New Ustilaginoidins from Rice False Smut Balls Caused by

Subscriber access provided by Binghamton University | Libraries

Article

New Ustilaginoidins from Rice False Smut Balls Caused by Villosiclava virens and Their Phytotoxic and Cytotoxic Activities Weibo Sun, Ali Wang, Dan Xu, Weixuan Wang, Jiajia Meng, Jungui Dai, Yang Liu, Daowan Lai, and Ligang Zhou J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 02 Jun 2017 Downloaded from http://pubs.acs.org on June 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 34

Journal of Agricultural and Food Chemistry

New Ustilaginoidins from Rice False Smut Balls Caused by Villosiclava virens and Their Phytotoxic and Cytotoxic Activities

Weibo Sun,† Ali Wang,† Dan Xu,† Weixuan Wang,† Jiajia Meng,† Jungui Dai, Liu,§ Daowan Lai,*,† and Ligang Zhou*,†

†

⊥

Yang

Key Laboratory of Pest Monitoring and Green Management of MOA, Department of Plant

Pathology, College of Plant Protection, China Agricultural University, Beijing 100193, China ⊥

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of

Materia Medica, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100050, China §

Key Laboratory of Agro-products Processing of MOA, Institute of Food Science and Technology,

Chinese Academy of Agricultural Sciences, Beijing 100193, China

*

Corresponding Author

Tel.: +86 10 62731199. Fax: +86 10 6273 1062. E-mail: [email protected].

1

ACS Paragon Plus Environment


Page 2 of 34

1

ABSTRACT

2

Ustilaginoidins are a class of bis-naphtho-γ-pyrones, typically produced by

3

Villosiclava virens, the pathogen of the rice false smut (RFS) which has been one of

4

the most destructive rice fungal diseases. Previously, we found that the ustilaginoidins

5

identified from the culture of V. virens on rice medium were less polar than those

6

reported from the RFS balls in general. In this study, we re-investigated the

7

HPLC-DAD-HRMS profile of the EtOAc extract of the RFS balls, and found several

8

interesting peaks that corresponding to new ustilaginoidins. As a result, eight new and

9

polar congeners, named ustilaginoidins Q-T, 1-4, 2,3-dihydroustilaginoidin T, 5, and

10

ustilaginoidins U-W, 6-8, were isolated. In addition, seventeen known ustilaginoidins,

11

including ustilaginoidins K-N, 9-12, P, 13, E1, 14, isochaetochromin B2, 15, and

12

ustilaginoidins A-J, 16-25, were re-isolated. The structures of the new compounds

13

were elucidated by comprehensive analysis of the spectroscopic data. Ustilaginoidins

14

Q, 1, and R, 2, feature an uncommon 2-hydroxypropyl substituted skeleton, and

15

biogenetically incorporate one more acetate unit than the common ustilaginoidins.

16

Ustilaginoidin

17

Ustilaginoidins R, 2, U, 6, B, 17, and I, 24, showed moderate inhibitory activities

18

toward the radicle or germ elongation of rice seeds. Ustilaginoidins R, 2, S, 3, V, 7, W,

19

8, B, 17, C, 18, and H-J, 23-25, were cytotoxic to the tested human cancer cell lines

20

(HCT116, NCI-H1650, BGC823, Daoy, and HepG2) with IC50 values in the range of

21

4.06~44.1 µM.

W,

8,

is

a

rare

formate-containing

bis-naphtho-γ-pyrone.

22 23

KEYWORDS: bis-naphtho-γ-pyrones, ustilaginoidins, rice false smut balls,

24

Villosiclava virens, phytotoxic activity, cytotoxicity

25 2


Page 3 of 34


26

INTRODUCTION

27

The rice false smut (RFS) caused by the pathogenic fungus Villosiclava virens

28

(Nakata) Tanaka and Tanaka (anamorph: Ustilaginoidea virens Takahashi) has

29

become an emerging, increasingly significant and worldwide fungal disease in many

30

rice (Oryza sativa L.) growing areas over the past few years.1, 2 A typical symptom of

31

this disease is the formation of ball-like colonies on rice panicles called RFS balls.

32

Two types of mycotoxins have been reported from the RFS balls, including the

33

colorless ustiloxins and colored ustilaginoidins.3-7 Ustilaginoidins are 9,9′-linked

34

bis-naphtho-γ-pyrones with an aR configuration.8-10 Up to now, ten ustilaginoidins

35

(A-J) have been reported from the RFS balls.3-5 Ustilaginoidins were also identified

36

from the culture of V. virens on rice media.11 These metabolites showed teratogenicity

37

towards mouse embryo limb bud and midbrain cells,12 cytotoxicity against human

38

epidermoid carcinoma cells (KB),13 and ovarian cancer cells (A2780), 11 inhibition of

39

ATP synthesis in mitochondria,14 antibacterial activity,11, 15 as well as phytotoxicity

40

against the radicle elongation of rice seeds.11

41

Previously, we characterized thirteen ustilaginoidins from the EtOAc extract of V.

42

virens fermented on rice medium.11 These metabolites are generally less polar than

43

those reported from the RFS balls,3-5 as they do not contain any oxygenated side chain

44

in the γ-pyrone rings of their structures. The HPLC profile of the crude extract of V.

45

virens fermented on rice medium is distinct from that of the RFS balls (Figure 1) for

46

having less peaks in the “polar” region (retention time: 0-25 min). On careful

47

investigation of the HPLC-DAD-HRMS profile of the crude extract of the RFS balls

48

(data not shown), we found several unidentified peaks that corresponding to new

49

ustilaginoidins. As a result, eight new bis-naphtho-γ-pyrones were isolated, together 3



Page 4 of 34

50

with 17 known congeners. Herein, we reported the isolation and structure elucidation

51

of the new ustilaginoidins, as well as their biological activities.

52

MATERIALS AND METHODS

53

General Experimental Procedures. Optical rotations were recorded on a Rudolph

54

Autopol IV automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ).

55

UV spectra were recorded on a TU-1810 UV/vis spectrophotometer (Beijing Persee

56

General Instrument Co., Ltd., Beijing, China). Circular dichroism (CD) spectra were

57

recorded on a J-810 CD spectrometer (JASCO Corp., Tokyo, Japan). Infrared (IR)

58

spectra were measured on a Nicolet Nexus 470 FT-IR spectrometer (Thermo Electron

59

Scientific Instrument Crop., Madison, WI). High-resolution electrospray ionization

60

mass spectrometry (HRESIMS) spectra were recorded on a LC 1260/Q-TOF-MS

61

6520 machine (Agilent Technologies, Santa Clara, CA). 1H,

62

(HSQC, HMBC) spectra were measured on an Avance 600 NMR spectrometer

63

(Bruker BioSpin, Zürich, Switzerland). Chemical shifts are expressed in δ (ppm)

64

referring to the internal standard TMS, or solvent residual peaks (δH 2.50, δC 39.5 for

65

DMSO-d6), and coupling constants (J) are in hertz. Silica gel (200-300 mesh)

66

(Qingdao Marine Chemical Inc., Qingdao, China) and Sephadex LH-20 (Pharmacia

67

Biotech, Uppsala, Sweden) were used for column chromatography. Preparative

68

high-speed counter-current chromatography (HSCCC) was performed on a TBE-300B

69

instrument (Tauto Biotech, Shanghai, China) equipped with three preparative coils, a

70

polytetrafluoroethylene tube (2.6 mm in diameter and total volume of 280 mL), and a

71

20 mL sample loop. The separation was carried out at 25 °C using the lower phase as

72

the mobile phase at a flow rate of 3.2 mL/min, revolution speed of 800 rpm, and

73

detection wavelength at 280 nm. HPLC-DAD analysis was performed using an

13

C, and 2D NMR

4


Page 5 of 34


74

LC-20A instrument with a SPD-M20A photodiode array detector (Shimadzu Corp.,

75

Tokyo, Japan) and an analytical column (250 mm×4.6 mm i.d., 5 µm, Luna, C18(2)

76

100A) (Phenomenex Inc., Torrance, CA). The mobile phase consisted of methanol (B),

77

and water contained 0.02% oxalic acid (A), using a gradient elution program (0 min

78

50% B, 5 min 50% B, 35 min 100% B, 40 min 100% B). Semi-preparative HPLC

79

separation was carried out on a Lumtech instrument (Lumiere Tech. Ltd., Beijing,

80

China) equipped with a K-501 pump (flow rate: 3 mL/min) and a K-2501 UV detector

81

using a C18 column (250 mm×10 mm i.d., 5µm, Luna, C18) (Phenomenex Inc.).

82

RFS Balls Materials. RFS balls were collected from Linyi County (118.24°E,

83

35.15°N), Shandong Province, China, in October 2011. The balls were left to dry in

84

shade at room temperature to a constant weight, and were stored at -20 °C before use.

85

Extraction and Isolation. The dry and powdered RFS balls (9.1 kg) were soaked in

86

deionized water at room temperature (3×30 L, 48 h each) and shaken vigorously

87

occasionally. After filtration, the residue was soaked in ethanol for another three times

88

(3×30 L, 48 h each). The ethanol extracts were combined and concentrated under

89

vacuum to obtain a black gum-like residue which was suspended in water and

90

successively partitioned with petroleum ether, ethyl acetate (EtOAc), and n-butanol.

91

The EtOAc fraction was concentrated to obtain a red residue (264.1 g).

92

The EtOAc extract was chromatographed over the 0.2% oxalic acid treated silica

93

gel (40 cm×8 cm i.d.), eluting with a gradient of CH2Cl2/EtOAc (from 100:0 to 0:100,

94

v/v) to give five fractions (Fr. 1-5). Fr. 2 (25 g) eluted with CH2Cl2/EtOAc (100:1, v/v,

95

10 L) was subjected to gel permeation chromatography on Sephadex LH-20 (70 cm×2

96

cm i.d.) using CHCl3/MeOH (1:1, v/v, 280 mL) as eluent to afford three subfractions

97

(Fr. 2.1-2.3), among which Fr. 2.2 (6 g) was repeatedly separated by HSCCC using

98

n-hexane/EtOAc/MeOH/H2O (6:4:6.5:6, v/v) to yield six subfractions (Fr. 2.2.1-2.2.6). 5



99

Page 6 of 34

Fr. 2.2.1 was purified by semi-preparative HPLC (80% MeOH/H2O, containing 0.02%

100

oxalic acid) to yield 16 (10.0 mg). Similarly, compound 22 (6.0 mg) was purified

101

from Fr. 2.2.2; 21 (2.5 mg), 13 (1.2 mg) and 10 (2.5 mg) were purified from Fr. 2.2.3;

102

14 (1.6 mg) and 12 (3.7 mg) were purified from Fr. 2.2.4; 20 (3.7 mg) and 9 (1.3 mg)

103

were purified from Fr. 2.2.5; 15 (2.6 mg), 19 (2.4 mg) and 11 (2.8 mg) were purified

104

from Fr. 2.2.6.

105

Fr. 3 (35 g) eluted with CH2Cl2/EtOAc (10:1, v/v, 10 L) was subjected to

106

chromatography over Sephadex LH-20 (70 cm×2 cm i.d.) eluting with CHCl3/MeOH

107

(1:1, v/v, 280 mL) to give three subfractions (Fr. 3.1-3.3), among which Fr. 3.2 (20 g)

108

was separated by HSCCC using n-hexane/EtOAc/MeOH/H2O (4:5:5:6, v/v) as the

109

solvent system to yield five subfractions (Fr. 3.2.1-3.2.5). Fr. 3.2.1 was purified by

110

semi-preparative HPLC (70% MeOH/H2O, containing 0.02% oxalic acid) to yield 24

111

(10.8 mg). Likewise, compounds 7 (7.5 mg), 17 (10.0 mg) and 1 (1.8 mg) were

112

purified from Fr. 3.2.2; 23 (6.3 mg) was purified from Fr. 3.2.3; 5 (1.0 mg) was

113

purified from Fr. 3.2.4; 8 (3.6 mg), 4 (1.8 mg) and 3 (2.9 mg) were purified from Fr.

114

3.2.5, respectively.

115

Fr. 4 (27 g) eluted with CH2Cl2/EtOAc (1:1, v/v, 8 L) was processed in the same

116

manner as Fr. 3, by chromatographing over Sephadex LH-20 (70 cm×2 cm i.d.) using

117

CHCl3/MeOH (1:1, v/v, 280 mL) to give three subfractions (Fr. 4.1-4.3), among

118

which Fr. 4.3 (10 g) was separated by HSCCC using n-hexane/EtOAc/MeOH/H2O

119

(3:5:4:6.7, v/v) as the solvent system to yield four subfractions (Fr. 4.3.1-4.3.4). Fr.

120

4.3.1 was purified by semi-preparative HPLC (55% MeOH/H2O, containing 0.02%

121

oxalic acid) to afford 6 (2.8 mg). Similarly, compound 25 (12.2 mg), 18 (4.6 mg), and

122

2 (3.3 mg) was obtained from Fr. 4.3.2, Fr. 4.3.3 and Fr. 4.3.4, respectively. The

123

oxalic acid in each sample was removed by washing with water. 6


Page 7 of 34


124

Ustilaginoidin Q, 1. Red amorphous powder; [α]D24 -83.9 (c 0.1, acetone); UV

125

(MeOH) λmax (log ε) 227 (4.67), 290 (4.74), 417 (3.89) nm; CD (MeOH) λ (∆ε) 218

126

(-2.88), 234 (+13.28), 242 (+10.30), 264 (+27.86), 292 (-39.33), 348 (+3.09), 376

127

(-0.31), 384 (-0.13) nm; IR (KBr) νmax 3383, 1676, 1653, 1439, 1367, 1262, 1231,

128

1122, 840, 783, 722 cm−1; 1H NMR (CD3COCD3, 600 MHz) and

129

(CD3COCD3, 150 MHz), Tables 1 and 2; HRESIMS m/z 557.1099 [M-H]- (calcd for

130

C30H21O11, 557.1089).

13

C NMR

131

Ustilaginoidin R, 2. Red amorphous powder; [α]D24 -58.7 (c 0.1, acetone); UV

132


133

(+4.19), 223 (-4.49), 262 (+49.75), 293 (-46.79), 340 (+2.34) nm; IR (KBr) νmax 3390,

134

2923, 1646, 1624, 1569, 1516, 1451, 1399, 1278, 1227, 1029, 914, 842, 722, 674, 573

135

cm−1; 1H NMR (CD3COCD3, 600 MHz) and

136

Tables 1 and 2; HRESIMS m/z 573.1015 [M-H]- (calcd for C30H21O12, 573.1038).

13

C NMR (CD3COCD3, 150 MHz),

137

Ustilaginoidin S, 3. Red amorphous powder; [α]D27 -90.3 (c 0.1, acetone); UV

138


139

(+4.49), 220 (+2.31), 234 (+22.18), 242 (+18.35), 262 (+48.33), 294 (-61.96), 348

140

(+4.87), 364 (-0.80) nm; IR (KBr) νmax 3726, 3385, 2921, 2851, 1653, 1616, 1590,

141

1469, 1372, 1273, 1229, 1149, 1087, 842, 582 cm−1; 1H NMR (CD3COCD3, 600 MHz)

142

and

143

[M-H]- (calcd for C29H19O11, 543.0933).

13

C NMR (CD3COCD3, 150 MHz), Tables 1 and 2; HRESIMS m/z 543.0951

144

Ustilaginoidin T, 4. Orange-yellow amorphous powder; [α]D27 -128.5 (c 0.1,

145

acetone); UV (MeOH) λmax (log ε) 230 (4.90), 292 (4.99), 418 (4.23) nm; CD (MeOH)

146

λ (∆ε) 210 (+5.94), 222 (-4.14), 262 (+69.50), 292 (-86.23), 344 (+5.43), 364 (-0.39),

147

372 (-0.17) nm; IR (KBr) νmax 3420, 2919, 2851, 1634, 1589, 1509, 1454, 1383, 1228,

148

1150, 1028, 907, 848, 675, 582 cm−1; 1H NMR (CD3COCD3, 600 MHz) and

13

C 7



Page 8 of 34

149

NMR (CD3COCD3, 150 MHz), Tables 1 and 2; HRESIMS m/z 545.1097 [M-H]-

150

(calcd for C29H21O11, 545.1089).

151

2,3-Dihydroustilaginoidin T, 5. Light-green amorphous powder; [α]D24 -54.4 (c

152

0.1, acetone); UV (MeOH) λmax (log ε) 234 (4.46), 293 (4.55), 327 (3.94), 417 (3.82)

153

nm; CD (MeOH) λ (∆ε) 224 (-3.80), 264 (+32.15), 294 (-28.87), 340 (+0.69), 365

154

(-0.26), 379 (-0.06) nm; IR (KBr) νmax 3420, 2919, 2851, 1746, 1632, 1554, 1509,

155

1455, 1385, 1233, 1121, 1032, 842, 699, 579 cm−1; 1H NMR (CD3COCD3, 600 MHz)

156

and

157

[M-H]- (calcd for C29H23O11, 547.1246).

13

C NMR (CD3COCD3, 150 MHz), Tables 1 and 2; HRESIMS m/z 547.1226

158

Ustilaginoidin U, 6. Light-green amorphous powder; [α]D27 -69.2 (c 0.1, acetone);

159

UV (MeOH) λmax (log ε) 233 (4.17), 270 (4.19), 294 (4.23), 327 (3.66), 419 (3.51) nm;

160

CD (MeOH) λ (∆ε) 210 (+8.07), 224 (-8.00), 262 (+82.60), 294 (-78.52), 340 (+3.63),

161

356 (-0.80) nm; IR (KBr) νmax 3376, 2924, 1661, 1635, 1447, 1384, 1229, 1154, 1027,

162

905, 850, 683, 579 cm−1; 1H NMR (CD3COCD3, 600 MHz) and

163


164

C28H21O12, 549.1038).

13

C NMR

165

Ustilaginoidin V, 7. Light-green amorphous powder; [α]D27 -154.1 (c 0.1,

166

acetone); UV (MeOH) λmax (log ε) 235 (4.25), 294 (4.29), 327 (4.01), 419 (3.94) nm;

167

CD (MeOH) λ (∆ε) 210 (+5.62), 224 (-5.56), 262 (+61.80), 294 (-58.48), 340 (+2.41),

168

362 (-0.64) nm; IR (KBr) νmax 3406, 2924, 1628, 1561, 1442, 1384, 1366, 1344, 1230,

169

1151, 1123, 1083, 874, 844, 665, 583 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C

170

NMR (DMSO-d6, 150 MHz), Tables 1 and 2; HRESIMS m/z 533.1108 [M-H]- (calcd

171

for C28H21O11, 533.1089).

172

Ustilaginoidin W, 8. Red amorphous powder; [α]D27 -86.9 (c 0.1, acetone); UV

173

(MeOH) λmax (log ε) 229 (4.53), 291 (4.65), 416 (3.86) nm; CD (MeOH) λ (∆ε) 210 8


Page 9 of 34


174

(+7.90), 224 (-3.38), 262 (+71.53), 294 (-84.81), 344 (+4.15), 364 (-0.50) nm; IR

175

(KBr) νmax 3391, 2923, 1730, 1633, 1588, 1508, 1455, 1385, 1363, 1272, 1153, 1084,

176

1024, 958, 885, 843, 675, 581 cm−1; 1H NMR (CD3COCD3, 600 MHz) and 13C NMR

177


178

C29H19O12, 559.0882).

179

Phytotoxic Activity. Compounds 2, 3, 6, 7, 8, 17, 18, and 23-25 were evaluated for

180

their inhibitory activities on the radicle and germ elongation of rice (Oryza sativa)

181

seeds as described previously.11 The other isolated compounds were not tested either

182

due to the limited amount available, or have been reported previously.11 The seeds of

183

two rice varieties (Lijiang, and Zhonghua 11), by courtesy of Prof. Zejian Guo

184

(Department of Plant Pathology, China Agricultural University), were used.

185

Five three-day-germinated rice seeds were placed in the well containing 200 µL

186

of working solution in a 24-well plate. The pure compounds were evaluated at 50, 100,

187

200, and 400 µg/mL dissolved in sterile distilled water containing a final

188

concentration of 2.5% DMSO. The 2.5% DMSO in distilled water was used as the

189

negative control and glyphosate (N-(phosphonomethyl)glycine) as the positive control.

190

Three replicates were used for each treatment. The plates were incubated in a moist

191

chamber at 25 °C in the dark. The lengths of radicle and germ of each seed were

192

measured after 48 h. The inhibition activity was calculated as follows: Inhibition (%)

193

= [(Lc-Lt)/Lc]×100, where Lc is the length of the control group, and Lt is that of the

194

treated.

195

Cytotoxic Activity. Cytotoxic activities of compounds 2, 3, 6, 7, 8, 17, 18, and 23-25

196

were tested against five human carcinoma cells using the microculture tetrazolium

197

(MTT) assay as described previously.11 The tested cell lines included HCT116 (colon 9



Page 10 of 34

198

cancer), NCI-H1650 (lung carcinoma), BGC823 (gastric cancer), Daoy (desmoplastic

199

cerebellar medulloblastoma), and HepG2 (liver hepatocellular carcinoma). Taxol was

200

used as the positive control.

201

RESULTS AND DISCUSSION

202

The EtOAc extract of the RFS balls was subjected to silica gel and Sephadex

203

LH-20 column chromatography, followed by separation and purification using

204

HSCCC and semi-preparative HPLC, which resulted in the isolation of eight new

205

bis-naphtho-γ-pyrones, namely, ustilaginoidins Q-T, 1-4, 2,3-dihydroustilaginoidin T,

206

5, and ustilaginoidins U-W, 6-8 (Figure 2), and seventeen known congeners (Figure

207

3).

208

Ustilaginoidin Q, 1, was isolated as a red amorphous powder. It exhibited a

209

prominent deprotonated peak at m/z 557.1099 [M-H]- in the HRESIMS spectrum,

210

indicating a molecular formula of C30H22O11. The IR spectrum displayed signals for

211

hydroxyl (3383 cm-1), and conjugated carbonyl (1676, 1653 cm-1) groups. The UV

212

spectrum showed maximum absorptions at 227, 290, 417 nm which was analogous to

213

those of ustilaginoidins A-C.5 The NMR data of 1 (Tables 1 and 2) were similar to

214

those of ustilaginoidin A, 16,5 however, a 2-hydroxy propyl group (CH2-11′: δC 44.8,

215

δH 2.70 dd, 2.63 dd; CH-12′: δC 65.6, δH 4.13 m; CH3-13′: δC 23.7, δH 1.18 d) in 1

216

replaced the methyl group in ustilaginoidin A, 16. This was corroborated by analysis

217

of the HMBC spectrum, in which correlations were observed from the methylene

218

protons (H2-11′) to C-2′ (δC 171.9), and C-3′ (δC 107.7), and from the doublet methyl

219

(CH3-13′) to C-11′, and C-12′ (Figure 4). Thus, compound 1 was identified as a

220

2′-(2-hydroxypropyl) derivative of ustilaginoidin A.

10


Page 11 of 34


221

Ustilaginoidin R, 2, was isolated as an analogue of ustilaginoidin Q, 1. Its

222

molecular formula was determined as C30H22O12 by HRESIMS, bearing one more

223

oxygen atom than that of 1. The NMR data were quite similar (Tables 1 and 2), except

224

that the signals for 2-methyl group in 1 was replaced by those of a hydroxymethyl

225

group (δH 4.43, 4.39, each d; δC 61.1) in 2. Thus, ustilaginoidin R, 2, was deduced as a

226

2-hydroxymethyl derivative of ustilaginoidin Q, 1.

227

Ustilaginoidins Q, 1, and R, 2, both contained a 2-hydroxypropyl substituent in

228

the γ-pyrone ring, which were not previously found among the reported

229

ustilaginoidins. The axial chirality of both compounds was deduced to be

230

R-configured, as they showed intense positive Cotton effect at around 264 nm, while

231

negative at around 292 nm (Figure 5), which were found in all the reported

232

ustilaginoidins.5, 9, 11 We attempted to determine the absolute configuration of C-12′ of

233

1 by applying the modified Mosher’s method,16 however we failed to obtain the

234

Mosher’s esters of the secondary alcohol at C-12′ due to the competitive esterification

235

of six phenolic hydroxyl groups. The scarcity of the material deterred further chemical

236

derivatization. Crystallization of this compound was not successful as yet. By the

237

same token, the absolute configuration of C-12′ of 2 was not determined. Thus, the

238

absolute configuration of C-12′ in 1 and 2 remained unclear.

239

Ustilaginoidin Q, 1, was structurally related to hypochromin B,17 which was

240

isolated from a marine-derived fungus Hypocrea vinosa, but they differed at C-12′

241

and the axial configurations. Hypochromin B had a ketone group at C-12′, and an aS

242

axial configuration, opposite to that of 1.

243

Ustilaginoidin S, 3, was isolated as a red amorphous powder with molecular

244

formula of C29H20O11 as established by HRESIMS measurement. Its UV, IR, and

245

NMR data were similar to those of ustilaginoidin L, 10,11 however, the signals for 11



Page 12 of 34

246

2-methyl group in 10 were replaced by those of a hydroxymethyl group (δH 4.39, 4.32,

247

each d; δC 61.0) in 3, implying that 3 was a 2-hydroxymethyl derivative of

248

ustilaginoidin L, 10. This was confirmed by the HMBC correlations observed from

249

the hydroxymethyl group to C-2 (δC 173.6), and C-3 (δC 104.2).

250

Ustilaginoidin T, 4, was obtained as an orange-yellow amorphous powder, which

251

showed pseudomolecular peak at m/z 545.1097, indicating a molecular formula of

252

C29H22O11, which had two more protons than that of 3. The NMR data of 4 resembled

253

those of 3 (Tables 1 and 2), however, two methine signals (δH 4.13, 2.77, each dq),

254

and two upfield-shifted methyl doublets (δH 1.37, 1.22) were observed in 4, instead of

255

the two olefinic methyl singlets (δH 2.28, 2.02) in 3, suggesting that 4 was a

256

2′,3′-dihydro derivative of 3. This was concluded by analysis of the HMBC spectrum,

257

in which 2′-CH3 (δH 1.37, d) showed cross-peaks to C-2′ (δC 79.0) and C-3′ (δC 46.8),

258

3′-CH3 (δH 1.22, d) showed correlations to C-2′, C-3′, and C-4′ (δC 201.7) (Figure 4).

259

The large coupling constant of H-2′ and H-3′ (10.8 Hz) revealed the trans relationship

260

between them.

261

2,3-Dihydroustilaginoidin T, 5, was isolated as an analogue of 4, whose

262

molecular formula was determined as C29H24O11 by HRESIMS, bearing two more

263

protons than that of 4. Careful comparison of the NMR data (Tables 1 and 2) revealed

264

their great similarities, but they differed in the upper pyrone units, in which signals for

265

a methine (δH 4.47, m; δC 78.6) and methylene groups (δH 3.03, 2.72, each dd; δC 38.6)

266

in 5 replaced those of the double bond in 4. This suggested that 5 was a 2,3-dihydro

267

derivative of 4, which was consistent with the observation that each proton of the

268

2-hydroxymethyl group in 5 appeared as a doublet of doublets (δH 3.78, 3.72) and

269

shifted to upfield when compared to those of 4. This deduction was supported by the

12


Page 13 of 34


270

HMBC correlations observed from the hydroxymethyl group to C-2 (δC 78.6) and C-3

271

(δC 38.6).

272

Ustilaginoidin U, 6, was obtained as a light-green amorphous powder, and had a

273

molecular formula of C28H22O12. The NMR data of 6 (Tables 1 and 2) contained only

274

one set of signals attributed to one naphtho-γ-pyrone unit, thus implying the

275

symmetric nature of 6. Detailed comparison of NMR data revealed the signals of 6

276

were almost superimposable on those of the upper unit in 5. Thus, 6 was determined

277

as a symmetric dimer of 2-hydroxymethyl-5,6,8-trihydroxyl-naphtho-γ-pyrone with a

278

9,9′-linkage. The structural elucidation was corroborated by analysis of the 2D NMR

279

(HSQC, HMBC) data. Ustilaginoidin U, 6, was structurally related to ustilaginoidin J,

280

25,5 but differed at C-2′ and C-3′, in which a double bond was present in

281

ustilaginoidin J, 25.

282

Ustilaginoidin V, 7, was isolated as a congener of 6. The molecular formula of 7

283

was deduced as C28H21O11 by HRESIMS analysis, with one less oxygen atom than

284

that of 6. The NMR data were similar to each other, and the main differences were

285

attributed to the substituents at C-2, in which a methyl group (δH 1.29; δC 20.4) in 7

286

replaced that of a hydroxymethyl group in 6. The HMBC correlations from 2-CH3 to

287

C-2 (δC 72.8), and C-3 (δC 42.7) supported this conclusion. Thus, ustilaginoidin V, 7,

288

was determined as a deoxy derivative of 6.

289

Ustilaginoidin W, 8, was isolated as a red amorphous powder, with a molecular

290

formula of C29H20O12. Its UV and NMR data (Tables 1 and 2) were similar to those of

291

ustilaginoidin I, 24,5 however, the additional signals including a deshielded singlet at

292

δH 8.13, and its corresponding carbon at δC 161.8, as revealed by the HSQC

293

experiment, suggested the presence of a formate group in 8. The HMBC correlations

294

from the formate proton (δH 8.13) to the oxygen-containing methylene group (δC 64.9), 13



Page 14 of 34

295

and from the methylene protons (δH 4.40, 4.37, each dd) to the formate carbonyl (δC

296

161.8), C-2 (δC 75.4), and C-3 (δC 38.4), revealed that the formate group was attached

297

to 2-methylene group via an ester bond (Figure 4). Thus, this compound was deduced

298

as a formate ester of ustilaginoidin I. Although such esters were found in many natural

299

products, such as prieurianin18 and gitaloxin,19 ustilaginoidin W, 8, represented the

300

first formate-containing bis-naphtho-γ-pyrone. It is worth noting that compound 8 was

301

not stable even stored at -20 °C, and gradually converted to ustilaginoidin I, 24.

302

The absolute configuration of the 9/9′ axis of 3-8, was determined as R by 5, 9, 11

303

comparing their CD spectra (Figure 5) with the reported ustilaginoidins,

304

showed negative first (~294 nm), and positive second (~262 nm) Cotton effects.

305

However,

306

2,3-dihydro-pyran-4-one ring in compounds 4−8 was not determined due to the

307

difficulties in crystallization, and the limited amount of material available for

308

chemical derivatization.

the

absolute

configuration

of

the

chiral

centers

of

which

the

309

The known compounds were identified as ustilaginoidins K-N, 9-12, P, 13, E1,

310

14, isochaetochromin B2, 15, and ustilaginoidins A-J, 16-25, by comparing their

311

physical and spectroscopic data with the literature.

312

compounds 9-15 were isolated from the RFS balls for the first time, though they were

313

previously reported in the fermentation products of V. virens on rice medium.

314

Among the isolated compounds, ustilaginoidins Q-T, 1-4, 2,3-dihydroustilaginoidin T,

315

5, ustilaginoidins U-W, 6-8, B, 17, C, 18, and H-J, 23-25, with oxygenated

316

substituent(s) at C-2 and/or C-2′, were relatively polar, while the others were less

317

polar.

5, 11

It is worth mentioning that

11

318

The polyketide pathway was reported to be involved in the biosynthesis of the

319

ustilaginoidins.11 In the case of ustilaginoidins Q, 1, and R, 2, it was obvious that one 14


Page 15 of 34


320

more acetate unit was involved in the biosynthesis, followed by reduction of the keto

321

group at C-12′ to give a hydroxyl group.

322

In our previous study, thirteen less polar ustilaginoidins (without any oxygenated

323

substituent in the γ-pyrone rings) were evaluated for their phytotoxic activities

324

towards the radicle elongation of rice seeds, and ustilaginoidins O, E, and F, and

325

isochaetochromin B2 were found to have moderate activities.11 In this study, ten polar

326

ustilaginoidins including compounds 2, 3, 6, 7, 8, 17, 18, and 23-25, were selected

327

and tested for their inhibitory activities on the radicle and germ elongation of rice

328

seeds. Among them, ustilaginoidins R, 2, U, 6, B, 17, and I, 24, showed inhibition

329

against the growth of radicle and germ at the tested concentrations of 50, 100, 200,

330

and 400 µg/mL, though not as effective as the positive control (glyphosate) (Table 3).

331

Interestingly, these compounds showed higher inhibition ratio against the elongation

332

of radicle than that of germ in most cases, however, ustilaginoidin I, 24, exhibited

333

similar or less effect on radicle than germ for the variety Zhonghua 11. In general, the

334

Lijiang variety was more susceptible to the tested compounds than that of Zhonghua

335

11. As far as the Lijiang variety was concerned, all the active compounds displayed

336

more than 50% inhibition ratio against the radicle growth at 200 and 400 µg/mL, and

337

ustilaginoidin B, 17, was the most active. Taking the previous results together,11 the

338

inhibition against the radicle growth of Lijiang variety at 200 µg/mL was in the order

339

of ustilaginoidin F (72.22%) > ustilaginoidin B (64.96%) > isochaetochromin B2

340

(61.35%) > ustilaginoidin R (60.97%) > ustilaginoidin E (60.14%) > ustilaginoidin V

341

(54.4%) > ustilaginoidin I (51.57%) > ustilaginoidin O (50.0%). However, no clear

342

structure activity relationship could be drawn.

343

We also evaluated the cytotoxic activities of the ten polar ustilaginoidins. All the

344

tested compounds, except ustilaginoidin U, 6, showed cytotoxicities against at least 15



Page 16 of 34

345

one human cancer cell line with IC50 values in the range of 4.06~44.1 µM, though not

346

as active as the positive control (taxol) (Table 4). Ustilaginoidin S, 3, displayed

347

moderate to weak inhibition against four cancer cell lines, including NCI-H1650,

348

BGC823, Daoy, and HepG2, with IC50 values of 16.4, 31.1, 44.1, and 40.6 µM,

349

respectively. Ustilaginoidins B, 17, and I, 24, exhibited cytotoxic activities against

350

three cancer cell lines with IC50 values of 4.06~38.7 µM. Ustilaginoidins R, 2, C, 18,

351

and H, 23, selectively inhibited the growth of NCI-H1650 cells with respective IC50

352

values of 30.1, 27.4, and 29.7 µM, while ustilaginoidins W, 8, and J, 25, selectively

353

inhibited that of BGC823 cells with IC50 values of 32.5 and 4.98 µM, respectively.

354

Ustilaginoidin V, 7, showed weak cytotoxicity towards HCT116 cells only (41.9 µM).

355

Among them, ustilaginoidin I, 24, displayed the strongest activity against HCT116

356

(IC50 4.06 µM) and Daoy (IC50 25.6 µM) cells, while ustilaginoidins B, 17, J, 25, and

357

S, 3, showed the strongest inhibition against NCI-H1650 (IC50 10.3 µM), BGC823

358

(IC50 4.98 µM) and HepG2 (IC50 40.6 µM) cells, respectively. Previously,

359

ustilaginoidins K and L were reported to have inhibitory activities against the A2780

360

cells (ovarian cancer) with IC50 values of 4.18 and 7.26 µM, respectively,11 while

361

ustilaginoidins A, D, E, and G were cytotoxic to KB cells (epidermoid carcinoma)

362

with IC50 values of 0.42~1.94 µM. 13

363

In conclusion, we have characterized eight new, polar bis-naphtho-γ-pyrones,

364

namely ustilaginoidins Q-T, 1-4, 2,3-dihydroustilaginoidin T, 5, and ustilaginoidins

365

U-W, 6-8, from the RFS balls caused by V. virens. These compounds all contain at

366

least one oxygenated substituent at C-2 and/or C-2′ of the γ-pyrone rings. Among

367

them, ustilaginoidins Q, 1, and R, 2, feature a 2-hydroxypropyl unit at C-2′, which

368

suggest an additional acetate unit being involved in their biosynthesis when compared

369

to the other ustilaginoidins. This kind of ustilaginoidins has not been reported 16


Page 17 of 34


370

previously. Ustilaginoidin W, 8, was a rare formate-containing ustilaginoidin. In

371

addition, 17 known congeners were re-isolated. All the isolated compounds were

372

revealed to have an aR configuration for the 9,9′-axis by studying their CD profiles.

373

Ten polar ustilaginoidins that bear at least one oxygenated side chain in the γ-pyrone

374

rings were selected and evaluated for their phytotoxic and cytotoxic activities. The

375

results showed that ustilaginoidins R, 2, U, 6, B, 17, and I, 24, exhibited inhibition

376

against the growth of radicle and germ of the rice seeds at the tested concentrations,

377

while ustilaginoidins R, 2, S, 3, V, 7, W, 8, B, 17, C, 18, and H-J, 23-25, were

378

cytotoxic to the tested human cancer cell lines (IC50 4.06~44.1 µM). In combination

379

with our previous study,11 it seems that the oxygenated substitution at C-2/C-2′ does

380

not have a clear relationship to the phytotoxic or cytotoxic activities. Further work

381

should be conducted to address the physiological and ecological roles of these

382

ustilaginoidins, especially in the pathogenesis of RFS, as well as their potential

383

hazards to grain production and food safety.

384

ASSOCIATED CONTENT

385

Supporting Information

386

HRESIMS, IR, NMR, and CD spectra for compounds 1−8. This material is available

387

free of charge via the Internet at http://pubs.acs.org.

388

AUTHOR INFORMATION

389

Corresponding Authors

390

*(L.Z.) Tel: +86 10 62731199. Fax: +86 10 62731062. E-mail: [email protected].

391

*(D.L.) Tel: +86 10 62733609. E-mail: [email protected].

392

Author Contributions

17



Page 18 of 34

393

L. Zhou, Y. Liu and D. Lai designed research; W. Sun and D. Lai performed HPLC

394

analysis, extracted and isolated the compounds, obtained and interpreted the NMR

395

spectra; A. Wang and D. Xu collected the RFS samples; W. Wang and J. Meng

396

performed the phytotoxicity test; J. Dai performed the cytotoxicity test; W. Sun, D.

397

Lai and L. Zhou analyzed data and wrote the paper. All authors revised and approved

398

the final version of the manuscript.

399

Funding

400

This work was supported by the National Natural Science Foundation of China

401

(31471729 and 31271996), the National Basic Research Program of China

402

(2013CB127805), and the Chinese Universities Scientific Fund (2017QC111).

403

Notes

404

The authors declare no competing financial interest.

405

REFERENCES

406

(1) Tanaka, E.; Ashizawa, T.; Sonoda, R.; Tanaka, C. Villosiclava virens gen. nov.,

407

comb. nov., teleomorph of Ustilaginoidea virens, the causal agent of rice false smut.

408

Mycotaxon 2008, 106, 491-501.

409

(2) Fan, J.; Yang, J.; Wang, Y.-Q.; Li, G.-B.; Li, Y.; Huang, F.; Wang, W.-M.

410

Current understanding on Villosiclava virens, a unique flower-infecting fungus

411

causing rice false smut disease. Mol. Plant Pathol. 2016, 17, 1321-1330.

412

(3) Shibata, S.; Ogihara, Y.; Ohta, A. Metabolic products of fungi. XXII. On

413

ustilaginoidins. (2). The structure of ustilaginoidin A. Chem. Pharm. Bull. 1963, 11,

414

1179-1182.

415

(4) Shibata, S.; Ogihara, Y. Metabolic products of fungi. XXIII. Ustilaginoidins. 3.

416

The structure of ustilaginoidins B and C. Chem. Pharm. Bull. 1963, 11, 1576-1578.

18


Page 19 of 34


417

(5) Koyama, K.; Natori, S. Further characterization of seven bis(naphtho-γ-pyrone)

418

congeners of ustilaginoidins, pigments of Claviceps virens (Ustilaginoidea virens).

419

Chem. Pharm. Bull. 1988, 36, 146-152.

420

(6) Koiso, Y.; Natori, M.; Iwasaki, S.; Sato, S.; Sonoda, R.; Fujita, Y.; Yaegashi, H.;

421

Sato, Z. Ustiloxin: a phytotoxin and a mycotoxin from false smuth balls on rice

422

panicles. Tetrahedron Lett. 1992, 33, 4157-4160.

423

(7) Koiso, Y.; Li, Y.; Iwasaki, S.; Hanaoka, K.; Kobayashi, T.; Sonoda, R.; Fujita, Y.;

424

Yaegashi, H.; Sato, Z. Ustiloxins, antimitotic cyclic peptides from false smut balls on

425

rice panicles caused by Ustilaginoidea virens. J. Antibiot. 1994, 47, 765-773.

426

(8) Shibata, S.; Ogihara, Y. Absolute configurations of ustilaginoidins. Tetrahedron

427

Lett. 1963, 4, 1777-1780.

428

(9) Koyama, K.; Natori, S.; Iitaka, Y. Absolute configurations of chaetochromin A

429

and related bis(naphtho-γ-pyrone) mold metabolites. Chem. Pharm. Bull. 1987, 35,

430

4049-4055.

431

(10) Lu, S.; Tian, J.; Sun, W.; Meng, J.; Wang, X.; Fu, X.; Wang, A.; Lai, D.; Liu, Y.;

432

Zhou, L. Bis-naphtho-γ-pyrones from fungi and their bioactivities. Molecules 2014,

433

19, 7169-7188.

434

(11) Lu, S.; Sun, W.; Meng, J.; Wang, A.; Wang, X.; Tian, J.; Fu, X.; Dai, J.; Liu, Y.;

435

Lai, D.; Zhou, L. Bioactive bis-naphtho-γ-pyrones from rice false smut pathogen

436

Ustilaginoidea virens. J. Agric. Food Chem. 2015, 63, 3501-3508.

437

(12) Tsuchiya, T.; Sekita, S.; Koyama, K.; Natori, S.; Takahashi, A. Effect of

438

chaetochromin A, chaetochromin D and ustilaginoidin A, bis(naphtho-γ-pyrone)

439

derivatives, on the mouse embryo limb bud and midbrain cells in culture. Congenital

440

Anomalies 1987, 27, 245-250.

19



Page 20 of 34

441

(13) Koyama, K.; Ominato, K.; Natori, S.; Tashiro, T.; Tsuruo, T. Cytotoxicity and

442

antitumor activities of fungal bis (naphtho-γ-pyrone) derivatives. J. Pharmacobio-Dyn.

443

1988, 11, 630-635.

444

(14) Kawai, K.; Hisada, K.; Mori, S.; Nozawa, Y.; Koyama, K.; Natori, S. The

445

impairing effect of chaetochromin A and related mycotoxins on mitochondrial

446

respiration. Proc. Jpn. Assoc. Mycotoxicol. 1991, 31-35.

447

(15) Kong, X.; Ma, X.; Xie, Y.; Cai, S.; Zhu, T.; Gu, Q.; Li, D. Aromatic polyketides

448

from a sponge-derived fungus Metarhizium anisopliae mxh-99 and their

449

antitubercular activities. Arch. Pharmacal Res. 2013, 36, 739-744.

450

(16) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. High-field FT NMR

451

application of Mosher's method. The absolute configurations of marine terpenoids. J.

452

Am. Chem. Soc. 1991, 113, 4092-4096.

453

(17) Ohkawa, Y.; Miki, K.; Suzuki, T.; Nishio, K.; Sugita, T.; Kinoshita, K.;

454

Takahashi, K.; Koyama, K. Antiangiogenic metabolites from a marine-derived fungus,

455

Hypocrea vinosa. J. Nat. Prod. 2010, 73, 579-582.

456

(18) Gullo, V. P.; Miura, I.; Nakanishi, K.; Cameron, A. F.; Connolly, J. D.;

457

Duncanson, F. D.; Harding, A. E.; McCrindle, R.; Taylor, D. A. H. Structure of

458

prieurianin, a complex tetranortriterpenoid. Nuclear magnetic resonance analysis at

459

nonambient temperatures and X-ray structure determination. J. Chem. Soc., Chem.

460

Commun. 1975, 345-346.

461

(19) Fujii, Y.; Ikeda, Y.; Yamazaki, M. High-performance liquid chromatographic

462

determination of secondary cardiac glycosides in Digitalis purpurea leaves. J.

463

Chromatogr. 1989, 479, 319-325.

464 20


Page 21 of 34


465

21



Page 22 of 34

Figure captions

Figure 1. HPLC chromatograms of the EtOAc extract of Villosiclava virens fermented on rice medium (A), and the EtOAc extract of the RFS balls (B) Figure 2. Structures of the new ustilaginoidins (1-8) isolated from the RFS balls Figure 3. Structures of the known ustilaginoidins (9-25) isolated from the RFS balls Figure 4. Selected HMBC (H→C) correlations of 1, 4, and 8 Figure 5. CD spectra of 1-8 (MeOH)

22


Page 23 of 34


Table 1. 1H NMR (600 MHz) Data for 1-8 a Position

1

2

3

4

2

2′ 3

6.13 s

6.32 s

3′

6.19 s

6.18 s

7

6.61 s

6.62 s

6.57 s

6

8b

7

4.47 m

4.46 m

4.49 dqd 6.2, 2.8)

4.21 dq (11.0, 6.2)

4.46 m

3.03 dd (17.5, 12.4) 2.72 dd (17.5, 2.9)

3.03 dd (17.5, 12.4) 2.71 dd (17.5, 2.9)

4.37 dddd (12.3, 5.3, 3.5, 2.9) 2.81 dd (17.4, 11.8) 2.69 dd (17.4, 2.8)

2.77 dq (10.8, 7.0)

2.78 dq (11.0, 7.0)

6.63 s

6.51 s c

3.03 dd (17.5, 12.4) 2.71 dd (17.5, 2.9) 6.51 s

2.91 dd (17.4, 12.3) 2.61 dd (17.4, 2.9) 6.430 s

6.54 s

c

6.51 s

6.434 s

6.62 s

4.13 dq (10.8, 6.2) 6.32 s

6.319 s

5

(11.8,

4.67 dddd (12.6, 5.5, 3.1, 2.8)

3.06 dd (17.4, 12.6) 2.80 dd (17.4, 2.8) 6.13 s

7′

6.62 s

6.64 s

6.54 s

6.51 s

6.50 s

10

6.39 s

6.38 s

6.38 s

6.39 s

5.94 s

5.94 s

5.68 s

5.97 s

10′

6.41 s

6.40 s

6.322 s

5.90 s

5.93 s

5.94 s

5.71 s

6.39 s

2-CH3/2-CH2OH

2.30 s

4.43 d (16.7) 4.39 d (16.7)

4.45 d (16.7) 4.41 d (16.7) 1.37 d (6.2)

3.78 dd (12.2, 3.2) 3.72 dd (12.2, 4.9) 1.40 d (6.2)

3.78 dd (12.1, 3.7) 3.72 dd (12.1, 4.9) 3.78 dd (12.1, 3.7) 3.72 dd (12.1, 4.9)

1.29 d (6.2)

2′-CH3/2′-CH2OH

4.39 d (16.7) 4.32 d (16.7) 2.28 s

3′-CH3

2.02 s

1.22 d (7.0)

1.22 d (7.0)

11′

2.70 dd (14.3, 4.6),

3.57 dd (12.1, 3.5) 3.52 dd (12.1, 5.3)

2.33 s

2.71 dd (14.3, 4.5), 23



12′

2.63 dd (14.3, 8.2) 4.13 m

Page 24 of 34

2.63 dd (14.3, 8.3) 4.12 m

13′ 1.18 d (6.2) 1.19 d (6.2) a Recorded in acetone-d6 for 1-6, and 8, and DMSO-d6 for 7. b

Chemical shifts for 2-CH2OCHO: δH 4.40 (1H, dd, J = 12.3, 3.1 Hz), 4.37 (1H, dd, J = 12.3, 5.5 Hz) (-CH2OCHO); 8.13 (1H, s, -CH2OCHO).

c

Assignments within a column may be interchanged.

24


Page 25 of 34


Table 2. 13C NMR (150 MHz) Data for 1-8 a Position

1

2

3

4

5

6

7

8b

2 2′ 3

171.0 C 171.9 C 106.7 CH

173.8 C 172.0 C 104.1 CH

173.6 C 166.5 C 104.2 CH

173.8 C 79.0 CH 104.1 CH

78.6 CH 79.0 CH 38.6 CH2

78.6 CH 78.6 CH 38.6 CH2

72.8 CH 77.3 CH 42.7 CH2

75.4 CH 171.1 C 38.4 CH2

3′

107.7 CH

107.7 CH

113.4 C

46.8 CH

46.8 CH

38.6 CH2

37.7 CH2

106.7 CH

4 4′ 4a

184.70 C 184.74 C 102.9 C

184.9 C 184.7 C 103.4 C

184.9 C 184.1 C 103.3 C

185.0 C 201.7 C 103.4 C

199.5 C 201.7 C 102.9 C

199.6 C 199.6 C 102.9 C

198.06 C 198.09 C 101.69 C

198.4 C 184.7 C 102.7 C

4a′ 5

103.1 C nd c

103.2 C nd

102.5 C 163.2 C

102.2 C 163.3 C d

102.2 C nd

102.9 C 165.2 C

101.74 C 164.6 C

102.9 C nd

5′ 5a

nd c 106.45 C

nd 106.4 C e

nd 106.5 C

nd 107.0 C

nd 105.3 C

165.2 C 105.3 C

164.7 C 104.2 C

nd 105.5 C

5a′ 6 6′ 7 7′ 8 8′ 9

106.53 C 159.7 C 159.7 C 101.5 CH 101.6 CH 160.7 C 160.7 C 107.0 C

106.6 C e 159.8 C 159.8 C 101.5 CH 101.7 CH 160.7 C f 160.8 C f 107.0 C

106.0 C 159.8 C 159.6 C 101.4 CH 101.1 CH 160.6 C 160.3 C 106.8 C

105.3 C 159.7 C 160.4 C 101.6 CH 100.8 CH 160.7 C 161.6 C 106.8 C

105.3 C 160.3 C 160.3 C 100.8 CH 100.8 CH 161.7 C g 161.6 C g 107.2 C d

105.3 C 160.4 C 160.4 C 100.8 CH 100.8 CH 161.6 C 161.6 C 107.2 C

104.2 C 158.7 C 158.7 C 99.9 CH 99.9 CH 160.0 C 160.0 C 107.1 C

106.5 C 160.5 C 159.7 C 101.1 CH 101.5 CH 160.7 C 159.8 C 106.9 C

9′ 9a

107.0 C 141.24 C

107.0 C 141.25 C h

106.7 C 141.3 C

106.7 C 141.3 C

107.2 C d 143.1 C

107.2 C 143.1 C

107.2 C 141.53 C

107.4 C 143.1 C 25



9a′ 10

141.25 C 99.8 CH

141.32 C h 99.7 CH

141.1 C 99.9 CH

143.1 C 99.8 CH

143.1 C 100.2 CH i

143.1 C 100.2 CH

141.56 C 98.5 CH

141.2 C 100.4 CH

10′ 10a 10a′

99.8 CH 153.4 C 153.5 C

99.9 CH 153.1 C 153.6 C

99.3 CH 152.93 C 152.85 C

100.0 CH 153.1 C 156.4 C

100.0 CH i 156.4 C j 156.3 C j

100.2 CH 156.5 C 156.5 C

98.6 CH 154.7 C 154.9 C

99.7 CH 155.8 C 153.5 C

2-CH3/2-CH2OH

20.7 CH3

61.1 CH2

61.0 CH2

61.1 CH2

64.2 CH2

64.2 CH2

20.4 CH3

18.8 CH3

19.8 CH3

19.8 CH3

64.2 CH2

62.8 CH2

9.0 CH3

10.0 CH3

10.1 CH3

2′-CH3/2′-CH2OH 3′-CH3

a c

Page 26 of 34

11′

44.8 CH2

44.8 CH2

12′

65.6 CH

65.6 CH

13′ 23.7 CH3 23.7 CH3 Recorded in acetone-d6 for 1-6, and 8, and DMSO-d6 for 7. nd: not detected.

d

Signals deduced from HMBC spectra.

b e-j

20.7 CH3

Chemical shifts for 2-CH2OCHO: δC 64.9 (-CH2OCHO), 161.8 (-CH2OCHO). assignments within a column may be interchanged.

26


Page 27 of 34


Table 3. Phytotoxic Activities of the Isolated Compounds on Radicle and Germ Growth of Rice Seeds O. sativa var. Zhonghua 11 Compound a

Ustilaginoidin R, 2

Ustilaginoidin U, 6

Ustilaginoidin B, 17

Ustilaginoidin I, 24

EtOAc extract Glyphosate (Positive control)

a

Concentration

O. sativa var. Lijiang Inhibition ratio

Inhibition ratio of

Inhibition ratio of

radicle growth (%)

germ growth (%)

50

13.55 ± 4.58 hij

4.67 ± 6.03 gh

100

16.00 ± 7.48 hij

6.64 ± 4.11 gh

200

19.41 ± 6.71 ghi

17.16 ± 5.92 ef

60.97 ± 9.38 de

37.13 ± 1.27 efg

400

23.32 ± 8.04 fgh

25.71 ± 6.34 de

65.53 ± 3.45 d

42.25 ± 6.70 def

(µg/mL)

of radicle growth (%) 49.00 ± 6.47 gh 59.54 ± 6.96 defg

Inhibition ratio of germ growth (%) 37.13 ± 5.52 efg 37.13 ± 1.27 efg

50

11.84 ± 2.57 ij

14.53 ± 7.47 fg

48.72 ± 7.60 h

34.94 ± 1.27 fg

100

36.51 ± 8.59 de

24.39 ± 6.34 def

49.00 ± 4.71 gh

38.60 ± 5.80 defg

200

39.93 ± 4.08 cde

32.94 ± 6.83 cd

400

49.45 ± 8.88 c

41.81 ± 7.45 c

66.24 ± 8.73 d

43.71 ± 6.33 def

50

19.66 ± 8.86 ghi

14.53 ± 4.96 fg

45.30 ± 8.24 h

40.79 ± 3.80 def

100

30.16 ± 8.07 efg

22.42 ± 8.21 def

50.43 ± 3.92 fgh

42.25 ± 8.86 def

200

33.09 ± 2.24 def

25.05 ± 9.04 de

64.96 ± 7.40 d

45.91 ± 6.70 de

400

42.12 ± 6.51 cd

28.99 ± 5.22 d

68.09 ± 3.24 cd

47.37 ± 7.91 d

54.42 ± 5.01 efgh

40.06 ± 2.53 def

50

6.72 ± 3.30 j

6.64 ± 4.11 gh

46.44 ± 5.69 h

29.82 ± 4.39 g

100

21.12 ± 4.65 ghi

27.68 ± 4.56 d

49.57 ± 5.60 gh

41.52 ± 1.27 def

200

23.08 ± 8.26 fghi

28.34 ± 4.96 d

400

36.51 ± 6.57 de

38.86 ± 5.22 c

60.40 ± 5.43 def

45.18 ± 7.91 de

500

13.10 ± 5.78 hij

0.21 ± 2.55 h

7.25 ± 2.74 i

0.86 ± 1.42 h

50

76.80 ± 1.84 b

67.78 ± 1.14 b

76.64 ± 1.97 bc

63.45 ± 2.53 c

100

84.37 ± 1.12 ab

76.99 ± 3.01 a

82.34 ± 1.78 ab

74.42 ± 4.57 b

200

87.55 ± 1.27 a

81.59 ± 3.01 a

85.47 ± 3.08 ab

81.73 ± 1.27 ab

400

90.96 ± 0.85 a

84.88 ± 2.28 a

90.60 ± 2.26 a

84.65 ± 2.19 a

51.57 ± 5.15 efgh

42.25 ± 4.57 def

The other tested compounds were inactive at the tested concentrations.

27



Page 28 of 34

Table 4. Cytotoxicities of the Isolated Compounds

Compound

IC50 (µM) HCT116

NCI-H1650

BGC823

Daoy

HepG2

Ustilaginoidin R, 2

>50.0

30.1

>50.0

>50.0

>50.0

Ustilaginoidin S, 3

>50.0

16.4

31.1

44.1

40.6

Ustilaginoidin U, 6

>50.0

>50.0

>50.0

>50.0

>50.0

Ustilaginoidin V, 7

41.9

>50.0

>50.0

>50.0

>50.0

Ustilaginoidin W, 8

>50.0

>50.0

32.5

>50.0

>50.0

Ustilaginoidin B, 17

>50.0

10.3

26.8

38.7

>50.0

Ustilaginoidin C, 18

>50.0

27.4

>50.0

>50.0

>50.0

Ustilaginoidin H, 23

>50.0

29.7

>50.0

>50.0

>50.0

Ustilaginoidin I, 24

4.06

>50.0

19.5

25.6

>50.0

Ustilaginoidin J, 25

>50.0

>50.0

4.98

>50.0

>50.0

Taxol (Positive control)

0.00190

1.10

0.000107

0.00504

0.0146

28


Page 29 of 34


mAU 2500 290nm,4nm (1.00)

A

2000 1500 1000 500 0 0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

min

mA U 290nm,4nm (1.00) 500

B 250

0 0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

min

Figure 1.

29



OH OH O 6

5

HO HO

9 9'

10 10' 9a' 5a'

8' 6'

OH OH O

4 4a 10a

5a 9a

8

O O

2

2'

10a' 4a' 4' 5'

11'

13'

HO HO

OH OH O Ustilaginoidin R (2)

OH

HO HO

OH OH O Ustilaginoidin U (6)

O O

OH

HO HO

O O

OH OH O

OH OH O

OH OH

HO HO

O O

OH

HO HO

O O

O

H O

OH OH O

OH OH O Ustilaginoidin V (7)

OH

OH OH O 2,3-Dihydroustilaginoidin T (5)

OH OH O Ustilaginoidin T (4)

OH OH O

O O

OH OH O

OH OH O

OH OH O Ustilaginoidin S (3)

HO HO

OH

OH

OH OH O

O O

O O

OH

OH OH O Ustilaginoidin Q (1)

HO HO

Page 30 of 34

Ustilaginoidin W (8)

Figure 2.

30


Page 31 of 34


Figure 3.

31



Page 32 of 34

Figure 4.

32


Page 33 of 34


Figure 5.

33



Page 34 of 34

Table of content graphic

34


New Ustilaginoidins from Rice False Smut Balls Caused by

Recommend Documents