Mycotoxins from the Fungus Botryotrichum ... - ACS Publications

Subscriber access provided by UNIV OF YORK

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22

Journal of Agricultural and Food Chemistry

1

Mycotoxins from the Fungus Botryotrichum piluliferum

2 3

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

7

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:

13

* Tel.: +66 043-009700 ext. 42174-5. Fax: +66-43-202373. E-mail: [email protected]

ACS Paragon Plus Environment


Page 2 of 22

2/22

14

ABSTRACT

15

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,

18

dimethylversicolorin A, 10, and 8-O-methylaverufin, 11 were isolated from the fungus

19

Botryotrichum piluliferum.

20

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

33 34 35 36 37 38


Page 3 of 22


3/22

39

INTRODUCTION

40

The fungus Botryotrichum piluliferum belongs to the family Chaetomiaceae.1

41

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

46

seed borne fungi of chili pepper.3

47

Botryotrichum has reported it to contain asterriquinone CT2 from Botryotrichum spp.4 and

48

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

51

Many fungi such as Aspergillus species,6 Aschersonia coffeae Henn. BCC 28712,7

52

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

55

metabolites from fungi isolated from Thai soil, crude n-hexane and EtOAc extracts of B.

56

piluliferum displayed cytotoxicity against the KB cell line with 85.5% and 59.7% inhibition,

57

respectively, at a concentration of 50 µg/mL.

58

cytotoxicity toward the MCF-7 cell line with 57.5% inhibition.

59

structural elucidation, and bioactivities of eleven mycotoxins from B. piluliferum are

60

presented.

Moreover, the EtOAc extract presented

61 62

MATERIALS AND METHODS

63

General Experimental Procedures


Herein, the isolation,


Page 4 of 22

4/22 64

Optical rotations were measured on a DIP-1000 digital polarimeter (JASCO, Easton, MD).

65

IR spectra were obtained using a Bruker Tenser 27 spectrophotometer (Bruker, Ettlingen,

66

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).

69

Micromass Q-TOF 2 hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer

70

(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).

73

Fungal Material

74

The fungus strain (no. Bp01) was isolated from forest soil collected from Three Pagoda Pass,

75

Sangkhla Buri, Kanchanaburi Province, Thailand in 2010. It was identified as the

76

Botryotrichum piluliferum by K. Soytong, one of the coauthors. The fungus was cultured at

77

25-28 °C for 9 weeks using the method described in our previous report.13 The biomass was

78

collected by filtering, air-dried and ground into a powder.

79

Extraction and Isolation

80

The biomass powder of B. piluliferum (240 g) was extracted successively with n-hexane (1L

81

x 3), EtOAc (1L x 3), and MeOH (1L x 3) by stirring at room temperature. The n-hexane

82

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,

84

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.

86

fractions, FH1-FH5. Recrystallization of fraction FH1 (76 mg) from CH2Cl2 yielded 9 (30

87

mg). Fraction FH2 (196 mg) was purified using preparative TLC, eluted with 101 mL of n-

88

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


Page 5 of 22


5/22 89

mg, Rf = 0.56). Fraction FH3 (681 mg) was further subjected to a silica gel FCC (45 x 3 cm)

90

eluting with 1.8 L of an isocratic system of n-hexane-CH2Cl2 (40:60) to give 10 (3 mg) and 8

91

(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

93

FH5 (102 mg) using preparative TLC with hexane-CH2Cl2-MeOH (5:95:2, developed 3

94

times). The EtOAc extract (9.3 g) was chromatographed on silica gel FCC (45 x 6 cm),

95

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,

97

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

101

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

103

4 cm), eluted with 2.4 L of an isocratic system of CH2Cl2-EtOAc (97:3) and pooled to two

104

subfractions, FE5.1 (102 mg) and FE5.2 (720 mg).

105

preparative TLC, eluted with 101 mL of CH2Cl2-EtOAc-MeOH (70:30:1, developed 6 times)

106

to yield 3 (15 mg, Rf = 0.64) and 4 (11 mg, Rf = 0.57). The MeOH extract (16.1 g) was

107

applied to silica gel FCC (45 x 6 cm), eluted with a mixture of n-hexane-EtOAc (100:0,

108

90:10, 80:20, 70:30, 60:40, 40:60, 20:80, 0:100 v/v, 300 mL each), and EtOAc-MeOH,

109

(90:10, 80:20, 70:30, 60:40, 50:50, 30:70, 0:100 v/v, 300 mL each), to give 32 collected

110

fractions. On the basis of their TLC characteristics, these gave four pooled fractions, FM1-

111

FM4.

112

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



Page 6 of 22

6/22 113

mg) by preparative TLC, using 101 mL of CH2Cl2-EtOAc-MeOH (75:25:1, developed 4

114

times), gave 1 (4 mg, Rf = 0.77) and 2 (3 mg, Rf = 0.72).

115

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)

117

νmax 2932, 2852, 1749, 1664, 1639, 1593, 1418, 1228, 1084 cm-1; 1H and

118

(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,

121

Oxisterigmatocystin F, 2: pale yellow solid; mp 184-185 oC; [α]25D -196.5 (c 0.1,

122


123

νmax 2928, 2853, 1752, 1659, 1634, 1591, 1452, 1222, 1080 cm-1; 1H and

124


125

for C21H17ClO8+H, 433.0690); m/z 435.0674 [M+2+H]+ (calcd for C21H17ClO8+2+H,

126

435.0661).

13

C NMR data

127

Oxisterigmatocystin G, 3: pale yellow solid; mp 202-203 oC; [α]25D -99.5 (c 0.1,

128


129

νmax 2929, 2863, 1752, 1659, 1635, 1591, 1462, 1223, 1081 cm-1; 1H and

130


131

for C21H18O8+H, 399.1080).

13

C NMR data

132

Oxisterigmatocystin H, 4: pale yellow solid; mp 180-182 oC; [α]25D -198.2 (c 0.1,

133


134

νmax 2848, 1752, 1659, 1641, 1600, 1471, 1267, 1100 cm-1; 1H and

135

400 MHz and 100 MHz) (Table 1); HR-ESI-TOFMS m/z 399.1097 [M+H]+ (calcd for

136

C21H18O8+H, 399.1080).


13

C NMR data (CDCl3,

Page 7 of 22


7/22 137

6-O-methylversicolorin A, 9: yellow needles; mp 233-235 oC; [α]25D -312.8 (c 0.1,

138


139

νmax 2927, 2852, 1629, 1613, 1579, 1383, 1305, 1212, 1155 cm-1; 1H NMR (CDCl3, 400

140

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-

141

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-

142

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),

144

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),

145

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),

146

and 190.3 (C-9); HR-ESI-TOFMS m/z 375.0481 [M+Na]+ (calcd for C19H12O7+Na,

147

375.0481).

148

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

151

Cytotoxicity assays

152

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

154

described by Hunt and co-workers15, as in our previous reported.13

155

RESULTS AND DISCUSSION

156

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

160

and F, 1 and 2, oxisterigmatocystins G and H, 3 and 4, sterigmatocystin, 5,16 N-0532B, 6,17

161

O-methylsterigmatocystin, 7,18 N-0532A, 8,17 6-O-methylversicolorin A, 9, 6,8-O-



Page 8 of 22

8/22 162

dimethylversicolorin A, 10,19 and 8-O-methylaverufin, 11.7 Compounds 3, 4, and 9 were

163

isolated from the natural source for the first time.

164

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

186

oxisterigmatocystin D.23 The methyl protons of the 4′-acetoxy group of 2 appeared at a


Page 9 of 22


9/22 187

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

193

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



Page 10 of 22

10/22 212

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


Page 11 of 22


11/22 237

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.

259

piluliferum. As mentioned above, the fungus B. piluliferum was found in the seeds of chili

260

pepper,3 and so the contamination of B. piluliferum in the food chain and agricultural soil

261

should be monitored.

These mycotoxins could be responsible for the toxicity of the fungus B.



Page 12 of 22

12/22 262 263

ACKNOWLEDGMENT

264

This work was supported by the Thailand Research Fund via the Royal Golden Jubilee Ph.D.

265

program (0353/2551), and the Thailand Research Fund and Khon Kaen University (grant

266

number RTA5980002). We thank the Center of Excellence for Innovation in Chemistry

267

(PERCH-CIC) for partial support and BIOTEC via the Bioresources Research Network

268

(BRN), Thailand for bioactivity tests.

269 270

Supporting Information

271

1

272

via the Internet at http://pubs.acs.org.

273

REFERENCES

274

(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.


Page 13 of 22


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.



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.


Page 15 of 22


15/22 334 335 336

(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.

339



Page 16 of 22

16/22

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


Page 17 of 22


17/22

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.


4

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


Page 18 of 22

18/22

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


0.16 4.7

2.5

Page 19 of 22


19/22

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


OCH3 O

OH

11


Page 20 of 22

20/22

Figure 2.


Page 21 of 22


21/22

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



Page 22 of 22

22/22

TABLE OF CONTENTS GRAPHIC

Botryotrichum piluliferum

New mycotoxins


Mycotoxins from the Fungus Botryotrichum ... - ACS Publications

Recommend Documents