Effects of different oligochitosans on isoflavone metabolites

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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

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Journal of Agricultural and Food Chemistry

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Effects of different oligochitosans on isoflavone metabolites, antioxidant activity

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

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germination

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

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aYunnan

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

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b

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

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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

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

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of China, 266101

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* Corresponding author and proofs

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Shengbao Cai

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

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Abstract

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

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

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

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

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

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

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

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significantly increased the aforementioned parameters in soybean seeds 72 h after

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

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

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

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

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

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application could dramatically stimulate the transcription of these genes.

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

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oligochitosan, soybean seeds


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

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

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

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

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

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

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

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

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

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

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

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flavonoid family encompasses thousands of compounds and can be classified into

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

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

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

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

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

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

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

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

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


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

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

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oligochitosan has been performed, and findings indicate that oligochitosans possess

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

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

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

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

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condition stress. Previous studies reported that oligochitosans could increase the

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photosynthesis of Dendrobium orchids by increasing their chlorophyll content and

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

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

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

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

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

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

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

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

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oligochitosans to induce accumulation of secondary metabolites, especially

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

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oligochitosan in flavonoid accumulation have yet to be illuminated.

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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

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

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

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

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

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

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were analyzed to delineate the underlying mechanisms of oligochitosan on isoflavone

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metabolite accumulation in soybean seeds during germination.

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MATERIALS AND METHODS

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Chemicals and reagents

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

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

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

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

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

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

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

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

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

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

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

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Soybean seed treatments

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


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

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

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

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

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

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

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

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

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

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

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identification and quantification of isoflavone metabolites and evaluation of TPC,

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

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

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

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(0.2 μm) for further analysis.

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Determination of TPC

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

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

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

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Determination of TFC

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

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

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milligrams of rutin equivalents per 100 g of DW.

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Evaluation of DPPH radical scavenging capacity

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

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

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

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thrice.

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Evaluation of ABTS radical scavenging capacity

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

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

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

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

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Identification and quantification of flavonoid metabolites by UHPLC–ESI–

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MS/MS

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The flavonoid metabolites of soybean seeds with and without chitohexaose

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

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

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

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

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

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

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

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


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

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

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

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flavonoid metabolites determined under the same conditions.

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Analysis of isoflavone biosynthetic gene expression by qRT–PCR

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Total RNA was extracted from soybean seeds with and without chitohexaose

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

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

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

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

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

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

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

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

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

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

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as an internal control.

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Statistical analysis

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

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

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

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

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RESULTS AND DISCUSSION

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TPC and TFC of soybean seeds during germination

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

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

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

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

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

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

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

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

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

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

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chitotetraose, chitopentaose, chitohexaose, and chitoheptaose significantly increased

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

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48 h of germination (p < 0.05).

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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germination.

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

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

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

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

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

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that both chitohexaose and chitoheptaose could markedly alleviate chilling stress in

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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activate defense signaling.33

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DPPH and ABTS radical scavenging activity of soybean seeds during

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germination

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

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

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soybean seeds treated with oligochitosans of different DPs during germination were

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

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

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

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

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

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

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Chitotriose and chitotetraose significantly increased the DPPH radical scavenging

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

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contrast, chitopentaose did not significantly affect the DPPH radical scavenging

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

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

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chitoheptaose showed the strongest effects on enhancing the DPPH radical

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

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respectively. In particular, chitohexaose treatment increased the DPPH radical

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

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germination.

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

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

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

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during which ABTS radical scavenging activity unexpectedly significantly decreased

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

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five oligochitosans significantly enhanced the ABTS radical scavenging activity of

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

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

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

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

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

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oligochitosans of different DPs on DPPH and ABTS radical scavenging activities

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during soybean seed germination may be attributed to the complicated compositions

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

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

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


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

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

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

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

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radical scavenging activity and between TFC and ABTS radical scavenging activity

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

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

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analysis results indicated that phenolic compounds, especially flavonoids, may

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contribute significantly to the antioxidant activity of soybeans during germination,

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consistent with the findings of many previous studies reporting that phenolic

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

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Identification and quantification of flavonoid metabolites

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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ethylene could significantly induce the accumulation of daidzin, genistin,

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malonyldaidzin, and malonylgenistin in soybean leaves; these isoflavones were

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dramatically upgraded by chitohexaose treatment in soybean seeds in the present

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

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

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produce functional foods or agricultural products because they exert relatively fewer

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

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

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applications to address cost issues.

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Expression of isoflavone biosynthetic genes

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

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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


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


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


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

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

427

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

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

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

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

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Dietary Isoflavones and Expression of Isoflavonoid Biosynthetic Genes in

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

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elicitor from Fusarium sambucinum and its elicitation of defensive responses in

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application of oligochitosan-nanosilica on the enhancement of soybean seed

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

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

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

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

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

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Funct Foods 2018, 40, 729-735.

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Antioxidant Properties, and Inhibition toward Digestive Enzymes with Molecular

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Docking Analysis of Different Fractions from Prinsepia utilis Royle Fruits.

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Molecules 2018, 23, 3373.

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

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

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

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

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

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

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

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

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

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1596-1603.

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

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Size effects of chitooligomers on the growth and photosynthetic characteristics of

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

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

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

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

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

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

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and antioxidant activity of phenolics in native and fermented Prinsepia utilis

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

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2017, 37, 354-362.

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(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

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

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

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

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

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isoflavone glycosides in Pueraria lobata cultures by tandem mass spectrometry.

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

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

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


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Page 26 of 38

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

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

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

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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

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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

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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.;

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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.

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(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

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560


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).



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.


Page 29 of 38


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.



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).


Page 31 of 38


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).



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


Page 33 of 38


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).



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Fig. 1


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Fig. 2



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Fig. 3


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Fig. 4



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TOC


Effects of different oligochitosans on isoflavone metabolites

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