Depsitinuside: A new depside galactoside from an endophytic fungus isolated from Viburnum tinus

Article abstract of DOI:10.1080/10286020.2011.606811

Chromatographic purification of the extract of an endophytic fungal culture yielded depsitinuside (1), a new phenolic ester together with ergosterol (2) and (22E,24S)-24-methyl-5-a-cholesta-7,22-diene-3b,5,6b-triol (3). The structure of 1 was elucidated based on 1D, 2D NMR spectroscopy and high-resolution mass spectrometry, whereas the known compounds (2 and 3) were identified by 1H NMR, mass spectrometry, and in comparison with the literature values. Compound 1 was evaluated for its enzyme inhibitory potential against acetylcholinesterase, butyrylcholinesterase and lipoxygenase, and was found inactive (10%-40% inhibition at a concentration of 2 mg/ml).

Full text of DOI:10.1080/10286020.2011.606811

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Depsitinuside: a new depside
galactoside from an endophytic fungus
isolated from Viburnum tinus
a
a
a
a
Mamona Nazir , Misbah Sultan , Naheed Riaz , Maria Hafeez
b
b
c
,
Hidayat Hussain , Ishtiaq Ahmed , Barbara Schulz , Siegfried
c
a
b
d
Draeger , Abdul Jabbar , Karsten Krohn , Muhammad Ashraf
a
&
a
Muhammad Saleem
Department of Chemistry , Baghdad-ul-Jadeed Campus, The
Islamia University of Bahawalpur , Bahawalpur, 63100, Pakistan
b
Department of Chemistry , University of Paderborn , Warburger
Strasse 100, 33098, Paderborn, Germany
c
Institute of Microbiology, University of Braunschweig ,
Spielmannstraße 7, 31806, Braunschweig, Germany
d
Department of Pharmacy , Faculty of Pharmacy and Alternative
Medicine, The Islamia University of Bahawalpur , Bahawalpur,
63100, Pakistan
Published online: 10 Oct 2011.
To cite this article: Mamona Nazir , Misbah Sultan , Naheed Riaz , Maria Hafeez , Hidayat Hussain ,
Ishtiaq Ahmed , Barbara Schulz , Siegfried Draeger , Abdul Jabbar , Karsten Krohn , Muhammad
Ashraf & Muhammad Saleem (2011) Depsitinuside: a new depside galactoside from an endophytic
fungus isolated from Viburnum tinus , Journal of Asian Natural Products Research, 13:11, 1056-1060,
DOI: 10.1080/10286020.2011.606811
To link to this article: http://dx.doi.org/10.1080/10286020.2011.606811
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Journal of Asian Natural Products Research
Vol. 13, No. 11, October 2011, 1056–1060
NOTE
Depsitinuside: a new depside galactoside from an endophytic fungus
isolated from Viburnum tinus
a
Mamona Nazir , Misbah Sultan , Naheed Riaz , Maria Hafeez , Hidayat Hussain ,
a
a
a
b
b
Ishtiaq Ahmed , Barbara Schulz , Siegfried Draeger , Abdul Jabbar , Karsten Krohn ,
c
c
a
b
d
Muhammad Ashraf and Muhammad Saleem *
a
a
Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur,
Bahawalpur 63100, Pakistan; Department of Chemistry, University of Paderborn, Warburger
b
c
Strasse 100, 33098 Paderborn, Germany; Institute of Microbiology, University of Braunschweig,
d
Spielmannstraße 7, 31806 Braunschweig, Germany; Department of Pharmacy, Faculty of
Pharmacy and Alternative Medicine, The Islamia University of Bahawalpur, Bahawalpur 63100,
Pakistan
(Received 12 April 2011; final version received 17 July 2011)
Chromatographic purification of the extract of an endophytic fungal culture yielded
depsitinuside (1), a new phenolic ester together with ergosterol (2) and (22E,24S)-24-
methyl-5-a-cholesta-7,22-diene-3b,5,6b-triol (3). The structure of 1 was elucidated
based on 1D, 2D NMR spectroscopy and high-resolution mass spectrometry, whereas
1
the known compounds (2 and 3) were identified by H NMR, mass spectrometry, and in
comparison with the literature values. Compound 1 was evaluated for its enzyme
inhibitory potential against acetylcholinesterase, butyrylcholinesterase and lipoxygen-
ase, and was found inactive (10%–40% inhibition at a concentration of 2 mg/ml).
Keywords: Viburnum tinus; endophytic fungus; depside; steroids; enzyme inhibition
1
.
Introduction
resistance to some types of abiotic stress.
Endophytes also could be potential sources
of novel natural products with agrochem-
ical, pharmaceutical, and industrial poten-
tial [5–8].
Investigation of endophytic fungi is a
relatively new and attractive area of
natural product chemistry and drug devel-
opment. Endophytes are wonderful natural
machinery to synthesize countless chemi-
cals of diverse structural classes with
potential activities [1–4]. Colonization
and propagation of endophytes may offer
a significant benefit to their host plants by
producing a plethora of metabolites that
provide protection and survival value to
the plants. These compounds could impact
the broader ecological community as plant
growth regulators, antimicrobials, antivir-
als, and insecticidals, or even mediate
In continuation of our research on
endophytic fungi to find medicinally
important active metabolites, we investi-
gated the culture extract of a nonsporulat-
ing endophytic fungus isolated from the
leaves of Viburnum tinus, and isolated a
new phenolic ester depsitinuside (1) and
two known steroids: ergosterol (2) [9] and
(22E,24S)-24-methyl-5-a-cholesta-7,22-
diene-3b,5,6b-triol (3) [10], which were
characterized spectroscopically (Figure 1).
*Corresponding author. Email: drsaleem_kr@yahoo.com; m.saleem@iub.edu.pk
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.606811
http://www.tandfonline.com

Journal of Asian Natural Products Research
1057
OH
5
CH3
OH OH
''
H C
CH3
3
6
A
13
CH
3
7
CH3
5
''
O
8
3
CH3
O
2
HO
2''
CH3
1''
Ha
Hb
OH
O
1
1
O
'
H
H
HO
B
3'
13'
CH3
2
HO
5'
7'
1
OH
OH
CH3
H C
CH3
3
CH3
CH3
CH3
CH3
O
O
O
O
CH3
HO
HO
CH3
HO
CH3
OH
OH
3
4
5
Figure 1. Metabolites (1–3) isolated from endophytic fungus, strain 8984.
2
.
Results and discussion
Five aromatic methines resonated as
1
broad singlets in the H NMR spectrum at
Compound 1 was isolated as a colorless
amorphous powder. The HR-FAB-MS
d 6.41, 6.38, 6.36, 6.29, and 6.22, whereas
the upfield region displayed three signals
for two methylene groups at d 2.57 (1H, dt,
(
2ve mode) of 1 exhibited the molecular
2
ion peak at m/z 589.3009 [M 2 H] ,
corresponding to the molecular formula
C H O with 10 double bond equival-
J ¼ 11.5, 7.5 Hz, H-8 ), 2.45 (1H, dt,
a
J ¼ 11.5, 7.5 Hz, H-8 ), and 2.35 (2H, t,
b
3
2 45 10
0
J ¼ 7.8 Hz, H-7 ). The downfield shift and
ences (DBE). The IR spectrum of 1
indicated the presence of carbonyl and
hydroxyl groups, besides exhibiting
stretching bands for aromatic and aliphatic
splitting of two germinal protons H-8
a,b
were due to the magnetic anisotropic effect
of the carbonyl function, helping us to fix
CH -8 at ring-A (Figure 1), and therefore,
1
13
2
systems. The H and C NMR spectral data
see Section 3) were the evidence for two
0
CH -7 could be fixed at ring-B. These data
2
(
indicated that 1 must be a depside-type
nucleus and more likely an identical
structure to decarboxynorimbricaric acid
benzene rings in the molecule, compensat-
ing the eight DBE; one DBE was attributed
1
to a carbonyl function. The H NMR
(
4), which is actually a 3-hydroxy-5-
spectrum displayed a methine doublet at d
4
propylphenyl ester of olivetolic acid [11].
The lengths of the aliphatic chains in 1
were found to be six and seven carbon
skeletons. The main difference was the
glycone moiety, which was fixed at C-3 on
ring-A based on HMBC correlations. The
anomeric methine (d 4.58) showed its
HMBC interaction with C-3 (d 156.6)
.58 (J ¼ 7.6 Hz), which correlated with a
1
3
carbon in the C NMR spectrum at d 102.8
for an anomeric center of sugar moiety. The
presence of the glycon unit accommodated
the remaining DBE and led to the idea that
the other part of the molecule could be an
acyclic aliphatic system.

1
058
M. Nazir et al.
and lipoxygenase, and was found to be
OH
inactive (10%–40% inhibition) at a con-
centration of 2 mg/ml.
OH
OH
O
CH3
O
HO
OH
3. Experimental
O
O
3.1 General experimental procedures
IRspectra wererecordedasKBrpelletsona
JASCO 320-A infrared spectrophotometer.
Mass spectra were recorded on Finnigan
HO
CH3
(
Varian MAT, Waldbronn, Germany) 112
Figure 2. HMBC correlations observed in the
spectrum of 1.
and Finnigan (Varian MAT) 312 and 112S
double-focusing mass spectrometers con-
nected to MASPEC data system-1 on PC
base or on a Jeol JMX-HX 110 mass
spectrometer. Linked scan and peak match-
ingexperiments werealso carried outonthe
(Figure 2). The HMBC correlations of
methylene protons (d 2.57 and 2.45) with
carbon signals at d 143.7, 110.9, and 114.6
confirmed one aliphatic chain at C-7 of
ring-A, whereas the triplet methylene (d
2.35) showed correlations with carbons at
d 145.3, 113.0, and 112.3 indicating the
1
13
same instruments. H and C NMR spectra
1
were recorded on a Bruker (500 MHzfor H
1
3
and 125 MHz for
C) spectrometer;
0
00
chemical shifts d are in parts per million,
while coupling constants J are in hertz. The
GC wascarriedoutonaShimadzuGC (GC-
attachment at C-5 of ring-B. H-4 of the
sugar unit displayed its position at d 3.69
as a doublet with a smaller coupling
constant (J ¼ 1.5 Hz), as an indication of
galactose. The idea was further supported
by the carbon shifts of the sugar moiety in
9
A) (3% OV-1 silanized chromosorb W,
column temperature 1808C, injection port
and detector temperature 275–3008C, flow
rate 35 ml/min, flame ionization detector).
Optical rotations were measured on a
JASCO DIP-360 polarimeter. Column
chromatography was carried out using
silica gel (GF254 type 60 of 70–230 mesh
E. Merck, Darmstadt, Germany) as a
stationary phase packed in glass columns
with organic solvents as the mobile phase.
Thin layer chromatography (TLC) was
done on pre-coated aluminum TLC plates
1
3
the C NMR spectrum. The acid hydroly-
sis of 1 provided various products, among
which the sugar could be separated and
identified as b-D-galactose through its
optical rotation sign and comparison of the
retention time of its trimethylsilyl (TMS)
ether with that of the standard in gas
chromatography (GC). The structure of 1
was finally confirmed by the combination
of COSY, HMQC, and HMBC data and
was named as depsitinuside.
(
GF254, 0.25 mm E. Merck). Ceric sulfate
solution was prepared in 65% sulfuric acid
and used as the spraying agent. The TLC
plates were visualized by spraying this
reagent with subsequent heating under UV
light at 254 and 366 nm.
Depsides with a phenyl benzoate
nucleus form the largest group of lichen
metabolites with minor structural vari-
ations [12–15]. Of this class of molecules,
lecanorin (5) has the lowest number of
carbons and is known as a phytotoxic agent
3
.2 Isolation of fungi and culture
[
14], whereas other depsides are also
reported to possess antiproliferative and
cytotoxic activities [15].
Following surface sterilization, the endo-
phytic fungus, internal strain No. 8984,
was isolated from the leaves of V. tinus
[16]. It was cultivated for 28 days at 208C
on biomalt agar medium [17].
Compound 1 was evaluated for its
enzyme inhibitory properties against
acetylcholinesterase, butyrylcholinesterase

Journal of Asian Natural Products Research
1059
0
0
0
3
.3 Isolation and purification
Twelve liters of the culture medium
fungus and agar medium) were extracted
with ethyl acetate to afford a crude extract
2 g) after the removal of the solvent under
31.5 (C-8 ), 29.2 (C-9 ), 28.8 (C-10 ), 31.1
(C-11 ), 22.3 (C-12 ), 13.5 (C-13 ), 102.8
0 0 0
00 00 00
C-1 ), 70.9 (C-2 ), 75.5 (C-3 ), 68.6 (C-
(
(
0
0
00 00
4
FAB-MS (2ve mode): m/z 589.3009
), 73.0 (C-5 ), and 61.3 (C-6 ); HR-
(
2
[
M 2 H]
(calcd for C H O ,
32 45 10
reduced pressure. The combined concen-
trated extract (2 g) was column-chromato-
graphed on silica gel by eluting with a
gradient of n-hexane/ethyl acetate (100:0–
5
89.3012).
3
.3.2 Acid hydrolysis of 1
0
:100) to obtain seven fractions (A–G).
A solution of 1 (4 mg) in MeOH (5 ml)
containing 1 N HCl (4 ml) was refluxed for
Fraction F (35.0 mg) on further repeated
silica gel column chromatography eluted
with an isocratic mobile phase system of
4 h, concentrated under reduced pressure,
and diluted with H O (8 ml). It was
2
3
0% ethyl acetate in n-hexane yielded 1
22.0 mg). Fraction C (45.0 mg) on further
extracted with EtOAc and the residue
recovered from the organic phase was
found to be a mixture of products. The
aqueous phase was concentrated and
D-galactose was identified by the sign of
(
repeated silica gel column chromatog-
raphy with 8% ethyl acetate in n-hexane
yielded 2 (11 mg), whereas 3 (2.5 mg) was
purified from fraction D (24.0 mg) by
silica gel column chromatography eluting
with 15% ethyl acetate in n-hexane.
2
0
its optical rotation (½aꢀ þ79.5). It was
D
also confirmed based on the retention time
of its TMS ether (a-anomer 3.8 min,
b-anomer 5.2 min) with that of standard
[18] in GC.
3
.3.1 Depsitinuside (1)
Colorless amorphous powder (22.0 mg);
IR (KBr) nmax: 3475 (OH), 3120 and 2920
(
CZH), 1720 (CvO) and 1610, 1560
1
Acknowledgements
(
aromatic ring);
00 MHz): d 6.41 (1H, br s, H-4), 6.38
H NMR (CDCl3,
Muhammad Saleem is thankful to the Alax-
ander von Humboldt (AvH) Foundation,
Germany, for financial support. He is also
obliged to Third World Academy of Science
5
0
0
(
(
(
1H, br s, H-6 ), 6.36 (1H, br s, H-2 ), 6.29
1H, br s, H-4 ), 6.22 (1H, br s, H-6), 4.58
0
(
TWAS), Italy, for providing some of the basic
00
1H, d, J ¼ 7.6 Hz, H-1 ), 3.69 (1H, d,
laboratory facilities in Chemistry Department
of IUB. We thank Kathrin Meier for excellent
technical assistance.
0
0
J ¼ 1.5 Hz, H-4 ), 3.66 (1H, dd, J ¼ 10.5,
0
0
3
2
3
.5 Hz, H -6 ), 3.56 (1H, t, J ¼ 7.6 Hz, H-
b
0
0
00
), 3.54 (1H, dd, J ¼ 10.5, 4.8 Hz, H -6 ),
a
00
.42 (1H, dd, J ¼ 7.6, 1.5, H-3 ), 3.36 (1H,
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