Why does chickpea germination improve antioxidative activity of

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Food and Beverage Chemistry/Biochemistry

Why does chickpea germination improve antioxidative activity of soluble phenolic compounds? Minwei Xu, Zhao Jin, Jae Bom Ohm, Paul Schwarz, jiajia rao, and Bingcan Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02208 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Journal of Agricultural and Food Chemistry

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Why does chickpea germination improve antioxidative activity

3

of soluble phenolic compounds?

4

Minwei Xu1, Zhao Jin1, Jae-Bom Ohm2, Paul Schwarz1, Jiajia Rao1, Bingcan Chen1*

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1. Department of Plant Sciences, North Dakota State University, Fargo, ND 58108, USA

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2. USDA-ARS, Red River Valley Agricultural Research Center, Cereal Crops Research Unit,

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Hard Spring and Durum Wheat Quality Lab, Fargo, ND 58108, USA

8 9 10

Resubmission to Journal of Agricultural and Food Chemistry

11 12 13 14 15 16 17 18 19 20

*

To

whom

correspondence

should

be

addressed.

[email protected]

ACS Paragon Plus Environment

Tel.

(701)

231-9450,

e-mail:


21

ABSTRACT:

22

Our recent study found that antioxidative activity of phenolic compounds extracted

23

from germinated chickpea was boosted both in an in vitro assays and in oil-in-water

24

emulsion.1 The purpose of this study was to elucidate the mechanism by which the

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composition of phenolic compounds extracted from chickpea germination enhances

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its antioxidative activity. Liquid chromatography coupled with electrospray ionization

27

quadrupole time of flight mass spectrometry (LC-ESI-QTOF-MS) and size-exclusion

28

chromatography with multi-angle light-scattering and refractive index detection

29

(SEC-MALS-RI) were employed to evaluate the phenolic composition of soluble

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phenolic compounds (free and bound) and molar mass of soluble bound phenolic

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compounds, respectively, over the period of 6 days germination. According to

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principal component analysis of the interrelationship between germination time and

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phenolic composition, it is revealed that protocatechuic acid 4-O-glucoside and

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6-hydroxydaidzein played a pivotal role in the soluble free phenolic compounds,

35

while gentisic acid and 7,3',4'-trihydroxyflavone were important in the soluble bound

36

phenolic compounds. Molar masses of soluble bound phenolic compounds were

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increased after 6 days germination. A protective and/or a dual antioxidative effects

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were proposed to explicate how antioxidative activity of soluble bound phenolic

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compounds in oil-in-water emulsions was improved with germination.

40 41

KEYWORDS: germination, chickpea, antioxidative activity, phenolic compounds,

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molar mass


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1. Introduction

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Chickpeas have high nutritional value and play an important role in traditional diets in

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India, Pakistan, and Mexico.2 Chickpea constituents are a well-recognized source of

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nutrients including proteins, carbohydrates, minerals and phytochemicals.3 Phenolic

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compounds, a class of critical phytochemicals, are mainly composed of phenolic acids

48

and flavonoids in chickpea.4 Two forms of phenolic compounds, i.e., insoluble and

49

soluble, are present in chickpea seeds. Soluble phenolic compounds can be directly

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extracted by polar solvent, and are characterized as either soluble free or soluble

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bound phenolic compounds. Soluble free phenolic compounds can be separated from

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soluble phenolic compounds with medium polarity solvents. Residues of the soluble

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phenolic compounds are termed as soluble bound phenolic compounds. Therefore,

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soluble free phenolic compounds can be considered as pure phenolic compounds

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without or with simple moieties such as protocatechuic acid or protocatechuic acid

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hexoside.5 Soluble bound phenolic compounds are phenolic compounds conjugated

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with relatively higher molecular weight of soluble moieties such as soluble

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polysaccharides, protein, and polar lipids.6,7

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Germination is an economical and effective way to improve the quantity of total

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phenolic content in chickpea.8 During germination, the cell wall is loosened to expose

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protein and starch to the protease and carbohydrase which are secreted into the

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endosperm. Decomposed protein and starch constituents, such as amino acids and

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glucose, are used for respiration or synthesis of new cell components for plant

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development, thus causing significant changes in the biochemical characteristics of



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dormant seeds. In the meantime. soluble free phenolic compounds can either be

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originated from glucose through glycolysis, oxidative pentose phosphate pathway, and

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shikimate pathway,9 or from amino acids, such as phenylalanine and tyrosine directly.

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The newly biosynthesized soluble free phenolic compounds can be transferred and

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conjugated to form soluble bound phenolic compounds, or even polymerized to

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produce insoluble bound phenolic compounds.10 Conversely, moieties of insoluble

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bound and soluble bound phenolic compounds can be decomposed to form soluble

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free phenolic compounds during germination.1 In addition, phenolic composition

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could also be altered during germination as Wu et al.8 reported that both diversity and

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content of isoflavones were improved by chickpea germination, and isoflavone

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contents increased by over 100 fold the first 4 days after germination.

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Our recent study investigated the antioxidative activity of soluble free and bound

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phenolic compounds extracted from germinated chickpea in an in vitro assays and in

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oil-in-water emulsion system. The results indicated that the in vitro antioxidative

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activity of both soluble free and soluble bound phenolic compounds had increased

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with increasing germination time.1 The antioxidative activity of soluble free phenolic

81

compounds was greater than soluble bound phenolic compounds in an in vitro assays.

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In contrast, soluble bound phenolic compounds had superior activity against lipid

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oxidation in oil-in-water emulsions. And longer germination time (6 days) exerted

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stronger boost effect on the antioxidative activity.

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With previous results, it was hypothesized that the enhanced antioxidative activity and


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the different activity between chickpea soluble free and soluble bound phenolic

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compounds in an in vitro assays and in oil-in-water emulsion may be related to the

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composition and structure change of phenolic compounds over the course of

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germination. Therefore, the objectives of this study were to (i) identify and

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semi-quantitatively interpret the main soluble phenolic compounds with liquid

91

chromatography–electrospray

92

spectrophotometer (LC-ESI-QTOF-MS); (ii) investigate the molar mass of soluble

93

bound phenolic compounds with size exclusion chromatography–multiangle laser

94

light scattering detector–refractive index detector (SEC-MALS-RI); (iii) propose a

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mechanism of how germination influence antioxidative activity of soluble phenolic

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compounds in chickpea.

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2. Materials and methods

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2.1 Chemicals

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Chickpea (Cicer aretinium L.) was gifted from AGT Food and Ingredients (Minot, ND,

100

USA). Sodium hydroxide, hydrochloric acid, acetonitrile, acetone, acetone, ethyl ether,

101

ethyl acetate, and other materials were purchased from VWR International. (West

102

Chester, Pa., U.S.A.).

103

2.2 Germination of Chickpea

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The method of chickpea germination was described by Xu and coworkers and applied

105

without modification.1

ionization–quadrupole


time

of

flight–mass


106

2.3 Extraction of Phenolic Compounds

107

Extraction of soluble free (CF0, CF2, CF4 and CF6) and soluble bound phenolic

108

compounds (CB0, CB2, CB4 and CB6) from chickpeas at 0, 2, 4, and 6 days after

109

germination was followed as described by Xu and coworkers without modification.1

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2.4 Hydrolyzation of soluble bound phenolic compounds

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Soluble bound phenolic compounds collected from section 2.3 were hydrolyzed for

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the evaluation of phenolic composition. Briefly, 30 mL soluble bound phenolic

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compounds were concentrated into 3 mL by freeze drying. Two hundred microliters of

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concentrated soluble bound phenolic compounds were hydrolyzed with 2 mL 3N

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sodium hydroxide, purged with nitrogen, and shaken for 1 h. After adjusting pH to 2-3,

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3 mL ethyl ether/ethyl acetate (1/1, v/v) was added to extract the phenolic compounds.

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The extraction was repeated thrice. Supernatants were collected and 3 mL of 6 N

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hydrochloric acid was added to the solution purged with nitrogen to hydrolyze the

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residues for 20 min with use of a 95 °C water bath. Aliquots of three milliliters of

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ethyl ether/ethyl acetate (1/1, v/v) were added to extract the phenolic compounds. The

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combined ethyl ether/ethyl acetate solvents were taken to dryness using a stream of

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nitrogen. Five hundred microliters of acetonitrile were added into each sample and

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stored at -8 °C for structure characterization.

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2.5 LC-ESI-QTOF-MS analysis

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Both soluble free phenolic compounds and hydrolyzed soluble bound phenolic

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compounds were tested with LC-ESI-QTOF-MS according to the method with minor


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modification, as described by Kadam and Lele (2017).11 An Agilent 1290 series

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Liquid Chromatography system utilizing a Kinetex(R) C18 (2.6 µm, 150 mm × 4.6

129

mm) column was used to separate phenolic compounds. The mobile phase comprised

130

of water (solvent A) and acetonitrile (solvent B) with the gradient: 0–5 min (A: 95%,

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B: 5%), 30–40 min (A: 0%, B: 100%), 41–45 min (A: 95%, B: 5%) system with a

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flow rate of 0.5 mL/min and a column temperature of 30 °C. The injection volume

133

was 20 µL with a total run time of 45 min.

134

A diode array detector (DAD) with a working range from 190 to 600 nm was

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employed to observe the UV absorption of the separated chickpea extracts. An Agilent

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G6540 UHD Accurate QTOF-MS was utilized for analysis of the chromatographed

137

chickpea extracts. Sample ionization was achieved using an Electrospray Ionization

138

(ESI) interface in negative ion mode. The gas and vaporizer temperatures were set to

139

300 °C, with a drying gas flow rate of 7 L/min. The nebulizer (N2) was set at 50 psig

140

with a fragmentor voltage set to 200 V, skimmer voltage at 65 V, octopole RF voltage

141

at 750 V, and a capillary voltage at 4000 V. The collision energy was set as 0, 10, 25,

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30 and 40 eV. The full scan mass of the mass spectrophotometer covered the range of

143

m/z from 100 to 1000.

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Data analysis was performed using MassHunter Qualitative Analysis software B.05.00

145

(Agilent technologies). The identification of the detected compounds proceeded by

146

generation of candidate molecular formula with a mass accuracy limit of 5 ppm and a

147

MS score >80 (related to the contribution to mass accuracy, isotope abundance and



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isotope spacing). Agilent personal Compound Database Library (PCDL) version

149

B.05.01 build 92 was employed to create the custom database. For the retrieval of

150

phenolic compound chemical structure, the following databases were consulted:

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ChemSpider

152

MassBank

153

(http://metlin.scripps.edu), and Phenol-Explorer (http://www.phenol-explorer.eu).

154

2.6 SEC-MALS-RI analysis

155

Soluble bound phenolic compounds collected from Section 2.3 were concentrated by

156

reducing the volume by 90%, and the resulting solutions filtered through a 0.2 µm

157

polytetrafluoroethylene (PTFE) disposable membrane filter prior to SEC-MALS-RI

158

analysis. SEC-MALS-RI analysis was carried out using a Yarra 3 µm SEC-4000, 300

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× 7.8 mm SEC column and an Agilent G1315 C DAD detector, an Agilent 1362 A RI

160

detector, and a DAWN® HELEOS™ II MALS detector. ASTRA® 7.1.2.5 software

161

was used to analyze the data. Deionized water was used as an eluent for the

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SEC-MALS-RI analysis. SEC conditions were set as follows: injection volume of 10

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µL, mobile phase flow rate of 0.4 mL/min, and a column temperature of 30 °C.

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Specific refractive index increments (dn/dc) of sample solutions with concentrations

165

in the range of 0.8–1.5 mg/mL were determined using an Agilent 1362 A RI detector.

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The dn/dc values of CB0, CB2, CB4, and CB6 were 0.147, 0.145, 0.127 and 0.125

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mL/g, respectively.12,13

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2.7 Statistical Analysis

(http://www.chemspider.com), (http://www.massbank.jp),

HMDB

METLIN


(http://www.hmdb.ca/), Metabolite

Database

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The experiments were performed at least twice and data were expressed as mean±

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standard deviation of duplicate or triplicate measurements. Data were statistically

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analyzed using statistical software, SAS version 9.4 (SAS institute Inc. Cary, NC).

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One-way analysis of variance (ANOVA) was conducted, and significant difference

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was defined at p < 0.05 by Tukey’s test. The relationships between germination time

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and phenolic compounds of chickpea extracts were determined by principal

175

component analysis (PCA) based on the counts of each phenolic compounds using

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SPSS 22.0 (SPSS Inc., Armonk, NY).

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3. Results and discussion

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3.1 Effect of germination time on major phenolic compounds in chickpea

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In total, 16 phenolic compounds from germinated chickpea were identified by m/z

180

value in conjunction with product ion and retention time (Table 1). Most of these

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phenolic compounds increased during the period of germination, with little decrease

182

being observed by virtue of ion counts (Table 2). However, germination time had a

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variable impact on soluble free and soluble bound phenolic compounds in terms of

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their content and composition.

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3.1.1 Soluble free phenolic compounds

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During germination, phenolic acids and flavonoids are synthesized for the quenching

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of reactive oxygen species or as precursors to synthesize plant tissue, such as lignin.14

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As a result, individual phenolic compounds in free form are anticipated to increase

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upon germination. A dramatic increase in the content of soluble free phenolic



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compounds was observed during chickpea germination in light of the remarkable

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increase of both peak area and peak numbers at 260 nm (Fig. 1A).

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Semi-quantification by LC-ESI-QTOF-MS also highlighted the content increase of

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hesperetin (9), 7,3',4'-trihydroxyflavone (7), 8-hydroxydihydrodaidzein (4), and

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6-hydroxydaidzein (8) during chickpea germination (Table 2). However, not all the

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soluble free phenolic compounds increased during germination, as seen from the data

196

in Table 2. The ion counts of afzelechin (5), prunetin (6), formononetin (14), and

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glycitein (16) declined during germination. Two reasons may account for this

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phenomenon. First, the depleted soluble free phenolic compounds may have be

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consumed by actively scavenging radicals generated from chickpea physiological

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activity during germination. Second, soluble free phenolic compounds may be

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transformed into soluble bound form by the transferase.15 This can be supported by

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the fact that the contents of afzelechin (5), prunetin (6), formononetin (14), and

203

glycitein (16) in soluble bound form were raised over the course of germination

204

(Table 2). The similar transformation between soluble and insoluble phenolic

205

compounds during lentil germination were reported by Yeo and Shahidi.16

206

Principal component analysis (PCA) was carried out to gain an overview of the

207

similarities and differences among compositions of soluble free phenolic compounds

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during germination. The first two principal components explained 43.65% and 30.13%

209

of the total variance in the data set (Fig. 2). CF2 and CF4 showed high correlation

210

with PC1, while CF6 had high relationship with PC2 (Fig. 2A). In regard to the

211

loading of phenolic compounds, protocatechuic acid 4-O-glucoside (1) and


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6-hydroxydaidzein (8) loaded heavily in PC2, while negatively loaded in PC1 (Fig.

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2B). Prunetin (6) and formononetin (14) were related with both PC1 and PC2.

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However, decreased amounts of prunetin (6) and formononetin (14) were observed

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with chickpea germination. Consequently, only protocatechuic acid 4-O-glucoside (1)

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and 6-hydroxydaidzein (8) were considered as main phenolic acids responsible for the

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enhanced in vitro antioxidative activity of CF6.

218

Protocatechuic acids and its derivatives are widely present in cereals and legumes,

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such as black rice, black wheat, oats, lentils, pinto beans, kidney bean, etc.17 Xu et

220

al.18 reported that protocatechuic acids had strong free radical scavenging activity in

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cultured neural cells. A natural source of 6-hydroxydaidzein was first identified by

222

Hirota

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2,2-diphenyl-1-picrylhydrazyl (DPPH)-radical scavenging activity as high as that of

224

α-tocopherol. In addition, 6-hydroxydaidzein was more effective antioxidant than

225

quercetin and ascorbic acid in oxygen radical absorbing capacity (ORAC) assay and

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to prevent the in vitro oxidation of low-density lipoproteins (LDL).20

227

3.1.2 Soluble bound phenolic compounds

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Soluble phenolic compounds of chickpea seeds are mainly present in bound form,

229

which can be synthesized through the conjugation of free phenolic compounds with

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the soluble moieties. Alternatively, soluble bound phenolic compounds can be

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produced through the hydrolysis of insoluble phenolic compounds such as by cell wall

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decomposition during germination.17

et

al.19

from

soybean

miso

and

was


proved

to

exhibit


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The bound phenolic compounds were observed to increase during chickpea

234

germination, as supported by the DAD results presented in Fig. 1B. Peaks were

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further investigated via LC-ESI-QTOF-MS (Table 1). Most of the identified soluble

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bound

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4-hydroxy-8-methoxy-2H-fruo[2,3-h]-1-benzopyran-2-one

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hesperetin (9), 7,3',4'-trihydroxyflavone (7), 8-hydroxydihydrodaidzein (4), prunetin

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(6), pseudobaptigenin (13), formononetin (14), 3',4',5,7-tetrahydroxy isoflavanone

240

(15), glycitein (16), and gentisic acid (2).

241

The level of soluble bound phenolic compounds can also be reduced by metabolic

242

activity by the break down into soluble free phenolic compounds or the conversion

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into insoluble bound phenolic compounds. For instance, the amount of

244

6-hydroxydaidzein (8) in soluble bound form was observed to decrease and may have

245

decomposed into free form based on the increase in soluble free form (Table 2). In

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contrast, isolicoflavonol (12) and 1-naphthyl acetate (11) may be transformed into

247

their insoluble form rather than free form, as little of them were detected in the

248

soluble free extracts.

249

Principal component analysis (PCA) was applied to gain an insight of the similarities

250

and differences among the compositions of soluble bound phenolic compounds during

251

germination. CB0 and CB2 had opposing influence on phenolic compounds

252

composition as compared with CB4 and CB6 based on their locations on PC1 and

253

PC2 score plots. This suggests that the composition of soluble bound phenolic

phenolic

compounds

had

increased,


(3),

such afzelechin

as (5),

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compounds had a significant change within 2 days after germination. As CB4 and

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CB6 were located further to the right of PC1 while to the middle of PC2, PC1 was the

256

dominant component rather than PC2. Gentisic acid (2), 7,3',4'-trihydroxyflavone (7),

257

and 1-naphthyl acetate (11) were heavily loaded on PC1, which means they should be

258

the major phenolic components of CB4 and CB6.

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In regard to our previous study, the antioxidative activity of soluble bound phenolic

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compounds increased with time after germination both in an in vitro assays and in

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oil-in-water emulsions.1 DPPH radical scavenging and ORAC value of soluble bound

262

phenolic compounds increased from 45.7 µmol TE/L and 0.61 µmol TE/mL at 0 day

263

to 75.7 µmol TE/L and 1.51 µmol TE/mL at 6 days germination, respectively.1 CB4

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and CB6 could also retard lipid oxidation of stripped soybean oil-in-water emulsion

265

by extending the lag phase to 12 and 14 days, respectively. Since the same trends were

266

observed on the content increase of gentisic acid (2) and 7,3',4'-trihydroxyflavone (7)

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during germination, they might be the principle phenolic compounds contributing to

268

the improved antioxidative activity of CB4 and CB6.

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Gentisic acid (2) and 7,3',4'-trihydroxyflavone (7) are both effective antioxidants as

270

reported by researchers. Villaño et al.21 reported that gentisic acid had a lower IC50

271

(the amount of antioxidant needed to decrease the DPPH radical concentration by

272

50%) than protocatechuic acid, siringic acid, caffeic acid, caftaric acid, and ferulic

273

acid. Jung et al.22 reported that 7,3',4'-trihydroxyflavone had a 2.20 µM IC50 value of

274

DPPH, lower than L-ascorbic acid (12.78 µM). In addition, 7,3',4'-trihydroxyflavone,



275

the major active constituent extracted from Albizzia julibrissin bark by ethyl acetate,

276

could quench 58.71 % hydroxyl radicals which was higher than the same amount of

277

L-Ascorbic acid.22

278

Although LC-ESI-QTOF-MS was able to identify 16 compounds, it should be pointed

279

out that there might still be other unknown phenolic compounds or the interactions

280

among the components in the extracts, which may contribute to the overall antioxidant

281

activity of the extracts.

282

3.2 Effect of germination time on molar mass of major soluble bound phenolic

283

compounds in chickpea

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SEC-MALS-RI was used to further study molecular mass change of soluble bound

285

phenolic compounds during chickpea germination. Size exclusion chromatography

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separates solutes based on the particle size or molecular weight of compounds: large

287

particle size or big molecular weight elutes out faster than small particle size. The UV

288

results (260 nm) shown in Fig. 3 indicate the peak area with larger particle size (RT

289

27-32 min) increased with germination time. In addition, SEC peaks attributed to

290

smaller particle size peaks were generated during the period of 4-6 days germination.

291

This phenomenon was attributed to the biosynthesis of soluble bound phenolic

292

compounds.

293

The detection principle of RI detector involves measuring the change in refractive

294

index of the column effluent passing through the detector. The main peak shown in

295

Fig. 3 (RT 27-31 min) indicates that most of the solutes of soluble bound phenolic


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compounds are eluted with the first peak. Similar results were reported in the

297

fractionation of lentil extracts using a SEC method.23 The average molar mass of the

298

first peak derived from the samples in different germination time was evaluated by the

299

combination of RI and MALS detector. These results were shown in Fig. 3 and Table

300

3, and indicated the increase of both number average molecular weight (Mn) and

301

weight average molecular weight (Mw) with increasing germination time. Six days

302

after germination, Mn and Mw of soluble bound phenolic compounds were

303

statistically larger than that in any of the shorter germination periods.

304

In the previous study,1 the antioxidative activity of soluble bound phenolic

305

compounds in oil-in-water emulsions increased during chickpea germination.

306

However, the soluble free phenolic compounds had little to no antioxidative effect in

307

the same emulsions. The molecular weight of soluble bound phenolic compounds may

308

be correlated to the antioxidative activity, as soluble bound phenolic compounds had

309

an increased molar mass with germination time, while soluble free phenolic

310

compounds can be considered as phenolic compounds with much lower molecular

311

weight compared to bound ones.

312

3.3 Proposed mechanisms for the enhanced antioxidative active of soluble

313

phenolic compounds after chickpea germination

314

Based on the current findings and previous results, we speculate that there are two

315

possible mechanisms to explain the different antioxidative activities between soluble

316

free and bound phenolic compounds extracted from germinated chickpea in an in vitro



317

assays and in oil-in-water emulsions. First, the transformation of phenolic compounds

318

in soluble free and bound fractions during germination may play a major role. As

319

indicated by LC-ESI-QTOF-MS results, composition of both soluble free and bound

320

phenolic compounds varies during germination. PCA analysis suggested that Gentisic

321

acid (2) and 7,3',4'-trihydroxyflavone (7) were the major compounds in soluble bound

322

phenolics extracted from chickpea after 6 days germination, and correlated to the

323

greater antioxidative activity in oil-in-water emulsions. Protocatechuic acid

324

4-O-glucoside (1) and 6-hydroxydaidzein (8) that extracted from chickpea after 6 days

325

germination were highly associated with the antioxidative activity of soluble free

326

fractions in an in vitro assays.

327

Second, a protective and/or a dual antioxidant effect stemmed from moieties might be

328

another factor contributing to the enhanced antioxidative activity of soluble bound

329

phenolic compounds extracted from chickpea after 6 days germination (Fig 4). It has

330

been reported that moieties of soluble bound phenolic compounds can protect

331

phenolic compounds from being oxidized.24,25 Free radicals are generated gradually in

332

emulsion system. Excessive phenolic compounds without protection, such as soluble

333

free phenolic compounds, can be rapidly oxidized and consumed by oxidants

334

including radicals (Fig. 4A); while soluble bound phenolic compounds can keep

335

viability under the protection of moieties to which they are attached against oxidants

336

(Fig. 4B). Conversely, free radicals were excessive at the very beginning in an in vitro

337

assays (Fig. 4C) and soluble free phenolic compounds had higher efficacy than

338

soluble bound phenolic compounds.1 This is because moieties of soluble bound


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phenolic compounds may impede the proximity of active phenolic group and free

340

radicals by steric hindrance (Fig. 4D). Moreover, reaction time of in an in vitro assays

341

is too short to release the entire antioxidative capacity of soluble bound phenolic

342

compounds. A well-known example for such protective mechanism is that of dietary

343

fiber which can negatively affect the release and absorption of phenolic molecules.26

344

In addition, moieties of soluble bound phenolic compounds can either be antioxidants

345

or pro-antioxidants that may impose synergistic or antagonistic effect with phenolic

346

compounds. Typically, polysaccharides and alcohol soluble proteins are believed to be

347

the moieties that phenolic compounds are conjugated with. Many researchers reported

348

that crude polysaccharides and protein possess potential free radical scavenging

349

capability.27–31 Soluble polysaccharides may donate hydrogen to the oxidized phenolic

350

compounds with their activated reducing ends. Consequently, the durable

351

antioxidative activity of soluble bound phenolic compounds may be caused by the

352

synergism of phenolic compounds and the moieties they attached.

353

4 Conclusion

354

During chickpea germination, compositions of soluble free and soluble bound

355

phenolic compounds varied markedly. Meanwhile, the molar masses of soluble bound

356

phenolic compounds were observed to be increased. These variations of phenolic

357

compounds were responsible for the antioxidative activities in both in an in vitro

358

assays and in oil-in-water emulsions. The positive correlation between molar mass

359

and antioxidative activity has profound guiding significance for improving the



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360

efficacy of natural antioxidants. A protective and/or a dual antioxidative effects are

361

speculated for the enhanced antioxidative activity of soluble bound phenolic

362

compounds from chickpea after germination.

363 364

Acknowledgment

365

This work is supported by the USDA National Institute of Food and Agriculture,

366

Hatch project number ND01593.

367 368

References

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468 469 470


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Page 23 of 31


471 472

Figure captions

473

Figure. 1 HPLC chromatograms of (A) soluble free phenolic compounds, and (B)

474

soluble bound phenolic compounds extracted from chickpea during chickpea

475

germination. All these extractions were separated with C18 column and monitored

476

with DAD at 260 nm. Numbers on the peaks stand for the proposed phenolic

477

compounds identified by Q-TOF-MS: Protocatechuic acid 4-O-glucoside (1), Gentisic

478

acid

479

8-Hydroxydihydrodaidzein (4), Afzelechin (5), Prunetin (6), 7,3',4'-Trihydroxyflavone

480

(7), 6-Hydroxydaidzein (8), Hesperetin (9), Kaempferol (10), 1-Naphthyl acetate (11),

481

Isolicoflavonol

482

3',4',5,7-Tetrahydroxy isoflavanone (15), and Glycitein (16)

483

Figure. 2 PCA (A) score plot, and (B) loading plot of phenolic compounds extracted

484

from chickpea during the period of germination. CF and CB denoting soluble free and

485

soluble bound phenolic compounds after 0, 2, 4, and 6 days germination; numbers

486

indicating the different phenolic compounds: Protocatechuic acid 4-O-glucoside (1),

487

Gentisic acid (2), 4-Hydroxy-8-methoxy-2H-fruo[2,3-h]-1-benzopyran-2-one (3),

488

8-Hydroxydihydrodaidzein (4), Afzelechin (5), Prunetin (6), 7,3',4'-Trihydroxyflavone

489

(7), 6-Hydroxydaidzein (8), Hesperetin (9), Kaempferol (10), 1-Naphthyl acetate (11),

490

Isolicoflavonol

491

3',4',5,7-Tetrahydroxy isoflavanone (15), and Glycitein (16)

492

Figure 3 Characteristics variation of soluble bound phenolic compounds during

493

chickpea germination. All soluble bound phenolic extractions were separated with

494

SEC column and monitored with DAD at 260 nm, refraction index detector, and

495

multi-angle laser light scattering detector

496

Figure 4 Schematic diagram for the speculated antioxidative mechanism of soluble

497

phenolic compounds extracted from germinated chickpea in an in vitro assays and in

498

oil-in-water emulsion system

(2),

4-Hydroxy-8-methoxy-2H-fruo[2,3-h]-1-benzopyran-2-one

(12),

Pseudobaptigenin

(12),

Pseudobaptigenin

(13),

(13),


Formononetin

Formononetin

(3),

(14),

(14),


499 500

Page 24 of 31

Table 1 Profiles of phenolic compounds in germinated chickpea extracts identified by LC-ESI-QTOF-MS* Peak

RT

Collision energy (ev)

Observed m/z [M-H]

Calculated -

-

Molecular

Diff.

Formula

(ppm)

Score

Product ions

Proposed compounds

No.

(min)

1

9.655

10

315.0730

315.0722

C13H16O9

-2.53

95.40

108.0209, 153.0190

Protocatechuic acid 4-O-glucoside

2

12.047

25

153.0193

153.0193

C7H6O4

0.01

99.80

109.0255, 135.0013

Gentisic acid

3

12.491

25

231.0310

231.0299

C12H8O5

-4.86

88.77

133.0162, 137.0101, 159.0298, 189.0008

4

15.906

10

271.0300

271.0612

C15H12O5

-1.58

81.75

109.0167, 119.0359, 150.9863

8-Hydroxydihydrodaidzein

5

16.894

30

273.0776

273.0768

C15H14O5

-2.86

96.28

109.0277, 125.0986, 241.1087

Afzelechin

6

17.590

30

283.0615

283.0612

C16H12O5

0.65

95.24

195.0228, 211.0157, 223.0137, 239.0073,

Prunetin

m/z [M-H]

4-Hydroxy-8-methoxy-2H-fruo[2,3-h]1-benzopyran-2-one

268.0064 7

17.955

40

269.0528

269.0455

C15H10O5

-1.40

97.36

195.0214, 211.0143, 223.0125, 239.0077

7,3',4'-Trihydroxyflavone

8

19.258

25

269.0531

269.0455

C15H10O5

-1.78

84.91

117.0350, 143.0506, 194.9254, 239.0354

6-Hydroxydaidzein

9

19.278

10

301.0723

301.0718

C16H14O6

-1.87

98.00

107.0013, 109.0166, 150.9857, 286.0143

Hesperetin

10

19.442

30

285.0407

285.0405

C15H10O6

-0.92

97.36

117.0340, 143.0514, 187.0381, 239.0375

Kaempferol

11

19.618

10

185.0610

185.0608

C12H10O2

0.19

98.27

141.0693

1-Naphthyl acetate

12

19.674

10

353.1039

353.1031

C20H18O6

-2.45

97.33

111.0082, 125.0241

Isolicoflavonol

13

20.294

25

281.0455

281.0455

C16H10O5

0.25

92.81

134.9930, 167.0307, 208.0270, 224.0211,

Pseudobaptigenin

14

20.564

25

267.0671

267.0663

C16H12O4

-3.06

98.13

104.0216, 135.0025, 167.0418, 195.0364,

253.0217 Formononetin

223.0302, 252.0316 15

21.858

30

287.0565

287.0561

C15H12O6

-1.52

89.09

109.0295, 125.0243

3',4',5,7-Tetrahydroxyisoflavanone

16

22.493

30

283.0615

283.0612

C16H12O5

-1.23

98.95

116.9955, 211.0400, 224.0409, 239.0352,

Glycitein

268.0375

501

*RT, retention time; Diff., difference between calculated m/z and observed m/z.


Page 25 of 31


502 503 504 505

Table 2 Dynamic changes of proposed phenolic compounds during chickpea germination* Peak No.

Proposed compounds

CF0

CF2

CF4

CF6

CB0

CB2

CB4

CB6

42.4 ± 0.8 e

N.A.

3.1 ± 0.2 a

2.8 ± 0.3 a

2.3 ± 0.1 a

5

(×10 counts) b

Protocatechuic acid 4-O-glucoside

18.3 ± 0.9

2

Phenolic acid

Gentisic acid

N.A.

N.A.

N.A.

N.A.

31.6 ± 0.0 b

21.0 ± 0.4 a

21.4 ± 1.9 a

35.4 ± 3.8 b

3

Coumarin

N.A.

N.A.

N.A.

N.A.

2.5 ± 0.3 a

3.6 ± 0.4 a

4.1 ± 0.3 ab

5.7 ± 0.6 b

4

Isoflavone

0.9 ± 0.1 a

4.6 ± 0.6 c

N.A.

0.4 ± 0.1 a

5.5 ± 0.5 c

3.1 ± 0.2 b

N.A.

2.3 ± 0.2

b

d

7.2 ± 0.1 e

N.A.

N.A.

[2,3-h]-1-benzopyran-2-one 8-Hydroxydihydrodaidzein

2.5 ± 0.1 b

3.0 ± 0.2 b

c

b

5

Flavanol

Afzelechin

3.5 ± 0.2

6

Isoflavone

Prunetin

73.6 ± 1.3 d

7

Flavone

7,3',4'-Trihydroxyflavone

Isoflavone

6-Hydroxydaidzein

a

N.A.

0.8 ± 0.1

22.3 ± 0.9 bc

11.2 ± 0.8 a

N.A.

2.8 ± 0.2 a

15.6 ± 0.2 b

13.0 ± 1.0 b

33.7 ± 2.5 c

5.9 ± 0.0

b

4.8 ± 0.4

ab

5.0 ± 0.3

ab

c

a

1.8 ± 0.1

ab

2.9 ± 0.0

b

9

Flavanone

Hesperetin

0.7 ± 0.1

10

Flavonol

Kaempferol

N.A.

2.2 ± 0.2

35.0 ± 1.7

d

Phenolic acid

4-Hydroxy-8-methoxy-2H-fruo

26.9 ± 3.3

c

1

8

506 507 508

Class

N.A.

11.3 ± 1.7 8.4 ± 1.2

N.A.

c

N.A. 5.1 ± 0.2

N.A. ab

N.A.

3.2 ± 0.1

ab

N.A.

N.A.

8.9 ± 0.7 a

8.4 ± 0.0 a

d

c

4.8 ± 0.1

17.8 ± 2.2 b

24.4 ± 1.1 c

2.5 ± 0.3 a

5.2 ± 0.6 a

2.7 ± 0.2

a

N.A.

0.8 ± 0.1

a

1.0 ± 0.1 ab

21.2 ± 1.0 b b

19.2 ± 1.7 b 0.4 ± 0.0 a

11

Naphthyl ester

1-Naphthyl acetate

N.A.

N.A.

N.A.

N.A.

8.2 ± 0.0

12

Isoflavonol

Isolicoflavonol

N.A.

N.A.

N.A.

N.A.

99.2 ± 0.4 d

52.0 ± 3.4 c

20.7 ± 2.6 b

3.5 ± 0.1 a

13

Isoflavone

Pseudobaptigenin

15.0 ± 1.4 d

N.A.

N.A.

7.5 ± 0.8 ab

10.9 ± 0.0 bc

23.4 ± 1.3 e d

c

14.1 ± 0.5 cd ab

6.2 ± 0.2 a ab

5.1 ± 0.5

1.9 ± 0.3

N.A.

4.2 ± 0.6

a

26.6 ± 2.4

bc

87.4 ± 2.1 d

14

Isoflavone

Formononetin

84.4 ± 10.4

15

Isoflavone

3',4',5,7-Tetrahydroxy isoflavanone

N.A.

N.A.

N.A.

1.4 ± 0.0 a

N.A.

0.5 ± 0.0 a

4.4 ± 0.5 b

7.5 ± 0.3 c

16

Isoflavone

Glycitein

232.1 ± 29.4 c

163.9 ± 16.7 b

31.2 ± 0.2 a

N.A.

2.1 ± 0.3 a

15.7 ± 1.9 a

26.2 ± 1.4 a

43.3 ± 2.2 a

42.0 ± 1.7

17.6 ± 1.8

11.7 ± 0.6

*CF and CB denoted soluble free phenolic compounds and soluble bound phenolic compounds extracted from germinated chickpea, followed with different germination time: 0, 2, 4, and 6 days, respectively. Different letters indicate statistically significant differences intraspecies (p < .05).



509 510 511 512 513 514 515

516 517 518 519 520 521 522 523 524 525 526 527 528 529

Table 3 Variation of average molecular weight of main soluble bound phenolic compounds in chickpea extracts* Germination time (days) Mn (g/mol) Mw (g/mol) Mw/Mn 0 4003±142 a 4830±54 a 1.21 2 4548±71 a 5258±115 ab 1.16 a a 4 4213±170 4786±642 1.14 6 5219±43 b 6230±60 bc 1.19 * Data points represent mean (n=2) ± standard deviation. Different letters indicate statistically significant differences intraspecies (p < .05).


Page 26 of 31

3000 2000 1000 0

CF6

3000 2000 1000 0

CF4

3000 2000 1000 0 3000 2000 1000 0

CF2

A

467

8 9 13 14 16 Intensity (mAu)

530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555


Intensity (mAu)

Page 27 of 31

CF0

0

5

10 15 20 Retention time (min)

25

30

3000 2000 1000 0

CB6

3000 2000 1000 0

CB4

3000 2000 1000 0

CB2

3000 2000 1000 0

CB0

0

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

5

10 15 20 Retention time (min)

25

30

Figure 1 HPLC chromatograms of (A) soluble free phenolic compounds, and (B) soluble bound phenolic compounds extracted from chickpea during chickpea germination. All these extractions were separated with C18 column and monitored with DAD at 260 nm. Numbers on the peaks stand for the proposed phenolic compounds identified by Q-TOF-MS: Protocatechuic acid 4-O-glucoside (1), Gentisic acid (2), 4-Hydroxy-8-methoxy-2H-fruo[2,3-h]-1-benzopyran-2-one (3), 8-Hydroxydihydrodaidzein (4), Afzelechin (5), Prunetin (6), 7,3',4'-Trihydroxyflavone (7), 6-Hydroxydaidzein (8), Hesperetin (9), Kaempferol (10), 1-Naphthyl acetate (11), Isolicoflavonol (12), Pseudobaptigenin (13), Formononetin (14), 3',4',5,7-Tetrahydroxy isoflavanone (15), and Glycitein (16)



6 CF

A

1.5 1.0 0.5

4 CF CF2

4 CB

6 CB

0.0 0 CF

-0.5

2 CB

-1.0

0 CB

B

2.0 Principal component 2 (30.13%)

2.0 Principal Component 2 (30.13%)

556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581

Page 28 of 31

1.5 8

1.0 14 1

6

10

3 13

0.5 0.0

72 11

12 9 5 4

-0.5

15 16

-1.0 -1.5

-1.5

-2.0

-2.0 -1.0

-0.5

0.0

0.5

1.0

Principal Component 1 (43.65%)

1.5

2.0

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

Principal component 1 (43.65%)

Figure 2 PCA (A) score plot, and (B) loading plot of phenolic compounds extracted from chickpea during the period of germination. CF and CB denoting soluble free and soluble bound phenolic compounds after 0, 2, 4, and 6 days germination; numbers indicating the different phenolic compounds: Protocatechuic acid 4-O-glucoside (1), Gentisic acid (2), 4-Hydroxy-8-methoxy-2H-fruo[2,3-h]-1-benzopyran-2-one (3), 8-Hydroxydihydrodaidzein (4), Afzelechin (5), Prunetin (6), 7,3',4'-Trihydroxyflavone (7), 6-Hydroxydaidzein (8), Hesperetin (9), Kaempferol (10), 1-Naphthyl acetate (11), Isolicoflavonol (12), Pseudobaptigenin (13), Formononetin (14), 3',4',5,7-Tetrahydroxy isoflavanone (15), and Glycitein (16)


Page 29 of 31

582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607


Figure 3 Characteristics variation of soluble bound phenolic compounds during chickpea germination. All soluble bound phenolic extractions were separated with SEC column and monitored with DAD at 260 nm, refraction index detector, and multi-angle laser light scattering detector



608

609 610 611 612 613

Figure 4 Schematic diagram for the speculated antioxidative mechanism of soluble phenolic compounds extracted from germinated chickpea in an in vitro assays and in oil-in-water emulsion system


Page 30 of 31

Page 31 of 31


614 615 616

TABLE OF CONTENTS GRAPHICS

617


Why does chickpea germination improve antioxidative activity of

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