Isolation and Identification of Three Î³-Glutamyl Tripeptides and Their

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Isolation and identification of three #–glutamyl tripeptides and their putative production mechanism in aged garlic extract Masashi Nakamoto, Takuto Fujii, Toshiaki Matsutomo, and Yukihiro Kodera J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05480 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 4, 2018

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Journal of Agricultural and Food Chemistry

COOH

COOH

S

H2N

CH3

S-Allylcysteine (2)

COOH

S-1-Propenylcysteine (3)

COOH

O

S

N H

S

H2N

COOH

O H2N

S

H2N

S-Methylcysteine (1)

COOH

H2N

CH3

COOH

γ-Glutamyl-S-methylcysteine (4)

S

N H

γ-Glutamyl-S-allylcysteine (5) 1’’

1

H2N

4

S

N H

COOH

COOH

COOH

O

H2N 2

4’

3

O

COOH

COOH

O

H 3’ 5 N 2’

5’ N

H

2’’

3’’

S 4’’ CH3

1’

γ-Glutamyl-S-1-propenylcysteine (6) 1

1’’

COOH 4

H2N 2

γ-Glutamyl- γ-glutamyl-S-methylcysteine (7)

3

O

H 3’ 5 N 2’ 4’

O

5’

COOH

COOH

N 2’’ 3’’ H

5’’

S 4’’

6’’

1’

γ-Glutamyl- γ-glutamyl-S-allylcysteine (8) 1

1’’

COOH H2N 2

O

H 4 3’ 5 N 2’ 4’

3

O

COOH

5’

COOH N 2’’ 3’’ H

5’’

S 4’’

6’’

1’

γ-Glutamyl- γ-glutamyl-S-1-propenylcysteine (9)

Figure 1　 1　Chemical structures of S-alk(en)ylcysteine and their γ-glutamyl peptides observed in raw garlic or aged garlic extract. The wave bond in the chemical structure of compound (9) indicate both cis and trans form.

ACS Paragon Plus Environment


Absorbance (mV)

SAMC

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GSAC

GS1PC

Time (min)

Figure 2　 2　Post-column HPLC chromatogram of aged garlic extract. Detection was performed at 500 nm absorbance using iodoplatinate reagent. Double line arrows indicate candidates of sulfur-containing compounds unidentified. SAMC: S-Allylmercaptocysteine, GSAC: γGlutamyl-S-allylcysteine, GS1PC: γ-Glutamyl-S-1 -propenylcysteine. 　


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350 GGSMC GGSAC GGS1PC

Content (µ g/g-dry)

300 250 200 150 100 50 0 0

5

10

15

20

25

Time (month) Figure 3 　Changes of γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteines in aged garlic extract during aging period. Values are mean ± SD, n = 3. The value of GGS1PC at each analytical point is sum of cis and trans forms. Quantitative analysis of each compound was performed by LC-MS using selective ion monitoring mode with mass tolerance for IS, GGSMC, GGSAC and GGS1PC setting to ± 10 ppm. GGSMC: γ-glutamyl-γ- glutamyl-Smethylcysteine, GGSAC: γ-glutamyl-γ-glutamyl-S-allylcysteine, GGS1PC: γglutamyl-γ-glutamyl-S-1-propenylcysteine. 　



(B) 10

350

9

300

Content (µg/g-wet)

Content (µg/g-wet)

(A) 8 7 6 5 4 3 2

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250 200 150 100 50

1 0

0 0

2

4

Storage period (week)

Content (µg/g-wet)

(C)

0

2

4


300 250 200 150 100 50 0 0

2

4


Figure 4 　Changes of γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteines in the early aging process. Changes in concentration of GGSMC (A), GGSAC (B) and GGS1PC (C) at 25ºC, respectively. Values are mean ± SD, n = 3. The Value of GGS1PC at each analytical point is sum of cis and trans forms. Quantitative analysis of each compound was performed by LC-MS using selective ion monitoring mode with mass tolerance for IS, GGSMC, GGSAC and GGS1PC setting to ± 10 ppm. GGSMC: γ-glutamyl-γglutamyl-S-methylcysteine, GGSAC: γ-glutamyl-γ-glutamyl-Sallylcysteine, GGS1PC: γ-glutamyl-γ-glutamyl-S-1-propenylcysteine. 　


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COOH

O H2N COOH

COOH

S

R1 _ 3

COOH

O H2N

N H

N H

S

COOH

R1 _ 3

GTPase

H2N O

COOH

O

H N COOH

N H

COOH H2N

S

R1 _ 3

Figure 5 　Plausible production pathway of γ-glutamyl-γ-glutamyl-Salk(en)yl-cysteines in aged garlic extract during aging process. R1: methyl, R2: allyl, R3: 1-propenyl. 　　　


S

R1 _ 3


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Aging O H2N COOH

COOH

COOH N H

S

R1 _ 3

O

H N

H2N O

COOH

COOH N H

S

R1 _ 3

γ–Glutamyl tripeptides γ–Glutamyl dipeptides

Graphic for Table of Content


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1

Isolation and identification of three γ–glutamyl tripeptides and their

2

putative production mechanism in aged garlic extract

3 4

Masashi Nakamoto1, Takuto Fujii2, Toshiaki Matsutomo2 and Yukihiro Kodera2*

5 6

Affiliations

7

1: Healthcare Research and Development Division, Wakunaga Pharmaceutical Co., Ltd.,

8

1624 Shimokotachi, Koda–cho, Akitakata–shi, Hiroshima 739-1195 Japan.

9

2: Central Research Institute, Wakunaga Pharmaceutical Co., Ltd., 1624 Shimokotachi,

10

Koda–cho, Akitakata–shi, Hiroshima 739-1195 Japan.

11 12

Authors

13

Masashi Nakamoto

E-mail: [email protected]

14

Takuto Fujii


15

Toshiaki Matsutomo


16

Yukihiro Kodera

E-mail: [email protected],

17 18

*: Correspondence should be sent to Yikihiro Kodera

19

Central Research Institute, Wakunaga Pharmaceutical Co., Ltd.

20

1624 Shimokotachi, Koda–cho, Akitakata–shi, Hiroshima 739-1195 Japan.

21

E-mail: [email protected],

22

Tel: +81 826 45 2331, Fax: +81 826 45 4351

23



24

Abstract

25

We analyzed aged garlic extract (AGE) to understand its complex sulfur chemistry

26

using post–column high-performance liquid chromatography with an iodoplatinate

27

reagent and liquid chromatography high resolution mass spectrometry (LC-MS). We

28

observed unidentified peaks of putative sulfur compounds. Three compounds were

29

isolated and identified as γ–glutamyl–γ–glutamyl–S–methylcysteine, γ–glutamyl–γ–

30

glutamyl–S–allylcysteine

31

cysteine (GGS1PC) by nuclear magnetic resonance and LC–MS analysis using based on

32

comparisons with chemically synthesized reference compounds. GGSAC and GGS1PC

33

were novel compounds. Trace amounts of these compounds were detected in raw garlic,

34

but the contents of these compounds increased during the aging process. Production of

35

these compounds was inhibited using a γ–glutamyl transpeptidase (GGT) inhibitor in

36

the model reaction mixtures. These findings suggest that γ–glutamyl tripeptides in AGE

37

are produced by GGT during the aging process.

(GGSAC)

and

γ–glutamyl–γ–glutamyl–S–1–propenyl–

38 39

Key words: aged garlic extract, garlic, γ–glutamyl transpeptidase, γ–glutamyl tripeptide

40 41 42 43 44 45 46 47


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48

Introduction

49 50

Allium plants produce organic sulfur compounds using the ultimate inorganic

51

source, sulfate (SO42–), and sulfur is incorporated into L–cysteine.1-3 Several reactions

52

ensure after sulfur fixation. These include glutamylation and glycylation to yield

53

glutathione, deglycylation to yield γ–glutamyl–S–alk(en)ylcysteines, and S–oxygenation

54

and deglutamylation to yield S–alk(en)ylcysteine sufloxides.1-3 Among these

55

compounds, γ–glutamyl–S–alk(en)ylcysteines and S–alk(en)ylcysteine sufloxides are

56

the main sulfur storage molecules. These compounds transform to S–alk(en)yl

57

sulfinothioates and S–alk(en)ylcysteines, such as allicin and S–allylcysteine (SAC),

58

when raw plants are crushed, sliced, or soaked in an aqueous alcoholic solution.4, 5

59

These products are further changed into various compounds through complicated

60

chemical reactions by themselves or with other compounds during storage.6-8 Earlier

61

studies to elucidate the complicated sulfur chemistry in garlic have helped us to

62

understand the properties of sulfur compounds, such as production of allylthiosulfinates

63

and polyallylsulfides, generation of S–alk(en)ylcysteines, antibiotic activity of allicin in

64

crushed fresh garlic, cyclooxygenase inhibition of allylsulfides in garlic oil, antiplatelet

65

activity of ajoenes in oil macerate, antihepatotoxic activity of SAC, and

66

immunomodulatory effect of S–1–propenylcysteine (S1PC).9, 10

67

The sulfur compounds mentioned above are divided into hydrophilic and

68

hydrophobic compounds. Hydrophobic compounds, such as allylpolysulfides, ajoenes,

69

and vinyldithiins, are mainly present in garlic oil or oil–macerate products, and are

70

generally volatile and have highly reactive properties, which reduce their content in

71

garlic preparations.9, 10 In contrast, hydrophilic compounds, such as SAC, S1PC and



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72

S–allylmercaptocysteine in aged garlic extract (AGE), are stable, odorless,11,12 and show

73

beneficial pharmacological properties.13-18 Only trace amounts of hydrophilic

74

compounds exists in fresh garlic, but they can be produced from precursor compounds,

75

γ–glutamyl–S–alk(en)ylcysteines, by γ–glutamyl transpeptidase (GGT), an endogenous

76

enzyme, during the aging process.19 We obtained evidences that γ–glutamyl–S–

77

alk(en)ylcysteines and S–alk(en)ylcysteines reacted with hydrophobic compounds

78

during the aging process;20 therefore, we analyzed AGE to understand its sulfur

79

chemistry and found several unidentified peaks of putative sulfur compounds in its

80

chromatograms using a newly developed analytical method, post–column high

81

performance liquid chromatography (HPLC) using an iodoplatinate reagent that can

82

specifically detect sulfur compounds.20 We focused on these peaks and identified one

83

known and two novel γ–glutamyl tripeptides, γ–glutamyl–γ–glutamyl–S–methyl–

84

cysteine

85

γ–glutamyl–γ–glutamyl–S–1–propenylcysteine (GGS1PC) (Figure 1). Furthermore, we

86

revealed the production mechanism of these compounds according to our hypothesis;

87

γ–glutamyl tripeptides were produced by GGT during the aging process. This study may

88

be useful for isolating and identifying sulfur compounds in garlic and garlic

89

preparations, and may provide an understanding of the sulfur chemistry in AGE.

(GGSMC),21

γ–glutamyl–γ–glutamyl–S–allylcysteine

(GGSAC),

and

90 91

Materials and methods

92

Chemicals

93 94

Chemicals were obtained from Wako Pure Chemical Industry (Tokyo, Japan) and

95

Tokyo Chemical Industry (Tokyo, Japan). Raw garlic was purchased from a local


Page 11 of 45


96

market. Aged garlic extract (AGE) was prepared according to the method described in a

97

previous report.22

98 99

Syntheses of γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteine derivatives

100 101

The reference compounds of S–substituted cysteine derivatives (S–methyl–,

102

S–allyl–, S–1–propenyl–, and S–3–butenyl–) and γ–glutamyl–S–alk(en)ylcysteine

103

derivatives (S–alk(en)yl: S–methyl–, S–allyl–, and S–1–propenyl–) were synthesized

104

according to previously described methods.20,

105

alk(en)ylcysteine derivatives were glutamylated according to previously

106

method.25 The synthesized γ–glutamyl–γ–glutamyl–S–alk(en)yl–cysteine derivatives

107

were purified by preparative HPLC using a Shimadzu HPLC system LC–10A

108

(Shimadzu Corporation, Kyoto, Japan) using the preparative HPLC 2nd conditions

109

described in the below section, “Isolation and identification of γ–glutamyl–γ–

110

glutamyl–S–alk(en)yl–cysteine derivatives.” The subject compound–containing

111

fraction was concentrated by a rotary evaporator and lyophilized using an FRD–50P

112

freeze–dryer (AGC Techno Glass, Shizuoka, Japan).

23-25

The obtained γ–glutamyl–S– reported

113

The chemical structure and purity of the synthesized compound were determined

114

by nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography–high

115

resolution mass spectrometry (LC–HRMS), and HPLC. 1H– and

116

the compounds were measured in deuterium oxide (D2O) using a VNMR-500

117

spectrometer (Varian Inc., Palo Alto, CA, USA) at 500 and 125 MHz in several

118

analytical modes (COSY, correlation spectroscopy; DEPT, distorsionless enhancement

119

by polarization transfer; HSQC, heteronuclear single quantum correlation; HMBC,


13

C–NMR spectra of


120

heteronuclear multiple bond coherence).

121

LC–HRMS analyses were carried out using an UltiMate 3000 chromatograph

122

(Dionex/Thermo Fisher Scientific, Waltham, MA, USA) coupled to a Q–Exactive mass

123

spectrometer (Thermo Fisher Scientific). The analytes were separated under the

124

following conditions: column, Cadenza CD–C18 (2.0 mm × 75 mm, 3 µm, Imtakt

125

Corporation, Kyoto, Japan); solvent A, water containing 0.1% (v/v) formic acid; solvent

126

B, 80% methanol (v/v) containing 0.1% (v/v) formic acid; gradient program (%B), 0–7

127

min (0%), 7–10 min (0→40%), 10–17 min (40%), 17–20 min (40→100%), 20–23 min

128

(100%), 23–23.01 min (100→0%); flow rate of the mobile phase, 0.2 mL/min. Mass

129

spectrometry (MS) was carried out under the following conditions: ionization mode,

130

ESI+ (positive mode); scan mode, Full–MS; resolution, 70,000; maximum IT, 200 ms;

131

isolation width, 4.0 m/z. The HPLC analysis was performed to determine sample purity

132

using a Nexera HPLC system (Shimadzu Corporation) using the same separation

133

conditions as those for mentioned in the LC–HRMS analysis.

134

The characterization data of synthesized compound (7), GGSMC, were as

135

follows: HRMS, calculated [M+H]+ = 394.1278, observed [M+H]+ = 394.1281;

136

1

137

H-4”), 2.22–2.29 (m, 1H, Hb–3’), 2.48 (t, J = 7.33 Hz, 2H, H–4’), 2.54 (ddd, J = 3.4,

138

5.4, 12.7 Hz, 2H, H–4), 2.90 (dd, J = 8.3, 14.1 Hz, 1H, Ha–3’’), 3.06 (dd, J = 4.6, 14.1

139

Hz, 1H, Hb–3”), 3.88 (t, J = 6.4 Hz, 1H, H–2), 4.37 (dd, J = 4.7, 9.4 Hz, 1H, H–2’),

140

4.60 (dd, J = 4.6, 8.4 Hz, 1H, H–2’’); 13C–NMR (in D2O) δ, 14.7 (C–4’’), 26.0 (C–3),

141

26.4 (C–3’), 31.2 (C–4), 31.6 (C–4’), 34.9 (C–3’’), 52.4 (C–2’), 52.5 (C–2’’), 53.6

142

(C–2), 173.3 (C–1), 174.6 (C–5), 174.8 (C–5’), 175.0 (C–1’’), 175.5 (C–1’).

143

Characterization NMR data of synthesized GGSMC were consistent with the previous

H–NMR (in D2O) δ, 2.02–2.08 (m, 1H, Ha–3’), 2.18–2.22 (m, 2H, H–3), 2.15 (s, 3H,


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144

report.21

145

The characterization data of synthesized compound (8), GGSAC, were as follows:

146

HRMS, calculated [M+H]+ = 420.1435, observed [M+H]+ = 420.1435; 1H–NMR (in

147

D2O) δ, 2.00–2.08 (m, 1H, Ha–3’), 2.16–2.20 (m, 2H, H–3), 2.21–2.28 (m, 1H, Hb–3’),

148

2.51–2.55 (m, 2H, H–4), 2.47 (t, J = 7.3 Hz, 2H, H–4’), 2.86 (dd, J = 8.2, 14.1 Hz, 1H,

149

Ha–3’’), 3.04 (dd, J = 4.8, 14.1 Hz, 1H, Hb–3’’), 3.22 (d, J = 7.1 Hz, 2H, H–4’’), 3.87 (t,

150

J = 4.9 Hz, 1H, H–2), 4.36 (dd, J = 4.9, 9.3 Hz, 1H, H–2’), 4.56 (dd, J = 4.8, 8.2 Hz, 1H,

151

H–2’’), 5.19 (dd, J = 9.3, 17.1 Hz, 2H, H–6’’), 5.81 (ddq, J = 7.3, 17.1, 10.3 Hz 1H,

152

H–5’’);

153

(C–4’), 34.1 (C–4’’), 52.5 (C–2’), 53.5 (C–2), 117.9 (C–6’’), 133.7 (C–5’’), 173.1 (C–1),

154

174.5 (C–5), 174.6 (C–5’), 174.8 (C–1’’), 175.4 (C–1’).

13

C-NMR (in D2O) δ, 26.0 (C–3), 26.4 (C–3’), 31.2 (C–4), 31.3 (C–3’’), 31.6

155

The characterization data of synthesized cis form of compound (9), cis–GGS1PC,

156

were as follows: HRMS, calculated [M+H]+ = 420.1435, observed [M+H]+ = 420.1434;

157

1

158

2.15–2.23 (m, 2H, H–3), 2.22–2.26 (m, 1H, Hb–3’), 2.44 (t, J = 7.6 Hz, 2H, H-4’),

159

2.51–2.55 (m, 2H, H–4), 3.07 (dd, J = 8.3, 14.2 Hz, 1H, Ha–3’’), 3.25 (dd, J = 4.4, 14.2

160

Hz, 1H, Hb–3’’), 3.85 (t, J = 6.1 Hz, 1H, H–2), 4.35 (dd, J = 4.9, 9.3 Hz, 1H, H–2’),

161

4.55 (dd, J = 4.4, 8.2 Hz, 1H, H–2’’), 5.78 (dq, J = 6.9, 9.3 Hz, 1H, H–5’’), 6.01 (dd, J =

162

1.2, 9.3 Hz, 1H, H–4’’);

163

31.3 (C–4), 31.8 (C–4’), 34.7 (C–3’’), 53.0 (C–2’), 53.8 (C–2’’), 53.9 (C–2), 123.8

164

(C–5’’), 126.9 (C–4’’), 173.6 (C–1), 174.6 (C–5), 174.8 (C–5’), 174.9 (C–1’), 175.9

165

(C–1’’).

H-NMR (in D2O) δ, 1.70 (dd, J = 1.0, 6.8 Hz, 3H, H–6’’), 1.99–2.06 (m, 1H, Ha–3’),

13

C–NMR (in D2O) δ, 13.9 (C–6’’), 26.1 (C–3), 26.6 (C–3’),

166

The characterization data of synthesized trans form of compound (9),

167

trans–GGS1PC, were as follows: HRMS, calculated [M+H]+ = 420.1435, observed



168

[M+H]+ = 420.1434; 1H–NMR (in D2O) δ, 1.75 (dd, J = 1.5, 6.6 Hz, 3H, H–6’’),

169

2.00–2.08 (m, 1H, Ha–3’), 2.16–2.21 (m, 2H, H–3), 2.21–2.27 (m, 1H, Hb–3’), 2.45 (t,

170

J = 7.3 Hz, 2H, H–4’), 2.51–2.56 (m, 2H, H–4), 3.02 (dd, J = 8.3, 14.3 Hz, 1H, Ha–3’’),

171

3.21 (dd, J = 4.4, 14.2 Hz, 1H, Hb–3’’), 3.88 (t, J = 6.4 Hz, 1H, H–2), 4.37–4.41 (m, 1H,

172

H–2’), 4.58 (dd, J = 4.4, 8.3 Hz, 1H, H–2’’), 5.89 (dq, J = 6.6, 14.9 Hz, 1H, H–5’’), 6.00

173

(dd, J = 1.5, 14.9 Hz, 1H, H–4’’); 13C–NMR (in D2O) δ, 17.6 (C–6’’), 26.0 (C–3), 26.5

174

(C–3’), 31.2 (C–4), 31.6 (C–4’), 33.9 (C–3’’), 52.6 (C–2’), 53.0 (C–2’’), 53.7 (C–2),

175

121.0 (C–5’’), 130.7 (C–4’’), 173.3 (C–1), 174.5 (C–5), 174.6 (C–5’), 174.9 (C–1’),

176

175.9 (C–1’’).

177 178

Analysis of sulfur compounds in aged garlic extract using post–column HPLC with

179

an iodoplatinate reagent

180 181

Methanol containing 1% (v/v) formic acid (8.5 mL) was added to 1.5 mL of AGE

182

(extract content: 14–20% (w/v)). This mixture was shaken vigorously for 1 min and

183

centrifuged at 1,750 g for 10 min. The supernatant was concentrated using a rotary

184

evaporator, and the resulting residue was applied to a preconditioned Sep–Pack Plus

185

C18 cartridges (500 mg, Waters Corporation, Milford, MA, USA), and the cartridge was

186

washed with 20 mL of water. The non–adhesion liquid portion and water–washed

187

fraction were combined and concentrated using a rotary evaporator. The residue was

188

dissolved in 1 mL of water, the mixture was filtered using a membrane filter (pore size:

189

0.45 µm), and the filtrate was used as the sample solution. The sample was analyzed

190

according to the method in a previous report20 with the following modifications: column,

191

Cadenza CD–C18 (4.6 mm × 250 mm, 3 µm, Imtakt Corporation); mobile phase,


Page 14 of 45

Page 15 of 45


192

5%(v/v) methanol containing 0.1% (v/v) formic acid; flow rate, 0.5 mL/min isocratic;

193

detection, 500 nm; post–column reagent, iodoplatinate reagent20; flow rate of the

194

post–column reagent, 0.2 mL/min.

195 196

Isolation

197

derivatives

and

identification

of

γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteine

198 199

Approximately 2.8 g of concentrated AGE (extract content: 38–42% (w/w)) was

200

mixed with a mixture of methanol (8.5 mL) and formic acid (0.1 mL). This mixture was

201

shaken vigorously for 10 min and centrifuged at 1,750 g for 10 min. The supernatant

202

was concentrated using a rotary evaporator, and 3 mL of water was added to the residue.

203

The resulting mixture was applied to a preconditioned Sep–Pack Plus C18 Cartridges

204

(500 mg, Waters Corporation), and the cartridge was washed with 20 mL of water. The

205

non–adhesion liquid portion and washed–water fraction were combined and

206

concentrated using a rotary evaporator. γ–Glutamyl tripeptides in the resulting residue

207

were separated and purified by preparative HPLC using an LC–10A HPLC system

208

(Shimadzu Corporation) under the following conditions: first preparative HPLC

209

(1st–HPLC) for GGSMC: column, Cadenza CD–C18 (28 mm × 250 mm, 5 µm, Imtakt

210

Corporation); solvent, 10% (v/v) methanol containing 0.1% (v/v) formic acid; flow, 9.0

211

mL/min; second preparative HPLC (2nd–HPLC) for GGSMC: column, Cadenza

212

CD–C18 (10 mm × 250 mm, 3 µm, Imtakt Corporation); solvent, 5% (v/v) methanol

213

containing 0.1% (v/v) formic acid; flow, 2.6 mL/min; 1st–HPLC for GGSAC: column,

214

Cadenza CD–C18 (28 mm × 250 mm, 5 µm, Imtakt Corporation); solvent, 15% (v/v)

215

methanol containing 0.1% (v/v) formic acid; flow, 9.0 mL/min; 2nd–HPLC for GGSAC:



216

column, Cadenza CD–C18 (10 mm × 250 mm, 3 µm, Imtakt Corporation); solvent, 15%

217

(v/v) methanol containing 0.1% (v/v) formic acid; flow, 2.6 mL/min; 1st–HPLC for

218

GGS1PC: column, Cadenza CD–C18 (28 mm x 250 mm, 5 µm, Imtakt Corporation);

219

solvent, 20% (v/v) methanol containing 0.1% (v/v) formic acid; flow, 9.0 mL/min;

220

2nd–HPLC for GGSAC: column, Cadenza CD–C18 (10 mm × 250 mm, 3 µm, Imtakt

221

Corporation); solvent, 20% (v/v) methanol containing 0.1% (v/v) formic acid; flow, 2.5

222

mL/min, respectively. All chromatographies were monitored at 220 nm.

223

The γ–glutamyl tripeptides–containing fraction was concentrated and lyophilized

224

using an FRD-50P freeze–dryer (AGC Techno Glass). Structural analyses of the

225

obtained compounds were performed using NMR and LC–HRMS according to the

226

conditions provided in the section “Syntheses of γ–glutamyl–γ–glutamyl–S–

227

alk(en)yl–cysteine derivatives.”

228

The isolated compounds were characterized using LC-MS and NMR analysis, and

229

these data were compared with the data for the synthesized compounds described above.

230

The characterization data of the isolated compound (7), GGSMC, were as follows:

231

HRMS, calculated [M+H]+ = 394.1278, observed [M+H]+ = 394.1277; 1H-NMR (in

232

D2O) δ, 1.99–2.06 (m, 1H, Ha–3’), 2.15–2.22 (m, 2H, H-3), 2.15 (s, 3H, H-4”),

233

2.20–2.28 (m, 1H, Hb–3’), 2.47 (t, J = 7.33 Hz, 2H, H–4’), 2.53 (ddd, J = 3.4, 5.4, 12.7

234

Hz, 2H, H–4), 2.89 (dd, J = 8.3, 14.1 Hz, 1H, Ha–3’’), 3.05 (dd, J = 4.7, 14.1 Hz, 1H,

235

Hb–3”), 3.85 (t, J = 6.4 Hz, 1H, H–2), 4.34 (dd, J = 4.9, 9.4 Hz, 1H, H–2’), 4.56 (dd, J

236

= 4.7, 8.4 Hz, 1H, H–2’’); 13C-NMR (in D2O) δ, 14.7 (C–4’’), 26.1 (C–3), 26.5 (C–3’),

237

31.3 (C–4), 31.7 (C–4’), 35.1 (C–3’’), 52.7 (C–2’), 52.8 (C–2’’), 53.8 (C–2), 173.5

238

(C–1), 174.6 (C–5), 174.9 (C–5’), 175.2 (C–1’’), 175.9 (C–1’). Characterization NMR

239

data of isolated GGSMC was consistent with the previous report.21


Page 16 of 45

Page 17 of 45


240

The characterization data of isolated compound (8), GGSAC, were as follows:

241

HRMS, calculated [M+H]+ = 420.1435, observed [M+H]+ = 420.1434; 1H-NMR (in

242

D2O) δ, 1.99–2.06 (m, 1H, Ha–3’), 2.16–2.22 (m, 2H, H–3), 2.20–2.27 (m, 1H, Hb–3’),

243

2.52–2.56 (m, 2H, H–4), 2.47 (t, J = 7.5 Hz, 2H, H–4’), 2.87 (dd, J = 8.1, 14.0 Hz, 1H,

244

Ha–3’’), 3.06 (dd, J = 4.7, 14.0 Hz, 1H, Hb–3’’), 3.23 (d, J = 7.3 Hz, 2H, H–4’’), 3.85 (t,

245

J = 6.2Hz, 1H, H–2), 4.35 (dd, J = 4.9, 9.3 Hz, 1H, H–2’), 4.56 (dd, J = 4.7, 8.2 Hz, 1H,

246

H–2’’), 5.19 (dd, J = 7.1, 13.7 Hz, 2H, H–6’’), 5.82 (ddq, J = 7.1, 17.1, 10.0 Hz, 1H,

247

H–5’’);

248

(C–4’), 34.1 (C–4’’), 52.8 (C–2’), 52.9 (C–2’’), 53.9 (C–2), 117.9 (C–6’’), 133.7 (C–5’’),

249

173.6 (C–1), 174.6 (C–5), 174.8 (C–5’), 175.0 (C–1’’), 175.9 (C–1’).

13

C–NMR (in D2O) δ, 26.0 (C–3), 26.5 (C–3’), 31.3 (C–4), 31.6 (C–3’’), 31.7

250

The characterization data of isolated trans form of compound (9), trans–GGS1PC,

251

were as follows; HRMS calculated [M+H]+ = 420.1435, observed [M+H]+ = 420.1433:

252

1

253

2.15–2.22 (m, 2H, H–3), 2.21–2.28 (m, 1H, Hb–3’), 2.40 (t, J = 7.1 Hz, 2H, H–4’),

254

2.51–2.54 (m, 2H, H–4), 3.02 (dd, J = 8.3, 14.2 Hz, 1H, Ha–3’’), 3.21 (dd, J = 4.4, 14.4

255

Hz, 1H, Hb–3’’), 3.89 (t, J = 6.4 Hz, 1H, H–2), 4.35 (dd, J = 5.2, 9.1 Hz, 1H, H–2’),

256

4.58 (dd, J = 4.4, 8.2 Hz, 1H, H–2’’), 5.85 (dq, J = 6.4, 14.9 Hz, 1H, H–5’’), 5.96 (dd, J

257

= 1.5, 15.1 Hz, 1H, H–4’’) ; 13C–NMR (in D2O) δ, 17.7 (C–6’’), 26.0 (C–3), 26.5 (C–3’),

258

31.2 (C–4), 31.7 (C–4’), 33.8 (C–3’’), 52.8 (C–2’), 52.4 (C–2’’), 53.5 (C–2), 121.0

259

(C–5’’), 130.7 (C–4’’), 173.1 (C–1), 174.3(C–5), 174.5 (C–5’), 174.8 (C–1’), 175.4

260

(C–1’’).

H–NMR (in D2O) δ, 1.71 (dd, J = 1.5, 6.6 Hz, 3H, H–6’’), 1.99–2.05 (m, 1H, Ha–3’),

261 262

Content of γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteine derivatives in aged garlic

263

extract



264 265

Approximately 1 g of AGE (extract content: 14–20% (w/w)) was mixed with 0.2

266

mL of the internal standard solution (IS, 0.5 mg/mL of S–3–butenylcysteine in 20 mM

267

HCl) and 0.1 mL formic acid. This mixture was shaken vigorously for 1 min and

268

centrifuged at 15,000 rpm for 10 min. The supernatant was filtered using membrane

269

filter (pore size: 0.45 µm), and the filtrate was used as the sample solution.

270

The quantitative analyses were performed using LC–HRMS according to the same

271

conditions outlined in the section “Syntheses of γ–glutamyl–γ–glutamyl–S–

272

alk(en)yl–cysteine derivatives.” The mass tolerances for IS, GGSMC, GGSAC and

273

GGS1PC were set to ± 10 ppm.

274 275

Production of γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteine during the early stages

276

of the aging process

277 278

Raw garlic cloves were sliced to a thickness of approximately 5 mm; 2–4 g of

279

these pieces were placed into a tube with a cap, and 10 mL of 15% ethanol was added.

280

Several sample tubes were prepared, stored at 25°C, and collected at a fixed time

281

interval (week 0, week 2, and week 4) to examine the changes in γ–glutamyl–γ–

282

glutamyl–S–alk(en)ylcysteines. Formic acid (0.2 mL) and the IS solution (0.2 mL; 0.5

283

mg/mL of S–3–butenylcysteine in 20 mM HCl) were added to the collected tubes and

284

shaken vigorously. The mixtures of the sample materials were treated using a

285

multi–beads shocker (Yasui Kikai Co., Osaka, Japan) to obtain the homogenates. The

286

resulting homogenate was transferred to a centrifuge tube and centrifuged at 1,750 g for

287

10 min. An aliquot of the supernatant was filtered using a membrane filter (pore size:


Page 18 of 45

Page 19 of 45


288

0.45 µm), and the filtrate was used as the sample solution.

289

To examine the content of γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteines in raw garlic,

290

5–6 g of sliced raw garlic were placed into a tube, and 20 mL of water, 0.2 mL of formic

291

acid and 0.2 mL of the IS solution (0.5 mg/mL of S–3–butenylcysteine in 20 mM HCl)

292

were added. The mixture was treated using a multi–beads shocker to obtain a

293

homogenate of raw garlic. The resulting homogenate was transferred to a centrifuge

294

tube and centrifuged at 1,750 g for 10 min. An aliquot of the supernatant was filtered

295

using a membrane filter (pore size: 0.45 µm), and the filtrate was used as the sample

296

solution.

297

The obtained sample solutions were analyzed using LC–HRMS as described in the

298

section “Syntheses of γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteine derivatives.” The

299

mass tolerances for IS, GGSMC, GGSAC, and GGS1PC were set to ± 10 ppm.

300 301

Model reactions for analysis of the production mechanism of γ–glutamyl–γ–

302

glutamyl–S–alk(en)ylcysteines

303 304

Model reaction using raw garlic

305 306

Raw garlic cloves were cut into pieces (approximately 2 × 3 × 5 mm); 3–4 g of garlic

307

pieces were placed in 15 mL tube, and 4 mL of 15% ethanol was added. Several sample

308

tubes containing mixture of garlic pieces and 15% ethanol were prepared, and the tubes

309

were divided into three groups: control group, boiled group, and GGT inhibitor–added

310

group. The tubes of the boiled group were dipped into boiling water for 10 min and

311

cooled to room temperature. GGsTop® (GGT inhibitor, Wako Pure Chemical, Osaka,

312

Japan) was dissolved in water and used as a GGT inhibitor (10 mM). A GGsTop®

313

solution (300 nmol) was added to the tubes of the GGT inhibitor–added group. The



314

tubes of each group were stored at 25°C and collected at fixed time intervals (day 0, day

315

5, day 10, day 20, day 30, and day 60) to examine the changes in the

316

S–alk(en)ylcysteines, γ–glutamyl–S–alk(en)ylcysteines, and γ–glutamyl–γ–glutamyl–S–

317

alk(en)ylcysteines. Approximately 8 mL of 20 mM HCl and 0.2 mL of the IS solution

318

(0.5 mg/mL of S–3–butenylcysteine in 20 mM HCl) were added to the collected tubes,

319

and shaken vigorously. The mixture was treated using the multi-beads shocker to obtain

320

the homogenate. The resulting homogenate was transferred to a centrifuge tube and

321

centrifuged at 1,750 g for 10 min. An aliquot of the supernatant was filtered using a

322

membrane filter (pore size: 0.45 µm), and the filtrate was used as the sample solution.

323

Quantitative analyses of the γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteines were

324

performed using LC–MS as described in the section “Synthesis of γ–glutamyl–

325

γ–glutamyl–S–alk(en)ylcysteine derivatives.”

326

Quantitative analyses of S-alk(en)ylcysteines and γ–glutamyl–S–alk(en)ylcysteines

327

were performed using an Aquity ultra performance LC (Waters Corporation) under the

328

following conditions: derivatization of amino group, AccQ·Tag Ultra Derivatization Kit

329

(Waters Corporation); column, AccQ–Tag UltraTM (2.1 mm × 100 mm, 1.7 µm, Waters

330

Corporation); solvent A, 20% (v/v) AccQ·Tag UltraTM Eluent A Concentrate Amino

331

acid analysis (Waters Corporation); solvent B, AccQ·Tag UltraTM Eluent B Amino acid

332

analysis (Waters Corporation); gradient program (%B), 0–0.54 min (0.1%), 0.54–5.74

333

min (0.1→9.1%), 5.74–7.74 min (9.1%), 7.74–8.04 min (9.1→10.6%), 8.04–9.54 min

334

(10.6%), 9.54–11.74 min (10.6→21.2%), 11.74–12.04 min (21.2→59.6%), 12.04–13.04

335

min (59.6%), 13.04–13.13 min (59.6→0.1%), 13.13–15.0 min (0.1%); flow, 0.7

336

mL/min; detection, 260 nm; injection volume, 1.0 µL.

337 338

Model reaction using the garlic protein fraction and GGT inhibitor


Page 20 of 45

Page 21 of 45


339 340

To examine the endogenous GGT activity in garlic involving the production of

341

γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteines, we performed a model reaction using the

342

garlic protein fraction and GGT inhibitor (GGsTop®, Wako Pure Chemical). The garlic

343

protein fraction was prepared using an ammonium sulfate precipitation method. The

344

outer skin of raw garlic was removed, and approximately 500 g of garlic cloves were

345

homogenized with 500 mL of water. The homogenate was filtered with cheesecloth, and

346

the filtrate was filtered using filter paper (Filter Paper No.1, Toyo Roshi Kaisha, Ltd.,

347

Tokyo, Japan). Ammonium sulfate was added to the filtrate to create a saturated solution

348

under cooling using an ice bath and with stirring. The obtained mixture was centrifuged

349

at 1,750 g for 15 min, and the supernatant was removed. The precipitate was transferred

350

into a dialysis tube (cutoff: 3.5 kDa) and dialyzed using purified water at 4°C for

351

approximately 15 h; dialysis was repeated twice. The inner fraction of the dialysis tube

352

was poured into plastic tubes and stored at –80°C before use. The protein content in the

353

inner fraction was determined by bicinchonic acid protein assay (BCA assay) with

354

bovine serum albumin as the standard protein; the protein content was 12.9 mg/mL.

355

The synthesized γ–glutamyl–S–alk(en)ylcysteines were dissolved in water (GSAC:

356

4.12 µmol/mL, GS1PC: 4.25 µmol/mL). Model reaction mixtures were prepared in 1.5

357

mL of plastic tube with the following composition: GSAC control mixture group, 50 µL

358

of GSAC solution (206 nmol), 100 µL of garlic protein fraction, 650 µL of 15% ethanol

359

solution; GSAC and inhibitor mixture group, 50 µL of GSAC solution (206 nmol), 100

360

µL of garlic protein fraction, 630 µL of 15% ethanol solution, 20 µL of GGsTop®

361

solution (200 nmol); GS1PC control mixture group, 50 µL of GS1PC solution (212

362

nmol), 100 µL of garlic protein fraction, 650 µL of 15% ethanol solution; GS1PC and



363

inhibitor mixture group, 50 µL of GS1PC solution (212 nmol), 100 µL of garlic protein

364

fraction, 630 µL of 15% ethanol solution, 20 µL of GGsTop® solution (200 nmol). The

365

tubes for each group were stored at 25°C and collected at fixed time intervals (day 0,

366

day 1, day 3, and day 5). IS solution (100 µL; 0.5 mg/mL of S–3–butenylcysteine in 20

367

mM HCl) and 100 µL of methanol containing 10% formic acid were added to each tube.

368

The mixture was shaken vigorously for approximately 10 s and centrifuged at 15,000

369

rpm for 10 min. An aliquot of the supernatant was filtered using a membrane filter

370

(cutoff: 3.0 kDa), and the filtrate was used as the sample solution. Quantitative analyses

371

of γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteines were performed on an LC–MS system

372

described in the section “Synthesis of γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteine

373

derivatives.” Quantitative analyses of S–alk(en)ylcysteines and γ–glutamyl–S–

374

alk(en)ylcysteines were performed using an Aquity ultra performance LC system

375

described in the section “Model reaction using raw garlic.”

376 377

Results

378 379

Analysis of the sulfur compounds in aged garlic extract using post–column HPLC

380

with an iodoplatinate reagent

381 382

We obtained evidences suggesting that the γ–glutamyl–S–alk(en)ylcysteines and

383

S–alk(en)ylcysteines reacted with hydrophobic sulfur compounds during the aging

384

process;20 therefore, we analyzed AGE to understand its sulfur chemistry by

385

post–column HPLC using an iodoplatinate reagent and LC–HRMS, using several

386

different gradient programs for the mobile phases. Figure 2 shows a chromatogram of


Page 22 of 45

Page 23 of 45


387

AGE using post–column HPLC; it contains several unidentified peaks of putative

388

sulfur–containing compounds. We first focused on the peak eluted at 87 min in the

389

post–column HPLC chromatogram, and analyzed the corresponding peak by

390

LC–HRMS because its peak intensity was stronger than that of the other peaks of

391

putative sulfur compounds, and no other peaks were observed from 70 to 100 min on

392

the post–column HPLC chromatogram. The mass signals of m/z 420.1435 and 422.1392

393

correspond to theoretical elemental compositions of C16H26O8N3S and C16H26O8N334S,

394

which indicate the presence of sulfur compounds based on the theoretical difference in

395

the exact mass and relative signal intensity between

396

34

32

S–containing ions (100%) and

S–substituted ions (4%).

397 398

Isolation

399

derivatives

and

identification

of

γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteine

400 401

To isolate and identify the putative sulfur compounds in AGE, we first focused on

402

the compound with [M+H]+ = 420.1435, which is likely tripeptide GGSAC or GGS1PC

403

based on its elemental composition. The elution time of the putative sulfur compound

404

during the HPLC analysis was similar to that of the synthesized GGSAC; therefore, we

405

attempted to isolate this compound using preparative HPLC by comparison with the

406

elution time of the synthesized GGSAC. Other two compounds, corresponding to

407

GGSMC and GGS1PC, were isolated in a similar way manner.

408

The characterization data for the isolated compounds were consistent with the data

409

for the synthesized compounds. In the 1H–NMR spectrum of isolated GGS1PC, two

410

methylene signals (–CH=CH–S–) in the S–propenyl group were observed at 5.85 ppm



411

(dq, J = 6.4, 14.9 Hz) and 5.96 ppm (dd, J = 1.5, 15.1 Hz), which indicated a trans

412

propenyl structure (JCH=CH–S– = 9–16 Hz).25 Furthermore, the weak signals at 5.75–5.82

413

ppm and 1.71 ppm, which were considered =CH–S– and methyl groups in the

414

cis–S–1–propenyl group, were also observed (data not shown). The estimated J value

415

for the =CH–S– group was 9.3 Hz, which is the characteristic J value for the cis form of

416

the olefin structure. The integration intensity of the methyl signal derived from the cis

417

form was around one tenth of that for the trans form, which indicated that the isolated

418

compound contained a mixture of the cis (minor component) and trans (major

419

component) forms. The chemical shifts of these methyl groups were consistent with

420

reported values.25 For the LC–HRMS analysis of GGS1PC from AGE with a mass

421

tolerance for GGS1PC set to ± 10 ppm, the peak corresponding to the elution time of

422

the cis form was observed, and its mass signals was m/z 420.1433, corresponding to the

423

theoretical elemental compositions of C16H26O8N3S (calculated [M+H]+ = 420.1435).

424 425

Content of γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteine derivatives in aged garlic

426

extract and raw garlic

427 428

The changes in the GGSMC, GGSAC and GGS1PC contents during the aging

429

process were determined using LC–HRMS. The contents of these compounds reached

430

maximum levels after approximately 4 months of aging, and gradually decreased during

431

the subsequent aging process (Figure 3). More than 80% of the maximum levels of

432

these compounds were produced within 1 month during the aging process. To examine

433

the changes in the γ–glutamyl tripeptides within 1 month, their contents in the test

434

preparations for the early aging process and raw garlic were analyzed. The contents of


Page 24 of 45

Page 25 of 45


435

the three γ–glutamyl tripeptides (GGSMC, GGSAC, and GGS1PC) in raw garlic were

436

less than 3, 70, and 60 nmol/g–fresh–weight, respectively. These compounds reached

437

maximum levels, approximately 8, 300, and 250 nmol/g–fresh–weight, respectively,

438

after 1 month (Figure 4).

439 440

Production of γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteines in model reaction

441

mixtures

442 443

GGSAC and GGS1PC were the major γ–glutamyl tripaptides in AGE; therefore,

444

we focused on the production mechanism of these two compounds using the model

445

reaction approach. Although the contents of GGSAC and GGS1PC in the GGT

446

inhibitor–added group slightly increased within 10 days and their contents were

447

maintained for 60 days (GGSAC: < 7 nmol/g–wet, GGS1PC: < 13 nmol/g–wet), their

448

contents in the control group increased significantly; the maximum contents during the

449

test period were 36 and 76 nmol/g–wet for GGSAC and GGS1PC, respectively (Figure

450

5). The contents of GGSAC and GGS1PC in the boiled group were almost the same as

451

that during the initial test period. Figure 6 shows the changes in the putative precursor

452

compounds for GGSAC and GGS1PC, and the changes in the contents of SAC and

453

S1PC produced from the corresponding γ–glutamyl–S–alk(en)ylcysteines by GGT. The

454

pattern of changes for GSAC and GS1PC were not different in the control group and

455

GGT inhibitor–added group (Figure 6 A and B). The boiling treatment seemed to

456

destroy GSAC and GS1PC because their initial contents in the boiled group were

457

approximately half the amounts of the control groups and GGT inhibitor–added groups.

458

The GGT inhibitor, GGsTop®, affected the GGT activity because the contents of SAC



459

and S1PC treated with GGsTop® did not increase, while their contents in the control

460

groups increased (Figure 6 C and D).

461

Figure 7 shows the model reaction using the garlic protein fraction and GGT

462

inhibitor. Productions of GGSAC and SAC were observed in the control group; the

463

content of GGSAC was twice as high as that of SAC at day 5. The contents of GGSAC

464

and SAC did not increase in the GSAC and inhibitor mixture group during the test

465

period. The same phenomenon was observed in the model reaction using GS1PC as a

466

substrate for GGT, while the production amount of GGS1PC was similar to GGSAC in

467

the control group, but that of S1PC was quite smaller than that of SAC (less than 0.1%

468

against initial GS1PC content).

469 470

Discussion

471 472

Allium plants are characterized by sulfur compounds, such as γ–glutamyl-

473

S–alk(en)ylcysteine and S–alk(en)ylcysteine sulfoxide, which accumulate as sulfur

474

storage molecules.1-3 S–Alk(en)ylcysteine sulfoxides were produced from the

475

corresponding precursor, γ–glutamyl–S–alk(en)ylcysteine sulfoxides, by GGT, or

476

γ–glutamyl–S–alk(en)ylcysteine that was transformed to S–alk(en)ylcysteine by GGT

477

and subsequently oxidized by an oxidase.25 In the Allium preparations, such as AGE,

478

S–alk(en)ylcysteines (e.g. SMC, SAC, and S1PC) are produced from the corresponding

479

precursor dipeptides, γ–glutamyl–S–alk(en)ylcysteines by endogenous GGT in plants.1-3,

480

9, 23

481

compound of S–methylcysteine sulfoxide and in its desulfoxide form,9 but there are no

482

reports for the identification of γ–glutamyl–γ–glutamyl tripeptide, GGSMC, in these

γ–Glutamyl–S–methylcysteine was found in onion and garlic as a precursor


Page 26 of 45

Page 27 of 45


483

Allium plants and their preparations, whereas other types of γ–glutamyl tripeptide,

484

glutathione and its derivatives, S–methylglutathione, and S–(β–carboxypropyl)–

485

glutathione, were confirmed as intermediate compounds in the biosynthesis pathway of

486

S–alk(en)ylcysteine sulfoxides.26 We identified three γ–glutamyl tripeptides, GGSMC,

487

GGSAC, and GGS1PC in AGE, which was prepared by soaking raw garlic in an

488

aqueous alcohol solution for more than 10 months at room temperature. Small amounts

489

of these compounds exist in raw garlic, but their amount in AGE increased during the

490

aging process. The content of these γ–glutamyl tripeptides reached a maximum level

491

after approximately 4 months of aging and gradually decreased thereafter. More than

492

80% of the amounts of these compounds present at 4 months were generated at around 2

493

months in AGE (Figure 3).

494

We hypothesized that γ–glutamyl tripeptides were produced from γ–glutamyl

495

dipeptides by the endogenous GGT in garlic during the aging period. The productions of

496

GGSAC and GGS1PC were inhibited in the model reaction mixtures using GGT

497

inhibitor GGsTop®, while their contents in the control groups increased (Figure 5). The

498

contents of GSAC, a putative precursor compound, quickly decreased within 10 days

499

and then gradually decreased in the control group and GGT inhibitor–added group

500

(Figure 6 A). The SAC content in GGT inhibitor–added group also slightly increased

501

during the early test period (Figure 6C). The change in the amounts of GSAC and SAC

502

were on the order of micromoles per gram of wet–weight–garlic and that of GGSAC

503

was on the order of nanomoles per gram of wet–weight–garlic. A similar phenomenon

504

was observed for GGS1PC, GS1PC, and S1PC. Additionally, the productions of

505

GGSAC, GGS1PC, SAC, and S1PC were confirmed in another model reaction using

506

garlic protein fraction as an enzyme fraction involving the GGT activity (Figure 7).



Page 28 of 45

507

These results suggest that γ–glutamyl tripeptides are produced via transfer of

508

γ–glutamyl–S–alk(en)ylcysteine to glutamic acid in other γ–glutamyl–S–alk(en)yl–

509

cysteines by an endogenous catalyst in garlic, which helps to simultaneously produce

510

γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteine and S–alk(en)ylcysteine during aging

511

process (Figure 8). Furthermore, the GGT affinity in raw garlic for the substrates may

512

be different or several types of GGT may be present in garlic because produced amounts

513

of

514

different during the aging process, and the time to maximum production of

515

S–alk(en)ylcysteines were longer than that of γ–glutamyl–γ–glutamyl–S–alk(en)yl–

516

cysteines (e.g. approximately 10 months for S-alk(en)ylcysteines19, 20 and within a few

517

months for γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteines) (Figure 3 and Figure 4). The

518

contents of GGSAC and GGS1PC on day 0 were different (Figure 4 and Figure 5). We

519

used different lots of raw garlic that were purchased over a span of more than 6 months

520

for these experiments. γ–Glutamyl tripeptide contents may vary depending on the origin

521

of the garlic, and γ–glutamyl tripeptides may be accumulated or consumed in raw garlic

522

while endogenous GGT in raw garlic involves the production of γ–glutamyl tripeptides.

γ–glutamyl–γ–glutamyl–S–alk(en)yl–cysteines

and

S–alk(en)ylcysteines

were

523

We observed both γ–glutamyl tripeptides, cis–GGS1PC and trans–GGS1PC in

524

AGE from the LC–HRMS analysis, and the content of the cis form was approximately

525

10% of the trans form (data not shown). In the Allium plants, the trans form of propenyl

526

group is the major component, and the trans form is the natural form of the compound.

527

Previously, we identified the cis form of S1PC in AGE, and its content was about

528

10–20% of that of the trans form.8, 20 S1PC was produced from the precursor GS1PC by

529

the presence of GGT in raw garlic. Herein, the content of the cis form of GS1PC in raw

530

garlic was less than 1% of that of the trans from. We revealed that the majority of the cis


Page 29 of 45


531

form observed in AGE was produced by isomerization of the trans form during the

532

aging process, and the isomerization reaction was reversible.8, 20 Based on the previous

533

report,8 the cis form of GGS1PC can be produced from the trans form by an

534

isomerization reaction during the aging process because the contents of the cis forms of

535

GS1PC and GGS1PC in raw garlic were much smaller than those in AGE.

536

In conclusion, the present study indicates that the aging process using an aqueous

537

alcoholic solution can provide the conditions to produce γ–glutamyl tripeptides,

538

γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteines that are produced by endogenous GGT in

539

raw garlic. These compounds may be the same as a marker compound for the aging

540

process using an aqueous alcoholic solution because the contents of these compounds in

541

raw garlic are less than 10–20% of the corresponding amount in AGE, and these

542

compounds are generated during the aging process.

543 544

Abbreviations Used

545

AGE, aged garlic extract; SAC, S–allylcysteine; S1PC, S–1–propenylcysteine;

546

GGSMC,

547

glutamyl–S–allylcysteine; GGS1PC, γ–glutamyl–γ–glutamyl–S–1–propenylcysteine;

548

GGT, γ–glutamyltranspeptidase.

γ–glutamyl–γ–glutamyl–S–methylcysteine;

GGSAC,

γ–glutamyl–γ–

549 550

Acknowledgment

551

The authors deeply thank Dr. Takami Oka of Wakunaga Pharmaceutical Co., Ltd, for his

552

helpful advice, encouragement and critical reading of the manuscript.

553



554

Reference

555

1. Lancaster J.E.; Show M.L. γ–Glutamyl peptides in the biosynthesis of

556

S–alk(en)yl–L–cysteine sulphoxides (flavor precursors) in Allium. Phytochem. 1989,

557

28, 455–460.

558

2. Yoshimoto N.; Yabe A.; Sugino Y.; Murakami S.; Sai–Ngam N.; Sumi S.;

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Tsuneyoshi T.; Saito K. Garlic γ–glutamyl transpeptidases that catalyze

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deglutamylation of biosynthetic intermediate of alliin. Front Plant Sci. 2015, 5, 758.

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doi: 10.3389 /fpls.2014.00758. eCollection 2014.

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3. Yoshimoto N.; Onuma M.; Mizuno S.; Sugino Y.; Nakabayashi R.; Imai S.;

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Tsuneyoshi T.; Sumi S.; Saito K. Identification of a flavin–containing

564

S–oxygenating monooxygenase involved in alliin biosynthesis in garlic. Plant J.

565

2015, 83(6), 941–951.

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4. Amagase H.; Petesch B.L.; Matsuura H.; Kasuga S.; Itakura Y. Intake of garlic and its bioactive components. J. Nutr. 2001, 131 (supplement 3), 955S–962S. 5. Amagase H. Clarifying the real bioactive constituents of garlic. J. Nutr. 2006, 136 (supplement 3), 716S–725S. 6. Block E. The organosulfur chemistry of the genus Allium – Implication for the organic chemistry of sulfur. Angew. Chem. Int. Ed. Engl. 1992, 31, 113–1178.

572

7. Lawson L.D.; Wang Z.Y.; Hughes B.G. γ–Glutamyl–S–alklycysteines in garlic and

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other Allium spp.: Precursor of age–dependent trans–S–1–propenyl thiosulfinates. J.

574

Nat. Prod. 1991, 54(2), 436–444.

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8. Kodera Y.; Matsutomo T.; Itoh K. The evidence for the production mechanism of

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cis-S–1–propenylcysteine in aged garlic extract based on a model reaction approach

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using its isomers and deuterated solvents. Planta Med. Lett. 2015, 2, e69–e72.


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9. Garlic and Other Aliums: The Lore and the Science, Block E. The Royal Society of Chemistry, Cambridge, UK, 2010.

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10. Suzuki J.; Yamaguchi T.; Matsutomo T.; Amano T.; Morihara N.; Kodera Y.

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S-1-Propenylcysteine promotes the differentiation of B cell into IgA–producing cells

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by the induction of Erk1/2–dependent Xbp1 expression in Peyer’s patches. Nutrition.

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2016, 32, 884–889.

584

11. Sumioka I.; Matsura T.; Yamada K. Therapeutic effect of S–allylmercaptocysteine

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on acetaminophen–induced liver injury in mice. Eur. J. Pharmacol. 2001, 21,

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433(2-3), 177–85.

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12. Amano H.; Kazamori D.; Itoh K. Pharmacokinetics and N–acetylation metabolism

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of S-methyl–L–cysteine and trans–S–1–propenyl–L–cysteine in rats and dogs.

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Xenobiotica 2016, 46(11), 1017–1025.

590 591

13. Kyo E.; Uda N.; Kasuga S.; Itakura Y. Immunomodulatory effects of aged garlic extract. J. Nutr. 2001, 131(Suppl 3), 1075S–1079S.

592

14. Fallah–Rostami F.; Tabari M.A.; Esfandiari B.; Aghajanzadeh H.; Behzadi M.T.

593

Immunomodulatory activity of aged garlic extract against implanted fibrosarcoma

594

tumor in mice. N. Am. J. Med. Sci. 2013, 5, 207–212.

595

15. Ishikawa H.; Saeki T.; Otani T.; Suzuki T.; Shimozuma K.; Nishino H.; Fukuda S.;

596

Morimoto K. Aged garlic extract prevents a decline of NK number and activity in

597

patients with advanced cancer. J. Nutr. 2006, 136(Suppl 3), 816S–820S.

598

16. Amano H.; Kazamori D.; Itoh K. Evaluation of the effects of S–allyl–L–cysteine,

599

S–methyl–L–cysteine, trans–S–1–propenyl–L–cysteine, and their N–acetylated and

600

S–oxidized metabolites on human CYP activities. Biol. Pharm. Bull. 2016, 39,

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1701–1707.



602

17. Nantz M.P.; Rowe C.A.; Muller C.E.; Creasy R.A.; Stanilka J.M.; Percival S.S.

603

Supplementation with aged garlic extract improves both NK and γδ–T cell function

604

and reduces the severity of cold and flu symptoms: a randomized, double–blind,

605

placebo–controlled nutrition intervention. Clin. Nutr. 2012, 31(3), 337–344.

606

18. Harauma A.; Moriguchi T. Aged garlic extract improves blood pressure in

607

spontaneous hypertensive rats more safely than raw garlic. J. Nutr. 2006, 136(3S),

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769S–773S.

609 610

19. The Science and Therapeutic Application of Allium Sativum L and Related Species, H. P. Koch and L. D. Lawson, Eds., Williams & Wilkins, Baltimore, Md, USA, 1996. 1996

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20. Matsutomo T.; Kodera Y. Development of an analytical method for sulfur

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compounds in aged garlic extract with the use of a postcolumn high performance

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liquid chromatography method with sulfur–specific detection. J. Nutr. 2016,

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146(Suppl), 450S–455S.

615 616

21. Kasai T.; Shiroshita Y.; Sakamura S. γ–Glutamyl peptides of Vigna radiata seeds. Phytochemistry, 1986, 25(3), 679–682.

617

22. Ryu K.; Ide N.; Matsuura H.: Itakura Y. Nα–(1–Deoxy–D–fructos–1–yl) –L–arginine,

618

an antioxidant compound identified in aged garlic extract. J Nutr. 2001, 131,

619

972S–976S.

620

23. Jayathilaka L.; Gupta S.; Huang JS.; Lee J.; Lee BS. Preparation of

621

(+)–trans-isoalliin and its isomers by chemical synthesis and RP–HPLC resolution.

622

J Biomol Tech. 2014, 25(3), 67–76.

623 624 625

24. Carson JF, Wong FF. Synthesis of cis-S-prop-1-enyl-L-cysteine. Chem Ind. 1963, 1963 2, 764–776.

25. King FE, Kidd DAA. A new synthesis of glutamine and of γ-dipeptides of glutamic


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626

acid from phthalylated intermediates. J Chem Soc. 1949, 1949 3315-3319.

627

26. Kodera Y.; Ushijima M.; Amano H.; Suzuki JI.; Matsutomo T. Chemical and

628

Biological Properties of S–1–Propenyl–l–Cysteine in Aged Garlic Extract.

629

Molecules, 2017, 22(4), pii: E570. doi: 10.3390/molecules22040570

630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649



Page 34 of 45

650

Figure Captions

651

Figure 1 Chemical structures of S–alk(en)ylcysteine and their γ–glutamyl peptides

652

observed in raw garlic or aged garlic extract.

653 654

Figure 2 Post-column HPLC chromatogram of aged garlic extract. Detection was

655

performed at 500 nm absorbance using an iodoplatinate reagent. Double line arrows

656

indicate putative sulfur-containing compounds unidentified. SAMC, S–Allylmercapto–

657

cysteine; GSAC, γ–Glutamyl–S–allylcysteine; GS1PC, γ–Glutamyl–S–1–propenyl–

658

cysteine.

659 660

Figure 3 Changes in γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteines in aged garlic

661

extract during the aging period. Values are means ± SD, n = 3. The values of GGS1PC

662

at each analytical point are sum of cis and trans forms. The quantitative analyses of

663

each compound were performed using LC–MS. GGSMC, γ–glutamyl–γ–glutamyl–S–

664

methylcysteine;

665

γ–glutamyl–γ–glutamyl–S–1–propenylcysteine.

GGSAC,

γ–glutamyl–γ–glutamyl–S–allyl–cysteine;

GGS1PC,

666 667

Figure 4 Changes in γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteines in the model

668

reaction mixtures. Changes in the content of GGSMC (A), GGSAC (B), and GGS1PC

669

(C) at 25ºC. Values are means ± SD, n = 3. The Value of GGS1PC at each analytical

670

point is sum of the cis and trans forms. The quantitative analyses of each compound

671

were performed using LC–MS. GGSMC, γ–glutamyl–γ–glutamyl–S–methylcysteine;

672

GGSAC, γ–glutamyl–γ–glutamyl–S–allylcysteine; GGS1PC, γ–glutamyl–γ–glutamyl–

673

S–1–propenylcysteine.


Page 35 of 45


674

Changes in γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteines in the model

675

Figure 5

676

reaction mixtures. Changes in concentration of GGSAC (A) and GGS1PC (B) at 25ºC,

677

respectively. The tubes of the boiled group were dipped into boiling water for 10 min

678

and cooled to room temperature. GGsTop® (GGT inhibitor) was dissolved in water and

679

used as a GGT inhibitor (10 mM). A GGsTop® solution (300 nmol) was added to the

680

tubes of the GGT inhibitor–added group. Values are means ± SD, n = 3. The Value of

681

GGS1PC at each analytical point is sum of the cis and trans forms. The quantitative

682

analyses of each compound were performed using LC-MS. GGSAC, γ-glutamyl–γ–

683

glutamyl–S–allylcysteine; GGS1PC, γ–glutamyl–γ–glutamyl–S–1–propenylcysteine.

684

Changes in γ–glutamyl–S–alk(en)ylcysteines and S–alk(en)yl-cysteines in

685

Figure 6

686

the model reaction mixtures. Changes in concentration of GSAC (A), GS1PC (B), SAC

687

(C) and S1PC (D) at 25ºC. The tubes of the boiled group were dipped into boiling water

688

for 10 min and cooled to room temperature. GGsTop® (GGT inhibitor) was dissolved in

689

water and used as a GGT inhibitor (10 mM). A GGsTop® solution (300 nmol) was

690

added to the tubes of the GGT inhibitor–added group. Values are means ± SD, n = 3.

691

The Value of GS1PC and S1PC at each analytical point is sum of the cis and trans

692

forms. GSAC, γ–glutamyl–S– allylcysteine; GS1PC, γ–glutamyl–S–1–propenyl–

693

cysteine; SAC, S–allylcysteine; S1PC, S–1–propenylcysteine.

694

Productions in γ–glutamyl–γ–glutamyl–S–alk(en)ylcysteines and

695

Figure 7

696

S–alk(en)ylcysteines in the model reaction mixtures using the garlic protein fraction.

697

GGsTop® (GGT inhibitor) was dissolved in water and used as a GGT inhibitor (10 mM).



698

A GGsTop® solution (200 nmol) was added to the tubes of the GGT inhibitor–added

699

group. Productions of GGSAC and SAC in the mixtures of GSAC and garlic protein

700

fraction with/without GGsTop® (A), productions of GGS1PC and S1PC in the mixtures

701

of GSAC and garlic protein fraction with/without GGsTop® (B), at 25ºC. Values are

702

means ± SD, n = 3. The Value of GS1PC and S1PC at each analytical point is sum of the

703

cis and trans forms. GGSAC, γ–glutamyl–γ–glutamyl–S–allylcysteine; GGS1PC,

704

γ–glutamyl–γ– glutamyl–S–1–propenylcysteine; GSAC, γ–glutamyl–S–allylcysteine;

705

GS1PC, γ–glutamyl–S–1–propenylcysteine; SAC, S–allylcysteine; S1PC,

706

S–1–propenylcysteine.

707

Plausible production pathway of γ–glutamyl–γ–glutamyl–S–alk(en)yl–

708

Figure 8

709

cysteines in aged garlic extract during aging process. R1, methyl; R2, allyl; R3,

710

1–propenyl.

711 712 713 714 715 716 717 718 719 720 721


Page 36 of 45

Page 37 of 45


722

Figures

723

Figure 1

724 COOH

COOH

S

H2N

CH3

S-Allylcysteine (2)

COOH

S-1-Propenylcysteine (3)

COOH

O

S

N H

S

H2N

COOH

O H2N

S

H2N

S -Methylcysteine ((1) 1) S-Methylcysteine

COOH

H2N

CH3

COOH

γγ-Glutamyl-S-methylcysteine -Glutamyl-S-methylcysteine ((4) 4)

S

N H

γ-Glutamyl-S-allylcysteine (5) 1

H2N

COOH

4

S

N H

1’’

COOH

COOH

O

H2N 2

H 3’ 5 N 2’ 4’

3

O

COOH

O

COOH

5’ N

2’’

H

3’’

S 4’’ CH3

1’

γ-Glutamyl-S-1-propenylcysteine (6) 1

COOH 4

H2N 2

3

H 3’ 5 N 2’ O

O

γ-Glutamyl-γ-glutamyl-S-methylcysteine (7) 1’’

COOH

5’ N 2’’ 4’ 3’’ H COOH

5’’

S 4’’

6’’

1’

γ-Glutamyl-γ-glutamyl-S-allylcysteine (8) 1

1’’

COOH 4

H2N 2

H 3’ 5 N 2’

3

O

O

COOH

5’ N 2’’ 4’ 3’’ H COOH

5’’

S 4’’

6’’

1’

γ-Glutamyl-γ-glutamyl-S-1-propenylcysteine (9)

725 726 727 728



729

Figure 2

730

Absorbance (mV)

SAMC

GSAC

GS1PC

Time (min)

731 732 733 734 735 736 737 738 739 740 741 742 743 744


Page 38 of 45

Page 39 of 45


745

Figure 3

746

Content (nmol/g-dry extract)

1000 GGSMC GGSAC GGS1PC

800 600 400 200 0 0

5

10

15

Time (month) 747 748 749 750 751 752 753 754 755 756 757 758


20

25


759

Page 40 of 45

Figure 4

760

B GGSAC content (nmol/g-wet)

GGSMC content (nmol/g-wet)

A 30

20

10

0 0

2 Time (week)

800 600 400 200 0

4

GGS1PC content (nmol/g-wet)

800 600 400 200 0 2

2

Time (week)

C

0

0

4

Time (week) 761 762 763 764 765 766 767


4

Page 41 of 45


768

Figure 5

769

A

B 100 Control (+) GGsTop®

40

GGS1PC content (nmol/g-wet)

GGSAC content (nmol/g-wet)

50

Boil

30 20 10

Control (+) GGsTop®

80

Boil

60 40 20 0

0 0

10

20

30

40

50

60

70

0

10

Time (Day)

20

30

40

Time (Day)

770 771 772 773 774 775 776 777 778 779 780 781 782 783


50

60

70


784

Page 42 of 45

Figure 6

785

A

B 14

Control (+) GGsTop® Boil

12 10

GS1PC content (µmol/g-wet)

GSAC content (µmol/g-wet)

14

8 6 4 2

10 8 6 4 2

0

0 0

10

20

30 40 Time (Day)

50

60

70

0

C

10

20

30 40 50 Time (Day)

60

70

60

70

D 3

3


S1PC content (µmol/g-wet)

SAC content (µmol/g-wet)


12

2

1

Control (+) GGsTop® Boil 2

1

0

0 0

10

20

30

40

50

60

70

0

10

Time (Day)

20

30

40

Time (Day)

786 787 788 789 790


50

Page 43 of 45


791

Figure 7

A

B

2.5

2.5 Production amount vs initial GS1PC (mol%)

Production amount vs initial GSAC (mol%)

792

GGSAC (-) GGsTop® GGSAC (+) GGsTop®

2.0

SAC (-) GGsTop® SAC (+) GGsTop®

1.5 1.0 0.5 0.0 0

2

4

6

GGS1PC (-) GGsTop® GGS1PC (+) GGsTop®

2.0

S1PC (-) GGsTop® S1PC (+) GGsTop®

1.5 1.0 0.5 0.0 0

2

4 Time (day)

Time (day)

793 794 795 796 797 798 799 800 801 802 803 804 805


6


806

Page 44 of 45

Figure 8

807 808 COOH

O H2N COOH

COOH

R1 _ 3

COOH

O H2N

S

N H

N H

S

COOH

R1 _ 3

GGT

H2N O

COOH H2N

S

R1 _ 3

809 810 811 812 813 814 815 816 817 818 819 820 821 822


COOH

O

H N COOH

N H

S

R1 _ 3

Page 45 of 45


823

TOC Graphic

824 Aging O H2N COOH

COOH

COOH N H

S

R1 _ 3

O

COOH

O

H N

H2N

COOH

N H

S

R1 _ 3

γ–Glutamyl tripeptides γ–Glutamyl dipeptides

825


Isolation and Identification of Three Î³-Glutamyl Tripeptides and Their

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