Isolation and Identification of Three γ-Glutamyl Tripeptides and Their

Mar 2, 2018 - allylmercaptocysteine in aged garlic extract (AGE), are stable, odorless,11,12 and ... yl-S-allylcysteine (GGSAC), and γ-glutamyl-γ-gl...
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Cite This: J. Agric. Food Chem. 2018, 66, 2891−2899

Isolation and Identification of Three γ‑Glutamyl Tripeptides and Their Putative Production Mechanism in Aged Garlic Extract Masashi Nakamoto,† Takuto Fujii,‡ Toshiaki Matsutomo,‡ and Yukihiro Kodera*,‡ †

Healthcare Research and Development Division and ‡Central Research Institute, Wakunaga Pharmaceutical Co., Ltd., 1624 Shimokotachi, Koda-cho, Akitakata-shi, Hiroshima 739-1195, Japan ABSTRACT: We analyzed aged garlic extract (AGE) to understand its complex sulfur chemistry using post-column highperformance liquid chromatography with an iodoplatinate reagent and liquid chromatography high resolution mass spectrometry (LC−MS). We observed unidentified peaks of putative sulfur compounds. Three compounds were isolated and identified as γglutamyl-γ-glutamyl-S-methylcysteine, γ-glutamyl-γ-glutamyl-S-allylcysteine (GGSAC) and γ-glutamyl-γ-glutamyl-S-1-propenylcysteine (GGS1PC) by nuclear magnetic resonance and LC−MS analysis based on comparisons with chemically synthesized reference compounds. GGSAC and GGS1PC were novel compounds. Trace amounts of these compounds were detected in raw garlic, but the contents of these compounds increased during the aging process. Production of these compounds was inhibited using a γ-glutamyl transpeptidase (GGT) inhibitor in the model reaction mixtures. These findings suggest that γ-glutamyl tripeptides in AGE are produced by GGT during the aging process. KEYWORDS: aged garlic extract, garlic, γ-glutamyl transpeptidase, γ-glutamyl tripeptide

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compounds, γ-glutamyl-S-alk(en)ylcysteines, by γ-glutamyl transpeptidase (GGT), an endogenous enzyme, during the aging process.19 We obtained evidence that γ-glutamyl-Salk(en)ylcysteines and S-alk(en)ylcysteines reacted with hydrophobic compounds during the aging process;20 therefore, we analyzed AGE to understand its sulfur chemistry and found several unidentified peaks of putative sulfur compounds in its chromatograms using a newly developed analytical method, post-column high performance liquid chromatography (HPLC) using an iodoplatinate reagent that can specifically detect sulfur compounds.20 We focused on these peaks and identified one known and two novel γ-glutamyl tripeptides, γ-glutamyl-γglutamyl-S-methyl-cysteine (GGSMC),21 γ-glutamyl-γ-glutamyl-S-allylcysteine (GGSAC), and γ-glutamyl-γ-glutamyl-S-1propenylcysteine (GGS1PC) (Figure 1). Furthermore, we revealed the production mechanism of these compounds according to our hypothesis; γ-glutamyl tripeptides were produced by GGT during the aging process. This study may be useful for isolating and identifying sulfur compounds in garlic and garlic preparations and may provide an understanding of the sulfur chemistry in AGE.

INTRODUCTION Allium plants produce organic sulfur compounds using the ultimate inorganic source, sulfate (SO42−), and sulfur is incorporated into L-cysteine.1−3 Several reactions ensure after sulfur fixation. These include glutamylation and glycylation to yield glutathione, deglycylation to yield γ-glutamyl-S-alk(en)ylcysteines, and S-oxygenation and deglutamylation to yield Salk(en)ylcysteine sufloxides.1−3 Among these compounds, γglutamyl-S-alk(en)ylcysteines and S-alk(en)ylcysteine sufloxides are the main sulfur storage molecules. These compounds transform to S-alk(en)yl sulfinothioates and S-alk(en)ylcysteines, such as allicin and S-allylcysteine (SAC), when raw plants are crushed, sliced, or soaked in an aqueous alcoholic solution.4,5 These products are further changed into various compounds through complicated chemical reactions by themselves or with other compounds during storage.6−8 Earlier studies to elucidate the complicated sulfur chemistry in garlic have helped us to understand the properties of sulfur compounds such as production of allylthiosulfinates and polyallylsulfides, generation of S-alk(en)ylcysteines, antibiotic activity of allicin in crushed fresh garlic, cyclooxygenase inhibition of allylsulfides in garlic oil, antiplatelet activity of ajoenes in oil macerate, antihepatotoxic activity of SAC, and immunomodulatory effect of S-1-propenylcysteine (S1PC).9,10 The sulfur compounds mentioned above are divided into hydrophilic and hydrophobic compounds. Hydrophobic compounds, such as allylpolysulfides, ajoenes, and vinyldithiins, are mainly present in garlic oil or oil−macerate products and are generally volatile and have highly reactive properties, which reduce their content in garlic preparations.9,10 In contrast, hydrophilic compounds, such as SAC, S1PC, and Sallylmercaptocysteine in aged garlic extract (AGE), are stable, odorless,11,12 and show beneficial pharmacological properties.13−18 Only trace amounts of hydrophilic compounds exist in fresh garlic, but they can be produced from precursor © 2018 American Chemical Society

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MATERIALS AND METHODS

Chemicals. Chemicals were obtained from Wako Pure Chemical Industry (Tokyo, Japan) and Tokyo Chemical Industry (Tokyo, Japan). Raw garlic was purchased from a local market. Aged garlic extract (AGE) was prepared according to the method described in a previous report.22 Syntheses of γ-Glutamyl-γ-glutamyl-S-alk(en)ylcysteine Derivatives. The reference compounds of S-substituted cysteine derivatives (S-methyl-, S-allyl-, S-1-propenyl-, and S-3-butenyl-) and Received: Revised: Accepted: Published: 2891

December 6, 2017 February 28, 2018 March 2, 2018 March 2, 2018 DOI: 10.1021/acs.jafc.7b05480 J. Agric. Food Chem. 2018, 66, 2891−2899

Article

Journal of Agricultural and Food Chemistry

The characterization data of synthesized compound (7), GGSMC, were as follows: HRMS, calculated [M + H]+ = 394.1278, observed [M + H]+ = 394.1281; 1H-NMR (in D2O) δ, 2.02−2.08 (m, 1H, Ha3′), 2.18−2.22 (m, 2H, H-3), 2.15 (s, 3H, 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, 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 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′), 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), 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 (C-2), 173.3 (C-1), 174.6 (C-5), 174.8 (C-5′), 175.0 (C-1′′), 175.5 (C-1′). Characterization NMR data of synthesized GGSMC were consistent with the previous report.21 The characterization data of synthesized compound (8), GGSAC, were as follows: HRMS, calculated [M + H]+ = 420.1435, observed [M + H]+ = 420.1435; 1H-NMR (in D2O) δ, 2.00−2.08 (m, 1H, Ha3′), 2.16−2.20 (m, 2H, H-3), 2.21−2.28 (m, 1H, Hb-3′), 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, 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, 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, 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, H-5′′); 13C NMR (in D2O) δ, 26.0 (C-3), 26.4 (C-3′), 31.2 (C-4), 31.3 (C-3′′), 31.6 (C4′), 34.1 (C-4′′), 52.5 (C-2′), 53.5 (C-2), 117.9 (C-6′′), 133.7 (C-5′′), 173.1 (C-1), 174.5 (C-5), 174.6 (C-5′), 174.8 (C-1′′), 175.4 (C-1′). The characterization data of synthesized cis form of compound (9), cis-GGS1PC, were as follows: HRMS, calculated [M + H]+ = 420.1435, observed [M + H]+ = 420.1434; 1H NMR (in D2O) δ, 1.70 (dd, J = 1.0, 6.8 Hz, 3H, H-6′′), 1.99−2.06 (m, 1H, Ha-3′), 2.15−2.23 (m, 2H, H-3), 2.22−2.26 (m, 1H, Hb-3′), 2.44 (t, J = 7.6 Hz, 2H, H4′), 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 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′), 4.55 (dd, J = 4.4, 8.2 Hz, 1H, H2′′), 5.78 (dq, J = 6.9, 9.3 Hz, 1H, H-5′′), 6.01 (dd, J = 1.2, 9.3 Hz, 1H, H-4′′); 13C-NMR (in D2O) δ, 13.9 (C-6′′), 26.1 (C-3), 26.6 (C3′), 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 (C-5′′), 126.9 (C-4′′), 173.6 (C-1), 174.6 (C-5), 174.8 (C-5′), 174.9 (C-1′), 175.9 (C-1′′). The characterization data of synthesized trans form of compound (9), trans-GGS1PC, were as follows: HRMS, calculated [M + H]+ = 420.1435, observed [M + H]+ = 420.1434; 1H-NMR (in D2O) δ, 1.75 (dd, J = 1.5, 6.6 Hz, 3H, H-6′′), 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, J = 7.3 Hz, 2H, H4′), 2.51−2.56 (m, 2H, H-4), 3.02 (dd, J = 8.3, 14.3 Hz, 1H, Ha-3′′), 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, 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 (dd, J = 1.5, 14.9 Hz, 1H, H4′′); 13C-NMR (in D2O) δ, 17.6 (C-6′′), 26.0 (C-3), 26.5 (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), 121.0 (C-5′′), 130.7 (C-4′′), 173.3 (C-1), 174.5 (C-5), 174.6 (C-5′), 174.9 (C-1′), 175.9 (C-1′′). Analysis of Sulfur Compounds in Aged Garlic Extract Using Post-Column HPLC with an Iodoplatinate Reagent. Methanol containing 1% (v/v) formic acid (8.5 mL) was added to 1.5 mL of AGE (extract content: 14−20% (w/w)). This mixture was shaken vigorously for 1 min and centrifuged at 1750g for 10 min. The supernatant was concentrated using a rotary evaporator, and the resulting residue was applied to a preconditioned Sep-Pack Plus C18 cartridges (500 mg, Waters Corporation, Milford, MA, USA); the cartridge was washed with 20 mL of water. The non-adhesion liquid portion and water-washed fraction were combined and concentrated using a rotary evaporator. The residue was dissolved in 1 mL of water, the mixture was filtered using a membrane filter (pore size: 0.45 μm), and the filtrate was used as the sample solution. The sample was analyzed according to the method in a previous report20 with the following modifications: column, Cadenza CD-C18 (4.6 mm × 250 mm, 3 μm, Imtakt Corporation); mobile phase, 5%(v/v) methanol containing 0.1% (v/v) formic acid; flow rate, 0.5 mL/min isocratic; detection, 500 nm; post-column reagent, iodoplatinate reagent;20 flow rate of the post-column reagent, 0.2 mL/min.

Figure 1. Chemical structures of S-alk(en)ylcysteine and their γglutamyl peptides observed in raw garlic or aged garlic extract. γ-glutamyl-S-alk(en)ylcysteine derivatives (S-alk(en)yl: S-methyl-, Sallyl-, and S-1-propenyl-) were synthesized according to previously described methods.20,23−25 The obtained γ-glutamyl-S-alk(en)ylcysteine derivatives were glutamylated according to previously reported method.25 The synthesized γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteine derivatives were purified by preparative HPLC using a Shimadzu HPLC system LC-10A (Shimadzu Corporation, Kyoto, Japan) using the preparative HPLC second conditions described in the below section, Isolation and Identification of γ-Glutamyl-γ-glutamyl-Salk(en)ylcysteine Derivatives. The subject compound-containing fraction was concentrated by a rotary evaporator and lyophilized using an FRD-50P freeze-dryer (AGC Techno Glass, Shizuoka, Japan). The chemical structure and purity of the synthesized compound were determined by nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography−high resolution mass spectrometry (LC− HRMS), and HPLC. 1H- and 13C-NMR spectra of the compounds were measured in deuterium oxide (D2O) using a VNMR-500 spectrometer (Varian Inc., Palo Alto, CA, USA) at 500 and 125 MHz in several analytical modes (COSY, correlation spectroscopy; DEPT, distorsionless enhancement by polarization transfer; HSQC, heteronuclear single quantum correlation; HMBC, heteronuclear multiple bond coherence). LC−HRMS analyses were carried out using an UltiMate 3000 chromatograph (Dionex/Thermo Fisher Scientific, Waltham, MA, USA) coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific). The analytes were separated under the following conditions: column, Cadenza CD-C18 (2.0 mm × 75 mm, 3 μm, Imtakt Corporation, Kyoto, Japan); solvent A, water containing 0.1% (v/v) formic acid; solvent B, 80% methanol (v/v) containing 0.1% (v/ v) formic acid; gradient program (%B), 0−7 min (0%), 7−10 min (0 → 40%), 10−17 min (40%), 17−20 min (40 → 100%), 20−23 min (100%), 23−23.01 min (100 → 0%); flow rate of the mobile phase, 0.2 mL/min. Mass spectrometry (MS) was carried out under the following conditions: ionization mode, ESI+ (positive mode); scan mode, FullMS; resolution, 70 000; maximum IT, 200 ms; isolation width, 4.0 m/ z. The HPLC analysis was performed to determine sample purity using a Nexera HPLC system (Shimadzu Corporation) using the same separation conditions as those for mentioned in the LC−HRMS analysis. 2892

DOI: 10.1021/acs.jafc.7b05480 J. Agric. Food Chem. 2018, 66, 2891−2899

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Journal of Agricultural and Food Chemistry Isolation and Identification of γ-Glutamyl-γ-glutamyl-Salk(en)ylcysteine Derivatives. Approximately 2.8 g of concentrated AGE (extract content: 38−42% (w/w)) was mixed with a mixture of methanol (8.5 mL) and formic acid (0.1 mL). This mixture was shaken vigorously for 10 min and centrifuged at 1750g for 10 min. The supernatant was concentrated using a rotary evaporator, and 3 mL of water was added to the residue. The resulting mixture was applied to a preconditioned Sep-Pack Plus C18 Cartridges (500 mg, Waters Corporation), and the cartridge was washed with 20 mL of water. The non-adhesion liquid portion and washed-water fraction were combined and concentrated using a rotary evaporator. γ-Glutamyl tripeptides in the resulting residue were separated and purified by preparative HPLC using an LC-10A HPLC system (Shimadzu Corporation) under the following conditions: first preparative HPLC (1st-HPLC) for GGSMC: column, Cadenza CD-C18 (28 mm × 250 mm, 5 μm, Imtakt Corporation); solvent, 10% (v/v) methanol containing 0.1% (v/v) formic acid; flow, 9.0 mL/min; second preparative HPLC (2ndHPLC) for GGSMC: column, Cadenza CD-C18 (10 mm × 250 mm, 3 μm, Imtakt Corporation); solvent, 5% (v/v) methanol containing 0.1% (v/v) formic acid; flow, 2.6 mL/min; first-HPLC for GGSAC: column, Cadenza CD-C18 (28 mm × 250 mm, 5 μm, Imtakt Corporation); solvent, 15% (v/v) methanol containing 0.1% (v/v) formic acid; flow, 9.0 mL/min; second-HPLC for GGSAC: column, Cadenza CD-C18 (10 mm × 250 mm, 3 μm, Imtakt Corporation); solvent, 15% (v/v) methanol containing 0.1% (v/v) formic acid; flow, 2.6 mL/min; first-HPLC for GGS1PC: column, Cadenza CD-C18 (28 mm × 250 mm, 5 μm, Imtakt Corporation); solvent, 20% (v/v) methanol containing 0.1% (v/v) formic acid; flow, 9.0 mL/min; second-HPLC for GGSAC: column, Cadenza CD-C18 (10 mm × 250 mm, 3 μm, Imtakt Corporation); solvent, 20% (v/v) methanol containing 0.1% (v/v) formic acid; flow, 2.5 mL/min, respectively. All chromatographies were monitored at 220 nm. The γ-glutamyl tripeptides-containing fraction was concentrated and lyophilized using an FRD-50P freeze-dryer (AGC Techno Glass). Structural analyses of the obtained compounds were performed using NMR and LC−HRMS according to the conditions provided in the section Syntheses of γ-Glutamyl-γ-glutamyl-S-alk(en)yl-cysteine Derivatives. The isolated compounds were characterized using LC−MS and NMR analysis, and these data were compared with the data for the synthesized compounds described above. The characterization data of the isolated compound (7), GGSMC, were as follows: HRMS, calculated [M + H]+ = 394.1278, observed [M + H]+ = 394.1277; 1H NMR (in D2O) δ, 1.99−2.06 (m, 1H, Ha-3′), 2.15−2.22 (m, 2H, H3), 2.15 (s, 3H, H-4′′), 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 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, 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 = 4.7, 8.4 Hz, 1H, H-2′′); 13C NMR (in D2O) δ, 14.7 (C-4′′), 26.1 (C3), 26.5 (C-3′), 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 (C-1), 174.6 (C-5), 174.9 (C-5′), 175.2 (C-1′′), 175.9 (C-1′). Characterization NMR data of isolated GGSMC were consistent with the previous report.21 The characterization data of isolated compound (8), GGSAC, were as follows: HRMS, calculated [M + H]+ = 420.1435, observed [M + H]+ = 420.1434; 1H NMR (in 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′), 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, 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, J = 6.2 Hz, 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, 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, H-5′′); 13CNMR (in D2O) δ, 26.0 (C-3), 26.5 (C-3′), 31.3 (C-4), 31.6 (C-3′′), 31.7 (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′′), 173.6 (C-1), 174.6 (C-5), 174.8 (C-5′), 175.0 (C-1′′), 175.9 (C-1′). The characterization data of isolated trans form of compound (9), trans-GGS1PC, were as follows: HRMS calculated [M + H]+ = 420.1435, observed [M + H]+ = 420.1433; 1H-NMR (in D2O) δ, 1.71 (dd, J = 1.5, 6.6 Hz, 3H, H-6′′), 1.99−2.05 (m, 1H, Ha-3′), 2.15−2.22

(m, 2H, H-3), 2.21−2.28 (m, 1H, Hb-3′), 2.40 (t, J = 7.1 Hz, 2H, H4′), 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 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′), 4.58 (dd, J = 4.4, 8.2 Hz, 1H, H2′′), 5.85 (dq, J = 6.4, 14.9 Hz, 1H, H-5′′), 5.96 (dd, J = 1.5, 15.1 Hz, 1H, H-4′′); 13C-NMR (in D2O) δ, 17.7 (C-6′′), 26.0 (C-3), 26.5 (C3′), 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 (C-5′′), 130.7 (C-4′′), 173.1 (C-1), 174.3(C-5), 174.5 (C-5′), 174.8 (C-1′), 175.4 (C-1′′). Content of γ-Glutamyl-γ-glutamyl-S-alk(en)ylcysteine Derivatives in Aged Garlic Extract. Approximately 1 g of AGE (extract content: 14−20% (w/w)) was mixed with 0.2 mL of the internal standard solution (IS, 0.5 mg/mL of S-3-butenylcysteine in 20 mM HCl) and 0.1 mL of formic acid. This mixture was shaken vigorously for 1 min and centrifuged at 15 000 rpm for 10 min. The supernatant was filtered using membrane filter (pore size: 0.45 μm), and the filtrate was used as the sample solution. The quantitative analyses were performed using LC−HRMS according to the same conditions outlined in the section Syntheses of γ-Glutamyl-γ-glutamyl-S-alk(en)yl-cysteine Derivatives. The mass tolerances for IS, GGSMC, GGSAC, and GGS1PC were set to ±10 ppm. Production of γ-Glutamyl-γ-glutamyl-S-alk(en)ylcysteine during Early Stages of the Aging Process. Raw garlic cloves were sliced to a thickness of approximately 5 mm; 2−4 g of these pieces was placed into a tube with a cap, and 10 mL of 15% ethanol was added. Several sample tubes were prepared, stored at 25 °C, and collected at a fixed time interval (week 0, week 2, and week 4) to examine the changes in γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteines. Formic acid (0.2 mL) and the IS solution (0.2 mL; 0.5 mg/mL of S-3butenylcysteine in 20 mM HCl) were added to the collected tubes and shaken vigorously. The mixtures of the sample materials were treated using a multi-beads shocker (Yasui Kikai Co., Osaka, Japan) to obtain the homogenates. The resulting homogenate was transferred to a centrifuge tube and centrifuged at 1750g for 10 min. An aliquot of the supernatant was filtered using a membrane filter (pore size: 0.45 μm), and the filtrate was used as the sample solution. To examine the content of γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteines in raw garlic, 5−6 g of sliced raw garlic was placed into a tube, and 20 mL of water, 0.2 mL of formic acid, and 0.2 mL of the IS solution (0.5 mg/mL of S-3-butenylcysteine in 20 mM HCl) were added. The mixture was treated using a multi-beads shocker to obtain a homogenate of raw garlic. The resulting homogenate was transferred to a centrifuge tube and centrifuged at 1750g for 10 min. An aliquot of the supernatant was filtered using a membrane filter (pore size: 0.45 μm), and the filtrate was used as the sample solution. The obtained sample solutions were analyzed using LC−HRMS as described in the section Syntheses of γ-Glutamyl-γ-glutamyl-Salk(en)ylcysteine Derivatives. The mass tolerances for IS, GGSMC, GGSAC, and GGS1PC were set to ±10 ppm. Model Reactions for Analysis of the Production Mechanism of γ-Glutamyl-γ-glutamyl-S-alk(en)ylcysteines. Model Reaction Using Raw Garlic. Raw garlic cloves were cut into pieces (approximately 2 × 3 × 5 mm3); 3−4 g of garlic pieces was placed in 15 mL tube, and 4 mL of 15% ethanol was added. Several sample tubes containing mixture of garlic pieces and 15% ethanol were prepared, and the tubes were divided into three groups: control group, boiled group, and GGT inhibitor-added group. The tubes of the boiled group were dipped into boiling water for 10 min and cooled to room temperature. GGsTop (GGT inhibitor, Wako Pure Chemical, Osaka, Japan) was dissolved in water and used as a GGT inhibitor (10 mM). A GGsTop solution (300 nmol) was added to the tubes of the GGT inhibitor-added group. The tubes of each group were stored at 25 °C and collected at fixed time intervals (day 0, day 5, day 10, day 20, day 30, and day 60) to examine the changes in the S-alk(en)ylcysteines, γglutamyl-S-alk(en)ylcysteines, and γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteines. Approximately 8 mL of 20 mM HCl and 0.2 mL of the IS solution (0.5 mg/mL of S-3-butenylcysteine in 20 mM HCl) were added to the collected tubes and shaken vigorously. The mixture was treated using the multibeads shocker to obtain the homogenate. The 2893

DOI: 10.1021/acs.jafc.7b05480 J. Agric. Food Chem. 2018, 66, 2891−2899

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resulting homogenate was transferred to a centrifuge tube and centrifuged at 1750g for 10 min. An aliquot of the supernatant was filtered using a membrane filter (pore size: 0.45 μm), and the filtrate was used as the sample solution. Quantitative analyses of the γglutamyl-γ-glutamyl-S-alk(en)ylcysteines were performed using LC− MS as described in the section Synthesis of γ-Glutamyl-γ-glutamyl-Salk(en)ylcysteine Derivatives. Quantitative analyses of S-alk(en)ylcysteines and γ-glutamyl-Salk(en)ylcysteines were performed using an Aquity ultra performance LC (Waters Corporation) under the following conditions: derivatization of amino group, AccQ·Tag Ultra Derivatization Kit (Waters Corporation); column, AccQ-Tag Ultra (2.1 mm × 100 mm, 1.7 μm, Waters Corporation); solvent A, 20% (v/v) AccQ·Tag Ultra Eluent A Concentrate Amino acid analysis (Waters Corporation); solvent B, AccQ·Tag Ultra Eluent B Amino acid analysis (Waters Corporation); gradient program (%B), 0−0.54 min (0.1%), 0.54−5.74 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 (10.6%), 9.54−11.74 min (10.6 → 21.2%), 11.74−12.04 min (21.2 → 59.6%), 12.04−13.04 min (59.6%), 13.04−13.13 min (59.6 → 0.1%), 13.13−15.0 min (0.1%); flow, 0.7 mL/min; detection, 260 nm; injection volume, 1.0 μL. Model Reaction Using Garlic Protein Fraction and GGT Inhibitor. To examine the endogenous GGT activity in garlic involving the production of γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteines, we performed a model reaction using the garlic protein fraction and GGT inhibitor (GGsTop, Wako Pure Chemical). The garlic protein fraction was prepared using an ammonium sulfate precipitation method. The outer skin of raw garlic was removed, and approximately 500 g of garlic cloves was homogenized with 500 mL of water. The homogenate was filtered with cheesecloth, and the filtrate was filtered using filter paper (Filter Paper No.1, Toyo Roshi Kaisha, Ltd., Tokyo, Japan). Ammonium sulfate was added to the filtrate to create a saturated solution under cooling using an ice bath and with stirring. The obtained mixture was centrifuged at 1750g for 15 min, and the supernatant was removed. The precipitate was transferred into a dialysis tube (cutoff: 3.5 kDa) and dialyzed using purified water at 4 °C for approximately 15 h; dialysis was repeated twice. The inner fraction of the dialysis tube was poured into plastic tubes and stored at −80 °C before use. The protein content in the inner fraction was determined by bicinchonic acid protein assay (BCA assay) with bovine serum albumin as the standard protein; the protein content was 12.9 mg/mL. The synthesized γ-glutamyl-S-alk(en)ylcysteines were dissolved in water (GSAC: 4.12 μmol/mL, GS1PC: 4.25 μmol/mL). Model reaction mixtures were prepared in 1.5 mL of plastic tube with the following composition: GSAC control mixture group, 50 μL of GSAC solution (206 nmol), 100 μL of garlic protein fraction, 650 μL of 15% ethanol solution; GSAC and inhibitor mixture group, 50 μL of GSAC solution (206 nmol), 100 μL of garlic protein fraction, 630 μL of 15% ethanol solution, 20 μL of GGsTop solution (200 nmol); GS1PC control mixture group, 50 μL of GS1PC solution (212 nmol), 100 μL of garlic protein fraction, 650 μL of 15% ethanol solution; GS1PC and inhibitor mixture group, 50 μL of GS1PC solution (212 nmol), 100 μL of garlic protein fraction, 630 μL of 15% ethanol solution, 20 μL of GGsTop solution (200 nmol). The tubes for each group were stored at 25 °C and collected at fixed time intervals (day 0, day 1, day 3, and day 5). IS solution (100 μL; 0.5 mg/mL of S-3-butenylcysteine in 20 mM HCl) and 100 μL of methanol containing 10% formic acid were added to each tube. The mixture was shaken vigorously for approximately 10 s and centrifuged at 15 000 rpm for 10 min. An aliquot of the supernatant was filtered using a membrane filter (cutoff: 3.0 kDa), and the filtrate was used as the sample solution. Quantitative analyses of γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteines were performed on an LC−MS system described in the section Synthesis of γGlutamyl-γ-glutamyl-S-alk(en)ylcysteine Derivatives. Quantitative analyses of S-alk(en)ylcysteines and γ-glutamyl-S-alk(en)ylcysteines were performed using an Aquity ultra performance LC system described in the section Model Reaction Using Raw Garlic.

Article

RESULTS Analysis of Sulfur Compounds in Aged Garlic Extract Using Post-Column HPLC with an Iodoplatinate Reagent. We obtained evidence suggesting that the γglutamyl-S-alk(en)ylcysteines and S-alk(en)ylcysteines reacted with hydrophobic sulfur compounds during the aging process;20 therefore, we analyzed AGE to understand its sulfur chemistry by post-column HPLC using an iodoplatinate reagent and LC−HRMS, using several different gradient programs for the mobile phases. Figure 2 shows a chromato-

Figure 2. Post-column HPLC chromatogram of aged garlic extract. Detection was performed at 500 nm absorbance using an iodoplatinate reagent. Double line arrows indicate putative sulfur-containing compounds unidentified. SAMC, S-allylmercaptocysteine; GSAC, γglutamyl-S-allylcysteine; GS1PC, γ-glutamyl-S-1-propenylcysteine.

gram of AGE using post-column HPLC; it contains several unidentified peaks of putative sulfur-containing compounds. We first focused on the peak eluted at 87 min in the postcolumn HPLC chromatogram and analyzed the corresponding peak by LC−HRMS because its peak intensity was stronger than that of the other peaks of putative sulfur compounds, and no other peaks were observed from 70 to 100 min on the postcolumn HPLC chromatogram. The mass signals of m/z 420.1435 and 422.1392 correspond to theoretical elemental compositions of C16H26O8N3S and C16H26O8N334S, which indicate the presence of sulfur compounds based on the theoretical difference in the exact mass and relative signal intensity between 32S-containing ions (100%) and 34Ssubstituted ions (4%). Isolation and Identification of γ-Glutamyl-γ-glutamylS-alk(en)ylcysteine Derivatives. To isolate and identify the putative sulfur compounds in AGE, we first focused on the compound with [M + H]+ = 420.1435, which is likely tripeptide GGSAC or GGS1PC based on its elemental composition. The elution time of the putative sulfur compound during the HPLC analysis was similar to that of the synthesized GGSAC; therefore, we attempted to isolate this compound using preparative HPLC by comparison with the elution time of the synthesized GGSAC. The other two compounds, corresponding to GGSMC and GGS1PC, were isolated in a similar way manner. The characterization data for the isolated compounds were consistent with the data for the synthesized compounds. In the 1 H-NMR spectrum of isolated GGS1PC, two methylene signals (−CH=CH−S−) in the S-propenyl group were observed at 5.85 ppm (dq, J = 6.4, 14.9 Hz) and 5.96 ppm (dd, J = 1.5, 15.1 Hz), which indicated a trans propenyl structure (JCHCH−S− = 9−16 Hz).25 Furthermore, the weak signals at 5.75−5.82 and 1.71 ppm, which were considered =CH−S− and methyl groups in the cis-S-1-propenyl group, were also observed (data not 2894

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Journal of Agricultural and Food Chemistry shown). The estimated J value for the =CH−S− group was 9.3 Hz, which is the characteristic J value for the cis form of the olefin structure. The integration intensity of the methyl signal derived from the cis form was around one tenth of that for the trans form, which indicated that the isolated compound contained a mixture of the cis (minor component) and trans (major component) forms. The chemical shifts of these methyl groups were consistent with reported values.25 For the LC− HRMS analysis of GGS1PC from AGE with a mass tolerance for GGS1PC set to ±10 ppm, the peak corresponding to the elution time of the cis form was observed, and its mass signals was m/z 420.1433, corresponding to the theoretical elemental compositions of C16H26O8N3S (calculated [M + H]+ = 420.1435). Content of γ-Glutamyl-γ-glutamyl-S-alk(en)ylcysteine Derivatives in Aged Garlic Extract and Raw Garlic. The changes in the GGSMC, GGSAC, and GGS1PC contents during the aging process were determined using LC−HRMS. The contents of these compounds reached maximum levels after approximately 4 months of aging and gradually decreased during the subsequent aging process (Figure 3). More than

Figure 3. Changes in γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteines in aged garlic extract during the aging period. Values are means ± SD, n = 3. The values of GGS1PC at each analytical point are sum of cis and trans forms. The quantitative analyses of each compound were performed using LC−MS. GGSMC, γ-glutamyl-γ-glutamyl-S-methylcysteine; GGSAC, γ-glutamyl-γ-glutamyl-S-allylcysteine; GGS1PC, γglutamyl-γ-glutamyl-S-1-propenylcysteine.

80% of the maximum levels of these compounds were produced within 1 month during the aging process. To examine the changes in the γ-glutamyl tripeptides within 1 month, their contents in the test preparations for the early aging process and raw garlic were analyzed. The contents of the three γ-glutamyl tripeptides (GGSMC, GGSAC, and GGS1PC) in raw garlic were less than 3, 70, and 60 nmol/g-fresh-weight, respectively. These compounds reached maximum levels, approximately 8, 720, and 600 nmol/g-fresh-weight, respectively, after 1 month (Figure 4). Production of γ-Glutamyl-γ-glutamyl-S-alk(en)ylcysteines in Model Reaction Mixtures. GGSAC and GGS1PC were the major γ-glutamyl tripaptides in AGE; therefore, we focused on the production mechanism of these two compounds using the model reaction approach. Although the contents of GGSAC and GGS1PC in the GGT inhibitoradded group slightly increased within 10 days and their contents were maintained for 60 days (GGSAC, < 7 nmol/g-

Figure 4. Changes in γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteines in the model reaction mixtures. Changes in the content of (A) GGSMC, (B) GGSAC, and (C) GGS1PC at 25 °C. Values are means ± SD, n = 3. The value of GGS1PC at each analytical point is the sum of the cis and trans forms. The quantitative analyses of each compound were performed using LC−MS. GGSMC, γ-glutamyl-γ-glutamyl-S-methylcysteine; GGSAC, γ-glutamyl-γ-glutamyl-S-allylcysteine; GGS1PC, γglutamyl-γ-glutamyl-S-1-propenylcysteine. 2895

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Figure 5. Changes in γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteines in the model reaction mixtures. Changes in concentration of (A) GGSAC and (B) GGS1PC at 25 °C, respectively. The tubes of the boiled group were dipped into boiling water for 10 min and cooled to room temperature. GGsTop (GGT inhibitor) was dissolved in water and used as a GGT inhibitor (10 mM). A GGsTop solution (300 nmol) was added to the tubes of the GGT inhibitor-added group. Values are means SD, n = 3. The value of GGS1PC at each analytical point is the sum of the cis and trans forms. The quantitative analyses of each compound were performed using LC−MS. GGSAC, γ-glutamyl-γ-glutamyl-S-allylcysteine; GGS1PC, γ-glutamyl-γglutamyl-S-1-propenylcysteine.

Figure 6. Changes in γ-glutamyl-S-alk(en)ylcysteines and S-alk(en)yl-cysteines in the model reaction mixtures. Changes in concentration of (A) GSAC, (B) GS1PC, (C) SAC, and (D) S1PC at 25 °C. The tubes of the boiled group were dipped into boiling water for 10 min and cooled to room temperature. GGsTop (GGT inhibitor) was dissolved in water and used as a GGT inhibitor (10 mM). A GGsTop solution (300 nmol) was added to the tubes of the GGT inhibitor-added group. Values are means SD, n = 3. The value of GS1PC and S1PC at each analytical point is the sum of the cis and trans forms. GSAC, γ-glutamyl-S-allylcysteine; GS1PC, γ-glutamyl-S-1-propenylcysteine; SAC, S-allylcysteine; S1PC, S-1-propenylcysteine.

during the initial test period. Figure 6 shows the changes in the putative precursor compounds for GGSAC and GGS1PC, and the changes in the contents of SAC and S1PC produced from the corresponding γ-glutamyl-S-alk(en)ylcysteines by GGT. The patterns of changes for GSAC and GS1PC were not

wet; GGS1PC, < 13 nmol/g-wet), their contents in the control group increased significantly; the maximum contents during the test period were 36 and 76 nmol/g-wet for GGSAC and GGS1PC, respectively (Figure 5). The contents of GGSAC and GGS1PC in the boiled group were almost the same as that 2896

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Figure 7. Productions in γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteines and S-alk(en)ylcysteines in the model reaction mixtures using the garlic protein fraction. GGsTop (GGT inhibitor) was dissolved in water and used as a GGT inhibitor (10 mM). A GGsTop solution (200 nmol) was added to the tubes of the GGT inhibitor-added group. (A) Productions of GGSAC and SAC in the mixtures of GSAC and garlic protein fraction with/without GGsTop, (B) productions of GGS1PC and S1PC in the mixtures of GSAC and garlic protein fraction with/without GGsTop, at 25 °C. Values are means SD, n = 3. The value of GS1PC and S1PC at each analytical point is the sum of the cis and trans forms. GGSAC, γ-glutamyl-γ-glutamyl-Sallylcysteine; GGS1PC, γ-glutamyl-γ-glutamyl-S-1-propenylcysteine; GSAC, γ-glutamyl-S-allylcysteine; GS1PC, γ-glutamyl-S-1-propenylcysteine; SAC, S-allylcysteine; S1PC, S-1-propenylcysteine.

and S-(β-carboxypropyl)-glutathione, were confirmed as intermediate compounds in the biosynthesis pathway of Salk(en)ylcysteine sulfoxides.26 We identified three γ-glutamyl tripeptides, GGSMC, GGSAC, and GGS1PC in AGE, which was prepared by soaking raw garlic in an aqueous alcohol solution for more than 10 months at room temperature. Small amounts of these compounds exist in raw garlic, but their amounts in AGE increased during the aging process. The content of these γ-glutamyl tripeptides reached a maximum level after approximately 4 months of aging and gradually decreased thereafter. More than 80% of the amounts of these compounds present at 4 months were generated at around 2 months in AGE (Figure 3). We hypothesized that γ-glutamyl tripeptides were produced from γ-glutamyl dipeptides by the endogenous GGT in garlic during the aging period. The productions of GGSAC and GGS1PC were inhibited in the model reaction mixtures using GGT inhibitor GGsTop, while their contents in the control groups increased (Figure 5). The contents of GSAC, a putative precursor compound, quickly decreased within 10 days and then gradually decreased in the control group and GGT inhibitor-added group (Figure 6A). The SAC content in GGT inhibitor-added group also slightly increased during the early test period (Figure 6C). The changes in the amounts of GSAC and SAC were on the order of micromoles per gram of wetweight-garlic and that of GGSAC was on the order of nanomoles per gram of wet-weight-garlic. A similar phenomenon was observed for GGS1PC, GS1PC, and S1PC. Additionally, the productions of GGSAC, GGS1PC, SAC, and S1PC were confirmed in another model reaction using garlic protein fraction as an enzyme fraction involving the GGT activity (Figure 7). These results suggest that γ-glutamyl tripeptides are produced via transfer of γ-glutamyl-S-alk(en)ylcysteine to glutamic acid in other γ-glutamyl-S-alk(en)ylcysteines by an endogenous catalyst in garlic, which helps to simultaneously produce γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteine and S-alk(en)ylcysteine during aging process (Figure 8). Furthermore, the GGT affinity in raw garlic for the

different in the control group and GGT inhibitor-added group (Figure 6A, B). The boiling treatment seemed to destroy GSAC and GS1PC because their initial contents in the boiled group were approximately half the amounts of the control groups and GGT inhibitor-added groups. The GGT inhibitor, GGsTop, affected the GGT activity because the contents of SAC and S1PC treated with GGsTop did not increase, while their contents in the control groups increased (Figure 6C, D). Figure 7 shows the model reaction using the garlic protein fraction and GGT inhibitor. Productions of GGSAC and SAC were observed in the control group; the content of GGSAC was twice as high as that of SAC at day 5. The contents of GGSAC and SAC did not increase in the GSAC and inhibitor mixture group during the test period. The same phenomenon was observed in the model reaction using GS1PC as a substrate for GGT, while the production amount of GGS1PC was similar to GGSAC in the control group, but that of S1PC was quite smaller than that of SAC (less than 0.1% against initial GS1PC content).

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DISCUSSION Allium plants are characterized by sulfur compounds, such as γglutamyl-S-alk(en)ylcysteine and S-alk(en)ylcysteine sulfoxide, which accumulate as sulfur storage molecules.1−3 S-Alk(en)ylcysteine sulfoxides were produced from the corresponding precursor, γ-glutamyl-S-alk(en)ylcysteine sulfoxides, by GGT, or γ-glutamyl-S-alk(en)ylcysteine that was transformed to Salk(en)ylcysteine by GGT and subsequently oxidized by an oxidase.25 In the Allium preparations, such as AGE, Salk(en)ylcysteines (e.g., SMC, SAC, and S1PC) are produced from the corresponding precursor dipeptides, γ-glutamyl-Salk(en)ylcysteines by endogenous GGT in plants.1−3,9,23 γGlutamyl-S-methylcysteine was found in onion and garlic as a precursor compound of S-methylcysteine sulfoxide and in its desulfoxide form,9 but there are no reports for the identification of γ-glutamyl-γ-glutamyl tripeptide, GGSMC, in these Allium plants and their preparations, whereas other types of γ-glutamyl tripeptide, glutathione and its derivatives, S-methylglutathione, 2897

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Figure 8. Plausible production pathway of γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteines in aged garlic extract during aging process. R1, methyl; R2, allyl; R3, 1-propenyl.

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substrates may be different or several types of GGT may be present in garlic because produced amounts of γ-glutamyl-γglutamyl-S-alk(en)ylcysteines and S-alk(en)ylcysteines were different during the aging process, and the times to maximum production of S-alk(en)ylcysteines were longer than that of γglutamyl-γ-glutamyl-S-alk(en)yl-cysteines (e.g., approximately 10 months for S-alk(en)ylcysteines19,20 and within a few months for γ-glutamyl-γ-glutamyl-S-alk(en)ylcysteines) (Figures 3 and 4). The contents of GGSAC and GGS1PC on day 0 were different (Figures 4 and 5). We used different lots of raw garlic that were purchased over a span of more than 6 months for these experiments. γ-Glutamyl tripeptide contents may vary depending on the origin of the garlic, and γ-glutamyl tripeptides may be accumulated or consumed in raw garlic, while endogenous GGT in raw garlic involves the production of γglutamyl tripeptides. We observed both γ-glutamyl tripeptides, cis-GGS1PC and trans-GGS1PC, in AGE from the LC−HRMS analysis, and the content of the cis form was approximately 10% of the trans form (data not shown). In the Allium plants, the trans form of propenyl group is the major component, and the trans form is the natural form of the compound. Previously, we identified the cis form of S1PC in AGE, and its content was about 10−20% of that of the trans form.8,20 S1PC was produced from the precursor GS1PC by the presence of GGT in raw garlic. Herein, the content of the cis form of GS1PC in raw garlic was less than 1% of that of the trans form. We revealed that the majority of the cis form observed in AGE was produced by isomerization of the trans form during the aging process, and the isomerization reaction was reversible.8,20 On the basis of the previous report,8 the cis form of GGS1PC can be produced from the trans form by an isomerization reaction during the aging process because the contents of the cis forms of GS1PC and GGS1PC in raw garlic were much smaller than those in AGE. In conclusion, the present study indicates that the aging process using an aqueous alcoholic solution can provide the conditions to produce γ-glutamyl tripeptides, γ-glutamyl-γglutamyl-S-alk(en)ylcysteines that are produced by endogenous GGT in raw garlic. These compounds may be the same as a marker compound for the aging process using an aqueous alcoholic solution because the contents of these compounds in raw garlic are less than 10−20% of the corresponding amount in AGE, and these compounds are generated during the aging process.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81 826 45 2331. Fax: +81 826 45 4351. ORCID

Yukihiro Kodera: 0000-0003-0538-0105 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Takami Oka of Wakunaga Pharmaceutical Co., Ltd, for the helpful advice, encouragement, and critical reading of the manuscript.

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ABBREVIATIONS USED AGE, aged garlic extract; SAC, S-allylcysteine; S1PC, S-1propenylcysteine; GGSMC, γ-glutamyl-γ-glutamyl-S-methylcysteine; GGSAC, γ-glutamyl-γ-glutamyl-S-allylcysteine; GGS1PC, γ-glutamyl-γ-glutamyl-S-1-propenylcysteine; GGT, γ-glutamyltranspeptidase

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REFERENCES

(1) Lancaster, J. E.; Shaw, M. L. γ-Glutamyl peptides in the biosynthesis of S-alk(en)yl-L-cysteine sulfoxides (flavor precursors) in Allium. Phytochemistry 1989, 28, 455−460. (2) Yoshimoto, N.; Yabe, A.; Sugino, Y.; Murakami, S.; Sai-Ngam, N.; Sumi, S.; Tsuneyoshi, T.; Saito, K. Garlic γ-glutamyl transpeptidases that catalyze deglutamylation of biosynthetic intermediate of alliin. Front. Plant Sci. 2015, 5, 758. (3) Yoshimoto, N.; Onuma, M.; Mizuno, S.; Sugino, Y.; Nakabayashi, R.; Imai, S.; Tsuneyoshi, T.; Sumi, S.; Saito, K. Identification of a flavin-containing S-oxygenating monooxygenase involved in alliin biosynthesis in garlic. Plant J. 2015, 83 (6), 941−951. (4) Amagase, H.; Petesch, B. L.; Matsuura, H.; Kasuga, S.; Itakura, Y. Intake of garlic and its bioactive components. J. Nutr. 2001, 131 (Suppl 3), 955S−962S. (5) Amagase, H. Clarifying the real bioactive constituents of garlic. J. Nutr. 2006, 136 (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, 1135−1178. (7) Lawson, L. D.; Wang, Z. Y.; Hughes, B. G. γ-Glutamyl-Salklycysteines in garlic and other Allium spp.: Precursor of agedependent trans-S-1-propenyl thiosulfinates. J. Nat. Prod. 1991, 54 (2), 436−444. (8) Kodera, Y.; Matsutomo, T.; Itoh, K. The evidence for the production mechanism of cis-S-1-propenylcysteine in aged garlic 2898

DOI: 10.1021/acs.jafc.7b05480 J. Agric. Food Chem. 2018, 66, 2891−2899

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Journal of Agricultural and Food Chemistry extract based on a model reaction approach using its isomers and deuterated solvents. Planta Med. Lett. 2015, 2, e69−e72. (9) Garlic and Other Aliums: The Lore and the Science, Block E; The Royal Society of Chemistry: Cambridge, U.K., 2010. (10) Suzuki, J.; Yamaguchi, T.; Matsutomo, T.; Amano, T.; Morihara, N.; Kodera, Y. S-1-Propenylcysteine promotes the differentiation of B cell into IgA-producing cells by the induction of Erk1/2-dependent Xbp1 expression in Peyer’s patches. Nutrition 2016, 32, 884−889. (11) Sumioka, I.; Matsura, T.; Yamada, K. Therapeutic effect of Sallylmercaptocysteine on acetaminophen-induced liver injury in mice. Eur. J. Pharmacol. 2001, 433 (2−3), 177−85. (12) Amano, H.; Kazamori, D.; Itoh, K. Pharmacokinetics and Nacetylation metabolism of S-methyl-L-cysteine and trans-S-1-propenylL-cysteine in rats and dogs. Xenobiotica 2016, 46 (11), 1017−1025. (13) Kyo, E.; Uda, N.; Kasuga, S.; Itakura, Y. Immunomodulatory effects of aged garlic extract. J. Nutr. 2001, 131 (Suppl 3), 1075S− 1079S. (14) Behzadi, M. T.; Fallah-Rostami, F.; Tabari, M. A.; Esfandiari, B.; Aghajanzadeh, H. Immunomodulatory activity of aged garlic extract against implanted fibrosarcoma tumor in mice. N. Am. J. Med. Sci. 2013, 5, 207−212. (15) Ishikawa, H.; Saeki, T.; Otani, T.; Suzuki, T.; Shimozuma, K.; Nishino, H.; Fukuda, S.; Morimoto, K. Aged garlic extract prevents a decline of NK number and activity in patients with advanced cancer. J. Nutr. 2006, 136 (Suppl 3), 816S−820S. (16) Amano, H.; Kazamori, D.; Itoh, K. Evaluation of the effects of Sallyl-L-cysteine, S-methyl-L-cysteine, trans-S-1-propenyl-L-cysteine, and their N-acetylated and S-oxidized metabolites on human CYP activities. Biol. Pharm. Bull. 2016, 39, 1701−1707. (17) Nantz, M. P.; Rowe, C. A.; Muller, C. E.; Creasy, R. A.; Stanilka, J. M.; Percival, S. S. Supplementation with aged garlic extract improves both NK and γδ−T cell function and reduces the severity of cold and flu symptoms: a randomized, double-blind, placebo-controlled nutrition intervention. Clin. Nutr. 2012, 31 (3), 337−344. (18) Harauma, A.; Moriguchi, T. Aged garlic extract improves blood pressure in spontaneous hypertensive rats more safely than raw garlic. J. Nutr. 2006, 136 (3S), 769S−773S. (19) The Science and Therapeutic Application of Allium Sativum L and Related Species; Koch, H. P.; Lawson, L. D., Eds.; Williams & Wilkins: Baltimore, MD, 1996. (20) Matsutomo, T.; Kodera, Y. Development of an analytical method for sulfur compounds in aged garlic extract with the use of a postcolumn high performance liquid chromatography method with sulfur-specific detection. J. Nutr. 2016, 146 (Suppl), 450S−455S. (21) Kasai, T.; Shiroshita, Y.; Sakamura, S. γ-Glutamyl peptides of Vigna radiata seeds. Phytochemistry 1986, 25 (3), 679−682. (22) Ryu, K.; Ide, N.; Matsuura, H.; Itakura, Y. Nα-(1-Deoxy-Dfructos-1-yl)-L-arginine, an antioxidant compound identified in aged garlic extract. J. Nutr. 2001, 131, 972S−976S. (23) Jayathilaka, L.; Gupta, S.; Huang, J. S.; Lee, J.; Lee, B. S. Preparation of (+)-trans-isoalliin and its isomers by chemical synthesis and RP−HPLC resolution. J. Biomol Tech 2014, 25 (3), 67−76. (24) Carson, J. F.; Wong, F. F. Synthesis of cis-S-prop-1-enyl-Lcysteine. Chem. Ind. 1963, 2, 764−776. (25) King, F. E.; Kidd, D. A. A. A new synthesis of glutamine and of γ-dipeptides of glutamic acid from phthalylated intermediates. J. Chem. Soc. 1949, 0, 3315−3319. (26) Kodera, Y.; Ushijima, M.; Amano, H.; Suzuki, J. I.; Matsutomo, T. Chemical and Biological Properties of S-1-Propenyl-L-Cysteine in Aged Garlic Extract. Molecules 2017, 22 (4), E570.

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