Isolation, Identification, and Activity Evaluation of Chemical

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Bioactive Constituents, Metabolites, and Functions

Isolation, Identification, and Activity Evaluation of Chemical Constituents from the Soil Fungus Fusarium avenaceum SF-1502 and Endophytic Fungus Fusarium proliferatum AF-04 Chun-Xiao Jiang, Jie Li, Jun-Min Zhang, Xiao-Jie Jin, Bo Yu, Jianguo Fang, and Quan-Xiang Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05576 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 28, 2019

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Journal of Agricultural and Food Chemistry

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Isolation, Identification, and Activity Evaluation of Chemical

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Constituents from the Soil Fungus Fusarium avenaceum SF-

3

1502 and Endophytic Fungus Fusarium proliferatum AF-04

4 5

Chun-Xiao Jiang,†,⊥,Δ Jie Li,†,Δ Jun-Min Zhang,‡ Xiao-Jie Jin,§ Bo Yu,† Jian-Guo

6

Fang,† and Quan-Xiang Wu*,†

7 8

† State

9

and Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s

Key Laboratory of Applied Organic Chemistry, College of Chemistry

10

Republic of China

11

‡

12

Republic of China

13

§

14

730000, People’s Republic of China

15

School of Pharmacy, Lanzhou University, Lanzhou 730000, People’s

⊥

College of Pharmacy, Gansu University of Chinese Medicine, Lanzhou

School of Advanced Study, Taizhou University, Taizhou 318000, People’s

16

Republic of China

17

Δ

These authors have the equal contribution in this study.

1

ACS Paragon Plus Environment


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ABSTRACT:

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Fusarium, a large genus of filamentous fungi, is widely distributed in soil and

20

plants. Fusarium is a prolific source of novel chemical constituents with

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various bioactivities. In search for antibiotics from soil and endophytic fungi,

22

the secondary metabolites of Fusarium avenaceum SF-1502 and Fusarium

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proliferatum AF-04 were investigated. An alkaloid (1), a depsipeptide (6), and

24

five sesquiterpenoids (7–11) were isolated from the extracts of the soil fungus

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F. avenaceum SF-1502. Three alkaloids (2–4), a depsipeptide (5), three

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sesquiterpenoids (9, 11, and 12), a diterpene (13), and four 1,4-

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naphthoquinones (14–17) were also separated from the extract of the green

28

Chinese onion derived fungus F. proliferatum AF-04. Fusaravenin (1)

29

represents the first example of a natural naphthoisoxazole type zwitter-ionic

30

alkaloid, a naphthoisoxazole formic acid connected with a morpholino carbon

31

skeleton. Cyclonerotriol B (7) is a new cyclonerane sesquiterpene. Another

32

new sesquiterpene, 3-hydroxy--acorenol (12), possesses an acorane

33

framework. The known compounds 9 and 11 were found from both fungi. The

34

structures of the new compounds were determined via extensive HR-ESI-MS

35

and comparison between experimental and calculated NMR results. The

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biological properties of 1–5 and 7–17 were evaluated against eight

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anthropogenic bacteria, while 1 and 7–11 were also screened for inhibitory

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effects against four plant pathogen bacteria. The known compounds 8, 9, and

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14–17 showed potent antibacterial activities toward some of the tested 2


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anthropogenic bacteria.

41 42

KEYWORDS:

43

Fusarium

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naphthoisoxazole amide, alkaloid, sesquiterpenoid, 1,4-naphthoquinone,

45

depsipeptide, antibacterial activity

avenaceum

SF-1502,

Fusarium

3


proliferatum

AF-04,


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INTRODUCTION

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Fusarium, a large genus of filamentous fungi comprising up to 1000 species,

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are relatively abundant members of the soil microbial family and associated

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with plants found in many tropical and temperate regions. Despite the fact that

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most species are apparently harmless, many of them may cause a range of

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opportunistic infections in humans, occurring in the nails, cornea, entire body,

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and bloodstream.1 Some Fusarium species are among the most important

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fungal pathogens of plants that cause crop diseases, including ear rot in maize

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and head blight in wheat, thus contributing to significant crop yield reduction.2

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Many animal diseases, such as feed refusal, weight loss, death of cattle and

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sheep as well as chicken mortality, are also caused by these fungi.3 They can

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produce different kinds of mycotoxins as secondary metabolites, such as

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fumonisins,

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beauvericin, and enniatins, containing plentiful structures including alkaloids,

60

polyketides, and terpenoids. The optimal conditions for toxin production are low

61

temperatures (5–8 oC), darkness, and a lightly acidic environment (pH around

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5.6).4,5 These compounds are of interest due to the broad spectrum of their

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biological activities, e.g., antibacterial, antifungal, insecticidal, and cytotoxic

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properties.6,7 Fusarium avenaceum AS 3.4594 and Fusarium proliferatum have

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been used as biotransformation fungi for structural modification of complicated

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natural products.8‒10 Polyketide derivates (in malt-extract liquid medium, F.

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proliferatum ZS07 isolated from the gut of long-horned grasshoppers),11

trichothecenes,

fusaric

acid,

moniliformin,

4


fusaproliferin,

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sesterterpene (in yellow corn kernels, F. proliferatum isolated from ear rot

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infected corn),12 and fumonisin derivatives (in corn cultures, F. proliferatum from

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Fusarium Research Center, the Pennsylvania State University)13 were isolated

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from F. proliferatum. The secondary metabolites of F. proliferatum in rice solid

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medium and F. avenaceum have not been reported.14

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Stimulated by the fruitful natural products chemistry of terrestrial alpine

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region fungi, our group has been engaged in the discovery of novel and diverse

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antimicrobial compounds from soil and endophytic fungi.15‒17 Recently, the

76

crude extracts (in potato sucrose liquid medium) of Fusarium avenaceum SF-

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1502 isolated from the root tip soil of Chlorophytum comosum, and the extract

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(in rice solid medium) of Fusarium proliferatum AF-04 separated from the green

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Chinese onion were analyzed. HPLC analysis and bioassay-guided

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separations were customized for the isolation of a series of alkaloids,

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terpenoids, and naphthoquinones. Herein, we report the details of isolation,

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structural elucidation by HR-ESI-MS, NMR spectral interpretation, and quantum

83

mechanical calculation, and biological evaluation of secondary metabolites of

84

these two fungi.

85 86

■ MATERIALS AND METHODS

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General. HR-ESI-MS spectra were obtained using an orbitrap Elite

88

spectrometer (Thermo, USA). UV spectra were obtained using an UV-

89

2800SPC UV/vis spectrometer (Shanghai, China). IR spectra (KBr pellets) 5



90

were recorded on a NEXUS 670 spectrometer (Nicolet, USA). NMR spectra

91

were measured on an INOVA-600 (Varian, USA) and an AVANCE III-400

92

(Bruker, Switzerland) spectrometers (TMS as internal standard,  in ppm, J in

93

Hz). The CD3OD, (CD3)2CO, and CDCl3 were used as the deuterated

94

solvents. Optical rotations were obtained using a Rudolph Research Analytical

95

(USA) A21202-T polarimeter. Semi-preparative HPLC were carried out on a

96

1525 liquid chromatograph (Waters, USA) with 2489 UV/Visible (254 nm)

97

(Waters, USA) peak detections using a Synergi Hydro-Rp 80A column

98

(Phenomenex, USA), C18, 4 m, 250 × 10 mm, and a CN-ES column (ACE,

99

England), CN-ES, 5 m, 250 × 10 mm. Column chromatography (CC) was

100

performed on Sephadex LH-20 (Mitsubishi), MCI resin (120 m, Mitsubishi),

101

and silica gel (200–300 mesh) (Qingdao Haiyang Chemical Group

102

Corporation, Qingdao, China). The HP20 resin (Mitsubishi, Japan) was used

103

to extract the broth. Thin-layer chromatography (TLC) was carried on

104

precoated silica gel plates (GF254, 10–40 m, Qingdao, China). Analytical

105

grade solvents were used for CC and TLC, and chromatographic grade

106

solvents were used on HPLC.

107

Fungal Material. The root tip soil of C. comosum (a decorative plant,

108

purchased from Flowers and Plants Market in Lanzhou, China) was used to

109

isolate the fungal strains in our laboratory. F. avenaceum SF-1502 was found

110

on Sep. 20, 2015, which was identified in accordance with morphological traits

111

and its 18S rRNA sequence (GenBank accession no. MG520365). The 6


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endophytic fungus F. proliferatum AF-04 (GenBank accession no. MF426031)

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was isolated from the green Chinese onion. The strains were stored in the

114

cryogenic refrigerator (– 80°C).

115

Extraction and Isolation. The strain (60 L, SF-1502) was grown in a liquid

116

medium with supplements (potato 120 g, sucrose 12 g, in 600 mL of purified

117

H2O in 1L Erlenmeyer flasks) with shaking (160 rpm) in two oscillation

118

incubators (26 °C, 21 d). The mycelia and broth were centrifuged at 4000

119

r/min for 30 min. The extract of the mycelia (4.1 g) was obtained by extracting

120

with EtOAc in an ultrasonic instrument (3 × 2.0 L × 30 min, 30 oC). The broth

121

was extracted with HP20 resin. The HP20 resin (4.8 L) was washed with, in

122

turn, H2O (10.0 L), 50% MeOH/H2O (10.0 L), MeOH (15.0 L), and i-PrOH

123

(15.0 L). The MeOH extract (2.9 g) was yielded. The extracts of the mycelia

124

and broth were combined to yield the final extract (7.0 g) after TLC and

125

HPLC-UV analyses.

126

This extract (7.0 g) was subjected to silica gel CC (260.0 × 42.0 mm) eluted

127

with gradient CH2Cl2/MeOH (1:0 (3.5 L), 40:1 (3.5 L), 20:1 (3.8 L), 10:1 (3.2

128

L), 8:1 (2.8 L), 5:1 (3.0 L), 2:1 (2.5 L), 1:1 (2.5 L), and 0:1 (3.0 L)) to give nine

129

fractions (F1–F9). Fractions F2, F4, and F6, composed mains of alkaloids and

130

sesquiterpenoids, were further chromatographed on different CC. F2 (2.1 g)

131

was separated via CC (235.0 × 35.0 mm, silica gel, CH2Cl2/acetone 20:1, 3.7

132

L) to yield eight sub-fractions (F2-1–F2-8). F2-1 (7.0 mg) was eluted via CC

133

(100.0 × 8.0 mm, silica gel, petroleum ether (PE)/acetone 10:1 (120.0 mL), 7



134

CH2Cl2/EtOAc 8:1 (100.0 mL), and PE/EtOAc 5:1 (100.0 mL)) to obtain 10

135

(1.5 mg). F2-5 (64.6 mg) was purified through repeated silica gel CC (160.0 ×

136

12.0 mm) with gradient PE/acetone (5:1 (250.0 mL), 4:1 (250.0 mL), and 3:1

137

(200.0 mL)) and PE/EtOAc (4:1 (150.0 mL), 2:1 (120.0 mL), and 1:1 (85.0

138

mL)) to yield 8 (15.6 mg). F2-6 (141.2 mg) was subjected to CC (145.0 × 15.0

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mm, silica gel) using gradient PE/acetone (5:1 (320.0 mL), 4:1 (220.0 mL),

140

and 3:1 (230.0 mL)), PE/EtOAc (4:1 (220.0 mL), 2:1 (260.0 mL), and 1:1

141

(260.0 mL)), and CH2Cl2/EtOAc (3:1 (220.0 mL) and 1:1 (180.0 mL)) to get 9

142

(1.5 mg). F2-7 (152.9 mg) was separated via CC (155.0 × 15.0 mm, silica gel,

143

PE/EtOAc 5:1, 550.0 mL; 1800.0 × 25.0 mm, Sephadex LH-20, CH2Cl2/MeOH

144

2:1, 650.0 mL) to find 6 (130.0 mg). F4 (401.2 mg) was subjected to CC

145

(100.0 × 30.0 mm, silica gel, CH2Cl2/MeOH 10:1, 720.0 mL) to yield three

146

sub-fractions (F4-1–F4-3). F4-2 (155.5 mg) was divided into six sub-fractions

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(F4-2-1–F4-2-6) using silica gel CC (140.0 × 15.0 mm) with PE/EtOAc at 1:2

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(550.0 mL). F4-2-5 (23.0 mg) was eluted via CC (130.0 × 10.0 mm, silica gel,

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PE/acetone 3:1 (220.0 mL) and CH2Cl2/EtOAc 1:2 (150.0 mL)) to obtain 11

150

(1.0 mg). F4-2-6 (106.2 mg) was separated via CC (145.0 × 13.0 mm, silica

151

gel, CH2Cl2/acetone 2:1 (230.0 mL) and 1:1 (200.0 mL)) to obtain 7 (2.5 mg).

152

F6 (160.4 mg) was divided into three sub-fractions. F6-1 (70.5 mg) was

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eluted, in turn, via silica gel CC (130.0 × 12.0 mm, CH2Cl2/acetone 3:1, 330.0

154

mL), Sephadex LH-20 CC (1600.0 × 25.0 mm, MeOH, 550 mL), and HPLC

155

(Synergi Hydro-Rp 80A, 60:40 MeOH/H2O (0.1% formic acid), 2 mL/min)) to 8


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obtain 1 (1.8 mg, tR = 13.2 min). A large-scale culture (104 flasks, AF-04) was grown on rice solid medium

158

(120 g rice in 150 mL purified H2O, 1L Erlenmeyer flasks) for 28 d in two

159

constant temperature oscillation incubators (26 °C). The medium with the

160

strain was extracted with EtOAc in an ultrasonic instrument (6 × 12 L × 30

161

min) at room temperature to yield an extract (88.6 g).

162

The crude extract was divided into seven fractions (F10–F16) via silica gel

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CC (150.0 × 120.0 mm) eluting with gradient PE/acetone (20:1 (5.2 L), 10:1

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(7.0 L), 5:1 (5.0 L), 3:1 (5.5 L), 1:1 (7.0 L), and 0:1 (10.0 L)). F11 (998.0 mg)

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was separated via CC (150.0 × 35.0 mm, silica gel, PE/CH2Cl2/acetone

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20:10:1 (1.5 L) and CH2Cl2/MeOH 100:1 (1.0 L)) to yield 4 (12.6 mg) and 9

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(5.3 mg). F12 (684.4 mg) was eluted via silica gel CC (150.0 × 25.0 mm) with

168

gradient PE/EtOAc at 3:1 (800.0 mL), 2:1 (600.0 mL), and 1:1 (500.0 mL) to

169

obtain 13 (26.1 mg) and F12-1. F12-1 (159.2 mg) was separated via silica gel

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CC (150.0 × 18.0 mm), in turn, with CH2Cl2/acetone 100:1 (303.0 mL),

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CH2Cl2/EtOAc 10:1 (220.0 mL), and CH2Cl2/MeOH 50:1 (255 mL), to yield 12

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(7.6 mg). F13 (6.9133 g) was divided into eight sub-fractions via CC (240.0 ×

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42.0 mm, silica gel, CH2Cl2/acetone 30:1 (3.0 L), 20:1 (3.2 L), and 10:1 (3.5

174

L)). Sub-fraction F13-3 (665.1 mg) was underwent further chromatography

175

(180.0 × 25.0 mm, silica gel, PE/acetone 4:1 (500.0 mL) and CH2Cl2/MeOH

176

80:1 (405.0 mL), separately) to yield 5 (82.1 mg). F13-5 (1.4500 g) was eluted

177

via CC (200.0 × 35.0 mm, silica gel, PE/acetone 3:1 (1.2 L), CH2Cl2/EtOAc 9



178

(0.2% acetic acid) 10:1 (1.2 L), and PE/EtOAc 1:1 (800.0 mL), separately) to

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obtain 2 (12.1 mg). F13-7 (557.6 mg) was separated via silica gel CC (200.0 ×

180

25.0 mm) eluting with PE/acetone 3:1 (800.0 mL) to yield 11 (1.1 mg). F13-8

181

(2.6585 g) was eluted via silica gel CC (240.0 × 38.0 mm) with

182

CH2Cl2/acetone 30:1 (5.2 L) and PE/EtOAc at 6:1 (6.0 L) to yield 3 (4.5 mg).

183

F14 (5.3000 g) was divided into nine sub-fractions (F14-1–F14-9) via CC

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(210.0 × 42.0 mm, silica gel, CH2Cl2/acetone 10:1 (3.0 L) and 7:1 (4.0 L)).

185

F14-2 (79.1 mg) was separated via CC (170.0 × 15.0 mm, silica gel,

186

CH2Cl2/EtOAc at 3:1 (280.0 mL); and 1600.0 × 25.0 mm, Sephadex LH-20,

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MeOH (1.0 L) to get 16 (5.9 mg) and 17 (1.7 mg). F14-5 (155.4 mg) was

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further eluted via CC (180.0 × 18.0 mm, silica gel, CH2Cl2/EtOAc/MeOH

189

40:1:1 (420.0 mL), CH2Cl2/EtOAc 3:1 (400.0 mL); and 1600.0 × 25.0 mm,

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Sephadex LH-20, MeOH (1.5 L) to obtain 14 (5.6 mg) and 15 (14.7 mg).

191

In order to contrast the features of the secondary metabolites from F.

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avenaceum SF-1502 and F. proliferatum AF-04, HPLC analysis (Figure 1) of

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the extracts (the EtOAc part of the mycelia and the MeOH part of the broth)

194

from SF-1502 (cultured in potato sucrose liquid medium) and the extract (the

195

EtOAc part) from AF-04 (cultured in rice solid medium) was carried out

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employing a CN-ES column. Elutions of the extracts were carried out using an

197

isocratic mobile phase (60% A 20 min and 75% A 30 min) consisting of (A)

198

MeOH and (B) 0.1% formic acid in H2O at 25 °C. The flow rate was 1 mL/min.

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The UV peak detection wave length was 254 nm. 10


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200

Fusavenin, 1: light yellow powder; [α] D + 21.4 (c 0.14, MeOH); IR (KBr)

201

max 3433, 2955, 2926, 2852, 2371, 1641, 1459, 1363, 1299, 1247, 1119, 1018,

202

779, 711, 618, 568 cm−1; UV (MeOH) max (log ) = 324 (3.19), 372 (2.97) nm;

203

HR-ESI-MS m/z 327.1336 [M + H]+, calcd for C18H19N2O4, 327.1339; 1H NMR

204

(600 MHz, TMS, CD3OD) and 13C NMR (150 MHz, TMS, CD3OD) see Table 2.

205

Cyclonerotriol B, 7: colorless oil; [α]26 D + 6.7 (c 0.15, MeOH); IR (KBr) max

206

3400, 2966, 2930, 1726, 1656, 1460, 1380, 1261, 1152, 1098, 1024, 976, 919,

207

885, 802 cm−1；HR-ESI-MS m/z 279.1925 [M + Na]+, calcd for C15H28O3Na

208

279.1931; 1H NMR (600 MHz, TMS, (CD3)2CO) and 13C NMR (150 MHz, TMS,

209

(CD3)2CO ) see Table 3.

210

3β-Hydroxy-β-acorenol, 12: colorless oil; [α]25 D - 50.0 (c 0.1, MeOH); HR-

211

ESI-MS m/z 261.1828 [M + Na]+, calcd for C15H26O2Na 261.1825; 1H NMR (400

212

MHz, TMS, CDCl3) and 13C NMR (100 MHz, TMS, CDCl3) see Table 3.

213

Antibacterial Bioassays. The bioactivities of compounds 1–5 and 7–17

214

were screened using the micro-broth dilution method according to the

215

National Committee for Clinical Laboratory Standards in 96-well culture plates

216

with Mueller-Hinton (MH) broth.18 The standard anthropogenic bacterial

217

strains Bacillus megaterium, Bacillus subtilis, Clostridium perfringens,

218

Escherichia coli, and a strain of MRSA (methicillin-resistant Staphylococcus

219

aureus), Mu50, Newman WT, and RN4220, were obtained from the Institute of

220

Microbiology, School of Life Sciences, Lanzhou University. The standard plant

221

pathogenic bacteria Erwinia carotovora, Pseudomonas syringae, Ralstonia 11



222

solanacearum, and Xanthomonas oxyzae, were provided by the Institute of

223

Pesticides, Northwest Agriculture and Forestry University. The antibacterial

224

drugs, acheomycin, ampicillin, erythromycin, levofloxacin, and streptomycin

225

(≥95% purity, Sigma, China), were used as the positive controls. DMSO used

226

for diluting the compounds served as the blank control. The tested bacteria

227

were incubated in the MH broth with shaking (190 rpm) in a constant

228

temperature oscillation incubator (30 °C, 12 h). The bacterial concentration

229

was adjusted to 1×105–1×106 cfu/mL with MH broth. The bacterial

230

suspension, 50 L per hole, was added into 96-well culture plate. The initial

231

compound concentration in DMSO is 200 g/mL. 50 L Inception compound

232

solution was added in the first empty space and mixed evenly. Using the

233

double dilution method, 50 L solution was transferred from the first hole to

234

the second hole and then shaken up as mixture uniform. The repetitive

235

operation was carried out from the second hole to the third one. Then the

236

analogy was repeated to the twelfth hole. The paralleling operations were

237

performed three times. After incubation (30 °C) for 24 h, the minimal inhibitory

238

concentrations (MIC) were determined.

239

■ RESULTS AND DISCUSSION

240

The HPLC results of the extracts from SF-1502 and AF-04 are show in Figure

241

1. The SF-1502 extract has similar absorption peaks as the AF-04 extract at the

242

retention time of 9–17 min. But there is not obvious peak of SF-1502 extract,

243

which was different with the AF-04 extract at the retention time of 17–50 min 12


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(Figure 1). The crude extracts were subject to biological evaluation toward eight

245

anthropogenic bacteria and four plant pathogenic bacteria. They showed

246

moderate antibacterial activities against some of the tested anthropogenic

247

bacteria. Then the isolation of the secondary metabolites from these two fungi

248

was initiated. An alkaloid, fusaravenin (1), the first natural naphthoisoxazole

249

amide derivative, a depsipeptide (6), and five sesquiterpenoids (7–11),

250

cyclonerotriol B (7), a new sesquiterpene with the cyclonerane framework, were

251

isolated from the crude extracts of the EtOAc part of the mycelia and the MeOH-

252

soluble part of the broth of the soil fungus F. avenaceum SF-1502. Three

253

alkaloids (2–4), a depsipeptide (5), three sesquiterpenoids (9, 11, and 12), 3-

254

hydroxy--acorenol (12), another new sesquiterpene containing the acorane

255

skeleton, a diterpene (13), and four 1,4-naphthoquinones (14–17), were

256

obtained from the crude extract of the endophytic fungus F. proliferatum AF-04.

257

Two sesquiterpenoids 9 and 11 were found from both fungi (Figure 2).

258

Compound 1 was obtained as a light brownish solid. High-resolution ESI-

259

MS yielded m/z 327.1336 for the [M + H]+ ion, which was consistent with a

260

molecular formula of C18H18N2O4 and accounts for 11 degrees of unsaturation.

261

Absorption bands at 1641 and 1459 cm−1 in the IR spectrum indicate the

262

existence of carbonyl and olefinic groups, and 3433 cm−1 implies the presence

263

of exchangeable protons.

264 265

The 13C NMR spectrum (Table 2) revealed 16 carbon signals. 11 signals, two connected with electrophilic groups, possessed chemical shifts in the 13



266

aromatic range of the spectrum; one signal represented a carboxyl (δC 166.3,

267

C-14); and four signals, of which two presented higher intensities, were shown

268

as sp3 hybridization carbon atoms. Following an inspection of the HSQC, 1H

269

NMR (Table 2), and DEPT-135 spectra, it was evident that each of the signals

270

at δC 54.8 and 67.4 represented two magnetically equivalent methylenes

271

(CH2-1′/CH2-4′ and CH2-2′/CH2-3′, respectively). With the help of HSQC

272

correlations, the 1H NMR data presented as six methylenes (δH 4.36, H2-12;

273

2.84, H2-13; 2.74, H2-1′ and H2-4′; and 3.70, H2-2′ and H2-3′) connected to

274

either the aromatic ring or substituted heteroatoms recognized according to

275

their chemical shifts, and five aromatic methines (δH 8.55, d, J = 7.8 Hz, H-2;

276

δH 7.73, t, J = 7.8 Hz, H-3; δH 8.62, d, J = 8.4 Hz, H-4; δH 8.42, d, J = 8.4 Hz,

277

H-8; δH 7.07, d, J = 7.8 Hz, H-9) identified as tri- and tetrasubstituted benzene

278

protons confirmed by typical coupling constants (J = 7.8 or 8.4 Hz) and 1H-1H

279

COSY correlations (H-2/H-3/H-4 and H-8/H-9) (Figure 3).

280

According to the HMBC correlations (Figure 3) from the methine (H-2) of

281

the trisubstituted benzene to C-10 (δC 131.3), H-3 to C-5 (δC 124.4), and from

282

the methine (H-8) of the tetrasubstituted benzene to C-10, and H-9 to C-5, it

283

was proved that tri- and tetrasubstituted benzenes constituted a naphthalene

284

nucleus. The existence of a carboxyl, isoxazole, and ethyl chain in 1

285

unambiguously confirmed by HMBC correlations, from H-2 to C-14 of the

286

carboxyl, and from H-8 to C-11 (δC 165.7) of the isoxazol, H-12 to C-11 and C-

287

13 (δC 57.4), and H-13 to C-12 (δC 37.5) of the ethyl chain. HMBC correlations 14


Page 14 of 36

Page 15 of 36


288

from H-2′ to C-1′ and C-3′, H-3′ to C-2′ and C-4′ gave a morpholine fragment

289

consistent with the chemical shifts.19 The morpholine was jointed by the

290

nitrogen atom with C-13 of the ethyl chain determined by HMBC correlations

291

from H-13 to C-1′ and C-4′.

292

In all, besides a naphthalene ring, a morpholine ring, and the carboxyl

293

group, the two degrees of unsaturation remaining in compound 1 were

294

ascribed to ring structures. Extensive 1D & 2D NMR and HR-ESI-MS

295

measurements allowed the identification of an isoxazole ring. It is shown that

296

C-6 (δC 162.9) and C-11 (δC 165.7), aromatic carbons, are connected to

297

oxygen or nitrogen atoms by their chemical shifts and the requirements of the

298

molecular formula. There should be a double bond between C-11 and a

299

heteroatom. This heteroatom should also be jointed to another heteroatom to

300

form an isoxazole ring, based upon the degrees of unsaturation and the

301

chemical shift of C-6. 20,21

302

In order to unambiguously determine the isoxazole ring, we also

303

performed quantum mechanical calculations of the 13C NMR chemical shifts of

304

1, using GIAO method at the B3LYP/6-311G(d,p) level. The calculated data

305

agreed well with the experimental (Table 2). This compound was named

306

fusaravenin (1), which is the first example of a natural naphthoisoxazole type

307

zwitterionic alkaloid, a new naphthoisoxazole formic acid connected with a

308

morpholino carbon skeleton.20,21 Owing to the presence of a basic nitrogen

309

and an acidic carboxyl group, fusaravenin should be as a zwitter-ion, 15



Page 16 of 36

310

corresponding to a naphthoisoxazole alkaloid with the positive morpholine ion

311

and the negative carboxylate ion.

312

Compound 7 was isolated as a colorless oil. Its molecular formula was

313

determined from the (+)-HR-ESI-MS ion at m/z 279.1925 [M + Na]+, calculated

314

for C15H28O3Na (279.1931), revealing two degrees of unsaturation. The infrared

315

spectrum showed strong absorption bands for a hydroxyl moiety (at 3400 cm−1)

316

and a carbon-carbon double bond (at 1656 and 1460 cm−1). The 1H NMR,

317

NMR (Table 3), and DEPT-135 spectra in combination with the HSQC spectrum,

318

exhibited resonances for five methyls, of which one was secondary (δH 1.05, δC

319

15.4, CH3-1) and four were tertiary (δH 1.33, δC 30.5, CH3-12; δH 1.26, δC 26.6,

320

CH3-13; δH 1.14, δC 26.0, CH3-14; δH 1.33, δC 30.5, CH3-15), three methylenes,

321

four methines of which two were olefinic (δH 5.72, δC 123.1, CH-9; δH 5.63,

322

δC142.7, CH-10), and three oxygenated quaternary carbons (δC 80.6, C-3; 74.5,

323

C-7; 70.2, C-11). Three hydroxyl proton signals at δH 3.41 (OH-11), 2.92 (OH-

324

7) and 2.85 (OH-3) were shown in 1H NMR spectrum.

325

13C

Interpretation of the 1H-1H COSY and HMBC spectra (Figure 4) led to the 1H-1H

326

construction of the planar structure of compound 7. The

327

correlations of H3-1/H-2/H-6/H2-5/H2-4 and H2-8/H-9/H-10 established the

328

linkage of CH3-1−CH-2−CH-6−CH2-5−CH2-4 and CH2-8−CH-9=CH-10. The

329

key HMBC correlations from H3-1 to C-2, C-3, and C-6 and from H3-13 to C-2,

330

C-3, and C-4 confirmed a cyclopentane ring. Using the HMBC correlations from

331

OH-3 to C-2, C-3, C-4, and C-13, the location of the first oxygenated quaternary 16


COSY

Page 17 of 36


332

carbon (C-3) was determined. The side chain with a carbon-carbon double

333

bond and two oxygenated quaternary carbons (C-7 and C-11) was determined

334

by the key HMBC correlations from H3-14 to C-7 and C-8, from olefinic proton

335

H-10 to C-8, C-9, C-11, C-12, and C-15, from OH-7 to C-6, C-7, C-8, and C-14,

336

and OH-11 to C-11, C-12, and C-15. The HMBC correlations from H3-14 and

337

OH-7 to C-6 connected the two fragments (a cyclopentane ring and the side

338

chain) through the C-6 to C-7 bond. This is a new sesquiterpenoid containing

339

the cyclonerane framework.22

340

The relative configuration of 7 was determined by analysis of the NOE data

341

(Figure 4) and 1H NMR coupling constants. The NOE correlations between H-

342

2/H3-13, and H-6/H3-1 indicated that OH-3, CH3-1, and H-6 were on the same

343

side of the cyclopentane ring. The NOE correlations of H-6 with H3-14 proved

344

that H-6 and CH3-14 are close to each other in stable conformation. Model

345

building of each (H-6/H3-14 and H-6/H3-14) via Chem3D energy

346

minimization calculations provided that the structure (Figure 2) is more stable.

347

The cis-configuration of double bond C-9=C-10 was elucidated by the JH-9,H-10

348

(10.4 Hz) value. It was named cyclonerotriol B.

349

Compound 12 was also obtained as a colorless oil. The molecular formula

350

was determined as C15H26O2 by (+)-HR-ESI-MS (m/z 261.1828 [M + Na]+,

351

calculated 261.1825) and NMR data, which indicated three degrees of

352

unsaturation. The

353

δC 135.4 (C-8) and 120.9 (CH-7) taking into account one unsaturation. Hence,

13C

NMR spectrum (Table 3) showed two alkene signals at

17



354

the two remaining degrees of unsaturation were attributed to two rings. The 1H

355

NMR (Table 3),

356

presence of three tertiary methyls (δH 1.21, δC 29.0, CH3-12; δH 1.23, δC 31.4,

357

CH3-13; δH 1.65, δC 23.4, CH3-15), one secondary methyl (δH 0.93, δC 8.7, CH3-

358

14), four methylenes, four methines [one oxygenated (δH 4.28, δC 73.8, CH-3)

359

and one olefinic (δH 5.38, H-7)], and three quaternary carbons [one oxygenated

360

(δC 73.6, C-11) and one olefinic (C-8)].

13C

NMR, DEPT-135, and HSQC spectra also indicated the

361

The 1H-1H COSY spectrum and proton chemical shifts revealed the spin

362

systems of three structural fragments, H-1/H2-2/H-3/H-4/H3-14, H2-6/H-7, and

363

H2-9/H2-10 (Figure 5). The skeleton was established from long range HMBC

364

correlations of the hydrogen atoms of the above fragments with some

365

quaternary carbons (Figure 5). According to the key HMBC correlations from H-

366

1 and H3-14 to C-3 and C-5, the cyclopentane ring with CH3-14 and OH-3 was

367

confirmed. The HMBC correlations of H-10 with C-5, C-6, C-8, and C-9; H-15

368

with C-7 and C-9 assigned the cyclohexane ring with a carbon-carbon double

369

bond and CH3-15. Based on the correlations mentioned two rings were

370

connected through C-5 as a spirane. Furthermore, HMBC correlations from H3-

371

12 and H3-13 to oxygenated quaternary carbon C-11 and methine carbon C-1

372

suggested that C-11 connected with C-1 directly. The planar structure of

373

compound 12 was determined as an acorane sesquiterpenoid.23

374

To confirm the relative configuration of 12, NOE experiments were carried

375

out. The correlations (Figure 5) of H-6a with H-3, H-6b with H3-13, H3-14 with 18


Page 18 of 36

Page 19 of 36


376

H-1 and H-10b suggested that CH2-6 and C-11 were on the same facial plane,

377

OH-3 and CH3-14 were on the opposite plane. Finally, the structure of

378

compound 12 was assigned as 3-hydroxy--acorenol. Comparing the NMR spectroscopic data with that reported in the literature,

379 380

the isolated known compounds were elucidated as indol-3-acetic acid (2),24

381

methyl indolyl-3-acetate (3),25 bassiatin (4),26 beauvericin (5),27 enniatin A

382

(6),28 cyclonerodiol (8),22 epicyclonerodiol oxide (9),29 cyclonerodiol oxide(10),

383

29

384

5-O-methyljavanicin (15),32 methyl ether fusarubin (16),31 and

385

anhydrojavanicin (17).33

cyclonerodiol lactone (11), 29 fusaproliferin (13), 30 5-O-methylsolaniol (14),31

386

In order to identify the various Gram-positive and -negative bacterial

387

inhibitors as candidates for severe bacterial infections of human beings or

388

plants, pharmacological evaluation of the isolated compounds was carried

389

out. The activities of compounds 1–5 and 7–17 (Table 1) was screened

390

against the anthropogenic bacteria B. megaterium, B. subtilis, C. perfringens,

391

E. coli, MRSA, Mu50, Newman WT, and RN4220. Compound 8 (MIC 12.5

392

μg/mL) exhibited stronger antibacterial activity against B. megaterium than the

393

positive controls, the clinical drugs, ampicillin, erythromycin, and streptomycin.

394

Compound 9 (MIC 50.0 μg/mL) displayed similar antibacterial activity against

395

B. megaterium with ampicillin and erythromycin, and stronger than

396

streptomycin. All of the 1,4-naphthoquinone derivatives 14–17 showed

397

selective antibacterial activities against B. megaterium, B. subtilis, C. 19



398

perfringens, E. coli, MRSA, and RN4220. None of the tested compounds

399

showed any significant inhibition of the strains Mu50 and Newman WT (Table

400

1). The biological activities of compounds 1 and 7–11 were also evaluated

401

against the plant pathogen bacteria E. carotovora, P. syringae, R.

402

solanacearum, and X. oxyzae. Unfortunately, the tested compounds did not

403

display any activity.

404

In conclusion, a new acorane sesquiterpene, 3-hydroxy--acorenol (12),

405

has been discovered, along with 11 known compounds from the green

406

Chinese onion-derived fungus F. proliferatum AF-04. We also found a novel

407

alkaloid, fusaravenin (1), representing a new naphthoisoxazole formic acid

408

connected with a morpholino carbon frame, the first example of a natural

409

naphthoisoxazole type zwitter-ionic alkaloid, and a new cyclonerane

410

sesquiterpene, cyclonerotriol B (7), together with four known compounds from

411

the soil fungus F. avenaceum SF-1502. Although the source of these two

412

Fusarium species was different, the known compounds 9 and 11, cyclonerane

413

sesquiterpenes, were found from both fungi. Similar cyclodepsipeptides 5 and

414

6 have also been isolated from these two fungi using different culture

415

mediums. After the biological evaluation against eight anthropogenic and four

416

plant pathogenic bacteria, 8, 9, and 14–17 showed potent antibacterial

417

activities against some of the tested anthropogenic bacteria. The bioactive

418

results contribute to the valuation of these two Fusarium species. The

419

importance of the source and the culture medium of these fungi are 20


Page 20 of 36

Page 21 of 36

420


demonstrated.

421 422

■ ASSOCIATED CONTENT

423

1D and 2D NMR, IR, UV, and HR-ESI-MS spectra of compounds 1, 7, and 12.

424

The Supporting Information is available free of charge on the ACS

425

Publications website at DOI: 10.1021/acs.jafc.

426 427

■ AUTHOR INFORMATION

428

Corresponding author

429

*(Q.-X. Wu) Fax: +86-931-8915557. E-mail: [email protected].

430

Funding

431

This work was financially supported by the National Natural Science

432

Foundation of China (Nos. 21672087 and 21202075).

433

Notes

434

The authors declare no competing financial interest.

435 436

■ REFERENCES

437

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Page 26 of 36

Page 27 of 36


553

Figure Captions:

554

Figure 1. HPLC analysis results (254 nm) of SF-1502 and AF-04.

555

Figure 2. Structures of compounds 1‒17.

556

Figure 3. Key 1H-1H COSY and HMBC correlations of 1.

557

Figure 4. Key 1H-1H COSY, HMBC, and NOE correlations of 7.

558

Figure 5. Key 1H-1H COSY, HMBC, and NOE correlations of 12.

27



559

Page 28 of 36

Table 1. Antibacterial Activity Data of Compounds 1–5 and 7–17 compounds

MIC (g/mL) BMa

BSa

ECa

CPa

MRSA

RN4220

Mu50

NewmanWT

1

>100

>100

>100

>100

>100

>100

>100

>100

2

50

100

>100

>100

>100

100

>100

100

3

>100

100

>100

>100

>100

>100

>100

100

4

100

50

>100

100

100

100

100

100

5

50

100

>100

>100

50

>100

>100

100

7

>100

>100

>100

>100

>100

>100

>100

>100

8

12.5

100

100

100

100

100

100

100

9

50

100

50

50

100

100

100

100

10

>100

>100

>100

>100

>100

>100

>100

>100

11

>100

>100

>100

>100

>100

>100

>100

>100

12

>100

100

>100

100

>100

>100

>100

100

13

50

100

>100

100

50

>100

100

100

14

25

50

25

50

12.5

50

100

100

15

25

50

25

12.5

12.5

50

100

100

16

25

50

50

50

12.5

50

100

50

17

25

100

25

>100

25

50

>100

50

levofloxacin

3.13

1.56

0.78

0.78

>100

>100

>100

>100

erythromycin

50

25

0.78

0.39

>100

>100

>100

>100

ampicillin

50

12.5

50

>100

>100

>100

>100

>100

streptomycin

100

100

6.25

6.25

>100

>100

>100

>100

acheomycin

1.56

1.56

0.20

0.20

>100

>100

>100

>100

a

BM is Bacillus megaterium, BS is Bacillus subtilis, CP is Clostridium perfringens, and

EC is Escherichia coli. 560

28


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561

Table 2. Experimental 1H NMR & 13C NMR and calculated 13C NMR Data

562

for Compound 1 position

C(Exp.) a

C(Cal.)

C(Cal.-Exp.)

1

123.3

136.1

12.8

2

132.7

137.5

4.8

8.55 (1H, d, J = 7.8 Hz)

3

126.4

130.3

3.9

7.73 (1H, t, J = 7.8 Hz)

4

130.7

131.6

0.9

8.62 (1H, d, J = 8.4 Hz)

5

124.4

124.5

0.1

6

162.9

166.5

3.6

7

114.0

123.1

9.1

8

135.1

125.5

-9.6

8.42 (1H, d, J = 8.4 Hz)

9

110.9

123.2

12.3

7.07 (1H, d, J = 7.8 Hz)

10

131.3

134.4

3.1

11

165.7

162.5

-3.2

12

37.5

36.1

-1.4

4.36 (2H, t, J = 6.4 Hz)

13

57.4

68.7

11.3

2.84 (2H, t, J = 6.4 Hz)

14

166.3

167

0.7

1’

54.8

64.1

9.3

2.74 (2H, brs)

2’

67.4

75.8

8.4

3.70 (2H, t, J = 4.4 Hz)

3’

67.4

75.4

8

3.70 (2H, t, J = 4.4 Hz)

4’

54.8

60.7

5.9

2.74 (2H, brs)

a

H(Exp.) b

Recorded at 150 MHz in CD3OD. b Recorded at 600 MHz in CD3OD.

563

29



564

Page 30 of 36

Table 3. NMR Data for Compounds 7 and 12 7

12

position

Ha

C (DEPT)b

Hc

C(DEPT)d

1

1.05 (d, 6.8)

15.4 (CH3)

2.07 (m)

54.3 (CH)

2

1.63 (m)

44.9 (CH)

2.05 (m), 1.81 (m)

36.4 (CH2)

3

-

80.6 (C)

4.28 (t, 6.4)

73.8 (CH)

4

1.70 (m), 1.60 (m)

41.6 (CH2)

1.96 (m)

46.1 (CH)

5

1.86 (m), 1.59 (m)

25.0 (CH2)

-

45.3 (C)

6

1.85 (m)

55.2 (CH)

2.40 (m), 1.91 (m)

31.8 (CH2)

7

-

74.5 (C)

5.38 (brs)

120.9 (CH)

8

2.19 (m)

44.9 (CH2)

-

135.4 (C)

9

5.72 (dt, 10.4, 4.8)

123.1 (CH)

1.99 (m)

28.5 (CH2)

10

5.63 (d, 10.4)

142.7(CH)

1.85 (m), 1.52 (dt, 13.6, 6.0)

31.6 (CH2)

11

-

70.2 (C)

-

73.6 (C)

12

1.33 (s)

30.5 (CH3)

1.21 (s)

29.0 (CH3)

13

1.26 (s)

26.6 (CH3)

1.23 (s)

31.4 (CH3)

14

1.14 (s)

26.0 (CH3)

0.93 (d, 7.2)

8.7 (CH3)

15

1.33 (s)

30.5 (CH3)

1.65 (s)

23.4 (CH3)

OH-3

2.85 (s)

-

OH-7

2.92 (s)

-

OH-11

3.41 (s)

-

a

Recorded at 600 MHz in (CD3)2CO. b Recorded at 150 MHz in (CD3)2CO. c Recorded

at 400 MHz in CDCl3. d Recorded at 100 MHz in CDCl3. 565

30


Page 31 of 36


566 SF-1502

567 AF-04

568 569

Figure 1.

31



570 571

Figure 2.

32


Page 32 of 36

Page 33 of 36


572 573

Figure 3.

33



574 575

Figure 4.

34


Page 34 of 36

Page 35 of 36

576 577


Figure 5.

35



578

Table of Contents Graphics

SF-1502

AF-04

579

36


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Isolation, Identification, and Activity Evaluation of Chemical

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