Antioxidative Maillard Reaction Products Generated in Processed

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Bioactive Constituents, Metabolites, and Functions

Antioxidative Maillard Reaction Products Generated in Processed Aged Garlic Extract Junichiro Wakamatsu, Timo D. Stark, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06907 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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Journal of Agricultural and Food Chemistry

1

Antioxidative

Maillard

Reaction

Products

2

Generated in Processed Aged Garlic Extract

3

Junichiro Wakamatsu, Timo D. Stark, and Thomas Hofmann*

4 5

Food Chemistry and Molecular Sensory Science, Technische Universität

6

München, Lise-Meitner-Straße 34, 85354 Freising, Germany

7 8 9 10 11 12 13 14 15 16

*

17

PHONE

+49-8161/71-2902

18

FAX

+49-8161/71-2949

19

E-MAIL

[email protected]

To whom correspondence should be addressed

20 21

1 ACS Paragon Plus Environment


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23

ABSTRACT

24

A powder formulation of aged garlic extract was heated at 100 oC for one day to

25

obtain higher antioxidant activity determined with ABTS radical scavenging

26

(ARS) and ORAC assays. Activity-guided fractionation afforded 12 new in vitro

27

antioxidative

28

hydroxymethyl)pyrrol-1-yl]arginine

29

dihydro-6H-pyrano[2,3-b] pyrazine-3-yl]butane-1,2,3-triol (4) and 4-[6-(1,2-

30

dihydroxyethyl)-6,7-dihydro-furo[2,3-b]pyrazin-3-yl]-butane-1,2,3-triol (5), -[(2-

31

formyl-5-hydroxymethyl)-pyrrol-1-yl]

32

dihydroxyethyl)-2-oxotetrahydrofuran-3-yl]-5-(hydroxymethyl)-1H-pyrrole-2-

33

carbaldehyde (14), 4-(6-ethyl-2-pyrazinyl)-1,2,3-butanetriol (17), -[(2-formyl-5-

34

hydroxymethyl)pyrrol-1-yl] glutamic acid (19), (S)-1-[(5-hydroxymethyl)furan-2-

35

yl]methyl]-5-oxopyrrolidine-2-carboxylic

36

(hydroxymethyl)furan-2-yl}methyl]-2,5-dioxo-3-pyrrolidine acetic acid (21), (E)-4-

37

(5-methylpyrazin-2-yl)but-3-ene-7,2-diol

38

(hydroxymethyl)picolinic acid (24), (E)-4-(6-methylpyrazin-2-yl)but-3-ene-1,2-

39

diol (26) and 14 known compounds (Figure 1, 1, 2, 6-11, 13, 15, 16, 18, 22 and

40

25) which were characterized via 1D/2D-NMR, CD spectroscopy, and mass

41

spectrometry. ARS and ORAC activities of these antioxidants ranged from 0.01

42

to 0.49 mol TE/mol and from 0.01 to 3.50 mol TE/mol, respectively.

43

Additionally, plausible formation pathways for the new organic acid-type

44

products (15, 20 and 21) were proposed based on proving their generation in

45

model reactions detected via LC-MS/MS.

Maillard-type

products (3),

(Figure

1),

-[(2-formyl-5-

4-[7-hydroxy-6-(hydroxymethyl)-7,8-

aspartic

acid

46


acid

(20),

(23),

(12),

1-[5-(1,2-

3-hydroxy-1H-[{5-

4-acetyl-6-

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KEYWORDS: aged garlic extract, Maillard reaction, antioxidant, glutamic acid,

48

citric acid

49 50

INTRODUCTION

51

Aerobic organisms, such as human being, animals, and some kinds of

52

bacteria utilize oxygen for energy metabolism to survive, and during oxidative

53

phosphorylation in cells, partial amounts of the oxygen could be incompletely

54

reduced into reactive oxygen species (ROS), e.g. hydrogen peroxide,

55

superoxide anion radicals and hydroxyl radicals which have important roles to

56

maintain cellular homeostasis.1 While ROS are essential molecules to operate

57

cellular systems, excessive ROS levels accumulated by smoking, overeating,

58

overdrinking, stressful daily life and aging could be also one of triggers for

59

undesirable cellular oxidation which is related to diseases like diabetes, cancer,

60

and Parkinson’s disease.2―4

61

Garlic (Allium Sativum. L) is recognized as a multifunctional food in the

62

world, and it has been used as a flavorful spice in dishes and for herbal therapy

63

due to its biological activities based on antiatherosclerotic, anticarcinogenic and

64

antioxidative effects.5-7 Recently, black colored garlic prepared via heating at

65

70-90 oC under high humidity (70-90%) for 30-45 days from raw garlic, namely

66

black garlic,8-11 has been reported as a special garlic preparation considerably

67

possessing higher antioxidant activity than raw garlic.8-9 Although S-allyl-L-

68

cysteine as major sulfur-containing amino acid and potential antioxidant in garlic

69

preparations was already identified and quantified in black garlic,10-11 other

70

antioxidative compounds generated by the thermal processing are still unclear.



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Aged garlic extract (AGE) is a well-known garlic preparation possessing

72

cardio-protective,12,13 liver-protecticve,14,15 and cancer-preventing effects as well

73

as antioxidative activity. 16-17 Since it is manufactured by extracting fresh garlic

74

with aqueous ethanol and maturing the extract for more than 10 months under

75

ambient temperatures, excessive constituents, such as sugars and amino acids

76

would predominantly remain in AGE, thus, assuming that a further thermal

77

processing of AGE will also induce a benefit of antioxidant activity through

78

browning reactions.

79

Therefore, the objective of the present study was to thermally process AGE,

80

to isolate and identify potential antioxidants by means of activity-guided

81

fractionation with ABTS radical scavenging (ARS) and oxygen radical

82

absorbance capacity (ORAC) assays, to evaluate the chemical antioxidant

83

activity of those compounds using both assays, and, finally to propose reaction

84

pathways for three novel compounds with a common pyrrolidone ring structure

85

and a unique carboxylic acid moiety.

86 87

MATERIALS AND METHODS

88

Chemicals. The following reagents were obtained commercially: 2,2’-

89

azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), (±)-

90

6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), fluorescein

91

sodium salt (FL), 2,2’-azobis(2-methylpropinamidine) (AAPH), quercetin,

92

ascorbic

93

phosphate, sodium hydroxide solution (Sigma Aldrich, Steinheim, Germany).

94

AGE and powdered AGE were produced and provided by Wakunaga

95

Pharmaceutical Co. Ltd. Water for chromatographic preparations was purified

acid,

potassium

dihydrogenphosphate,


dipotassium

hydrogen-

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96

with a Milli-Q Gradient A10 system (Millipore, Schwalbach, Germany), and

97

solvents used were of HPLC-grade (Merck, Darmstadt, Germany). Deuterated

98

solvents were obtained from Euriso-Top (Saarbrücken, Germany).

99

General Experimental Procedures. 1D/2D-NMR spectroscopy 1H, 1H-1H

100

COSY, 1H-13C HSQC and HMBC, and

13C

101

on an Avance III 500 MHz spectrometer with a CTCI probe and an Avance III

102

400 MHz spectrometer with a BBO probe (Bruker, Rheinstetten, Germany),

103

respectively. Topspin software (version 2.1; Bruker) as well as MestReNova

104

software (version 5.2.3; Mestrelab Research, Santiago de Compostella, Spain)

105

was used to process the NMR data. Mass spectra of the compounds were

106

measured on a Waters Synapt G2-S HDMS mass spectrometer (Waters,

107

Manchester, UK) coupled to an Acquity UPLC core system (Waters, Milford, MA,

108

USA). For circular dichroism (CD) spectroscopy, sample solutions of

109

compounds were analyzed by means of a Jasco J-810 spectropolarimeter

110

(Hachioji, Japan). A Büchi Sepacore system (Flawil, Switzerland) was employed

111

for medium pressure liquid chmromatography (MPLC) separation using a

112

polypropylene cartridge (id. 40 mm, l. 150 mm) and LiChroprep RP18, 25-40 m

113

mesh material (Merck, Darmstadt). HPLC separations were performed on a

114

preparative HPLC system (Jasco, Gross-Umstadt) consisting of two PU-2087

115

Plus pumps, a DG-2080-53 degasser, a LG-2080-02 gradient unit and a 2010

116

Plus multiwavelength detector.

NMR experiments were performed

117

Preparation and thermal processing of powdered AGE. Briefly, ethanol

118

was removed from liquid AGE (pH 5.8) using an evaporator, and it was

119

continuously powdered for one hour with a laboratory spray-drying machine

120

including a rotary disk system for atomization (Ohkawara Kakohki Co., Ltd. 5 ACS Paragon Plus Environment


121

Japan) and fine powder was obtained with 3.5% water content for powder

122

processing. Thereafter, the water content of three batches of powdered AGE

123

were individually adjusted to 5.4, 8.0 and 10.5% (w/w) via a closed chamber

124

(PR-4K, ESPEC, Japan) which was electronically controlled in the following

125

conditions; temperature: 25

126

confirmed by using an infrared moisture meter (MOC-120H, Shimazu, Japan).

127

Subsequently, liquid AGE (50 mL) and four powder formulations (10 g each)

128

containing 3.5, 5.4, 8.0, and 10.5 % water were put into glass vials with caps

129

(50 mL), and they were thermally processed in a laboratory oven at 80 and 100

130

oC.

131

2, 3, 4 and 5 at 100 oC. For antioxidative activity measurements by means of

132

ARS and ORAC assays in duplicate, aliquots (100 mg) of each heated material

133

were dissolved in water (10 mL), followed by filtration using 0.45 m syringe

134

filters.

oC,

humidity: 60%. The water content was

Samplings were performed on day 1, 3, 5, 10, 15 and 30 at 80 oC and day 1,

135

Ultrafiltration. Powdered AGE containing 3.5 % water was thermally

136

processed at 100 oC for 1 day, and an aliquot (1000 mg) was dissolved in water

137

(250 mL). The solution was ultrafiltrated using a 5 kDa cutoff membrane

138

(polyethersulfone, Sartorius stedim biotech GmbH, Germany) and a filtration

139

device (Vivacell 250, Sartorius stedim biotech GmbH) under ambient

140

temperature. The filtrate and the residue were dried by lyophilization, yielding

141

704 and 298 mg, respectively. Next, an aliquot of the dried filtrate (480 mg) was

142

dissolved in water (100 mL), and a further ultrafiltration step using 1 kDa cutoff

143

membrane (regenerated cellulose, EMD Millipore Corporation, USA) and a

144

device (Stirred Ultrafiltration Cells model 8400, EMD Millipore Corporation) was

145

conducted under ambient temperature. The filtrate and the residue were freeze6 ACS Paragon Plus Environment

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dried, which resulted in 225 and 241 mg, respectively. Finally, the filtrates and

147

the residues as well as a recombined solution based on natural ratios were

148

evaluated on antioxidant activities using ARS and ORAC assays.

149

MPLC separation. Aliquots (500 mg) of the low molecular weight fraction

150

(97%, and, thereafter, antioxidative activities of the three

340

fractions, a recombined solution of each fraction using natural ratios, and the

341

whole extract were evaluated employing ARS and ORAC assays (Figure 4). In

342

both assays, the recombined solution showed comparable activities as the

343

whole extract, thus indicating that no significant activity losses took place during

344

the ultrafiltration. Although the ARS activity of the 1-5 kDa fraction relatively


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345

exhibited the highest value, differences between the three fractions were

346

marginal. In contrast, the ORAC assay revealed that the low molecular weight

347

fraction (< 1 kDa) relatively possessed the highest contribution by a factor of

348

0.55 compared to the whole activity, followed by 1-5 kDa and >5 kDa fractions.

349

Therefore, the low molecular weight fraction was further fractionated by means

350

of chromatographic approaches.

351

Isolation, Purification and Structure Elucidation. The low molecular

352

weight fraction (< 1 kDa) was further separated by means of MPLC into 17

353

fractions (M1-17) and evaluated on antioxidant activities using the ARS and

354

ORAC assays (Figure 5). The sum of yields of the 17 fractions was 96.2%

355

exhibiting a good recovery. In the ARS assay (Figure 5B), fraction M4

356

highlighted the highest activity, followed by seven fractions (M5-M11). The sum

357

of activities of M4-M11 accounted for 89% of the whole activity of the low

358

molecular weight fraction. Further, the ORAC assay also indicated that fraction

359

M4 has the highest activity, and seven fractions from M5 to M11 as well as M2

360

showed relatively high activities (Figure 4B). The sum of the ORAC activities of

361

the eight fractions (M2 and M5-M11) accounted for 80% of the whole low

362

molecular weight fraction. Taking both assessments into consideration, eight

363

fractions between M4 and M11 should preferentially be focused on, and herein,

364

the investigation of the four fractions M4-M7 is reported.

365

M4-M7 were purified via HPLC, thus affording totally 26 compounds (Figure

366

1). NMR spectroscopy and high-resolution MS of the isolated compounds

367

enabled the identification of the known compounds as 1,2,3-butanetriol,4,4’-

368

(2,5-pyrazinediyl)bis- (1), 1,2,3-butanetriol,4,4’-(2,6-pyrazinediyl)bis- (2), DDMP

369

(6), 1,2,3-butanetriol-4-(6-methyl-2-pyrazinyl) (7),


5-hydroxymethyl-2-furan-


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370

carboxylic acid (8), hydroxymethylfurfural (9), thymidine (10), 5-hydroxymethyl-

371

1H-pyrrole-2-carbaldehyde

372

acid (13), (2S)-1-[2-(furan-2-yl)-2-oxoethyl]-5-oxopyrrolidine-2-carboxylic acid

373

(15), 5-hydroxy-3,4-dimethyl-2(5H)-furanone (16), 5-hydroxymaltol (18), -[(2-

374

formyl-5-hydroxymethyl)pyrrol-1-yl]alanine (22), cordyrrole A (25), respectively.

375

Although compounds 1, 2, 7 and 22 are literature known compounds, so far the

376

identification was proposed only by mass spectrometry,19,20,31 herein, their

377

structure elucidation by means of NMR and TOF MS (ESI) data are presented

378

for the first time (SI). The spectroscopic data for compounds 6, 8-11, 13, 15, 16,

379

18 and 25 were well in line with literature data (SI).21-30

(11),

2-formyl-5-(hydroxymethyl)-pyrrole-1-acetic

380

Compounds 4 and 5 were obtained from M4 as brown amorphous powders.

381

UPLC-TOF MS in the ESI+ mode revealed the same protonated molecule of m/z

382

287.1252 [M+H]+, suggesting a molecular formula of C12H18N2O6 and implying

383

isomers. Also the 1H and

384

Four nonequivalent methylene protons resonating at 2.50, 3.66, 2.90 and 3.55

385

ppm [H-12a, -12b, -15a, -15b] as well as two heteroatom-bearing methine

386

protons at 3.74 and 3.34 ppm [H-13,-14] were assigned for 4. From the

387

NMR spectrum and heteronuclear HSQC correlations, two methylene carbons

388

resonating at 38.0 and 63.3 ppm [C-12,-15] and two methine carbons at 71.2

389

and 74.9 ppm [C-13,-14] were determined. These assignments indicated the

390

presence of a 1,2,3-butanetriol moiety, which was supported by homonuclear

391

COSY connectivities of H-12/H-13, H-13/H-14 and H-14/H-15. In addition, an

392

aromatic carbon at 137.0 ppm [C-7] and three aromatic quaternary carbons at

393

135.4 [C-5], 151.7 [C-8], and 155.8 ppm [C-10] were observed

394

pyrazine system, of which the assignment was achieved by the HMBC

13C

NMR spectra showed quite similar data (Table 1).


13C

forming a

Page 17 of 43


395

correlations of H-7C-5,8 and of H-7C-10. Then, a linkage between the

396

above two moieties was confirmed to be at C-8 via the HMBC signal of H-7C-

397

12. Furthermore, two methine proton resonances at 4.12 [H-2] and 4.07 ppm

398

[H-3] and two nonequivalent methylene protons at 2.80 [H-4a] and 3.08 ppm [H-

399

4b] exhibited HMBC cross peaks of H-2C-3,4,10, H-3C-5 and H-4C-

400

2,3,5,10. This interpretation as well as the HSQC spectrum of carbons of C-2, -

401

3 and -4 revealed the presence of a tetrahydropyran moiety linked to the

402

pyrazine ring at C-10 and C-5. Besides, the COSY and HMBC spectra indicated

403

a hydroxymethyl function connected at C-2 based on correlations of H-11C-

404

2,3. These assignments enabled to determine the chemical structure of 4 as 4-

405

[7-hydroxy-6-(hydroxymethyl)-7,8-dihydro-6H-pyrano[2,3-b]pyrazine-3-

406

yl]butane-1,2,3-triol, a new compound.

407

In contrast to the NMR data of 4, 5 showed different resonances of the

408

aromatic carbons at 143.2 and 161.9 ppm [C-4, -9], and the two carbons

409

revealed HMBC connectivities to three aliphalic protons [H-2C-3,4,9, H-3C-

410

2,4,9]. These differences indicated that 5 has a tetrahydrofuran moiety bonded

411

with the pyrazine ring at C-9 and C-4. Then, two aliphalic methylene protons

412

resonating at 3.41 ppm [H-11] and a heteroatom-bearing methine at 3.83 ppm

413

[H-10] showed in the HMBC spectrum cross peaks of H-11C-2,10 and H-

414

10C-2,3,11, thus highlighting the presence of a dihydroxyethyl function

415

connected to carbon C-2. Therefore, the structure of 5 was established as 4-[6-

416

(1,2-dihydroxyethyl)-6,7-dihydrofuro[2,3-b]pyrazin-3-yl]-butane-1,2,3-triol, which

417

has not been reported before in literature.

418

Additionally, further alkyl-and alkenyl-pyrazines (compounds 17, 23 and 26)

419

were isolated from M7. The mass spectra of 17 showed a protonated molecule



420

of m/z 213.1242 [M+H]+, thus suggesting a two-nitrogen-containing molecular

421

formula of C10H16N2O3. Comparing the 1H and

422

of 4 and 5, the presence of a 1,2,3-butanetriol moiety linked to a pyrazine ring at

423

carbon C-2 was deduced. An ethyl group proposed from the 1H and HSQC

424

spectra was located at C-6, and this assignment was supported by the HMBC

425

spectrum showing connectivities of H-11C-5,6,12 and H-12C-6,11. Thus,

426

the chemical structure of 17 was established as 4-(6-ethyl-2-pyrazinyl)-1,2,3-

427

butanetriol.

13C

NMR spectra of 17 to those

428

High-resolution mass spectrometry for compounds 23 and 26 indicated the

429

same protonated molecule of m/z 181.0982 [M+H]+, suggesting their elemental

430

compositions of C9H12N2O2. Also, 23 and 26 showed almost similar chemical

431

NMR shifts and slightly different from compound 17, thus implying to be

432

constitution isomers. The 1H NMR spectra and HSQC correlations of 23 and 26

433

enabled to assign H 6.69 and 6.86 for 23 and H 6.69 and 6.92 for 26 as

434

olefinic protons at C-7 and C-8 with coupling constants of 15.9 and 15.8 Hz,

435

respectively, resulting in the olefinic (E) arrangements. In 23 and 26, the

436

presence of methyl functions were confirmed via the 1H spectrum and HSQC

437

correlations, and positions of the methyl functions and the alkenyl residues were

438

determined by further interpretation of the HMBC connectivities of H-5C-3,6,

439

H-7C-2,3 and H-11C-5,6 for 23 and H-6C-2,5,11, H-7C-2,3 and H-

440

11C-5,6 for 26. Subsequently, the structures were disclosed as (E)-4-(5-

441

methylpyrazin-2-yl)but-3-ene-7,2-diol (23) and (E)-4-(6-methylpyrazin-2-yl)but-

442

3-ene-1,2-diol (26).

443

Pyrrole-type compounds 3, 12, 14 and 19 were isolated as brown

444

amorphous powders, respectively. For these compounds, corresponding NMR 18 ACS Paragon Plus Environment

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signals for 5-hydroxymethyl-1H-pyrrole-2-carbaldehyde residues were observed

446

(Table 2). Coupling constants of two olefinic protons at C-3 and C-4 exhibited

447

J=3.9-4.0 Hz, typical values for a pyrrole system,28 and the moiety was

448

undoubtedly confirmed via the heteronuclear HMBC connectivities of H-3C-

449

2,4,5,6, H-4C-2,3,5,7, H-6C-2,3 and H-7C-4,5. In compound 12, H of an

450

aliphatic methine proton resonating at 5.48 ppm [H-6] and two nonequivalent

451

methylene at 2.81 and 3.30 ppm [H-7a/-7b] and C of a methylene carbon at

452

37.1 ppm [C-7], a methine at 54.8 ppm [C-6] and two quaternary at 170.2 [C-8]

453

and 172.0 ppm [C-11] indicating the existence of an aspartic acid motif and

454

substantiated via the HMBC and COSY correlations of H-C-7C-6,8,11 and H-

455

7ab/H-6. In contrast, 3 and 19 showed additional signals of a nonequivalent

456

methylene (H: 1.83 and 2.01, C: 35.1) or two nonequivalent methylene and a

457

quaternary carbon (H: 1.01, 1.42, 2.95 and 3.07, C: 26.2, 40.3 and 157.1). For

458

3, an arginine residue was established via 2D-NMR signals of H-9C-8,14, H-

459

10C-8,9 and H-11C-9,10,13 as well as a broad singlet (9.11 ppm) assigned

460

for H-N-12. A glutamic acid moiety was determined by interpretation of the

461

COSY and HMBC cross peaks of H-9ab/H-10ab, H-9C-8,10,11,12 and H-

462

10C-8,9,11 for 19. NMR signals were not observed to determine the linkages

463

between the amino acid moieties and pyrrole rings for the compounds 3, 12 and

464

19, but this phenomenon was in line with S-allyl-L-cysteine type compounds in

465

the previous study.32 Also, their expected deprotonated molecules of m/z

466

281.1240 [M-H]-, 240.0511 [M-H]- and 254.0668 [M-H]- were detected by means

467

of LC-ESI-TOF-MS, and consequently the identification of the chemical

468

structures was confirmed as -[(2-formyl-5-hydroxymethyl)pyrrol-1-yl]arginine

469

(3), -[(2-formyl-5-hydroxymethyl)pyrrol-1-yl]aspartic acid (12), and -[(2-formyl19 ACS Paragon Plus Environment


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470

5-hydroxymethyl) pyrrol-1-yl]glutamic acid (19). So far, three analogs of glycine,

471

alanine and S-allyl-L-cysteine were reported in literature,20,

472

new compounds of this structure type are described.

29, 32

herein, three

473

Besides, in compound 14 a -butyrolactone moiety was identified based on

474

the HMBC connectivities of H-8C-9,11, H-10C-9,11 and H-11C-8,9,10.

475

Further interpretation of the HMBC spectrum clarified a C-8-N-1 linkage

476

between the pyrrole and -butyrolactone moieties due to signals of H-8C-2,5.

477

Moreover, a 1,2-ethyldiol moiety consisted of carbon resonances at 61.8 [C-13]

478

and 72.0 ppm [C-12] and proton resonances at 3.38 [H-13] and 3.78 ppm [H-12]

479

was assigned based on the COSY correlation of H-12/H-13 for the -

480

butyrolactone residue, generating a C-10-C-12 linkage determined via the

481

HMBC connectivities of H-12C-10,11 and H-13C-10,12. The NMR

482

interpretation and high resolution mass spectrometry exhibiting the protonated

483

molecule of m/z 270.0978 [M+H]+ revealed the identification of 14 as 1-[5-(1,2-

484

dihydroxyethyl)-2-oxotetrahydrofuran-3-yl]-5-(hydroxymethyl)-1H-pyrrole-2-

485

carbaldehyde. To the best of our knowledge, this compound has not been

486

previously reported in literature. Compounds 20 and 21 were obtained as brown amorphous powders. By

487 488

comparing the 1H and

13C

489

6.2 and 6.2, C: 56, 108, 109, 148 and 155) for a hydroxymethylfuran moiety like

490

in compounds 8 and 9 were observed. In compound 20, two quaternary carbons

491

at 173.2 and 174.1 ppm [C-14, -13], two methylene carbons at 22.4 and 29.0

492

ppm [C-11,-12] and a methine carbon at 58.2 ppm [C-10] were assigned by the

493

13C

494

corresponding protons of H-10C-11,12,14, H-11C-12,13,14 and H-12C-

NMR spectra (Table 3), similar resonances (H: 4.3,

NMR and HSQC spectra. These carbons showed HMBC cross peaks with


Page 21 of 43


495

13 indicating the presence of a pyroglutamic acid residue. Further interpretation

496

of the HMBC correlations confirmed a N-9-C-8 methylene bridge between both

497

moieties via the signals of H-8C-4,5,10,13, and, consequently, the relative

498

structure was established in accordance to its expected elemental composition

499

of C11H13NO5 and the detected deprotonated molecule of m/z 238.0715 [M-H]-.

500

Using the S-configured compound 15 as a reference compound, the absolute

501

configuration of 20 was deduced by means of CD spectroscopy as S

502

(Supporting Information, Figure S183), and, therefore, 20 could be elucidated

503

as (S)-1-[(5-hydroxymethyl)furan-2-yl]methyl]-5-oxopyrrolidine-2-carboxylic acid.

504

For the structure determination of 21, three quaternary carbons resonating

505

at 71.7 174.4 and 177.9 ppm [C-11, -13, -10] and a methylene carbon at 42.0

506

ppm [C-12] were determined by the HSQC spectrum. The HMBC correlations of

507

H-11C-10,11,13 indicated the presence of a 2,5-dioxopyrrolidine moiety

508

consisted of carbons [C-10-C13] and a nitrogen. Also, two methylene protons at

509

2.81 and 2.90 ppm [H-14ab] showed HMBC cross peaks with a quaternary

510

carbon resonating at 171.5 ppm [C-15] and the three above mentioned carbons

511

[C-10-C12]

512

connectivities clarified that an ethyl carboxylic acid is located at C-11. A

513

methylene bridge for a linkage between both motifs was confirmed through

514

further HMBC signals of H-8C-4,5,10,13. The presence of a hydroxyl function

515

at the chiral center C-11 was proposed based on the elemental composition of

516

C12H13NO7 that was expected from the deprotonated molecule of m/z 282.0619

517

[M-H]-. These assignments enabled to determine the structure of 21 as 3-

518

hydroxy-1H-[5-(hydroxymethyl)furan-2-methyl]-2,5-dioxo-3-pyrrolidine

of

the

2,5-dioxopyrrolidine

motif,


and

consequently

these

acetic


Page 22 of 43

519

acid. To the best of our knowledge, compounds 20 and 21 have not yet been

520

reported in literature.

521

Compound 24 was obtained from M7 as brown oil. UPLC-ESI-TOF MS

522

showed a deprotonated molecule of m/z 194.0457 [M-H]-, suggesting a

523

molecular formula of C9H9NO4. The following 1H and

524

and 8.25, C: 120.3, 120.8, 144.5, 148.9 and 163.7) were assigned for the

525

pyridine ring, which was supported by HMBC connectivities of H-3)C-2,4,5

526

and H-5C-3,6. In addition, methyl protons resonating at 2.68 ppm [H-10] and

527

methylene protons at 4.71 ppm [H-7] were observed. Their positions were

528

confirmed at C-2 and C-4 of the pyridine ring, respectively, via the HMBC

529

spectrum of H-7C-2,3 and H-10C-4,8 as a hydroxymethyl and an acetyl

530

group. A carboxylic group linked at C-6 was determined based on the

531

suggested molecular formula and the HMBC cross peak between the

532

quaternary carbon resonance at 165.8 ppm [C-9] and the proton resonance for

533

H-5. Therefore, the chemical structure was established as 4-acetyl-6-(hydroxy-

534

methyl)picolinic acid (24), a new structure.

13C

NMR signals (H: 8.07

535

Proposed Reaction Pathways for the Formation of Compounds 15, 20

536

and 21. As compounds 15, 20 and 21 contain glutamic acid or citric acid

537

moieties and organic acid-type Maillard products have not previously been

538

reported in literature, their formation were demonstrated via model reactions

539

using D-glucose and L-glutamic acid or citric acid and confirmed by means of

540

UPLC-ESI-MS/MS. (Supporting Information, S184). Consequently, the following

541

reaction pathways for Maillard-type products 15, 20 and 21 could be proposed

542

(Figure 6).


Page 23 of 43


543

A reaction cascade was supposed starting from the 3-deoxyhexosone (1) and

544

glucosone (5) generated from glucose in course of the Maillard reaction.

545

Reaction of 1 with amino acids yields the 1-amino-1-deoxy-fructose (2) that

546

reacts with the citric acid via water elimination resulting in the amide-type

547

conjugation (3). After intramolecular dehydration forming the diketo-pyrrolidine

548

ring (4), the alkyl chain in 4 is further cyclized by a reaction of the hydroxyl

549

group with the ketone to reveal the tetrahydrofuran ring, followed by the

550

elimination of two water molecules yielding compound 21. Also, 1 could react

551

with L-glutamic acid rising the pyroglutamic acid and furan systems for

552

compound 20 via the same reaction cascade as mentioned above. Besides,

553

generation of compound 15, an analog for 20, starts from the reaction of L-

554

glutamic acid with 5 upon water elimination. Affording the pyroglutamic acid

555

type intermediate (6) like in the case of compound 20 and 21, it transforms into

556

a further intermediate (7) via tautomerization and dehydration. The terminal

557

hydroxyl group of the alkyl chain of 7 attacks nucleophilic to the ketone group

558

for the formation of the tetrahydrofuran ring, followed by dehydration of two

559

molecules of water, thus yielding compound 15 (Figure 6).

560

In Vitro Antioxidant Activities of Isolated Compounds. All isolated

561

compounds were assessed by ARS and ORAC assays (Table 4). Ascorbic acid

562

and quercetin, well-known antioxidants in literature, exhibited comparable

563

activities to literature data in the both assays.17 Among the compounds, 18

564

showed the highest ARS as well as ORAC activity of 0.49 mol and 3.50 mol

565

TE/mol. Interestingly, both activities of DDMP (6), a structural similar

566

compound, were lower by a factor of four for ARS and 22 for ORAC activity,

567

which may be caused by the lack of the second double bond. Activities of the 23 ACS Paragon Plus Environment


Page 24 of 43

568

other compounds ranged from 0.01 to 0.14 mol for ARS and 0.01 to 0.41 mol

569

TE/mol for ORAC assay.

570

In summary, activity-guided fractionation using ARS and ORAC assays

571

enabled the identification of 26 compounds in thermally processed AGE powder

572

and their chemical antioxidant activities were determined. In particular, 12 new

573

structures -[(2-formyl-5-hydroxymethyl) pyrrol-1-yl]arginine (3), 4-[7-hydroxy-6-

574

(hydroxymethyl)-7,8-dihydro-6H-pyrano

575

(4)

576

1,2,3-triol (5), -[(2-formyl-5-hydroxy-methyl)-pyrrol-1-yl] aspartic acid (12), 1-[5-

577

(1,2-dihydroxyethyl)-2-oxotetra-hydrofuran-3-yl]-5-(hydroxymethyl)-1H-pyrrole-

578

2-carbaldehyde (14), 4-(6-ethyl-2-pyrazinyl)-1,2,3-butanetriol (17), -[(2-formyl-

579

5-hydroxymethyl)pyrrol-1-yl] glutamic acid (19), (S)-1-[(5-hydroxymethyl)furan-

580

2-yl]methyl]-5-oxopyrrolidine-2-carboxylic

581

(hydroxymethyl)furan-2-yl}methyl]-2,5-dioxo-3-pyrrolidine acetic acid (21), (E)-4-

582

(5-methylpyrazin-2-yl)but-3-ene-7,2-diol

583

(hydroxymethyl)picolinic acid (24), (E)-4-(6-methylpyrazin-2-yl)but-3-ene-1,2-

584

diol (26) were described. Additionally, a formation pathway for three unique

585

organic acid-type products was proposed starting from 3-deoxyhexosone and

586

glucosone with L-glutamic acid or citric acid. As a next step, biological studies

587

will be performed, which may reveal physiological activities of the isolated

588

compounds and highlight the biological as well as flavourful value of garlic

589

products.

and

[2,3-b]pyrazine-3-yl]butane-1,2,3-triol

4-[6-(1,2-dihydroxyethyl)-6,7-dihydro-furo[2,3-b]pyrazin-3-yl]-butane-

acid

590 591

Author information

592

Corresponding author 24 ACS Paragon Plus Environment

(20),

(23),

3-hydroxy-1H-[{5-

4-acetyl-6-

Page 25 of 43


593

Phone:

+49-8161-71-2902.

594

[email protected]

Fax:

+49-8161-71-2949.

E-mail:

595 596

Funding

597

We are grateful to Wakunaga Pharmaceutical Co. Ltd., for financial support.

598 599

Notes

600

The authors declare no competing financial interest.

601 602

Acknowledgments

603

We thank the NMR team, which is managed by Dr. Oliver Frank, of the Chair of

604

Food Chemistry and Molecular Sensory Science for performing the NMR

605

measurements on the isolated compounds.

606 607

Supporting information

608

Analytical conditions for HPLC purification of the isolated compounds, 1D/2D

609

NMR and mass spectra of all compounds, CD spectra of compounds 15 and 20,

610

MS analysis of model reactions and antioxidative assays are summarized in the

611

Supporting Information. This information is available free of charge via the

612

Internet http://pubs.acs.org.

613



615

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616

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Sasaki, J.; Lu, Chao.; Machiya, E.; Tanahashi, M.; Hamada, K. Processed

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10. Bae, S. E.; Cho, S. Y.; Won Y. D.; Lee, S. H.; Park, H. J. A comparative

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11. Bae, S. E.; Cho, S. Y.; Won Y. D.; Lee, S. H.; Park, H. J. Changes in S-allyl

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Rosen, R. T.; Amagase, H. Inhibiting progression of coronary calcification

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14. Nakagawa, S.; Kasuga, S.; Matsuura, H. Prevention of liver damage by

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15. Wang, B. H.; Zuzel, K. A.; Rahman, K.; Billington, D. Protective effects of

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18. Stark, T. D.; Salger, M.; Frank, O.; Balemba, O. B.; Wakamatsu, J.;

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19. Magaletta, R. L.; Ho, C. T. Effect of roasting time and temperature on the

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generation

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21. Hwang, I. G.; Kim, H. Y.; Woo, K. S.; Lee, S. H.; Lee, J.; Jeong, H. S.

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Isolation and identification of the antioxidant DDMP from heated Pear

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22. Kosuge, T.; Tsuji, K.; Nukaya, H.; Terada, A.; Ochiai, M.; Wakabayashi, K.;

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Nagao, M.; Sugimura, T. Isolation and identification of 5-hydroxymaltol, a

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mutagenic substance in glucose pyrolysate. Agric. Biol. Chem. 1983, 47(4),

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23. Surmont, R.; Verniest, Guido.; Kimpe, N. D. Short synthesis of the seed

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24. Xiao, Y.; Wang, Y. L.; Gao, S. X.; Sun, C.; Zhou, Z. Y. Chemical

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composition of hydrilla Verticillatta (L.f) Royle in Taihu lake. Chinese J.

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25. Wang, Y. C.; Zhang, Y. W.; Zheng, L. H.; Bao, Y. L.; Wu, Y.; Yu, C. L.;

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Huang, Y. X.; Sun, L. G.; Zhang, Y.; Jia, X. J.; Li, Y. X. Four new alkaloids

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26. Quiroz-Florentino, H.; Aguilar, R.; Santoyo, B. M.; Diaz, F.; Tamariz, J.

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Total systheses of natural furan derivatives Rehmanones A, B, and C.

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27. Mitsukura, K.; Sato, Y.; Yoshida, T.; Nagasawa, T. Oxidation of

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heterocyclic and aromatic aldehydes to the corresponding carboxylic acids

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by Acetobacter and Serratia strains. Biotechnol. Lett. 2004, 26, 1643 ―

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29. Olsson, K.; Pernemalm, P. A.; Theander, O. Formation of aromatic

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Page 30 of 43

Page 31 of 43


Table 1. 1H and 13C NMR Data of Alkylpyrazins 4 and 5 no.

compound 4 　

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

H (J values in Hz) 4.12, m 4.07, m 2.80, dd (16.9, 6.4) 3.08, dd (16.9, 4.7)

compound 5

　 C 81.5 61.7 35.0

H (J values in Hz)

C

4.99, ddd (10.0, 7.0, 3.5) 3.22, m, 2H

81.2 28.2 143.2

7.80, s

135.6 150.9

135.4 7.99, s

3.66, m 2H 2.50, dd (13.9, 2.9) 2.90, dd (13.9, 2.9) 3.74, m 3.34, m 3.66, m 3.55, dd (10.4, 3.4)

137.0 151.7 155.8 60.7 38.0 71.2 74.9 63.3

161.9 71.9 62.0 38.3

3.83, m 3.41, m, 2H 2.55, dd (13.9, 9.6) 2.88, dd (13.8, 2.9) 3.71, m 3.32, m 3.39, m 　3.55, dd (10.7, 3.6)

71.2 74.9 63.3

Both compounds were analyzed in DMSO-d6 (1H: 400 MHz, 13C: 100 MHz).



Page 32 of 43

Table 2. 1H and 13C NMR Data of Pyrrole derivatives 3, 12, 14 and 19 no. 2 3 4 5 6a 6b 7 8 9a 9b

compound 3 　 H (J values in Hz)a

 Ca

compound 12

　 H (J values in Hz)b

　

　 H (J values in Hz)a Ca 　　 6.27, d (3.9) 7.12, d (3.9)

4.41, d (13.9) 4.47, d (13.9) 9.44, s 179.3 5.59, m 60.3 1.86, ddt 30.4 (15.1, 10.9, 5.7, 5.7)

4.55, s (2H) 9.33, s 5.48, m

178.4 54.8

2.81, dd (16.9, 7.7)

37.1

2.43, m

3.30, m

1.01, tt 26.2 (11.6, 11.6, 5.7, 5.7) 1.42, tt 10b (11.3, 11.3, 5.5, 5.5) 11a 2.95, m 40.3 11b 3.07, m 12 9.11, br, NH 13 157.1 14 　　 173.3

6.20, d (3.9) 7.05, d (4.0)

10a

　　

compound 14

 Cb 144.6 109.2 125.9 131.6 55.3

　 6.20, d (4.0) 6.89, d (3.9)

144.7 109.9 122.8 132.4 55.7

　

9.36, s 5.49, t (10.2, 10.2)

179 54.4

9.30, s

172.9

29.7 2.30, m 2.54, dddd (13.7, 8.9, 7.3, 4.4) 1.83, ddd 35.1 (15.0, 9.0, 5.7) 2.01, dt (15.8, 8.0, 8.0) 182.8

78.1

172.0

2.27, m 2.67, m 3.78, m 3.38, s 2H 　　

27.9

In DMSO-d6 (1H: 500 MHz, 13C: 125 MHz)

b

In a mixture of D2O/methanol-d4 (9/1, 1H: 400 MHz, 13C: 100 MHz)

　 H (J values in Hz)b Cb

4.51, s (2H)

4.79, m

a

compound 19

144.4 109.9 125.8 131.0 55.0

170.2

　

　


72.0 61.8 　

　　 6.41, d (4.1) 7.19, d (4.1) 4.64, s 2H

145.4 111.7 128.1 132.9 56.5 181.7 61.4

178.0 　　

　

Page 33 of 43


Table 3. 1H and 13C NMR Data of Compounds 20 and 21 no.

compound 20 　 H (J values in Hz)a

2 3 4 5 6 7 8a 8b 10 11a 11b 12a 12b 13 14a 14b 15 　

6.20, d (3.2) 6.21, d (3.2) 4.33, s 2H 5.17, br OH 3.95, d (15.6) 4.77, d (15.6) 3.97, m 1.93, m 2.29, m 2.30, m, 2H

　  Ca

155.5 107.6 109.3 148.7 55.6 37.7

H (J values in Hz)b 　 6.18, d (3.2) 6.20, d (3.2) 4.32, s, 2H 5.15, br, OH 4.52, d (15.8) 4.56, d (15.8)

58.2 22.4 29.0 174.1 173.2

　

compound 21

　

 Cb 154.1 107.8 108.3 148.0 55.6 35.0 177.9 71.7

2.65, d (18.2) 3.05, d (18.2) 2.81, d (16.9) 2.90, d (17.0) 　　

a


a


42.0 174.4 40.6 171.5



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Table 4. Antioxidant Activity of Isolated Compounds 1―26 ARS [mol TE/mol] compounds 1 0.01 ± 0.001 2 0.02 ± 0.001 3 0.13 ± 0.019 4 0.02 ± 0.002 5 0.01 ± 0.001 6 0.12 ± 0.008 7 0.03 ± 0.007 8 0.01 ± 0.001 9 0.01 ± 0.002 10 0.13 ± 0.008 11 0.02 ± 0.009 12 0.01 ± 0.002 13 0.01 ± 0.001 14 0.05 ± 0.005 15 0.04 ± 0.002 16 0.14 ± 0.007 17 0.01 ± 0.001 18 0.49 ± 0.018 19 0.06 ± 0.003 20 0.03 ± 0.003 21 0.02 ± 0.001 22 0.01 ± 0.001 23 0.04 ± 0.002 24 0.01 ± 0.001 25 0.01 ± 0.001 26 0.01 ± 0.001 ascorbic acid 1.24 ± 0.013 quercetin 3.85 ± 0.052 Data represents the mean ±S.D.

　

　

ORAC [mol TE/mol] 0.01 ± 0.001 0.06 ± 0.001 0.10 ± 0.002 0.02 ± 0.002 0.02 ± 0.001 0.16 ± 0.003 0.11 ± 0.005 0.01 ± 0.001 0.01 ± 0.001 0.03 ± 0.002 0.08 ± 0.002 0.05 ± 0.004 0.05 ± 0.003 0.10 ± 0.005 0.09 ± 0.002 0.41 ± 0.018 0.11 ± 0.006 3.50 ± 0.152 0.13 ± 0.009 0.35 ± 0.007 0.33 ± 0.004 0.05 ± 0.003 0.11 ± 0.001 0.02 ± 0.002 < 0.001 0.03 ± 0.005 0.78 ± 0.046 6.45 ± 0.029



Figure Legends

Figure 1.

Chemical structures of antioxidants 1-26 identified in the thermally processed AGE powder.

Figure 2.

Changes of antioxidant activity on AGE formulations processed by heating at (A) 80 oC and (B) 100 oC. Data represents fold-changes to their initial activity by mean±S.D, (◆) 3.5 % powder, (□) 5.4 % powder, (△) 8.0 % powder, (×) 10.5 %, and (○) liquid.

Figure 3.

ARS　(white bars) and ORAC (black bars)　activities of the driest powder (3.5 % water) which was thermally processed at 80 oC

for 3 and 5 days or 100 oC for 1 and 2 days. Data represents

fold-changes to the initial activity by mean±S.D. Figure 4.

Activity distribution in fractions separated from powdered AGE heated at 100 oC for 1 day via the two-step ultrafiltration. The evaluation was done by means of ARS (white bars) and ORAC assays (black bars), and data represents ratios to values of the whole extract by mean±S.D.

Figure 5.

MPLC separation of the low molecular weight fraction of thermally processed AGE powder (A), and ARS (B, black bars) and ORAC activities (B, white bars) of its subfractions. Data represents mean ±S.D.

Figure 6.

Reaction pathways proposed for the formation of compounds 15, 20 and 21.


Page 36 of 43

Page 37 of 43


Figure 1 (Wakamatsu et al.) O OH

OH

HO

N OH

OH

8

11

O

2

15

3

OH

7

N

5

14

OH

4

6

7

8

1

N

13

7

HO

OH

6

N

4

2

8

3 OH

OH

OH

6 O 7

O

HO O

COOH

8

CHO

O

HO

3

NH

NH

N

1

HO

10

COOH COOH

6

11

9

8

2

HO

10

5

11

12

O

O 4

N

COOH

1

N

2

HO

HO

7

5

3

10

9

6

O

O

12

OH

O

O

O

15

3

HO

OH O

1

N

2

HO

8

9

COOH

6

2

19 OH 2

9 10

N

6

4

8 3

23

N 4

N

2

N

5

11

O

N 8

10

9

14

OH

O

7

OH

9

COOH

N

8

HO


25

N H

1

7

HO O

24

11

21

5

10

5

COOH

12

13

5

O

1 6

O

4

1

O O

2 3

1

7

COOH

8

6

13

9

3

20 HO

HO

12 3

4

HO 12

N

5

O

11

6

17 7

11 10

1

COOH

O

N

HO

11

4

N

15

HOOC 3

12

18

2

OH

16

7

10 6

10

1

7

14

7

5

8

9

O

O 4

OH HO

O

N

14 O

HO

COOH

OH

13

11

8

13

22

4

O

N

9

HO

N

7

O HO

O

N

O

5

HO

12

HO

HO

5

4

NH2

NH

10

14

OH

OH

3

13

COOH

6

O

10

11

9

N

2

11

O

9

NH

1

3

OH

N

12

HO

OH

10

OH

5

2 OH

1

N

13

15

OH

OH

9

12

14

4

N

OH

1 HO

HO

N

OH

OH

2

9 10

11

N

6

8 3

26

N 4

5


Page 38 of 43

Figure 2 (Wakamatsu et al.)

Activity change (/day 0 )

18

A

15 12 9 6 3 0 0

Activity change (/day 0)

18

3

6

9

12

15

18

21

24

27

30

B

15 12 9 6 3 0 0

1

2

3

Heating time (day)


4

5

Page 39 of 43


Figure 3 (Wakamatsu et al.) 20

Activity change ( / day 0)

16

12

8

4

0 Initial

80 ℃ 3 days

80 ℃ 5 days


100 ℃ 1 day 100 ℃ 2 days


Figure 4 (Wakamatsu et al.) 1.2

Activty distribution ratio

1.0 0.8 0.6 0.4 0.2 0.0 1 kDa >

1-5 kDa

5 kDa

Antioxidative Maillard Reaction Products Generated in Processed

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