Nigrosphaerin A a new isochromene derivative from the endophytic fungus Nigrospora sphaerica

Article abstract of DOI:10.1016/j.phytol.2013.09.001

Nigrosphaerin A, a new isochromene derivative (1), was isolated from the endophytic fungus Nigrospora sphaerica and chemically identified as 3-(3,4-dihydroxyphenyl)-4,6,8-trihydroxy-1H-isochromen-1-one-6-O-β-d- glucopyranoside. In addition nineteen known compounds (2-20) were isolated from the same fungus and chemically identified. Compounds (1-3, 5, and 7-16) were isolated for the first time from this fungus. In vitro antileukemic, antileishmanial, antifungal, antibacterial and antimalarial activities of (1-20) were examined. Compounds 5, 7, 9 and 10 showed good antileukemic activity against HL60 cells with IC₅₀ values of 0.03, 0.39, 0.2 and 0.4 μg/mL, respectively and against K562 cells with IC₅₀ values of 0.35, 0.35, 0.49 and 0.01 μg/mL, respectively. Compounds 3, 4 and 6 showed moderate antileishmanial activity with IC₅₀ values of 30.2, 26.4 and 36.4 μg/ml, respectively. Compound 7 showed moderate antifungal activity against Cryptococcus neoformans with IC₅₀ value of 14.8 μg/mL.

Full text of DOI:10.1016/j.phytol.2013.09.001

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PHYTOL 596 1–5
Phytochemistry Letters xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
1
2
3
Q1 Nigrosphaerin A a new isochromene derivative from the endophytic
fungus Nigrospora sphaerica
4
5
Q2 Ahmed M. Metwaly a,b, Hazem A. Kadry b, Atef A. El-Hela b, Abd-Elsalam I. Mohammad b,
Guoyi Ma a, Stephen J. Cutler c, Samir A. Ross a,d,
*
6
7
8
9
a National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
b Department of Pharmacognosy, Faculty of Pharmacy, University of Al-Azhar, Cairo, Egypt
c Department of Medicinal Chemistry, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
d Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
Nigrosphaerin A, a new isochromene derivative (1), was isolated from the endophytic fungus Nigrospora
sphaerica and chemically identified as 3-(3,4-dihydroxyphenyl)-4,6,8-trihydroxy-1H-isochromen-1-
Received 27 June 2013
Received in revised form 23 August 2013
Accepted 3 September 2013
Available online xxx
one-6-O-b-D-glucopyranoside. In addition nineteen known compounds (2–20) were isolated from the
same fungus and chemically identified. Compounds (1–3, 5, and 7–16) were isolated for the first time
from this fungus. In vitro antileukemic, antileishmanial, antifungal, antibacterial and antimalarial
activities of (1–20) were examined. Compounds 5, 7, 9 and 10 showed good antileukemic activity against
Keywords:
HL60 cells with IC50 values of 0.03, 0.39, 0.2 and 0.4
values of 0.35, 0.35, 0.49 and 0.01 g/mL, respectively. Compounds 3, 4 and 6 showed moderate
antileishmanial activity with IC50 values of 30.2, 26.4 and 36.4 g/ml, respectively. Compound 7 showed
moderate antifungal activity against Cryptococcus neoformans with IC50 value of 14.8 g/mL.
mg/mL, respectively and against K562 cells with IC50
Nigrospora sphaerica
Nigrosphaerin A
Isochromene
m
m
m
Antileukemic
ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Antileishmanial
10
11
1. Introduction
isochromene derivative (1), along with nineteen known com- 30
pounds (2–20). The antileukemic, antileishmanial, antifungal and 31
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Endophytic fungi are prospective producers of an abundant
source of bioactive chemically novel compounds with potential for
exploitation in a wide variety of medical areas (Tenguria et al.,
2011). Fungi belonging to the genus Nigrospora have been a rich
source of bioactive secondary metabolites, such as nigrosporolides
which found to have plant growth-inhibiting activity (Kim et al.,
2001), phomalactones with good anti plant pathogenic fungi effect
(Kim et al., 2001), phytotoxic antibacterial nigrosporins (Tanaka
et al., 1997), phytotoxic lactones (Fukushima et al., 1998),
epoxydons and pyrones (Trisuwan et al., 2008).
The fungus Nigrospora sphaerica has been reported as an
endophyte in several plants and marine organisms (Zhang et al.,
2009). N. sphaerica has been found to be a source of biologically
active secondary metabolites, including diterpenes (Turner and
Aldridge, 1983), diketopiperazines (Cutler et al., 1991), lactones
(Kim et al., 2001) and nigrosporolides (Zhang et al., 2009).
Chemical and biological investigation for the endophytic fungus
N. sphaerica (Fig. 1), led to the isolation of Nigrosphaerin A, a new
antibacterial activities of the isolated compounds were studied.
32
2. Results and discussion
33
Compound 1 (Fig. 2A) was isolated as a greenish yellow 34
amorphous powder. The molecular formula C21H20O12 was 35
determined by HR-ESI-MS (+ve mode) showing molecular ion 36
peak [M+H]+ at m/z 465.0990 (calcd. for C21H21O12, 465.1033) 37
indicating twelve degrees of unsaturation. The IR spectrum 38
showed that 1 contained hydroxyl (
nmax 1648 cmꢀ1) functional groups. The UV spectrum showed 40
n
max 3405 cmꢀ1), and carbonyl 39
(
absorption bands at lmax 255, 290 (sh) and 365.0 nm.
The 1H NMR spectroscopic data of 1 displayed signals at
41
d
H 7.59 42
(1H, d, J = 2.0 Hz, H-20),
d
H 7.45 (1H, dd, J = 8.4, 2.0 Hz, H-60) and
d
43
6.86H (1H, d, J = 8.4, H-50) indicating a 1,3,4-trisubstituted benzene 44
ring. These protons were found to be correlated to dC 114.8, dC 45
119.7 and
coupled aromatic protons resonating at
5) and H 6.75(1H, d, J = 1.6 Hz, H-7) were correlated to
d
C 115.8 in the HMQC spectrum, respectively. Two meta 46
H 6.64(1H, d, J = 1.6 Hz, H- 47
C 97.5 and 48
d
d
d
to dC 103.4 in the HMQC spectrum, respectively. The hydroxyl 49
proton (C-8) found to be resonating at dH 13.48 due to 50
intermolecular hydrogen bonding with the carbonyl group (C-1). 51
The 13C NMR, DEPT and HMQC spectroscopic data of 1 displayed 21 52
signals, including one methylene, ten methine and ten quaternary 53
*
Corresponding author at: National Center for Natural Products Research, School
Q3
of Pharmacy, The University of Mississippi, University, MS 38677, USA.
Tel.: +1 662 915 1031; fax: +1 662 915 7989.
E-mail address: sross@olemiss.edu (S.A. Ross).
1874-3900/$ – see front matter ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
http://dx.doi.org/10.1016/j.phytol.2013.09.001
Please cite this article in press as: Metwaly, A.M., et al., Nigrosphaerin A a new isochromene derivative from the endophytic fungus
Nigrospora sphaerica. Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2013.09.001

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Fig. 1. Nigrospora sphaerica.
Fig. 2. Compound 1 (A) and key HMBC (H ! C) of 1 (B).
54
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60
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71
72
73
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75
76
77
78
79
80
81
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84
85
86
87
88
carbons. The carbon resonating at
d
C 171.8 ppm is characteristic for
uridine (11) (Mantsch and Smith, 1973), adenosine (12) (Liu et al.,
2011), 3-benzylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (13)
(Fdhila et al., 2003), 3-methylhexahydro pyrrolo[1,2-a]pyrazine-
1,4-dione (14) (Hendea et al., 2006) and methyl 4-hydroxybenzo-
ate (15) (Li et al., 2008b), 4-(2-hydroxyethyl)phenol (16) (Li et al.,
2012). Four fatty acids (17–20) have been also isolated and
chemically identified as stearic acid (17), oleic acid (18), palmitic
acid (19), and myristic acid (20) using 1H NMR and GC/MS after
methylation.
89
90
91
92
93
94
95
96
carbonyl group (C-1). The 13C, HMQC and HMBC NMR data further
established four phenolic hydroxyl groups at C-4 (
d
C 157.4), C-8 (dC
163.3), C-30 (dC 145.3) and C-40 (dC 147.5). Six carbons for sugar
were found to be resonating at dC 103.9 (C-100), dC 73.8 (C-200), dC
77.6 (C-300), dC 69.8 (C-400), dC 75.8 (C-500) and dC 60.8 (C-600).
Anomeric proton at dH 4.77 found to be correlated to C-100 at dC
103.9 in the HMQC spectrum and to dC 158.5 in the HMBC
spectrum, indicating that the sugar is attached to C-6. The large
coupling constant of the anomeric proton J = 7.2 Hz indicated the
97
98
99
Compounds 5, 7, 9 and 10 showed good antileukemic activity
(Table 1 and Fig. 4) against acute HL60 cells with IC50 values of 0.03,
b
-configuration of the sugar (Avilov et al., 2003; Gao et al., 2008).
The following correlations have been found in the HMBC spectrum
(Fig. 2B): H-7 ( H 6.75) to C-5 ( C 97.5) and C-9 ( C 106.1), from H-5
H 6.64) to C-4 ( C 157.4) and C-9 ( H 7.59) to
C 106.1), from H-20 (
C-3 (dC 143.6), C-40 (dC 147.5) and C-60 (dC 119.7), from H-50 (dH
6.86) to C-10 ( C 122.2) and C-30 ( C 145.3) and from H-60 (
H 7.45)
to C-20 ( C 114.8) and C-40 (
C 147.5). The hydrolysis of 1 with 0.5 N
0.39, 0.2 and 0.4
cells with IC50 values of 0.35, 0.35, 0.49 and 0.01
respectively. Standard taxol showed IC50 values of 0.0005
mL and 0.0023 M/mL, respectively. Compounds 3, 4 and 6
showed moderate antileishmanial activity with IC50 values of 30.2,
26.4 and 36.4 g/mL, respectively. (Value of IC50 for standard
pentamidine was found to be 1.01 g/mL). Compound 7 showed
moderate antifungal activity against Cryptococcus neoformans with
IC50 value of 14.81 g/mL. It was found to be 0.28 g/mL for
m
M/mL, respectively and against chronic K562
M/mL,
M/
100
101
102
103
104
105
106
107
108
109
d
d
d
m
(
d
d
d
d
m
m
d
d
d
d
d
m
HCl yielded 3-(3,4-dihydroxyphenyl)-4,6,8-trihydroxy-1H-iso-
chromen-1-one and glucose which was identified by co-chroma-
tography with standard sugars using TLC. Based on the above
evidences, the structure of 1 was established as 3-(3,4-dihydrox-
yphenyl)-4,6,8-trihydroxy-1H-isochromen-1-one-6-O-
pyranoside.
Fifteen compounds (2–16, Fig. 3) have been isolated and were
chemically identified using 1D NMR (1H, 13C, DEPT135) and 2D
NMR (COSY, HMQC, HMBC) as well as HR-ESI-MS and found to be
m
m
m
standard amphotericin B.
3. Experimental
3.1. General
b-D-gluco-
110
111
ergosta-6,22-diene-3
b
,5
-ol (3) (Gong et al., 2010), ergosta-
-ol (4) (Pang and Sterner, 1993), ergos-
ta-4,6,8(14),22-tetraene-3one (5) (Lee et al., 2005), ergosta-
5(6),7,22-triene-3 -ol (6) (Li et al., 2007), ergosta-7,9(14),22-
triene-3 -ol (7) (Li et al., 2008a), ergosta-7,22-epidioxy-3 -ol (8)
a
,8
a
-triol (2) (Cateni et al., 2007),
NMR spectra were recorded on a Bruker Avance DRX-500
instrument at 500 (1H) and 125 MHz (13C), and a Varian Mercury
400 MHz spectrometer at 400 (1H) and 100 MHz (13C). The HR-ESI-
MS spectra were measured using a Bruker Bioapex-FTMS with
electrospray ionization (ESI). The GC–MS was interfaced to a HP
5973 quadrupole mass selective detector. The injector tempera-
ture was 250 8C, and 1 mL injections were performed in the split
(1:10) mode using helium as carrier gas. Column chromatographic
separation was performed on silica gel 60 (0.04–0.063 mm) and
112
113
114
115
116
117
118
119
120
ergosta-7,22-diene-3
b
4,6,8(14),22-tetraene-3
b
b
b
b
(Cateni et al., 2007), 4-(hydroxymethyl)-3,5-dimethyl dihydro-
furan-2(3H)-one (9) (Rukachaisirikul et al., 2009), 3-(1-hydro-
xyethyl)-4-methyl dihydrofuran-2(3H)-one (10) (Choi et al., 2008),
Please cite this article in press as: Metwaly, A.M., et al., Nigrosphaerin A a new isochromene derivative from the endophytic fungus
Nigrospora sphaerica. Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2013.09.001

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3
Fig. 3. Compounds 2–16.
Fig. 4. The concentration-dependent effects of compounds 5, 7, 9 and 10 on the growths of acute leukemia HL60 (A) and chronic leukemia K562 (B) cells. Cells were treated
with compounds for 48 h and then cell numbers were determined by the trypan blue exclusion test. Results shown are mean ꢂ SD (bars) of triplicate experiments.
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122
123
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sephadex LH-20 (0.25–0.1 mm, Merck). TLC was performed on
precoated TLC plates with silica gel 60 F254 (0.2 mm, Merck). Semi
preparative HPLC (Waters Delta Prep 4000) was performed using
3.2. Fungal material
125
The fungus N. sphaerica was isolated from surface sterilized 126
fresh leaves of an apparently healthy vinca rosea (Apocynaceae) 127
Luna1 RP-18 (250,10 mm ꢁ 5
mm; flow rate 5 mL/min).
Please cite this article in press as: Metwaly, A.M., et al., Nigrosphaerin A a new isochromene derivative from the endophytic fungus
Nigrospora sphaerica. Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2013.09.001

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A.M. Metwaly et al. / Phytochemistry Letters xxx (2013) xxx–xxx
Table 1
chromatographed on sephadex LH-20 eluting with MeOH to give
compound 12 (15.9 mg). Subfraction 4 was chromatographed with
semi-preparative HPLC with linear gradient elution 20–85%
aqueous methanol to obtain compounds 13 (7.8 mg), 14
(4.3 mg) and 16 (1.4 mg).
179
180
181
182
183
Inhibitory effects of compounds 5, 7, 9 and 10 on the growth of human leukemia
cells in vitro (48 h drug exposure for HL60 and K562 cells). Experiments were carried
out in triplicate.
Compounds
Estimated IC50
HL60 cells
(mM)
K562 cells
3.4. 3-(3,4-Dihydroxyphenyl)-4,6,8-trihydroxy-1H-isochromen-1-
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185
Compound 5
Compound 7
Compound 9
Compound 10
Taxol
0.03 ꢂ 0.05
0.39 ꢂ 0.03
0.2 ꢂ 0.001
0.4 ꢂ 0.001
0.35 ꢂ 0.002
0.35 ꢂ 0.002
one-6-O-b-D-glucopyranoside (1)
0.49 ꢂ 0.0012
0.01 ꢂ 0.003
Greenish yellow amorphous powder; UV (MeOH):
nm: 255 (3.93), 290(sh) (3.71) and 365.0 (3.92); IR
l
max (log
e)
186
187
188
189
190
191
192
193
194
195
196
197
198
0.0005 ꢂ 0.00008
0.0023 ꢂ 0.0005
nmax: 3405,
1648 and 1024 cmꢀ1; HR-ESI-MS m/z 465.0990 [M+H]+ (calcd. for
C
21H21O12, 465.1033). 1H NMR (DMSO,
d, 400 MHz, ppm): 6.64
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135
136
137
138
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143
144
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collected in March 2010 in Cairo, Egypt. The leaves were rinsed
with water and followed by surface sterilization in 70% EtOH for
1 min, rinsed with sterilized water, then cut into small pieces (2 cm
in length and width) and deposited in on a petri dish containing
PDA medium (200 g potato, 20 g glucose, and 15 g agar in 1 L
distilled water, supplemented with 100 mg/L chloramphenicol)
and cultivated at 28 8C for 3 days. The hypha tips were observed
and transferred to new PDA plates and subcultured until pure
culture was obtained. The fungus was identified by the regional
center for mycology and biotechnology, Cairo, Egypt. Identification
was based on The Data Base Identification Program of the Regional
Center for Mycology and Biotechnology (RCMB) for fungi, using an
Image Analysis System and on current universal keys (Fisher and
Cook, 1998; Hoog et al., 2000). After purification the fungus was
grown on PDA at 28 8C for 5 days. Ten pieces (0.5 ꢁ 0.5 cm2) of
mycelial agar plugs were inoculated into ten 1000 mL Erlenmeyer
flasks containing sterilized (100 g Asian rice and 100 ml distilled
water) at room temperature for 40 days.
(1H, d, J = 1.6 Hz, H-5), 6.75 (1H, d, J = 1.6 Hz, H-7), 7.59 (1H, d,
J = 2.0 Hz, H-20), 7.45 (1H, dd, J = 8.4, 2.0 Hz, H-60), 6.86 (1H, d,
J = 8.4, H-50), 4.77 (1H, d, J = 7.2 Hz, H-100), 3.33 (1H, H-200), 3.30 (1H,
H-300), 3.18 (1H, H-400), 3.27 (1H, H-500), 3.65 and 3.70 (2H, H-600);
13C NMR (DMSO,
d, 125 MHz, ppm): 171.9 (C-1), d143.6 (C-3),
157.4 (C-4), 97.5 (C-5), 158.5 (C-6), 103.4 (C-7), 163.3 (C-8), 106.1
(C-9), 137.4 (C-10), 122.2 (C-10), 114.8 (C-20), 145.3 (C-30), 147.5 (C-
40), 115.8 (C-50), 119.7 (C-60), 103.9 (C-100), 73.8 (C-200), 77.6 (C-300),
69.8 (C-400), 75.8 (C-500), 60.8 (C-600).
3.5. Acid hydrolysis of compound 1
199
2 mg of compound 1 was refluxed with HCl (0.5 N, 2 mL) for 2 h.
The hydrolyzed product was extracted with CH2Cl2/H2O. The sugar
was extracted from the aqueous layer using pyridine (1 mL). The
200
201
202
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sugar was identified as
D-glucose by co-chromatography with
authentic samples of different sugars using silica gel TLC using
solvent system EtOAc/MeOH/HOAc/H2O (11:2:2:2) followed by
spraying with anisaldehyde/H2SO4 and heating at 100 8C.
146
3.3. Extraction and Isolation
3.6. Antileukemic assay
207
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157
158
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169 Q5
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The fungus was extracted by adding 2 L EtOAc to each flask and
homogenized. The homogenized suspensions were collected,
filtrated, concentrated under vacuum and partitioned with
distilled water. Ethyl acetate portion was evaporated to dryness
and fractionated using hexane and 90% MeOH to afford hexane
fraction (7.6 g) and MeOH fraction (8.0 g). Water portion was
fractionated against n-butanol to afford water fraction (36 g) and
butanol fraction (7 g). N. sphaerica MeOH fraction (8 g) was
subjected to Si gel VLC eluted with hexane, EtOAc and finally
MeOH. Six fractions were collected (500 mL each). Fractions 2–3
(410.9 mg) were chromatographed on sephadex LH-20 using
eluent MeOH:CHCl3 (5:5) to yield nine subfractions. Subfraction 2
was chromatographed with Si-SPE column eluted with CHCl3,
MeOH in a matter of increasing polarity to afford compound 2
(3 mg), compound 4 (5 mg) and compound 5 (4 mg). Subfraction 3
was chromatographed on Si-SPE column eluted with CHCl3, MeOH
to give compounds 6 (12.6 mg) and 8 (1.3 mg). Subfraction 4 was
chromatographed on sephadex LH-20 using eluent MeOH:CHCl3
(5:5) to afford compound 7 (2.4 mg) and compound 3 (3.9 mg).
Subfractions 5–6 was chromatographed on Si gel eluted with
Human acute leukemia HL60 cells and human chronic leukemia
562 cells were purchased from American Type Culture Collection,
Rockville MD, USA. Both cell lines were grown in suspension
culture at 37 8C in RPMI-1640 medium supplemented with 10%
208
209
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212
213
214
215
216
217
218
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non-dialysed fetal bovine serum (FBS), 2 mM
L-glutamine,
100 units/mL of penicillin and 10 g/mL of streptomycin. For
m
the cell growth inhibition assay, HL60 and K562 cells were set up at
1 ꢁ 105 cells/well in Costar 24-well plates. Cells were allowed to
grow undisturbed for 24 h before addition of ‘‘compounds’’. After
48 h incubation with drugs at 37 8C, viable cell counts were made
by using the trypan blue exclusion method to assess cell viability
(Roper and Drewinko, 1976).
3.7. Antimicrobial assay
220
Crude extracts and isolated compounds were tested for
antimicrobial activity against Candida albicans ATCC 90028,
Candida glabrata ATCC90030, Candida krusei ATCC 6258, Asper-
igillus fumigates ATCC 90906, Methicillin-resistant Staphylococcus
aureus ATCC 33591, Cryptococcus neoformans ATCC 90113,
Staphylococcus aureus ATCC 2921, Escherichia coli ATCC 35218,
Pseudomonus aeruginosa ATCC 27853, Mycobacterium intracellulare
ATCC 23068, Ciprofloxacin and Amphotericin B were used as
positive standards (Bharate et al., 2007; Radwan et al., 2009).
221
222
223
224
225
226
227
228
229
CHCl3, MeOH in
a matter of increasing polarity to afford
compounds 9 (5.8 mg) and compound 10 (2.7 mg).
The butanol fraction (7.0 mg) was subjected to Si gel VLC
gradient eluted with CHCl3/MeOH and finally MeOH. Eight
fractions were collected (500 mL each). Fraction 2 (17.8 mg) was
chromatographed on sephadex LH-20 eluting with MeOH/H2O,
followed by purification with semi-preparative HPLC eluted with
75% MeOH/H2O to give compound 1 (6.3 mg). Fractions 3–4
(702.9 mg) were chromatographed on sephadex LH-20 eluting
with MeOH to yield 5 subfractions. Subfraction 2 was chromato-
graphed on RP-Si-SPE (C18) column using eluent H2O:MeOH (6:4)
to give compounds 11 (6.7 mg) and 15 (7.3 mg). Subfraction 3 was
3.8. Antimalarial assay
230
Crude extracts were tested on chloroquine sensitive (D6,
Sierraleon) and resistant (W2, Indo-china) strains of Plasmodium
falciparum using previously reported method; Artemisinin and
Chloroquine were used as positive standards (Bharate et al., 2007).
231
232
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Please cite this article in press as: Metwaly, A.M., et al., Nigrosphaerin A a new isochromene derivative from the endophytic fungus
Nigrospora sphaerica. Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2013.09.001

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Fisher, F.W., Cook, N.B., 1998. Fundamentals of Diagnostic Mycology. WB Saunders, 277
Philadelphia, PA.
Fukushima, T., Tanaka, M., Gohbara, M., Fujimori, T., 1998. Phytotoxicity of three 279
lactones from Nigrospora sacchari. Phytochemistry 48, 625–630.
Gao, Z., Ali, Z., Khan, I.A., 2008. Glycerogalactolipids from the fruit of Lycium 281
barbarum. Phytochemistry 69, 2856–2861.
Gong, Q., Zhang, Y., Tan, N., Chen, Z., 2010. Chemical constituents of three poisonous 283
mushrooms. Tianran Chanwu Yanjiu Yu Kaifa 22, 185–188.
Hendea, D., Laschat, S., Baro, A., Frey, W., 2006. Diastereoselective alkylation of a 285
proline-derived bicyclic lactim ether. Helv. Chim. Acta 89, 1894–1909.
Hoog, G.D., Guarro, J., Gene´, J., Figueras, M., 2000. Atlas of Clinical Fungi. Centraal- 287
bureau voor Schimmelcultures (CBS), Baarn.
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3.9. Antileishmanial assay
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The antileishmanial activity of the isolated metabolites was
tested in vitro against a culture of L. donovani promastigotes;
Pentamidine and Amphoterecin B were used as positive standards
(Abdel-Mageed et al., 2012).
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Acknowledgments
Kim, J.-C., Choi, G.J., Park, J.-H., Kim, H.T., Cho, K.Y., 2001. Activity against plant 289
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We are grateful to theEgyptian Government and National Center
for Natural Products Research, School of Pharmacy, University of
Mississippi, for financial support. We are also thankful for Dr.
Baharthi Avula for HRMS, Drs. Melissa Jacob, Babu Tekwani and
Shabana Khan for antimicrobial, antileishmanial and antimalarial
assays. This investigation was conducted in part in a facility
constructed with support from the research facilities improvement
program C06 RR-14503-01 from the NIH NCRR. This work is
supported in part by United States Department of Agriculture ARS
Specific Cooperative Agreement No. 58-6408-2-0009.
pathogenic fungi of phomalactone isolated from Nigrospora sphaerica. Pest 290
Manag. Sci. 57, 554–559.
291
Lee, W.Y., Park, Y., Ahn, J.-K., Park, S.-Y., Lee, H.-J., 2005. Cytotoxic activity of ergosta- 292
4,6,8(14),22-tetraen-3-one from the sclerotia of Polyporus umbellatus. Bull. 293
Korean Chem. Soc. 26, 1464–1466.
294
Li, D., Li, X., Cui, C., Wang, B., 2008a. Chemical constituents of endophytic fungus 295
Hypocreales sp. derived from the red alga Symphyocladia latiuscula. Haiyang 296
Kexue 32, 51–55.
297
Li, P., Zuo, T., Wang, X., Zhu, L., Wu, S., Zhang, G., 2008b. Chemical constituents 298
of Scutellaria barbata D . Don (II). Zhongguo Yaowu Huaxue Zazhi 18, 299
374–376.
300
Li, X., Han, L., Cao, Y.-R., Liu, J., Jiang, Y., Huang, X.-S., 2012. Separation and 301
identification of chemical constituents from a Streptomyces sp. isolated from 302
sika deer feces. Huaxue Yu Shengwu Gongcheng 29 (31/32) 49.
303
Li, X., Sun, G.-Z., Zheng, Y.-N., Lin, W.-H., Sattler, I., 2007. Separation and structures 304
251
Appendix A. Supplementary data
of two steroids from mangrove endophyte Penicillium. Tianran Chanwu Yanjiu 305
Yu Kaifa 19, 420–422.
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(1H NMR, 13C NMR, DEPT 135, HMQC, HMBC, HR-ESI-MS, UV and
IR) spectral data of compound 1.
Liu, D., Wang, S., Zhang, L., Li, L., 2011. Chemical composition of n-BuOH extract of 307
Potentilla anserina L. and its protective effect of EAhy926 endothelial cells under 308
hypoxia. Lat. Am. J. Pharm. 30, 1889–1894.
309
Mantsch, H.H., Smith, I.C.P., 1973. Solvent effects on the carbon-13 nuclear mag- 310
netic resonance spectra of cholesterol pyridine, and uridine. Can. J. Chem. 51, 311
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytol.2013.09.001.
1384–1391.
312
-ol from 313
314
Pang, Z., Sterner, O., 1993. The isolation of ergosta-4,6,8(14),22-tetraen-3
b
injured fruit bodies of Marasmius oreades. Nat. Prod. Lett. 3, 193–196.
256
References
Radwan, M.M., Rodriguez-Guzman, R., Manly, S.P., Jacob, M., Ross, S.A., 2009. 315
Sepicanin A—a new geranyl flavanone from Artocarpus sepicanus with activity 316
against methicillin-resistant Staphylococcus aureus (MRSA). Phytochem. Lett. 2, 317
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
Abdel-Mageed, W.M., Backheet, E.Y., Khalifa, A.A., Ibraheim, Z.Z., Ross, S.A., 2012.
Antiparasitic antioxidant phenylpropanoids and iridoid glycosides from Tecoma
mollis. Fitoterapia 83, 500–507.
141–143.
Roper, P.R., Drewinko, B., 1976. Comparison of in vitro methods to determine drug- 319
induced cell lethality. Cancer Res. 36, 2182–2188.
318
Avilov, S.A., Antonov, A.S., Silchenko, A.S., Kalinin, V.I., Kalinovsky, A.I., Dmitrenok,
P.S., Stonik, V.A., Riguera, R., Jimenez, C., 2003. Triterpene glycosides from the
Far Eastern sea cucumber Cucumaria conicospermium. J. Nat. Prod. 66, 910–916.
Bharate, S.B., Khan, S.I., Yunus, N.A.M., Chauthe, S.K., Jacob, M.R., Tekwani, B.L., Khan,
I.A., Singh, I.P., 2007. Antiprotozoal and antimicrobial activities of O-alkylated
and formylated acylphloroglucinols. Bioorg. Med. Chem. 15, 87–96.
Cateni, F., Doljak, B., Zacchigna, M., Anderluh, M., Piltaver, A., Scialino, G., Banfi, E.,
2007. New biologically active epidioxysterols from Stereum hirsutum. Bioorg.
Med. Chem. Lett. 17, 6330–6334.
Choi, J.-H.,Horikawa, M.,Okumura,H.,Kodani,S.,Nagai,K., Hashizume, D., Koshino, H.,
Kawagishi, H., 2008. Endoplasmic reticulum (ER) stress protecting compounds
from the mushroom Mycoleptodonoides aitchisonii. Tetrahedron 65, 221–224.
Cutler, H.G., Hoogsteen, K., Littrell, R.H., Arison, B.H., 1991. Epoxyexserohilone, a
novel metabolite from Nigrospora sphaerica. Agric. Biol. Chem. 55, 2037–2042.
Fdhila, F., Vazquez, V., Sanchez, J.L., Riguera, R., 2003. dd-Diketopiperazines: anti-
biotics active against Vibrio anguillarum isolated from marine bacteria associ-
ated with cultures of Pecten maximus. J. Nat. Prod. 66, 1299–1301.
320
Rukachaisirikul, V., Arunpanichlert, J., Sukpondma, Y., Phongpaichit, S., Sakayaroj, J., 321
2009. Metabolites from the endophytic fungi Botryosphaeria rhodina PSU-M35 322
and PSU-M114. Tetrahedron 65, 10590–10595.
323
Tanaka, M., Fukushima, T., Tsujino, Y., Fujimori, T., 1997. Nigrosporins A and B, new 324
phytotoxic and antibacterial metabolites produced by a fungus Nigrospora 325
oryzae. Biosci. Biotechnol. Biochem. 61, 1848–1852.
326
Tenguria, R.K., Khan, F.N., Quereshi, S., 2011. Endophytes—mines of pharmacologi- 327
cal therapeutics. World J. Sci. Technol. 1, 127–149.
328
Trisuwan, K., Rukachaisirikul, V., Sukpondma, Y., Preedanon, S., Phongpaichit, S., 329
Rungjindamai, N., Sakayaroj, J., 2008. Epoxydons and a pyrone from the marine- 330
derived fungus Nigrospora sp. PSU-F5. J. Nat. Prod 71, 1323–1326.
Turner, W.B., Aldridge, D.C., 1983. Fungal Metabolites, vol. II. Academic Press, New 332
York.
331
333
Zhang, Q.-H., Tian, L., Zhou, L.-D., Zhang, Y., Li, Z.-F., Hua, H.-M., Pei, Y.-H., 2009. Two 334
new compounds from the marine Nigrospora sphaerica. J. Asian Nat. Prod. Res. 335
11, 962–966.
336
Please cite this article in press as: Metwaly, A.M., et al., Nigrosphaerin A a new isochromene derivative from the endophytic fungus
Nigrospora sphaerica. Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2013.09.001