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Journal of Agricultural and Food Chemistry is published by the American ChemicalSociety. 1155 Sixteenth Street N.W., Washington, DC 20036Published by American Chemical Society. Copyright © American Chemical Society.However, no copyright claim is made to original U.S. Government works, or worksproduced by employees of any Commonwealth realm Crown government in the courseof their duties.
Article
Isolation and identification of potential allelochemicals from aerialparts of Avena fatua L. and their allelopathic effect on wheat
Xingang Liu, Fajun Tian, Yingying Tian, Yanbing Wu, Fengshou Dong, Jun Xu, and Yongquan ZhengJ. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05498 • Publication Date (Web): 14 Apr 2016
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Isolation and identification of potential allelochemicals from aerial parts of 1
Avena fatua L. and their allelopathic effect on wheat 2
3
Xingang Liu†, Fajun Tian
†,‡, Yingying Tian
†, Yanbing Wu
‡, Fengshou Dong
†, Jun Xu
†, 4
Yongquan Zheng*,†
5
†State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of 6
Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China 7
‡Henan Institute of Science and Technology, Xinxiang, 453003, China 8
9
*Correspondence: Dr. Yongquan Zheng, State Key Laboratory for Biology of Plant 10
Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of 11
Agricultural Sciences, Beijing, 100193,P.R. China; Phone: 86-10-62815938; Fax: 12
86-10-62815938; E-mail: zhengyongquan@ippcaas.cn 13
14
15
16
17
18
19
20
21
22
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ABSTRACT: Five compounds (syringic acid, tricin, acacetin, syringoside and 23
diosmetin) were isolated from the aerial parts of wild oats (Avena fatua L.) using 24
chromatography columns of silica gel and Sephadex LH-20. Their chemical structures 25
were identified by means of ESI and HR-MS (electrospray ionization and 26
high-resolution mass spectrometry) as well as 1H NMR and
13C NMR spectroscopic 27
analyses. Bioassays showed that the five compounds had significant allelopathic 28
effects on the germination and seedling growth of wheat (Triticum aestivum L.). The 29
five compounds inhibited fresh wheat as well as the shoot and root growth of wheat 30
by approximately 50% at a concentration of 100 mg/kg, except for tricin and 31
syringoside for shoot growth. The results of activity testing indicated that the aerial 32
parts of wild oats had strong allelopathic potential and could cause different degrees 33
of influence on surrounding plants. Moreover, these compounds could be key 34
allelochemicals in wild oats-infested wheat fields and interfere with wheat growth via 35
allelopathy. 36
KEYWORDS: wild oats, wheat, allelopathy, allelochemicals, inhibitory effect, 37
mechanism 38
39
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41
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45
INTRODUCTION 46
Allelopathy is defined as the phenomenon that plants can affect (by inhibition or 47
promotion) the growth and development of other plants in the surrounding 48
environment by releasing chemicals into the surrounding environment. Allelopathy 49
have an important significance in the functioning of the natural community because 50
allelopathy also includes the effects on microorganisms and fauna.1-3
In recent years, 51
to make a thorough inquiry about the mechanisms of exotic plants, allelopathy has 52
become a research hotspot.4, 5
Many studies have shown that weeds had allelopathic 53
effects and interfered with the growth and development of plants nearby through the 54
release of allelochemicals.3, 6-10
Additionally, the allelopathy of exotic weeds plays an 55
important role in the invasion process. 56
Allelochemicals are secondary metabolites that have evolved in plants for defense 57
purposes.11
The most important role of allelochemicals is to disrupt seed germination 58
and the normal growth of plants. Allelochemicals are mainly derived from the 59
secretions of living plant tissues or decomposition of plant residues.12, 13
And they 60
have a strong effect even in small dosages and have potential as templates of 61
herbicides for new herbicide classes.8 Therefore, in-depth exploitation and 62
identification of potent allelochemicals as well as a thorough inquiry into the 63
allelopathic mechanisms are of the greatest importance to develop new 64
environmentally safe biological control strategies for sustainable agriculture. 65
Wild oat (Avena fatua L.) is an annual weeds and one of the major competitive 66
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weed species in wheat fields.14
It may affect the germination or growth of neighboring 67
plants such as wheat through allelopathy.15
There are few studies on the allelopathic 68
effect of wild oats on wheat. Schumacher confirmed that the root exudates of wild 69
oats had a significant effect on the growth of spring wheat in early 1983.16
Afterwards, 70
Pérez collected the root exudates of wild oats and identified that they contained the 71
compounds hydroxybenzoic acid, vanillic acid and coumarin by high performance 72
liquid chromatography (HPLC). This study also found that the root exudates of wild 73
oats exhibited significant inhibitory effects in the growth of the roots and coleoptile of 74
spring wheat.17
However, these studies were mainly concentrated on the root exudates 75
of wild oats, and most of the compounds with allelopathic activity were not identified. 76
Additionally, the allelopathy of the aerial parts of wild oats was not studied. In recent 77
years, studies on the allelopathic effect of the aerial parts of wild oats on wheat were 78
rare. In 2006, a Zhang study proved that the water extract of the entire wild oat plant 79
had an inhibitory effect on wheat.18
Other research was mainly concentrated in a 80
survival competition between wild oats and wheat.19
These studies reported the 81
inhibition of wheat germination, seedling growth and yield increased with an increase 82
in the density of wild oats. Meanwhile, wild oats produced and released 83
allelochemicals inhibiting wheat germination and seedling growth in the process of 84
competition to obtain a competitive advantage. Therefore, allelopathy seemed to be 85
the significant factor for the successful interference of wild oats with wheat. Some 86
studies have also had shown that wild oats had allelopathic effects on wheat.16, 17
Over 87
many years, various types of allelochemicals have been isolated and identified from 88
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hundreds of plants.3, 20-23
However, it has never been reported that a certain 89
allelochemical was isolated and identified from the aerial parts of wild oats nor were 90
further studies of its allelopathy performed. Moreover, the allelopathic mechanism of 91
the wild oats against wheat is largely unknown. 92
The allelochemicals of aerial parts, due to rain, dew and fog washing the plant 93
surface and falling into the soil, inhibited or promoted the surrounding biological 94
species. These compounds affected the structure and function of soil microorganisms 95
as well as further affecting the nearby plants. Therefore, the separation and 96
identification of allelochemicals has important significance to the study of allelopathy. 97
Accordingly, the main objective of the study described herein was to isolate and 98
identify key allelochemicals from the aerial parts of wild oats, and evaluated the 99
effects of these allelochemicals on wheat. These studies could provide evidence of the 100
interference of wild oats with wheat through allelopathy. Meanwhile, we also studied 101
the release mechanism of these allelochemicals. 102
MATERIALS AND METHODS 103
Plant material. The Avena fatua L. was collected at physiological maturity from a 104
wheat field located in the Zhengzhou district of the Henan province (Central China, N 105
34°76', E 113°65'), in May 2011. The aerial parts and underground portion of Avena 106
fatua L. were separated. The aerial parts of the plant were air dried in shade, ground 107
to a fine powder, passed through a 2 mm sieve and stored in a dark glass flask at 4 °C. 108
The selected wheat (Triticum aestivum L.) cultivars was Duokang 1, which is an 109
important cultivar grown in China, and was obtained from the Institute of Crop 110
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Research of the Chinese Academy of Agricultural Sciences. 111
Choice of the extraction solvent. Petroleum ether, ethanol, chloroform and 112
methanol were used to extract the aerial parts of Avena fatua L.( 150 g each, 3×333 113
ml of solvent), on three days. When performing the ethanol extraction, the first time 114
anhydrous ethanol was used, followed by 80% ethanol and then 70% ethanol. 115
Afterwards, the same extraction solutions were merged and filtered through filter 116
paper to remove larger impurities. Subsequently, the filtrates were concentrated to a 117
small volume by rotary evaporation and immediately transferred into a 150 mL flask. 118
They were prepared at 1000 mg/mL using the original solvent in accordance with the 119
plant dry weight calculation. Several solutions at 100 mg/mL, 10 mg/mL, and 1 120
mg/mL were also prepared by serially diluting the above solution. All solutions were 121
stored in the refrigerator at 4 °C. Then, the wheat germination and seedling growth 122
were determined by bioassays. The fraction with the strongest inhibition on wheat 123
germination and growth was further separated. 124
Initial separation of the allelochemicals. According to the above tests results, 125
twenty kilograms of powder from the aerial parts of Avena fatua L., which has the 126
strongest inhibition on wheat germination and growth, were soaked and extracted 127
three times with 150 L of the extraction solvent. The procedure was the same as that 128
described in the previous section. The filtrate was subsequently concentrated at 35 °C 129
in vacuo to give an aqueous residue. A small amount of distilled water was used to 130
dissolve the residues. Petroleum ether, ethyl acetate and chloroform were chosen as 131
the extraction solvents, and the volume of each extraction solvent was three times that 132
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of the sample volume. For each extraction, the samples were shaken vigorously for 15 133
min to ensure complete extraction, and then, they were distilled until the full 134
hierarchical analysis. The extracts from the same extraction solvent were combined 135
and concentrated into a paste in a rotary evaporator. A 1.5 g aliquot of extracts was 136
dissolved in 150 ml of methanol, and then, compounds at concentrations of 0.5 137
mg/mL, 1 mg/mL, and 10 mg/mL were prepared by serial dilution with methanol for 138
the wheat bioassays. 139
Bioassay procedure. Seed germination test. The seed germination test steps were 140
as follows: the test wheat seeds were surface sterilized by immersion in 5% sodium 141
hypochlorite for 10 min. The seeds were entirely rinsed three times with autoclaved 142
deionized water and then dried on a clean bench. Afterwards, the seeds were soaked 143
in deionized water at 21°C for 12 h and gently blotted with paper towels. A 50 g 144
portion of the soil media was evenly placed in a Petri dish (10 cm diameter). Then, 10 145
ml of the paste solution was added to each Petri dish. The corresponding pure solvent 146
served as the control. After the organic solvent was volatilized at room temperature, 147
20 mL of distilled water was added. Thirty seeds for each treatment were put on the 148
soil surface. Next, the Petri dishes were placed in an illuminated growth chamber kept 149
at 25°C ± 1°C, 80 ± 2% RH (relative humidity) and a 12/12 h L/D photoperiod. Three 150
replicates were performed for each treatment. The seeds were considered germinated 151
when there was a visible radicle protrusion through the lemma and palea. The 152
germination count was recorded every day for seven days and used to calculate the 153
germination rate. The germination percentage was recorded on the seventh day. The 154
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entire experiment was repeated twice. 155
Seedling growth tests. The seeding growth test steps were as follows: uniformly, 156
germinated seedlings (30 seedlings with radicle lengths of approximately 1 mm) from 157
the bioassay wheat species were placed into glass beakers (150 ml) containing 150 g 158
of the soil media. The soil media was moistened with 30 ml of the solution paste; the 159
corresponding pure solvent served as the control. Three replicates were performed for 160
each treatment. The beakers containing the various bioassay wheat seedlings were 161
placed into a growth chamber kept at 25°C during the 12 h light period and 20°C 162
during the 12 h dark period at 80 ± 2% RH and a 12/12 h L/D photoperiod. The wheat 163
fresh weight, root length and shoot length were measured after 14 days. 164
Bioassay-guided fractionation and purification. One thousands grams of silica 165
gel was dissolved using chloroform, and then placed into the column. Afterwards, the 166
bubbles were removed, and the balance of the liquid was retained. The appropriate 167
volume of chloroform was used to wash the column. Then, the fraction (20 g) that had 168
the strongest inhibition on wheat germination and growth in the initial separation of 169
the allelochemicals was dissolved using 60 ml of methanol, and then, 60 g of silica 170
gel was added. They were uniformly mixed, concentrated to dryness, and then ground 171
with a mortar. We added dry samples into the aforementioned column and subjected 172
them to silica gel column chromatography (80 cm × 6 cm) using a 173
chloroform/methanol (99:1, 97:3, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30 and 50:50; 174
v/v) mixture as the elution solvent. Finally, pure methanol was used for elution. Each 175
150 mL was taken as a fraction and applied to thin-layer chromatography (TLC). The 176
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chromatogram was visualized under UV light, and nineteen main fractions were 177
obtained (fraction 1 to fraction 19). Concentrated fractions at 500 mg/kg were 178
prepared according to the weight of the mixture calculation for the wheat bioassay. 179
The fractions with the strong inhibition or promotion of wheat growth using bioassays 180
were further purified. 181
The fractions of 5(6), 6(7), 7(8), 8(9) and 12(13) showed obviously bioactivity in 182
the wheat germination and seedling growth and were chosen for further isolation. 183
Fraction 5 was subjected to silica gel column chromatography (70 cm × 3 cm) using a 184
chloroform/methanol (5:1, 2:1 and 1:1; v/v) mixture as the elution solvent. Finally, 185
chloroform was used for elution. Each 150 ml was collected as a fraction and applied 186
to TLC. The chromatogram was visualized under UV light and combined with the 187
same fractions. Among the six fractions, 5-3 was purified by a prepared Sephadex 188
LH-20 column (70 cm × 2 cm) using a chloroform/ethyl acetate (5:1, 4:1, 2:1 and 1:1; 189
v/v) mixture as the elution solvent. Each 50 ml was collected as a fraction, applied to 190
TLC and was visualized under UV light. Finally, 5-3-6 was further isolated by a 191
LH-20 column and some crystals appeared (compound 1). Fraction 6 was subjected to 192
a Sephadex LH-20 column (70 cm × 2 cm) directly using a chloroform/methanol (1:1; 193
v/v) mixture as the elution solvent. Each 50 ml was collected as a fraction, applied to 194
TLC and visualized under UV light. Finally, some yellow crystal appeared 195
(compound 2). Fractions 7 and 8 were subjected to silica gel column chromatography 196
using chloroform/ethyl acetate (2:1, 1:1, 1:0; v/v) and ethyl acetate/methanol (9:1, 8:2, 197
5:5 and 0:10; v/v) mixtures as the elution solvent. Each 150 ml was collected as a 198
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fraction and applied to TLC. The chromatogram was visualized under UV light, and 199
the same fractions were combined. Each fraction was divided into five fractions. 200
Among these five fractions, 7-2 was subjected to a silica gel column using an ethyl 201
acetate/methanol (99:1, 98:2, 95:5, 90:10 and 85:15; v/v) mixture as the eluting 202
solvent. Fraction 7-2-2 was further separated by a Sephadex LH-20 column, and some 203
crystals appeared in fraction 7-2-2-5 (compound 3). Fraction 8-2 was subjected to a 204
Sephadex LH-20 column directly using a chloroform/methanol (1:1; v/v) mixture as 205
the elution solvent. Each 10 ml was collected as a fraction, applied to TLC and 206
visualized under UV light. From fraction 8-2-5 some yellow powder appeared 207
(compound 4). Fraction 12 was subjected to silica gel column chromatography using 208
an ethyl acetate/methanol (99:1. 95:5, 90:10, 80:20 and 50:50; v/v) mixture as the 209
elution solvent, and six fractions were obtained. Fraction 12-2 was further purified by 210
a silica gel column and a Sephadex LH-20 column twice using a chloroform/ethyl 211
acetate mixture in different proportions. Then, compound 5 appeared in fraction 212
12-2-5-6 by cutting the TLC board. 213
Mass spectrometric and NMR spectroscopic analyses. The isolated compounds 214
were characterized based on the data of high-resolution mass spectrometry (HR-MS), 215
1H NMR and
13C NMR spectroscopy (300Hz, d6-DMSO, TMS as an internal standard) 216
and optical rotation as described below. 217
Instruments: High-resolution mass spectrometry experiments were carried out on a 218
Finnigan LTQ XL linear ion trap mass spectrometer using electrospray ionization 219
(ESI-MS). The NMR spectra were measured in deuterated dimethyl sulfoxide with 220
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JEOL-ECA-600 NMR spectrometers (300 MHz for 1H, 500 MHz for
13C). All 221
chemical shifts are reported as a value relative to TMS. 222
Bioassay of the five compounds. The five identified compounds were bought and 223
dissolved in methanol. Concentrations of 30 mg/kg, 150 mg/kg, and 200 mg/kg were 224
added into the Petri dishes (10 cm diameter) containing a certain quantity of the soil 225
media. The concentrations of the compounds were 10, 50, 100 and 150 mg/kg in soil 226
for the wheat bioassay. Meanwhile, distilled water served as the control. The 227
biological activities of these substances were determined as described above. 228
Study on the allelochemical release mechanism. To further study the release 229
mechanism of these compounds from the aerial parts of Avena fatua L. into the 230
surrounding environment, the following experiments were carried out. The aerial parts 231
of wild oats (3.5 kg) were immersed into distilled water for 48 h (v/v = 1:5). Then, 232
they were filtered through filter paper to remove larger impurities. The filtrates were 233
washed with petroleum ether, ethyl acetate, and chloroform, and then, they were 234
extracted by a 1: 2 extraction of the amount of soaking liquid to the amount of 235
organic solvent. The filtrates were concentrated in vacuo at reduced pressure. The dry 236
residues were dissolved in acetonitrile and filtered with 0.22 µm syringe filters for 237
UHPLC-MS/MS analysis. 238
Statistical analysis. All of the bioassays were performed twice with at least three 239
replicates. The germination count, fresh weight, root and shoot lengths were subjected 240
to one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls 241
test to determine significant differences among mean values at the probability level of 242
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0.05. All of the data were analyzed using the Statistical Package for Social Sciences 243
(SPSS; version 17.0 for Windows). All data were transformed to a percentage of 244
inhibition. 245
RESULTS AND DISCUSSION 246
Bioassay-guided fractionation and purification. Choice of the extraction solvent. 247
It is necessary to demonstrate the inhibition of target plants for allelopathic substances 248
to be considered to be effective.24
Methanol and ethanol are strongly hydrophilic 249
extraction agents, whereas petroleum ether and chloroform are strongly lipophilic 250
extraction agents. During the screening process with various extraction solvents, the 251
ethanol extract had a significant inhibitory effect on the germination and seedling 252
growth of wheat at concentrations as low as 100 mg/mL. At a concentration of 1 g dry 253
weight powder of aerial parts of Avena fatua L. equivalent mL-1
, the ethanol fraction 254
caused 96.17%, 47.69%, 45.08% and 74.76% inhibition of the germination, root 255
length, shoot length and fresh weight of wheat, respectively, compared with the 256
control tests (Fig. 1). The inhibitory effect of methanol was slight lower than that of 257
ethanol, and the toxicity of ethanol was lower than those of the other three organic 258
solvents. Therefore, from Fig. 1, we concluded that the ethanol phase had the 259
strongest inhibition on wheat germination and seedling growth, and thus, it was 260
selected as the extraction solvent for further extraction and separation in the 261
experiment. We also concluded that the inhibitory effect was correlated with the 262
concentration of the extract. 263
Initial separation of the allelochemicals. The ethanol fraction was separated by 264
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petroleum ether, ethyl acetate and chloroform extraction, and their biological 265
activities were determined. Petroleum ether, ethyl acetate and chloroform were used 266
in ascending order with a polar solvent. All three fractions had inhibitory effects on 267
the germination, root length, shoot length, and fresh weight of wheat, while the 268
inhibitory effect of ethyl acetate was the most significant (Fig. 2). The inhibitory rates 269
of the germination, root length, shoot length and fresh weight of wheat at 0.5 mg/mL 270
were 91.37%, 64.89%, 61.53% and 85.89%, respectively, compared with the control 271
tests. The inhibition rates of other extraction agents were obviously lower than that of 272
ethyl acetate at a low concentration. Therefore, the purification and isolation of 273
allelochemicals were carried out only using the ethyl acetate fraction for further 274
studies. The potential inhibitory effects of these compounds mainly depended on the 275
concentration of the extracts and the types of plant secondary metabolites. In addition, 276
these compounds could also inhibit radicle and seedling growth when they inhibited 277
seed germination. The results of this experiment and those in the literatures reported 278
different types of other plants with similar results.25 279
Separation and purification of the allelochemicals. The separation and purification 280
of the allelochemicals are the most basic problems in the study of allelopathy and are 281
difficult tasks. In the process of separation, this experiment selected higher activity 282
fractions and easy to separate fractions for further separation. However, for high 283
polarity and complex fractions, even though they showed strong allelopathic 284
inhibition in the activity assay, we abandoned them because we also considered the 285
difficultly of the separation in the experimental process. We only separated the 286
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fractions that they were easy to separate and had some activity. Therefore, the ethyl 287
acetate fraction was purified and isolated by silica gel and Sephadex LH-20 288
chromatography columns. The active fractions were collected by using different 289
solvent mixtures for elution and applied to TLC. The chromatogram was visualized 290
under UV light, and nineteen main fractions were obtained (fraction 1 to fraction 19). 291
Fig. 3 shows that fractions 5, 6, 7, 8 and 12 obviously inhibited or promoted wheat 292
growth by bioassays, and these fractions were further purified and isolated using 293
different mixtures of elution solvents. Finally, the process allowed the isolation of five 294
compounds, all obtained as solids withstanding crystallization. 295
Identification of the purified bioactive allelochemicals. Plant interspecific and 296
intraspecific allelopathy were achieved through specific chemicals termed 297
allelochemicals. Therefore, allelochemicals are the most basic and important problem 298
in the research of allelopathy. However, the separation and identification of 299
allelochemicals is a difficult task, but their identification is an unavoidable problem in 300
the research of allelopathy. The simplest and most reliable method to determine the 301
purity of a compound was to determine the melting point or boiling point; the shorter 302
the melting process or boiling range, the higher the purity of the compound. The 303
molecular formulas of the five active compounds, which were separated and purified, 304
were determined by high resolution ESI MS. The high resolution mass spectra of the 305
five compounds had protonated and sodiated forms (Table 1). The 1H NMR and
13C 306
NMR spectra of the substances are as follows: 307
Compound 1: white crystalline powder, MP (melting point) 204-207 °C. The 308
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molecular formula of compound 1 was determined to be C9H10O5 (m/z 199.05995 309
[M+H]+; calculated for 199.06010) based on its high-resolution mass spectrum. The 310
1H NMR spectrum (d6-DMSO) showed the following peaks: δ 12.61 (1H, s, -COOH), 311
9.21 (1H, s, -OH), 7.20 (2H, s, H-2,6), and 3.81 (6H, s, H-3, 5-OCH3); The 13
C NMR 312
spectrum (d6-DMSO) showed the following: δ 167.29 (-COOH), 147.43 (C-3, 5), 313
140.17 (C-4), 120.34 (C-1), 106.79(C-2, 6), 55.96 (OCH3). From the comparison of 314
these data with those reported in the literature,26
the substance was identified as 315
4-hydroxy-3,5-dimethoxybenzoic acid (syringic acid). 316
Compound 2: faint yellow needle-shaped crystal, MP 291-292 °C. Compound 2 317
was assigned the molecular formula C17H14O7, as determined by its high-resolution 318
mass spectrum (m/z 331.08145 [M+H]+; calculated for 331.08123). The
1H NMR
319
spectrum (d6-DMSO, 300 M Hz) showed the following peaks: δ 12.97 (1H, s, 5-OH), 320
10.8 (1H, s, 7-OH), 9.35 (1H, s, 4’-OH), 7.33 (2H, s, H2’, 6’), 6.99 (1H, s, H-3), 6.56 321
(1H, s, H-8), 6.20 (1H, s, H-6), and 3.89 (6H, s, 3’,5’-OCH3); The 13
C NMR spectrum 322
(d6-DMSO) showed the following: δ 181.83 (C-4), 164.13 (C-2), 163.67 (C-7), 323
161.41 (C-5), 157.34 (C-9), 148.17 (C-3’,5’), 139.80 (C-4’), 120.37 (C-1’), 104. 324
30(C-2’,6’), 103.74 (C-10), 103.59 (C-3), 98.83 (C-6), 94.21 (C-8), 56.34 325
(C-3’,5’-O-Me). Accordingly, the substance was established as 326
5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-5,6-dihydro-4H-chromen-4-one 327
(tricin) from comparison of these data with those reported in the literature.27
328
Compound 3: yellowish powder, MP 260-265 °C. Compound 3 displayed an 329
[M+H]+ ion at m/z 285.07556 (calculated for 285.07575) in the high-resolution mass
330
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spectrum, consistent with a molecular formula of C16H12O5. The 1H NMR spectrum
331
(d6-DMSO) showed the following peaks: δ 12.93 (1H, s, 5-OH), 10.86 (1H, s, 7-OH), 332
8.05 (2H, d, J = 9.0 Hz, H-2’, 6’), 7.12 (2H, d, J = 12.0 Hz, H-3’, 5’), 6.87 (1H, s, 333
H-3), 6.51 (1H, d, J = 2.1 Hz, H-8), 6.20 (1H, d, J = 2.1 Hz, H-6), and 3.86 (6H, s, 334
OCH3); The 13
C NMR spectrum (d6-DMSO) showed the following: δ 181.80 (C-4), 335
164.24 (C-2), 163.28 (C-7), 162.30 (C-4’), 161.45 (C-5), 157.33 (C-9), 128.32 336
(C-2’,6’), 122.81 (C-1’), 114.57 (C-3’, 5’), 103.76 (C-10), 103.52 (C-3), 98.90 (C-6), 337
94.04 (C-8), and 55.56 (OCH3-4’). Compound 3 was thus established as 338
5,7-dihydroxy-2-(4-methoxyphenyl)-4-benzopyrone (acacetin) by the comparison of 339
these data with those reported in the literature.28
340
Compound 4: white needle-shaped crystal, MP 192 °C. The molecular formula of 341
compound 4, C17H24O9, was determined from its high-resolution mass spectrum (m/z 342
[M+Na]+
395.13099; calculated for 395.13125). The 1H NMR spectrum (d6-DMSO)
343
showed the following: δ 6.72 (2H, s, H-3, H-5), 6.49 (1H, d, J = 16.0 Hz, =CH), 6.34 344
(1H, m, =CH), 4.92 (1H, dd, J = 2.4, 7.2 Hz), 4.28 (1H, t, J = 5.76 Hz, -CH), 4.12 (2H, 345
t, J = 4.95 Hz,-CH2), 3.83 ( 6H, s, -OCH3), and 3.61~3.00 (6H, m, -OH); The 13
C 346
NMR spectrum (d6-DMSO) showed the following: δ152.72 (C-2,6), 133.77 (C-1’), 347
132.61 (C-4), 130.19 (C-2’), 128.45 (C-1), 104.40 (C-3,5), 61.49 (C-3’), 102.52 348
(glc-1), 77.25 (glc-5), 76.55 (glc-3), 74.18 (glc-2), 69.92 (glc-4), 61.49 (glc-6). 349
Compound 4 was determined to be 350
(2R,3S,4S,5R,6S)-2-(hydroxymethyl)-6-[4-[(E)-3-hydroxyprop-1-enyl]-2,6-dimethox351
yphenoxy]-oxane-3,4,5-triol (syringoside). 352
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Compound 5: yellow powder, MP 260-265 °C. The high-resolution mass spectrum 353
of compound 5 exhibited an [M+H]+
ion peak at m/z 301.07016 (calculated for 354
301.07066), corresponding to a molecular formula of C16H12O6. The 1H NMR
355
spectrum (d6-DMSO) showed the following: δ 12.94 (1H, s, 5-OH), 10.8 (1H, s, 356
7-OH), 9.35 (1H, s, 4’-OH), 7.53 (1H, dd, J = 2.3, 8.5 Hz, H-6’), 7.43 (1H, d, J = 2 357
Hz, H-2’), 7.07 (1H, d, J = 8.7 Hz, H-5’), 6.75 (1H, s, H-3), 6.46 (1H, d, J = 2.0 Hz, 358
H-8), 6.20 (1H, d, J = 2 Hz, H-6), and 3.85 (3H, s, OCH3); The 13
C NMR spectrum 359
(d6-DMSO) showed the following: δ 181.72 (C-4), 164.26(C-2), 163.52 (C-7), 161.48 360
(C-5), 157.33 (C-9), 151.13 (C-4’), 146.78 (C-3’),123.00 (C-1’), 118.74 (C-6’), 361
112.94 (C-2’), 112.12 (C-5’),103.75 (C-3), 98.90 (C-6), 93.93 (C-8) and 55.76 362
(OCH3). Consequently, compound 5 was elucidated as 363
3',5,7-trihydroxy-4'-methoxyflavone (diosmetin).29
364
The compounds amount of syringic acid, tricin, acacetin, syringoside and diosmetin 365
were 32.1 mg, 1.21 g, 51.4 mg, 10.8 mg and 28.9 mg, respectively. The structures of 366
the five compounds were shown in Fig.4. The isolation and identification of tricin, 367
acacetin and diosmetin belonged to flavonoid compounds. While syringic acid and 368
syringoside were phenolic and glycoside compounds, respectively. The 369
allelochemicals were divided into 14 kinds including flavonoids and phenols. And 370
phenols was one of the most common form of allelochemicals. This is the first to 371
report the five compounds which were isolated and identified from the aerial parts of 372
Avena fatua L. Hydroxy benzoic acid, vanillic acid, scopoletin and coumarin were 373
identified and reported from the root exudates of wild oats in the Previous studies, 374
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while the related reports of these compounds as allelochemicals were in other plants, 375
such as Parthenium hysterophorus L., watermelon seeds, Zosima absinthifolia, 376
Rorippa sylvestris, etc..17, 30-32
Therefore, the activities of the potential allelopathic 377
substances were verified, and the mechanism of their action was the focus of further 378
research. 379
Biological activities of the five compounds. The biological activities of the five 380
compounds were determined using wheat germination and seedling growth. The five 381
compounds from Avena fatua L. had different inhibitory effects on the germination 382
and seedling growth of wheat. These experimental results showed that tricin had a 383
significant inhibitory effect on wheat seed germination, while the inhibitory effect of 384
diosmetin was not particularly evident (Fig. 5, A). In seeding growth experiments, we 385
compared the influence of different compounds on the same index and the same 386
compounds on different indices. The results showed that the five compounds had 387
conspicuous inhibitory effects on the root length of wheat at concentrations as low as 388
10 mg/kg, and the inhibitory effects were enhanced when the concentrations of the 389
compounds increased (Fig. 5, B). The inhibitory rates of all of the compounds were 390
more than 50% at 100 mg/kg. In the determination of the impact of the five 391
compounds on the shoot length of wheat, we found that the inhibitory effects of 392
acacetin and diosmetin were enhanced when the concentrations of the compounds 393
increased (Fig. 5, C). Nevertheless, for syringic acid and syringoside, the inhibitory 394
effects were stronger at low concentrations compared with high concentrations. 395
However, the mechanism of this phenomenon was unknown and needed further 396
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research. Tricin showed some promotion effect at the minimum concentration, and 397
differences in the inhibitory rates were not obvious between the different 398
concentrations. The inhibitory effects of the five compounds on the fresh weight of 399
wheat also gradually enhanced when the concentrations of the compounds increased 400
except for diosmetin (Fig. 5, D). At the minimum concentration, the inhibitory effects 401
of diosmetin, syringic acid and tricin were weak, and the inhibitory rates were 402
approximately 20%. Nevertheless, when the concentrations of the five compounds 403
were 100 mg/kg, the inhibitory effects were manifested. The inhibitory rates of 404
syringic acid, tricin, acacetin, syringoside and diosmetin were 65.87%, 48.81%, 405
77.47%, 75.09% and 61.43%, respectively, at 100 mg/kg. Experiments to further 406
validate that the aerial parts of wild oats had strong allelopathic potential and could 407
cause different degrees of influence on the surrounding plants by allelochemicals 408
were performed. 409
Avena fatua L. allelopathy can be achieved by allelochemicals produced and 410
released into the environment. The allelopathic activity test showed that the five 411
compounds from Avena fatua L. could play a role as allelochemicals for the defense 412
of wheat against other plants. Nevertheless, there was insufficient evidence of 413
allelopathic activity to explain how other plants are affected in the process of the wild 414
oats growth, and it is not known what allelochemicals besides these five compounds 415
can be released into the ambient environment by wild oats. 416
Allelochemical release mechanism. The identification was performed by using 417
UHPLC-MS/MS, and the identities of the analytes were confirmed by the retention 418
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times and major mass signals of the mass spectra of standards that had been identified 419
in the identification step of the purified bioactive allelochemical. All of the 420
parameters for MRM transitions, and cone voltage collision energy were optimized to 421
acquire the highest sensitivity and resolution (Table 2). Syringoside acid was 422
determined in the chloroform and ethyl acetate phase. Acacetin and tricin were found 423
in the ethyl acetate phase. The results showed that syringoside acid, acacetin and 424
tricin could be released into the surrounding environment by washing due to rain, dew 425
or fog. However, it was unclear if the five compounds were released over the entire 426
life cycle of wild oats and how they affect other plants. Additionally, we need to 427
know the dynamics of the five compounds as well as their fate and activity under field 428
conditions. 429
In this study, the results showed that the aerial parts of wild oats had allelopathic 430
compounds. Five compounds were extracted, isolated and identified for the first time 431
as potential allelochemicals produced by Avena fatua L. plants. These compounds 432
were obtained by the extraction, purification and characterization steps of the aerial 433
parts of wild oats. Syringic acid, tricin, acacetin, syringoside and diosmetin, identified 434
as allelochemicals from wild oats, had significant allelopathy on the germination and 435
seedling growth of wheat. Syringoside acid, acacetin and tricin could be released into 436
the surrounding environment by washing due to rain, dew or fog. 437
ACKNOWLEDGMENT 438
This work was supported by National Natural Science Foundation of China 439
(31371970, 30900951 and 31201528). 440
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526
527
Figure captions 528
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Figure 1. Allelopathic effect of four solvent extract of the aerial part of Avena fatua L. 529
on the germination and seedling growth of tested wheat (Duokang 1). 530
Figure 2. Allelopathic effect of initial separation of the aerial part of Avena fatua L. 531
on the germination and seedling growth of tested wheat (Duokang 1). 532
Figure 3. Allelopathic effect of 19 fractions of the first time column separations of 533
ethyl acetate phase of the aerial part of Avena fatua L. extract on the germination and 534
seedling growth of tested wheat (Duokang 1). 535
Figure 4. Structure of five compounds isolated from the aerial parts of Avena fatua L. 536
Figure 5. Allelopathic effect of 5 identified compounds on the germination and 537
seedling growth of tested wheat (Duokang 1). 538
539
Table captions 540
Table 1. The molecular formula of the five purified active compounds using 541
high-resolution mass spectrometry. 542
Table 2. Mass-spectrometric conditions to determine five compounds. 543
544
545
Table 1. The molecular formula of the five purified active compounds using 546
high-resolution mass spectrometry. 547
Observed Species Observed m/z Expected m/z Mass Error (ppm)
Compound 1 [M+H]+ 199.05995 199.06010 -0.75
Compound 2 [M+H]+ 331.08145 331.08123 -0.66
Compound 3 [M+H]+ 285.07556 285.07575 -0.67
Compound 4 [M+Na]+ 395.13099 395.13125 -0.66
Compound 5 [M+H]+ 301.07016 301.07066 -1.66
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548
549
Table 2. Mass-spectrometric conditions to determine five compounds. 550
551
Compound Molecular
formula
Molecular
weight
tR
(min)
CV
(V)
Quantification ion
transition
CE1
(eV)
Confirmatory ion
transiton
CE2
(eV)
Ion ratio
Syringic
acid
C9H10O5 198.05 0.80 23 197.0→153.0 12 197.0→182.0 12 5.74
Tricin C17H14O7 330.29 1.80 36 329.3→299.2 31 329.3→314.3 28 1.87
Acacetin C16H12O5 284.07 1.45 32 283.2→268.0 23 283.2→239.5 32 10.06
Syringoside C17H14O9 372.06 1.10 40 395.4→232.0 23 395.4→217.0 30 14.13
Diosmetin C16H12O6 300.06 1.20 34 299.0→284.0 24 299.0→256.0 30 3.15
Notes: CV is cone voltage and CE is collision energy. 552
553
554
555
556
557
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558
Abstract photograph 559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
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577
Figure 1. Allelopathic effect of four solvent extract of the aerial part of Avena fatua L. 578
on the germination and seedling growth of tested wheat (Duokang 1). 579
580
581
Figure 2. Allelopathic effect of initial separation of the aerial part of Avena fatua L. 582
on the germination and seedling growth of tested wheat (Duokang 1). 583
584
Figure 3. Allelopathic effect of 19 fractions of the first time column separations of 585
ethyl acetate phase of the aerial part of Avena fatua L. extract on the germination and 586
seedling growth of tested wheat (Duokang 1). 587
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588
Figure 4. Structure of five compounds isolated from the aerial parts of Avena fatua L. 589
590
591
Figure 5. Allelopathic effect of 5 identified compounds on the germination and 592
seedling growth of tested wheat (Duokang 1). 593
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