Changes of S-Allylmercaptocysteine and γ-Glutamyl- S-allylmercaptocysteine Contents and Their Putative Production Mechanisms in Garlic Extract during the Aging Process

Article abstract of DOI:10.1021/acs.jafc.8b02541

γ-Glutamyl-S-allylmercaptocysteine (GSAMC), a putative precursor compound of S-allylmercaptocysteine (SAMC), was isolated and identified from aged garlic extract (AGE). We analyzed the change of their contents in AGE during the aging process, chronologica

Full text of DOI:10.1021/acs.jafc.8b02541

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Food and Beverage Chemistry/Biochemistry
Changes of S-allylmercaptocysteine and #-glutamyl-S-
allylmercaptocysteine contents and their putative production
mechanisms in garlic extract during the aging process
Takuto Fujii, Toshiaki Matsutomo, and Yukihiro Kodera
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02541 • Publication Date (Web): 18 Sep 2018
Downloaded from http://pubs.acs.org on September 19, 2018
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Changes of Sꢀallylmercaptocysteine and γꢀglutamylꢀSꢀallylmercaptocysteine contents
and their putative production mechanisms in garlic extract during the aging process
Takuto Fujii, Toshiaki Matsutomo and Yukihiro Kodera*
Affiliations
Central Research Institute, Wakunaga Pharmaceutical Co., Ltd., 1624 Shimokotachi,
Koda–cho, Akitakata–shi, Hiroshima 739ꢀ1195 Japan.
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Authors
Takuto Fujii
Eꢀmail: fujii_tk@wakunaga.co.jp
Eꢀmail: matsutomo_t@wakunaga.co.jp
Eꢀmail: kodera_y@wakunaga.co.jp,
Toshiaki Matsutomo
Yukihiro Kodera
*: Correspondence should be sent to Yukihiro Kodera
Central Research Institute, Wakunaga Pharmaceutical Co., Ltd.
1624 Shimokotachi, Koda–cho, Akitakata–shi, Hiroshima 739ꢀ1195 Japan.
Eꢀmail: kodera_y@wakunaga.co.jp,
Tel: +81 826 45 2331, Fax: +81 826 45 4351
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Abstract
γꢀGlutamylꢀSꢀallylmercaptocysteine (GSAMC), a putative precursor compound of
SꢀAllylmercaptocysteine (SAMC), was isolated and identified from aged garlic extract
(AGE). We analyzed the change of their contents in AGE during the aging process,
chronologically 1 month to 22 months. The formation of these compounds occurred
mostly during the early stage of the aging period: SAMC content reached a maximum at
approximately 4 months, whereas GSAMC content reached a maximum at 1 month and
then decreased during the subsequent aging period. In order to assess the possible
relationship between the change of the two compounds during the aging process, we set
up the model reactions with the hypothesis that GSAMC is produced from
γꢀglutamylꢀSꢀallylcysteine (GSAC)/γꢀgultamylꢀSꢀ1ꢀpropenylcysteine (GS1PC), and that
SAMC is produced from GSAMC by endogenous γꢀglutamyl transpeptidase (GGT) in
garlic during the early stage of the aging process. We found that in the model reactions,
SAMC was produced from GSAMC by the garlic protein fraction having GGT activity,
and its production was suppressed by a GGT inhibitor. Furthermore, the production of
GSAMC from allicin and GSAC/GS1PC was found in another model reaction. The
reaction between allicin and GS1PC was faster than that between allicin and GSAC, and
thus may be involved in the production of GSAMC in the early stage of aging process.
Key words: Sꢀallylmercaptocystine, γꢀglutamylꢀSꢀallylmercaptocystine, aged garlic
extract, GGT, allicin
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Introduction
Garlic preparations contain various sulfur compounds that are divided into two
types, hydrophobic and hydrophilic. Hydrophobic sulfur compounds, such as
allylpolysulfides, vinyldithiins, and ajoenes, are the main constituents of garlic oil and
oilꢀmacerated products, and are produced from allicin, a highly reactive, transient
1–3
compound.
In contrast, hydrophilic sulfur compounds, such as Sꢀmethylcysteine
(SMC), Sꢀallylcysteine (SAC), and Sꢀ1ꢀpropenylcysteine (S1PC) are odorless and stable,
and present in aged garlic extract (AGE) that is prepared by soaking and aging in
1
–4
aqueous alcoholic solution. SMC, SAC, and S1PC are present in small amounts in
raw garlic and are produced when raw garlic is soaked and aged in aqueous alcoholic
solution from the respective precursors, γꢀglutamylꢀSꢀmethylcysteine, γꢀglutamylꢀSꢀ
allylcysteine (GSAC), and γꢀglutamylꢀSꢀ1ꢀpropenylcysteine (GS1PC), by endogenous
1
, 4, 5
γꢀglutamyl transpeptidase (GGT) in garlic.
AGE contains another hydrophilic sulfur compound, Sꢀallylmercaptocysteine
(SAMC), which shows the protective effect against acetoaminophenꢀinduced liver
6
7
injury, antiproliferative effects against colon cancer, antiꢀmetastatic activity against
8
9
prostate cancer, and attenuation of gentamicinꢀinduced ototoxicity. SAMC is
chemically synthesized by a reaction between allicin and cysteine, that the reaction
1
, 10
proceeds very rapidly even at room temperature.
This reaction is thought to occur in
garlic preparations during processing because γꢀglutamylꢀSꢀallylmercaptocysteine
(GSAMC), a putative precursor of SAMC, like GSAC, a precursor of SAC, and GS1PC,
1
a precursor of S1PC, is not detectable in raw garlic. When raw garlic is crushed or
sliced, allicin having highly reactive and unstable properties is quickly and abundantly
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produced (12ꢀ37 ꢁmol/g fresh weight) from the precursor alliin. However, cysteine, the
1
other raw material for SAMC, is not detectable in whole raw garlic and crushed garlic.
SAMC content gradually increases during aging and the cysteine content gradually
increases during aging while the origin of cysteine is unknown and allicin disappears in
1
the early stage of the aging process. These changes in the content of allicin and
cysteine suggest that SAMC may be produced from other substances, besides cysteine
and allicin. One possible mechanism is that SAMC is produced by a reaction between
11
SAC/S1PC and diallyldisulfide (DADS). However, the amount of allylpolysulfides,
including DADS in AGE is small, i.e. 0.01ꢀ0.05 mg/g, while the SAC content is > 1
12
mg/g. Furthermore, the reaction between SAC/S1PC and DADS proceeds very slowly
11
at room temperature, the manufacturing condition for the AGE production. Recently,
Matsutomo et al. showed the presence of GSAMC in AGE using HPLC analysis
specific for sulfur detection method and LCꢀMS analysis and that GSAMC is produced
11
during the aging process. In the present study, we tested the hypothesis that GSAMC
is produced from GSAC/GS1PC in the early stage of the aging period, and that it serves
as a substrate to produce SAMC by endogenous GGT. To this end, we first isolated and
identified GSAMC from AGE to confirm its existence, measured its content during
aging and then set up model reactions to determine the possible mode of SAMC and
GSAMC productions. In this study, we present the evidence to support our hypothesis
concerning the formation of SAMC and GSAMC in AGE.
Materials and methods
Chemicals
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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
13
previous report.
Reference compound synthesis
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00
01
The reference compounds Sꢀsubstituted cysteine derivatives (Sꢀallylꢀ,
02 Sꢀ1ꢀpropenylꢀ, and Sꢀ3ꢀbutenylꢀ) and γꢀglutamylꢀSꢀalkenylcysteine derivatives
03 (Sꢀalkenyl: Sꢀallylꢀ and Sꢀ1ꢀpropenylꢀ) were synthesized according to previously
11, 14ꢀ16
04 described methods.
SAC was oxidized with hydrogen peroxide to yield
1
4, 15
05 Sꢀallylcysteine sulfoxide (alliin) as described previously.
SAMC was synthesized by
GSAMC was synthesized as
1
, 6, 10
06 reacting allicin and cysteine as described previously.
16
07 previously described, and the synthesized GSAMC was purified by preparative HPLC
08 using a Shimadzu HPLC system LC–10A (Shimadzu Corporation, Kyoto, Japan) using
09 the preparative HPLC conditions described in the section, “Isolation and identification
10 of GSAMC.” The GSAMCꢀcontaining fraction was concentrated with a rotary
11 evaporator and lyophilized using an FRD–50P freeze–dryer (AGC Techno Glass,
10
12 Shizuoka, Japan). Allicin was synthesized and purified as previously described and
13 stored at –80°C until use.
14
The chemical structure and purity of the synthesized compounds were determined
15 by nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography–high
1
13
16 resolution mass spectrometry (LC–HRMS), and HPLC. H– and C–NMR spectra of
2
17 the compounds were measured in deuterium oxide (D O) using a VNMRꢀ500
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18 spectrometer (Varian Inc., Palo Alto, CA, USA) at 500 and 125 MHz in several
19 analytical modes (COSY, correlation spectroscopy; DEPT, distorsionless enhancement
20 by polarization transfer; HSQC, heteronuclear single quantum correlation; HMBC,
21 heteronuclear multiple bond coherence).
22
LC–HRMS analyses were carried out using an UltiMate 3000 chromatograph
23 (Dionex/Thermo Fisher Scientific, Waltham, MA, USA) coupled to a Q–Exactive mass
24 spectrometer (Thermo Fisher Scientific). The analytes were separated under the
25 following conditions: column, Cadenza CD–C18 (2.0 mm × 75 mm, 3 ꢁm, Imtakt
26 Corporation, Kyoto, Japan); solvent A, water containing 0.1% (v/v) formic acid; solvent
27 B, 80% methanol (v/v) containing 0.1% (v/v) formic acid; gradient program (%B), 0–1
28 min (5%), 1–20 min (5→100%), 20–22 min (100%), 22.01–30 min (5%); flow rate of
29 the mobile phase, 0.2 mL/min. Mass spectrometry (MS) was carried out under the
+
30 following conditions: ionization mode, ESI (positive mode); scan mode, Full–MS;
31 resolution, 70,000; maximum IT, 200 ms; isolation width, 4.0 m/z. Purity of synthesized
32 compounds was determined by the HPLC analysis using a NEXERA X2 HPLC system
33 (Shimadzu Corporation) using the same separation conditions as those mentioned in the
34 LC–HRMS analysis.
35
The characterization data of synthesized GSAMC were as follows: HRMS,
+
+
1
2
36 calculated [M+H] = 323.0730, observed [M+H] =323.0730; HꢀNMR (in D O) δ,
37 2.12ꢀ2.21 (m, 2H, Hꢀ3), 2.50ꢀ2.54 (m, 2H, Hꢀ4), 3.00 (dd, J = 9.0, 14.1 Hz, 1H, Haꢀ3’),
38 3.27 (dd, J = 4.4, 14.2 Hz, 1H, Hbꢀ3’), 3.38 (d, J = 6.6 Hz, 2H, Hꢀ4’), 3.83 (t, J = 6.1
39 Hz, 1H, Hꢀ2), 4.64 (dd, J = 4.4, 8.9 Hz, 1H, Hꢀ2’), 5.21 (dd, J = 9.2, 25.2 Hz, 1H, Hꢀ6’),
1
3
2
40 5.89 (ddq, J = 7.3, 10.0, 17.0 Hz, 1H, Hꢀ5’); CꢀNMR (in D O) δ, 29.2 (Cꢀ3), 34.5
41 (Cꢀ4), 42.0 (Cꢀ3’), 43.9 (Cꢀ4’), 55.9 (Cꢀ2), 56.8 (Cꢀ2’), 121.8 (Cꢀ6’), 136.5 (Cꢀ5’),
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42 176.5 (Cꢀ1), 177.5 (Cꢀ5), 178.1 (Cꢀ1’).
43
44 Isolation and identification of GSAMC
45
46
Approximately 100 g of concentrated AGE (extract content: 38–42% (w/w)) was
47 mixed with a mixture of methanol (300 mL) and formic acid (3.5 mL). This mixture
48 was shaken vigorously for 10 min and centrifuged at 1,750 g for 10 min. The
49 supernatant was concentrated using a rotary evaporator, and 30 mL of water was added
50 to the residue. The resulting mixture was mixed with a mixture of methanol (300 mL)
51 and formic acid (3.5 mL). This mixture was shaken vigorously for 10 min and filtered
52 with filter paper (Advantec No.2, Toyo Roshi Kaisha, Ltd., Tokyo, Japan). The filtrate
53 was concentrated using a rotary evaporator and 10 mL of water was added to the residue.
54 Resulting solution was applied to a preconditioned Sep–Pack Plus C18 Cartridges (500
55 mg, Waters Corporation), and the cartridge was washed with 20 mL of water. The
56 non–adhesion liquid portion and washed–water fraction were combined and
57 concentrated using a rotary evaporator. GSAMC in the resulting residue were separated
58 and purified by preparative HPLC using an LC–10A HPLC system (Shimadzu
59 Corporation) under the following conditions: column, Cadenza CD–C18 (28 mm × 250
60 mm, 5 ꢁm, Imtakt Corporation); solvent, 20% (v/v) methanol containing 0.1% (v/v)
61 formic acid; flow, 8.0 mL/min; detection, 220 nm. The GSAMC containing fraction was
62 concentrated and lyophilized using an FRDꢀ50P freeze–dryer (AGC Techno Glass).
63
The isolated compounds were characterized using LCꢀHRMS and NMR
64 according to the conditions provided in the section “Reference compound
65 synthesis,” and these data were compared with the data for the synthesized compounds
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66 described above.
The characterization data of isolated GSAMC were as follows: HRMS, calculated
67
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2
68 [M+H] = 323.0730, observed [M+H] =323.0735; HꢀNMR (in D O) δ, 2.13ꢀ2.22 (m,
69 2H, Hꢀ3), 2.22ꢀ2.54 (m, 2H, Hꢀ4), 3.00 (dd, J = 9.0, 14.2 Hz, 1H, Haꢀ3’), 3.27 (dd, J =
70 4.2, 14.2 Hz, 1H, Hbꢀ3’), 3.38 (d, J = 7.3 Hz, 2H, Hꢀ4’), 3.84 (t, J = 6.1 Hz, 1H, Hꢀ2),
71 4.65 (dd, J = 4.2, 8.9 Hz, 1H, Hꢀ2’), 5.21 (dd, J = 9.1, 25.9 Hz, 1H, Hꢀ6’), 5.89 (ddq, J
1
3
2
72 = 7.3, 9.8, 17.1 Hz, 1H, Hꢀ5’); CꢀNMR (in D O) δ, 29.2 (Cꢀ3), 34.4 (Cꢀ4), 41.9 (Cꢀ3’),
73 43.9 (Cꢀ4’), 55.9 (Cꢀ2), 56.9 (Cꢀ2’), 121.8 (Cꢀ6’), 136.5 (Cꢀ5’), 176.5 (Cꢀ1), 177.5
74 (Cꢀ5), 178.0 (Cꢀ1’).
75
76 Changes in SAMC and GSAMC during the aging process
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78
AGE samples were collected at the factory in Wakunaga Pharmaceutical Co., Ltd.
79 at fixed intervals, 1, 2, 4, 10, 16, and 22 months and stored at –80°C before use.
80 Approximately 1 g of AGE (extract content: 14–20% (w/w), 100 grams of liquid AGE
81 contains 14ꢀ20 grams of solid materials derived from garlic) was mixed with 0.2 mL of
82 the internal standard solution (IS, 0.5 mg/mL of Sꢀ3ꢀbutenylcysteine in 20 mM HCl) and
83 0.1 mL formic acid. This mixture was shaken vigorously for 1 min and centrifuged at
84 15,000 rpm for 10 min. The supernatant was filtered using membrane filter (pore size:
85 0.45 ꢁm), and the filtrate was used as the sample solution.
86
The quantitative analyses of GSAMC were performed using LC–HRMS according
87 to the same conditions outlined in the section “Reference compound synthesis.” The
88 mass tolerances for IS and GSAMC were set to ± 10 ppm. Quantitative analyses of
89 SAMC was performed using an Acquity ultra performance LC (Waters Corporation)
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90 under the following conditions: derivatization of amino group, AccQ·Tag Ultra
TM
91 Derivatization Kit (Waters Corporation); column, AccQ–Tag Ultra (2.1 mm × 100
TM
92 mm, 1.7 ꢁm, Waters Corporation); solvent A, 20% (v/v) AccQ·Tag Ultra Eluent A
TM
93 Concentrate Amino acid analysis (Waters Corporation); solvent B, AccQ·Tag Ultra
94 Eluent B Amino acid analysis (Waters Corporation); gradient program (%B), 0–0.54
95 min (0.1%), 0.54–5.74 min (0.1→9.1%), 5.74–7.74 min (9.1%), 7.74–8.04 min
96 (9.1→10.6%), 8.04–9.54 min (10.6%), 9.54–11.74 min (10.6→21.2%), 11.74–12.04
97 min (21.2→59.6%), 12.04–13.04 min (59.6%), 13.04–13.13 min (59.6→0.1%),
98 13.13–15.0 min (0.1%); flow, 0.7 mL/min; detection, 260 nm; injection volume, 1.0 ꢁL.
99
00 Laboratory experiment to mimic aging using raw garlic and a GGT inhibitor
01
02
A laboratory experiment mimicking the aging process of garlic extract was
03 performed using raw garlic and a GGT inhibitor according to previously described
5
04 method. Raw garlic cloves were cut into pieces (approximately 2 × 3 × 5 mm). Then,
05 3–4 g of the garlic pieces were placed in a 15 mL tube, and 4 mL of 15% ethanol was
06 added. Several sample tubes containing garlic pieces and 15% ethanol were prepared,
07 and the tubes were divided into three groups: the control, boiled, and GGT
08 inhibitorꢀadded groups. The tubes for the boiled group were dipped in boiling water for
09 10 min and then cooled to room temperature. For the GGT inhibitorꢀadded group,
10 GGsTop® (GGT inhibitor, Wako Pure Chemical, Osaka, Japan) was dissolved in water
11 (10 mM), and GGsTop® solution (300 nmol) was added to the tubes. The tubes were
12 stored at 25°C and the content of SAMC and GSAMC was examined at fixed time
13 intervals (day 0, day 5, day 10, day 20, day 30, and day 60). Approximately 8 mL of 20
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14 mM HCl and 0.2 mL of the IS solution (0.5 mg/mL Sꢀ3ꢀbutenylcysteine in 20 mM HCl)
15 were added to the tubes collected at fixed intervals, and then shaken vigorously. The
16 mixture was treated with a multiꢀbeads shocker to obtain the pulverized mixture. The
17 resulting mixture was transferred to a centrifuge tube and centrifuged at 1,750 g for 10
18 min. An aliquot of the supernatant was filtered using a membrane filter (pore size: 0.45
19 ꢁm), and this filtrate was used as the sample solution. Quantitative analyses of SAMC
20 and GSAMC were performed using UPLC and LC–HRMS as described in the section
21 “Changes of SAMC and GSAMC during the aging process.”
22
23 Model reaction for SAMC production using GSAMC, a garlic protein fraction, and
24 a GGT inhibitor
25
26
To examine the endogenous SAMCꢀproducing GGT activity in garlic, we set up a
27 model reaction using a garlic protein fraction (GPF) and a GGT inhibitor (GGsTop®,
28 Wako Pure Chemical). The GPF was prepared using an ammonium sulfate precipitation
29 method, and the protein content in the fraction was determined by the bicinchonic acid
5
30 (BCA) assay as described previously. The protein content was estimated to be 12.9
31 mg/mL, using bovine serum albumin as a standard. The GPF was stored in a plastic tube
32 at –80°C until use.
33
The synthesized GSAMC was dissolved in water (2.83 ꢁmol/mL). GlycylGlycine
34 (GlyGly), threonine, and methionine were dissolved in water and used as the substrates
35 for GGT (GlyGly, 2.40 ꢁmol/mL; threonine, 4.39 ꢁmol/mL; methionine, 1.85
36 ꢁmol/mL). Model reaction mixtures were prepared in 1.5 mL plastic tubes with the
37 following composition: GGsTop® added group, 50 ꢁL of GSAMC solution (141 nmol),
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38 25 ꢁL of GlyGly solution (60 nmol), 25 ꢁL of threonine solution (110 nmol), 25 ꢁL of
39 methionine solution (46 nmol), 100 ꢁL of GPF, 10 ꢁL of GGsTop® solution (100 nmol),
40 665 ꢁL of 5% ethanol solution; GGsTop® nonꢀadded group (control), 50 ꢁL of GSAMC
41 solution (141 nmol), 25 ꢁL of GlyGly solution (60 nmol), 25 ꢁL of threonine solution
42 (110 nmol), 25 ꢁL of methionine solution (46 nmol), 100 ꢁL of GPF, 675 ꢁL of 5%
43 ethanol solution. The tubes for each group were stored at 25°C and collected at fixed
44 time intervals (1 min, 15 min, 30 min, 1 h, 3 h, 5 h, and 24 h). IS solution (100 ꢁL of 0.5
45 mg/mL Sꢀ3ꢀbutenylcysteine in 20 mM HCl) and 100 ꢁL of methanol containing 10%
46 (v/v) formic acid were added to each tube. The mixture was shaken vigorously for
47 approximately 10 s and centrifuged at 15,000 rpm for 10 min. An aliquot of the
48 supernatant was filtered using a membrane filter (cutoff: 3.0 kDa), and the filtrate was
49 used as the sample solution. The quantitative analysis of SAMC was performed using an
50 Acquity ultra performance LC system as described in the section “Changes in SAMC
51 and GSAMC during the aging process”
52
53 Model reaction to produce GSAMC using γꢀglutamylꢀSꢀalkenylcysteines, alliin,
54 and GPF
55
56
γꢀGlutamylꢀSꢀalkenylcysteine was dissolved in water (GSAC, 479 nmol/mL;
57 GS1PC, 719 nmol/mL), and alliin was dissolved in water (1.34 ꢁmol/mL). Model
58 reaction mixtures were prepared in 1.5 mL plastic tubes with the following composition:
59 GSAC and alliin group, 100 ꢁL of GSAC solution (47.9 nmol), 100 ꢁL of alliin solution
60 (134 nmol), 500 ꢁL of GPF; GS1PC and alliin group, 100 ꢁL of GS1PC solution (71.9
61 nmol), 100 ꢁL of alliin solution (134 nmol), 500 ꢁL of GPF. The tubes for each group
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2
62 were stored at 25°C and collected at approximately 5 s and 1 min. At collection, 200 ꢁL
63 of methanol containing 10% (v/v) formic acid was added, immediately. The mixture
64 was shaken vigorously for approximately 10 s and then centrifuged at 15,000 rpm for 10
65 min. An aliquot of the supernatant was filtered using a membrane filter (cutoff: 3.0 kDa),
66 and the filtrate was used as the sample solution. Quantitative analyses of GSAMC were
67 performed using an NEXERA X2 HPLC system (Shimadzu Corporation) under the
68 following conditions: column, Cadenza CDꢀC18 (2.0 mm × 75 mm, 3 ꢁm, Imtakt);
69 solvent A, 0.1% (v/v) formic acid; solvent B, 80% (v/v) methanol containing 0.1% (v/v)
70 formic acid; gradient program (%B), 0–5.0 min (0%), 5.0–25.0 min (0→30%),
71 25.0–35.0 min (30→100%), 35.0–45.0 min (100%), 45.0–45.01 min (100→0%, stop);
72 flow, 0.2 mL/min; detection, 220 nm; injection volume, 2.0 ꢁL.
73
74 Model reaction for GSAMC production using γꢀglutamylꢀSꢀalkenylcysteines and
75 allicin
76
77
γꢀGlutamylꢀSꢀalkenylcysteines were dissolved in water (GSAC, 479 nmol/mL;
78 GS1PC, 719 nmol/mL), and allicin was dissolved in water (1.92 ꢁmol/mL). Model
79 reaction mixtures were prepared in 1.5 mL plastic tubes with following composition:
80 GSAC and allicin group, 100 ꢁL of GSAC solution (47.9 nmol), 200 ꢁL of allicin
81 solution (384 nmol), 400 ꢁL of purified water; GS1PC and allicin group, 100 ꢁL of
82 GS1PC solution (71.9 nmol), 200 ꢁL of allicin solution (384 nmol), 400 ꢁL of purified
83 water. The tubes were stored at 25°C and collected at approximately 5 s, 1 day, and 3
84 days. To each tube, 200 ꢁL of methanol containing 10% (v/v) formic acid was added.
85 The mixtures were shaken vigorously for approximately 10 s and then centrifuged at
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86 15,000 rpm for 10 min. An aliquot of the supernatant was filtered using a membrane
87 filter (cutoff, 3.0 kDa), and the filtrate was used as the sample solution. Quantitative
88 analyses of GSAMC were performed using a LC as described in the section “Model
89 reaction for the production of GSAMC using γꢀglutamylꢀSꢀalkenylcysteines, alliin,
90 and GPF.”
91
92 Results
93
94 Isolation and identification of GSAMC in aged garlic extract, and changes of
95 SAMC and GSAMC during the aging process
96
97
GSAMC in AGE was separated and purified by preparative HPLC. The isolated
98 GSAMC was characterized using LCꢀHRMS and NMR and confirmed as GSAMC by
99 comparison with the data for synthesized GSAMC.
00
The changes in SAMC and GSAMC contents in AGE during the aging process
01 were examined. GSAMC content reached a maximum at 1 month and then decreased
02 during the subsequent aging process, whereas SAMC content reached a maximum after
03 approximately 4 months of aging process and then gradually decreased during the
04 subsequent aging process. The contents of GSAMC and SAMC in three different lots
05 tested showed similar changes during aging process (Figure 2, Supplement Figures 1
06 and 2).
07
08
Laboratory experiment to mimic aging of garlic extract using raw garlic and a
09 GGT inhibitor
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11
To examine whether the endogenous GGT activity in raw garlic is involved in
12 SAMC production, a laboratory experiment mimicking the industrial aging process was
13 set up using raw garlic and a GGT inhibitor. The SAMC content in the control group
14 increased during the test period. The SAMC content in GGT inhibitorꢀadded group
15 increased slightly until 10 days, and then did not change during the subsequent test
16 period. In the boiled group, the initial SAMC content was approximately five times
17 higher than that of the other groups and then gradually decreased during the test period
18 (Figure 3A). The GSAMC contents in the control and the GGT inhibitorꢀadded groups
19 increased, and the amounts in both groups were similar during the subsequent test
20 period. Although GSAMC was not detected in raw garlic, it was detected in the boiled
21 group at the first time point, and the amount did not change during the rest of the test
22 period (Figure 3B). The amount of SAMC produced in control group was
23 approximately 10% of the GSAMC produced during the test period.
24
25 Model reactions for SAMC production using GPF and a GGT inhibitor
26
27
To confirm the production of SAMC from GSAMC by endogenous GGT in garlic,
28 we examined a model reaction using synthesized GSAMC and GPF. SAMC production
29 was observed in the control group at 15 min of incubation, and then increased during
30 subsequent incubation, but was slightly decreased at 24 h compared to the amount at 5 h.
31 SAMC production was also observed in the GGT inhibitorꢀadded group. However, the
32 amount produced was much less than that in the control group, and SAMC contents
33 were reduced to trace amounts by 24 h (Figure 4).
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35 Model reactions for GSAMC production using γꢀglutamylꢀSꢀalkenylcysteines, alliin,
36 GPF, and allicin
37
38
In the model reactions using γꢀglutamylꢀSꢀalkenylcysteines, alliin, and GPF,
39 GSAMC was not detected in the GSAC/alliin group, but it was detected in the
40 GS1PC/alliin group (Figure 5A). The amount of GSAMC produced in the GS1PC/alliin
41 group at 5 s and 1 min did not differ significantly (Student’s tꢀtest, P = 0.39). GSAMC
42 production was observed in the GSAC/allicin group on days 1 and 3 but was not
43 observed at 5 s. GSAMC was detected in the GS1PC/allicin group even at 5 s, and the
44 content increased during subsequent incubation (Figure 5B). The amounts of GSAMC
45 produced at 5 s in the GS1PC/alliin (Figure 5A) and GS1PC/allicin (Figure 5B) groups
46 did not differ significantly (Student’s tꢀtest, P = 0.34).
47
48 Discussion
49
50
SAMC is formed in AGE as a characteristic hydrophilic compound, and its
1
–3
51 biological properties as an active component in AGE have been evaluated. However,
52 this compound is not detected in fresh whole garlic, and only a trace amount is found
1
53 when garlic is crushed (approximately 0.01 ꢁmol/g fresh garlic). This compound can
54 be chemically synthesized by the reaction between cysteine and allicin that proceeds
1
, 10
55 very rapidly even at room temperature.
This reaction is thought to occur during the
56 processing of garlic and to be a major SAMCꢀproducing mechanism in garlic
1
57 preparations. Alliin, a precursor of allicin is a substrate for SAMC production, and is
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58 abundant in fresh garlic as a sulfur storage molecule (28ꢀ79 ꢁmol/g fresh garlic). One
59 molecule of allicin is produced from two molecules of alliin when garlic is processed by
1
60 crushing, slicing, mincing, and soaking in aqueous alcoholic solution. However,
61 cysteine, the other substrate for SAMC production, is not detectable in fresh whole
1
62 garlic or crushed garlic. Based on these differences in the amount of raw materials, we
63 hypothesized other reactions for SAMC production. One possible reaction for SAMC
1
1
64 production is the one between SAC/S1PC and DADS. However, this reaction
11
65 proceeded very slowly under the condition to prepare AGE. Another reaction involves
66 GSAMC, a putative precursor of SAMC, but it is not detectable in raw garlic. However,
67 Matsutomo et al. recently demonstrated the presence of GSAMC in AGE using
11
68 postꢀcolumn HPLC with a sulfurꢀspecific detection reagent and LCꢀHRMS. This
69 finding led us to hypothesize that GSAMC is produced from GSAC/GS1PC in the early
70 stage of the aging process, and that SAMC is produced from GSAMC by endogenous
71 GGT.
72
In the present study, we first confirmed that GSAMC was not detectable in fresh
73 garlic, but was present in AGE. The GSAMC content in aged garlic extract reached
74 maximum levels within one month and then gradually decreased (Figure 2). The SAMC
75 content increased while GSAMC decreased, and reached maximum levels at 4 months
76 and then gradually decreased during the subsequent aging process (Figure 2). These
77 results indicated that most of the GSAMC and SAMC were produced during the early
78 stage of the aging process. Since the increase in SAMC was related to the decrease in
79 GSAMC, we used the experimental aging reactions with raw garlic and a GGT inhibitor
80 to test our hypothesis for SAMC production. GSAMC production was observed after 5
81 days of incubation, and GSAMC content increased in both the control and GGT
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82 inhibitorꢀadded groups, while SAMC content increased in the control group but not in
83 the GGT inhibitorꢀadded group (Figure 3). Further, GPF which had GGT activity,
84 produced SAMC from synthesized GSAMC, while the presence of GGT inhibitor
85 suppressed SAMC production (Figure 4). These results strongly suggest that GGT in
86 raw garlic is involved in SAMC production, namely that SAMC was produced from
87 GSAMC by GGT during the aging process. Since GSAMC is not detectable in raw
1
88 garlic, we examined GSAMC production in model reactions using GSAC, GS1PC,
89 alliin, allicin and GPF: GPF also contained the CꢀS lyase alliinase (the data not shown),
90 which lyses alliin to produce 2ꢀpropenesulfenic acid (2ꢀPSA), that is rapidly condensed
1
91 to allicin (Reaction A in Figure 6). GSAMC production was observed at 5 s in both the
92 GS1PC/alliin/GPF and GS1PC/allicin model reaction groups, and there was no
93 significant difference in the amounts of GSAMC produced between these two groups
94 (Student’s tꢀtest, P = 0.34). GSAMC production was observed on day 1 and day 3 in the
95 GSAC/allicin reaction, while production of GSAMC was not observed in the
96 GSAC/alliin/GPF reaction group. These results suggested that 2ꢀPSA does not react
97 with GSAC/GS1PC (Reaction B in Figure 6), although 2ꢀPSA is a very unstable
1
98 compound, and that allicin reacts with GSAC/GS1PC after very rapid condensation of
99 2ꢀPSA to allicin. According to our observations, we postulate that SAMC is produced
00 during the aging process in the following manner: first, when garlic is soaked in to
01 aqueous alcoholic solution, alliin is transformed to allicin by alliinase (Reaction A in
02 Figure 6). Next, GSAMC is produced by the reaction between allicin and
03 GSAC/GS1PC, and then SAMC is produced from GSAMC by GGT (Reaction C in
04 Figure 6). The changes in GSAMC and SAMC content in aged garlic indicate that these
05 reactions occur in the early stage of aging, i.e., within a few months (Figure 2).
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06
Previously, Matsutomo et al. showed that SAMC was produced by the reaction
07 between SAC/S1PC and DADS using a model reaction approach. SAMC production in
08 these model reactions was temperature dependent, e.g., the amount of SAMC produced
09 at 25°C was approximately half of that produced at 40°C and was one sixth of that
11
10 produced at 60°C. The DADS content in aged garlic during the early stage of the
11 aging process was < 1.2 ꢁmol/gꢀdry (unpublished data). Since the aging process
12 normally proceeds at room temperature, the contribution of this reaction involving
13 DADS (Reactions D and E in Figure 6) towards total SAMC production seems to be
14 less than that in the Reaction C pathway in Figure 6 during the early stage of the aging
15 process, although it may occur during the late stage of the aging process. The alternative
16 mechanisms of SAMC production, i.e. the reaction between ajoene and cysteine as well
17 as the reaction between diallyltrisulfide (DATS) and cysteine, were presented in the
1
18 previous report. However, the contribution of the reaction between ajoene and cysteine
19 may be small for SAMC formation in AGE because ajoene is hardly produced in a
1
20 highꢀpolarity solvent being used for AGE production. Indeed, ajoene content in AGE is
12
21 reported to be approximately 0.04 ꢁmol/g. In addition, production of DATS proceeds
1
22 slowly in water at room temperture and thus, contribution of this compound for SAMC
23 formation may be also small.
24
In conclusion, our study suggests that GSAMC and SAMC in AGE are produced
25 through the combination of chemical and enzymatic reactions during the aging process.
26 Both hydrophilic and hydrophobic compounds such as alliin, GSAC, GS1PC and allicin
27 may serve as the substrates. These results provide the important insight to help
28 understanding of the complex sulfur chemistry in AGE and during the aging process.
29
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30 Reference
31 1. The Science and Therapeutic Application of Allium Sativum L and Related Species,
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H. P. Koch and L. D. Lawson, Eds., Williams & Wilkins, Baltimore, Md, USA,
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34 2. 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.
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36 3. Amagase H. Clarifying the real bioactive constituents of garlic. J. Nutr. 2006, 136
(supplement 3), 716S–725S.
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38 4. Kodera Y.; Ushijima M.; Amano H.; Suzuki JI.; Matsutomo T. Chemical and
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40
Biological Properties of Sꢀ1ꢀPropenylꢀlꢀCysteine in Aged Garlic Extract. Molecules,
2017, 22(4), pii: E570. doi: 10.3390/molecules22040570
41 5. Nakamoto, M.; Fjii, T.; Matsutomo, T.; Kodera, Y. Isolation and identification of
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three γꢀglutamyl tripeptides and their putative production mechanism in aged garlic
extract. J. Agric. Food Chem. 2018, 66, 2891–2899.
44 6. Sumioka, I.; Matsura, T.; Yamada, K. Therapeutic effect of Sꢀallylmercaptocysteine
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46
on acetaminophenꢀinduced liver injury in mice. Eur. J. Pharmacol. 2001, 433 (2–3),
177–185.
47 7. Shirin, H.; Pinto, J. T.; Kawabata, Y.; Soh, J.W.; Delohery, T.; Moss, S.F.; Murty, V.;
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Rivlin, R. S.; Holt, P. R. Weinstein, I. B. Antiproliferative effects of
Sꢀallylmercaptocysteine on colon cancer cells when tested alone or in combination
with sulindac sulfide. Cancer Res. 2001, 61(2), 725–731.
51 8. Howard, E. W.; Ling, M. T.; Chua, C. W.; Cheung, H. W.; Wang, X.; Wong, Y. C.
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53
Garlicꢀderived Sꢀallylmercaptocysteine is a novel in vivo antimetastatic agent for
androgenꢀ independent prostate cancer. Clin. Cancer Res. 2007,13(6), 1847–56.
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G. O. The Effect of Garlic Derivatives (SꢀAllylmercaptocysteine, Diallyl Disulfide,
and SꢀAllylcysteine) on Gentamicin Induced Ototoxicity: An Experimental Study.
Clin. Exp. Otorhinolaryngol. 2016, 9(4), 309–313.
58 10. Albrecht F.; Leontiev R.; Jacob, Claus.; Slusarenko, A. J. An optimized facile
procedure to synthesize and purify allicin. molecules. 2017, 22, 770.
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60 11. 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 postꢀcolumn high performance
liquid chromatography method with sulfur–specific detection. J. Nutr. 2016,
146(Suppl), 450S–455S.
64 12. Kodera, Y.; Matsuura, H.; Sumiyoshi, H.; Sumi S. Garlic Chemistry: Chemical and
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biological properties of sulfurꢀcontaining compounds derived from garlic. In Food
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Society: Washington, DC, 2003; pp 346–357.
69 13. Ryu K.; Ide N.; Matsuura H.: Itakura Y. N
α
ꢀ(1ꢀDeoxyꢀDꢀfructosꢀ1ꢀyl) –Lꢀarginine,
an antioxidant compound identified in aged garlic extract. J Nutr. 2001, 131,
972S–976S.
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71
72 14. Jayathilaka L.; Gupta S.; Huang JS.; Lee J.; Lee BS. Preparation of (+)ꢀtransꢀ
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isoalliin and its isomers by chemical synthesis and RP–HPLC resolution. J Biomol
Tech. 2014, 25(3), 67–76.
75 15. Carson JF, Wong FF. Synthesis of cisꢀSꢀpropꢀ1ꢀenylꢀLꢀcysteine. Chem Ind. 1963, 2,
764–776.
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77 16. King FE, Kidd DAA. A new synthesis of glutamine and of γꢀdipeptides of
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glutamic acid from phthalylated intermediates. J Chem Soc. 1949, 3315–3319.
80 Acknowledgment
81 The authors deeply thank Dr. Takami Oka of Wakunaga Pharmaceutical Co., Ltd, for his
82 helpful advice, encouragement and critical reading of the manuscript.
83
84 Conflict of interest
85 The authors declare no conflict of interests.
86
87 Author Contributions
88 All authors worked equally on the preparation and editing of the MS.
89
90 Abbreviations used
91 AGE, aged garlic extract; SMC, Sꢀmethylcysteine; SAC, Sꢀallylcysteine; S1PC,
92 Sꢀ1ꢀpropenylcysteine; SAMC, Sꢀallylmercaptocysteine; GSAC, γꢀglutamylꢀSꢀallylꢀ
93 cysteine; GS1PC, γꢀglutamylꢀSꢀ1ꢀpropenylcysteine; GSAMC, γꢀglutamylꢀSꢀ
94 allylmercaptocysteine; DADS, diallyldisulfide; DATS, diallyltrisulfide; 2ꢀPSA,
95 2ꢀpropensulfenic acid; GGT, γꢀglutamyl transpeptidase; GPF, garlic protein fraction
96
97
98
99
00
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02 Figure Captions
03
04 Figure 1 Chemical structures of sulfur compounds observed in raw garlic and aged
05 garlic extract.
5
06
5
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07 Figure 2
Changes of Sꢀallymercaptocysteine and γꢀglutamylꢀSꢀallylmercaptoꢀ
08 cystine in aged garlic extract during aging period. ꢁmol/gꢀdry: content in dryꢀAGE.
09
10 Figure 3
Changes of Sꢀallylmercaptocysteine and γꢀglutamylꢀSꢀallylmercaptoꢀ
11 cysteine in the model reaction mixtures. Changes in concentration of Sꢀallylmercaptoꢀ
12 cysteine (A), and γꢀglutamylꢀSꢀallylmercaptocysteine (B) at 25ºC, respectively. GGsTop
13 (Wako Pure Chemical Industry) was used as GGT inhibitor. Values are mean ± SD, n =
14 3. ꢁmol/gꢀwet: content in fresh weight of garlic.
15
16 Figure 4 Production of Sꢀallylmercaptocysteine in model reaction. Garlic protein
17 fraction (GPF) was added to the mixture of γꢀglutamylꢀSꢀallylmercaptocysteine
18 with/without GGsTop, and reaction was stopped at fixed interval. GGsTop (Wako Pure
19 Chemical Industry) was used as GGT inhibitor. (ꢀ) GGT inhibitor, control group: (+)
20 GGT inhibitor, GGT inhibitor added group. GPF was used as a transpeptidase having
21 GGT activity. Values are mean ± SD, n = 3.
22
23 Figure 5
Production of γꢀglutamylꢀSꢀallylmercaptocysteine in model reaction.
24 Alliinase solution was added to the mixture of γꢀglutamylꢀSꢀalkenylcysteines (GSAC or
25 GS1PC) and alliin, and reaction was stopped within five seconds, or at 1minute after
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26 addition of GPF (A), and allicin solution was added to the solution of
27 γꢀgultamylꢀSꢀalkenylcysteines (GSAC or GS1PC), and reaction was stopped within 5
28 seconds, at one day, or at 3 days after addition of allicin (B). GPF was used as a
29 CSꢀlyase alliinase to produce allicin from alliin. Values are mean ± SD, n = 3.
30
31 Figure 6 Plausible reaction pathway on Sꢀallylmercaptocysteine production in aged
32 garlic extract during aging process. 2PSA, 2ꢀPropenesulfenic acid; DADS,
33 Diallyldisulfide; GSAC, γꢀGlutamylꢀSꢀallylcysteine; GS1PC, γꢀGlutamylꢀSꢀ1ꢀ
34 propenylcysteine; SAC, SꢀAllylcysteine; S1PC, Sꢀ1ꢀPropenylcysteine; SAMC, SꢀAllylꢀ
35 mercaptocysteine; GSAMC, γꢀGlutamylꢀSꢀallylmercaptocysteine; GGT, γꢀGlutamyl
36 transpeptidase.
5
37
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50 Figures
51
52 Figure 1
53
O
S
COOH
Alliin
COOH
S
COOH
S
H N
H N
H N
2
2
2
S
-Allylcysteine (SAC)
S
-1-Propenylcysteine (S1PC)
O
COOH
S
COOH
H N
2
S
N
H N
S
H
2
COOH
γ
-Glutamyl-S-allylcysteine (GSAC)
S-Allylmercaptocysteine (SAMC)
O
COOH
O
1‘ COOH
5
‘
H N
S
H N
2
4
3
‘
S
2
2
3
5
2‘
4‘
6‘
N
N
S
H
H
COOH
1
COOH
γ-Glutamyl-
S-1-propenylcysteine (GS1PC)
γ
-Glutamyl-
S
-allylmercaptocysteine (GSAMC)
O
S
S
S
OH
S
S
2-Propenesulfenic acid
Diallyldisulfide (DADS)
Allicin
5
5
5
5
5
5
54
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58
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60 Figure 2
61
62
1
0
SAMC in Lot 1
8
6
4
2
0
SAMC in Lot 2
SAMC in Lot 3
GSAMC in Lot 1
GSAMC in Lot 2
GSAMC in Lot 3
0
5
10
15
20
25
Time (month)
5
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5
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73 Figure 3
74
(
A)
0
0
0
0
0
0
.5
.4
.3
.2
.1
.0
Control
Add GGT inhibitor
Heat treatment
0
10
20
30
40
50
60
70
Time (day)
(
B)
5
Control
Add GGT inhibitor
Heat treatment
4
3
2
1
0
0
10
20
30
40
50
60
70
Time (day)
5
5
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78 Figure 4
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4
3
3
22
1
1
0
0
0
1
15 30
min)
1
3
(hr)
5
24
0
1
3
(hr)
5
24
(
(
-) GGT inhibitor (control)
(+) GGT inhibitor
5
5
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92
(
A)
12
10
8
6
4
2
0
5
sec.
1 min.
5
sec.
1 min.
GSAC/Alliin + GPF
GS1PC/Alliin + GPF
(
B)
12
1
0
8
6
4
2
0
5
sec.
1 day
3 days
5 sec.
1 day
3 days
GSAC + Allicin
GS1PC + Allicin
5
5
5
5
5
93
94
95
96
97
ACS Paragon Plus Environment

Page 29 of 30
Journal of Agricultural and Food Chemistry
5
5
98 Figure 6
99
Reaction A
Alliinase
Alliin
2PSA
Allicin
DADS
Reaction B
GGT
GGT
GSAC
GS1PC
2PSA
Allicin
GSAMC
SAMC
Reaction C
GSAC
GS1PC
GSAMC
GSAMC
SAMC
Reaction D
GGT
DADS
GGT
GSAC
GS1PC
SAMC
SAMC
2
PSA or
Allicin or
DADS
Reaction E
GSAC
GS1PC
SAC
S1PC
6
6
6
6
6
00
01
02
03
04
ACS Paragon Plus Environment