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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: [email protected] 13

14

15

16

17

18

19

20

21

22

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ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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

40

41

42

43

44

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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

Page 17 of 29



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

Page 18 of 29



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

Page 19 of 29



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

Page 20 of 29



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Fitotherpia 1994, 1, 88. 517

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seeds, with allelopatic effects. EurAsia. J. Biosci 2010, 4, 17-22. 519

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526

527

Figure captions 528

Page 24 of 29



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


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


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

Page 25 of 29



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

Page 26 of 29



558

Abstract photograph 559

560

561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

Page 27 of 29



577

Figure 1. Allelopathic effect of four solvent extract of the aerial part of Avena fatua L. 578


580

581

Figure 2. Allelopathic effect of initial separation of the aerial part of Avena fatua L. 582


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


Page 28 of 29



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


Page 29 of 29



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