New Î²-Lactone with Tea Pathogenic Fungus ... - ACS Publications

Subscriber access provided by MIDWESTERN UNIVERSITY

Bioactive Constituents, Metabolites, and Functions

New #-Lactone with Tea Pathogenic Fungus Inhibitory Effect from Marine-derived Fungus MCCC3A00957 Xi-Xiang Tang, Xia Yan, Wen-Hao Fu, Lu-Qi Yi, Bo-Wen Tang, Li-Bo Yu, Meijuan Fang, Zhen Wu, and Ying-Kun Qiu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00228 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 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 30

Journal of Agricultural and Food Chemistry

1

New β-Lactone with Tea Pathogenic Fungus Inhibitory Effect from

2

Marine-derived Fungus MCCC3A00957

3

Xi-Xiang Tang, 1, † Xia Yan, 3, † Wen-Hao Fu, 2 Lu-Qi Yi, 2 Bo-Wen Tang, 2 Li-Bo Yu, 1 Mei-Juan

4

Fang, 2 Zhen Wu, 2 and Ying-Kun Qiu 2, *

5

1

Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography State,

6

Ministry of Natural Resources, Da-Xue Road, Xiamen 361005, China;

7

E-Mails: [email protected] (X.-X. Tang), [email protected] (L.-B. Yu)

8

2

Fujian Provincial Key Laboratory of Innovative Drug Target Research, School of Pharmaceutical Sciences, Xiamen University, South Xiang-An Road, Xiamen, 361102, China;

9 10

E-Mails: [email protected] (W.-H. Fu); [email protected] (L.-Q. Yi); [email protected]

11

(B.-W. Tang); [email protected] (M.-J. Fang); [email protected] (Z. Wu);

12

[email protected] (Y.-K. Qiu)

13

3

Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, Ningbo University, Ningbo 315832, China; [email protected] (X.Y.)

14 15 16 17

* Corresponding author:

18

Tel.: +86-592-2189868; Fax: +86-592-2189868;

19

E-mail address: [email protected]

20

†

These authors contributed equally to this paper

21

ACS Paragon Plus Environment


23

Page 2 of 30

ABSTRACT

24

Fusarium solani H915 (MCCC3A00957), a fungus originating from mangrove sediment, showed

25

potent inhibitory activity against tea pathogenic fungus Pestalotiopsis theae. Successive

26

chromatographic separation on an ethyl acetate (EtOAc) extract of F. solani H915 resulted in the

27

isolation of five new alkenoic diacid derivatives, fusarilactones A–C (1–3), fusaridioic acids B (4) and

28

C (5), in addition to seven known compounds (6–12). The chemical structures of these metabolites

29

were elucidated on the basis of UV, IR, HR-ESI-MS and NMR spectroscopic data. The antifungal

30

activity of the isolated compounds was evaluated. Compounds with a β-lactone ring (1, 2 and 7)

31

exhibited potent inhibitory activities, while none of the other compounds show activity. The ED50

32

values of the compounds 1, 2 and 7 were 38.14 ± 1.67 µg/mL, 42.26 ± 1.96 µg/mL, and 18.35 ± 1.27

33

µg/mL respectively. Additionally, inhibitory activity of these compounds against 3-hydroxy-3-

34

methylglutaryl-CoA (HMG-CoA) synthase gene expression was also detected by real time RT-PCR.

35

Results indicated that compounds 1, 2 and 7 may inhibit the growth of P. theae by interfering with the

36

biosynthesis of ergosterol by down-regulating the expression of HMG-CoA synthase.

37 38

KEYWORDS: Fusarium solani H915; fusarilactones A, B, C; fusaridioic acids B, C; HMG-CoA

39

synthase; tea pathogenic fungus inhibitory effect.


Page 3 of 30

41


INTRODUCTION

42

Tea [Camellia sinensis O. Kuntze] is an important economic crop in many countries. However,

43

the growth and production of tea can be severely disturbed by various factors, especially fungal

44

diseases that infects the leaves. 1-2 Infected tea leaves often exhibit severe damage of the blade tissue

45

and discoloration of the leaf, such as blight (Exobasidium vexans Massee), grey blight (Pestalotiopsis

46

theae (Sawada) Steyaert), brown blight (Colletotrichum camelliae Massee), sooty mold (Capnodium

47

theae Boedijn), and red rust (Cephaleuros parasiticus Karst). 1, 3-7

48

Fungicides and biocontrol agents have demonstrated potential against these pathogenic fungi.

49

Synthetic chemical fungicides such as thiophanatemethyl, carbendazim and contact fungicides such as

50

mancozeb and, copper oxychloride have been effectively used in the field.

51

including Bacillus subtilis, 9 Trichoderma viride, 10 Ochrobactrum anthropic, 11 and Streptomyces spp.

52

12

53

fungicides can lead to many problems, such as phytopathogen resistance, food safety threat and

54

environmental pollution. 13-15 Therefore, the development of new natural product derivative fungicides

55

is of great importance to overcome these shortcomings．

8

Biocontrol agents,

have also been used in antagonizing these tea pathogenic fungi. However, long-term use of chemical

56

To date there have only been a few reports on active compounds against tea pathogenic fungi.

57

Essential oil-based β-methoxyacrylate derivatives have been synthesized and showed remarkable

58

inhibitory activities against P. theae. 16 Major tea leaf volatile constituents, including geraniol, linalool,

59

methyl salicylate, benzyl alcohol, and 2-phenylethanol were found to exhibit significant antifungal

60

activities toward Colletorichum camelliae Massea.

61

temperature, pressure, salt concentration and pH, are known to be a rich resource for bioactive natural

62

products.

18-19

17

Marine environments, with a wide range of

Previously, we identified a new macrolactin

20

form Bacillus subtilis B5, a bacteria



Page 4 of 30

63

isolated from sea sediment 3000 metres below the Pacific Ocean, which exhibited antifungal activity

64

against P. theae and Colletorichum gloeosporioides. Mangrove ecosystems are coastal wetlands with

65

high primary production rates, equal to those of tropical humid evergreen forests and coral reefs.

66

Sediment microorganisms help create and maintain mangrove ecosystems by decomposition of organic

67

matter and are critical for the cycling of nutrients. 22 Mangrove-associated microorganisms are thought

68

to be of great value for obtaining bioactive natural products. 23-24 Endophytic Fusarium sp have also

69

been shown to produce various secondary metabolites, such as cyclic pentapeptides, cyclic

70

lipopeptides, 25 α-pyridone derivatives, ceramide derivatives, 15 azaphilone derivatives, 26 isocoumarin

71

derivatives,

72

These metabolites showed various bioactivities. We herein report the isolation, structural

73

determination, and antifungal activity of the metabolites from an extract of Fusarium solani H915

74

(MCCC3A00957), a fungus originating from mangrove sediment.

27

trehalose-containing glycolipids,

28

and polyketide-derived isoquinoline alkaloids.

21

29

75 76

MATERIALS AND METHODS

77

General experimental procedures

78

Open-column separations were carried out using silica gel (Yantai Chemical Industry Research

79

Institute, Yantai, China) or Cosmosil 75 C18-OPN (75 μm, Nakalai Tesque Co. Ltd., Kyoto, Japan).

80

The preparative HPLC was performed with a preparative Cosmosil ODS column (250 mm × 20.0 mm

81

i.d., 5 m, Cosmosil, Nakalai Tesque Co. Ltd., Kyoto, Japan), via a Varian binary gradient LC system

82

(Varian Inc. Corporate, Santa Clara, CA, USA). The HR-ESI-MS spectra were acquired on a Q-

83

Exactive Mass spectrometer (Thermo Fisher Scientific Corporation, Waltham, MA, USA). UV spectra

84

were recorded on a Shimadzu UV-260 spectrometer (Shimadzu Corporation, Tokyo, Japan). IR spectra


Page 5 of 30


85

in KBr pellets were determined using a Perkin-Elmer 683 infrared spectrometer (PerkinElmer, Inc.,

86

Waltham, MA, USA). Optical rotations were measured on a JASCO P-200 polarimeter (JASCO

87

Corporation, Tokyo, Japan), equipped with a 5-cm cell. The NMR spectra of the compounds dissolved

88

in DMSO-d6 were run on a Bruker Avance III 600 FT NMR spectrometer (Bruker Corporation,

89

Billerica, MA, USA).

90

Circular Dichroism (CD) spectra and Electronic Circular Dichroism (ECD) Calculations

91

The circular dichroism (CD) spectra were acquired on a Chirascan circular dichroism

92

spectrometer (Applied Photophysics Ltd., Leatherhead, UK). The theoretical electronic circular

93

dichroism (ECD) spectra of the isolated compounds were calculated on the basis of the relative

94

configurations determined by their NOESY spectra and J values in the 1H NMR. Conformational

95

analyses and density functional theory (DFT) calculations were used to generate and optimize the

96

conformers with energy. The ECD calculations were performed following a method descripted

97

previously [25].

98

Fungus and carbohydrate fermentation

99

The H915 strain was isolated from the mangrove sediments at the estuary of Zhangjiangkou

100

Mangrove National Nature Reserve (23° 55′35.37″N, 117°24′50.93″E), Fujian province, China using

101

a tablet pour method. The internal transcribed spaces (ITS) region was amplified and sequenced by

102

using the general primers ITS1 and ITS4. The ITS region of the fungus was a 576 bp DNA sequence

103

(GenBank accession number KY978583) which had 99% identity to Fusarium solani. The strain was

104

deposited at the China Center for Type Culture Collection (CCTCC, M2017150) and Marine Culture

105

Collection of China (MCCC, 3A00957). Carbohydrate fermentation was carried out by subculturing

106

the fungus onto a rice-artificial sea water medium, incubated at 28°C for 30 days in a standing position.



Page 6 of 30

107

P. theae (ITS GenBank accession number HQ832793) was isolated from foliar lesions of the tea leaf

108

and its pathogenicity to tea leaves was verified both in vitro and in vivo (unpublished data).

109

Extraction and isolation

110

The rice-artificial sea water culture (10 kg) of F. solani H915 was extracted with ethyl acetate

111

(EtOAc, 20 L) three times and concentrated under reduced-pressure at 40 ºC to yield 16.4 g crude

112

extract. Then, 15.0 g of the EtOAc extract was divided into 10 fractions (Fr. 1 – Fr. 10) over a silica

113

gel (300 g) column eluted with petroleum ether : ethyl acetate (v/v) (20:1; 10:1; 5:1; 2:1; 1:1, 1.0 L

114

each) and chloroform : methyl alcohol (v/v) (50:1; 20:1; 10:1; 5;1; 2:1; 0:1, 1.0 L each). Except for the

115

low-yielding non-polar (Fr. 1 – Fr. 4) and extreme high-polar fractions (Fr. 10), most of the fractions

116

were subjected to ODS columns for further separation. Fr. 5 (1.1 g) was separated over 20 g ODS and

117

eluted with 10%, 30%, 50%, 70% and 100% CH3OH/H2O (0.2 L each) to give five subfractions (subFr.

118

5.1 – subFr. 5.5). SubFr. 5.5 was purified by preparative HPLC ODS column and isocratic eluted with

119

acetonitrile-H2O (27:73, v/v) to yield compounds 9 (15 mg) and 10 (15 mg). Fr. 6 (4.6 g) was subjected

120

to an ODS (100 g) column and eluted with 10%, 30%, 50%, 70% and 100% CH3OH/H2O (0.5 L each)

121

to give nine subfractions (subFr. 6.1 – subFr. 6.9). Then, subFr. 6.9 (1.3 g) was purified by preparative

122

HPLC and isocratic eluted with acetonitrile-H2O (42:58, v/v) to give compounds 1 (7 mg), 2 (7 mg)

123

and 7 (300 mg). Fractionation of Fr. 7 (1.3 g), resulting in 8 subfractions (subFr. 7.1 – subFr. 7.8), was

124

conducted via an ODS (30 g) column eluted with 10%, 30%, 50%, 70% and 100% CH3OH/H2O (0.3

125

L each). SubFr. 7.8 (80 mg) was purified by preparative HPLC, isocratic eluted with acetonitrile-H2O

126

(45:55, v/v), to afford compounds 3 (10 mg) and 5 (10 mg). Fr. 8 (1.5 g) was separated using an ODS

127

(30 g) column, eluted with CH3OH/H2O (10% – 100%, 0.3 L each), to give a 10 subfractions (subFr.

128

8.1 – subFr. 8.10). Preparative HPLC with isocratic elution of acetonitrile : H2O (44:56, v/v) was used


Page 7 of 30


129

for subFr. 8.7 (105 mg), leading to the isolation of compound 6 (10 mg). Fr. 9 (3.8 g) was also separated

130

using an ODS (100 g) column, eluting with 0%, 30%, 50%, 70% and 100% CH3OH/H2O (0.5 L each),

131

to give nine subfractions (subFr. 9.1 – subFr. 9.9). SubFr. 9.7 (325 mg) was purified by preparative

132

HPLC isocratic eluted with acetonitrile-H2O (45:55, v/v) to obtain compounds 4 (6 mg), 8 (8 mg), 11

133

(14 mg) and 12 (19 mg).

134

Fusarilactone A (1)

135

Isolated as a white amorphous powder; HRESIMS m/z 345.1668 [M+Na]+ (calcd. for C18H26O5Na,

136

345.1672) in the positive mode, and m/z 321.1711 [M-H]- (calcd. for 321.1707 C18H25O5) in the

137

negative mode; [α]D25 = +16° (c = 0.5, CH3OH), IR (KBr) (νmax): 3406, 1654, 1436 cm−1. UV (CH3OH)

138

λmax (log ε): 201 (3.91), 233 (3.58), 266 (3.80) nm. 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150

139

MHz, DMSO-d6) data are listed in Table 1 and Table 2.

140

Fusarilactone B (2)

141


142


143

negative mode; [α]D25 = 0° (c = 0.5, CH3OH). UV (CH3OH) λmax (log ε): 216 (4.38), 267 (4.03) nm.

144

IR (KBr) (νmax): 3385, 1646, 1436 cm−1. 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz,

145

DMSO-d6) data are listed in Table 1 and Table 2.

146

Fusarilactone C (3)

147


148


149

negative mode; [α]D25 = 2° (c = 0.5, CH3OH). UV (CH3OH) λmax (log ε): 228 (3.53) and 266 (3.85)

150

nm. IR (KBr) (νmax): 3406, 2927, 1560, 1430 cm−1. 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150

151

MHz, DMSO-d6) data are listed in Table 1 and Table 2.

152

Fusaridioic acid B (4)



Page 8 of 30

153


154


155

negative mode; [α]D25 = 0° (c = 0.5, CH3OH), IR (KBr) νmax): 3406, 1570, 1430 cm−1. UV (CH3OH)

156

λmax (log ε): 231 (3.57), 266 (3.86) nm. 1H NMR (600 MHz, DMSO-d6) and

157


13C

NMR (150 MHz,

158

Fusaridioic acid C (5)

159


160


161

negative mode; [α]D25 = 0° (c = 0.5, CH3OH), IR (KBr) (νmax): 3416, 1570, 1430 cm−1. UV (CH3OH)

162

λmax (log ε): 229 (3.47), 268 (3.93) nm. 1H NMR (600 MHz, DMSO-d6) and

163


164

Antifungal activity assay

165

13C

NMR (150 MHz,

The antifungal assay of all the isolated compounds was performed against tea pathogenic fungus 30

166

P. theae using a previously described method.

167

which a 0.6 cm diameter piece of tested fungal strains cylinder agar was placed on the center and sterile

168

blank paper discs of 0.5 cm diameter were placed at a distance of 2 cm away from the growing mycelial

169

colony. Approximately 20 μg compound was added to each paper disc. DMSO without compound was

170

used as the blank control and commercial fungicide hexaconazole was used as the positive control.

171

The plates were incubated at 28 °C until mycelial growth covered the control discs.

172

ED50 Detection.

173

As reported previously,

174

31

The tests were carried out in PDA Petri plates in

compounds with final concentrations of 150–4.688 µg/mL (two-fold

dilution) were mixed with PDA medium and poured into a set of PDA Petri plates. P. theae mycelial


Page 9 of 30


175

cylinder agar (0.6 cm) was placed in the center of each treated Petri dish. Treated Petri dishes were

176

then incubated at 28°C until the fungal growth covered the blank control plates. DMSO was used as

177

the blank control and hexaconazole was used as the positive control. Mycelial growth of fungus (cm)

178

in both treated (T) and control (C) were measured diametrically. The mean and standard deviation

179

were calculated to determine the percentage inhibition of growth (I%) with the formula

180

− T)/C × 100. Corrected inhibition (%) = [(% I − C.F.)/(100 − C.F.)] × 100. Correction

181

Factor (CF) = [(90 − C)/C] × 100 as described previously. 31 From the concentration (μg/mL) and

182

corresponding corrected percent growth inhibition data, the ED50 (μg/mL) value was calculated

183

statistically by Probit analysis, using the Probit Package of MSTATC software. The experiment was

184

repeated three times.

185

Total RNA Isolation

I (%) = (C

186

P. theae cells cultured in PDA medium for 3 days at 28°C were treated with compounds 1–12 at

187

10 µg/mL for 16 h. Cells were harvested by centrifuging at 6000 rpm for 5 min. Cells were then

188

homogenized in liquid nitrogen and total RNA was extracted with Spin Column Fungal Total RNA

189

Purification Kit (Sangon Biotech, China) and stored at -80 °C until use.

190

Real-Time RT-PCR Analysis of HMG-CoA synthase expression

191

The effect of compounds 1–12 on the mRNA expression of HMG-CoA synthase of P. theae cells

192

at a concentration of 10 µg/mL was analyzed by Real-Time PCR. DMSO and abscisic acid at 10 µg/mL

193

32

194

HMG-CoA synthase (forward: TACTCGCTCACCTGCTACAC, reverse: GCGTACGACTTCTG

195

GACGAC), and GAPDH (forward: CATGTCCATGCGTGTCCCTA, reverse: CAGTGGAGA

196

CAACCTCGTCC) was determined by real-time RT-PCR. The cDNA was synthesized from total RNA

197

using PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan). TaKaRa SYBR® Premix Ex

were used as the blank and positive controls, respectively. The expression of mRNA transcripts of



Page 10 of 30

198

Taq™ II (Takara, Japan) and Stepone Real-Time PCR Detection System (Applied Biosystems, USA)

199

were used for Real-Time PCR analysis.

200 201

RESULTS AND DISCUSSION

202

Structural Elucidation

203

A series of chromatographic methods was used during the isolation of an extract from the rice-artificial

204

sea water medium of F. solani H915. As a result, 12 compounds were isolated, including five new

205

compounds, fusarilactone A (1), fusarilactone B (2), fusarilactone C (3), fusaridioic acid B (4),

206

fusaridioic acid C (5), and seven known compounds (6–12, Figure 1).

207 208

Insert Figure 1 here

209 210

Compound 1 (fusarilactone A) was isolated as an amorphous white powder. The molecular

211

formula C18H26O5, which gave six degrees of unsaturation, was established by positive and negative

212

HRESIMS ion peaks at m/z 345.1668 [M+Na]+ and 321.1711 [M-H]-, respectively. The UV maximum

213

absorption bands at max (log ε): 233 (3.58) nm and 266 (3.80) were assigned to an unconjugated

214

carbonyl and a conjugated carbonyl, respectively. In the low-field region of the 13C NMR, two carboxyl

215

carbon signals were observed at δC 170.8 (C-14) and δC 168.6 (C-1, which is conjugated with the

216

double bond system). In the low field region of the 1H NMR, two olefinic protons were observed with

217

br. s peaks at δH 5.55 (H-2) and 5.71 (H-4). In addition, a pair of trans-alkene hydrogens were observed

218

at δH 5.34 (H-8) and 5.40 (H-9), with a coupling constant of 15.4 Hz. Their corresponding olefinic

219

carbon signals were found in the sp2 region of the 13C NMR spectrum at δC 119.2 (C-2), 152.4 (C-3),


Page 11 of 30


220

129.9 (C-4), 140.4 (C-5), 136.8 (C-8), and 127.1 (C-9). The sp3 high-field region of the 1H NMR

221

spectrum showed three methyl proton signals. Two br.s methyl peaks at δH 2.13 (3-CH3) and 1.76 (5-

222

CH3) were assigned as being linked to quaternary olefinic carbons. The other methyl at δH 0.91 (d, J =

223

6.6 Hz, 7-CH3) is connected to a methylene group. In the 1H-1H COSY spectrum, the proton at δH 4.52

224

(m) showed correlations with those at δH (1.85 & 1.78, H-11) and δH 3.51 (H-13). The HSQC spectrum,

225

signal at δH 4.52 (m) showed a correlation with the carbon at δC 74.3, which was attributed to the 12-

226

CH. The two dd peaks at δH 3.71 (J = 11.6, 3.9 Hz) and 3.61 (J = 11.6, 3.5 Hz), which form a typical

227

ABX coupling system with H-13 (δH 3.51), were assigned to the two protons at 13-CH2OH.

228

Elucidation of HSQC, 1H-1H COSY and HMBC spectra indicated that the planar structure of 1 was

229

almost identical to that of hymeglusin (7), 33-34 a typical β-lactone antibiotic isolated from a culture of

230

Scopulariopsis sp. F-244. Most of the 1D NMR spectral data of 1 were very similar to those of

231

hymeglusin (7), except for the signals at positions 8 and 9. The molecular formula of 1 has two fewer

232

hydrogen atoms than 7, and the pair of olefinic 1H and 13C signals that appeared in the sp2 low field

233

region, indicated that 1 was the dehydrogenation product of hymeglusin (7) at positions 8 and 9. The

234

configuration of the double bonds was also revealed by a NOESY experiment, where correlations

235

between H-4 (δH 5.71) and H-2 (δH 5.55), as well as H-4 and H-6 (δH 2.02, 1.97), were observed.

236

Moreover, the NOESY correlation between H-12 and 13-CH2OH indicated the trans-configuration of

237

C-11 and 13-CH2OH, which was confirmed by the 1H and 13C NMR similarity of C-11, C-12, C-13

238

and C-14 between compounds 1 and 7. As a result, the structure of 1 was elucidated and called as

239

fusarilactone A (Figure 2).

240

The theoretical electronic circular dichroism (ECD) spectra of 7S, 12R, 13R -1 and 7R, 12R, 13R -1

241

were also calculated and compared with the experimental values to determine the absolute



Page 12 of 30

242

configuration. As shown in Figure 3a, the experimental ECD spectrum was similar to both of the

243

calculated ECD spectra. Considering that the absolute configuration of C-7 cannot be determined by

244

the CD spectrum and ECD calculation, since carbon chain flexibility. The absolute configuration of 1

245

was determined to be 12R, 13R (Figure 3).

246 247

Insert Table 1 here

248 249

Insert Table 2 here

250 251


252 253


254 255

Compound 2 (fusarilactone B) was obtained as an amorphous white powder. Positive and

256

negative HRESIMS suggested its molecular formula to be C17H26O5, which gave five degrees of

257

unsaturation. The 1H, 13C NMR and DEPT spectra, in which two carbonyls, two ethylenic bonds, two

258

methyls and seven methylenes were found, indicated that the structure of 2 was similar to that of

259

hymeglusin (7), except for the absence of a methyl at C-7. In the 13C NMR of 2, the carbon signal at δ

260

19.5 assigned to 7-CH3 of hymeglusin (7) was not present. The 13C chemical shift of C-7 changed from

261

δC 30.8 to 28.9, and the 13C NMR signals assigned to C-6 and C-8 were also shifted from δC 48.8 to

262

40.7 and from δC 36.6 to 28.9, respectively. The structure of 2, including its relative configuration, was

263

further supported by the correlations found in 1H-1H COSY, HMBC and NOESY spectra (Figure 2).


Page 13 of 30


264

Compound 3 (fusarilactone C) was isolated as an amorphous white powder. The molecular

265

formula of C20H32O5, suggested by positive and negative HRESIMS, gave five degrees of unsaturation.

266

Two carbonyl carbons and two ethylenic bonds could be found in the low field region of the 13C NMR.

267

The high field region of the 1H, 13C NMR and DEPT spectra showed two oxygenated methines, three

268

methines without an oxygen-link, six methylenes and four methyls. Compound 3 also has a similar

269

structure to hymeglusin (7). However, the 1H-1H COSY correlations of H-12/H-13, H-13/H-14 and H-

270

14/H-15, 15-CH3, together with the HMBC correlations from H-12 to C-16, and from H-15 to C-16,

271

supported the presence of a six-membered lactone ring in 3, which is different from the β-lactone ring

272

in compound 1, 2 and 7. The relative configuration of 3 was further assigned using NOESY

273

correlations between H-12 (δH 4.55) and H-14 (δH 3.94), H-13e (δH 1.91), and between H-14 and H-

274

13e, 15-CH3 (δH 1.10), as well as the correlations induced by the protons at the opposite side of the six-

275

membered ring: between H-15 (δH 2.50) and H-13a (δH 1.70). The configuration of the double bonds

276

was similar with those in compounds 1 and 2, as shown in Figure 2.

277

Compound 4 (fusaridioic acid B) was isolated as an amorphous white powder with positive and

278

negative HRESIMS ion peaks at m/z 351.1768 [M+Na]+, and 327.1806 [M-H]-, respectively. Its

279

molecular weight is higher than that of 2 by 18 mass units, indicating that 4 could be the ring-opened

280

analog of 2. Most of the 1H, 13C NMR and DEPT data of 4 were similar to those of 2, except for the

281

carbon signals assigned to C-12, 13 and 14. In detail, the chemical shift of the 14-carbonyl was shifted

282

from δC 170.9 to 175.4, while that of C-12 was shifted from δC 74.8 to 69.3. 1H-1H COSY, HMBC and

283

NOESY spectra helped to confirm the structure of 4 as the ring-opened analog product of 2. As

284

described in a previous report 35, hydrogen bonds between the C-14 carbonyl oxygen and 12-OH, and

285

between the 14-carboxyl hydroxyl and 13-CH2OH helped to elucidate the relative configuration



Page 14 of 30

286

between C-12 and C-13 by using the coupling constant between H-12 and H-13. In compound 4, the

287

trans-coplanar position induced an H12-H13 coupling constant of 7–8 Hz (Figure 2). Thus, the coupling

288

splitting of H-13 in 4 presented as a td peak with J values of 7.0 Hz (× 2) and 4.6 Hz (× 1) (Table 1).

289

Compound 5 (fusaridioic acid C) was also isolated as an amorphous white powder. The molecular

290

formula of C18H30O6, was established by the HRESIMS positive and negative ion peaks at m/z

291

349.1985 [M+Na]+ and 325.2030 [M-H]-. The

292

carbons and four olefinic carbons belonging to two ethylenic bonds, in the sp2 low field region. The

293

sp3 high field region of the 1H NMR and 13C NMR spectra showed the existence of two methine, five

294

methylenes and four methyls. The 13C NMR data of the C-14 carbonyl was closer to that of compound

295

4, rather than compounds 1 and 2, indicating that compound 5 was also a ring-opened structure.

296

Comparing the NMR data with those of 4, signals of a methylene bearing an oxygen [(δC 60.3 and δH

297

3.49, 3.45), 13-CH2OH] disappeared and a set of methyl signal emerged at [δC 13.1 and δH 0.98 (3H,

298

d, J = 8.3 Hz), 11-CH3]. The 1H-1H COSY, HMBC and NOESY spectra confirmed the loss of the 13-

299

CH2OH and presence of the methyl at C-11, and the structure of 5 was elucidated as shown in Figure

300

2.

13C

NMR and DEPT spectra, showed two carbonyl

301

The known compounds 6–12 were characterized by HRESIMS and NMR spectra. Only the

302

NMR data of hymeglusin (7) 33-34, and L-660282 (11), 36-37 had been reported previously, and they were

303

identified by comparison with the known NMR data. Fusaridioic acid A (12) was a new compound,

304

recently reported by our group.

305

using 2D-NMR and identified as: (2E,4E,7S)-12,14-dihydroxy-3,5,7-trimethyl-tetradeca-2,4-dienoic

306

acid

307

trienedioic acid (8), (2E,5E)-3,5,7-trimethylocta-2,5-dienedioic acid (9), and (2E,4E)-3,5,7-

(6),

35

Chemical structures of other known compounds were elucidated

(2E,4E,7S,8E,12S,13S)-12-hydroxy-13-(hydroxymethyl)-3,5,7-trimethyltetradeca-2,4,8-


Page 15 of 30


308

trimethylocta-2,4-dienedioic acid (10). Their 1H-NMR data were given in Table S1 of the supporting

309

information, and their 13C-NMR data were listed in Table 2.

310 311

Evaluation of antifungal activity

312

The antifungal activity of the isolated compounds was evaluated using a paper disc inhibition

313

assay and ED50 detection. Compounds 1, 2, and 7 demonstrated significant activities against the tea

314

pathogenic fungus P. theae (Figure 4), however, none of the other compounds exhibited this effect.

315

The ED50 values of new compounds 1 and 2 and known compound 7 were 38.14 ± 1.67 µg/mL, 42.26

316

± 1.96 µg/ mL, 18.35 ± 1.27 µg/ mL to P. theae respectively, while the positive control hexaconazole

317

was 16.34 ± 1.25 µg/ mL. All 12 compounds were also tested for cytotoxic effects against mice 3T3-

318

L1 cells through CCK8 methods, but they did not show significant cytotoxicity (IC 50 > 100 µM).

319 320


321 322

Concerning the structure-activity relationship, the results showed that the β-lactone ring seemed

323

to be important for the anti-fungal inhibitory activity. Compounds 1, 2, and 7, which contain a β-

324

lactone ring, showed potent activity, while their corresponding open ring derivatives, compounds 8, 4,

325

11, and 12, showed no inhibitory activity against P. theae. The number of double bonds, and the methyl

326

side chain did not influence the activity. The fact that compound 9 and 10 did not show activity

327

indicated that the long main aliphatic chain may be necessary for the anti-fungal activity.

328

The most potent compounds in this study were hymeglusin (7) and its derivatives fusarilactone A

329

(1) and fusarilactone B (2). Hymeglusin (7) was reported as a 3-hydroxy-3-methylglutaryl-CoA



Page 16 of 30

330

(HMG-CoA) synthase specific inhibitor by covalently binding through the β-lactone to the active Cys

331

129 residue of the enzyme

332

distributed in eukaryotes (vertebrates, insects, plants and fungi), archaea, and certain bacteria 40-41 and

333

takes part in three metabolic pathways: synthesis and degradation of ketone bodies, valine, leucine and

334

isoleucine degradation, and butanoate metabolism.

335

potential for antiviral, 42-43 anti-bacterial, 39 cardiovascular protection. 44-45

336

38-39

and forming of a thioester adduct. HMG-CoA synthase is widely

42

Inhibition of HMG-CoA synthase has shown

It is known that fungisterol, mainly refers to ergosterol, is an important and specific component 46.

337

of the fungal cell membrane

338

mevalonate biosynthesis, farnesyl-PP biosynthesis and ergosterol biosynthesis. HMG-CoA synthase

339

(ERG13) is the second key enzyme that catalyzes the condensation of a third acetyl-CoA to

340

acetoacetyl-CoA to yield 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) during mevalonate

341

biosynthesis

342

cubense tropical race4 (Foc TR4), an important lethal pathogen of bananas, both the mRNA and the

343

protein of HMG-CoA synthase were significantly upregulated.

344

potential new target for the effective inhibition of Foc R4 early growth for controlling Fusarium wilt

345

of bananas. A previous report indicated that statins which target HMG-CoA reductase, the third key

346

enzyme of mevalonate biosynthesis, can strongly inhibit the growth of the Candida species and

347

Aspergillus fumigatus, while these effects could be reversed by supplementation of the

348

culture with ergosterol.

349

potent inhibitory activity against tea pathogenic fungus P. theae and significant HMG-CoA synthase

350

gene expression downregulation (Figure 5). These results implied that inhibition of HMG-CoA

351

synthase gene expression by compounds 1, 2 and 7 could inhibit the growth of tea pathogenic fungi

47.

The biosynthesis of ergosterol can be divided into three parts:

It was reported that during the conidial germination of Fusarium oxysporum f. sp.

49

48

This protein is thought to be a

In our tests, the new compounds 1, 2 and known compound 7 exhibited


Page 17 of 30

352


by interrupting ergosterol biosynthesis.

353


354 355 356

Acknowledgements

357

The project was supported by the COMRA Project of China (DY135-B2-16), National Basic

358

Research Program of China (973 Program) (No. 2015CB755901), the Xiamen Ocean Economic

359

Innovation and Development Demonstration Project (16PZP001SF16), Fujian Key Science and

360

Technology Program (No.2018N0017), Scientific Research Foundation of Third Institute of

361

Oceanography, SOA. (No.2016002, 2017002) and Xiamen Science and Technology Program

362

(No.3502Z20172009 and 3502Z20182029).

363

References

364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383

1.

Saravanakumar, D.; Vijayakumar, C.; Kumar, N.; Samiyappan, R., PGPR-induced defense responses in the tea plant

against blister blight disease. Crop Protection 2007, 26 (4), 556-565. 2.

Saha, D.; Dasgupta, S.; Saha, A., Antifungal activity of some plant extracts against fungal pathogens of tea (Camellia

sinensis). Pharmaceutical Biology 2005, 43 (1), 87-91. 3.

Gunasekera, T.; Paul, N.; Ayres, P., The effects of ultraviolet-B (UV-B: 290-320 nm) radiation on blister blight disease

of tea (Camellia sinensis). Plant Pathology 1997, 46 (2), 179-185. 4.

Ponmurugan, P.; Baby, U.; Rajkumar, R., Growth, photosynthetic and biochemical responses of tea cultivars infected

with various diseases. Photosynthetica 2007, 45 (1), 143-146. 5.

Sanjay, R.; Ponmurugan, P.; Baby, U., Evaluation of fungicides and biocontrol agents against grey blight disease of

tea in the field. Crop Protection 2008, 27 (3-5), 689-694. 6.

Sarkar, S.; Ajay, D.; Pradeepa, N.; Balamurugan, A.; Premkumar, R., Evaluation of chemical and neem pesticides

against Pestalotiopsis theae causing grey blight disease of tea. Annals of Plant Protection Sciences 2009, 17 (1), 252-253. 7.

Chakraborty, B.; Basu, P.; Das, R.; Saha, A.; Chakraborty, U., Detection of cross reactive antigens between

Pestalotiopsis theae and tea leaves and their cellular location. Annals of applied biology 1995, 127 (1), 11-21. 8.

Sanjay, R.; Ponmurugan, P.; Baby, U., Evaluation of fungicides and biocontrol agents against grey blight disease of

tea in the field. Crop Protection 2008, 27 (3), 689-694. 9.

Kim, G. H.; Lim, M.-T.; Hur, J.-S.; Yum, K.-J.; Koh, Y.-J., Biological control of tea anthracnose using an antagonistic

bacterium of Bacillus subtilis isolated from tea leaves. Plant Pathol J 2009, 25, 99-102. 10. Naglot, A.; Goswami, S.; Rahman, I.; Shrimali, D.; Yadav, K. K.; Gupta, V. K.; Rabha, A. J.; Gogoi, H.; Veer, V., Antagonistic potential of native Trichoderma viride strain against potent tea fungal pathogens in North East India. The



384 385

plant pathology journal 2015, 31 (3), 278.

386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427

anthropi BMO‐111 against blister blight disease of tea. Journal of applied microbiology 2013, 114 (1), 209-218.

Page 18 of 30

11. Sowndhararajan, K.; Marimuthu, S.; Manian, S., Biocontrol potential of phylloplane bacterium Ochrobactrum 12. Elango, V.; Manjukarunambika, K.; Ponmurugan, P.; Marimuthu, S., Evaluation of Streptomyces spp. for effective management of Poria hypolateritia causing red root-rot disease in tea plants. Biological Control 2015, 89, 75-83. 13. Marrone, P. G., Barriers to adoption of biological control agents and biological pesticides. Integrated pest management. Cambridge University Press, Cambridge 2009, 163-178. 14. Yoon, M.-Y.; Kim, Y. S.; Ryu, S. Y.; Choi, G. J.; Choi, Y. H.; Jang, K. S.; Cha, B.; Han, S.-S.; Kim, J.-C., In vitro and in vivo antifungal activities of decursin and decursinol angelate isolated from Angelica gigas against Magnaporthe oryzae, the causal agent of rice blast. Pesticide biochemistry and physiology 2011, 101 (2), 118-124. 15. Xiao, J.; Zhang, Q.; Gao, Y. Q.; Tang, J. J.; Zhang, A. L.; Gao, J. M., Secondary metabolites from the endophytic Botryosphaeria dothidea of Melia azedarach and their antifungal, antibacterial, antioxidant, and cytotoxic activities. J Agric Food Chem 2014, 62 (16), 3584-90. 16. Su, H.; Wang, W.; Bao, L.; Wang, S.; Cao, X., Synthesis and Evaluation of Essential Oil-Derived β-Methoxyacrylate Derivatives as High Potential Fungicides. Molecules 2017, 22 (5), 763. 17. Zhang, Z.-Z.; Li, Y.-B.; Qi, L.; Wan, X.-C., Antifungal activities of major tea leaf volatile constituents toward Colletorichum camelliae Massea. Journal of agricultural and food chemistry 2006, 54 (11), 3936-3940. 18. Kiuru, P.; DʼAuria, M. V.; Muller, C. D.; Tammela, P.; Vuorela, H.; Yli-Kauhaluoma, J., Exploring marine resources for bioactive compounds. Planta medica 2014, 80 (14), 1234-1246. 19. Gerwick, W. H.; Fenner, A. M., Drug discovery from marine microbes. Microbial ecology 2013, 65 (4), 800-806. 20. Li, W.; Tang, X.-X.; Yan, X.; Wu, Z.; Yi, Z.-W.; Fang, M.-J.; Su, X.; Qiu, Y.-K., A new macrolactin antibiotic from deep sea-derived bacteria Bacillus subtilis B5. Natural product research 2016, 30 (24), 2777-2782. 21. Alongi, D. M., Carbon cycling and storage in mangrove forests. Ann Rev Mar Sci 2014, 6, 195-219. 22. Chen, Q.; Zhao, Q.; Li, J.; Jian, S.; Ren, H., Mangrove succession enriches the sediment microbial community in South China. Sci Rep 2016, 6, 27468. 23. Wang, K. W.; Wang, S. W.; Wu, B.; Wei, J. G., Bioactive natural compounds from the mangrove endophytic fungi. Mini Rev Med Chem 2014, 14 (4), 370-91. 24. Xu, D. B.; Ye, W. W.; Han, Y.; Deng, Z. X.; Hong, K., Natural products from mangrove actinomycetes. Mar Drugs 2014, 12 (5), 2590-613. 25. Li, G.; Kusari, S.; Golz, C.; Strohmann, C.; Spiteller, M., Three cyclic pentapeptides and a cyclic lipopeptide produced by endophytic Fusarium decemcellulare LG53. RSC Advances 2016, 6 (59), 54092-54098. 26. Yang, S. X.; Gao, J. M.; Laatsch, H.; Tian, J. M.; Pescitelli, G., Absolute configuration of fusarone, a new azaphilone from the endophytic fungus Fusarium sp. isolated from Melia azedarach, and of related azaphilones. Chirality 2012, 24 (8), 621-7. 27. Yang, S. X.; Gao, J. M.; Zhang, Q.; Laatsch, H., Toxic polyketides produced by Fusarium sp., an endophytic fungus isolated from Melia azedarach. Bioorg Med Chem Lett 2011, 21 (6), 1887-9. 28. Yang, S. X.; Wang, H. P.; Gao, J. M.; Zhang, Q.; Laatsch, H.; Kuang, Y., Fusaroside, a unique glycolipid from Fusarium sp., an endophytic fungus isolated from Melia azedarach. Org Biomol Chem 2012, 10 (4), 819-24. 29. Yang, S.-X.; Xiao, J.; Laatsch, H.; Holstein, J. J.; Dittrich, B.; Zhang, Q.; Gao, J.-M., Fusarimine, a novel polyketide isoquinoline alkaloid, from the endophytic fungus Fusarium sp. LN12, isolated from Melia azedarach. Tetrahedron Letters 2012, 53 (47), 6372-6375. 30. Woo, J.-H.; Kitamura, E.; Myouga, H.; Kamei, Y., An antifungal protein from the marine bacterium Streptomyces sp. strain AP77 is specific for Pythium porphyrae, a causative agent of red rot disease in Porphyra spp. Applied and environmental microbiology 2002, 68 (6), 2666-2675.


Page 19 of 30

428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471


31. Kundu, A.; Saha, S.; Walia, S.; Shakil, N. A.; Kumar, J.; Annapurna, K., Cadinene sesquiterpenes from Eupatorium adenophorum and their antifungal activity. Journal of environmental science and health. Part. B, Pesticides, food contaminants, and agricultural wastes 2013, 48 (6), 516-22. 32. Alex, D.; Bach, T. J.; Chye, M.-L., Expression of Brassica juncea 3-hydroxy-3-methylglutaryl CoA synthase is developmentally regulated and stress-responsive. The Plant Journal 2001, 22 (5), 415-426. 33. Tomoda, H.; Kumagai, H.; Takahashi, Y.; Tanaka, Y.; Iwai, Y.; Omura, S., F-244 (1233A), a specific inhibitor of 3hydroxy-3-methylglutaryl coenzyme A synthase: taxonomy of producing strain, fermentation, isolation and biological properties. J. Antibiot. 1988, 41 (2), 247-9. 34. Kumagai, H.; Tomoda, H.; Omura, S., Biosynthesis of antibiotic 1233A (F-244) and preparation of [14C]1233A. J. Antibiot. 1992, 45 (4), 563-7. 35. Liu, S. Z.; Yan, X.; Tang, X. X.; Lin, J. G.; Qiu, Y. K., New Bis-Alkenoic Acid Derivatives from a Marine-Derived Fungus Fusarium solani H915. Mar Drugs 2018, 16 (12), 483. 36. Greenspan, M. D.; Yudkovitz, J. B.; Lo, C. Y. L.; Chen, J. S.; Alberts, A. W.; Hunt, V. M.; Chang, M. N.; Yang, S. S.; Thompson, K. L.; et, a., Inhibition of hydroxymethylglutaryl-coenzyme A synthase by L-659,699. Proc. Natl. Acad. Sci. U. S. A. 1987, 84 (21), 7488-92. 37. Aldridge, D. C.; Giles, D.; Turner, W. B., Antibiotic 1233A, a fungal β-lactone. J. Chem. Soc. C 1971,

(23), 3888-91.

38. Tomoda, H.; Ohbayashi, N.; Morikawa, Y.; Kumagai, H.; Ōmura, S., Binding site for fungal β-lactone hymeglusin on cytosolic 3-hydroxy-3-methylglutaryl coenzyme A synthase. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids 2004, 1636 (1), 22-28. 39. Skaff, D. A.; Ramyar, K. X.; McWhorter, W. J.; Barta, M. L.; Geisbrecht, B. V.; Miziorko, H. M., Biochemical and structural basis for inhibition of Enterococcus faecalis hydroxymethylglutaryl-CoA synthase, mvaS, by hymeglusin. Biochemistry 2012, 51 (23), 4713-4722. 40. Bahnson, B. J., An atomic-resolution mechanism of 3-hydroxy-3-methylglutaryl–CoA synthase. Proceedings of the National Academy of Sciences of the United States of America 2004, 101 (47), 16399-16400. 41. Bearfield, J.; Keeling, C.; Young, S.; Blomquist, G.; Tittiger, C., Isolation, endocrine regulation and mRNA distribution of the 3‐hydroxy‐3‐methylglutaryl coenzyme A synthase (HMG‐S) gene from the pine engraver, Ips pini (Coleoptera: Scolytidae). Insect molecular biology 2006, 15 (2), 187-195. 42. Liao, P.; Wang, H.; Hemmerlin, A.; Nagegowda, D. A.; Bach, T. J.; Wang, M.; Chye, M.-L., Past achievements, current status and future perspectives of studies on 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) in the mevalonate (MVA) pathway. Plant cell reports 2014, 33 (7), 1005-1022. 43. Peng, L. F.; Schaefer, E. A.; Maloof, N.; Skaff, A.; Berical, A.; Belon, C. A.; Heck, J. A.; Lin, W.; Frick, D. N.; Allen, T. M. J. J. o. I. D., Ceestatin, a novel small molecule inhibitor of hepatitis C virus replication, inhibits 3-hydroxy-3-methylglutarylcoenzyme A synthase. 2011, 204 (4), 609-616. 44. HEGARDT, F. G., Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a control enzyme in ketogenesis. Biochemical Journal 1999, 338 (3), 569-582. 45. Greenspan, M. D.; Bull, H.; Yudkovitz, J.; Hanf, D. P.; Alberts, A. W., Inhibition of 3-hydroxy-3-methylglutaryl-CoA synthase and cholesterol biosynthesis by β-lactone inhibitors and binding of these inhibitors to the enzyme. Biochemical Journal 1993, 289 (3), 889-895. 46. Prasad, R.; Shah, A. H.; Rawal, M. K. J. O. T. t. T. X., Antifungals: Mechanism of Action and Drug Resistance. 2016, 892, 327-349. 47. Hu, Z.; He, B.; Ma, L.; Sun, Y.; Niu, Y.; Zeng, B. J. I. J. o. M., Recent Advances in Ergosterol Biosynthesis and Regulation Mechanisms in Saccharomyces cerevisiae. 2017, 57 (3), 270. 48. Deng, G. M.; Yang, Q. S.; He, W. D.; Li, C. Y.; Yang, J.; Zuo, C. W.; Gao, J.; Sheng, O.; Lu, S. Y.; Zhang, S. J. A. M.; Biotechnology, Proteomic analysis of conidia germination in Fusarium oxysporum f. sp. cubense tropical race 4 reveals



472 473 474

Page 20 of 30

new targets in ergosterol biosynthesis pathway for controlling Fusarium wilt of banana. 2015, 99 (17), 7189-7207. 49. IG, M.; G, J.; T, S.; Letters, M. P. J. F. M., Growth inhibition of Candida species and Aspergillus fumigatus by statins. 2010, 262 (1), 9-13.

475


Page 21 of 30

477


Figure Legends

478 479

Figure 1. Structures of the isolated alkenoic acids from Fusarium solani H915.

480 481

Figure 2. Key 1H-1H COSY, HMBC and NOESY correlations of the new alkenoic acids.

482 483

Figure 3. Calculated and experimental electronic circular dichroism (ECD) spectra of the compound

484

1.

485 486

Figure 4. Inhibitory activity of compounds 1, 2, and 7 against tea pathogenic fungus Pestalotiopsis

487

theae.

488 489

Figure 5. Regulation effects of compounds 1–12 on the mRNA expression of HMG-CoA synthase.

490

The gene expression level was determined by real-time RT-PCR. DMSO (B) and abscisic acid (P)

491

were used as the blank and positive controls, respectively. GAPDH was used as reference gene.

492

Values represent the mean ± SD of three independent experiments. ***P < 0.001.

493



495

Table Captions

496 497

Table 1. 1H NMR data (600 MHz, DMSO-d6) of compounds 1–5.

498 499

Table 2. 13C NMR data (150 MHz, DMSO-d6) of compounds 1–12.

500 501


Page 22 of 30

Page 23 of 30


Figures

502

9

HOH2C 13 12

14

5

8

O

O

7

1

3 4

COOH 2

HOH2C 13 14

O

5 12

4

O

HOOC

2OH

5

1

3 4

OH

COOH 2

HOOC

14

7

12

14

O

5

12

4

O

1

3

COOH 2

13

14

CH2OH 12

OH

7

5

HOOC 8

503 504

6

4

1

3 4

COOH 2

14

HOH2C

13

7

1 2

9

7

1

3

COOH

13 CH

HOOC

14

2OH

COOH 2

5

1

3 4

12

4

COOH 2

7

COOH

5

HOOC

2

1

3

COOH

4

8

2

9

5

1

3 4

OH

1

3

6

8

7

5

OH

8

2

10

5

11

HOOC

1

3

O

5

7

7

16

15

5

3

OH

4

HOH2C 13

14

7

1 2

2

12

14

COOH 2

13

HO

O

1

13 CH

1

3

COOH 2

11

Figure 1

505


13 CH

HOOC

14

2OH

7

12

5 4

OH 12

1

3

COOH 2


Page 24 of 30

506

7

HOH2C

13

9

12

O 14

O

3

5

1

3

HOH2C

COOH

13

8

O 14

507 508

12

COOH

3 5

1

COOH

OH

HOH2C 11

H12 14 COOH 13 N OH H13

5

HO 14 15

12 1 6

O

O

2

CH2OH 14

5

12

1

O

1

HOOC 13

NOESY

HMBC

COSY

3

7

HOOC 14

12

OH 5

4

Figure 2

509 510


5

3

1

COOH

3

1

COOH

Page 25 of 30


Expt. Compd. 1 Calcd. 7S, 12R, 13R-1 Calcd. 7R, 12R, 13R-1

511 512

Figure 3

513 514 515 516



517

518 519

Figure 4


Page 26 of 30

Page 27 of 30


520 521

Figure 5

522 523



524

Tables

525

Table 1. Position

1

2

3

Page 28 of 30

4

5

7

2

5.55 br. s

5.56 br. s

5.57 br. s

5.51 br. s

5.58 br. s

5.57 br. s

4

5.71 br. s

5.74 br. s

5.73 br. s

5.68 br. s

5.73 br. s

5.73 br. s

2.06 dd (13.0,

2.07 dd (13.2, 6.1)

6a 6b

2.02 dd (12.8, 8.0) 1.98 dd (12.8, 2.33 spt (6.7)

8a

5.34 dd (15.4,

8b

6.8)

9a

5.40 dt (15.4,

9b

6.2)

10b 11a 11b 12 13a 13b

2.05 br. t (7.4)

7.5)

7

10a

2.07 dd (12.9,

2.04 m 1.85 dt (14.0, 6.9) 1.78 dt (14.0, 6.8) 4.52 m

6.0)

1.98 br. t

6.2)

1.83 dd (12.9,

(7.4)

1.83 dd (13.0,

8.5) 1.26 m

1.65 m 1.24 m

1.24 m

1.08 m

1.41 m

1.28 m

1.33 m

1.34 m

1.79 m

1.60 m

8.2) 1.33 m 1.17 m 1.17 m

1.81 m

1.64 m

1.65 m

1.26 m

1.27 m

1.07 m

1.10 m

1.37 m 1.26 m

1.32 m

1.33 m

1.35 m

1.37 m

1.18 m

1.27 m

1.29 m

1.32 m

1.81 m 2.34 m

1.73 m 4.53 td (6.7, 4.2)

1.52 m

1.23 m

4.55 m

3.53 m

1.74 m 3.57 br. t

4.53 td (6.7,

(5.9)

4.3)

1.91 dt (13.7,

3.51, covered by

3.51 br. dd

3.5)

2.30 td (7.0,

residual H2O

(7.8, 3.9)

1.70 br.t

4.6)

signal

1.26 m

3.50 br. dd (7.7, 3.9)

(12.7)

14

3.93 m

15

2.50 m

3-CH3

2.13 br. s

2.14 br. s

2.16 br. s

2.08 br. s

2.16 br. s

2.16 d (1.1)

5-CH3

1.76 br. s

1.78 br. s

1.77 br. s

1.71 br. s

1.76 d (0.7)

1.76 d (1.1)

7-CH3

0.91 d (6.6)

0.80 d (6.6)

0.80 d (6.6)

0.81 d (6.4)

11-CH3

0.98 d (7.0)

15-CH3

1.10 d (7.0)

13-

3.71 br. dd

3.71 dd (11.7,

3.49 br. d

3.72 dd (11.7,

CH2OH

(11.6, 3.9)

4.2)

(9.9)

4.2)

3.61 dd (11.6,

3.62 dd (11.7,

3.5)

3.5)

　

　

3.45 dd (9.9, 5.3)

526


　

3.62 dd (11.7, 3.3)

Page 29 of 30

527


Table 2. Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 3-CH3 5-CH3 7-CH3 11-CH3 15-CH3 13-CH2OH

1 168.6 119.2 152.4 129.9 140.4 48.5 34.6 136.8 127.1 28 33.6 74.3 58.7 170.8

2 168.7 119.3 152.4 128.3 142.1 40.7 28.9 28.9 27.6 24.8 33.5 74.8 58.7 170.9

19.5 18.7 20.4

19.5 18.5

56.6

56.7

3 168.3 118.8 152.8 129.7 141.1 48.8 30.8 36.6 26.6 25.1 35.9 76.3 36.5 66.3 41.6 174 19.5 18.6 19.7 13.3 　

528 529


4 168.2 118.8 152.9 128.3 142.3 40.7 27.6 29.0 29.3 25.6 35.1 69.3 55.2 175.4

5 168.2 118.7 153.0 129.6 141.2 48.9 30.7 36.6 25.8 33.6 46.3 71.9 26.8 176.8

7 168.1 118.6 153.1 129.6 141.3 48.8 30.7 36.6 26.5 25.1 33.6 74.8 58.8 170.8

19.5 18.6

19.5 18.6 19.7 13.1

19.7 18.6 19.5

60.3

　

56.6


530

TOC

531 532

Table of Contents

HOH2C O

HOH2C

COOH O

O

COOH O

1

2 HOH2C O

Page 30 of 30

COOH O 7

533 534 535


New Î²-Lactone with Tea Pathogenic Fungus ... - ACS Publications

Recommend Documents