Avenanthramide Aglycones and Glucosides in Oat Bran: Chemical

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Bioactive Constituents, Metabolites, and Functions

Avenanthramide Aglycones and Glucosides in Oat Bran: Chemical Profile, Levels in Commercial Oat Products, and Cytotoxicity to Human Colon Cancer Cells Wenbin Wu, Yao Tang, Junli Yang, Emmanuel Idehen, and Shengmin Sang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02767 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 9, 2018

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Journal of Agricultural and Food Chemistry

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Avenanthramide Aglycones and Glucosides in Oat Bran: Chemical Profile,

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Levels in Commercial Oat Products, and Cytotoxicity to Human Colon

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Cancer Cells

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Wenbin Wu†, Yao Tang†, Junli Yang†, Emmanuel Idehen, and Shengmin Sang*

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Laboratory for Functional Foods and Human Health, Center for Excellence in Post-Harvest

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Technologies, North Carolina Agricultural and Technical State University, North Carolina

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Research Campus, 500 Laureate Way, Kannapolis, North Carolina 28081, United States

9



These authors contributed equally to this study.

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Running title: AVAs in Oat

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Corresponding authors (Tel: 704-250-5710; Fax: 704-250-5729; E-mail: [email protected] or

13

[email protected])

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ABSTRACT:

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Avenanthramides (AVAs), unique phytochemicals in oat, have attracted an increasing amount of

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attention due to their outstanding health benefits. However, the chemical profile and the levels of

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AVAs in commercial oat products as well as their health benefits have not been examined in

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detail. In the present study, a total of twenty-nine AVA aglycones and AVA glucosides were

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identified and characterized from oat bran, using NMR (1D- and 2D-NMR) and LC-MS

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techniques. Among them, seventeen novel AVA glucosides were reported in oat bran for the first

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time. The most abundant AVA glucoside, 2c-3'-O-glc, had a similar growth inhibitory activity

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with the major AVA, 2c, against HCT-116 and HT-29 human colon cancer cells, indicating

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glucosylation does not affect the growth inhibitory effects of AVAs. Furthermore, the levels of

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all individual AVAs in thirteen commercial oat products were analyzed using HPLC-MS/MS.

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The total AVAs contents in various oat products ranged from 9.22 to 61.77 mg/kg (fresh weight).

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KEYWORDS: Oat bran, avenanthramides, structural elucidation, cytotoxicity, quantification

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Journal of Agricultural and Food Chemistry

INTRODUCTION

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Oat (Avena sativa L.) is an environmental friendly, drought tolerant and nitrogen efficient

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crop. As the third most important grain crop in the United States (1.3 tonnes in 2016, reported by

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United States Department of Agriculture (USDA)),1 oat seeds are good for human health and an

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abundant source of health promoting compounds. USDA and Food and Agriculture Organization

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of the United Nation (FAO) recommended whole grains including oat as healthy food for human

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routine consumption.2-3 The potential bio-effects of oat consist of anti-oxidant, anti-cancer, anti-

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inflammatory, anti-obesity, and anti-atherosclerosis activities based on cell, tissue, animal, and

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human experiments that have been previously stated.2-3 Whole grain oat diet has been reported to

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improve insulin sensitivity and plasma cholesterol profile associated with the changes in cecal

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microbiota composition in mice,4 and also improve vasodilator function in systemic and cerebral

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arteries in older adults.5 The health promoting compounds in oat include not only macros protein

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and dietary fiber but also micros phytochemicals, such as phenolic acids,6-7 vitamins,8 saponins,9

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flavonoids,10 and avenanthramides (AVAs).

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Among these compounds, AVAs have been considered as the signature compounds in oat.

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Oat is the only cereal that contains AVAs.11-13 AVAs are located in the outer layer of the oat

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grain (groats and hulls), and were first characterized by Collins11 using HPLC-MS-NMR

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techniques and synthetic standards. These compounds are substituted N-cinnamoyl anthranilic

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acids consisting of anthranilic acid and cinnamic acid moieties. The AVAs differ in the

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substitution pattern on the two moieties. Dimberg developed a systematic nomenclature of AVAs,

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assigning the anthranilate derivatives a number and the accompanying cinnamate derivatives the

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following letters: c for caffeic acid,

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anthranilic acid moiety consists of one anthranilic acid (1), 5-hydroxyanthranilic acid (2), 5-

f for ferulic acid, and p for p-coumaric acid.14 The

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acid

(3),

4-hydroxyanthranilic

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hydroxy-4-methoxyanthranilic

acid

(4),

or

4,5-

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dihydroxyanthranilic acid (5). The most common AVAs are esters of 5-hydroxyanthranilic acid

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with p-coumaric (2p), caffeic (2c), or ferulic (2f) acid.15-16 AVAs have been found to exhibit

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anti-inflammatory, anti-oxidant, anti-itching, anti-irritating, anti-atherogenic, and anti-cancer

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activities.2, 17-25 Studies have shown that the amount of AVAs in oat ranges from 0.55 to 775.5

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mg/kg (fresh weight), which covered a wide variation of concentrations.26-28 Notwithstanding the

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extensive research on AVAs, there are no detailed studies on the chemical profile of AVAs in oat.

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In addition, there exists little information about the AVA content in commercial oat products

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manufactured in North America. The nutrients in oat, including AVAs, would be influenced

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inevitably by processing, packaging and shelf-life from raw oat grains to products.29-31 Moreover,

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the reported AVA levels were usually quantified on the basis of the three main AVAs, 2p, 2c and

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2f, without considering the contributions of other AVAs. Some scientific reports used ferulic

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acid or 2p as reference standards for evaluating the total AVAs content equivalently.32-33 The

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responses of 2c, 2p, and 2f are significantly different from each other, which would induce

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significant errors when AVAs are quantified only based on one standard curve. Therefore, the

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objective of the present study was to explore a comprehensive profile of AVAs in oat and then

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quantify their levels in commercial oat products using LC-MS.

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MATERIALS AND METHODS

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Materials. Silica gel (230−400 mesh) (Sorbent Technologies Inc., Atlanta, GA) and Diaion HP-

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20 (Mitsubishi Chemical, Japan) were used for open column chromatography (CC).

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Chromatographic separations were monitored by analytical thin-layer chromatography (TLC) on

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250 µm thick, 2-25 µm particle size glass-backed silica gel plates (Sigma-Aldrich, St. Louis,

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MO). All analytical grade solvents and LC-MS grade solvents were obtained from Thermo

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Fisher Scientific (Waltham, MA). Oat bran (20 kg) was purchased from Kalyx (www.kalyx.com).

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All thirteen oat commercial products were purchased online at Amazon.com.

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HPLC-MS Analysis. HPLC-MS was performed with a Thermo-Finnigan Spectra System

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consisting of an Ultimate 3000 degasser, an Ultimate 3000 RS pump, an Ultimate 3000 RS auto-

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sampler, an Ultimate 3000 RS column compartment, and an LTQ Velos Pro ion trap mass

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spectrometer (Thermo Electron, San Jose, CA) incorporated with an electrospray ionization (ESI)

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interface. The column used was a 150 mm × 3.0 mm i.d., 5 µm, Gemini 5µ NX-C18

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(Phenomenex, Torrance, CA). The mobile phase consisted of 5% methanol in water containing

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0.1% formic acid (mobile phase A) and 5% water in methanol with 0.1% formic acid (mobile

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phase B). The gradient elution was carried out for 45 min at a flow rate of 0.3 mL/min. A

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gradient eluting system was applied: 0−10 min, 30−45% B; 10−30 min, 45−70% B; 30-35min,

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70−100% B; 35−41 min, 100% B; 41−42 min, 30% B. The column was then re-equilibrated with

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30% B from 42 to 45 min. The injection volume was 10 µL for each sample. The HPLC eluent

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was introduced into the ESI interface. For mass spectrometric parameter optimization, the

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purified compound in 50% methanol solution (10 µg/mL) was infused in the ESI source and

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analyzed in negative ion mode to obtain the following optimized parameters: spray voltage, 3.6

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kV; sheath gas (nitrogen) flow rate, 34 (arbitrary units); capillary voltage, −13 V; capillary

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temperature, 300 oC; tube lens offset, −60 V. For the quantification of the fifteen AVAs, target

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ions listed in Figure 1 were monitored using selected reaction monitoring (SRM) mode. For the

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identification of AVAs, the collision-induced dissociation (CID) was conducted with isolation

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width of 1.0 Da, and the normalized collision energy was set to 35% for MSn analysis. The mass

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range was measured between m/z 50-700. Data acquisition and analysis were performed with

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Xcalibur 2.0 version (Thermo Electron, San Jose, CA).

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Nuclear Magnetic Resonance (NMR) Analysis. 1H (600 MHz),

13

C (150 MHz), 1H-1H

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COSY (homonuclear correlation spectroscopy), HSQC (heteronuclear single quantum

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correlation), and HMBC (heteronuclear multiple bond correlation) NMR spectra were recorded

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on a Bruker 600 MHz NMR instrument. All samples were dissolved in methanol-d4 containing

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TMS (tetramethylsilane) as the internal standard.

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Extraction and Enrichment of AVAs. Oat bran was initially defatted by n-hexane (3

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days/time for 3 times, shaking at room temperature), then continued to extract with 80%

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methanol (Voat:Vmethanol = 1:5) at room temperature for three times (4 days/time). The methanol

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extraction was concentrated under reduced pressure to yield a crude residue (630 g). This residue

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was suspended in water and partitioned with n-hexane, ethyl acetate (EtOAc), and n-butanol (n-

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BuOH). After concentrated in vacuo, the EtOAc fraction (17.2 g) was suspended in ethanol and

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applied to Sephadex LH-20 column (7.5 cm i.d. × 60 cm, conditioned with 30% ethanol in water

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containing 0.1% acetic acid) eluted with 30% ethanol in water containing 0.1% formic acid,

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followed by 70% ethanol in water containing 0.1% formic acid (5 L each) to afford two fractions.

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The latter fraction was concentrated in vacuo to give the AVA-enriched fraction. The butanol

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fraction (170.1 g) was reconstituted in methanol and applied to Diaion HP-20 column (7.5 cm i.d.

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× 65 cm, conditioned with water containing 0.1% formic acid) eluted with 20%, 60%, and 100%

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methanol in water containing 0.1% formic acid (5 L each) to afford three fractions. The second

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fraction was concentrated in vacuo to get the AVA-glucoside-enriched fraction. The combination

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of fractions was monitored by TLC (chloroform/methanol/water, 140:70:11, v/v/v). The spots on

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TLC were visualized after burning with a H2SO4/ethanol (5:95, v/v) solution.

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Characterization of AVAs. Repeated purification of AVA-enriched fraction and AVA-

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glucoside-enriched fraction by silica gel column, Sephedex LH-20 column, and semi-preparative

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HPLC, afforded four AVAs, 3f (3.7 mg), 1c (0.6 mg), 1p (0.4 mg), 4f (0.4 mg), and two new

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AVA-glucosides, 2c-3'-O-glc (2.3 mg) and 3f-4'-O-glc (0.2 mg). The three major AVAs, 2p, 2c

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and 2f were obtained from PepsiCo Inc. as a gift.

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1

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8.82 Hz, H-3), 7.02 (1H, dd, J = 9.0, 2.7 Hz, H-4), 7.50 (1H, d, J = 2.7 Hz, H-6), 7.56 (1H, d, J =

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1.4 Hz, H-2'), 6.88 (1H, d, J = 8.2 Hz, H-5'), 7.19 (1H, dd, J = 1.3, 8.2 Hz, H-6'), 7.54 (1H, d, J =

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15.7 Hz, H-7'), 6.56 (1H, d, J = 15.6 Hz, H-8'), 4.82 (1H, m, H-1''), and 3.21-3.63 (6H, m, ranged

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from H-2'' to H-6''); δC 118.2 (C-1), 128.3 (C-2), 123.6 (C-3), 122.1 (C-4), 150.7 (C-5), 118.1 (C-

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6), 128.7 (C-1'), 117.8 (C-2'), 147.0 (C-3'), 150.6 (C-4'), 117.4 (C-5'), 125.9 (C-6'), 142.7 (C-7'),

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120.5 (C-8'), 104.2 (C-1''), 74.8 (C-2''), 77.6 (C-3''), 73.3 (C-4''), 78.4 (C-5''), and 61.5 (C-6'').

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1

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(1H, s, H-6), 7.31 (1H, br s, H-2'), 7.20 (1H, br s, H-5'), 7.20 (1H, br s, H-6'), 7.56 (1H, d, J =

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15.6 Hz, H-7'), 6.66 (1H, d, J = 15.6 Hz, H-8'), 3.40-3.95 (6H, m, ranged from H-2'' to H-6''),

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3.93 and 3.92 (each for 3H, OMe × 2).

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1

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br s, H-6), 7.03 (1H, d, J = 1.2 Hz, H-2'), 6.79 (1H, d, J = 8.5 Hz, H-3'), 6.75 (1H, d, J = 8.5 Hz,

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H-5'), 7.03 (1H, d, J = 8.5 Hz, H-6'), 7.48 (1H, d, J = 15.6 Hz, H-7'), 6.49 (1H, d, J = 15.6 Hz, H-

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8'), 3.85 and 3.86 (each for 3H, OMe × 2).

140

1

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(1H, m, H-4), 7.14 (1H, m, H-5), 8.11 (1H, dd, J = 7.9, 1.6 Hz, H-6), 7.09 (1H, d, J = 2.0 Hz, H-

H and 13C NMR data (600 MHz, methanol-d4) for 2c-3'-O-glucoside (2): δH 8.42 (1H, d, J =

H NMR data (600 MHz, methanol-d4) for 3f-4'-O-glucoside (20): δH 8.39 (1H, s, H-3), 7.55

H NMR data (600 MHz, methanol-d4) for 3f (12): δH 8.31 (1H, d, J = 1.2 Hz, H-3), 7.49 (1H,

H NMR data (600 MHz, methanol-d4) for 1c (7): δH 8.66 (1H, dd, J = 8.4, 0.9 Hz, H-3), 7.54

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2'), 6.80 (1H, d, J = 8.2 Hz, H-5'), 6.99 (1H, dd, J = 8.2, 2.0 Hz, H-6'), 7.55 (1H, d, J = 15.6 Hz,

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H-7'), 6.49 (1H, d, J = 15.6 Hz, H-8')

144

1

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7.54 (1H, ddd, J = 8.4, 7.4, 1.6 Hz, H-4), 7.14 (1H, m, H-5), 8.22 (1H, dd, J = 7.9, 1.6 Hz, H-6),

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7.51 (2H, d, J = 8.6 Hz, H-2', H-6'), 6.83 (2H, d, J = 8.6 Hz, H-3', H-5'), 7.61 (1H, d, J = 15.6 Hz,

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H-7'), 6.56 (1H, d, J = 15.6 Hz, H-8')

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1

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dd, J = 8.8, 2.4 Hz, H-5), 7.97 (1H, d, J = 8.8 Hz, H-6), 7.24 (1H, d, J = 1.8 Hz, H- 2'), 6.83 (1H,

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d, J = 8.1 Hz, H-5'), 7.11 (1H, dd, J = 8.1, 1.8 Hz, H-6'), 7.60 (1H, d, J = 15.6 Hz, H-7'), 6.56

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(1H, d, J = 15.6 Hz, H-8'), 3.93 (3H, s, OMe)

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Preparation of AVA Standards and the Extracts of Commercial Oat Products. The

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respective stock solutions (100 mM) of 2p, 2c, 2f, 3f, and 2c-3'-O-glc were dissolved in DMSO

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and then prepared in 50% (v/v) aqueous methanol solution and stored at −80 oC before use. The

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above stock solutions were diluted with 50% methanol to prepare varied concentrations ranging

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from 0.39 to 400 nM for 2p, 2f and 3f, and 3.9-4000 nM for 2c and 2c-3'-O-glc, respectively. All

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the samples were freshly prepared before use. Quantification was performed with external

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standards using standard curves generated ranging from 3.125-400 µM for 2p, 2f, and 3f, and

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15.625-2000 µM for 2c and 2c-3'-O-glc (r2 > 0.9997) (Table 1). The AVAs named with -p, -c or

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-f were quantified based on the standard curves of 2p, 2c, and 2f, respectively (except the 3f),

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while the contents of the AVA glucosides with -p or -f were also calculated with the standard

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curves of 2p and 2f. The quantification for caffeic acid derived glucosides were calculated by 2c-

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3'-O-glc, and 3f was applied as standard for 3f-4'-O-glc (Table 1).

H NMR data (600 MHz, methanol-d4) for 1p (14): δH 8.67 (1H, dd, J = 8.4, 0.9 Hz, H-3),

H NMR data (600 MHz, methanol-d4) for 4f (13): δH 8.23 (1H, d, J = 2.4 Hz, H-3), 6.54 (1H,

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At least three independent samples of each oat product were used in this study. Solvents,

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extraction tools and time ranges were considered as three major factors for optimizing the

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extraction conditions for AVAs (data not shown). One gram of each oat product was accurately

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weighed and extracted four times with 50 mL of 50% EtOH in water for 12 h, and then

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centrifuged at 16,100 g for 15 min. Supernatants from the four extractions were combined and

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concentrated to dryness at 35 oC under reduced pressure. The residue was reconstituted in 2.0

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mL of 50% methanol and centrifuged at 16,100 g for 15 min. The supernatants of each sample

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were diluted 20 times with 50% methanol before injection. Each sample was analyzed in

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triplicate. The remaining supernatants were stored in -80 oC freezer.

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Growth Inhibitory Effects of 2c and 2c-3'-O-glc on Human Colon Cancer Cells. Cell

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growth inhibition was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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bromide (MTT) colorimetric assay. Human colon cancer cells HCT-116 and HT-29 were seeded

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in 96-well microplates with 6000 cells/well and allowed to attach for 48 h at 37 °C. The tested

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compounds (in DMSO) were added to cell culture medium and further diluted to desired

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concentrations (0, 50, 100, 150, 200 and 400 µM). The final DMSO concentrations for control

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and treatments were 0.1% (n = 8-16). After 48 h, the medium was aspirated, and cells were

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treated with 100 µL of fresh medium containing 2.41 mM MTT. After incubation for 3 h at

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37 °C, the medium containing MTT was aspirated, 100 µL of DMSO was added to solubilize the

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formazan precipitate, and the plates were shaken gently for 1 h at room temperature. Absorbance

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values were derived from the plate reading at 550 nm on a Biotek Microtiter plate reader

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(BioTek, Winooski, VT). The reading reflected the number of viable cells and was expressed as

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a percentage of viable cells in the control. Both HCT-116 and HT-29 cells were cultured in

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McCoy’s 5A medium. All of the above media were supplemented with 10% fetal bovine serum,

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1% penicillin/streptomycin, and 1% glutamine, and the cells were kept in a 37 °C incubator with

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95% humidity and 5% CO2. IC50 values were obtained using GraphPad Prism version 7.0

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(GraphPad Software, San Diego, CA). For simple comparisons between treatments and control,

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two-tailed Student’s t-test was used. A p-value of less than 0.05 was considered statistically

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significant in all the tests.

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RESULTS AND DISCUSSION

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Purification and Structural Elucidation of AVAs. Four AVA aglycones (3f, 1c, 1p, and 4f)

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along with two new AVA glucosides (2c-3'-O-glc and 3f-4'-O-glc) (Figures 1 and 3) were

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isolated from oat bran by means of chromatographic methods, including silica gel, Diaion HP-20,

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and Sephadex LH-20 chromatography. The structures of AVA 3f, 1c, 1p, and 4f were confirmed

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by comparing their NMR data with those reported in literature.12, 34-35

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The new compound 2c-3'-O-glc had a molecular ion at m/z 476 [M-H]-, which is 162 mass

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units higher than that of 2c (m/z 314 [M-H]-), indicating it is the glucoside of 2c. The MS/MS

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spectrum of 2c-3'-O-glc showed the characteristic fragments at m/z 314 [M-162]- (corresponding

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to the loss of a glucose unit), m/z 297 [M-179]- (corresponding to the breakdown of the bond

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between C-1' and C-7') and m/z 178 [M-298]- (corresponding to the breakdown of the bond

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between C-8' and C-9'). The 1H- and 13C-NMR spectra of 2c-3'-O-glc were similar to those of 2c,

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except a series of signals that belonged to a glucose unit were present in 2c-3'-O-glc, further

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confirmed that this compound is a glucoside of 2c. The connection of the sugar residue to 2c

207

moiety was confirmed at C-3' by the observation of the crosspeak between anomeric proton (δH

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4.82) of the sugar unit to C-3' (δC 147.0) in the HMBC spectrum (Figure 2). All of these

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spectroscopic features suggest that 2c-3'-O-glc is a glucoside of 2c as shown in Figures 1-3, a

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novel compound from oat.

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The second new compound 3f-4'-O-glc gave a deprotonated ion at m/z 520.1 [M - H]- in its

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LC-MS spectrum. The MS/MS spectrum of the precursor ion at m/z 520.1 displayed a fragment

213

ion at m/z 358.1, generated by the loss of a glucose unit. 1H-NMR spectrum of this compound

214

showed glycone signals similar to those of 3f. Therefore, 3f-4'-O-glc was tentatively determined

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as a glucoside of 3f (Figures 1 and 3).

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Establishing the Chemical Profile of AVA Aglycones and Their Glucosides in

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Oat by LC-ESI/MSn. In order to establish the chemical profile of the major AVA aglycones

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and their glucosides in oat, we initially analyzed the fragmentation patterns of the nine standards

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(2c, 2f, 2p, 3f, 1c, 1p, 4f, 2c-3'-O-glc and 3f-4'-O-glc). Five additional AVA aglycones

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(compounds 5-6, 10-11, 15) and fifteen additional AVA glucosides (compounds 1, 3, 16-19, and

221

21-29) were tentatively characterized by analyzing their respective tandem mass spectra,

222

following the fragmentation patterns observed from the standards. Therefore, a total of twelve

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AVA aglycones and seventeen AVA glucosides were characterized from oat bran.

224

Fragmentation patterns of AVA aglycone and AVA glucoside standards. The fragmentation

225

patterns of AVAs were identified by analyzing the tandem mass of the nine AVAs standards: 1c

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(7), 1p (14), 2c (4), 2p (8), 2f (9), 3f (12), 4f (13), 2c-3'-O-glc (2), and 3f-4'-O-glc (20) (Figure 3).

227

All nine compounds had the fragment ion that lost one CO2 unit on ring A (pattern a) (Figure 3).

228

This pattern together with the molecular ions of the compounds could help us separate AVAs

229

from other type of compounds in oat. Patten b is the breakdown of C-C bond between the amide

230

group and the olefin group among all nine compounds (Figure 3). Pattern b is very useful to

231

determine the structure of the anthranilic acid unit. Fragment ions m/z 162, 178, 208, and 194 are

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typical negative fragmentation for 1-, 2-/4-, 3-, and 5-AVA analogues, respectively. The 2- and

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4-AVA analogues have almost the same tandem mass spectrum (9 and 13 in Figure 3), however,

234

the identification still can be done by considering their retention times and concentrations. For

235

example, the relative retention times of 2p, 2c, and 2f are earlier than those of 4p, 4c, and 4f,

236

respectively, when using C18 column, the concentrations of 2p, 2c, and 2f are much higher than

237

those of 4p, 4c, and 4f in oat. Both 2f (9) and 4f (13) were isolated and characterized by NMR in

238

this study. The data of these two compounds supported this identification method, and similar

239

results were also reported previously by Wise and Collins.13, 36-37 Pattern c is the loss of the

240

glucose unit in 2c-3'-O-glc and 3f-4'-O-glc (Figure 3), which can assist us to determine the

241

structures of AVA glucosides.

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Identification of AVA aglycons with cinnamic acid derivatives. Using the fragmentation

243

patterns identified above, we were able to determine the structures of five additional AVA

244

aglycones, compounds 5, 6, 10, 11, and 15. Compound 5 had the deprotonated ion at m/z 314,

245

which was 16 mass units higher than that of 2p, indicating 5 has one more hydroxyl group than

246

2p. It also possessed fragment ions at m/z 270, corresponding to the loss of CO2 (pattern a), and

247

m/z 194, formed by the breakdown of C-C bond between amide group and olefin group (pattern

248

b), indicating 5 is 5p (Figure 4).37-38 Likewise, compound 6 showed fragment ions at m/z 300,

249

following pattern a, and m/z 194, following pattern b, indicating 6 is 5f (Figure 4).36,

250

Compound 10 had the same molecular weight (m/z 328) as 2f. It possessed fragment ions at m/z

251

284, following pattern a, and m/z 208, following pattern b, suggesting 10 is 3p (Figure 4).31, 40

252

Compound 11 had the same molecular weight (m/z 298) and a similar tandem mass spectrum

253

(Figures 3 and 4), but different retention time (Table 2) as those of 2p. It possessed fragment ions

254

at m/z 254, corresponding to the loss of CO2 (pattern a), and m/z 178, formed by the breakdown

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of C-C bond between amide group and olefin group (pattern b), in its MS/MS spectrum,

256

suggesting 11 is 4p (Figure 4). Compound 15 had fragment ion at m/z 268, following pattern a,

257

and m/z 162, following pattern b, indicating 15 is 1f (Figure 4).38

258

Identification of AVA glucosides. Using the fragmentation patterns identified above, we were

259

able to determine the structures of fifteen additional AVA aglycones, compounds 1, 3, 16-19,

260

and 21-29. All the AVA glucosides were novel compounds. Compound 1 had a molecular weight

261

at m/z 492 [M-H]-, which was 162 mass units higher than that of 5c (m/z 330 [M-H]-), signifying

262

1 is the glucoside of 5c. This was supported by the observation of fragment ion m/z 330 [M-162-

263

H]-, corresponding to the loss of a glucose unit (pattern c), in the MS/MS spectrum of 1 (Figure

264

4). Typical fragment ions at m/z 286, corresponding to the loss of CO2 (pattern a), and m/z 194,

265

formed by the breakdown of C-C bond between amide group and olefin group (pattern b), in the

266

aglycone of 1 were similar to those of 5c. In addition, the characteristic fragmentation patterns of

267

5c-3'-O-glc were similar to those of 2c-3'-O-glc and 3f-4'-O-glc (Figures 3 and 4). All of these

268

features suggest that 1 is the glucoside of 5c. Compound 3 (m/z 460 [M-H]-) had characteristic

269

fragment ions at m/z 298, following pattern c, and m/z 162, following pattern b, suggesting 3 is a

270

glucoside of 1c, 1c-3'-O-glc (Figures 1 and 4). Using similar strategies, we tentatively identified

271

the structures of the rest AVA glucosides as shown in Figure 1.

272

Validation of the Quantitative HPLC-MS Method. The quantitative HPLC-MS method

273

was validated in terms of linearity, precision, and accuracy (Table 1). Calibration curves were

274

constructed by plotting the integrated peak areas (x) of chromatography versus the corresponding

275

concentrations of the injected standard solutions (y). The limits of quantification were 33.19,

276

8.06, 3.39, 9.78, 34.15 nM for 2p, 2c, 2f, 3f, and 2c-3'-O-glc, respectively. The intra-day

277

variation was determined by analyzing the known concentrations of 2p, 2f, 2c, 3f, and 2c-3'-O-

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glc in six replicates during a single day, while inter-day variation was determined in duplicate on

279

three consecutive days, respectively. The overall intra- and inter-day variations were less than

280

1.85%, indicating satisfactory precision of the instrumentation and stability of the samples were

281

achieved. Recovery tests were performed to examine the accuracy of the analytical method.

282

Accurate amounts of authentic standards with three different concentration levels (low, middle

283

and high, n = 3) were added into the oat bran product (commercial oat sample, number 1) before

284

the samples were extracted and analyzed by the HPLC-MS method. The mean extraction

285

recovery was ranged from 94.11 to 104.0% among these five AVA standards (Table 1),

286

indicating that this method was consistent, reproducible, and acceptable.

287

Quantification of the Contents of the Major AVAs in Thirteen Commercial Oat

288

Products by HPLC-MS. The content of AVAs in commercial oat is influenced not only by

289

plant species and geographic environments, but also by the postharvest and processing

290

conditions.31 Although the AVA content in raw samples has been discussed before, no report on

291

the content of AVAs in commercial oat products in North America could be found. In this study,

292

we developed an HPLC-MS method to analyze the AVAs in thirteen commercial oat products

293

including six oat brans, three oatmeal, and four cold oat cereals. Figure 5 shows the total ion

294

chromatogram (TIC) of the fifteen major AVA aglycones and glucosides in three kinds of oat

295

products extract generated from negative HPLC-ESI/MS.

296

The contents of the major AVA aglycones and glucosides in these commercial oat products

297

are summarized in Table 2. In general, the levels of these compounds in cold oat cereal are much

298

lower than those in oat bran or oatmeal. The total amount of AVAs in the commercial oat bran

299

samples measured in this study varies from 36.49 to 61.77 mg/kg (fresh weight), which is higher

300

than what reported in many raw oats of different varieties.41-42 The total amount of AVA

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glucosides is in the range of 0.92 to 5.77 mg/kg, which contributes to about 5.2-10.11 % of the

302

total AVAs level and is about 5.48-11.24% of the total amount of AVA aglycones. The total

303

amount of 2c, 2f, and 2p is about 78.48-88.00% of the total AVAs level indicating results based

304

on the quantification of these three AVA aglycones significantly underestimate the total levels of

305

AVAs. Among all oat products, the AVAs contents in oat brans (samples number 1-6) are about

306

four times higher than those in cold oat cereal (samples number 10-13). Oat bran, oat flour and

307

rolled oat are typical oat products. Cold oat cereals were made of oat groats, whereas hot or

308

common oat cereals were added with oat bran. AVAs mainly exist in oat bran, their contents

309

would be influenced by processes such as steaming, flaking and autoclaving slightly.12, 31

310

Due to the similarity of chemical structure, it was a challenge to accurately quantify total

311

contents of AVAs. This is the first study to quantify individual AVA aglycones together with

312

their newly identified glucosides using LC-MS. The HPLC with UV or ECD detection was

313

frequently used to identify and quantify the AVA aglycones. The UV-spectrum of the AVA

314

aglycones are characteristically with peaks at λmax 330-350 nm and valleys at λmin 270-280 nm

315

and could be used for separating AVA aglycones form other compounds.12, 34 However, lower

316

detection limits and higher selectivity are needed for HPLC-UV, especially when analyzing

317

complex matrices containing small amounts of different AVAs. The phytochemicals such as

318

flavonoids in oat extracts would also interfere with UV-based HPLC identification of AVAs. The

319

HPLC-ECD possesses higher sensitivity than HPLC with UV detection. However, the responses

320

of individual AVAs to different ECD channels are similar and their retention times are close to

321

each other, which make it incredibly difficult to confirm the structures of individual AVAs.

322

Therefore, the identification of AVAs could not be achieved without authenticated standards by

323

HPLC-ECD method.43

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324

The rapid development of LC-MS and tandem mass in the agricultural and environmental field

325

transformed this combined technique into a valuable tool for the determination of nutrients or

326

biohazards compounds.9, 31, 36-37, 39-40, 44 For identification purpose, LC-MS can be employed to

327

identify minor or unknown AVAs without purified standards for comparisons if the

328

fragmentation patterns of AVAs have been established.31,

329

quantification purpose, the selection of AVA standards for estimating the total AVAs levels

330

should be taken into consideration due to the responses of some AVAs are significantly different

331

from others under the LC-MS condition. For example, we observed that 2c is significantly less

332

sensitive than 2f and 2p, and 2f is around 1.7 folds more sensitive than 3f at the same

333

concentration level. Therefore, 2c could not be quantified based on the standard curve of 2f or 2p,

334

and 3f content would be underestimated if based on the standard curve of 2f. The response area

335

of AVA 2c-3'-O-glc was found comparable with that of 2c. Consequently, the amount of AVA

336

glucoside derivatives could be estimated roughly by their corresponding AVA aglycones. In this

337

study, we used 5 AVA standards to quantify different types of AVAs. Therefore, our results are

338

much more accurate than what have been reported in the literatures using LC-MS simply based

339

on either three major AVA aglycones (2c, 2p and 2f) or corresponding caffeic acid, p-coumaric

340

acid and ferulic acid equivalently.32-33, 41-42

341

Cell Growth Inhibition by 2c and 2c-3'-O-glc. We have demonstrated that 2c could

342

significantly inhibit the growth of HCT-116 human colon cancer cells.17 In order to determine

343

whether glucosylation affects the toxicity of AVAs to cancer cells, AVA 2c and 2c-3'-O-glc were

344

evaluated for their growth inhibitory effects against HCT-116 and HT-29 human colon cancer

345

cells in the present study. Our results showed that AVA 2c-3'-O-glc had comparable inhibitory

346

effects with 2c in both cell lines and the IC50s of 2c-3'-O-glc in HT-29 and HCT-116 cells are

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36-37, 39-40, 42, 45

However, for

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347

389.9 and 301.1 µM, and both 2c and 2c-3'-O-glc could significantly inhibit the growth of both

348

cells (Figure 6), indicating glucosylation did not significantly influent the cell growth inhibition

349

activities of 2c. The mechanism of cell growth inhibition of AVAs was still unknown. In our

350

previous study, we found that 2c at 200 µM could not induce apoptosis in HCT-116 cells.17

351

However, it has been reported that 2c at 400 µM could induce apoptosis on MDA-MB-231

352

breast cancer cells.25

353

In conclusion, twenty-nine AVA aglycones and glucosides were characterized by NMR and/or

354

HPLC-ESI/MSn techniques. Among them, AVA glucosides are novel compounds. Using the

355

AVAs standards, we have outlined the comprehensive profile of both AVA aglycones and

356

glucosides in oat and have determined the levels of the major AVAs in thirteen commercial oat

357

products. Besides, our results showed that both 2c and 2c-3'-O-glc possessed similar inhibitory

358

effect against the growth of human colon cancer cells indicating glucosylation does not affect the

359

activities of AVAs. AVAs are unique in oat, and have the potential to be used as the exposure

360

markers to reflect whole grain oat intake. It is likely that the AVA glucosides can be hydrolyzed

361

by gut microbiota to generate the AVA aglycones and therefore contribute to the total levels of

362

AVA aglycones in vivo. However, this needs to be further confirmed experimentally.

363

ACKNOWLEDGEMENT

364

This work was partially supported by USDA NIFA R01 grant 2018-03084 to S. Sang.

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365

REFERENCES

366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409

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18. Chen, C.-Y. O.; Milbury, P. E.; Collins, F. W.; Blumberg, J. B., Avenanthramides are bioavailable and have antioxidant activity in humans after acute consumption of an enriched mixture from oats. J. Nutr. 2007, 137 (6), 1375-1382. 19. Lee-Manion, A. M.; Price, R. K.; Strain, J.; Dimberg, L. H.; Sunnerheim, K.; Welch, R. W., In vitro antioxidant activity and antigenotoxic effects of avenanthramides and related compounds. J. Agric. Food Chem. 2009, 57 (22), 10619-10624. 20. Yang, J.; Ou, B.; Wise, M. L.; Chu, Y., In vitro total antioxidant capacity and antiinflammatory activity of three common oat-derived avenanthramides. Food Chem. 2014, 160, 338-345. 21. Koenig, R. T.; Dickman, J. R.; Wise, M. L.; Ji, L. L., Avenanthramides are bioavailable and accumulate in hepatic, cardiac, and skeletal muscle tissue following oral gavage in rats. J. Agric. Food Chem. 2011, 59 (12), 6438-6443. 22. Anderson, J. W.; Gilinsky, N. H.; Deakins, D. A.; Smith, S. F.; O'Neal, D. S.; Dillon, D. W.; Oeltgen, P. R., Lipid responses of hypercholesterolemic men to oat-bran and wheat-bran intake. Am. J. Clin. Nutr. 1991, 54 (4), 678-683. 23. Sang, S.; Chu, Y., Whole grain oats, more than just a fiber: Role of unique phytochemicals. Mol. Nutr. Food Res. 2017, 61 (7). 24. Junsheng, F.; Yingdong, Z.; Aaron, Y.; L., W. M.; Jodee, J.; YiFang, C.; Shengmin, S., Oat avenanthramides induce heme oxygenase‐1 expression via Nrf2‐mediated signaling in HK‐2 cells. Mol. Nutr. Food Res. 2015, 59 (12), 2471-2479. 25. Hastings, J.; Kenealey, J., Avenanthramide-C reduces the viability of MDA-MB-231 breast cancer cells through an apoptotic mechanism. Cancer Cell Int. 2017, 17 (1), 93. 26. Shi, Y.; Johnson, J.; O'Shea, M.; Chu, Y.-F., The bioavailability and metabolism of phenolics, a class of antioxidants found in grains. Cereal Food World 2014, 59 (2), 52-58. 27. Hitayezu, R.; Baakdah, M. M.; Kinnin, J.; Henderson, K.; Tsopmo, A., Antioxidant activity, avenanthramide and phenolic acid contents of oat milling fractions. J. Cereal Sci. 2015, 63, 35-40. 28. Fontes‐Candia, C.; Ramos‐Sanchez, V.; Chavez‐Flores, D.; Salmeron, I.; Perez‐ Vega, S., Extraction of different phenolic groups from oats at a nonthermal pilot scale: Effect of solvent composition and cycles. J. Food Process Eng. 2017, 41 (2), e12651. 29. Nayak, B.; Liu, R. H.; Tang, J., Effect of Processing on Phenolic Antioxidants of Fruits, Vegetables, and Grains—A Review. Crit. Rev. Food Sci. Nutr. 2015, 55 (7), 887-918. 30. Min, S. C.; Kim, Y. T.; Han, J. H., 18 Packaging and the Shelf Life of Cereals and Snack Foods. Food Packaging and Shelf Life 2010, 339. 31. Bryngelsson, S.; Dimberg, L. H.; Kamal-Eldin, A., Effects of commercial processing on levels of antioxidants in oats (Avena sativa L.). J. Agric. Food Chem. 2002, 50 (7), 1890-1896. 32. Skoglund, M.; Peterson, D. M.; Andersson, R.; Nilsson, J.; Dimberg, L. H., Avenanthramide content and related enzyme activities in oats as affected by steeping and germination. J. Cereal Sci. 2008, 48 (2), 294-303. 33. Verardo, V.; Serea, C.; Segal, R.; Caboni, M. F., Free and bound minor polar compounds in oats: Different extraction methods and analytical determinations. J. Cereal Sci. 2011, 54 (2), 211-217. 34. Bratt, K.; Sunnerheim, K.; Bryngelsson, S.; Fagerlund, A.; Engman, L.; Andersson, R. E.; Dimberg, L. H., Avenanthramides in Oats (Avena sativa L.) and Structure−Antioxidant Activity Relationships. J. Agric. Food Chem. 2003, 51 (3), 594-600.

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35. Fagerlund, A.; Sunnerheim, K.; Dimberg, L. H., Radical-scavenging and antioxidant activity of avenanthramides. Food Chem. 2009, 113 (2), 550-556. 36. Wise, M. L., Effect of Chemical Systemic Acquired Resistance Elicitors on Avenanthramide Biosynthesis in Oat (Avena sativa). J. Agric. Food Chem. 2011, 59 (13), 70287038. 37. Collins, F. W.; Burrows, V. D. Method for increasing concentration of avenanthramides in oats. 2010. 38. Bratt, K.; Sunnerheim, K.; Bryngelsson, S.; Fagerlund, A.; Engman, L.; Andersson, R. E.; Dimberg, L. H., Avenanthramides in oats (Avena sativa L.) and structure-antioxidant activity relationships. J. Agric. Food Chem. 2003, 51 (3), 594-600. 39. Ishihara, A.; Kojima, K.; Fujita, T.; Yamamoto, Y.; Nakajima, H., New series of avenanthramides in oat seed. Biosci. Biotechnol. Biochem 2014, 78 (12), 1975-1983. 40. Okazaki, Y.; Ishihara, A.; Nishioka, T.; Iwamura, H., Identification of a dehydrodimer of avenanthramide phytoalexin in oats. Tetrahedron 2004, 60 (22), 4765-4771. 41. Xie, Z.; Mui, T.; Sintara, M.; Ou, B.; Johnson, J.; Chu, Y.; O'Shea, M.; Kasturi, P.; Chen, Y., Rapid quantitation of avenanthramides in oat-containing products by high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC-TQMS). Food Chem. 2017, 224, 280-288. 42. Multari, S.; Pihlava, J.-M.; Ollennu-Chuasam, P.; Hietaniemi, V.; Yang, B.; Suomela, J.P., Identification and Quantification of Avenanthramides and Free and Bound Phenolic Acids in Eight Cultivars of Husked Oat (Avena sativa L) from Finland. J. Agric. Food Chem. 2018, 66 (11), 2900-2908. 43. Chen, C.-Y.; Milbury, P. E.; Kwak, H.-K.; Collins, F. W.; Samuel, P.; Blumberg, J. B., Avenanthramides and Phenolic Acids from Oats Are Bioavailable and Act Synergistically with Vitamin C to Enhance Hamster and Human LDL Resistance to Oxidation. J. Nutr. 2004, 134 (6), 1459-1466. 44. Hernández, F.; Sancho, J. V.; Ibáñez, M.; Guerrero, C., Antibiotic residue determination in environmental waters by LC-MS. Trends Anal. Chem. 2007, 26 (6), 466-485. 45. Pridal, A. A.; Böttger, W.; Ross, A. B., Analysis of avenanthramides in oat products and estimation of avenanthramide intake in humans. Food Chem. 2018, 253, 93-100.

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Figure Captions Figure 1. Chemical structures with ESI-MS/MS fragment ions of the major AVA aglycones and glucosides (1-15) and minor AVA glucosides (16-29) in oat Figure 2. 1H-1H COSY (▬) and HMBC (H

C) correlations for the new AVA glucoside, 2c-

3'-O-glc Figure 3. The MS/MS spectra and fragmentation patterns of the standards of nine AVAs (2c, 2p, 2f, 3f, 1c, 1p, 4f, 2c-3'-O-glc, and 3f-4'-O-glc) Figure 4. The MS/MS spectra and fragmentation patterns of the representative AVA aglycones (1f, 3p, 4p, 5p, 5f) and glucosides (5c-3'-O-glc, 1c-3'-O-glc). Figure 5. Total ion chromatogram (TIC) of the fifteen major AVAs in Cold Oat Cereal (A), Oatmeal (B); and oat bran (C) extract generated from negative HPLC-ESI/MS. 1, 5c-3'-O-glc; 2, 2c-3'-O-glc; 3, 1c-3'-O-glc; 4, 2c; 5, 5p; 6, 5f; 7, 1c; 8, 2p; 9, 2f; 10, 3p; 11, 4p; 12, 3f; 13, 4f; 14, 1p; 15, 1f. Figure 6. Cell growth inhibition in HCT-116 and HT-29 human colon cancer cell lines by 2c and 2c-3'-O-glc. Asterisk (*) denotes a significant difference of the 2c/2c-3'-O-glc groups in comparison with the control group. (Mean ± SD, n = 8-16).

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Table 1. The standard curves, limits of detection (LOD) and quantification (LOQ), interday and intraday precision, and recovery of the five AVA standards, and the coverage of the AVA compounds by each standard Analytes

Standard curves

R2

LOD (nM)

LOQ (nM)

Interday precision (%)

Intraday precision (%)

Recovery (%)

Coverage

2c

y = 333.86x 1014.8

0.9999

9.95

33.19

0.97

1.84

94.11-103.15

1c, 2c, 3c, 4c, 5c

2p

y = 6174.5x 3776.4

0.9998

2.41

8.06

1.19

1.72

96.04-103.3

1p, 2p, 3p, 4p, 5p and their glcs

2f

y = 26147x 1875.4

0.9999

1.13

3.39

0.78

1.42

95.51-104.0

1f, 2f, 4f, 5f and their glcs

3f

y = 8181.3x 4030.8

0.9998

2.93

9.78

0.51

1.10

96.17-102.13

3f and 3f glc

2c-3'-Oglc

y = 499.65x 114.86

1

10.25

34.15

0.84

1.55

95.99-103.03

(1c, 2c, 3c, 4c, 5c)-glcs

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Table 2. Contents of AVAs in Thirteen Commercial Oat Products (µg/kg of products) a,b Oat

5c-3'-O-glc

2c-3'-O-glc

1c-3'-O-glc

2c

5p

5f

1c

2p

1

158.11±7.13

3421.97±264.94

147.62±2.87

16473.44±13.41

358.80±4.08

286.67±2.16

1347.29±17.47

14633.92±247.13

2

124.03±0.90

3407.07±127.70

178.51±13.82

10924.68±206.33

140.03±1.82

72.30±3.47

842.03±0.69

12334.76±155.97

3

123.26±5.51

3746.14±134.08

185.58±7.08

16625.61±700.48

248.16±11.63

155.64±4.25

1229.31±32.69

15823.60±12.88

4

211.77±8.17

5279.59±95.11

279.09±9.99

18361.18±138.49

448.06±11.91

318.54±14.36

1495.23±63.00

18869.02±356.38

5

112.32±5.35

2718.96±74.74

96.34±1.74

15553.21±747.13

337.64±14.23

220.12±1.61

836.76±6.31

15813.15±174.98

6

169.35±5.55

3518.34±157.16

167.72±4.61

21949.73±518.07

504.38±3.80

304.83±11.63

1696.65±81.5

19107.79±484.10

7

39.45±1.48

980.70±23.15

55.78±2.68

6634.70±75.53

159.37±2.10

121.94±3.24

561.87±11.59

8005.39±308.56

8

97.66±3.72

2960.33±80.82

167.16±6.27

9373.16±103.72

110.21±0.09

53.18±1.26

681.12±12.86

8484.44±111.95

9

66.07±1.82

2408.96±91.87

127.46±0.93

11566.99±307.58

113.74±2.88

72.93±2.73

921.59±43.20

9191.96±119.16

10

trace

527.00±10.23

35.54±0.84

3367.73±43.66

54.43±1.12

31.35±1.32

268.88±3.06

2534.05±97.67

11

trace

875.67±39.48

56.40±1.54

4407.12±111.66

67.94±1.28

46.68±0.91

353.24±3.91

6240.49±128.71

12

121.47±2.19

497.69±3.63

31.09±1.40

3653.50±171.24

120.57±1.33

71.34±5.52

358.66±9.09

3256.95±37.08

13

trace

846.83±32.67

37.08±1.66

5865.43±113.88

81.72±3.93

42.77±1.53

500.17±11.81

5992.38±280.87

a

Values expressed as mean ± standard deviation.

b

Oat bran: 1-6; Oatmeal: 7-9; and Cold Oat Cereal: 10-13.

23 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 24 of 31

Table 2. Contents of AVAs in Thirteen Commercial Oat Products (µg/kg of products) a,b, continued Oat

2f

4p

3f

4f

1p

1f

3p

Total AVAs

1

11362.50±532.58

605.76±12.49

3399.17±90.39

303.16±12.77

286.78±3.78

149.17±3.78

334.84±4.42

50335.52±946.52

2

7915.52±210.48

429.72±4.89

2278.91±96.02

223.54±1.69

277.12±5.72

109.16±5.12

424.19±8.75

36491.64±709.87

3

10279.74±199.59

687.97±17.43

2212.53±171.30

203.21±7.62

346.57±3.84

164.71±3.89

246.36±2.73

48927.59±1143.05

4

13983.66±1082.67

759.06±35.58

4941.45±185.21

519.16±14.17

417.38±10.57

198.15±3.85

595.77±15.09

61772.22±1900.45

5

12078.50±452.72

427.02±11.35

4717.09±128.78

232.20±10.47

282.12±13.22

143.98±11.15

633.91±29.71

52041.56±1621.54

6

11870.94±324.09

650.20±27.39

3118.41±140.60

233.26±1.70

461.42±12.27

203.56±7.63

383.94±10.21

61260.77±1556.54

7

5861.30±64.86

452.57±5.72

2219.57±56.23

182.79±8.57

175.57±0.22

83.68±1.58

258.09±4.36

25100.41±534.93

8

6626.39±279.19

348.12±4.51

2490.21±18.78

182.77±8.78

169.34±1.93

69.53±1.85

426.57±4.86

29492.24±567.19

9

6824.09±161.07

181.26±2.01

1466.05±28.46

71.04±5.50

226.50±4.28

117.28±5.63

146.29±2.76

31343.61±661.65

10

1605.81±40.68

503.52±6.64

388.62±18.21

138.07±3.67

89.46±1.13

35.86±0.40

47.97±0.61

9223.11±221.96

11

4650.93±35.08

161.85±0.13

1207.07±57.98

82.10±1.94

145.44±1.89

34.95±1.47

132.93±1.72

17736.44±351.38

12

2311.13±111.02

243.14±4.59

1104.54±26.07

83.36±1.62

81.77±0.07

114.72±0.87

146.51±0.12

11750.09±365.53

13

3911.53±176.36

229.61±1.73

1233.73±9.00

84.98±3.24

144.64±6.09

60.65±1.66

155.09±6.53

18554.67±612.47

a

Values expressed as mean ± standard deviation.

b

Oat bran: 1-6; Oatmeal: 7-9; and Cold Oat Cereal: 10-13.

24 ACS Paragon Plus Environment

Page 25 of 31

Journal of Agricultural and Food Chemistry

Compound No.

tR(min)

Compound

R1

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

9.97 11.99 19.54 15.10 15.36 16.04 16.13 18.20 19.43 19.87 20.24 20.58 21.09 26.33 26.91

5c-3'-O-glc 2c-3'-O-glc 1c-3'-O-glc 2c 5p 5f 1c 2p 2f 3p 4p 3f 4f 1p 1f

OH H H H OH OH H H H OCH3 OH OCH3 OH H H

16 17 18 19 20 21 22 23 24 25 26 27 28 29

11.58 11.83 11.92 12.43 13.15 13.73 13.92 14.40 14.51 14.89 19.21 19.32 19.45 19.83

2f-4'-O-glc 2p-5-O-glc 5f-4'-O-glc 2f-5-O-glc 3f-4'-O-glc 3c-3'-O-glc 3p-4'-O-glc 4c-3'-O-glc 3f-5-O-glc 4p-4-O-glc 5f-4/5-O-glc 1f-4'-O-glc 1c-4'-O-glc 1p-4'-O-glc

H H OH H OCH3 OCH3 OCH3 OH OCH3 Oglc OH/Oglc H H H

R2

R3

R4

[M - H]-

Major AVA aglycones and glucosides Oglc OH 492 Oglc OH 476 Oglc OH 460 OH OH 314 H OH 314 OCH3 OH 344 OH OH 298 H OH 298 OCH3 OH 328 H OH 328 H OH 298 OCH3 OH 358 OCH3 OH 328 H OH 282 OCH3 OH 312 Minor AVA glucosides OH OCH3 Oglc 490 Oglc H OH 460 OH OCH3 Oglc 506 Oglc OCH3 OH 490 OH OCH3 Oglc 520 OH Oglc OH 506 OH H Oglc 490 H Oglc OH 476 Oglc OCH3 OH 520 H H OH 460 OH/Oglc OCH3 OH 506 H OCH3 Oglc 474 H OH Oglc 460 H H Oglc 444 OH OH H OH OH OH H OH OH OH H OH H H H

Figure 1

25 ACS Paragon Plus Environment

MS/MS IONS

447.93(53), 330.14(47), 286.05(B), 193.95(77) 432.27(1), 323.09(B), 314.08(74), 297.10(49), 178.00(85) 416.10(21), 323.02(B), 298.02(75), 297.10(66), 161.94 (47) 270.06(1), 178.00(B) 270.10(B), 194.00(56), 178.00(31) 300.13(B), 194.03(37) 254.17(B), 162.02 (65) 254.10(B), 178.01(30) 284.08(B), 178.01(10) 284.08(B), 268.08(25), 208.03(64) 254.11(B), 178.01(83) 314.16(B), 299.11(37), 298.11(27), 208.03(28), 176.03(28) 284.13(B), 178.02(28) 238.05(B), 161.97(69), 144.98(32) 268.08(B), 162.02(19) 446.30(1), 328.14(88), 284.14(B), 269.10(16), 178.00(45), 175.05(20) 340.09(B), 298.17(6), 254.13(4), 178.00(7) 344.12(B), 300.10(17), 193.99(34) 446.14(1), 340.07(B), 328.08(15), 284.07(17), 177.98(11) 476.33(1), 358.05(91), 314.06(32), 207.98(B) 462.24(11), 437.92(11), 344.12(45), 323.10(35), 297.10(40), 207.99(B) 370.08(B), 328.06(20), 284.07(9), 207.97(10) 323.11(58), 314.08(70), 297.12(26), 178.00(B) 476.13(1), 370.13(B), 358.16(27), 314.15(17), 208.04(19) 340.05(61), 298.04(36), 254.04(B), 178.00(98) 462.24(15), 356.06(B), 344.07(48), 330.04(11), 300.07(50), 193.95(13) 312.12(B), 268.11(67), 253.11(27), 175.02(29), 161.98(48) 323.10(48), 298.09(B), 297.11(41), 162.00(61) 400.18(24), 376.11(26), 282.13(B), 238.16(73), 162.10(57)

Journal of Agricultural and Food Chemistry

Page 26 of 31

HO O

OH O

N H HO

O

OH OH

O

OH

Figure 2

26 ACS Paragon Plus Environment

OH

Page 27 of 31

Journal of Agricultural and Food Chemistry

254.17 298.21

80

Relative Abundance

Relative Abundance

184.04 162.02 60 40

283.05 20

234.27 226.21

121.10

326.06 345.22 0 100

150

200

250 m/z

300

399.30

350

Relative Abundance

Relative Abundance 161.03

200

250 m/z

300

264.18 313.16 200

300 m/z

500

254.10

40

178.01

20

211.22

0 400

226.11

160.06

100

150

280.18 297.21

200

250 m/z

300

350

400

284.13

100 Relative Abundance

80 60 299.11

40 176.03 208.03 20

343.10

121.10 149.01 0 100 150

246.36 286.14

193.05

80 60 40

178.02

20

134.01

269.10 313.07

0 200

250 m/z

300

350

400

100

200

300

400

m/z

323.09 178.00

80

100

314.08

Relative Abundance

Relative Abundance

100

60 297.10 40 20 135.05 207.12

0 100

150

200

270.14 250

340.07 386.07 300 m/z

350

400

432.27 450

207.96

358.05

80 60 314.06

40

299.02

20

400.12

505.09

599.45

0 500

200

300

400 m/z

284.10

100

60

134.02 350

400

314.16

100

Relative Abundance

210.04

118.00

80

242.09 270.06 286.09

185.05

150

144.98

20

100

40

0 100

40

100

60

134.01

161.97

60

400

80

20

80

0

178.00

100

238.05

100

Relative Abundance

100

500

Figure 3 27 ACS Paragon Plus Environment

600

80 60 40 20 121.12 160.02 0 100 150

178.01 193.15 200

251.06

269.09

250 m/z

313.07 300

350

400

Journal of Agricultural and Food Chemistry

284.08

100

268.08

80 60 40 162.02

20

253.08

144.05

80 60 40 269.07 20

176.03

313.06

144.99

297.01

256.08

192.98

0 200

300

400

100

500

150

200

250 m/z

m/z

270.10

80 60

194.00

40 178.01 20

242.09

145.02

296.10

100

400

194.00 150.01

20

285.08

200

300

100

400

200

326.09 400

Relative Abundance

60

447.93

330.14 40

424.01 356.12

80

298.02

60 161.94 40 391.92 416.10 20

402.06

256.03

500

323.02

100

193.95

176.05

300 m/z

286.05

100

477.86

445.13

135.02

340.09 380.10

254.10 272.00

0

0 200

300

400 m/z

500

150

200

250

80 60 40 145.04

283.08 226.09

20

298.25

300 m/z

350

400

Figure 4 28 ACS Paragon Plus Environment

450

500

366.13

431.13

0 200

300 m/z

60 40

178.01

100

80

m/z

20

350

0

0

80

300

300.09

100

Relative Abundance

100

Relative Abundance

314.12

0

100

254.11

100 208.03

Relative Abundance

Relative Abundance

Relative Abundance

100

Relative Abundance

Page 28 of 31

400

Journal of Agricultural and Food Chemistry

Relative Abundance

Page 29 of 31

100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0

A (min)

9 6, 7

B

8

5 1

11

2

10

15

13

3

14

12

4

(min)

C (min)

8

10

12

14

16

18

20

Figure 5

29 ACS Paragon Plus Environment

22

24

26

28

30

Journal of Agricultural and Food Chemistry

Figure 6

30 ACS Paragon Plus Environment

Page 30 of 31

Page 31 of 31

Journal of Agricultural and Food Chemistry

Graphic Abstract:

31 ACS Paragon Plus Environment