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Mycotoxins from the fungus Botryotrichum piluliferum Oue-artorn Rajachan, Kwanjai Kanokmedhakul, Kasem Soytong, and Somdej Kanokmedhakul J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05522 • Publication Date (Web): 30 Jan 2017 Downloaded from http://pubs.acs.org on February 5, 2017
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Journal of Agricultural and Food Chemistry
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Mycotoxins from the Fungus Botryotrichum piluliferum
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Oue-artorn Rajachan,† Kwanjai Kanokmedhakul,†,* Kasem Soytong,‡
4
and Somdej Kanokmedhakul†
5 6
†
Natural Products Research Unit, Department of Chemistry and Center of Excellence for
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Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002,
8
Thailand
9
‡
10
Department of Plant Production Technology, Faculty of Agricultural Technology, King
Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand
11 12
*Corresponding author:
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* Tel.: +66 043-009700 ext. 42174-5. Fax: +66-43-202373. E-mail:
[email protected] ACS Paragon Plus Environment
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ABSTRACT
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Two new sterigmatocystin derivatives, oxisterigmatocystins E and F, 1 and 2, along with nine
16
known compounds, oxisterigmatocystins G and H, 3 and 4, sterigmatocystin, 5, N-0532B, 6,
17
O-methylsterigmatocystin,
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dimethylversicolorin A, 10, and 8-O-methylaverufin, 11 were isolated from the fungus
19
Botryotrichum piluliferum.
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spectroscopic evidence. Among these, compounds 3, 4, and 9 were discovered as natural
21
products for the first time. Compounds 1, 3, and 4 displayed antimalarial activity toward
22
Plasmodium falciparum (IC50 7.9 - 23.9 µM). In addition, compounds 1-6 and 8-11 exhibited
23
cytotoxicity against KB, MCF-7, and NCI-H187 cell lines (IC50 0.38-78.6 µM). However,
24
compounds 1-9 showed cytotoxic effects against the Vero cell line (IC50 0.65-15.9 µM). This
25
finding should promote awareness of the contamination of B. piluliferum in the food chain
26
and agricultural soil.
7,
N-0532A,
8,
6-O-methylversicolorin
A,
9,
6,8-O-
The structures of these mycotoxins were elucidated by
27 28 29 30 31 32
Keywords: Botryotrichum piluliferum, Chaetomiaceae, sterigmatocystin, mycotoxin, cytotoxicity
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INTRODUCTION
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The fungus Botryotrichum piluliferum belongs to the family Chaetomiaceae.1
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grown on potato dextrose agar are white when young and turned to pale brown when mature,
42
at 30 oC in 7 d, with septate mycelia, branches and setae. Conidia are chain-like on hyaline
43
conidiophores, globose, 12.50 – 15.50 µm diameter, with irregularly thick walls 3.0-3.5µm.
44
Teleomorphs were not found in this isolate. It was morphologically identified according to
45
Domsch et al.,1 and Downing.2 The fungus B. piluliferum has been reported as one of the
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seed borne fungi of chili pepper.3
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Botryotrichum has reported it to contain asterriquinone CT2 from Botryotrichum spp.4 and
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botryolides A - E,5 decarestrictine D,5 and sterigmatocystin5 from Botryotrichum sp.
49
(NRRL38180).5 However, no reports on the chemical constituents and bioactivity of B.
50
piluliferum have been found.
Colonies
Previous chemical investigation on the genus
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Many fungi such as Aspergillus species,6 Aschersonia coffeae Henn. BCC 28712,7
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Penicillium chrysogenum,8 including Botryotrichum sp. (NRRL 38180)5 have been reported
53
to produce mycotoxins. Mycotoxins have been reported as mutagenic and having
54
carcinogenic effects in animals and humans.9,10 In our continuing investigation on bioactive
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metabolites from fungi isolated from Thai soil, crude n-hexane and EtOAc extracts of B.
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piluliferum displayed cytotoxicity against the KB cell line with 85.5% and 59.7% inhibition,
57
respectively, at a concentration of 50 µg/mL.
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cytotoxicity toward the MCF-7 cell line with 57.5% inhibition.
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structural elucidation, and bioactivities of eleven mycotoxins from B. piluliferum are
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presented.
Moreover, the EtOAc extract presented
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MATERIALS AND METHODS
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General Experimental Procedures
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Optical rotations were measured on a DIP-1000 digital polarimeter (JASCO, Easton, MD).
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IR spectra were obtained using a Bruker Tenser 27 spectrophotometer (Bruker, Ettlingen,
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Germany). UV spectra were recorded on an Agilent 8453 UV–visible spectrophotometer
67
(Agilent Technologies, Santa Clara, CA). NMR spectra were acquired on a Varian Mercury
68
Plus 400 spectrometer (Varian, Polo Alto, CA).
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Micromass Q-TOF 2 hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer
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(Micromass, Manchester, UK). Silica gel 60 (Merck, Darmstadt, Germany) 230–400 mesh
71
was used for CC. TLC was performed on precoated silica gel 60 PF254 (Merck, Darmstadt,
72
Germany).
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Fungal Material
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The fungus strain (no. Bp01) was isolated from forest soil collected from Three Pagoda Pass,
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Sangkhla Buri, Kanchanaburi Province, Thailand in 2010. It was identified as the
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Botryotrichum piluliferum by K. Soytong, one of the coauthors. The fungus was cultured at
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25-28 °C for 9 weeks using the method described in our previous report.13 The biomass was
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collected by filtering, air-dried and ground into a powder.
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Extraction and Isolation
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The biomass powder of B. piluliferum (240 g) was extracted successively with n-hexane (1L
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x 3), EtOAc (1L x 3), and MeOH (1L x 3) by stirring at room temperature. The n-hexane
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extract (5.7 g) was separated by silica gel flash column chromatography (FCC) (45 x 5 cm),
83
eluting with a mixture of n-hexane-EtOAc (95:5, 90:10, 80:20, 70:30, 60:40, 40:60, 20:80,
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0:100 v/v, 300 mL each) and EtOAc-MeOH (90:10, 80:20, 70:30, 50:50, 30:70, 0:100 v/v,
85
300 mL each), to give 36 fractions.
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fractions, FH1-FH5. Recrystallization of fraction FH1 (76 mg) from CH2Cl2 yielded 9 (30
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mg). Fraction FH2 (196 mg) was purified using preparative TLC, eluted with 101 mL of n-
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hexane-CH2Cl2-MeOH (40:60:1, developed 6 times) to give 5 (44 mg, Rf = 0.47) and 6 (13
HRESI-TOFMS were recorded on a
Analysis of TLC characteristics gave five pooled
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mg, Rf = 0.56). Fraction FH3 (681 mg) was further subjected to a silica gel FCC (45 x 3 cm)
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eluting with 1.8 L of an isocratic system of n-hexane-CH2Cl2 (40:60) to give 10 (3 mg) and 8
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(41 mg). Fraction FH4 (154 mg) was subjected to Sephadex LH-20 (45 x 2 cm), eluting with
92
400 mL of MeOH to yield 11 (17 mg). Compound 7 (9 mg, Rf = 0.42) was obtained from
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FH5 (102 mg) using preparative TLC with hexane-CH2Cl2-MeOH (5:95:2, developed 3
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times). The EtOAc extract (9.3 g) was chromatographed on silica gel FCC (45 x 6 cm),
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eluting with a mixture of n-hexane-acetone (100:0, 90:10, 80:20, 70:30, 60:40, 40:60, 20:80,
96
0:100 v/v, 500 mL each) followed by acetone-MeOH (90:10, 80:20, 70, 60:40, 50:50, 30:70,
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0:100 v/v, 300 mL each) to give 41 collected fractions.
On the basis of their TLC
98
characteristics, these gave six pooled fractions, FE1-FE6.
Fraction FE4 (325 mg) was
99
chromatographed over silica gel FCC (45 x 3 cm), eluting with 1.5 L of n-hexane-CH2Cl2
100
(20:80) and pooled to two subfractions, FE4.1 (62 mg) and FE4.2 (115 mg). Fraction FE4.1 was
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purified using preparative TLC, eluted with 100 mL of CH2Cl2-MeOH (98:2, developed 3
102
times) to afford 1 (7 mg, Rf = 0.49). Fraction FE5 (1.25 g) was placed on silica gel FCC (45x
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4 cm), eluted with 2.4 L of an isocratic system of CH2Cl2-EtOAc (97:3) and pooled to two
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subfractions, FE5.1 (102 mg) and FE5.2 (720 mg).
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preparative TLC, eluted with 101 mL of CH2Cl2-EtOAc-MeOH (70:30:1, developed 6 times)
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to yield 3 (15 mg, Rf = 0.64) and 4 (11 mg, Rf = 0.57). The MeOH extract (16.1 g) was
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applied to silica gel FCC (45 x 6 cm), eluted with a mixture of n-hexane-EtOAc (100:0,
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90:10, 80:20, 70:30, 60:40, 40:60, 20:80, 0:100 v/v, 300 mL each), and EtOAc-MeOH,
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(90:10, 80:20, 70:30, 60:40, 50:50, 30:70, 0:100 v/v, 300 mL each), to give 32 collected
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fractions. On the basis of their TLC characteristics, these gave four pooled fractions, FM1-
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FM4.
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isocratic system of CH2Cl2-EtOAc (70:30, 1 L) to give 11 (6 mg). Purification of FM3 (150
Subfraction FE5.1 was purified using
Fraction FM2 (97 mg) was chromatographed on silica gel FCC, eluting with an
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mg) by preparative TLC, using 101 mL of CH2Cl2-EtOAc-MeOH (75:25:1, developed 4
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times), gave 1 (4 mg, Rf = 0.77) and 2 (3 mg, Rf = 0.72).
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Oxisterigmatocystin E, 1: pale yellow solid; mp 223-224 oC; [α]25D -121.7 (c 0.1,
116
CHCl3); UV (MeOH) λmax (log ε) 224 (3.59), 245 (3.67), 307 (3.19) nm; IR (film, CH2Cl2)
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νmax 2932, 2852, 1749, 1664, 1639, 1593, 1418, 1228, 1084 cm-1; 1H and
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(CDCl3, 400 MHz and 100 MHz) (Table 1); HR-ESI-TOFMS m/z 433.0677 [M+H]+ (calcd
119
for C21H17ClO8+H, 433.0690); m/z
120
435.0661).
13
C NMR data
435.0666 [M+2+H]+ (calcd for C21H17ClO8+2+H,
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Oxisterigmatocystin F, 2: pale yellow solid; mp 184-185 oC; [α]25D -196.5 (c 0.1,
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CHCl3); UV (MeOH) λmax (log ε) 220 (3.50), 243 (3.69), 309 (3.10) nm; IR (film, CH2Cl2)
123
νmax 2928, 2853, 1752, 1659, 1634, 1591, 1452, 1222, 1080 cm-1; 1H and
124
(CDCl3, 400 MHz and 100 MHz) (Table 1); HR-ESI-TOFMS m/z 433.0689 [M+H]+ (calcd
125
for C21H17ClO8+H, 433.0690); m/z 435.0674 [M+2+H]+ (calcd for C21H17ClO8+2+H,
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435.0661).
13
C NMR data
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Oxisterigmatocystin G, 3: pale yellow solid; mp 202-203 oC; [α]25D -99.5 (c 0.1,
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CHCl3); UV (MeOH) λmax (log ε) 207 (3.43), 238 (3.53), 307 (3.13) nm; IR (film, CH2Cl2)
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νmax 2929, 2863, 1752, 1659, 1635, 1591, 1462, 1223, 1081 cm-1; 1H and
130
(CDCl3, 400 MHz and 100 MHz) (Table 1); HR-ESI-TOFMS m/z 399.1099 [M+H]+ (calcd
131
for C21H18O8+H, 399.1080).
13
C NMR data
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Oxisterigmatocystin H, 4: pale yellow solid; mp 180-182 oC; [α]25D -198.2 (c 0.1,
133
CHCl3); UV (MeOH) λmax (log ε) 205 (3.70), 237 (3.72), 309 (3.42) nm; IR (film, CH2Cl2)
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νmax 2848, 1752, 1659, 1641, 1600, 1471, 1267, 1100 cm-1; 1H and
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400 MHz and 100 MHz) (Table 1); HR-ESI-TOFMS m/z 399.1097 [M+H]+ (calcd for
136
C21H18O8+H, 399.1080).
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C NMR data (CDCl3,
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6-O-methylversicolorin A, 9: yellow needles; mp 233-235 oC; [α]25D -312.8 (c 0.1,
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CHCl3); UV (MeOH) λmax (log ε) 229 (3.34), 288 (3.54), 445 (3.06) nm; IR (film, CH2Cl2)
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νmax 2927, 2852, 1629, 1613, 1579, 1383, 1305, 1212, 1155 cm-1; 1H NMR (CDCl3, 400
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MHz) δ 3.92 (3H, s, OMe-6), 4.72 (1H, dt, J = 7.2, 2.4 Hz, H-2′), 5.44 (1H, t, J = 2.4 Hz, H-
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3′), 6.50 (1H, t, J = 2.4 Hz, H-4′), 6.65 (1H, d, J = 2.4 Hz, H-7), 6.81 (1H, d, J = 7.2 Hz, H-
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1′), 7.32 (1H, d, J = 2.4 Hz, H-5), 7.33 (1H, s, H-4), 12.26 (1H, s, OH-8), and 12.38 (1H, s,
143
OH-1); 13C NMR (CDCl3, 100 MHz) δ 48.3 (C-2′), 56.2 (OMe-6), 101.9 (C-3′), 103.6 (C-4),
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107.0 (C-7), 108.6 (C-5), 110.0 (C-8a), 112.0 (C-9a), 113.1 (C-1′), 120.7 (C-2), 135.1 (C-4a),
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136.0 (C-10a), 145.7 (C-4′), 159.6 (C-1), 164.8 (C-3), 165.2 (C-8), 166.5 (C-6), 181.5 (C-10),
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and 190.3 (C-9); HR-ESI-TOFMS m/z 375.0481 [M+Na]+ (calcd for C19H12O7+Na,
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375.0481).
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Antimalarial assay
149
The Antimalarial assay was carried out using a method described by Trager and Jensen,11 and
150
Desjardins et al.12 as described in our previous report.13
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Cytotoxicity assays
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Cytotoxicity assays against three cancer cell lines, KB, MCF-7, and NCI-H187 were carried
153
out by O’Brien’s method.14 The cytotoxicity assays against Vero cell used the method
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described by Hunt and co-workers15, as in our previous reported.13
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RESULTS AND DISCUSSION
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The chromatographic separation of biomass powder of B. piluliferum gave two new
157
sterigmatocystin derivatives, 1 and 2, and nine known compounds, 3-11 (Figure 1). Their
158
structures were identified by spectroscopic data and by comparing the data obtained to those
159
of related known compounds published in the literature. They were oxisterigmatocystins E
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and F, 1 and 2, oxisterigmatocystins G and H, 3 and 4, sterigmatocystin, 5,16 N-0532B, 6,17
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O-methylsterigmatocystin, 7,18 N-0532A, 8,17 6-O-methylversicolorin A, 9, 6,8-O-
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dimethylversicolorin A, 10,19 and 8-O-methylaverufin, 11.7 Compounds 3, 4, and 9 were
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isolated from the natural source for the first time.
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Compounds 1 and 2 had the molecular formula C21H17ClO8, derived from 13C NMR
165
and HR-ESI-TOFMS, signifying 13 indices of hydrogen deficiency. The IR spectra of
166
compounds 1 and 2 showed absorption bands for ester (1749/1752 cm-1), aromatic ketone
167
(1664/1659 cm-1), and aromatic (1418/1452 cm-1) groups. The 13C NMR and DEPT spectra
168
of these two compounds indicated 21 carbon signals attributable to three methyl (two
169
methoxy and an acetoxy groups), a methylene, three sp2 methine, three sp3 methine and
170
eleven sp2 quaternary (including two carbonyl) carbons. The 1H and 13C NMR spectroscopic
171
data of 1 (Table 1) agreed with those of isolated N-0532A, 8, 17 except that the double bond at
172
C-3′ was saturated by a proton at C-3ʹ [δH/C 2.55 (m, H2-3′)/37.1] and an acetoxy group at C-
173
4ʹ [δH/C 2.11(s)/21.2, δC 169.8]. The three resonances of aromatic protons appeared at δ 6.38
174
(s, H-2), 7.08 (d, J = 9.0 Hz, H-5) and 7.58 (d, J = 9.0 Hz, H-6). Correlations of H-1′/H-
175
2′/H2-3′/H-4′ in the COSY spectrum confirmed the lack of a double bond at C-3′ of the
176
bishydrofuran unit. The HMBC also indicated the connectivity of an acetoxy group through
177
C-4′ by showing correlations of H3-6′ to C-5′ and C-4ʹ, and H-4′ to C-5′ (Figure 2).
178
The 1H and
13
C NMR spectroscopic data of 2 (Table 1) were similar to those of 1,
179
except that the resonance of methyl protons of the acetoxy group at C-4′ of 2 (δ 1.69)
180
appeared at a higher field than that of 1 (δ 2.11). The coupling constants between H-1′ and
181
H-2′ (J = 6.0 Hz) and the NOESY correlations of the two protons of 1 and 2 revealed a cis
182
ring fusion, the same as in the sterigmatocystin, 5,18,20,21 which allowed assignment of the
183
absolute configurations at both C-1′ and C-2′ as S. The assignment of configurations at C-4′
184
of 1 and 2 as R and S were determined by comparing the 1H NMR resonances of an acetoxy
185
group to those reported for related analogues, dothistromin pentaacetate22 and
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oxisterigmatocystin D.23 The methyl protons of the 4′-acetoxy group of 2 appeared at a
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higher field (δH 1.69) than in 1 (δH 2.11), agreeing with that reported for endo dothistromin
188
pentaacetate (δH 1.67)22 which was due to the strong shielding effect of xanthone (Figure 3).
189
On the other hand, the 4′-acetoxy group of 1 showed a resonance at δH 2.11, suggesting the
190
exo arrangement (Figure 3), which corresponds to that of oxisterigmatocystin D (δH 2.07).23
191
Furthermore, the optical rotation value of 1 was different from 2, suggesting the different
192
configurations at C-4′ of the two compounds. Based on the above evidence, the structure of
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1, oxisterigmatocystin F, was determined as a new sterigmatocystin derivative. Compound 2,
194
oxisterigmatocystin F was identified as the C-4′ epimer of 1 (Figure1). Compounds 3 and 4 possessed a molecular formula, C21H18O8, from
195
13
C NMR and
196
HR-ESI-TOFMS, indicating 13 degrees of hydrogen deficiency. IR spectra of both 3 and 4
197
showed bands for ester (1752/1752 cm-1), aromatic ketone (1659/1659 cm-1), and aromatic
198
(1462/1471 cm-1) groups. Their
199
attributable to three methyls (two methoxy groups and an acetoxy group), a methylene, four
200
sp2 methine, three sp3 methine and ten sp2 quaternary (including two carbonyl) carbons. The
201
1
H NMR,
13
13
C NMR and DEPT spectra displayed 21 carbon signals
C NMR and DEPT spectroscopic data of 3 (Table 1) were similar to those of 1,
202
except for the presence of an additional sp2 methine proton at C-7 [δH 6.75 (d, J = 8.4 Hz, H-
203
7)]. This information, together with the absence of Cl isotope in the MS data, established that
204
the chlorine atom was displaced by an sp2 methine proton. The COSY spectrum showed
205
correlations of H-5/H-6/H-7, indicating trisubstitution of the aromatic ring. The HMBC
206
spectrum of 3 clearly demonstrated correlations of H-7 to C-8a and C-5, H-6 to C-8 and C-
207
10a, and H-5 to C-7, C-8a and C-10a, confirming the structure of 3. Since the resonances of
208
acetoxyl groups of 3 (δH 2.10) and 4 (δH 1.67) appeared at low and high fields in the same
209
manner of those of 1 and 2, the configurations at C-4′ of 3 and 4 were assigned as R and S,
210
respectively. Moreover, the optical rotation values of 3 and 4 were comparable to those of 1
211
and 2, respectively. However, compounds 3 and 4 have been previously reported as synthetic
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mixture products during the preparation of O-methylsterigmatocystin (7).24 This is the first
213
isolation of compounds 3 and 4 from a natural source and they have been named
214
oxisterigmatocystin G, 3, and oxisterigmatocystin H, 4. Their spectroscopic data was also
215
reported.
216
Compound 9 had the molecular formula C19H12O7, deduced from 13C NMR and HR-
217
ESI-TOFMS, requiring 14 degrees of hydrogen deficiency. The IR spectrum displayed
218
absorption bands for aromatic ketones (1629 and 1613 cm-1) and aromatic (1579 cm-1)
219
groups. The 13C NMR and DEPT spectra displayed 19 carbon signals for a methoxy, five sp2
220
methine, two sp3 methine and eleven sp2 quaternary (including two carbonyl) carbons.
221
Careful examination of 1D and 2D NMR data indicated that the structure of 9 was similar to
222
that of 6,8-O-dimethylversicolorin A, which has been previously reported as a methylation
223
product of versicolorin A.25 The HMBC spectrum revealed correlations of hydroxyl proton at
224
C-8 to C-7, C-8 and C-8a confirming the position of the hydroxyl group at C-8. Besides, the
225
correlations of H-4 to C-2, C-3, C-10, and C-9a, H-5 to C-7, C-8a, and C-10, H-7 to C-5, C-6,
226
C-8, and C-8a, H-1′ to C-3, C-2′, C-3′, and C-4′, H-3′ to C-1′ and C-2′, H-4′ to C-2′, methoxyl
227
protons at C-6 to C-6, and hydroxyl proton (OH-1) to C-1, C-2 and C-9a indicated the
228
structure of 9. The absolute configuration of 9 was assigned to be the same as that of 6,8-O-
229
dimethylversicolorin A, 10, by comparing their optical rotations, -320 (c 0.12, dioxane) for
230
1019 and -312.8 (c 0.1, CHCl3) for 9. Thus, this is the first report of 6-O-methylversicolorin
231
A, 9, isolated from a natural source.
232
Compounds 1, 3 and 4 showed antimalarial activity against P. falciparum with IC50
233
values of 7.9, 14.7 and 23.9 µM, respectively. Compounds 1-4 exhibited cytotoxicity against
234
three cancer cell lines (KB, MCF-7 and NCI-H187) with IC50 values ranging from 3.5 to 78.6
235
µM. Compound 3 showed significant cytotoxicity against KB cells with IC50 value of 3.5
236
µM, which is lower than the control drug, ellipticine. Furthermore, 3 was cytotoxic against
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the MCF-7 cell line with IC50 value of 6.9 µM, which is lower than the control drug,
238
doxorubicin. However, 3 was highly cytotoxic toward the normal cell line (Vero cell) with
239
IC50 value of 1.6 µM, which is lower than the control drug, ellipticine 2.5 µM. Among
240
compounds 1-4, the influence of an exo- versus an endo- arrangement was noticed. The exo-
241
arrangements in 1 and 3 showed both higher antimalarial activity and cytotoxicity against
242
cancer cells than their endo- analogues, 2 and 4. It should be noted that the missing double
243
bond at C-3ʹ and the presence of an acetoxy group at C-4ʹ of sterigmatocystin derivatives 1-4
244
would play an important role for cytotoxicity enhancement when comparing to derivatives 5-
245
8. Moreover, the double bond at C-3ʹ and C-4ʹ of sterigmatocystin derivatives 5-8 exhibited
246
no cytotoxicity (>100 µM) toward KB and MCF-7, which corresponds to the results for
247
related structures in a previous report.7 In addition, compounds 9 and 10 showed strong
248
cytotoxicity against the NCI-H187 cell line with IC50 values of 2.1 and 0.38 µM,
249
respectively, which were lower than the control drug, ellipticine. By comparison between
250
compounds 9 and 10, the presence of a methoxy group at C-8 led to a decrease in cytotoxicity
251
toward normal cells. Interestingly, compound 10 was not cytotoxic against Vero cells and
252
should be further studied in detail. However, most of the isolated compounds showed
253
cytotoxicity against Vero cells with IC50 values in the range from 0.65 to 12.3 µM (Table2).
254
Sterigmatocystin, 5, has been reported to contaminate food stuffs such as wheat, rice,
255
coffee beans, corn and red pepper.
Furthermore, it has been discovered in cheese
256
contaminated with Aspergillus versicolor.9 Based on our results, most isolated compounds
257
from B. piluliferum are mycotoxins with structures metabolically related to the aflatoxin
258
carcinogen.
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piluliferum. As mentioned above, the fungus B. piluliferum was found in the seeds of chili
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pepper,3 and so the contamination of B. piluliferum in the food chain and agricultural soil
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should be monitored.
These mycotoxins could be responsible for the toxicity of the fungus B.
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ACKNOWLEDGMENT
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This work was supported by the Thailand Research Fund via the Royal Golden Jubilee Ph.D.
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program (0353/2551), and the Thailand Research Fund and Khon Kaen University (grant
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number RTA5980002). We thank the Center of Excellence for Innovation in Chemistry
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(PERCH-CIC) for partial support and BIOTEC via the Bioresources Research Network
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(BRN), Thailand for bioactivity tests.
269 270
Supporting Information
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1
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via the Internet at http://pubs.acs.org.
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REFERENCES
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(1) Domsch, K. H.; Gams, W.; Anderson, T.-H. Compendium of soil fungi, 1st ed. Germany:
275
H and 13C NMR and 2D NMR spectra for compounds 1−4 and 9 are available free of charge
IHW-Verl: Eching, 1993; pp 859.
276
(2) Downing, M. H. Botryotrichum and Coccospora. Mycologia 1953, 45, 934–940.
277
(3) Chigoziri, E.; Ekefan, E. J. Seed borne fungi of chili pepper (Capsicum frutescens) from
278
pepper producing areas of Benue State, Nigeria. Agric. Biol. J. North Am. 2013, 4, 370–
279
374.
280
(4) Mocek, U.; Schultz, L.; Buchan, T.; Baek, C.; Fretto, L.; Nzerem, J.; Sehl, L.; Sinha, U.
281
Isolation and structure elucidation of five new asterriquinones from Aspergillus,
282
Humicola and Botryotrichum species. J. Antibiot. 1996, 49, 854–859.
283
(5) Sy, A. A.; Swenson, D. C.; Gloer, J. B.; Wicklow, D. T. Botryolides A−E,
284
decarestrictine analogues from a fungicolous Botryotrichum sp. (NRRL 38180). J. Nat.
285
Prod. 2008, 71, 415–419.
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Journal of Agricultural and Food Chemistry
13/22 286
(6) Jurjević, Ž.; Peterson, S. W.; Solfrizzo, M.; Peraica, M. Sterigmatocystin production by
287
nine newly described Aspergillus species in section Versicolores grown on two different
288
media. Mycotoxin Res. 2013, 29, 141–145.
289
(7) Kornsakulkarn, J.; Saepua, S.; Srichomthong, K.; Supothina, S.; Thongpanchang, C.
290
New mycotoxins from the scale insect fungus Aschersonia coffeae Henn. BCC 28712.
291
Tetrahedron 2012, 68, 8480–8486.
292
(8) Maskey, R. P.; Grün-Wollny, I.; Laatsch, H. Isolation, structure elucidation and
293
biological activity of 8-O-methylaverufin and 1,8-O-dimethylaverantin as new
294
antifungal agents from Penicillium chrysogenum. J. Antibiot. 2003, 56, 459–463.
295
(9) Veršilovskis, A.; De Saeger, S. Sterigmatocystin: Occurrence in foodstuffs and
296
analytical methods – An overview. Mol. Nutr. Food Res. 2010, 54, 136–147.
297
(10) Mori, H.; Kawai, K.; Ohbayashi, F.; Kuniyasu, T.; Yamazaki, M.; Hamasaki, T.;
298
Williams, G. M. Genotoxicity of a variety of mycotoxins in the hepatocyte primary
299
culture/DNA repair test using rat and mouse hepatocytes. Cancer Res. 1984, 44, 2918–
300
2923.
301 302 303
(11) Trager, W.; Jensen, J. B. Human malaria parasites in continuous culture. Science 1976, 193, 673–675. (12) Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Quantitative assessment
304
of antimalarial activity in vitro by a semiautomated microdilution technique.
305
Antimicrob. Agents Chemother. 1979, 16, 710–718.
306
(13) Rajachan, O.-A.; Kanokmedhakul, S.; Kanokmedhakul, K.*; Soytong, K. Bioactive
307
depsidones from the fungus Pilobolus heterosporous. Planta. Medica. 2014, 80, 1635-
308
1640.
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Page 14 of 22
14/22 309
(14) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Investigation of the Alamar Blue
310
(resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J.
311
Biochem. 2000, 267, 5421–5426.
312
(15) Hunt, L.; Jordan, M.; De Jesus, M.; Wurm, F. M. GFP-expressing mammalian cells for
313
fast, sensitive, noninvasive cell growth assessment in a kinetic mode. Biotechnol. Bioeng.
314
1999, 65, 201–205.
315
(16) Fremlin, L. J.; Piggott, A. M.; Lacey, E.; Capon, R. J. Cottoquinazoline A and
316
cotteslosins A and B, metabolites from an Australian marine-derived strain of
317
Aspergillus versicolor. J. Nat. Prod. 2009, 72, 666–670.
318
(17) Nishino, Y.; Takawa, N; Mitsumori, N.; Seki, T. Chemical compound N-0532, the
319
manufacturing method and application. Jpn. Kokai Tokkyo Koho 1999, JP 11080162 A,
320
March 26, 1999.
321
(18) Cox, R. H.; Cole, R. J. Carbon-13 nuclear magnetic resonance studies of fungal
322
metabolites, aflatoxins, and sterigmatocystins. J. Org. Chem. 1977, 42, 112–114.
323
(19) Hatsuda, Y.; Hamasaki, T.; Ishida, M.; Kiyama, Y. 6, 8-O-Dimethylversicolorin A, a
324
new metabolite from Aspergillus versicolor. Agric. Biol. Chem. 1971, 35, 444.
325 326 327 328 329 330
(20) Zhu, F.; Lin, Y. Three xanthones from a marine-derived mangrove endophytic fungus. Chem. Nat. Compd. 2007, 43, 132–135. (21) Cai, S.; Zhu, T.; Du, L.; Zhao, B.; Li, D.; Gu, Q. Sterigmatocystins from the deep-seaderived fungus Aspergillus versicolor. J. Antibiot. 2011, 64, 193–196. (22) Gallagher, R. T.; Hodges, R. The chemistry of dothistromin, a difuronanthraquinone From Dothistroma pini. Aust. J. Chem. 1972, 25, 2399-2407.
331
(23) Zhao, H.; Wang, G.-Q.; Tong, X.-P.; Chen, G.-D.; Huang, Y.-F.; Cui, J.-Y.; Kong, M.-
332
Z.; Guo, L.-D.; Zheng, Y.-Z.; Yao, X.-S.; Gao, H. Diphenyl ethers from Aspergillus sp.
333
and their anti-Aβ42 aggregation activities. Fitoterapia 2014, 98, 77–83.
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(24) Rance, M. J.; Roberts, J. C. Total synthesis of (±)-O-methylsterigmatocystin. Tetrahedron Lett. 1970, 11, 2799–2802. (25) Gorst-Allman, C. P.; Steyn, P. S.; Wessels, P. L.; Scott, D. B. Carbon-13 nuclear
337
magnetic resonance assignments and biosynthesis of versicolorin A in Aspergillus
338
parasiticus. J. Chem. Soc., Perkin Trans. 1. 1978, No. 9, 961.
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FIGURE CAPTIONS Figure 1. Structures of the isolated compounds 1-11 Figure 2. Selected HMBC correlations of compound 1 Figure 3. Structures of 1 (exo- form) and 2 (endo- form), energy minimized using MM2
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Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1-4 Position
1 δH
1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 1' 2'
6.38 s
7.08 d (9.0)a 7.58 d (9.0)
3' 4' 5' 6' 1-OMe 8-OMe a
6.51 d (6.0) 4.30 ddd (9.6, 7.2, 6.0) 2.55 m 6.39 dd (9.6, 4.0) 2.11 s 3.96 s 4.03 s
2
δC 163.7 90.9 163.7 105.9 153.6 113.7 134.2 124.7 156.2 119.1 173.9 108.4 154.7 112.6 42.6 37.1 98.6 169.8 21.2 56.9 62.0
δH
3
δC
6.38 s
7.14 d (9.0) 7.61 d (9.0)
6.55 d (6.0) 4.26 ddd (7.2, 6.0, 2.4) 2.54 dd (7.2, 4.0) 6.46 brd (4.0) 1.69 s 3.97 s 4.05 s
163.6 90.4 163.9 106.2 153.3 113.8 134.3 124.8 156.3 119.2 174.2 108.2 154.8 113.8 42.7 37.0 98.7 169.8 21.1 56.9 62.0
δH 6.33 s
6.89 d (8.4) 7.48 t (8.4) 6.75 d (8.4)
6.48 d (6.0) 4.28 ddd (10.4, 6.0, 4.0) 2.54 td (8.4, 4.0) 6.38 t (4.0) 2.10 s 3.91 s 3.95 s
Values in parentheses are coupling constants in Hz.
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δC 163.4 90.6 163.1 105.6 153.5 109.0 133.7 106.4 160.7 113.9 175.2 109.1 156.7 112.4 42.7 37.1 98.6 169.8 21.2 56.7 56.5
δH 6.33 s
6.93 d (8.4) 7.49 t (8.4) 6.77 d (8.4)
6.51d (6.0) 4.25 dd (10.4, 6.0) 2.54 dd (6.0, 2.4) 6.43 brd (2.4) 1.67 s 3.91 s 3.95 s
δC 163.3 90.2 163.3 106.0 153.2 109.1 133.7 106.5 160.8 114.0 175.3 108.9 156.8 113.6 42.7 37.0 98.7 169.8 21.1 56.5 56.7
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Table 2. Biological Activity of Compounds 1-11 Compound 1 2 3 4 5 6 7 8 9 10 11 Dihydroartemisinin Doxorubicin Ellipticine
Antimalarial IC50 (µM) 7.9 >100 14.7 23.9 >100 >100 >100 >100 >100 >100 >100 0.004
KBa 7.7 25.5 3.5 25.1 >100 >100 >100 >100 >100 >100 >100 0.83 4.9
a
Human epidermoid carcinoma in the mouth
b
Human breast adenocarcinoma
c
Cytotoxicity IC50 (µM) MCF-7b NCI-H187c Vero celld 78.6 10.9 9.7 38.7 25.7 4.3 6.9 11.6 1.6 33.6 22.8 6.0 >100 70.3 0.82 >100 62.2 0.65 >100 >100 12.3 >100 35.3 2.3 36.1 2.1 2.5 >100 0.38 >100 >100 44.9 >100 17.0
Human small cell lung cancer
d
African green monkey kidney
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2.5
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Figure 1. 6'
CH3
CH3 5'
O
O
O
O 4' 3'
O
O 2'
5
R
10 H
6
O
7
10a 8a
4 4a 9a 9
8
H 1'
R
2
R OCH3 O
OCH3
OCH3
OH
O H
H O
H3CO
4
10 4a 10a 9a 8a
6 7
R OCH3 O
OCH3
7 R=H 8 R = Cl
R
8
O
9
O
OCH3
O
O 5
O
5 R=H 6 R = Cl
2 R = Cl 4 R=H
1 R = Cl 3 R=H
O
O
O
3
1
OCH3 O
H
H
O
O
O
O H
H
1
O 3 2
OH
H
O 4'
HO
1' 2'
CH3
O O
3'
H
9 R=H 10 R = CH3
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OCH3 O
OH
11
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Figure 2.
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Figure 3.
7
6 5
8 7
6
8a
10a
8
10a 4a
9 5
9
1
10
4a
9a
4
2ʹ 4
2
3
3ʹ
1ʹ
2ʹ
1 2
3
1ʹ
3ʹ 5ʹ
6ʹ 4ʹ
4ʹ
5ʹ 6ʹ
1
2
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TABLE OF CONTENTS GRAPHIC
Botryotrichum piluliferum
New mycotoxins
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