Cytochalasins and an Abietane-Type Diterpenoid with Allelopathic

Subscriber access provided by - Access paid by the | UCSB Libraries

Bioactive Constituents, Metabolites, and Functions

Cytochalasins and an Abietane-Type Diterpenoid with Allelopathic Activities from the Endophytic Fungus Xylaria sp. Wen-Bo Han, Yi-Jie Zhai, Hui-Yi Zhou, Jian Xiao, Jin-Ming Gao, Yuqi Gao, and Gennaro Pescitelli J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00273 • Publication Date (Web): 15 Mar 2019 Downloaded from http://pubs.acs.org on March 17, 2019

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 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 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.

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 31

Journal of Agricultural and Food Chemistry

1

Cytochalasins and an Abietane-Type Diterpenoid with Allelopathic

2

Activities from the Endophytic Fungus Xylaria sp.

3

Wen-Bo Han,†,# Yi-Jie Zhai,†,# Yuqi Gao,† Hui-Yi Zhou,† Jian Xiao,‡ Gennaro

4

Pescitelli,§

5

†Shaanxi

6

Chemistry and Pharmacy, Northwest A&F University, Yangling 712100, P. R. China

7

‡

8

Engineering, Baoji University of Arts and Sciences, Baoji 721013, P. R. China

9

§ Dipartimento

10

and Jin-Ming Gao*†

Key Laboratory of Natural Products & Chemical Biology, College of

Shaanxi Key Laboratory of Phytochemistry, College of Chemistry and Chemical

di Chimica e Chimica Industriale, Università di Pisa, via Risorgimento

35, I-56126 Pisa, Italy

11

12

Corresponding author:

13

Prof. Dr. Jin-Ming Gao. Tel.: +86-29-87092335

14

E-mail: [email protected]

15 16 17

1

ACS Paragon Plus Environment


19

ABSTRACT: Bioactivity-guided isolation of the cultures of the endophytic fugus

20

Xylaria sp. XC-16 residing in a deciduous tree Toona sinensis led to the discovery of

21

four new allelochemicals (1–4) including three cytochalasins, epoxycytochalasin Z17

22

(1), epoxycytochalasin Z8 (2), epoxyrosellichalasin (3), and an abietane-type

23

diterpenoid, hydroxyldecandrin G (4), along with four known analogues,

24

10-phenyl-[12]-cytochalasins Z16 (5) and Z17 (6), cytochalasin K (7), and cytochalasin

25

E (8). The structures of these compounds were elucidated by comprehensive

26

spectroscopic methods, and their absolute configurations were determined by

27

electronic circular dichroism (CD) and X-ray diffraction. All the chemicals were

28

tested for their allelopathic effects on the turnip (Raphanus sativus) and wheat

29

(Triticum aestivum). Notably, compounds 3, 4 and 7 strongly inhibited wheat shoot

30

elongation, and compounds 5, 7 and 8 inhibited wheat root elongation, showing

31

comparable IC50 values to the positive control glyphosate. Meanwhile, compound 8

32

was a potential inhibitor on turnip root elongation with the IC50 value of 1.57 ± 0.21

33

μM, which was 50-fold more potent than glyphosate. Nevertheless, compounds 5 and

34

7 stimulated the turnip shoot elongation at lower concentrations.

35

KEYWORDS: Xylaria sp.; cytochalasins; diterpenoid; allelopathic activities;

36

herbicides; phytotoxicity; endophyte

37 38 39

2


Page 2 of 31

Page 3 of 31


40 41

INTRODUCTION

42

Endophytes are defined as endosymbiotic microorganisms that inhabit in the tissue of

43

plant without causing any damage to their hosts. They are widely distributed

44

mutualists that protect the host plants from pathogenic microbial attack or other

45

hostile environments by producing a great diversity of secondary metabolites or

46

hydrolytic enzymes, which play an important role in agrochemical and

47

pharmaceutical industries.1−3 The genus Xylaria comprises several hundreds of

48

species and most of them are known from collections of stromata on dead wood.4,5

49

Fungi of this genus are widespread in vascular/nonvascular plants in their asexual

50

stage (known as fungal endophytes), which synthesizes various bioactive secondary

51

metabolites viz. terpenoids, cytochalasins, alkaloids, polyketides, volatile organic

52

compounds and many more.5 These metabolites have exhibited a wide spectrum of

53

potential activities such as cytotoxic, antibacterial, antimalarial and α-glucosidase

54

inhibitory activity.5

55

The

cytochalasins

represent

a

diverse

group

of

fungal

polyketide

56

synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid metabolites with

57

multiple biological functions that have been isolated from various fungi including

58

Aspergillus, Phomopsis, Chaetomium, Xylaria, Rosellinia, Zygosporium.2,6 Many

59

cytochalasins, such as cytochalasin A and B, the first characterized compounds, were

60

reported to inhibit the polymerization of actin, cytochalasin H was shown to regulate

61

plant growth, cytochalasin D was demonstrated to inhibit protein synthesis and 3



62

cytochalasin E was shown to prevent angiogenesis.7 Recently, some cytochalasins

63

have been shown to inhibit the biofilm formation of Staphylococcus aureus,8 and to

64

disrupt the actin cytoskeleton of eukaryotic cells.9 During our research on the

65

endophytic fungi for new agrochemicals,10−15 an extract derived from the endophytic

66

fungus Xylaria sp. XC-16, isolated from a deciduous tree Toona sinensis (family

67

Meliaceae),16 based on the OSMAC (one strain–many compounds) strategy,17 was

68

found to be allelopathic towards two herbaceous plants turnip (Raphanus sativus) and

69

wheat (Triticum aestivum) used for allelochemical assays.17,18 To identify more potent

70

allelochemicals with unforeseeable frameworks, a repetitive chromatographic

71

fractionation of the extract from the title fungus gave three new cytochalasin

72

derivatives (1−3) and a new abietane-type diterpenoid (4), along with four known

73

cytochalasins (5−8) (Figure 1). Herein, we present the isolation, the structure

74

determination and the allelopathic activities of these compounds.

75

MATERIALS AND METHODS

76

General Experimental Procedures

77

Optical rotations were recorded on an Autopol III automatic polarimeter (Rudolph

78

Research Analytical, NJ, USA). UV measurements were obtained using a UV−vis

79

Evolution 300 spectrometer. (Thermo Fisher Scientific Inc., MA, USA). IR spectra

80

were measured on a Bruker Tensor 27 spectrophotometer (Bruker Optics,

81

Rheinstetten, Germany) with KBr pellets. ECD spectra were performed on a

82

Chirascan spectrometer (Applied Photophysics Ltd., Leatherhead, Surrey, U.K.).

83

NMR spectra were obtained on a Bruker Avance III 500 Spectrometers (Bruker 4


Page 4 of 31

Page 5 of 31


84

BioSpin, Rheinstetten, Germany) with tetramethylsilane (TMS) as an internal

85

standard at room temperature. High-resolution (HR) ESI-MS spectra were recorded

86

on an Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer. Silica gel (200−300

87

or 300–400 mesh, Qingdao Marine Chemical Ltd., China), RP-18 gel (20–45 μm, Fuji

88

Silysia Chemical Ltd., Japan) and Sephadex LH-20 (Amersham Biosciences Inc.,

89

Shanghai, China) were used for column chromatography (CC). Fractions were

90

monitored by TLC, and compounds were visualized by spraying with 10% H2SO4 in

91

ethanol followed by heating. Semi-preparative RP-HPLC was analyzed by a Waters

92

1525EF (Waters Corp., MA, USA) liquid chromatography system equipped with a

93

Hypersil BDS C18 column (4.6 mm × 250 mm; 10.0 mm × 250 mm). All other

94

chemicals used in this study were of analytical grade.

95

Cultivation and Extraction

96

The strain Xylaria sp. XC-16 used in this investigation was described earlier.16 The

97

producing strain was cultured on a plate of potato dextrose agar (PDA) medium at 28

98

± 0.5 °C for 5 days. Then one piece (approximately 5 mm2) of mycelium was

99

vaccinated aseptically with 100 mL Erlenmeyer flasks each containing 30 mL of PD

100

liquid medium. and the seed liquids were cultivated at 28 °C for 3 days on a shaking

101

table at 120 rpm. A suspension (200 μL) of the seed liquid was inoculated cultivated

102

to a KM solid medium (corn pieces 32.9%, rice hull 49.3%, wheat bran 16.7%,

103

MgSO4 0.6%, KH2PO4 0.6%, with 30 mL of distilled water) in 300 Erlenmeyer flasks

104

(500 mL each). After a subsequent fermentation at 28 °C for 28 days, cultures were

105

ultrasonically extracted four times with methanol and acetone. The solvent was 5



106

removed and dried under reduced pressure to yield a crude extract. The extract was

107

dissolved in 90% MeOH-H2O (3 L) and further treated three times with petroleum

108

ether, and the remaining layer was extracted by EtOAc and concentrated under

109

vacuum to give a crude extract (58.2 g).

110

Isolation of metabolites 1−8

111

The crude extract was subjected to chromatography over a silica gel column

112

followed by a stepwise gradient elution with CHCl3-MeOH (v/v, 100:0 → 0:100) to

113

provide four fractions A~E. Fraction B was further purified by a RP-18 column eluted

114

with a gradient of MeOH-H2O (v/v, 30% → 100%) to obtain four fractions (B-1~B-4).

115

Fraction B-3 was subjected to silica gel CC eluting with a gradient of CHCl3-MeOH

116

(70:1 →10:1) and Sephadex LH-20 (MeOH), and further purified by RP-HPLC with

117

MeOH-H2O (42:58) to yield compound 4 (tR = 21.0 min, 11.0 mg). Fraction C was

118

further chromatographed on Sephadex LH-20 eluted with MeOH to obtain seven

119

fractions (C-1~C-7). Fraction C-4 was separated by a RP-18 column eluted with a

120

gradient of MeOH-H2O (v/v, 20% → 100%), and further purified by RP-HPLC with

121

MeCN-H2O (65:35) to yield compounds 2 (tR = 19.0 min, 3.4 mg) and 7 (tR = 21.5

122

min, 3.8 mg). Fraction C-5 was subjected to a RP-18 column eluted with a gradient of

123

MeOH-H2O (v/v, 20% →100%) and next purified by RP-HPLC with MeCN-H2O

124

(70:30) to afford compounds 1 (tR = 15 min, 4.2 mg) and 5 (tR = 16.5 min, 4.4 mg).

125

Fraction C-6 was isolated on the Sephadex LH-20 and then applied to RP-HPLC with

126

MeCN-H2O (65:35) to give 8 (tR = 19 min, 4.5 mg), 6 (tR = 22.5 min, 3.9 mg) and 3

127

(tR = 24 min, 3.1 mg). 6


Page 6 of 31

Page 7 of 31


128

Epoxycytochalasin Z17 (1): colorless prisms; [α]D20 +48 (c 0.013, CH3OH); UV

129

(MeOH) λmax (log ε) 206 (3.65), 243 (3.10); IR (KBr) νmax 3425, 2921, 2349, 1713,

130

1660, 1448, 1317, 1222, 1027, 981 cm−1; HR-ESI-MS: m/z: 502.2206 [M + Na]+

131

(calcd. for C28H33NO6Na, 502.2200); 1 H and

132

Table 1.

13

C NMR data assigned and listed in

133

Epoxycytochalasin Z8 (2): colorless needles; [α]D20 +4.4 (c 0.050, CH3OH); UV

134

(MeOH) λmax (log ε) 208 (2.91); IR (KBr) νmax 3437, 2939, 2351, 2246, 1707, 1454,

135

1234, 1112, 1022 cm−1; HR-ESI-MS: m/z: 504.2353 [M + Na]+ (calcd. for

136

C28H35NO6Na, 504.2357); 1 H and 13 C NMR data assigned and listed in Table 1.

137

Epoxyrosellichalasin (3): white solid powder; [α]D20 +4.0 (c 0.025, CH3OH); UV

138

(MeOH) λmax (log ε) 205 (3.57), 228 (3.13); IR (KBr) νmax 3420, 2923, 2854, 2350,

139

1712, 1665, 1451, 1380, 1232, 1032 cm−1; HR-ESI-MS: m/z: 502.2201 [M + Na]+

140

(calcd. for C28H33NO6Na, 502.2200); 1 H and

141

Table 1.

13

C NMR data assigned and listed in

142

Hydroxyldecandrin G (4): light-yellow solid; [α]D20 +16.7 (c 0.030, CH3OH); UV

143

(MeOH) λmax (log ε) 209 (3.63), 249 (3.19), 293 (2.44); IR (KBr) νmax 3428, 2965,

144

2349, 2247, 1666, 1558, 1211, 1105, 1024, 651 cm−1; HR-ESI-MS: m/z: 355.1885 [M

145

+ Na]+ (calcd. for C20H28O4Na, 355.1880); 1 H and 13 C NMR data assigned and listed

146

in Table 1.

147

Crystal data of epoxycytochalasin Z17 (1): C28H33NO6, Mr =479.55 prism from

148

MeOH, space group I 1 2 1, a = 26.2205(6) Å, b = 7.5143(1) Å, c = 27.6723(5) Å, V =

149

5045.40 (17) Å3, Z = 8, μ = 0.719 mm-1 and F(000) = 2048.0; T = 172.99(10); crystal 7



Page 8 of 31

150

dimensions: 0.10 × 0.08 × 0.05 mm3; R = 0.037, wR = 0.102, S = 1.022; Flack

151

parameter = 0.23(7); Crystallographic

152

the

data

for

1

has

been

deposited

at

Cambridge Crystallographic Data Center with the number CCDC-1886353.

153

Crystal data of epoxycytochalasin Z8 (2): C28H35NO6, Mr =481.57 needle from

154

MeOH, space group P 1 21 1, a = 7.2257(2) Å, b = 14.0039(4) Å, c = 13.4952(3) Å, V

155

= 1324.42(6) Å3, Z = 2, μ = 0.685 mm-1 and F(000) = 516.0; T = 173(2); crystal

156

dimensions: 0.13 × 0.05 × 0.04 mm3; R = 0.041, wR = 0.107, S = 1.107; Flack

157

parameter = 0.01(15); CCDC-1886352.

158

Determination of the Absolute Configuration of the 2, 3-Diol Units in Compound

159

4 by Snatzke’s and Frelek’s Method.

160

According to the published procedure,19,20 a 1:1.2 mixture of diol/Mo2(OAc)4 was

161

subjected to ECD measurements at a concentration of 0.5 mg/mL for compound 4.

162

The first ECD spectrum was recorded immediately after mixing, and its evolution was

163

monitored over time until the signal was stationary (approximately 2 h after mixing).

164

The inherent ECD was subtracted. The observed sign of the diagnostic band at 310 −

165

340 nm in the induced ECD spectrum was correlated to the absolute configuration of

166

the 2, 3-diol unit.

167

Allelopathic Activity Bioassay.

168

The seeds of two herbaceous plants, turnip (Raphanus sativus) and wheat (Triticum

169

aestivum) were used for the bioassay according to the previously reported

170

procedure.11,17 The plant seeds were washed by running water for 2 h, dipped in 0.5%

171

KMnO4 for 20 min, and washed until they were colorless. Glyphosate was selected as 8


Page 9 of 31


172

the positive control, the compounds and blank solvent MeOH were added to 12-well

173

plates to make the final concentrations of 100 and 6.25 μM. After the evaporation of

174

MeOH, the plant seeds were put into the 12-well plates and irrigated with deionized

175

water. Triplicate experiments were conducted. The plates were then cultivated 48 h

176

under 25 °C. The germination rates were calculated according to eq 1, and the

177

allelopathic effects [response index (RI)] were calculated according to the equation 2:

178

germination rate (%) = (number of germinated seeds) / (total number of seeds) (1)

179

If T > C, then RI = 1 − C/T; if T < C, then RI = T/C – 1

(2)

180

where T is the length of the treatment, C is the length of the blank control, and RI is

181

the response index (RI > 0 means stimulation effect, and RI < 0 means inhibition

182

effect). RIs are expressed as averages ± standard deviation (SD) for three replicates.

183

The data analysis was performed using SPASS 17.0 software.

184

Computational Section.

185

Molecular mechanics and preliminary Density Functional Theory (DFT) calculations

186

were run with Spartan’16 (Wavenfunction, Irvine, CA, USA, 2016) with default

187

parameters, default grids and convergence criteria; DFT and Time-Dependent DFT

188

(TDDFT) calculations were run with Gaussian’16 (Revision A.03; Gaussian Inc.:

189

Wallingford, CT, USA, 2016) with default grids and convergence criteria. The

190

conformational search was run with the Monte Carlo algorithm implemented in

191

Spartan’16 using Merck molecular force field (MMFF). All structures obtained

192

thereof were optimized with DFT at the B97X-D/6-31G(d) level in vacuo, and

193

reoptimized at the B97X-D/6-311+G(d,p) level including the polarizable continuum 9



Page 10 of 31

194

model (PCM) for MeOH in its Integral Equation Formalism (IEF) formulation. The

195

above procedure afforded 3 minima for compound 3, the most stable of which had a

196

population of >97%. TDDFT calculations were run with B3LYP and CAM-B3LYP

197

functionals and def2-TZVP basis set, including PCM for MeOH. Average ECD

198

spectra were computed by weighting the ECD spectrum calculated for each conformer

199

with Boltzmann factors at 300K estimated from DFT internal energies. ECD spectra

200

were plotted using the program SpecDis (version 1.71; Berlin: Germany, 2017;

201

http:/specdis-software.jimdo.com). Similarity factors were also estimated with

202

SpecDis.

203 204

RESULTS AND DISCUSSION

205

Structure Identification of New Compounds

206

Epoxycytochalasin Z17 (1) was obtained as colorless prisms, which was evidenced to

207

have a molecular formula of C28H33NO6 from its Na+-liganded molecular ion at m/z

208

502.2206 (C28H33NO6Na requires 502.2200) in its high-resolution electrospray

209

ionization mass spectrometry (HR-ESI-MS). The 1H and

210

revealed four methyls, three methenes and five methines. The 1H NMR resonance

211

signals at δH 7.35 (t, J = 7.1 Hz), 7.28 (t, J = 7.4 Hz), 7.18 (d, J = 7.1 Hz) disclosed

212

the presence of a monosubstituted benzene moiety in the molecule, which was

213

supported by the chemical shifts at δc 127.3, 129.1 and 129.2 in the

214

spectrum (Table 1). A pair of double bonds were indicated by the 1H NMR signals at

215

δH 5.62 (ddd, J =15.1, 11.0, 3.8 Hz), 5.77 (ddd, J = 15.1, 10.4, 1.4 Hz) and 6.40 (t, J = 10


13C

NMR spectra for 1

13C

NMR

Page 11 of 31


216

8.0 Hz) (Table 1). In the 13C NMR spectrum, the signals at δc 168.7, 170.4 and 205.1

217

indicated the presence of three carbonyl groups in 1. The subsequent analysis of its 2D

218

NMR spectra including HSQC, COSY, and HMBC unambiguously pinpointed that 1

219

was a cytochalasin derivative. Comparison of the 1H and

220

those of the known cytochalasin Z17 21 demonstrated that the double bond between C-5

221

and C-6 was epoxidized in 1. This was confirmed by the upfield chemical shifts of

222

C-5 and C-6 at δc 62.8 and 64.2 in the

223

correlations of CH3-11with C-4, C-5 and C-6, and of CH3-12 with C-6 and C-7 could

224

further witness this change. The relative configuration of 1 was next established by

225

NOESY experiment, coupled with comparison of the coupling constants described for

226

the corresponding centers in cytochalasin Z17.21 Specially, the stereochemistry of

227

5,6-epoxide function as shown in 1 was evidenced by the NOE correlations from

228

CH3-11 to H-3, and from CH3-12 to H-7. The large coupling constant of 10.4 Hz

229

between H-7 and H-8 established the location of H-8 at an axial position. In addition,

230

the NOE correlation from H-4 to H-8 indicated they were cofacial. The structure of 1

231

was confirmed by the single-crystal X-ray analysis (Cu Kα) leading to the

232

determination of its absolute configuration as (+)-(3S,4R,5R,6S,7S,8S,9R,16S)-1

233

(Figure 2).

13C

13C

NMR data of 1 with

NMR spectrum. Moreover, the HMBC

234

Epoxycytochalasin Z8 (2) isolated as colorless needles possessed a molecular

235

formula of C28H35NO6 from its Na+-liganded molecular ion at m/z 504.2353

236

(C28H35NO6Na requires 504.2357) in its high-resolution electrospray ionization mass

237

spectrometry (HR-ESI-MS). In other words, its formula has two more hydrogen 11



238

atoms than that of compound 1. The 1H NMR and

239

similar to those of cytochalasin Z8 (Table 1).22 However, the molecular formula of 2

240

allowed one extra O added compared to that of cytochalasin Z8. Specifically, the

241

singlets at δH 1.25 and 1.38 ascribed to CH3-11 and CH3-12 were both upfield

242

compared to those in cytochalasin Z8, which demonstrated that 2 was a 5,6-epoxide

243

cytochalasin Z8 derivative. These afore-mentioned structure elements were

244

subsequently substantiated by the HSQC, NOESY, and HMBC experiments, leading

245

to the unequivocal assignment of all 1H and

246

stereochemistry of 2 was next established by NOESY experiment and coupling

247

constants. A large diaxial coupling constant (10.4 Hz) between H-7 and H-8 indicated

248

an axial configuration for H-7. The NOE correlations from H-7 to CH3-12, from H-4

249

to H-8, and from CH3-11 to H-3 demonstrated that H-4 and H-8 were cofacial, and

250

H-3, H-7, CH3-11 and CH3-12 were on the opposite side of the cyclohexane ring.

251

Additionally, the NOE correlations from CH3-22 to H-17, and from CH3-23 to H-16

252

indicated the same orientation of CH3-22, H-17 and H-18. Thus, the structure of

253

compound 2 was determined as shown, and the absolute configuration of 2 was finally

254

confirmed as (+)-(3S,4R,5R,6S,7S,8S,9R,16S,17R,18S)-2 by single-crystal X-ray

255

diffraction analysis (Cu Kα) (Figure 2).

13C

13C

NMR spectra of 2 were very

NMR signals. The relative

256

Epoxyrosellichalasin (3) obtained as a white powder, exhibited a quasi-molecular

257

ion at m/z 502.2201 ([M+Na]+) in its high-resolution ESI mass spectrum

258

(HR-ESI-MS), demonstrating its molecular formula to be C28H33NO6. The 1H NMR

259

spectrum was very similar to that of rosellichalasin,21 a metabolite produced by the 12


Page 12 of 31

Page 13 of 31


260

phytopathogen Rosellinia necatrix,23 except for the signals ascribable to oxymethines

261

at δH 3.48 and δC 58.3, and at δH 2.87 and δC 60.9, respectively (Table 1). This

262

observation, along with a set of 2D NMR experiments (COSY, NOESY, HMQC and

263

HMBC) demonstrated that it was rosellichalasin21 bearing an epoxide function

264

between C-13 and C-14. This assumption was subsequently supported by the key

265

HMBC correlations of H-7 and H-15 with C-13, and of H-8 and H-16 with C-14. The

266

relative configuration of 3 was determined by interpretation of its NOESY spectrum

267

(Figure 3) and coupling constants. The NOE correlations from H-5 to H-4 and H-8,

268

from H-10 to H-4, and from CH3-12 to CH3-11 and H-7 oriented H-4, H-5 and H-8 on

269

the same side of the cyclohexane ring. The small vicinal coupling constant (1.7 Hz)

270

between H-13 and H-14 suggested the trans-oriented epoxy ring as depicted in 3,24

271

which resembled that in 19,20-epoxycytochalasin R.25 Thus, the stereochemistry of

272

13,14-epoxide was established as shown (Figure 1). Simultaneously, strong NOE

273

correlations from H-7 to CH3-12 and H-13, and from H-14 to H-8 and H-16 tamped

274

this speculation much further. The NOE correlations from H-20 to CH3-23 revealed

275

the E configuration of the C-18/C-19 double bond. A single crystal of 3 for X-ray

276

diffraction could not be obtained because of paucity of the sample. The absolute

277

configuration of 3 was then assigned by means of a well-established procedure26,27

278

based on the comparison of experimental and calculated CD spectra, as shown in

279

Figure 4. The CD spectrum recorded in methanol was well reproduced by calculations

280

run

281

CAM-B3LYP/def2-TZVP level, including a solvent model (IEF-PCM) for methanol.

with

time-dependent

density

functional

theory

13


(TDDFT)

at

the


Page 14 of 31

282

The input structures were obtained after a conformational search with molecular

283

mechanics and DFT geometry optimization at the B97X-D/6-311+G(d,p) level with

284

PCM for methanol. The preferred folding adopted by the macrocyle in the optimized

285

structures (see lowest-energy structure in Figure 3) is in keeping with NMR data, in

286

particular the NOE between H-16 and H-19. A good agreement between experimental

287

and calculated CD spectra was observed for the (3S)-3 absolute configuration. The

288

similarity factor28 for this enantiomer was 0.87, while it was only 0.0012 for the

289

opposite enantiomer. Thus, the absolute configuration of 3 was safely assigned as

290

(+)-(3S,4S,5S,6R,7S,8S,9S,13R,14R,16S)-3.

291

Hydroxyldecandrin G (4) was obtained as a light-yellow solid with a molecular

292

formula of C20H28O4, as accommodated by its Na+-liganded molecular ion at m/z

293

355.1885 (C20H28O4Na requires 355.1880) in its high-resolution electrospray

294

ionization mass spectrometry (HR-ESI-MS). The 1H and

295

similar to those of the decandrin G,29 if exempting the proton signal at δH 3.75 (ddd, J

296

= 12.1, 9.7, 4.6 Hz) and δC 69.2 (Table 2). This observation could only be explained

297

by assuming the attachment to C-2 of the hydroxyl group based on the consideration

298

of its molecular formula. This assumption was subsequently supported by the

299

interpretation of its 2D NMR spectra (HSQC, COSY and HMBC), which allowed the

300

exact assignment of all 1H and

301

CH3-18 to H-5 and H-3, and from H-2 to CH3-19 and CH3-20, along with the

302

coupling constant between H-2 and H-3 (J = 9.7 Hz) revealed the relative

303

configuration of 4. According to the in situ dimolybdenum CD method developed by

13C

13C

NMR data of 4 were

NMR signals. The NOESY correlations from

14


Page 15 of 31


304

Snatzke and Frelek,19,20 the absolute configurations of C-2 and C-3 in 4 were

305

determined as 2S,3S by the positive Cotton effect observed at 310 nm in their

306

Mo2(OAc)4-induced circular dichroism (ICD) spectrum, which ultimately permitted

307

assignment of the (+)-(2S,3S,5S,10R)-4 configuration (Figure 5).

308

The four known cytochalasins were identified as 10-phenyl-[12]-cytochalasins Z16

309

(5) and Z17 (6),21 cytochalasin K (7),30 and cytochalasin E (8),31 by comparison of 1H

310

and 13C NMR as well as mass spectrometric data with published data.

311 312

Allelopathic Activity.

313

All the isolated compounds 1−8 were evaluated for the allelopathic activities against

314

turnip (Raphanus sativus) and wheat (Triticum aestivum) according to our previously

315

reported methods.11,17 As shown in Table 3, all the tested compounds, to certain extent,

316

displayed potential inhibition on the shoot and root elongation of T. aestivum (RI

317

values ranging from − 0.02 to − 0.87 at 6.25 and 100 μM, respectively). It was rather

318

remarkable that compounds 3, 4 and 7 exhibited strong inhibition against the shoot

319

elongation with the IC50 values of 18.92 ± 0.80, 23.58 ± 0.43, and 24.02 ± 0.51 μM,

320

and compounds 5, 7 and 8 showed the similar inhibitory effects on the root elongation

321

with the IC50 values of 17.35 ± 0.05, 22.58 ± 0.58 and 19.74 ± 0.09 μM, respectively.

322

In particular, these compounds were tested to be comparable to glyphosate, a

323

commercial herbicide co-assayed as the positive control (Table 4).

324

The tested compounds 7 and 8 indicated the excellent inhibition toward the root

325

elongation of R. sativus with the IC50 values of 36.75 ± 0.09 and 1.57 ± 0.21 μM, 15



326

respectively, which were more potent than the positive control (Table 4). The strong

327

inhibitory effect of compound 8 on root elongation of R. sativus was in accordance

328

with our previous results.16 However, both compounds revealed weak growth

329

inhibition on shoot elongation of R. sativus with the IC50 values > 100 μM. In addition,

330

other compounds 1−6 were demonstrated to have no inhibition on both shoot and root

331

elongation of R. sativus by the inhibition rates of almost < 30% at 100 μM (data not

332

shown). Interestingly enough, compounds 5 and 7 exhibited the growth-promoting

333

activities on the shoot elongation of R. sativus with the RI values of 0.10 and 0.18 at

334

the lower concentrations of 6.25 and 12.5 μM, respectively.

335

In conclusion, this work describes seven cytochalasins derivatives including three

336

new structures, and a new abietane-type diterpenoid from the endophytic fungus

337

Xylaria sp. XC-16 and clarifies in particular the allelopathic activity of these

338

chemicals. Notably, compound 8 showed more than 50-fold inhibition effect on root

339

elongation of R. sativus compared with the positive control, and compound 5 strongly

340

suppressed the root elongation of T. aestivum without any inhibition of R. sativus,

341

indicating the good selectivity toward different herbaceous plants. In addition, both

342

compounds 5 and 7 can stimulate shoot elongation of R. sativus at lower

343

concentration. Recent study revealed the disruption of actin cytoskeleton by

344

cytochalasins,9 which indicated the significant cytotoxicity to eukaryotic cells, and

345

precluded the use of these metabolites in biotechnological application. Thus, it would

346

be necessary to find an agent with low cytotoxicity and strong allelopathic effects.

347

The present work not only offers the potential allelochemicals, but also provides 16


Page 16 of 31

Page 17 of 31


348

structure templates for pharmaceutical and synthetic chemists to develop more potent

349

lead compounds in the herbicide discovery efforts.

350 351

ASSOCIATED CONTENT

352

Supporting Information

353

1D and 2D NMR spectra of 1–4. This material is available free of charge via the

354

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

355

AUTHOR INFORMATION

356

Corresponding Author

357

* (J.-M.G.) Phone: +86-29-87092335. E-mail: [email protected].

358

ORCID

359

Jin-Ming Gao: 0000-0003-4801-6514

360

Gennaro Pescitelli: 0000-0002-0869-5076

361

Author Contributions

362

# W.

363

Funding

364

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

365

China (21702169), Natural Science Basic Research Plan in Shaanxi Province of China

366

(2018JQ2009, 2014JZ2-001), the Program of Unified Planning Innovation

367

Engineering of Science & Technology in Shaanxi Province (No.2015KTCQ02-14),

368

and Scientific Research Foundation of Northwest A&F University (Z111021702).

369

Acknowledgements

B. Han and Y. J. Zhai contributed equally to this work.

17



370

G.P. acknowledges the CINECA award under the ISCRA initiative for the availability

371

of high-performance computing resources and support.

372

Notes

373

The authors declare no competing financial interest.

374

REFERENCES

375

(1) Gao, J.-M., Yang, S.-X.; Qin, J.-C. Azaphilones: Chemistry and Biology. Chem. Rev. 2013,

376

113, 4755-4811.

377

(2) Zhang, Q.; Li, H. Q.; Zong, S. C.; Gao, J. M.; Zhang, A. L. Chemical and bioactive diversities

378

of the genus Chaetomium secondary metabolites. Mini-Rev. Med. Chem. 2012, 12, 127 – 148.

379

(3) Kumar, S.; Kaushik, N. Metabolites of endophytic fungi as novel source of biofungicide: a

380

review. Phytochem. Rev. 2012, 11, 507–522.

381

(4) Helaly, S. E.; Thongbai, B.; Stadler, M. Diversity of biologically active secondary metabolites

382

from endophytic and saprotrophic fungi of the ascomycete order Xylariales. Nat. Prod. Rep. 2018,

383

35, 992 – 1014.

384

(5) Song, F.; Wu, S. H.; Zhai, Y. Z.; Xuan, Q. C.; Wang, T. Secondary metabolites from the genus

385

xylaria and their bioactivities. Chem. Biodivers. 2014, 11, 673 – 694.

386

(6) Scherlach, K.; Boettger, D.; Remme, N.; Hertweck, C. The chemistry and biology of

387

cytochalasans. Nat. Prod. Rep. 2010, 27, 869 – 886.

388

(7) Qiao, K.; Chooi, Y. H.; Tang, Y. Identification and engineering of the cytochalasin gene

389

cluster from Aspergillus clavatus NRRL 1. Metab. Eng. 2011, 13, 723 – 732.

390

(8) Yuyama, K. T.; Wendt, L.; Surup, F.; Kretz, R.; Chepkirui, C.; Wittstein, K.; Boonlarppradab,

391

C.; Wongkanoun, S.; Luangsa-Ard, J.; Stadler, M, Abraham, W. R. Cytochalasans act as inhibitors 18


Page 18 of 31

Page 19 of 31


392

of biofilm formation of Staphylococcus aureus. Biomolecules 2018, 8, 129.

393

(9) Kretz, R.; Wendt, L.; Wongkanoun, S.; Luangsa-Ard, J. J.; Surup, F.; Helaly, S. E.; Noumeur,

394

S. R.; Stadler, M.; Stradal, T. E. B. The effect of cytochalasans on the actin cytoskeleton of

395

eukaryotic cells and preliminary structure-activity relationships. Biomolecules 2019, 9, 73.

396

(10) Xiao, J.; Zhang, Q.; Gao, Y. Q.; Tang, J.-J.; Zhang, A.-L.; Gao, J.-M. Secondary metabolites

397

from the endophytic Botryosphaeria dothidea of Melia azedarach and their antifungal,

398

antibacterial, antioxidant, and cytotoxic activities. J. Agric. Food Chem. 2014, 62, 3584 – 3590.

399

(11) Li, H.; Xiao, J.; Gao, Y. Q.; Tang, J. J.; Zhang, A. L.; Gao. J. M. Chaetoglobosins from

400

Chaetomium globosum, an endophytic fungus in Ginkgo biloba, and their phytotoxic and

401

cytotoxic activities. J. Agric. Food Chem. 2014, 62, 3734 – 3741.

402

(12) Li, X. J.; Tang, H.Y.; Duan, J. L.; Gao, J. M.; Xue, Q. H. Bioactive alkaloids produced by

403

Pseudomonas brassicacearum subsp neoaurantiaca, an endophytic bacterium from Salvia

404

miltiorrhiza. Nat. Prod. Res. 2013, 27, 496 – 499.

405

(13) Yuan, Y.; Tian, J. M.; Xiao, J.; Shao, Q.; Gao, J. M. Bioactive metabolites isolated from

406

Penicillium sp YY-20, the endophytic fungus from Ginkgo biloba. Nat. Prod. Res. 2014, 28, 278

407

–281.

408

(14) Xiao, J.; Zhang, Q.; Gao, Y. Q.; Shi, X. W.; Gao, J. M. Antifungal and antibacterial

409

metabolites from an endophytic Aspergillus sp. associated with Melia azedarach. Nat. Prod. Res.

410

2014, 28, 1388 – 1392.

411

(15) Li, H.; Wei, J.; Pan, S. Y.; Gao, J. M.; Tian, J. M. Antifungal, phytotoxic and toxic

412

metabolites produced by Penicillium purpurogenum. Nat. Prod. Res. 2014, 28, 2358 – 2361.

413

(16) Zhang, Q.; Xiao, J.; Sun, Q. Q.; Qin, J. C.; Pescitelli, G.; Gao, J. M. Characterization of

414

cytochalasins from the endophytic Xylaria sp. and their biological functions. J. Agric. Food Chem. 19



415

2014, 62, 10962 – 10969.

416

(17) Zhang, Q.; Wang, S. Q.; Tang, H. Y. Li, X. J.; Zhang, L.; Xiao, J.; Gao, Y. Q.; Zhang, A. L.;

417

Gao, J. M. Potential allelopathic indole diketopiperazines produced by the plant endophytic

418

Aspergillus fumigatus using the one strain-many compounds method. J. Agric. Food Chem. 2013,

419

61, 11447 – 11452.

420

(18) Liu, X.; Tian, F.; Tian, Y.; Wu, Y.; Dong, F.; Xu, J.; Zheng, Y. Isolation and identification of

421

potential allelochemicals from aerial parts of Avena fatua L. and their allelopathic effect on wheat.

422

J. Agric. Food Chem. 2016, 64, 3492 – 3500.

423

(19) Di Bari, L.; Pescitelli, G.; Pratelli, C.; Pini, D.; Salvadori, P. Determination of absolute

424

configuration of acyclic 1,2-diols with Mo2(OAc)4. 1. Snatzke's method revisited. J. Org. Chem.

425

2001, 66, 4819 – 482.

426

(20) Górecki, M.; Jabłońska, E.; Kruszewska, A.; Suszczyńska, A.; Urbańczyk-Lipkowska, Z.;

427

Gerards, M.; Morzycki, J. W.; Szczepek, W. J.; Frelek, J. Practical method for the absolute

428

configuration assignment of tert/tert 1,2-diols using their complexes with Mo2(OAc)4. J. Org.

429

Chem. 2007, 72, 2906 – 2916.

430

(21) Zhang, H. W.; Zhang, J.; Hu, S.; Zhang, Z. J.; Zhu, C. J.; Ng, S. W.; Tan, R. X. Ardeemins

431

and cytochalasins from Aspergillus terreus residing in Artemisia annua. Planta Med. 2010, 76,

432

1616 – 1621.

433

(22) Liu, R.; Gu, Q.; Zhu, W.; Cui, C.; Fan, G.; Fang, Y.; Zhu, T.; Liu, H.

434

10-Phenyl-[12]-cytochalasins Z7, Z8, and Z9 from the marine-derived fungus Spicaria elegans. J.

435

Nat. Prod. 2006, 69, 871 – 875.

436

(23) Kimura, Y.; Nakajima, H.; Hamasaki, T. Structure of rosellichalasin, a new metabolite 20


Page 20 of 31

Page 21 of 31


437

produced by Rosellinia necatrix. Agric. Biol. Chem. 1989, 53, 1699 – 1701.

438

(24) Tori, K.; Nakagawa, T.; Komeno, T. Nuclear Magnetic Resonance studies on steroids. 111.

439

steroidal epoxides and episulfides. J. Org. Chem. 1964, 29, 1136 – 1141.

440

(25) Espada, A.; Rivera-Sagredo, A.; de la Fuente, J. M.; Hueso-Rodriguez, J. A.; Elson, S. W.

441

New cytochalasins from the fungus Xylaria hypoxylon. Tetrahedron, 1997, 53, 6485 – 6492.

442

(26) Pescitelli, G.; Bruhn, T., Good computational practice in the assignment of absolute

443

configurations by TDDFT calculations of ECD spectra. Chirality 2016, 28, 466 – 474.

444

(27) Superchi, S.; Scafato, P.; Gorecki, M.; Pescitelli, G. Absolute configuration determination by

445

quantum mechanical calculation of chiroptical spectra: basics and applications to fungal

446

metabolites. Curr. Med. Chem. 2018, 25, 287 – 320.

447

(28) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. SpecDis: Quantifying the

448

comparison of calculated and experimental electronic circular dichroism spectra. Chirality 2013,

449

25, 243 – 249.

450

(29) Wang, H.; Li, M. Y.; Satyanandamurty, T.; Wu, J. New diterpenes from a Godavari

451

mangrove, Ceriops decandra. Planta Med. 2013, 79, 666 – 672.

452

(30) Steyn, P. S.; van Heerden, F. R.; Rabie, C. J. Cytochalasins E and K, toxic metabolites from

453

Aspergillus clavatus. J. Chem. Soc. Perkin Trans. 1 1982, 541 – 544.

454

(31) Büchi, G.; Kitaura, Y.; Yuan, SS.; Wright, H. E.; Clardy, J.; Demain, A. L.; Ginsukon, T.;

455

Hunt, N.; Wogan, G. N. Structure of cytochalasin E, a toxic metabolite of Aspergillus clavatus. J.

456

Am. Chem. Soc. 1973, 95, 5423 – 5425.

457 458 21



459 460 461 462 463 464

Figure captions

465 466

Figure 1. Structures of 1 – 8.

467

Figure 2. X-ray crystallography of 1 (upper) and 2 (bottom).

468

Figure 3. Selected 1H−1H COSY, HMBC (up) and NOESY correlations of 3 (down).

469

Figure 4. Experimental CD spectrum of (+)-3 in methanol (2.1 mM, 0.5 cm cell)

470

compared with the spectrum calculated on (3S,4S,5S,6R,7S,8S,9S,13R,14R,16S)-3 at

471

the TD-CAM-B3LYP/def2-TZVP level as Boltzmann-weighted average over 3

472

conformers optimized at the ɷB97X-D/6-311+G(d,p) level, including PCM solvent

473

model for MeOH. Plotting parameters: Gaussian band-width 0.35 eV; wavelength

474

shift +3 nm; scaled by a factor 2.

475

Figure 5. ICD spectrum of 4 in DMSO containing Mo2(OAc)4 with the inherent CD

476

subtracted (up), and Newman projection of Mo-complexes of 4 (down).

22


Page 22 of 31

Page 23 of 31


478 479 12

O

11 1'

3'

H

10

OH

7

O

22

5 13

9 3

HN

1

5'

15

H

O 17

O 21 O O

O

OH

HN

19 23

1

H

OH

HN

O O O

O

H H

H

O

O O O 3

2 16 13

11 20

1

HO

9

5 18

7

H

O

HN

19

4

O

O

O

O

O

HN

OH

O O

O O

8

481 482 483

O O O O 6

H

7

480

HN

O O O

OH O O

H

5

H HN

OH

OH

17

14

H

10

3

HO

OH 15

Figure 1

23


O OH


485

486 487

1

488 489 490

2 Figure 2

491 492 493

24


Page 24 of 31

Page 25 of 31


O O

H

H

O O

HN

O

COSY

O HMBC

NOE

494 495 496

Figure 3

25



498 499

Figure 4

26


Page 26 of 31

Page 27 of 31


501

502 503 504

Figure 5

505 506 507 508 509 510 511 512 513 514 515 27



Page 28 of 31

516 517

Table 1. NMR Spectroscopic Data of 1 − 3 in CDCl3 1 δC 1

2

δH (mult, J in Hz)

δC

170.4

172.1 5.93, br s

2

δH (mult, J in Hz)

3 δC

δH (mult, J in Hz)

170.5 6.04 br s

5.79, br s

3

56.6

3.58, m

56.9

3.74 ddd (7.6, 7.2, 1.8)

54.7

3.68, m

4

48.0

3.19 d (1.8)

47.5

3.19 d (1.8)

50.6

2.55, t (4.1)

5

62.8

62.2

36.2

2.00, qd (7.3, 4.1)

6

64.2

64.4

56.1

7

69.2

3.59, d (10.4)

70.5

3.68 d (10.4)

58.1

2.88, d (5.2)

8

46.8

3.40 t (10.4)

46.2

3.39 t (10.4)

48.8

1.61, dd (8.8, 5.2)

9

83.2

10

43.0

84.0 3.02, dd (13.3, 8.8)

43.3

2.92, dd (13.3, 5.6)

83.7 2.96, dd (13.2, 7.2)

43.5

2.92, dd (13.2, 7.6)

3.08, dd (13.4, 9.5) 2.90, dd (13.4, 4.4)

11

20.2

1.33 s

20.7

1.25, s

13.6

1.13, d (7.3)

12

14.1

1.42, s

14.5

1.38, s

20.1

1.31, s

13

124.8

5.77 ddd (15.1, 10.4, 1.4)

123.6

6.11, dd (15.1, 10.4)

58.3

3.48, dd (8.8, 1.7)

137.9

5.62, ddd (15.1, 11.0, 3.8)

140.7

5.27, ddd (15.1, 10.1,

60.9

2.87, m

38.4

2.08, br d (15.2)

14 15

4.2) 39.8

2.26 ddd (14.0, 3.8, 2.0)

42.8

2.10, m

2.08 dt (14.0, 11.0) 16

39.5

17

1.42, m 32.0

1.62, m

36.1

205.1

78.6

3.82, t (3.7)

204.6

18

142.8

42.7

2.76, m

142.1

19

132.0

6.40, t (8.0)

156.5

7.00, dd (16.0, 4.8)

130.9

6.75, t (8.2)

20

36.6

3.30, dd (12.4, 8.0)

121.3

5.71, dd (16.0, 1.7)

35.4

3.29, dd (14.4, 8.2)

3.30, m

3.13, dd (12.4, 8.0)

3.51, m

3.22, dd (14.4, 8.2)

21

168.7

166.5

168.8

22

17.6

1.17, d (6.8)

18.4

0.98, d (7.4)

18.7

1.17, d (6.8)

23

12.6

1.84, s

8.7

1.05, d (6.6)

12.2

1.89, s

1’

136.9

2’

129.2

7.18 d (7.1)

129.2

7.17 d (7.1)

129.2

7.21, d (7.3)

3’

129.1

7.35 t (7.1)

129.0

7.34 t (7.1)

129.1

7.35, t (7.3)

4’

127.3

7.28 t (7.1)

127.1

7.27 t (7.1)

127.3

7.29, t (7.3)

5’

129.1

7.35 t (7.1)

129.0

7.34 t (7.1)

129.1

7.35, t (7.3)

6’

129.2

7.18 d (7.1)

129.2

7.17 d (7.1)

129.2

7.21, d (7.3)

137.0

137.3

518 519 28


Page 29 of 31


520 521 522

Table 2. NMR Spectroscopic Data of 4 in MeOH-d4 4

1

δC

δH, multi (J in Hz)

45.6

1.48, t (12.1) 2.57, dd (12.1, 4.6)

2

69.2

3.75, ddd (12.1, 9.7, 4.6)

3

83.4

2.95, d (9.7)

4

40.4

5

50.1

1.83, dd (13.9, 4.0)

6

36.9

2.59, dd (18.1, 4.0) 2.68, dd (18.1, 13.9)

7

201.2

8

131.1

9

154.9

10

39.9

11

125.0

7.33, d (8.3)

12

132.4

7.64, dd (8.3, 2.2)

13

149.4

14

124.1

15

72.6

16

31.7

1.42, s

17

31.7

1.42, s

18

28.5

0.98, s

19

16.7

0.88, s

20

24.6

1.20, s

7.98, d (2.2)

523

29



Page 30 of 31

525 526

Table 3. Allelopathic effects on wheat (Triticum aestivum) of Compounds 1 − 8 a germination rate compds

6.25 μM

root elongation (RI)

100 μM

6.25 μM

100 μM

6.25 μM

1

0.83 ± 0.06

0.92 ± 0.06

-0.47 ± 0.05

-0.30 ± 0.05

-0.52 ± 0.03

-0.02 ± 0.02

2

0.67 ± 0.05

0.83 ± 0.06

-0.54 ± 0.05

-0.07 ± 0.01

-0.51 ± 0.04

-0.04 ± 0.02

3

0.92 ± 0.06

0.92 ± 0.05

-0.69 ± 0.04

0.00 ± 0.00

-0.87 ± 0.07

-0.09 ± 0.05

4

0.83 ± 0.05

0.92 ± 0.05

-0.66 ± 0.04

-0.38 ± 0.10

-0.62 ± 0.02

-0.13 ± 0.04

5

0.67 ± 0.06

0.83 ± 0.05

-0.58 ± 0.05

-0.16 ± 0.06

-0.74 ± 0.02

-0.36 ± 0.04

6

0.92 ± 0.00

0.67 ± 0.05

-0.45 ± 0.02

-0.14 ± 0.00

-0.41 ± 0.03

-0.27 ± 0.02

7

0.50 ± 0.02

0.92 ± 0.05

-0.72 ± 0.06

-0.44 ± 0.00

-0.78 ± 0.02

-0.07 ± 0.02

8

0.83 ± 0.06

0.83 ± 0.05

-0.65 ± 0.05

-0.07 ± 0.01

-0.80 ± 0.02

-0.29 ± 0.02

0.83 ± 0.06

0.92 ± 0.05

-0.53 ± 0.06

0.14 ± 0.04

-0.57 ± 0.02

-0.07 ± 0.01

0.92 ± 0.05

0.83 ± 0.00

Glyphosate b Ck

527

100 μM

shoot elongation (RI)

a

c

Mean ± SD. b Positive control. c Blank control.

528 529 530 531

Table 4. Inhibitory effects on turnip (Raphanus sativus) and wheat (Triticum aestivum) of

532

Compounds 1 – 8 (IC50 in μM) a

533

shoot elongation compds

534 535 536 537

turnip

wheat

turnip

>100

NA

wheat

1

NA

2

NA

98.70 ± 0.19

NA

3

NA

18.92 ± 0.80

NA

40.28 ± 0.19

4

NA

23.58 ± 0.43

NA

52.06 ± 0.16

5

>100

84.05 ± 0.27

>100

17.35 ± 0.05

6

NA

7

99.42 ± 0.16

8 Glyphosate a

b

root elongation

>100 c

Mean ± SD.

99.88 ± 5.31 b

>100

79.27 ± 0.26 >100

NA

>100

24.02 ± 0.51

36.75 ± 0.09

22.58 ± 0.58

46.58 ± 0.19

1.57 ± 0.21

19.74 ± 0.09

42.31 ± 0.66

83.09 ± 0.09

38.11 ± 0.29

Not activity (Inhibition rates < 30% at 100 μM).

control.

30


c

Positive

Page 31 of 31


539

TOC 540 541 OH

12

O

11 1'

10

OH

7 9

3

HN O

1

13

15

O 21 19 O O 1

542

O 23

2

Xylaria sp. XC-16

O 4

OH O O O

H

HO

17

OH

H HN

HO

22

5

H

eight allelochemicals

543 544 545

31


Cytochalasins and an Abietane-Type Diterpenoid with Allelopathic

Recommend Documents