent, and inhibited the activity of DNA polymerase alpha,19 and
the latter has been found to be an antifungal antibiotic which
was isolated in 1991 from the fungus Trichoderma koningii.20
Biogenetically, both steroids have been known to be biosynthezied
from ergosterol, a ubiquitous fungal sterol.
Glycolipids are membrane components and occur in all king-
doms of living organisms, i.e., bacteria, plants, and animals. They
are usually divided into three major groups, glycoglycerolipids,
glycosphingolipids, and isoprenoid glycosides, dependent on their
lipid moiety. A number of glycolipids occur, however, that cannot
be classified in any of these groups. These compounds are made of a
glycosyl residue attached to the hydroxyl group of a fatty alcohol or
a hydroxy fatty acid, or to the carboxyl group of a fatty acid (ester
linkage). They typically exist in bacteria and yeast and exhibit
interesting biological properties.21 To the best of our knowledge,
in the present study, fusaroside (1), which is a glycolipid made
of an unusual branched fatty acid and an a,a-trehalose, is quite
rare and has not been reported previously. Notably, this family
In conclusion, fusaroside (1), the first example of a novel class
of glycolipids, which was produced by the endophytic fungus
Fusarium sp., has been revealed to consist of an uncommon
polyunsaturated long-chain fatty acid with three separate olefinic
structure elements, a conjugated diene, a conjugated ketone and
a monoene, and a trehalose residue. Moreover, six previously
reported metabolites 2–7 were also obtained from this fungus.
Compounds 2–7 (except 6) have been described from endophytic
fungi for the first time. The five lipids 1–5 were found to exert some
toxic effects on brine shrimp larvae.
Experimental section
General
The optical rotations were measured on a Perkin-Elmer polarime-
ter 241 polarimeter at the sodium D line. IR spectra were recorded
on a Perkin-Elmer 1600 Series FT-IR spectrometer with KBr
pellets, peaks are reported in cm-1 on Perkin–Elmer 1600 Series
FT-IR. UV/vis spectra were recorded on a Varian Cary 100 UV-
visible spectrophotometer; peak wavelengths are reported in nm.
1H NMR spectra were recorded on Varian Inova 500 (499.8 MHz),
and Varian Inova 600 (600 MHz). Coupling constants (J) in Hz.
Abbreviations: s = singlet, d = doublet, dd = doublet doublet, t =
triplet, q = quartet, m = multiplet, br = broad. 13C NMR spectra
were recorded with Bruker Avance 500 (125.7 MHz). Chemical
shifts were measured relative to tetramethylsilane (TMS) as the
internal standard. 2D NMR spectra: H, H COSY spectra (1H,1H
Correlated Spectroscopy), HMBC spectra (Heteronuclear Multi-
ple Bond Connectivity), HSQC spectra (Heteronuclear Multiple
Quantum Coherence) and NOESY spectra (Nuclear Overhauser
Effect Spectroscopy). Mass spectra were recorded with EI MS
at 70 eV with Varian MAT 731, Varian 311A, AMD-402, high
resolution with perflurokerosine as the standard. ESI mass spectra
were recorded on a Quattro Triple Quadrupol mass spectrometer,
with a Finnigan TSQ 7000 with nano-ESI API ion source. High-
resolution ESI mass spectra were measured on a Micromass LCT
mass spectrometer coupled with a HP 1100 HPLC with a diode
array detector.
GC-MS was performed on a Thermo Finnigan Trace GC-MS
system (Autosampler AS 2000), using a Varian column CP-Sil
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8CB for amines (30 m ¥ 0.25 mm i.d., 0.25 mm film thickness).
The temperature was programmed from 40 ◦C (held for 1 min)
to 300 ◦C (held for 4 mins), ramping the temperature up at
and 19.85 min. D-Glucose (18.58, 19.16, 19.93 min), D-mannose
(18.20, 19.26, 20.01 min), and D-galactose (18.30, 18.76, 19.22 min)
as standards were analyzed as above.
◦
10 C min-1. The derivatized sugar residues of 1 were identified
Fusaroside (1). Amorphous solid. [a]2D0 = +179 (c = 0.186,
MeOH); EI-MS (70 eV): m/z (%) 491(4), 347 (2), 301(4), 275
(3), 259 (8), 231 (9), 232(13), 175 (6), 163 (19), 145 (27), 137 (17),
127 (18), 109 (30), 110 (16), 111 (8), 85 (34), 81 (43), 73 (75), 55
by comparison of their retention times and fragmentation pattern
with standards.
Silica gel (Merck) 60–120 mesh for column chromatography and
pre-coated TLC sheets (layer thickness 0.2 mm) and preparative
TLC plate (layer thickness 1.25 mm) of silica gel 60 GF254 were
used. Spots were detected on TLC under UV light or by heating
after spraying with 5% H2SO4 in methanol.
◦
(100); MS (ESI) (180 C): m/z (%) 693 [M+Na]+, 705 [M+Cl]+,
1363 [2 M+Na]+. ESI-MS/MS: m/z (%) 693 (27), 532 (10), 531
(100), 513 (34), 347 (12), 231 (5). HR-MS (ESI): m/z calcd. for
1
C34H54O13Na: 693.3456; found: 693.3457 [M+Na]+; H- and 13C-
NMR spectroscopic data, see Table 1.
Fungal material
Brine shrimp (Artemia salina) bioassay
The endophytic fungal strain was isolated from the fresh leaves
of the tree M. azedarach L. growing in the campus of Northwest
A&F University, Yangling, Shaanxi province, China. The isolate
was identified as Fusarium sp. LN-11 by morphological analysis
and was deposited at the Research Centre for Natural Medicinal
Chemistry, Northwest A&F University.
The brine shrimp toxicity was assayed by small modified
microtiter-plate method using brine shrimp Artemia salina as a
test organism. Briefly, approximately 30 nuclei larvae hatched
from eggs of A. salina in 0.2 ml of artificial sea water which were
incubated with a sample (5 ml in DMSO solution) in a deep-well
microtiter plate at room temperature. After 24 h, the dead larvae
were determined by counting the number of the dead animals in
each well under a microscope. To each test row, a blind sample was
accompanied by adding DMSO. The mortality rate was calculated
using the formula: M = [(A-B-N)/(G-N)]¥100
M = percent of the dead larvae after 24 h; A = number of the
dead larvae after 24 h; B = average number of the dead larvae in
the blind samples after 24 h; N = number of the dead larvae before
starting the test; G = number of selected larvae for test.
Cultivation
◦
After growing on potato dextrose agar (PDA) medium at 28 C
for 5 days, the fungus was inoculated in liquid medium containing:
CaCl2 0.5 g, KH2PO4 0.1 g, KCl 0.05 g, MgSO4·7H2O 0.1 g, glucose
20.0 g, peptone 15.0 g, 1000 ml H2O. The pH was adjusted to 6.0
before autoclaving. Fermentation was carried out in 1000 ml flasks
each containing 200 ml medium on a rotary shaker at 150 rpm at
28 ◦C for 5 days.
Acknowledgements
Extraction and isolation
This work was supported by the National Natural Science
Foundation of China (21102114), the Program for New Century
Excellent Talents in University (NCET-05-0852), and the program
for Huainan Union University (LZD1002). We would also like
to thank H. Frauendorf and R. Machinek, in the laboratory of
Institution for Organic and Biomolecular Chemistry, University
of Go¨ttingen, Germany, for the MS and NMR measurements.
The culture broth (20 L) of Fusarium sp. LN-11 was filtered to
give the mycelium and water phase. The mycelium was dried at
50 ◦C, and smashed directly with ultrasonic extraction by ethyl
acetate and acetone three times. These two parts were combined
after TLC, and defatted with cyclohexane after being dissolved
in methanol to get a crude extract (2.7 g). The crude extract
was subjected to a CC over silica gel and eluted with CH2Cl2–
MeOH (100 : 0–50 : 50) in a gradient elution manner to provide
seven fractions (Fr1.–Fr7.). Fr.6 (0.3 g) was purified by Sephadex
LH-20 column chromatography (MeOH) and reversed phase RP-
18 column chromatography (MeOH–H2O) to yield compounds 1
(1.8 mg) and 2 (5.1 mg). Fr.5 (0.5 g) was separated by Sephadex
LH-20 CC using MeOH, followed by preparative TLC with
CH2Cl2–MeOH (6 : 1) to afford compounds 3 (4 mg), 4 (6 mg)
and 5 (5 mg). Fr.4 (0.4 g) was rechromatographed on a Sephadex
LH-20 column (CH2Cl2–MeOH, 6 : 4), and further purified by
preparative TLC (CH2Cl2–MeOH, 9 : 1) to give compounds 6
(7 mg) and 7 (8 mg).
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