Effects of different oligochitosans on isoflavone metabolites

6. Kunming, Yunnan Province, People's Republic of China, 650500;. 7 b College of Food Science and Technology, Hebei Agricultural University, Baoding,...
0 downloads 0 Views 926KB Size
Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Functional Structure/Activity Relationships

Effects of different oligochitosans on isoflavone metabolites, antioxidant activity and isoflavone biosynthetic genes in soybean (Glycine max) seeds during germination Yijia Jia, YanLi Ma, Ping Zou, Gui-Guang Cheng, Jiexin Zhou, and Shengbao Cai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07300 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 1, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

1

Effects of different oligochitosans on isoflavone metabolites, antioxidant activity

2

and isoflavone biosynthetic genes in soybean (Glycine max) seeds during

3

germination

4

Yijia Jiaa, Yanli Mab, Ping Zouc, Guiguang Chenga, Jiexin Zhoua, Shengbao Caia*

5

aYunnan

6

Kunming, Yunnan Province, People’s Republic of China, 650500;

7

b

8

Hebei Province, People’s Republic of China, 071001;

9

c Marine

Institute of Food Safety, Kunming University of Science and Technology,

College of Food Science and Technology, Hebei Agricultural University, Baoding,

Agriculture Research Center, Tobacco Research Institute of Chinese

10

Academy of Agricultural Sciences, Qingdao, Shandong Province, People’s Republic

11

of China, 266101

12

* Corresponding author and proofs

13

Shengbao Cai

14

E-mail address: [email protected]/[email protected]

1 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 38

15

Abstract

16

Five oligochitosans with increasing degrees of polymerization (DPs), i.e., from

17

chitotriose to chitoheptaose, were examined to clarify the structure–bioactivity

18

relationship between the DPs of oligochitosans and their effects on the isoflavone

19

metabolites, total phenolic and flavonoid contents (TPC and TFC, respectively), and

20

antioxidant activity of soybean (Glycine max) seeds during germination.

21

Oligochitosans of different DPs exhibited varying influences on the TPC, TFC, and

22

antioxidant activities of soybean seeds. Chitohexaose exerted a strong effect and

23

significantly increased the aforementioned parameters in soybean seeds 72 h after

24

germination. Genistin, malonylgenistin, and genistein were the main isoflavones

25

found, and the genistin and genistein contents were significantly enhanced by 67.32%

26

and 131.38%, respectively, after chitohexaose treatment. Several critical genes

27

involved in the isoflavone biosynthesis (i.e., PAL, CHS, CHI, IFS) of soybeans treated

28

with and without chitohexaose were analyzed, and results suggested that chitohexaose

29

application could dramatically stimulate the transcription of these genes.

30

Keywords: Degree of polymerization, gene expression, germination, isoflavones,

31

oligochitosan, soybean seeds

2 ACS Paragon Plus Environment

Page 3 of 38

Journal of Agricultural and Food Chemistry

32 33

INTRODUCTION In the natural environment, plants are constantly exposed to biological and

34

non-biological factors, such as diseases, insects, ultraviolet light, saline-alkaline soil,

35

and low temperature, during their growth. Plants also produce hydrogen peroxide

36

during photosynthesis, which can generate hydroxyl radicals in the presence of

37

transition metals, leading to oxidative stress and cell damage.1 To cope with these

38

factors and ensure their survival and growth, plants have developed several defense

39

mechanisms. 2 Among these innate defense measures, synthesis of secondary

40

metabolites, such as alkaloids, terpenes, and phenolics, is considered an important

41

means for plants to adapt to the environment.3 Plants can be protected from natural

42

hazards by synthesizing secondary metabolites. Flavonoids belong to a large family of

43

plant phenolics that are widely accumulated in plants as secondary metabolites. The

44

flavonoid family encompasses thousands of compounds and can be classified into

45

several groups, such as isoflavones, flavones, flavonols, and flavanols.4, 5 These

46

secondary metabolites not only play extremely important roles in the plant host to

47

prevent chemical and physical damage but also confer health benefits to its

48

consumers.6 Therefore, many studies to increase plant flavonoid contents have been

49

conducted. Research has found that a number of abiotic and biotic elicitors could

50

effectively increase the flavonoid content of some crops.7-9

51

Oligochitosan, a degradation product of chitosan, is mainly derived from the

52

wastes of shellfish and crustaceans; it is composed of homo- or heterooligomers of

53

D-glucosamine and N-acetyl-D-glucosamine.10 Oligochitosan has much better water 3 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 38

54

solubility than chitosan and, thus, is, more suitable and convenient than the latter for

55

practical applications. 11, 12 Extensive research to investigate the bioactivities of

56

oligochitosan has been performed, and findings indicate that oligochitosans possess

57

abundant functional properties, such as antitumor, antioxidant, and antimicrobial

58

activities.13-16 These bioactivities allow the material to be applied to many fields, such

59

as agriculture, biomedicine, and food.17 In agriculture, oligochitosans effectively elicit

60

plant innate immunity, promote plant growth, and improve plant tolerance to adverse

61

condition stress. Previous studies reported that oligochitosans could increase the

62

photosynthesis of Dendrobium orchids by increasing their chlorophyll content and

63

improve the chilling and salt stress tolerance of wheat seedlings.12, 18, 19 Zou et al.12

64

and Zhang et al.19 revealed that the tolerance of wheat seedlings to chilling or salt

65

stress closely depended on the degree of polymerization (DP) of the oligochitosan

66

applied and that oligochitosans of DP = 6 or 7 showed better bioactivity than those of

67

other DPs. Oligochitosans, when used as an elicitor, have been proven to promote the

68

accumulation of secondary metabolites, such as stilbenes and isoflavones, in

69

plants.20-23 Despite their helpful results, however, most previous reports use

70

oligochitosan mixtures with different DPs; thus, the ability of individual

71

oligochitosans to induce accumulation of secondary metabolites, especially

72

flavonoids, in plants, remains unclear. Moreover, the underlying mechanisms of

73

oligochitosan in flavonoid accumulation have yet to be illuminated.

74 75

In the present work, an experiment was designed to clarify the structure– bioactivity relationship between the DPs of oligochitosans and their effects on the 4 ACS Paragon Plus Environment

Page 5 of 38

Journal of Agricultural and Food Chemistry

76

isoflavone metabolites, total phenolic and flavonoid contents (TPC and TFC,

77

respectively), and antioxidant activity of soybean (Glycine max) seeds during

78

germination. And the main isoflavones induced by five fully deacetylated single

79

oligochitosans, i.e., from 3 (chitotriose) to 7 (chitoheptaose), in soybean sprouts were

80

further identified and quantified. Finally, a series of isoflavone biosynthetic genes

81

were analyzed to delineate the underlying mechanisms of oligochitosan on isoflavone

82

metabolite accumulation in soybean seeds during germination.

83

MATERIALS AND METHODS

84

Chemicals and reagents

85

Acetonitrile, methanol, and Folin - Ciocalteu reagent were obtained from Merck

86

(Darmstadt, Germany). Standard samples of naringenin-7-O-glucoside(≥98.0%),

87

genistein (≥98.0%), genistin (≥98.0%), daidzein (≥98.0%), daidzin (≥98.0%), glycitin

88

(≥98.0%), glycitein (≥98.0%), and (-)-epigallocatechin (≥98.0%) were purchased

89

from Chengdu Must Bio-technology Co., Ltd. (Chengdu, Sichuan, China). Chitotriose

90

(≥95.0%), chitotetraose (≥95.0%), chitopentaose (≥95.0%), chitohexaose (≥95.0%),

91

and chitoheptaose (≥95.0%) were obtained from Qingdao BZ Oligo Bio-technology

92

Co., Ltd. (Qingdao, Shandong, China). Real-time fluorescence-based qRT–PCR

93

reagents were purchased from Tiangen Biotech (Beijing) Co., Ltd. (Beijing, China).

94

Soybean seeds were purchased from a local market (Kunming, Yunnan, China). All

95

other chemical reagents used in this work were of analytical grade.

96

Soybean seed treatments

97

Soybean seeds (G. max) were surface sterilized with distilled water for 3 min at 5 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

98

75 °C and then wiped with sterilized gauze.24 The seeds were divided into six groups

99

and separately immersed in distilled water containing 0.01% (m/v, 0.1 mg/mL)

Page 6 of 38

100

oligochitosans (i.e., chitotriose, chitotetraose, chitopentaose, chitohexaose, or

101

chitoheptaose; treatment groups) or distilled water (control group) for 6 h. The

102

concentrations of the oligochitosans were selected according to a previous study.12

103

The seeds were then transferred to a germination device for germination at 25 °C for

104

0, 24, 48, 72, or 96 h in the dark. The humidity was set to 85% ± 2%. After

105

germination, some of the germinated seeds in each group were immediately frozen at

106

–80 °C for further qRT–PCR assay; the other germinated seeds were lyophilized

107

(Alpha 1-2 LD plus, Christ, Germany) and extracted with 80% methanol for

108

identification and quantification of isoflavone metabolites and evaluation of TPC,

109

TFC and antioxidant activity. Briefly, dried and powdered samples (0.30 g) were

110

ultrasonically extracted with 15.0 mL of 80% methanol for 1 h at 40 °C. The extracted

111

slurry was centrifuged at 4000 × g, and the supernatant was filtered by a syringe filter

112

(0.2 μm) for further analysis.

113

Determination of TPC

114

The TPC of each group of soybean seeds (soybean sprouts) with and without

115

oligochitosan treatment was measured by a previously described method.25 TPC was

116

expressed as milligrams of gallic acid equivalents per 100 g of dry weight (DW).

117

Determination of TFC

118

The TFC of each group of soybean seeds (soybean sprouts) with and without

119

oligochitosan treatment was measured as described earlier.26 TFC was expressed as 6 ACS Paragon Plus Environment

Page 7 of 38

Journal of Agricultural and Food Chemistry

120

milligrams of rutin equivalents per 100 g of DW.

121

Evaluation of DPPH radical scavenging capacity

122

The DPPH radical scavenging capacity of each sample was determined based on a

123

previously reported method,26 and calculated using the following formula: DPPH

124

scavenging capacity (%) = [(Acontrol – Asample)/Acontrol] × 100. All tests were conducted

125

thrice.

126

Evaluation of ABTS radical scavenging capacity

127

To determine the ABTS radical scavenging capacity of each sample, a previously

128

reported method was applied.26ABTS radical scavenging capacity was calculated

129

using the following formula: ABTS scavenging capacity (%) = [(Acontrol –

130

Asample)/Acontrol] × 100. All tests were conducted thrice.

131

Identification and quantification of flavonoid metabolites by UHPLC–ESI–

132

MS/MS

133

The flavonoid metabolites of soybean seeds with and without chitohexaose

134

treatment were identified and quantified at 72 h of germination by a Thermo Fisher

135

Ultimate 3000 UHPLC System with a Q-Exactive Orbitrap mass spectrometer

136

(Thermo Fisher Scientific, Bremen, Germany). An Agilent ZORBAX SB-C18 column

137

(2.1 mm × 100 mm, 1.7 μm, USA) was used to separate the flavonoid metabolites in

138

the filtrate of each sample at 30 °C. The gradients of the mobile phases, including

139

water acidified with 0.5% (v/v) formic acid (phase A) and acetonitrile (phase B), were

140

as follows: 0–1 min, 5% B; 1–5 min, 5%–15% B; 5–10 min, 15%–25% B; 10–15 min,

141

25%–45% B; 15–16 min, 45%–5% B; 16–20 min, 5% B. The injection volume of the 7 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 38

142

sample was 3.0 μL, and the flow rate was 0.2 mL/min. The mass spectrometer was

143

operated in negative mode (3.3 kV). Other related parameters were fixed according to

144

an earlier report,27,28 and the appropriate standards were used to identify and quantify

145

flavonoid metabolites determined under the same conditions.

146

Analysis of isoflavone biosynthetic gene expression by qRT–PCR

147

Total RNA was extracted from soybean seeds with and without chitohexaose

148

treatment at 72 h of germination using an RNAprep Pure Plant kit (TianGen)

149

according to the manufacturer’s instructions. The cDNA was obtained from 600 ng of

150

the total RNA using a Fast King RT kit (TianGen). Gene primers were designed by

151

Sangon Biotech (Shanghai, China) based on a previous work7 and are summarized in

152

Table S1. qRT–PCR was performed using the Talent qPCR PreMix SYBR Green kit

153

(Tiangen) with an Applied Biosystems StepOnePlus™ Real-Time PCR system

154

according to a method reported earlier with minor modifications.7 In the present work,

155

the reactions were performed over 40 cycles of 95 °C/5 s and extension of 60 °C/15 s.

156

Data were analyzed using ABI StepOnePlus™ software version 2.3. The transcript

157

level of each gene was normalized against the soybean β-TUB gene, which was used

158

as an internal control.

159

Statistical analysis

160

Each experiment was repeated thrice, and data are presented as mean ± standard

161

deviation (SD). All data were analyzed by one-way ANOVA using Origin 8.5

162

software (OriginLab, Northampton, MA, USA), and Tukey's test was used to

163

determine significant differences (p < 0.05). 8 ACS Paragon Plus Environment

Page 9 of 38

Journal of Agricultural and Food Chemistry

164

RESULTS AND DISCUSSION

165

TPC and TFC of soybean seeds during germination

166

In the present study, the TPC and TFC of soybean seeds during germination

167

increased significantly after oligochitosan treatment. The five oligochitosans (i.e.,

168

chitotriose, chitotetraose, chitopentaose, chitohexaose, and chitoheptaose) showed

169

different effects on the TPC and TFC of soybean seeds 96h after germination (Tables

170

1 and 2). As shown in Table 1, in the control group, the TPC of soybean seeds did not

171

significantly change over the first 2 d of germination (p > 0.05) but increased by

172

8.05% and 10.10% at 72 and 96 h of germination (p < 0.05), respectively, when

173

compared with that of soybean seeds at 0 h of germination. Compared with the

174

control group, all five single oligochitosans did not dramatically increase the TPC of

175

soybeans at 0 and 24 h of germination (p > 0.05). However, application of chitotriose,

176

chitotetraose, chitopentaose, chitohexaose, and chitoheptaose significantly increased

177

the TPC of soybeans by 7.09%, 11.96%, 7.51%, 11.34%, and 7.22% , respectively, at

178

48 h of germination (p < 0.05).

179

The TPCs in soybean seeds peaked at72 h of germination in all five oligochitosan

180

treatment groups and then decreased at 96 h of germination. Some previous studies on

181

the effects of elicitors on the TPC of plants have reported a similar phenomenon.21, 24

182

Xu et al.,21 for example, reported that the TPCs of Vitis vinifera cell cultures treated

183

with oligochitosan or sodium alginate peaked at 36 h and then decreased with further

184

increases in treatment time. Liu et al.24 also found that mung bean sprouts treated with

185

different concentrations of ethephon exhibited peak phenolic contents at 48 h, after 9 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 38

186

which the TPCs of all sprouts dramatically decreased. In the present work, no

187

statistically significant difference was observed in the TPCs of soybeans between the

188

control, chitotriose, chitotetraose, chitopentaose, and chitoheptaose groups at 96 h of

189

germination (p > 0.05), although the TPC of the chitohexaose treatment group

190

remained significantly higher than those of the other groups (p < 0.05). In general, the

191

TPC results indicate that, among the tested oligochitosans, chitohexaose exerts the

192

strongest effects on the synthesis of phenolic compounds in soybean seeds during

193

germination.

194

The TFCs of soybean seeds treated by oligochitosans with different DPs during

195

germination are summarized in Table 2. In the control group, TFCs significantly

196

increased by 21.12%, 31.82%, and 43.68% at 48, 72, and 96 h of germination (p
3 was essential to improve

219

the growth and photosynthesis of wheat seedlings and that chitoheptaose exhibited the

220

strongest activity among oligochitosans of other DPs (DP: 2–8). Zou et al.12 found

221

that both chitohexaose and chitoheptaose could markedly alleviate chilling stress in

222

wheat seedlings. A previous study found that chitooctaose promoted the growth of

223

wheat seedlings suffering from salt stress best when compared with oligochitosans of

224

other DPs.19 The TPC and TFC results of the present work clearly indicate that DP

225

plays a critical role in promoting the synthesis of phenolic compounds, especially that

226

of flavonoids, in soybean seeds during germination and that chitohexaose possesses

227

the strongest activity among the oligochitosans studied. According to the results of

228

earlier studies and the current data, the optimal DP of oligochitosan varies among

229

plants and their applications and could range from 5 to 8. Oligochitosans must first be 11 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 12 of 38

230

recognized and then bound by their receptors on cell membranes to exert their

231

bioactivity and trigger various defense responses in plants. However, the structures of

232

receptors on cell membranes may vary among plants and functions, resulting in

233

differences in the optimal DP. A previous study reported that the rice receptor

234

chitin-elicitor binding protein preferably bound long-chain chitin oligosaccharides,

235

such as heptamers or octamers, and then formed a unique sandwich-type dimer to

236

activate defense signaling.33

237

DPPH and ABTS radical scavenging activity of soybean seeds during

238

germination

239

Anti-oxidation is an important physiological function for biological organisms to

240

cope with environmental stress. In the present study, the antioxidant activities of

241

soybean seeds treated with oligochitosans of different DPs during germination were

242

evaluated by the DPPH and ABTS radical scavenging methods; the results are

243

summarized in Tables 3 and 4. A concentration of 20.0 mg of dry soybean seeds/mL

244

was used in the DPPH radical scavenging test. In the control group, DPPH radical

245

scavenging activity did not change significantly during germination (p > 0.05).

246

Compared with the control, the five oligochitosans tested displayed different effects

247

on the DPPH radical scavenging activity of soybean seeds during germination.

248

Chitotriose and chitotetraose significantly increased the DPPH radical scavenging

249

activity of soybean seeds at 72 and 24 h of germination, respectively (p < 0.05). By

250

contrast, chitopentaose did not significantly affect the DPPH radical scavenging

251

activity of soybean seeds during the entire process of germination when compared 12 ACS Paragon Plus Environment

Page 13 of 38

Journal of Agricultural and Food Chemistry

252

with the control (p > 0.05). Among the five oligochitosans applied, chitohexaose and

253

chitoheptaose showed the strongest effects on enhancing the DPPH radical

254

scavenging activity of soybean seeds at 72 and 48 h of germination (p < 0.05),

255

respectively. In particular, chitohexaose treatment increased the DPPH radical

256

scavenging activity of the soybean seeds by approximately 20.72% at 72 h of

257

germination.

258

In the ABTS radical scavenging test, a concentration of 10.0 mg of dry soybean

259

seeds/mL was used. In the control group, ABTS radical scavenging activity did not

260

change significantly during germination (p > 0.05), except at 24 h of germination,

261

during which ABTS radical scavenging activity unexpectedly significantly decreased

262

(p < 0.05). In contrast to the results of the DPPH radical scavenging test, nearly all

263

five oligochitosans significantly enhanced the ABTS radical scavenging activity of

264

soybean seeds over 4 d of germination when compared with that of the control group

265

(p < 0.05). Among the five oligochitosans tested, chitohexaose and chitoheptaose

266

showed the strongest effects on increasing the ABTS radical scavenging activity of

267

soybeans at 72 h of germination (p < 0.05); no significant difference between these

268

two oligochitosans (p > 0.05) was noted. Some discrepancies in the effects of

269

oligochitosans of different DPs on DPPH and ABTS radical scavenging activities

270

during soybean seed germination may be attributed to the complicated compositions

271

of antioxidant compounds in soybean seeds and/or differences in the evaluation

272

methods. In general, regardless of the radical scavenging activity assessed, soybean

273

seeds treated with chitohexaose showed the highest antioxidant activity after 72 h of 13 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

274 275

Page 14 of 38

germination (p < 0.05). According to the results in Tables1–4, good correlations between TPC and

276

DPPH radical scavenging activity, as well as between TPC and ABTS radical

277

scavenging activity, were observed (r = 0.383, p < 0.05; r = 0.572, p < 0.01,

278

respectively). Moreover, Pearson’s correlation analyses between TFC and DPPH

279

radical scavenging activity and between TFC and ABTS radical scavenging activity

280

revealed that the TFCs of the samples were closely related to their antioxidant activity

281

(r = 0.478, p < 0.01; r = 0.604, p < 0.01, respectively). All Pearson’s correlation

282

analysis results indicated that phenolic compounds, especially flavonoids, may

283

contribute significantly to the antioxidant activity of soybeans during germination,

284

consistent with the findings of many previous studies reporting that phenolic

285

compounds are the major antioxidants of the corresponding plant materials.34, 35

286

Identification and quantification of flavonoid metabolites

287

Since soybean seeds treated with chitohexaose showed the highest TPC and TFC

288

and the strongest antioxidant activity at 72 h of germination, the phenolic

289

composition, especially that of flavonoids, of this sample and the corresponding

290

control sample were comparatively investigated by UHPLC–ESI–HRMS/MS in

291

negative mode. The related ion current chromatograms are illustrated in Figure 1, and

292

Table 5 summarizes the mass data, including compound names, molecular formulas,

293

retention times (Rt), [M-H]- m/z, MS/MS ion fragments, and errors (ppm). As shown

294

in Fig. 1 and Table 5, a total of 12 phenolic compounds, all of which were flavonoids,

295

were tentatively or positively identified based on the mass data of available authentic 14 ACS Paragon Plus Environment

Page 15 of 38

Journal of Agricultural and Food Chemistry

296

standards or previous reports;36, 37, 38 among the flavonoids found, 10 were

297

isoflavones. The ion current chromatograms of the control and chitohexaose-treated

298

groups were very similar at 72 h of germination, although the intensity of some peaks

299

differed (Fig. 1). This result suggests that chitohexaose treatment does not change the

300

phenolic composition of soybean seeds but affects their contents. Compounds 8, 9,

301

and 12 showed high peak areas in the chromatograms of the control and

302

chitohexaose-treated groups, thereby indicating that these phenolic compounds may

303

be the main compounds in the two samples. Compound 8 ([M-H]- m/z = 431.0973)

304

was positively identified as genistin by an authentic standard; this compound

305

produced a characteristic ion fragment at m/z = 268.0372 due to the loss of glucose

306

moiety (Fig. 2). Compound 9 was tentatively characterized as malonylgenistin

307

([M-H]- m/z = 517.0980); the characteristic ion fragment (m/z = 269.0450) of this

308

compound was produced by the loss of a malonyl–glucose moiety (Fig. 2).

309

Compound 12 ([M-H]- m/z = 269.0451) was positively characterized as genistein;

310

here, cleavage of ring C formed two characteristic ion fragments (m/z = 107.0123 and

311

m/z = 133.0281), as shown in Fig.2

312

The quantitative results of the 12 flavonoid metabolites in the control and

313

chitohexaose-treated groups are presented in Table 5. In the present work, eight of the

314

identified flavonoids (compounds 1, 2, 3, 4, 7, 8, 11, and 12) were quantified by their

315

corresponding commercial standards. Compounds 5 and 6, which were identified as

316

malonyldaidzin and acetyldaidzin, respectively, were quantified by daidzin, and

317

compounds 9 and 10, which were identified as malonylgenistin and acetylgenistin, 15 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 16 of 38

318

respectively, were quantified by genistin. As shown in Table 5, genistin,

319

malonylgenistin, and genistein were the predominant flavonoids detected in both the

320

control and chitohexaose-treated groups. In the control group, genistin,

321

malonylgenistin and genistein respectively accounted for about 21.67%, 34.41%, and

322

10.26% of the total contents of the 12 identified flavonoids; by comparison, the ratios

323

of these three flavonoids in the chitohexaose-treated group were approximately

324

26.55%, 23.20%, and 17.38%, respectively. After chitohexaose treatment, the

325

contents of nearly all flavonoids in the soybeans increased significantly, except that of

326

malonylgenistin (Table 5). The contents of genistin and genistein in the

327

chitohexaose-treated group increased by about 67.32% from 149.74 μg/g to 250.54

328

μg/g and by about 131.38% from 70.90 μg/g to 164.05 μg/g, respectively. By contrast,

329

the content of malonylgenistin decreased by about 7.92% from 237.76 μg/g to 218.94

330

μg/g. In general, the total contents of the 12 flavonoids in soybean seeds significantly

331

increased by about 36.57% at 72 h of germination after treatment with chitohexaose.

332

Isoflavones, as a subgroup of plant flavonoids, are primarily synthesized in

333

leguminous plants, especially in soybean seeds, and have structures similar to that of

334

17-β-estradiol. Isoflavones can bind to estrogen receptors to activate the estrogen

335

response, which is believed to exert health benefits when isoflavone-containing

336

products are consumed as a dietary supplement.7 Previous studies report that dietary

337

consumption of soy isoflavones exerts clear positive effects on the risk factors of

338

diseases associated with estrogen levels, such as hormone-dependent cancer and

339

osteoporosis.39 The main dietary isoflavones are glycitein, daidzein, genistein, and 16 ACS Paragon Plus Environment

Page 17 of 38

Journal of Agricultural and Food Chemistry

340

their glycosides, all of which were detected in the soybean seeds with and without

341

chitohexaose treatment in the present work (Table 5). Yuk et al.7 reported that

342

ethylene could significantly induce the accumulation of daidzin, genistin,

343

malonyldaidzin, and malonylgenistin in soybean leaves; these isoflavones were

344

dramatically upgraded by chitohexaose treatment in soybean seeds in the present

345

work. Compared with the study of Yuk et al.,7 the oligochitosans used in the present

346

work as elicitors of isoflavone accumulation may be more suitable than ethylene to

347

produce functional foods or agricultural products because they exert relatively fewer

348

side effects and, thus, could be considered safer. However, oligochitosan mixtures

349

rich in chitohexaose instead of pure chitohexaose should be used in practical

350

applications to address cost issues.

351

Expression of isoflavone biosynthetic genes

352

Oligochitosans are widely distributed in plant pathogen and often considered as a

353

signal of pathogen invasion in plants. The immune system of plant is activated when

354

the oligochitosan receptors in their cell membrane surface of plant was bound with

355

oligochitosans. Then, innate defense measures are taken. Synthesis of secondary

356

metabolites, such as phenolics and isoflavone metabolites, is considered an important

357

defense mechanism for plants to adapt to the environment (Fig. 3a). In the present

358

work, oligochitosans of different DPs, especially chitohexaose, could serve as

359

elicitors to improve the contents of flavonoid metabolites, which chiefly consist of

360

isoflavones, in soybean seeds. Isoflavones, such as genistein and daidzein, and their

361

glycosides are synthesized in a specific branch of the phenylpropanoid pathway (Fig. 17 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 18 of 38

362

3b). Several key genes, including phenylalanine ammonia-lyase (PAL), isoflavone

363

synthase (IFS), chalcone synthase (CHS), and chalcone isomerase (CHI) are known to

364

be involved in the phenylpropanoid and isoflavone pathways during isoflavone

365

biosynthesis.40, 41 Thus, to illustrate the underlying mechanism of isoflavone

366

accumulation during chitohexaose application, the expressions of several critical

367

isoflavone biosynthetic genes in soybean seeds treated with and without chitohexaose

368

were analyzed by qRT–PCR. Results showed that all critical genes, including PAL,

369

CHS7, CHI1, CHI2, IFS1, and IFS2, were significantly up-regulated in soybean seeds

370

treated by chitohexaose by approximately 1.25-fold (CHS7) to 4-fold (IFS1) when

371

compared with those of the control (p < 0.05) (Fig. 4). Such findings indicate that

372

chitohexaose may induce the transcriptional expression of critical genes involved in

373

isoflavone biosynthesis, resulting in increases in the isoflavone content of soybean

374

seeds at 72 h of germination. The enzyme PAL catalyzes the first step of the

375

phenylpropanoid pathway, which transforms L-phenylalanine to produce cinnamate,

376

which, in turn, is used as a precursor for various secondary metabolites, such as

377

tannins, lignans, flavones, and isoflavones.40 The genes CHS6 and CHS7 catalyze

378

proteins with a particularly vital role in flavonoid and isoflavone biosynthesis. Several

379

reports have found a positive correlation between the expression of CHS genes and

380

the genistein and total isoflavone contents.42 The enzyme CHI converts naringenin

381

and isoliquiritigenin chalcones to their corresponding flavanones.43 As shown in Fig.

382

4, the expressions of CHI1 and CHI2 genes exhibited similarly sensitive responses to

383

chitohexaose treatment; only a slight difference in expression was observed between 18 ACS Paragon Plus Environment

Page 19 of 38

Journal of Agricultural and Food Chemistry

384

this work and a previous study that reported that the CHI1 gene is more responsive to

385

ethephon treatment than the CHI2 gene.7 This discrepancy may be due to the different

386

elicitors and plant parts used in the previous and current studies. In the soybean

387

genome, IFS presents as two species,44 namely, IFS1 and IFS2, which synthesize the

388

corresponding enzymes belonging to cytochrome P450 monooxygenase and could

389

transform naringenin and liquiritigenin into genistein and daidzein, respectively. After

390

treatment with chitohexaose, the expressions of the IFS1 and IFS2 genes showed

391

significantly up-regulated tendencies (p < 0.05). The qRT–PCR results also revealed

392

that IFS1 is more sensitive to chitohexaose than IFS2, which suggests that IFS1

393

enzyme may play a key role in isoflavone biosynthesis in response to chitohexaose

394

signals; this finding differs from the results of a previous study that used ethylene as

395

an elicitor.7 The IFS gene expression results demonstrate that the phenylpropanoid

396

pathway, which produces isoflavones, is activated after chitohexaose treatment.

397

Chitohexaose treatment could improve the expressions of IFS1/2 and CHI1/2, which

398

are the main contributors to the formation of isoflavones, thereby resulting in

399

significant increments in TFC and TPC in soybean seeds. The expression of genes in

400

different isoforms showed differences in metabolite sensitivity and localization, which

401

may play differential roles in regulating isoflavone metabolism.45

402

In summary, this work demonstrated an effective and safe method of increasing

403

flavonoid contents in soybean seeds during germination. Oligochitosans of different

404

DPs exhibited different influences on the TPC, TFC, and antioxidant activities of

405

germinating soybean seeds, and chitohexaose could significantly increase the 19 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 20 of 38

406

aforementioned parameters in soybean seeds at 72h of germination. Using UHPLC–

407

ESI–HRMS/MS, genistin, malonylgenistin, and genistein were identified as main

408

substances. The contents of genistin and genistein were significantly enhanced by

409

chitohexaose treatment. Moreover, a set of structural genes of soybean seeds treated

410

with and without chitohexaose were analyzed by qRT–PCR, and results suggested

411

that chitohexaose application could dramatically stimulate the transcription of genes

412

involved in isoflavone biosynthesis.

413

Supporting Information Available: Gene primers used for qRT-PCR in the present

414

work.

415

Funding

416

The present work was financially supported by the National Natural Science

417

Foundation of China (Grant No. 31660461) and the key lab of marine bioactive

418

substance and modern analytical technique, SOA (Grant No. MBSMAT-2016-06)

419

References

420

(1) Foyer, C. H. Reactive oxygen species, oxidative signaling and the regulation of

421

photosynthesis. Environ. Exp. Bot. 2018, 154, 134-142.

422

(2) Züst, T.; Agrawal, A. A. Trade-Offs Between Plant Growth and Defense Against

423

Insect Herbivory: An Emerging Mechanistic Synthesis. Annu. Rev. Plant Biol.

424

2017, 68, 513-534.

425

(3) Mouden, S.; Klinkhamer, P. G. L.; Choi, Y. H.; Leiss, K. A. Towards

426

eco-friendly crop protection: natural deep eutectic solvents and defensive

427

secondary metabolites. Phytochem. Rev. 2017, 16, 935-951. 20 ACS Paragon Plus Environment

Page 21 of 38

Journal of Agricultural and Food Chemistry

428

(4) Hichri, I.; Barrieu, F.; Bogs, J.; Kappel, C.; Delrot, S.; Lauvergeat, V. Recent

429

advances in the transcriptional regulation of the flavonoid biosynthetic pathway.

430

J. Exp. Bot. 2011, 62, 2465-2483.

431 432

(5) Xiao, J. B. Dietary flavonoid aglycones and their glycosides: Which show better biological significance? Crit. Rev. Food Sci. 2015, 57, 1874-1905.

433

(6) Maag, D.; Erb, M.; Köllner, T. G.; Gershenzon, J. Defensive weapons and

434

defense signals in plants: some metabolites serve both roles. Bioessays 2015, 37,

435

167-174.

436

(7) Yuk, H. J.; Song, Y. H.; Curtis-Long, M. J.; Kim, D. W.; Woo, S. G.; Lee, Y. B.;

437

Uddin, Z.; Kim, C. Y.; Park, K. H. Ethylene Induced a High Accumulation of

438

Dietary Isoflavones and Expression of Isoflavonoid Biosynthetic Genes in

439

Soybean (Glycine max) Leaves. J. Agric. Food Chem. 2016, 64, 7315-7324.

440

(8) Al Tawaha, A. M.; Seguin, P.; L. Smith, D. ; Beaulieu, C. Biotic elicitors as a

441

means of increasing isoflavone concentration of soybean seeds. Ann. Appl. Biol.

442

2015, 146, 303-310.

443

(9) Ghasemzadeh, A.; Ashkani, S.; Baghdadi, A.; Pazoki, A.; Jaafar, H. Z.; Rahmat,

444

A.

445

Pharmaceutical Quality of Sweet Basil (Ocimum basilicum L.) by Ultraviolet-B

446

Irradiation. Molecules 2016, 21, 1203.

447 448 449

Improvement

in

Flavonoids

and

Phenolic

Acids

Production

and

(10)Onesippe, C.; Lagerge, S. Study of the complex formation between sodium dodecyl sulfate and chitosan. Colloid. Surface A 2008, 317, 100-108. (11)Yin, H.; Zhao, X. M.; Du, Y. G. Oligochitosan: a plant diseases vaccine-a review. 21 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

450

Page 22 of 38

Carbohyd. Polym. 2010, 82, 1-8.

451

(12)Zou, P.; Tian, X. Y.; Dong, B.; Zhang, C. S. Size effects of chitooligomers with

452

certain degrees of polymerization on the chilling tolerance of wheat seedlings.

453

Carbohyd. Polym. 2017, 160, 194-202.

454

(13) Yu, Z. J.; Zhao, L. H.; Ke, H. P. Potential role of nuclear factor-kappaB in the

455

induction of nitric oxide nd tumor necrosis factor-alpha by oligochitosan in

456

macrophages. Int. Immunopharmacol. 2004, 4, 193-200.

457 458 459

(14)Il’ina,

A.

V.;

Varlamov,

V.

P.

In

vitro

antitumor

activity

of

heterochitooligosaccharides. Appl. Biochem. Micro. 2015, 51, 1-10. (15)Nguyen, N. T.; Hoang, D. Q.; Nguyen, N. D.; Nguyen, Q. H.; Nguyen, D. H.

460

Preparation,

characterization,

and

antioxidant

activity

461

oligochitosan. Green Process Synth. 2017, 6, 461-468.

of

water-soluble

462

(16)Verlee, A.; Mincke, S.; Stevens, C. V. Recent developments in antibacterial and

463

antifungal chitosan and its derivatives. Carbohyd. Polym. 2017, 164, 268-283.

464

(17)Yin, H.; Du, Y. G.; Dong, Z. M. Chitin oligosaccharide and chitosan

465

oligosaccharide: two similar but different plant elicitors. Front. Plant Sci. 2016,

466

7, 522.

467

(18)Limpanavech, P.; Chaiyasuta, S.; Vongpromek, R.; Pichyangkura, R.; Khunwasi,

468

C.; Chadchawan, S.; Lotrakul, P.; Bunjongrat, R.; Chaidee, A.; Bangyeekhun, T.

469

Chitosan effects on floral production, gene expression, and anatomical changes in

470

the Dendrobium orchid. Sci. Hortic-Amaterdam. 2008, 116, 65-72.

471

(19)Zhang, X. Q.; Li, K. C.; Liu, S. Z.; Zou, P.; Xing, R. E.; Yu, H. H.; Chen, X. L.; 22 ACS Paragon Plus Environment

Page 23 of 38

Journal of Agricultural and Food Chemistry

472

Qin, Y. K.; Li, P. C. Relationship between the degree of polymerization of

473

chitooligomers and their activity affecting the growth of wheat seedlings under

474

salt stress. J. Agric. Food Chem. 2017, 65, 501-509.

475

(20)Tawaha, A.; Seguin, P.; Smith, D.; Beaulieu, C. Biotic elicitors as a means of

476

increasing isoflavone concentration of soybean seeds. Annu. Appl. Biol. 2005,

477

146, 303-310.

478

(21)Xu, A.; Zhan, J. C.; Huang, W. D. Oligochitosan and sodium alginate enhance

479

stilbene production and induce defense responses in Vitis vinifera cell suspension

480

cultures. Acta Physiol. Plant 2015, 37, 144-156.

481

(22)Li, P. Q.; Linhardt, R. J.; Cao, Z. M. Structural characterization of oligochitosan

482

elicitor from Fusarium sambucinum and its elicitation of defensive responses in

483

Zanthoxylum bungeanum. Int. J. Mol. Sci. 2016, 17, 2076-2096.

484

(23)Van Phu, D.; Du, B. D.; Van Tam, H.; Hien, N. Q. Preparation and foliar

485

application of oligochitosan-nanosilica on the enhancement of soybean seed

486

yield. Int. J. Agric. Biol. 2017, 2, 421-428.

487

(24)Liu, H. K.; Cao, Y.; Huang, W. N.; Guo, Y. D.; Kang, Y. F. Effect of ethylene on

488

total phenolics, antioxidant activity, and the activity of metabolic enzymes in

489

mung bean sprouts. Eur. Food Res. Technol. 2013, 237, 755-764.

490

(25)Cai, S. B.; Wang, O.; Wu, W.; Zhu, S. J.; Zhou, F.; Ji, B. P.; Gao, F. Y.; Zhang,

491

D.; Liu, J.; Cheng, Q. Comparative study of the effects of solid-state fermentation

492

with three filamentous fungi on the total phenolics content (TPC), flavonoids, and

493

antioxidant activities of subfractions from oats (Avena sativa L.). J. Agric. Food 23 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

494

Page 24 of 38

Chem. 2011, 60, 507-513.

495

(26)Sun, D.; Huang, S. Q.; Cai, S. B.; Cao, J. X. Han, P. Digestion property and

496

synergistic effect on biological activity of purple rice (Oryza sativa L.)

497

anthocyanins subjected to a simulated gastrointestinal digestion in vitro. Food

498

Res. Int. 2015, 78, 114-123.

499

(27)Zhang, C. T.; Ma, Y. L.; Gao, F. D.; Zhao, Y. X.; Cai, S. B.; Pang, M. J. The free,

500

esterified, and insoluble-bound phenolic profiles of Rhus chinensis Mill. fruits

501

and their pancreatic lipase inhibitory activities with molecular docking analysis. J

502

Funct Foods 2018, 40, 729-735.

503

(28)Zhang, X.; Jia, Y. J.; Ma, Y. L.; Cheng, G. G.; Cai, S. B. Phenolic Composition,

504

Antioxidant Properties, and Inhibition toward Digestive Enzymes with Molecular

505

Docking Analysis of Different Fractions from Prinsepia utilis Royle Fruits.

506

Molecules 2018, 23, 3373.

507

(29)Yoshiara, L. Y.; Mandarino, J. M. G.; Carrão-Panizzi, M. C.; Madeira, T. B.;

508

Bonifácio da Silva, J.; Costa de Camargo, A.; Shahidi, F.; Ida, E. I. Germination

509

changes the isoflavone profile and increases the antioxidant potential of soybean.

510

J. Food Bioact. 2018, 3, 144-150.

511

(30)Huang, X. Y.; Cai, W. X.; Xu, B. J. Kinetic changes of nutrients and antioxidant

512

capacities of germinated soybean (Glycine max L.) and mung bean (Vigna

513

radiata L.) with germination time. Food Chem. 2014, 143, 268-276.

514

(31)Yang, R.; Jiang, Y.; Xiu, L. L.; Huang, J. Y. Effect of chitosan pre-soaking on the

515

growth and quality of yellow soybean sprouts. J. Sci. Food Agric. 2019, 99, 24 ACS Paragon Plus Environment

Page 25 of 38

516

Journal of Agricultural and Food Chemistry

1596-1603.

517

(32)Zhang, X. Q.; Li, K. C.; Liu, Q. L.; Xing, R. E.; Yu, H. H.; Chen, X. L.; Li, P. C.

518

Size effects of chitooligomers on the growth and photosynthetic characteristics of

519

wheat seedlings. Carbohyd. Polym. 2016, 138, 27-33.

520

(33)Masahiro, H.; Rita, B.; Roberta, M.; Alba, S.; Miyu, K.; Yoshitake, D.; Sakiko,

521

A.; Flavia, S.; Alessia, R.; Ken, T. Chitin-induced activation of immune signaling

522

by the rice receptor CEBiP relies on a unique sandwich-type dimerization. Proc.

523

Natl. Acad. Sci. 2014, 111, 404-413.

524

(34)Huang, S. Q.; Ma, Y. L.; Zhang, C. T.; Cai, S. B.; Pang, M. J. Bioaccessibility

525

and antioxidant activity of phenolics in native and fermented Prinsepia utilis

526

Royle seed during a simulated gastrointestinal digestion in vitro. J. Funct. Foods

527

2017, 37, 354-362.

528

(35)Zhang, C. T.; Ma, Y. L.; Zhao, Y. X.; Hong, Y. Q.; Cai, S. B.; Pang, M. J.

529

Phenolic composition, antioxidant and pancreatic lipase inhibitory activities of

530

Chinese sumac (Rhus chinensis Mill.) fruits extracted by different solvents and

531

interaction between myricetin ‐ rhamnoside and quercetin-rhamnoside. Int. J.

532

Food Sci.Tech. 2018, 53, 1045-1053.

533

(36)Prasain, J. K.; Reppert, A.; Jones, K.; Barnes, S.; Lila, M. A. Identification of

534

isoflavone glycosides in Pueraria lobata cultures by tandem mass spectrometry.

535

Phytochem. Anal. 2010, 18, 50-59.

536

(37)Gu, E. J.; Kim, D. W.; Jang, G. J.; Song, S. H.; Lee, S.B.; Kim, B. M.; Cho, Y.;

537

Lee, H. J.; Kim, H. J. Mass-based metabolomic analysis of soybean sprouts 25 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

538

Page 26 of 38

during germination. Food Chem. 2017, 217: 311-319.

539

(38)Zhang, S.; Zheng, Z. P.; Zeng, M. M.; He, Z. Y.; Tao, G. J.; Qin, F.; Chen, J. A

540

novel isoflavone profiling method based on UPLC-PDA-ESI-MS. Food Chem.

541

2017, 219: 40-47.

542 543

(39)Pilšáková, L.; Riečanský, I.; Jagla, F. The physiological actions of isoflavone phytoestrogens. Physiol. Res. 2010, 59, 651-664.

544

(40)Huang, J. L.; Gu, M.; Lai, Z. B.; Fan, B. F.; Shi, K.; Zhou, Y. H.; Yu, J. Q.;

545

Chen, Z. X. Functional Analysis of the Arabidopsis PAL Gene Family in Plant

546

Growth, Development, and Response to Environmental Stress. Plant Physiol.

547

2010, 153, 1526-1538.

548

(41)Jiao, C. F.; Yang, R. Q.; Gu, Z. X. Cyclic ADP-ribose mediates nitric

549

oxide-guanosine 3′,5′-cyclic monophosphate-induced isoflavone accumulation in

550

soybean sprouts under UVB radiation. Can. J. Plant Sci. 2018, 98, 47-53.

551

(42)Yu, O.; Shi, J.; Hession, A. O.; Maxwell, C. A.; Mcgonigle, B.; Odell, J. T.

552

Metabolic engineering to increase isoflavone biosynthesis in soybean seed.

553

Phytochemistry 2003, 63, 753-763.

554

(43)Gutierrez-Gonzalez, J. J.; Guttikonda, S. K.; Lam-Son Phan, T.; Aldrich, D. L.;

555

Rui, Z.; Oliver, Y.; Nguyen, H. T.; Sleper, D. A. Differential expression of

556

isoflavone biosynthetic genes in soybean during water deficits. Plant Cell

557

Physiol. 2010, 51, 936-948.

558

(44)Sohn, S. I.; Kim, Y. H.; Kim, S. L.; Lee, J. Y.; Oh, Y. J.; Chung, J. H.; Lee, K. R.

559

Genistein production in rice seed via transformation with soybean IFS genes. 26 ACS Paragon Plus Environment

Page 27 of 38

560

Journal of Agricultural and Food Chemistry

Plant Sci. 2014, 217-218, 27-35.

561

(45)Kim, J. A.; Chung, I. M. Change in isoflavone concentration of soybean ( Glycine

562

max L.) seeds at different growth stages. J. Sci. Food Agr. 2010, 87, 496-503.

563

Figure captions

564

Fig.1 Negative ion current chromatograms of the control (a) and chitohexaose-treated

565

(b) soybean seeds at 72 h of germination.

566

Fig.2 MS/MS spectra and fragmentation patterns of three predominant isoflavones

567

detected in the control and chitohexaose-treated soybean seeds at 72 h of germination

568

by Q-Exactive Orbitrap Mass: genistin (a), malonylgenistin (b) and genistein (c).

569

Fig. 3 Schematic diagram of action mechanism. A diagram of a branch of

570

phenylpropanoid pathway (a). Several key genes used in present work: PAL:

571

phenylalanine ammonialyase; CHS: chalcone synthase; CHI: chalcone isomerase; and

572

IFS: isoflavone synthase; hypothetical model of the activation by oligochitosans of

573

different DPs on soybean seeds (b).

574

Fig. 4 Relative expression of isoflavonoid biosynthetic genes in control and

575

chitohexaose-treated soybean seeds at 72h of germination. (a): PAL, phenylalanine

576

ammonialyase; (b) and (c): CHS6 and CHS7, respectively (CHS, chalcone synthase);

577

(d) and (e): CHI1 and CHI2, respectively (CHI, chalcone isomerase); (f) and (g): IFS1

578

and IFS2, respectively (IFS, isoflavone synthase). *Significant difference between the

579

control and chitohexaose-treated soybean seeds (p < 0.05).

27 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 28 of 38

Table 1Total phenolic contents (mg/100g of DW*) of soybean seeds (Glycine max) with different treatments during germination period 0h 24h 48h 72h 96h Control

108.06 ± 2.87aA

106.55 ± 3.63aA

109.03 ± 2.12aA

116.76 ± 3.41aB

118.97 ± 1.95aB

Chitotriose

113.15 ± 4.22aAB

109.14 ± 2.38aA

116.76 ± 3.33bB

118.28 ± 3.13aB

119.13 ± 2.67aB

Chitotetraose

111.91 ± 5.32aA

112.90 ± 2.26aA

122.07 ± 2.03cB

124.58 ± 2.16bB

119.05 ± 3.67aB

Chitopentaose

112.19 ± 1.86aA

109.79 ± 3.84aA

117.22 ± 3.87bB

120.30 ± 2.93bB

116.35 ± 4.38aB

Chitohexaose

113.34 ± 3.98aA

110.22 ± 4.11aA

121.40 ± 3.56cB

138.44 ± 1.95cC

129.19 ± 1.55bD

Chitoheptaose

112.29 ± 2.32aAB

109.37 ±2.94aA

116.90 ± 2.17bB

125.46 ± 2.32dC

119.43 ± 1.97aD

Values were expressed as the mean ± SD of three replicates; values with different lower case indicated significant differences between different samples at the same germination time(p < 0.05); values with different upper case letters indicated significant differences between the same sample at different germination time(p < 0.05);*DW: dry weight of soybean sprout.

28 ACS Paragon Plus Environment

Page 29 of 38

Journal of Agricultural and Food Chemistry

Table 2 Total flavonoid contents (mg/100g of DW) of soybean seeds (Glycine max) with different treatments during germination period 0h 24h 48h 72h 96h Control

31.11 ± 0.89aA

31.50 ± 2.43aA

37.68 ± 2.64aB

41.01 ± 1.39aC

44.70 ± 1.54aD

Chitotriose

29.85 ± 0.79aA

36.89 ± 1.89bB

40.43 ± 3.64aC

43.44 ± 2.32aC

43.64 ± 1.94aC

Chitotetraose

29.95 ± 1.33aA

35.71 ± 0.64cB

38.83 ± 2.83aC

42.10 ± 1.34aC

41.10 ± 2.95aC

Chitopentaose

32.55 ± 0.55aA

37.43 ± 0.79cB

39.50 ± 1.14aB

47.33 ± 0.96bC

44.32 ± 1.42aD

Chitohexaose

44.71 ± 1.98bA

51.62 ± 1.63dA

53.34 ± 1.95bA

80.04 ± 2.08cB

62.29 ± 0.50bC

Chitoheptaose

36.97 ± 0.75cA

47.19 ± 1.75eB

53.86 ± 2.39bC

70.29 ± 1.77dD

57.54 ± 1.34cC

Values were expressed as the mean ± SD of three replicates; values with different lower case indicated significant differences between different samples at the same germination time(p < 0.05); values with different upper case letters indicated significant differences between the same sample at different germination time(p < 0.05); *DW: dry weight of soybean sprout.

29 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 30 of 38

Table 3 DPPH radical scavenging activities of soybean seeds (Glycine max) with different treatments during germination period 0h 24h 48h 72h 96h DPPH radical scavenging activity (%) at 20.0 mg of dry soybean seeds/mL Control

58.90 ± 3.84aA

54.68 ± 4.22aA

56.60 ± 1.18aA

57.67 ± 0.93aA

55.26 ± 2.22aA

Chitotriose

60.03 ± 1.56aA

54.17 ± 2.19aB

56.48 ± 0.93aB

60.16 ± 0.87bA

55.05 ± 0.96aB

Chitotetraose

62.28 ± 3.52aA

60.32 ± 1.11bA

55.14 ± 2.29aB

56.89 ± 1.97aB

57.44 ± 3.28aB

Chitopentaose

60.25 ± 0.75aA

51.08 ± 0.88aB

54.97 ± 3.09aB

54.88 ± 2.27aB

53.79 ± 1.20aB

Chitohexaose

59.86 ± 2.12aA

59.31 ± 0.29bA

56.21 ± 2.72aA

69.62 ± 1.45cB

65.68 ± 2.01cC

Chitoheptaose

61.01 ± 2.92aA

63.67 ± 1.78cA

64.38 ± 3.26bA

61.22 ± 2.80bA

54.98 ± 1.43aB

Values were expressed as the mean ± SD of three replicates; values with different lower case indicated significant differences between different samples at the same germination time(p < 0.05); values with different upper case letters indicated significant differences between the same sample at different germination time(p < 0.05).

30 ACS Paragon Plus Environment

Page 31 of 38

Journal of Agricultural and Food Chemistry

Table 4 ABTS radical scavenging activities of soybean seeds (Glycine max) with different treatments during germination period 0h 24h 48h 72h 96h 3.17aA

ABTS radical scavenging activity (%) at 10.0 mg of dry soybean seeds/mL 62.93 ± 3.98aB 68.68 ± 1.60aA 67.26 ± 2.98aA 68.28 ± 1.92aA

Control

71.06 ±

Chitotriose

79.88 ± 1.01bA

68.93 ± 2.93bB

76.77 ± 2.45bA

70.84 ± 1.98aB

74.99 ± 2.28bA

Chitotetraose

77.80 ± 2.43bA

75.16 ± 1.78cA

77.68± 3.32bA

76.16 ± 2.48bA

69.02 ± 3.21aB

Chitopentaose

72.67 ± 1.52aA

63.67 ± 2.66aB

75.27 ± 1.54bA

79.39 ± 1.12bC

80.41 ± 2.60cD

Chitohexaose

80.39 ± 2.28bAC

76.74 ± 1.93cB

74.04 ± 2.35bB

85.71 ± 2.43cD

83.90 ± 1.17cC

Chitoheptaose

78.04 ± 1.39bA

77.95 ± 2.31cA

80.10 ± 1.06cA

83.16 ± 3.45cB

81.26 ± 2.48cA

Values were expressed as the mean ± SD of three replicates; values with different lower case indicated significant differences between different samples at the same germination time(p < 0.05); values with different upper case letters indicated significant differences between the same sample at different germination time(p < 0.05).

31 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 32 of 38

Table 5 Identification and quantification of isoflavones in control (CG) and chitohexaose -treated (CHxG) groups at 72h of germination CG CHxG Rt* Error Peak No. Compounds Formula [M-H]Fragments References μg/g of D.W. (min) (ppm) 1

Naringenin-7-O-glucoside

C21H21O10

7.63

433.1138

151.0100(5), 270.0508(77)

1.955

Standard

15.56±0.89 a

25.87±1.27 b

2

(-)-epigallocatechin

C15H14O7

8.37

305.0696

165.0902(5), 305.0695(5)

9.819

Standard

0.60±0.03a

1.21±0.10b

3

Daidzin

C21H20O9

9.79

415.1027

252.0421(100), 415.1027(5)

0.798

Standard

34.20±0.68a

49.39±1.97b

4

Glycitin

C22H22O10

10.13

445.1131

283.0573(25), 445.1131(5)

0.330

Standard

0.22±0.01a

0.52±0.02b

1.272

36, 37

87.88±2.17a

105.71±3.86b

5

Malonyldaidzin#

C24H22O12

11.74

501.1034

253.0499(40), 295.0609(5), 457.1130(54), 501.1034(100)

6

Acetyldaidzin#

C23H22O10

12.01

457.1129

253.0492(80), 457.1129(5)

0.095

36, 37

0.92±0.05a

2.01±0.14b

C16H12O5

12.06

283.0608

268.0371(50)

2.332

Standard

13.58±0.84a

18.47±0.66b

7

Glycitein

32 ACS Paragon Plus Environment

Page 33 of 38

Journal of Agricultural and Food Chemistry

8

Genistin

C21H10O10

12.16

268.0372(100), 431.0973 269.0450(63), 431.0973(60)

0.317

Standard

1.329

37, 38

149.74±4.37a

250.54±7.67b

Malonylgenistin#

C24H22O13

13.75

268.0372(98), 269.0450(100), 517.0980 473.1082(8), 517.0980(30)

10

Acetylgenistin#

C23H22O11

13.91

473.1080

269.0451(100), 473.1080(10)

0.228

37, 38

1.07±0.07a

4.70±0.10b

11

Daidzein

C15H10O4

14.83

253.0500

117.0331(5), 253.0500(100)

1.678

Standard

78.48±1.85a

102.20±3.19b

12

Genistein

C15H10O5

16.73

269.0451

107.0123 (20), 133.0281(55), 269.0451(100)

2.305

Standard

70.90±2.02a

164.05±5.21b

690.91

943.61

9

Total content *Rt:

237.76±11.02 a

218.94±8.11b

Retention time; # daidzin was used for the semi-quantification of both acetyldaidzin and malonyldaidzin, and genistin was used for the

semi-quantification of both acetylgenistin and malonylgenistin; Values were expressed as the mean ± SD of three replicates; values with different lower case in the same row indicated significant differences between control and chitohexaose -treated groups (p < 0.05).

33 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 34 of 38

Fig. 1

34 ACS Paragon Plus Environment

Page 35 of 38

Journal of Agricultural and Food Chemistry

Fig. 2

35 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 36 of 38

Fig. 3

36 ACS Paragon Plus Environment

Page 37 of 38

Journal of Agricultural and Food Chemistry

Fig. 4

37 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 38 of 38

TOC

38 ACS Paragon Plus Environment