Flavonolignans from Elymus natans L. and Phytotoxic Activities

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Flavonolignans from Elymus natans L. and Phytotoxic Activities Quan Liu, Chenghui Wu, Aifeng Peng, Kun Gao, Jianjun Chen, Ya Li, and Hua Fu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04162 • Publication Date (Web): 30 Jan 2017 Downloaded from http://pubs.acs.org on February 6, 2017

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Journal of Agricultural and Food Chemistry

Flavonolignans from Elymus natans L. and Phytotoxic Activities

Quan Liu‡, Chenghui Wu†, Aifeng Peng‡, Kun Gao*,†, Jianjun Chen†, Ya Li†, Hua Fu ‡

†

State Key Laboratory of Applied Organic Chemistry, College of Chemistry and

Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of China ‡

State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture

Science and Technology, Lanzhou University, Lanzhou 730020, People’s Republic of China

ACS Paragon Plus Environment

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1

Abstract：：

2

Elymus natans, a perennial gramineous grass, plays an important role in animal

3

husbandry and environmental sustenance in the Qinghai-Tibet plateau due to its high

4

forage quality and good adaptability to the local environment. Bioassay showed that the

5

extracts of green grasses of E. natans (GG) exhibited stronger phytotoxic activities than

6

withered grasses (WG) against crops and grasses. In view of the secondary metabolites

7

which may be responsible for the resistance of the plant, the chemical components of GG

8

were investigated. The flavone tricin, E1, and ten flavonolignans, E2-E11, including

9

three new ones, E2, E10 and E11, were isolated and identified. As far as we know, this is

10

the first report on the chemical constitutions of the plant until now. The contents of

11

compounds E1 and E4-E7 in GG were significantly higher than those in WG in HPLC

12

analysis and they also showed observably phytotoxic activities against lettuce and

13

Festuca arundinacea.

14 15

Keywords:

16

Elymus natans; flavonoids; phytotoxic activities; flavonolignans

17


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18


Introduction

19

The genus Elymus is the largest genus in the Triticeae family, and there are

20

approximately 150 species of this genus distributed in most temperate regions of the

21

world.1 Elymus natans L. is a perennial typical gramineous grass in the genus, which

22

widely used as important forage in natural grassland and cultivated pastures, particularly

23

in the Qinghai-Tibet plateau, due to its excellent cold, salt and drought tolerance.2

24

It was reported that, for Elymus natans distributed in the Qinghai-Tibet Plateau, the

25

biomass and nutritional values (contents of crude protein, water soluble carbohydrate,

26

neutral detergent fiber and acid detergent fiber) were significantly different in different

27

regions or in different growth stages.2-4 The secondary metabolites produced by plants

28

play an important role in the interaction of plants with their environment, and in

29

particular with their biotic environment, where they may serve as attractants for

30

pollinators or seed dispersers, in defense against natural enemies or as allelochemicals

31

against competitors. Moreover, the natural compounds can have beneficial or detrimental

32

effects on the health of humans or livestock upon ingestion, and provide an important

33

basis for pharmaceutical research.5 However, to the best of our knowledge, the

34

phytochemical investigation of E. natans has not been reported.

35

In light of the ecological benefits, the phytotoxic activities and investigation of

36

chemical constituents of E. natans were processed in the study. Eleven flavonoids,

37

E1-E11 (Figure 1), including three new flavonolignans, E2, E10 and E11, were isolated

38

from the green grasses of E. natans (GG). Furthermore, the contents of isolated

39

compounds were analyzed and their phytotoxic activities were evaluated. These results

40

would be helpful to understand how E. natans to enhance the competitive advantage of


3


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the plant against other plants in nature.

42 43

MATERIALS AND METHODS

44

General Experimental Instruments Procedures. HRESIMS was performed with a

45

Bruker APEX-II mass spectrometer (Bruker, Ltd., Karlsruhe, Germany), and results are

46

presented in terms of m/z. IR spectra were obtained with a Nicolet FT-IR-360

47

spectrometer (Thermo Nicolet, Inc., Waltham, MA). Ultraviolet (UV) spectra were

48

obtained with a Shimadzu UV-260 spectrophotometer (Shimadzu Instruments Co., Ltd.,

49

Tokyo, Japan). The high-performance liquid chromatography (HPLC) system used was a

50

Waters instrument (Waters, Milford, MA, USA), with a binary pump (Model 1525), a

51

photodiode array detector (Model 2998), manual injector and Waters BreezeTM

52

workstation for identifications and integrations of chromatography peaks. The

53

chromatographic separation for sample preparation was performed on the Atlantis C18

54

column (10 µm, 150×10 mm, I.D.) (Waters, Milford, MA, USA). The XBridgeTM C18

55

column (5 µm, 250×4.6 mm I.D.) (Waters, Milford, MA, USA) was used for

56

chromatographic separation of analysis.

57

1

H and

13

C NMR spectra were determined with a Bruker AM-400BB (400 MHz)

58

spectrometer (Bruker, Ltd., Karlsruhe, Germany). Optical rotations were recorded on a

59

PerkinElmer 341 polarimeter (PerkinElmer, Ltd., Waltham, MA, USA). ECD was

60

determined with an Olis DSM 1000 spectrometer (Olis, Atlanta, USA). Column

61

chromatography employed silica gel (200−300 mesh, Qingdao Marine Chemical Factory,

62

China), HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan), MCI gel (CHP20P,

63

Mitsubishi Chemical Corporation, Tokyo, Japan), RP-18 (Merck, Darmstadt, Germany),


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64

and Sephadex LH-20 (Amersham Pharmacia Biotech, Tokyo, Japan). Thin-layer

65

chromatography (TLC) employed silica gel GF 254 plates (10−40 µm, Qingdao Marine

66

Chemical Factory, China), and spots on the plates were observed under UV light of 254

67

nm and visualized by spraying with 5% H2SO4 in EtOH (v/v), followed by heating.

68

Plant Material. Two kinds of plant of E. nutans, green grass (GG) and withered grass

69

(WG), were collected in July and November 2014 from an alpine meadow (101°53′ E,

70

33°40′ N, altitude 3534 m) in Maqu Contry in Gannan Tibetan autonomous prefecture,

71

Gansu province, China. The voucher specimens (EnG-1 and EnG-2) were deposited in

72

the state key laboratory of applied organic chemistry, Lanzhou University. The seeds of

73

wheat, rape, lettuce, Poa annua and Festuca arundinacea were bought from the seed

74

market in Lanzhou. Wheat, rape and lettuce were the common crops in the Tibet, and the

75

other plants were associated plants of E. nutans.

76

Sample Preparation. The same weight (500 g) of GG and WG were smashed and

77

extracted with MeOH three times at room temperature respectively. The MeOH extracts

78

were dried under reduced pressure at 35℃ to yield two brown syrups. In order to remove

79

water soluble carbohydrate and protein, each of the concentrated brown syrup was

80

suspended in water and extracted with an equal volume of EtOAc three times. Then two

81

EtOAc extracts were separately dissolved with 100 mL methanol in volumetric flask and

82

then filtered through a 0.45 µm filter membrane to be ready for biology assays and HPLC

83

analysis.

84

Phytotoxicity Bioassay. Inhibitory activities on the growth of crops and other plants

85

were evaluated using the plate-culture method.6 After the seed germinates of the tested

86

plant, three-day-old seedlings of rape were transferred to 12-well plates (VWR Scientific


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87

Inc., Shanghai, China) supplemented with 1/2 MS liquid medium. Then extracts and

88

compounds were diluted with ethyl alcohol and added into each of the treated pores to

89

prepare the final concentrations of 200, 100, 50 and 25 µg/mL, respectively. Per treatment

90

were performed 4 replicates. Then the plants were in a constant temperature humidity

91

chamber at 22 °C for 4 days, under 16h light/ 8h dark photoperiod conditions. The root

92

length, plant height and fresh weight were measured at the end of the experiment. The

93

inhibitory ratios (%) were calculated by reference to the control. Control bioassays only

94

contained the same amount of ethyl alcohol. Significant differences were evaluated by

95

Duncan’s test (p=0.05). Similar procedures were taken to evaluate the phytotoxic

96

activities against other plants. Two-day-old seedlings of wheat and lettuce were

97

monitored for 3 days. Six-day-old seedlings of F. arundinacea were monitored for 5 days.

98

Fourteen-day-old seedlings of P. annua were monitored for 8 days.

99

Extraction, Isolation, and Purification Process of the Flavonolignans. The dried

100

and powdered aerial parts of GG (11.8 kg) were extracted with MeOH three times at

101

room temperature to yield 980 g of the crude extract. The crude extract was suspended in

102

water and extracted with EtOAc, acquiring 190 g EtOAc extract.

103

The EtOAc extract was applied to a HP-20 column and eluted with H2O/MeOH (70:30,

104

50:50, 20:80 and 0:100) to give four portions. The H2O/MeOH (20:80) portion (40 g) was

105

separated by MCI gel with using a gradient of H2O/MeOH (70:30-10:90) to give four

106

fractions labeled Fr.1−Fr.4. Then Fr.3 was chromatographed on a silica gel (petroleum

107

ether/acetone, 20:1-1:1) to yield four subfractions Fr.3a-d.

108 109

Fr.3d was separated by suction filtration washing them with methanol to give soluble portion and insoluble portion.


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110

The soluble portion of Fr.3d was applied to Sephdex LH-20 gel and eluted with MeOH

111

to yield compound E1 (20 mg). The remaining part was subjected to silica gel column

112

chromatography (CHCl3/MeOH, 10:1) and HPLC eluted by H2O/MeOH (45:55) at 2.0

113

mL/min to produce compounds E2 (4 mg, 4.2 min) and E3 (3 mg, 5.7 min).

114

The insoluble portion of Fr.3d was separated by column chromatography on a RP-18

115

column (H2O/Acetone, 70:30−0:100) to yield four fractions Fr.3d1-4. Fr.3d2 was applied

116

to a silica gel column (CHCl3/MeOH, 10:1), a preparative TLC (CHCl3/MeOH, 10:1) and

117

then preparative HPLC (H2O/MeOH, 0.9:1.1) at 2.0 mL/min to yield compounds E4 (9

118

mg, 13.5 min), E5 (9 mg, 9.1 min), E6 (10 mg, 10.0 min) and E7 (10 mg, 14.8 min).

119

Fr.3d3 was subjected to Sephdex LH-20 gel (CHCl3/MeOH, 1:1) and preparative HPLC

120

(H2O/CH3CN, 1.25:0.75) at 2.0 mL/min to get the mixture of compounds E8 and E9 (12

121

mg, 10.2 min), compounds E10 (2 mg, 11.4 min) and E11 (2 mg, 8.6 min).

122

Natansin A (E2): [α]25D = −20.9 (c = 0.1, MeOH). UV (MeOH) λmax 272.3, 363.9 nm.

123

HRESIMS m/z 501.1152 [M + Na]+ (calcd for C26H22O9Na, 501.1156). 1H and 13C NMR

124

data are in Table 1. Tricin-4′-O-[(7′′R,8′′R)-β-4′′-hydroxy-phenyl-(7′′-O-methyl)-glyceryl] ether (E10): [α]

125 126

25 D

127

[M + Na]+ (calcd for C27H26O10Na, 533.1418). 1H and 13C NMR data are in Table 1.

= +26.4 (c = 0.1, MeOH). UV (MeOH) λmax 269.9, 340.1 nm. HRESIMS m/z 533.1414

Tricin -4′-O- [(7′′R,8′′S)-β-4′′-hydroxy-phenyl-(7′′-O-methyl)-glyceryl] ether (E11): [α]

128 129

25 D

130

[M + H]+ (calcd for C26H22O9H, 511.1599). 1H and 13C NMR data are in Table 1.

= −46.2 (c = 0.1, MeOH). UV (MeOH) λmax 269.9, 340.1 nm. HRESIMS m/z 511.1596

131

HPLC Analysis. HPLC analysis was measured on a Waters HPLC apparatus. UV

132

spectra were recorded on 200-400 nm, and the compounds were monitored at 275 nm.


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133

The mobile phase was composed of (A) 0.1% aqueous acetic acid and (B) acetonitrile,

134

and programmed with isocratic elution at 25% B. The flow rate was 1.0 mL/min with an

135

injection volume of 20 µL at the temperature of 30 °C.

136

Identifications of the compounds in two kinds of E. natans were completed by

137

comparing the retention times and the UV spectra with the standards of compounds

138

E1-E7. The contents of E2 and E3 were too low to be detected in the samples of GG and

139

WG. The quantitation of compounds E1, E4, E5, E6 and E7 were performed by means of

140

one point external standard method. Standard stock solutions of these compounds with

141

concentrations of 1.0 mg/mL were prepared. Then five various concentrations of the

142

standard solutions were detected by continuous dilutions of the stock solution. The

143

amount of each compound was finally expressed as milligram per gram dry weight

144

(mg/kg).

145 146

RESULTS AND DISCUSSION

147

Phytotoxic Effects of E. natans on Crops and Grasses. Wheat and rape are common

148

crops in Tibet Plateau. The samples of GG and WG were assessed phytotoxic effect

149

against crops (wheat, rape and lettuce) and grasses (P. annua and F. arundinacea).

150

Bioassay showed that the extracts had dose-dependently inhibited effects on seedlings

151

growth of the receptor plants (Table 2-4), indicating that sufficient quantities of the

152

secondary metabolites produced by the plant to influence other plants. Among five tested

153

plants treatment with the extracts of GG or WG, the activities on root lengths were higher

154

than plant heights and fresh weights. Interestingly, it was found that the activities of GG

155

on plant heights and fresh weights were significantly higher than WG at concentration of


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156

100 µg/mL or less. But there was no significant difference of inhibit effects on root

157

lengths between GG and WG, except the inhibit activities against P. annua at greater than

158

50 µg/mL. In general, the extracts of GG showed higher phytotoxic activities than WG.

159

Phytotoxic Metabolites Isolated from E. natans. On account of the higher phytotoxic

160

activities than WG, the extract of GG was subjected to multiple chromatographic steps to

161

afford three new flavonolignans (E2, E10 and E11), along with tricin (E1) and seven

162

known flavonolignans (E3-E9) (Figure 1). The structures of these obtained compounds

163

were determined by combining the extensive spectroscopic analysis, optical rotation, CD

164

analyses and comparison with the data in the literatures.

165

Compound E2 was obtained as yellow amorphous powder. It exhibited a [M + Na]+

166

peak at m/z 501.1152 in the HRESIMS (calcd for 501.1156), corresponding to a

167

molecular formula of C26H22O9. The UV spectrum exhibited maxima at 272 and 364 nm,

168

which was the typical characteristic of flavonoid derivatives. The 1H, 13C NMR (Table 1)

169

and HSQC spectra data indicated the presence of seven aromatic methine groups, two

170

methoxy groups, one oxygenated methylene group, and two aliphatic methine groups.

171

Comprehensive analysis of 1H NMR spectrum, the signals at δH 13.02 (1H, s, 5-OH), 6.22

172

(1H, d, J = 1.6 Hz, H-6) and 6.55 (1H, d, J = 1.6 Hz, H-8) indicated that the A-ring of

173

flavonoid was 5,7-dihydroxy substituted. The four aromatic signals at 6.88 (2H, d, J = 8.4

174

Hz, H-15 and 19) and 6.64 (2H, d, J = 8.4 Hz, H-16 and 18) revealed the presence of a

175

4-hydroxyphenyl fragment. The 1H and

176

with those of compound E3,7 except lacking the signals of methoxy group and oxygenated

177

aromatic quaternary carbon. The compound E2 should have a skeleton which was based

178

on a flavonolignans-type flavonoid. Analysis of 1H-1H COSY spectrum confirmed the

13

C NMR spectra of compound E2 were similar


9


of

C(11a)H-C(11b)H,

C(11a)H-C(12)H,

C(12)H-C(13)H

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179

correlations

and

180

C(15)H-C(16)H. In the HMBC spectrum, H-13 showed correlations with C-11, C-15,

181

C-1′, C-19, C-3′, C-12, C-2′ and C-14, whereas H-12 showed correlations only with C-2,

182

C-14, C-2′ and C-3. So H-12 and H-13 should be differentiated. In addition, the key

183

correlations of H-6′ with C-2、C-5′、C-4′ and C-2′, 3′-OCH3 (δH 3.61) with C-3′, and

184

5′-OCH3 (δH 4.00) with C-5′ in the HMBC spectrum were used to assigned H-6′ and two

185

methoxyl groups. Thus, the planar structure of compound E2 was constructed as a

186

flavonolignan in which the flavonoid and phenylpropanoid moiety were linked by C-3

187

and C-2′, respectively, to C-12 and C-13, to form a cyclohexane bridge.

188

The relative configuration of compound E2 was deduced from NMR data. Due to H-13

189

(δ 4.94) appeared as a broad singlet, it was obviously that H-12 and H-13 were in the

190

same facial plane. The relative structure of E2 was also in accordance with the known

191

flavonolignans E3, which was reported in a literature isolated from Avena sativa

192

belonging to the same family Gramineae.7 Furthermore, the absolute configuration of E2

193

was determined by ECD spectral analysis applying the aromatic quadrant rule. The

194

electronic circular dichroism curve clearly showed a negative cotton effect at 260 nm,

195

which was also similar with E3, whose absolute configurations were 12S, 13S. The ECD

196

spectrum calculation using the TDDFT (time-dependent density functional theory)

197

method at the B3LYP/6-311++G(d,p) level was used to confirm this conclusion. The

198

results showed that the experimental ECD spectrum of E2 was similar to the calculated

199

spectrum of (12S,13S) (Figure 2). Consequently, the absolute configuration of E2 at C-12

200

and C-13 could both be determined to be S as shown. Finally, compound E2 was named

201

as natansin A.


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202

The molecular backbone of this new flavonolignan is rather rare. Before this study,

203

there were only two natural compounds of the type of skeleton with a tricin moiety and a

204

coniferyl alcohol moiety linked though cyclohexane ring of C-C bonds.7-9 Compound E2

205

will enter into the architectural diversity of the flavonolignan family.

206

Compound E10 was obtained as yellow amorphous powder. It determined a molecular

207

formula of C27H26O10 in HRESIMS at m/z 533.1414 ([M+ Na]+ calcd for 533.1418), and

208

was speculated to have 15 degrees of unsaturation. UV absorption spectra exhibited

209

maxima at 269.9 and 340.1 nm, which was the typical characteristic for flavonoid

210

derivatives. The 1H NMR spectra (Table 1) showed signals at δH 12.95 (1H, s, 5-OH),

211

6.83 (1H, s, overlap, H-3), 6.28 (1H, br s, H-6), 6.59 (1H, br s, H-8), 7.41 (2H, br s, H-2′,

212

6′), 4.00 (6H, s, 3′, 5′-OCH3) and 3.16 (3H, s, 7′′-OCH3), indicating the presence of a

213

tricin (E1) fragment with notable differences in the higher chemical shifts of C-1′, 3′ and

214

5′ found in E10. The other 1H NMR signals at δH 7. 23 (2H, d, J = 8.4 Hz, H-2′′, 6′′), 6.84

215

(2H, d, J = 8.4 Hz, H-3′′, 5′′), 4.62 (1H, d, J = 6.8 Hz, H-7′′), 4.38 (1H, m, H-8′′), 3.86

216

(1H, dd, J = 11.6, 4.0 Hz, H-9′′a), 3.58 (1H, dd, overlapped, H-9′′b) and 3.16 (3H, s,

217

7′′-OCH3) were predicted to be 1-(4-hydroxyphenyl) propane-1-methoxy-2,3-diol. The

218

13

219

20 aromatic/olefinic carbons (δC 94.9−164.9), 3 methoxy carbons (δC 56.8, 56.8 and 56.9),

220

2 oxygenated methine carbons (δC 87.0 and 84.6), and an oxygenated methylene carbon

221

(δC 62.0). Furthermore, the 1H and

222

showed similarities with the known compound E8,10 except for the absence of a methoxy

223

signal at C-3′′. Above evidences confirmed that E10 was a flavonolignan which

224

possessed a tricin and a 1-(4-hydroxyphenyl) propane-1- methoxy-2,3-diol which were

C NMR spectrum contained 27 signals, including a conjugated ketonic carbon (δ 183.0),

13

C NMR chemical shifts of E10 were found to


11


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225

linked by 4′-O-8′′ to form an ether. Moreover, the coupling constant between H-7′′ and

226

H-8′′ was 6.8 Hz, corresponding to a threo form of chiral centers at C-7′′ and C-8′′ based

227

on empirical observation.11

228

Compound E11 had the same molecular formula with E10 as C27H26O10 according to

229

HRESIMS data (m/z 511.1596 [M + H] +，calcd for C26H22O9H，511.1599). Moreover, the

230

1

231

implying that E11 had the same planar structure with a tricin and a 1-(4- hydroxyphenyl)

232

propane-1-methoxy-2,3-diol. In further analysis of NMR data, E11 was determined to be

233

an erythro type rather than threo type due to the coupling constants between H-7′′ and

234

H-8′′ was 6.0 Hz.

H and 13C NMR spectroscopic data of E11 (Table 1) were very similar to those of E10,

235

To determine the absolute configurations of E10 and E11, the electronic circular

236

dichroism (ECD) spectra for (7′′R,8′′R)-E10a and (7′′R,8′′S)-E11a, as well as their

237

enantiomers (7′′S,8′′S)-E10b and (7′′S,8′′R)-E11b (Figure 3) were calculated using

238

TDDFT theory at the B3LYP/6-311++G(d,p) level and were compared with the

239

experimental data for E10 and E11, respectively. The calculated ECD curve for

240

(7′′R,8′′R)-E10a agreed well with the experimental spectrum for E10 (Figure 4) and the

241

calculated ECD curve for (7′′R,8′′S)-E11a agreed well with the experimental spectrum

242

for

243

tricin-4′-O-[(7′′R,8′′R)-β-4′′-hydroxy-phenyl-(7′′-O-methyl)-glyceryl] ether and E11 was

244

unambiguously

245

[(7′′R,8′′S)-β-4′′-hydroxy-phenyl-(7′′-O-methyl)-glyceryl] ether.

E11

(Figure

5).

Therefore,

assigned

E10

was

as

deduced

to

be

tricin-4′-O-

246

The other compounds were characterized by the spectropic methods (MS, 1H and 13C

247

NMR spectra). By analysing the data and comparing them with related literature data,


12

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248


their structures were identified as tricin, E1,12 E3,7 E4,11 E5,11 E6,13 E7,13 E8,7 and E9.7

249

Compounds E10 and E11 which comprised of a tricin skeleton with a phenylpropanoid

250

moiety linked by an ether bond were diastereomers of each other in plant. This

251

phenomenon was usually found in other tricin-type flavonolignans (E4-E9) in reported

252

literatures.7,10,11 May be there was a balance of chemical reaction that two stereo-isomers

253

transformed to each other because of their instabilities.

254

In addition, many of these compounds have several pharmacological activities.

255

Compound E1 was reported to inhibit COX activity in mice, PGE2 generation in colon

256

cells and murine plasma, and adenoma formation.14 Compounds E4 and E5 showed

257

significant vasodilatory potencies and potent hydroxyl radical scavenging activities.11

258

Compound E9 showed strong anti-inflammatory and anti-allergic activities.10 These

259

results suggest these metabolites can be useful potential agents for allergic inflammatory

260

or other diseases, and E. natans was ascertained as a possible therapeutic source first

261

time.

262

Quantitation of Compounds in the GG and WG. To understand the changes of the

263

chemical components during different growth stages, HPLC analysis were successfully

264

used in this study to identify these metabolites in the samples of GG and WG. The

265

chromatogram was measured at 275 nm at which most of the compounds had absorbance.

266

The results of the analysis indicated the plant was rich in compounds E1, E4, E5, E6

267

and E7. The contents of all five compounds in GG were much higher than in WG, which

268

demonstrate that these secondary metabolites were closely linked with the resistance of

269

the plant. It is noteworthy that the concentration of tricin, E1, was highest among these

270

compounds in GG, maybe it was the precursor of the biogenesis of other flavonolignans.


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271

It is very interesting to note the retention time of each erythro was earlier than threo in

272

HPLC chromatograms (Table 5), and the concentrations of each erythro were lower than

273

those of threo in GG and WG (E4>E5 and E7>E6). The optimal conformations of the

274

diastereomers were threo E4 and E7 with minimize energy at 37.3037 and 38.2486

275

kcal/mol, respectively, calculated by using MM2 force field calculations for energy

276

minimization with the molecular modeling program Chem3D Ultra 10.0.

277

Phytotoxic Activities of the Purified Metabolites. Compounds E1, E4, E5, E6 and

278

E7 isolated and purified from E. natans were evaluated by the bioassays of the phytotoxic

279

activities against seedlings of rape and F. arundinacea. No further bioactivity studies of

280

other isolated compounds could be performed due to the low yields. Results indicated

281

that the growths of the tested plant seedlings were obviously suppressed by all of five

282

compounds to different degrees (Table 6). The phytotoxic effects of these compounds

283

showed dose-dependent alterations. The seedlings of F. arundinacea were more sensitive

284

than rape, especially at low concentrations (≦50 µg/mL). The activities of flavonolignans

285

derived from tricin were stronger than tricin. The inhibitory activities of each erythro

286

were more or less stronger than threo. For example, the inhibit effects of E6 on root

287

length and fresh weight against rape and F. arundinacea were stronger than E7 at all

288

testing concentrations.

289

In addition, compounds E6 and E7 were more potent than E4 and E5, indicating that

290

the additional 3′′-methoxyl group on the aromatic ring of phenylpropanoid moiety could

291

enhance phytotoxic activities. It was reported that the ortho-methoxyl group on the

292

aromatic ring could increase stabilities of the phenoxy radicals and terminate free radical

293

chain reactions by their electron-donating properties.11,15 These findings are also


14

Page 15 of 30

294


consistent with previous reports on the influence of methoxyl group.16

295

It was found that these compounds didn't kill other plants, mainly inhibited the growths

296

of their roots. These results may be induced by these compounds through decreasing the

297

root activities or inhibiting the synthesis and transport of auxin, which should be

298

demonstrated by further researches.

299 300

E. natans was an important grass for livestock and grassland in Tibet Plateau. Bioassay

301

showed that the extracts of GG and WG exhibited observably phytotoxic activities. In the

302

study, the flavone tricin and ten compounds belonging to two types of flavonolignan

303

skeleton were isolated from GG and elucidated on the basis of spectroscopic analysis. It

304

is well known that plants usually synthesize secondary metabolites used to adapt to the

305

environment and enable their survival and well-being. But the compounds of

306

flavonolignans type were usually reported to exhibited varied pharmacological activities,

307

such as anti-inflammatory and anti-allergy activities.10 The phytotoxic activities of E4-E7

308

were reported for the first time in this paper. The phytotoxic activities of E1 were similar

309

to the results in literatures,17,18 which also were reported to exhibit strong fungicidal

310

activites.17,19

311

The result of phytochemical investigation would furthermore provided a useful

312

evidence for the chemotaxonomy research on the genus Elymus. There were many

313

controversial questions on the definition and classification of the genus to need be cleared

314

up.20

315

Of particular interest is the observation that the contents of flavonolignans E6 and E7

316

were over two times E4 and E5 without 3′′-methoxyl group, especially in sample of WG.


15


Page 16 of 30

317

Additionally, E6 and E7 exhibited stronger inhibit effects than E4 and E5. The

318

replacement of the methoxyl group at the C-3′′ position would improve the stabilities and

319

enhance phytotoxic activities for the type of flavonolignans with the 4′-O-8′′ ether

320

linkage between flavanone and phenylpropanoid units. Moreover, stereo conformation

321

also has an impact on the stabilities and phytotoxic activities. The inhibitory activities of

322

each erythro were stronger than threo, although less stable than threo. The phytotoxic

323

activity of erythro flavonolignan E6 was strongest, though the content of diastereomer E7

324

was highest.

325

There was 16 mg EtOAc extract in 1 g GG of E. natans, which is usually as the major

326

dominant. It is easy to reach up to 200 µg/mL in nature. The high contents and phytotoxic

327

activities of the extracts and main ingredients were useful to improve the population

328

competitions, indicating that sufficient quantities of the secondary metabolites produced

329

by the plant may participate in the defense against other plants and pathogens.17-19

330 331

AUTHOR INFORMATION

332

Corresponding Autohor

333

Tel.: +86-931-8912592. Fax: +86-931-8912582. E-mail: [email protected].

334

Author Contributions

335

Quan Liu and Chenghui Wu contributed equally to this work and should be considered as

336

co-first authors. Quan Liu designed research, performed experiments, analysed data and

337

wrote the paper; Chenghui Wu isolated and identified these compounds from the plant.

338

Funding

339

This work was funded by National Basic Research Program of China (2014CB138703)


16

Page 17 of 30


340

and the National Natural Science Foundation of China (31472144).

341

Notes

342

The authors declare no competing financial interest.

343

REFERENCES

344

(1) Zhou, Q.; Luo, D.; Ma, L. C.; Xie, W. G.; Wang, Y.; Wang, Y. R.; Liu, Z. P.

345

Development and cross-species transferability of EST-SSR markers in Siberian wildrye

346

(Elymus sibiricus L.) using Illumina sequencing. Scientific Reports 2016, 6:20549, DOI:

347

10.1038/srep20549.

348

(2) Zhou, H. J.; Mao, Z. X.; Huang, D. J.; Fu, H. Analysis on nutritional quality of

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Elymus nutans among different populations on Qinghai-Tibet Plateau. Pratacultural

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Science 2011, 28, 1198-1202.

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(3) Yan, X. B.; Wand, X.; Guo, Y. X.; Zhang, D. G. Biomass and feeding value

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dynamics of Elymus nutans pasture in cold alpine pastoral areas. Pratacultural Science

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2003, 20, 14-18.

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(4) Zhao, Y. Y.; Huang, D. J.; Mao, Z. X.; Nie, B.; Fu, H. A study on forage nutritional

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quality of Elymus nutans from different populations in the Qinghai-Tibet Plateau. Acta

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Prataculturae Sinica 2013, 22, 38-45.

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(5) Kroymann, J. Natural diversity and adaptation in plant secondary metabolism. Current Opinion in Plant Biology 2011, 14, 246–251.

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(6) Liu, Q.; Xu, R.; Yan, Z. Q.; Jin, H.; Cui, H. Y.; Lu, L. Q.; Zhang, D. H.; Qin, B.

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Phytotoxic allelochemicals from roots and root exudates of Trifolium pratense. J. Agric.

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Food Chem. 2013, 61, 6321−6327.

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(7) Wenzig, E.; Kunert, O.; Ferreira, D.; Schmid, M.; Schühly, W.; Bauer, R.;


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Hiermann, A. Flavonolignans from Avena sativa. Journal of Natural Products 2005, 68,

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289-292. (8) Parthasarathy, M. R.; Ranganathan, K. R.; Sharma, D. K.

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C NMR of

flavonolignans from Hydnocarpus wightiana. Phytochemistry 1979, 18, 506-508. (9) Sharma, D. K.; Ranganathan, K. R.; Parthasarathy, M. R.; Bhushan, B.; Seshadri, T.

367 368

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R. Flavonolignans from Hydnocarpus wightiana. Planta Med. 1979, 37, 79-83.

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(10) Lee, S. S.; Baek, Y. S.; Eun, C. S.; Yu, M. H.; Baek, N. I.; Chung, D. K.; Bang, M.

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H.; Yang, S. A. Tricin derivatives as anti-inflammatory and anti-allergic constituents from

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the aerial part of Zizania latifolia. Bioscience, biotechnology, and biochemistry 2015, 79,

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700-706. (11) Chang, C. L.; Wang, G. J.; Zhang, L. J.; Tsai, W. J.; Chen, R. Y.; Wu, Y. C.; Kuo, Y.

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H.

Cardiovascular

protective

flavonolignans

and

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quiquesetinervius. Phytochemistry 2010, 71, 271-279.

flavonoids

from

Calamus

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(12) Bai, N.; He, K.; Roller, M.; Lai, C. S.; Bai, L.; Pan, M. H. Flavonolignans and

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other constituents from Lepidium meyenii with activities in anti-inflammation and human

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cancer cell lines. J. Agric. Food Chem. 2015, 63, 2458-2463.

379 380

(13) Bouaziz, M.; Veitch, N. C.; Grayer, R. J.; Simmonds, M. S. J.; Damak, M. Flavonolignans from Hyparrhenia hirta. Phytochemistry 2002, 60, 515-520.

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(14) Boolbol, S. K.; Dannenberg, A. J.; Chadburn, A.; Martucci, C.; Guo, X. J.;

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Ramonetti, J. T.; Abreu-Goris, M.; Newmark, H. L.; Lipkin, M. L.; DeCosse, J. J.;

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Bertagnolli, M. M. Cyclooxygenase-2 overexpression and tumor formation are blocked

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by sulindac in a murine model of familial adenomatous polyposis. Cancer Res. 1996, 56,

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2556-2560.


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(15) Srinivasan, M.; Sudheer, A. R.; Menon, V. P. Ferulic acid: therapeutic potential through its antioxidant property. J. Clin. Biochem. Nutr. 2007, 40, 92-100.

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(16) Eklund, P. C.; Langvik, O. K.; Warna, J. P.; Salmi, T. O.; Willfor, S. M.; Sjoholm,

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R. E. Chemical studies on antioxidant mechanisms and free radical scavenging properties

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of lignans. Org. Biomol. Chem. 2005, 3, 3336-3347.

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(17) Kong, C. H.; Xu, X. H.; Zhou, B.; Hu, F.; Zhang, C. X.; Zhang, M. X. Two

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compounds from allelopathic rice accession and their inhibitory activity on weeds and

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fungal pathogens. Phytochemistry 2004, 65, 1123-1128.

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(18) Kong, C. H.; Liang, W. J.; Xu, X. H.; Hu, F.; Wang, P.; Jiang, Y. Release and

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activity of allelochemicals from allelopathic rice seedlings. J. Agric. Food Chem. 2004,

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52, 2861-2865.

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(19) Kong, C. H.; Xu, X. H.; Zhang, M.; Zhang, S. Z. Allelochemical tricin in rice hull

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and its aurone isomer against rice seedling rot disease. Pest Manag. Sci. 2010, 66,

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1018–1024.

400 401

(20) Zhang, T. L.; Yan, G. S. Taxonomic studies a perplexity of Elymus genus. Acta Agrestia Sinica, 2012, 20, 207-212.


19


Page 20 of 30

Figure captions Figure 1. Structures of compounds E1−E11 isolated from E. natans. Figure 2. Experimental ECD and the calculated ECD spectra of E2. Figure 3. Matrix of possible E10 and E11 configurations. Figure 4. Experimental ECD spectrum of E10 and the calculated ECD spectra of (7′′R,8′′R)-E10a and (7′′S,8′′S)-E10b. Figure 5. Experimental ECD spectrum of E11 and the calculated ECD spectra of (7′′R,8′′S)-E11a and (7′′S,8′′R)-E11b.


20

Page 21 of 30


Table 1. 1H and 13C NMR Data of compounds E2a, E10a and E11b. E2 no. 2 3 4 5 6 7 8 9 10 11 12

δH

6.22 d (1.6) 6.55 d (1.6)

3.69 dd (10.4, 4.4) 3.23 t (10.4) 3.55 br dd (10.4, 4.4) 4.94 br s

13 14 15 16 17 18 19 1′ 2′ 3′ 4′ 5′ 6′ 3′-OMe

7.51 s 3.61 s

5′-OMe 5-OH

4.00 13.02 s

6.88 d (8.4) 6.64 d (8.4) 6.64 d (8.4) 6.88 d (8.4)

E10 δC 159.9 112.0 181.5 163.2 99.5 164.8 94.7 158.1 105.1 62.6

no. 2 3 4 5 6 7 8 9 10 1′

δH

6.28 br s

42.1

2′

7.41 br s

36.7 138.8 129.2 115.9 156.6 115.9 129.2 118.8 128.6 147.1 148.8 144.7 103.6 60.5

3′ 4′ 5′ 6′ 3′,5′-OMe 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 7′′ 8′′ 9′′

56.7

7′′-OMe 5-OH

6.83 s

6.59 br s

7.41 br s 4.00 s 7.23 d (8.4) 6.84 d (8.4) 6.84 d (8.4) 7.23 d (8.4) 4.62 d (6.8) 4.38 m 3.86 dd (11.6, 4.0) 3.58 overlapped 3.16 s 12.92 s

E11 δC 164.5 105.8 183.0 162.9 99.7 164.9 94.9 158.8 105.4 126.9 105.0 154.4 141.6 154.4 105.0 56.8 130.3 129.7 115.7 157.9 115.7 129.7 84.6 87.0 62.0 56.9

δH 6.77 s

6.26 br s 6.55 br s

7.35 br s

7.35 br s 3.94 s 7.24 d (8.4) 6.84 d (8.4) 6.84 d (8.4) 7.24 d (8.4) 4.57 d (6.0) 4.30 m 3.86 dd (12.0, 4.0) 3.60 dd (12.0,2.8) 3.24 s 12.90 s

δC 162.8 104.7 181.6 161.4 99.5 166.0 94.7 157.6 103.2 125.5 104.1 153.0 139.0 153.0 104.1 56.3 128.8 128.6 114.7 156.8 114.7 128.6 82.1 85.2 59.8 56.4

a.1H (400 MHz) and 13C (125 MHz) NMR, TMS, were measured in Acetone-d6. b.1H (400 MHz) NMR, TMS, was measured in Acetone-d6 and 13C (125 MHz) NMR was in DMSO-d6.


21


Page 22 of 30

Table 2. Relative inhibition ratios of the extracts of GG and WG on root lengths of five plants sample

GG

WG

Concentration (µg/mL) 25

12.76±1.67dA

12.13±1.07deA

13.59±0.91fA

8.75±1.08efB

7.10±1.73dB

50

16.78±1.89cdB

13.92±0.62cdBC

21.94±1.63cA

14.89±1.88cdBC

12.14±1.37cC

100

25.90±2.75bA

18.92±2.22bB

23.51±0.91bcA

16.31±1.23bcBC

14.88±0.69bC

lettuce

F. arundinacea

rape

P. annua

wheat

200

33.53±3.13aA

25.35±1.63aB

27.42±0.90aB

21.04±1.08aC

19.69±1.19aC

25

16.96±1.11cdA

10.05±1.87eB

10.93±1.89gB

6.80±0.78fC

6.69±1.02dC

50

18.81±2.30cA

15.87±1.652cA

16.12±1.25eA

9.67±1.19eB

12.30±2.01cB

100

26.19±1.69bA

19.48±1.65bB

19.40±1.25dB

13.32±0.45dD

15.62±0.00bC

200

31.72±2.30aA

23.46±0.63aB

25.68±1.25abB

17.50±1.19bC

19.19±0.77aC

Note: Each value represents the mean ± standard deviation with ten replicates. Means (±SDs) followed by the same lowercase letters are not significantly different for each treatment concentrations (P=0.05) in Duncan’s test. The different capital letter indicates the significantly different for each treatment plants (P=0.05) in Duncan’s test.


22

Page 23 of 30


Table 3. Relative inhibition ratios of the extracts of GG and WG on plant heights of five plants sample

GG

WG


lettuce

F. arundinacea

rape

12.42±2.26cAB

13.60±0.61dA

9.92±0.82cdCB

8.57±1.35fC

9.07±1.26dC

50

17.65±1.96bA

17.09±1.60cA

11.81±0.82cB

12.46±2.02deB

12.29±0.60cB

100

22.22±1.31aA

22.66±3.77bA

15.58±0.82bB

15.19±0.67cB

15.32±1.60bB

200

24.84±2.26aB

27.89±2.09aA

20.77±0.00aC

23.75±0.67aB

18.97±0.61aC

25

8.35±0.11eAB

9.77±1.59eA

7.87±3.21dAB

7.71±0.66fAB

6.39±0.79eB

50

11.66±1.12cB

17.78±1.59cA

11.57±2.12cB

11.18±1.34eB

9.59±1.37dB

100

18.16±1.95bAB

20.22±0.61bcA

16.20±0.80bBC

13.50±0.66cdCD

15.07±1.19bD

200

22.71±1.12aB

27.19±1.59aA

17.59±0.80bD

20.45±0.67bBC

18.49±1.81aCD

P. annua

wheat



23


Page 24 of 30

Table 4. Relative inhibition ratios of the extracts of GG and WG on fresh weights of five plants sample

GG

WG


lettuce

F. arundinacea

rape

P. annua

wheat

10.27±1.70eAB

12.21±2.73dA

8.19±1.01gB

7.74±1.54eB

9.94±0.58dAB

50

19.47±2.79cA

13.04±1.31dBC

10.78±0.89eBC

10.10±1.75dC

13.63±1.10cB

100

24.61±1.19bA

17.44±1.98cB

16.29±1.09cB

16.50±0.58bB

16.82±0.98bB

200

29.81±1.18aA

26.69±0.35aB

21.47±0.14aD

23.23±1.01aC

19.35±1.28aE

25

9.69±1.92eB

12.59±0.79dA

7.04±0.63gC

7.25±0.60eC

9.73±1.03dB

50

15.78±1.23dA

13.81±2.12dA

9.56±0.52fB

10.36±2.15dB

13.47±0.71cA

100

18.83±2.23cA

17.30±1.52cAB

14.76±0.29dCD

13.46±0.60cD

16.12±0.53bBC

200

27.58±0.13abA

23.37±1.03bB

18.37±0.38bC

18.29±0.00bC

18.46±1.07aC



24

Page 25 of 30


Table 5. The contents of five compounds in the extracts of GG and WG Retention time (min) 25.37

E1

GG Content (mg/kg) 37.94

WG Content (mg/kg) 12.81

28.55

E5

20.02

7.41

30.38

E6

22.49

16.02

35.88

E4

22.59

8.59

38.81

E7

29.94

18.63

Compound


25


Page 26 of 30

Table 6. Phytotoxic effects of five compounds isolated from E. natans on rape and F. arundinacea

sample

root length

Concentratio n (µg/mL)

rape

F. arundinacea

25

8.89±2.56j

16.34±2.26h

plant height

fresh weight

rape

F. arundinacea

rape

F. arundinacea

7.16±0.00j

11.85±0.74j

11.10±1.66m

10.65±1.04m

19.75±1.13h

15.61±1.86k

16.21±1.33k

22.96±0.74fg

22.56±1.95gh

19.80±1.65gh

11.58±0.96gh i 14.90±0.96de f

50

17.28±1.13hi

22.22±1.49fg

100

21.48±3.22fg

27.78±3.71de

200

27.90±1.86c d

28.43±0.98de

18.77±3.31c

26.17±1.54de

27.97±0.58de

21.56±1.00de

25

9.56±0.75j

17.56±1.72h

7.47±1.37j

14.54±1.62i

12.78±2.69lm

10.45±2.24lm

50

15.34±1.15i

23.52±1.99fg

10.14±0.00hij

19.47±1.96h

16.88±1.49jk

17.49±0.99jk

100

23.37±2.65ef

29.15±2.86cd

13.35±0.00ef g

24.93±0.89ef

23.24±1.09fgh

19.84±3.54fg h

200

28.90±1.74c

30.47±1.72cd

22.63±2.45b

28.30±2.81cd

28.66±2.02cd

24.33±0.91cd

25

10.65±1.13j

18.25±0.58h

7.59±1.62j

15.25±0.00i

10.85±2.80m

13.88±3.52m

20.90±1.76gh

17.63±1.79ijk

15.82±1.57ijk

25.71±0.98de

23.56±1.00fg

20.86±1.81fg

E1

E4

50

11.91±0.94fg h 14.07±2.81ef g

15.60±0.43i

24.33±2.11fg

100

24.76±1.13e

27.37±1.17de

200

30.20±1.28b c

31.77±1.54bc

23.80±1.62b

29.10±1.29bc

30.02±0.40bcd

25.69±0.93bc d

25

16.96±1.53hi

21.53±0.60g

8.27±0.95j

19.29±1.23h

17.67±1.20ijk

14.05±1.42ijk

50

19.17±2.25g h

27.78±2.40de

12.69±0.95fg h

26.00±0.80de

22.52±0.57gh

19.66±0.31gh

100

32.68±0.42b

30.21±1.80cd

18.77±1.66c

28.41±1.61cd

25.70±1.65ef

22.84±2.07ef

200

37.84±1.13a

36.46±1.04a

29.27±0.95a

32.43±1.61a

34.19±0.34a

31.62±0.99a

25

14.88±1.14i

18.67±1.53h

10.05±0.91hij

16.61±1.65i

16.12±1.21k

13.79±0.93k

25.33±0.58ef

13.76±2.42ef g

19.25±1.37h

20.38±2.42hi

17.29±1.83hi

24.80±2.03fg

22.74±1.96fg

32.42±1.65ab

29.47±0.47ab

E5

E6

50

17.52±0.86hi

E7 100

20.59±0.86g

28.00±1.00de

17.46±2.75cd

27.43±0.46cd e

200

36.97±0.42a

34.67±1.15ab

25.40±1.58b

31.92±0.79a

Note: Values are presented as percentage of the mean compared to the control (n=10). Each value represents the mean ± standard deviation. Means (±SDs) followed by the same letters are not significantly different for each treatment concentrations (P=0.05) in Duncan’s test.


26

Page 27 of 30


Figure graphics:

OCH3 4' OH

OCH3 OH HO

HO

O

8

7

O

9

2 1'

3'

H

OCH3

OCH3

HO

O

18 5

4

OH O

12 11

H OH

17

OH O

OH

16

E2*

E1

HO

H

O

H3CO

O

HO

O

HO

O

OH O

OCH3

OH OCH3

E6 erythro E7 threo

OH O

OH OCH3 H 6'' 9'' H3CO 5'

O

O OCH3

OH OCH3 H H

H

OH

E4 threo E5 erythro

H3CO

OH OCH3

OH OH H

OCH3

OH O

H OH E3

OH OH H H3CO

OCH3

H

13

6

OH O

OCH3 OH

H

OH OCH3

E8 threo E9 erythro

5''

8'' 7'' O 2'' 3''

HO

O

7

2 1'

5

OH O

3

3'

OH

OCH3 E10* threo E11* erythro

Figure 1


27


Page 28 of 30

Figure 2

OH H3CO 5'

OCH3 H 6''

(R)

H

5''

8'' (R)

O 3''

HO 7

O 2 1'

O

HO

O

OCH3 H (S)

O

OH

OCH3

(Z)

O

OH

E10a

H3CO

OH

H

OH

OCH3

OH

HO

H3CO

(S)

3

5

OH

3'

OH

E10b

OCH3 H

(S)

H

O

OH H3CO

(R)

O OCH3

OH HO

O

O

OH

(R)

H

OCH3 H (S)

O

OH

OCH3

O

E11a

E11b

Figure 3


28

Page 29 of 30


Figure 4

Figure 5


29


Page 30 of 30

TABLE OF CONTENTS GRAPHICS


30

Flavonolignans from Elymus natans L. and Phytotoxic Activities

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