A tissue homogenate method to prepare gram-scale allium thiosulfinates and their disulfide conjugates with cysteine and glutathione

Article abstract of DOI:10.1021/jf4003818

The health benefits of Allium vegetables are widely attributed to the enzyme-derived organosulfur compounds called thiosulfinates (TS). However, the lack of a suitable method to prepare TS in good yields has hampered the evaluation of their biological activities. This paper describe a simple enzymatic method using Allium tissue homogenates as a reaction system to prepare gram-scale TS, including those enriched in 1-propenyl groups, which are particularly difficult to obtain. This method is simple, easy to scale up, and requires no column purification step, making it suitable for practical large-scale production of Allium TS. The prepared TS were further utilized to prepare the disulfide conjugates with cysteine and glutathione (CySSR and GSSR, R = methyl, ethyl, propyl, 1-propenyl, and allyl), which are the presumptive metabolites of TS. Among all of the Allium CySSR and GSSR conjugates, the newly prepared glutathione conjugate with 1-propenyl TS, GSSPe, showed the most potent effect to induce quinone reductase (QR, a representative phase II enzyme) in murine hepatoma cells (Hepa 1c1c7) and inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophage cells (RAW 264.7).

Full text of DOI:10.1021/jf4003818

Article
pubs.acs.org/JAFC
A Tissue Homogenate Method To Prepare Gram-Scale Allium
Thiosulfinates and Their Disulfide Conjugates with Cysteine and
Glutathione
Guodong Zhang* and Kirk L. Parkin*
,†
Department of Food Science, Babcock Hall, University of WisconsinMadison, 1605 Linden Drive, Madison, Wisconsin 53706,
United States
*
S Supporting Information
ABSTRACT: The health benefits of Allium vegetables are widely attributed to the enzyme-derived organosulfur compounds
called thiosulfinates (TS). However, the lack of a suitable method to prepare TS in good yields has hampered the evaluation of
their biological activities. This paper describe a simple enzymatic method using Allium tissue homogenates as a reaction system to
prepare gram-scale TS, including those enriched in 1-propenyl groups, which are particularly difficult to obtain. This method is
simple, easy to scale up, and requires no column purification step, making it suitable for practical large-scale production of Allium
TS. The prepared TS were further utilized to prepare the disulfide conjugates with cysteine and glutathione (CySSR and GSSR,
R = methyl, ethyl, propyl, 1-propenyl, and allyl), which are the presumptive metabolites of TS. Among all of the Allium CySSR
and GSSR conjugates, the newly prepared glutathione conjugate with 1-propenyl TS, GSSPe, showed the most potent effect to
induce quinone reductase (QR, a representative phase II enzyme) in murine hepatoma cells (Hepa 1c1c7) and inhibit nitric
oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophage cells (RAW 264.7).
KEYWORDS: Allium, thiosulfinate, alliinase, thiosulfinate conjugates, S-1-propenylmercaptocysteine (CySSPe),
S-1-propenylmercaptoglutathione (GSSPe)
INTRODUCTION
Chemical synthesis of TS is difficult; in particular, the 1-
■
propenyl TS, which are the major TS species in onions, are very
Epidemiological studies support that a diet rich in Allium
1
1,12
challenging to prepare.
An alliinase-based enzymatic
vegetables is correlated with reduced risks of many human
1
,2
method is very attractive; however, it can generate only
semipreparative quantities due to the very low yields of this
diseases. The health benefits of Allium vegetables are widely
attributed to the alliinase-transformed organosulfur compounds
called thiosulfinates (TS) and related organosulfur species
13,14
enzymatic reaction.
The reasons for the low reaction yields
include (1) the enzymatic reaction reportedly stopping after a
limited number of cycles because alliinase is inactivated by its
(
1
chemical structures of representative TS are shown in Figure
). For example, allicin, which is a major TS derived from
garlic, has been reported to have a variety of health-promoting
effects including anti-inflammation, up-regulation of phase II
enzymes, antiproliferation and induction of apoptosis,
cardioprotection via relaxation of pulmonary arteries and
3
14
own product TS; (2) the instability of both alliinase and its
product TS; and (3) the major impurities in the synthetic
alliinase substrates, S-alk(en)yl-L-cysteines (CySR), are inhib-
15
itors of alliinase. Recently an immobilized alliinase method
was reported to continuously produce TS via separation of the
newly generated TS from alliinase enzyme; however, the
3
inhibition of platelet aggregation, and antibacterial activity.
TS and related organosulfur compounds are unstable in vivo
and are conjugated with cysteine (Cys) and glutathione (GSH)
to generate putative metabolites named S-alk(en)ylmercapto-
cysteine (CySSR) and S-alk(en)ylmercaptoglutathione
GSSR), respectively (chemical structures of CySSR and
GSSR are shown in Figure 1).
metabolites of TS are widely believed to contribute to the
health-promoting effects of Allium vegetables. For example, S-
allylmercaptocysteine (CySSA), which is the cysteine disulfide
conjugate with allicin, has been shown to exert potent
anticancer activities in vitro and in vivo and has been marketed
14
procedure was complicated and time-consuming. Until now
there is no report of the use of alliinase enzyme for large-scale
preparation of TS.
For preparation of CySSR and GSSR, in our previous study
we have reported the chemical synthesis of non-1-propenyl
conjugates CySSR and GSSR (R = methyl, ethyl, propyl, and
(
4
−7
These presumptive
16
7
allyl). However, this multistep organic synthesis method was
difficult to carry out and cannot prepare the disulfide conjugate
of 1-propenyl TS (CySSPe and GSSPe, the chemical structures
are shown in Figure 1). The 1-propenyl TS are the major TS
3
8
−10
species in some Allium vegetables such as onions and have
as a dietary supplement for a decade.
Having a simple and reliable method to prepare TS in good
yields would help to investigate their biological activities and
potential therapeutic applications. However, until now there is
no satisfactory method to prepare TS. Direct solvent extraction
of crushed Allium tissues generates a complex mixture.
Received: December 18, 2012
Revised: March 4, 2013
Accepted: March 6, 2013
Published: March 6, 2013
©
2013 American Chemical Society
3030
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Journal of Agricultural and Food Chemistry
Article
Figure 1. Chemical structures of TS, CySSR, and GSSR in this study.
been shown to have the most potent phase II enzyme inducing
ethyl acetate (EtOAc) twice. Three to four grams of synthetic ACSO
17
(
R = methyl, ethyl, propyl, and allyl) dissolved in 10 mL water was
added into the resulting aqueous layer with a total reaction volume of
100 mL. The reaction mixture was incubated at room temperature
activities among major Allium TS species. Therefore, the
biological activities of CySSPe and GSSPe may be critical to our
understanding of the health benefits of onion and other Allium
vegetables.
In this paper, we describe a simple enzymatic method using
Allium tissue homogenates to prepare gram-scale TS, including
those enriched in 1-propenyl groups, which are particularly
difficult to obtain. The prepared TS were further utilized to
prepare CySSR and GSSR (R = methyl, ethyl, propyl, and allyl)
via a one-step process without any chromatography purification
process and CySSPe and GSSPe via a one-step process with
HPLC purification.
∼
for 40−60 min, and the formed TS were then extracted with an equal
amount of CH Cl . The CH Cl layer was dried over anhydrous
2
2
2
2
MgSO and evaporated to dryness under a gentle nitrogen stream at
4
2
0−22 °C to obtain the final product. The dried TS were used directly
for HPLC analysis or preparation of CySSR and GSSR without any
purification.
To check the purity of the prepared TS, the CH Cl extract was
2
2
analyzed on a 250 × 4.6 mm, 5 μm, silica gel column (Supelco,
Bellefonte, PA, USA) eluted with a gradient of 2-propanol in hexane
from 2:98 (v/v) held for 6 min to 10:90 for the next 10 min followed
by a 9 min hold, with a flow rate of 1.8 mL/min and the detector set at
1
8
MATERIALS AND METHODS
254 nm. The structures of the prepared TS were confirmed by NMR
■
1
and MS analysis; H NMR data of TS were reported in our previous
Materials and General Experimental Procedures. Chemicals
were obtained from Sigma-Aldrich (Milwaukee, WI, USA), and
solvents for extraction or HPLC analysis were purchased from Fisher
Scientific (Fair Lawn, NJ, USA) unless otherwise noted. (±)-S-
Alk(en)yl-L-cysteine sulfoxides (ACSO), including MCSO, ECSO,
PCSO, and 2-PeCSO, were prepared as described in our earlier
1
3
study.
Tissue Reaction To Enrich 1-Propenyl TS. White onions (200
g) were cut into two pieces for each onion bulb and heated in 100 mL
of boiling water for 4−5 min. After the heat-inactivated onion tissues
were cooled, they were homogenized with 10 g of peeled fresh garlic
for 1−2 min at 4 °C. The homogenized mixture was incubated at
room temperature for 40−60 min and filtered through a cheese cloth;
13
paper. Cell culture medium and supplements were from Invitrogen
Carlsbad, CA, USA). Garlic and onion were purchased from a local
(
the filtrate was extracted with 100 mL of CH Cl . The CH Cl extract
retailer in Madison, WI, USA. NMR data were collected on Varian
Unity-Inova 400 and 500 MHz NMR spectrometers (Analytical
Instrumentation Center, School of Pharmacy, UWMadison). High-
resolution ESI-MS was collected on an Agilent ESI-TOF mass
spectrometer (Mass Spectrometry/Proteomics Facility, Biotechnology
Center, UWMadison).
2
2
2
2
was dried over anhydrous MgSO and evaporated to dryness under a
4
gentle nitrogen stream at room temperature. HPLC analysis was
carried out using the conditions as described above.
Preparation of Non-1-propenyl CySSR and GSSR (R =
Methyl, Ethyl, Propyl, and Allyl). The prepared TS were
reconstituted in water, 1.5−2.0 M Cys or GSH dissolved in water
was added (1 M TS reacts with 2 M Cys or GSH, thus, extra TS were
used in the reaction), and the reaction mixture was stirred at room
temperature for 2 h. After the reaction, the reaction product was dried
using a rotary evaporator under vacuum or a freeze-dryer, and the
Preparation of Gram-Scale Non-1-propenyl TS. Peeled garlic
(
100 g) in 20−90 mL of water was homogenized using a kitchen
blender for 1−2 min at 4 °C, and the garlic homogenate was then
incubated at 20−22 °C for 30 min−1 h and filtered through a
cheesecloth; the filtrate was rapidly extracted with equal amounts of
3
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dried reaction mixture was then washed with CH Cl to remove
blots were incubated with horseradish peroxidase-conjugated secon-
dary antibody for 1 h at 20−22 °C and washed three times with TBST
buffer. The secondary antibody on the blot was detected by a
chemiluminescence reagent (Pierce, Rockford, IL, USA) coupled to
autoradiography using Kodak BioMax film.
Statistics. Results are expressed as the mean ± SD of at least three
separate experiments, each with three to four replicates for each
condition assessed. Group comparisons were carried out using
Student’s t test. P values of <0.05 were considered to be statistically
significant.
2
2
1
residual TS; no purification step was required. The H NMR data of
1
6
CySSR and GSSR were reported in our previous study.
Preparation of CySSPe and GSSPe. The above prepared 1-
propenyl-contaning TS were reconstituted in water, 1.5−2.0 M Cys or
GSH dissolved in water was added, and the reaction mixture was
stirred at room temperature for 1−2 h. The reaction mixture was
purified by HPLC to obtain pure CySSPe and GSSPe using a 250 × 10
mm C18 HPLC column (Phenomenex, Torrance, CA, USA) eluted
with 35% methanol in water with 0.5% acetic acid, flow rate of 3.2 mL/
min, and detection at 254 nm.
The structure of CySSPe was confirmed by comparing its NMR and
RESULTS AND DISCUSSION
Preparation of Gram-Scale Non-1-propenyl TS (RSS-
O)R, R = Methyl, Ethyl, Propyl, and Allyl). To achieve
■
(
19
1
MS data with a previous paper. H NMR of CySSPe (D O, 500
2
MHz): δ 4.35 (dd, J = 4.0, 8.0 Hz, 1H, H-2), 3.37 (dd, J = 4.0, 15.5 Hz,
1
H, H-3α), 3.21 (dd, J = 8.0, 15.0 Hz, 1H, H-3β), 6.17 (m, 2H, H-4
+
gram-scale preparation of TS using alliinase enzyme, we
developed a simple method for large-scale preparation of
crude alliinase. After garlic tissues were homogenized and
incubated to allow the conversion of endogenous garlic ACSO
to TS, the garlic homogenate was washed with an organic
solvent such as EtOAc. After EtOAc washing, alliinase enzyme
in the resulting aqueous layer was found to be still active.
Addition of gram-scale synthetic (±)-ACSO (MCSO, ECSO,
PCSO, or 2-PeCSO) into the aqueous layer generated the
corresponding pure TS after 30 min−1 h of incubation at room
temperature with a yield of 42−99% (Table 1 shows the yields
and H-5), 1.81 (m, 3H, H-6). HRESIMS, m/z, [M + H] at 194.0308
−
(
calcd 194.0309), [M − H] at 192.0166 (calcd 192.0153).
The structure of GSSPe was confirmed by a series of 1D and 2D
1
13
1
NMR, including H, C, COESY, HMQC, and HMBC. H NMR of
GSSPe (D O, 500 MHz): δ 3.82 (tr, J = 6.5 Hz, 1H, H-2), 2.19 (qr, J =
2
7
.5 Hz, 1H, H-3α), 2.20 (qr, J = 7.5 Hz, 1H, H-3β), 2.56 (tr, J = 7.5
Hz, 1H, H-4α), 2.58 (tr, J = 7.5 Hz, 1H, H-4β), 4.72 (dd, J = 4.5, 9.5
Hz, 1H, H-6), 3.01 (dd, J = 9.5, 14.5 Hz, 1H, H-7α), 3.27 (dd, J = 4.5,
1
1
(
5
4.5 Hz, 1H, H-7β), 3.97 (s, 2H, H-9), 6.12 (m, 2H, H-11 and H-12),
.78 (br d, J = 5.0 Hz, 3H, H-13). 13C NMR (D O, 125 MHz): δ 17.7
2
C-13), 26.4 (C-3), 31.6 (C-4), 38.4 (C-7), 42.0 (C-9), 53.0 (C-6),
4.2 (C-2), 123.5 (C-12), 133.5 (C-11), 172.9−175.1 (C-1, C-5, C-8,
+
and C-10). HRESIMS, m/z [M + H] at 380.0933 (calcd 380.0950),
−
[
M − H] at 378.0802 (calcd 378.0794).
Table 1. Yields and Scales of Reactions To Convert ACSO to
TS
Quinone Reductase (QR) Induction Assay. A bioassay based on
Hepa 1c1c7 cells (ATCC, Rockville, MD, USA) was used to assess QR
20
3
induction essentially as described earlier. Hepa 1c1c7 cells (5 × 10
cells/well) were plated in 96-well plates in 200 μL of MEM
supplemented with 10% fetal bovine serum and antibiotics (100
units/mL penicillin and 100 μg/mL streptomycin) at 37 °C in 5%
CO in air and allowed to attach for 24 h. The medium was then
2
replaced with fresh MEM containing test compounds for 48 h. A
standard assay cocktail was prepared, and the QR activity was
determined by measuring the absorbance of the reduced tetrazolium
dye over a 10 min period at 490 nm. The cell viability was determined
by crystal violet assay at 610 nm. The degree of QR induction was
calculated as the ratio of QR activity in the treated (induced) sample
relative to the untreated control sample. The concentration required
for doubling the specific activity of QR relative to nontreated control
cells was used as an indicator of inducer potency, expressed as CD
values.
Nitric Oxide (NO) Assay. Mouse macrophage RAW 264.7 cells
ATCC) were cultured in Dulbecco’s Modified Eagle’s Medium
DMEM, Invitrogen) supplemented with 15% fetal bovine serum with
(
(
antibiotics (a mixture of penicillin and streptomycin) at 37 °C in 5%
5
CO in a 96-well plate (5 × 10 cells/well) for 24 h. The medium was
2
then replaced with fresh medium containing test compounds and 1
μg/mL LPS (Sigma-Aldrich, St. Louis, MO, USA). After 24 h of
treatment, a 100 μL sample was taken from the culture supernatant of
each well and mixed with an equal amount of freshly prepared Griess
reagent and incubated for 10 min, and then the optical density was
measured at 542 nm. The cell viability was measured by a MTT (3-
and scales of the reactions). The yields are calculated on the
basis of the dry weight of the final product (note volatile losses
of TS during solvent evaporation). A relative yield of 2-PeCSO
(yield = 99%) > ECSO (yield = 67%) ≈ ECSO (yield = 65%) >
MCSO (yield = 42%) was obtained (Table 1), consistent with
(
5
4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay at
50 nm. IC50 values for NO inhibition were determined using XLfit
21
software (IDBS, Surrey, UK).
13
Western Blot. Hepa 1c1c7 and RAW 264.7 cells were plated in 6-
well plates and allowed to attach for 24 h and then treated with test
compounds for 24−48 h. Cell protein samples (50 μg) were resolved
using 10% SDS−polyacrylamide gel electrophoresis and transferred
onto a nitrocellulose membrane. The nitrocellulose membrane was
blocked with 5% skim milk solution in Tris-buffered saline Tween-20
the substrate selectivity of alliinase enzyme. The almost
complete conversion of (±)-2-PeCSO to allicin (yield = 99%)
indicated the slow and recalcitrant (−)-2-PeCSO in the
synthetic substrate was also converted by the crude alliinase.
Without any purification, the prepared TS were found be to
essentially without impurity as assessed by HPLC (Figure 2)
(
TBST) for 1 h and then incubated with primary antibodies against
1
and H NMR analysis (data not shown). The structures of the
inducible nitric oxide synthase (iNOS) and QR (Santa Cruz
Biotechnology, Santa Cruz, CA, USA) in 3% skim milk in TBST
buffer at 4 °C overnight. After three washings with TBST buffer, the
prepared TS were confirmed by comparing the NMR data with
13
our previous results.
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Figure 2. HPLC analysis of TS prepared by the washed garlic homogenate method.
Figure 3. (A) HPLC analysis of organosulfur profile of crushed fresh onions (top panel), fresh garlic (middle panel), and the blend of boiled onion
and fresh garlic (bottom panel). Tissue blending increased the proportion of 1-propenyl TS (peaks 3, 4, and 8) without scarifying the total yield.
Peaks: 1, PTSO; 2, benzyl alcohol as internal standard; 3, AllSS(O)Pe; 4, AllS(O)SPe; 5, AllSS(O)All (allicin); 6, MeSS(O)All; 7, MeS(O)SAll; 8,
MeS(O)SPe. (B) Proposed mechanism for the enrichment of 1-propenyl TS. Two key points for the enrichment of 1-propenyl organosulfur
compounds are that (i) inactivation of PTSO synthase in onion (via heating) diminished the production of PTSO and (ii) the enzyme-generated 1-
Pe-SOH needs to be rapidly captured by other sulfenic acids; otherwise they would react with each other to form di-1-propenyl TS (PeSS(O)Pe),
which would rapidly rearrange to form cyclized sulfur compounds.
3
Alliinase is a major enzyme in Allium tissues; for example, it
homologous (RS(O)SR) or heterologous (RS(O)SR′) TS.
However, using purified alliinase enzyme, the yields of this
enzymatic reaction were low, and only semipreparative
22,23
comprises ∼10% of total protein in garlic.
It catalyzes the
carbon−sulfur (C−S) bond cleavage of ACSO to form sulfenic
1
3,14
acids (RSOH), which rapidly react with each other to generate
quantities of TS were obtained.
Here we have developed
3
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Figure 4. (A) HPLC analysis of reaction product of Cys or GSH with tissue enriched 1-propenyl TS (analytical C18 HPLC was eluted by 30%
methanol/water solution with 0.5% acetic acid, FR = 0.8 mL/min, detection at 254 nm). Peaks: 1, Cys; 2, CySSM; 3, CySSA; 4, CySSPe; 5, GSH; 6,
GSSM; 7, GSSA; 8, GSSPe. (B) Selected COESY and HMBC correlations of GSSPe.
a simple method to prepare a large amount of active alliinase
enzyme. Compared with a previous method such as
Pe), which quickly rearranges to form cyclized sulfur
1
2,26
compounds;
(3) 1-Pe-SOH reacts with other sulfenic
(
NH ) SO precipitation, which took 1−2 days, this method
acids (such as Me-SOH, Pr-SOH, and 2-Pe-SOH) to form
heterologous 1-propenyl TS (1-Pe-S(O)S-R and R-S(O)S-1-
4
2
4
can prepare a large amount of crude alliinase in 30 min−1 h;
thus, this is very easy to scale-up. Due to the simple operations,
a large amount of alliinase enzyme can be used in the reaction
system, leading to high reaction yields in gram-scale reactions
1
2,26
Pe).
Among all TS, 1-propenyl TS are the most challenging
to prepare because they are present in low abundance in onions
or garlic; the organic synthesis involves multistep synthesis,
(
Table 1). During the preparation of the crude alliinase,
12,26
which is very difficult.
The washed garlic homogenate
endogenous TS and other compounds soluble in organic
solvent from garlic were removed by the first step organic
solvent washing (using EtOAc). Thus, the second-step organic
solvent extraction (using CH Cl ) after the enzymatic reaction
recovered only the newly formed TS derived from the
exogenously added synthetic ACSO. This leads to high purity
of the generated TS, even without any purification step (Figure
method described above cannot be used to prepare 1-propenyl
TS; addition of 1-PeCSO or a combination of 1-PeCSO and
another ACSO to the washed garlic homogenate generated
only the cyclized sulfur compounds dervied from di-1-propenyl
disulfide S-oxide (data not shown).
Here we developed a tissue reaction method to enrich 1-
propenyl TS, using mixed blending of fresh garlic and heat-
inactivated onion tissues. Figure 3A shows the HPLC analysis
of the organosulfur compounds derived from fresh onion (top
panel), fresh garlic (middle panel), and a mixture of fresh garlic
and heat-inactivated onion tissues (bottom panel). PTSO (peak
2
2
2
). For each reaction, the gram-scale enzymatic conversion can
be completed in 30 min−1 h; the short reaction time minimizes
the decomposition of relatively unstable TS.
Tissue Reaction To Enrich 1-Propenyl TS (1-PeS(O)SR
or 1-PeSS(O)R). S-1-Propenyl-L-cysteine sulfoxide (1-PeCSO)
is the dominant ACSO species in some Allium vegetables such
as onions. Upon tissue crushing, 1-PeCSO is converted by
alliinase to form S-1-propenyl sulfenic acid (1-Pe-SOH), which
has at least three fates: (1) 1-Pe-SOH is converted to a
lachrymatory factor propanethial S-oxide (PTSO) via the
1
) is the dominant organosulfur compound from fresh onion;
2
4
the other organosulfur compounds are minor. Allicin (peak 5)
is the dominant organosulfur compound from fresh garlic, no
PTSO was observed, and 1-propenyl TS (peaks 3 and 4)
comprise <5% of the total garlic TS. Using the mixed blending
strategy, the proportion of 1-propenyl TS (peaks 3, 4, and 8)
increased to 50% of the total TS, whereas no PTSO formation
25
catalytic activity of PTSO synthase; (2) 1-Pe-SOH reacts with
itself to form di-1-propenyl disulfide S-oxide (1-Pe-S(O)S-1-
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was observed. The total TS yields of the mixture were similar to
that of garlic tissues (data not shown).
CySSPe was confirmed by comparing its NMR and MS data
with previous results. GSSPe was not prepared before, and
19
Figure 3B shows the fate and flux of the S-1-propenyl group
in reaction processes to explain the enrichment of 1-propenyl
species. PTSO formation requires PTSO synthase, which exists
in onion but not in garlic; thus, PTSO formation in the
mixture was diminished by inactivation of onion enzyme. 1-
PeCSO in onions was converted by the garlic alliinase to form
the structure of GSSPe was confirmed by a series of 1D and 2D
1
13
NMR techniques including H, C, COESY, HMQC, and
HMBC (the NMR data of GSSPe are shown in Table 2;
selected COESY and HMBC correlations are shown in Figure
25
4
B).
1-Pe-SOH, which reacted with other sulfenic acids (mostly 2-
Table 2. H (500 MHz), HMBC (125 MHz), and 13C (125
1
Pe-SOH derived from garlic) to form the heterologous 1-
propenyl TS (the bottom panel in Figure 3A shows high levels
of AllSS(O)Pe and AllS(O)SPe from the mixture). Garlic tissue
contains a high amount of alliinase and no PTSO synthase;
also, it contains a high amount of 2-PeCSO, which will be
rapidly converted to All-SOH to capture onion-derived 1-Pe-
SOH. These properties make garlic a suitable candidate for
tissue reactions.
MHz) NMR Data for S-1-Propenylmercapto-L-glutathione
a
(
GSSPe) in D O
2
HMBC
position
δH
2J
3J
δC
1
2
3
175.1 or 172.9
54.2
3.82 (tr, 6.5)
2.19 (qr, 7.5)
C-1, C-3
C-2, C-4
C-4
26.4
Preparation of CySSR and GSSR (R = Methyl, Ethyl,
Propyl, and Allyl). Our previous study has reported the
chemical synthesis of non-1-propenyl CySSR and GSSR using
the reaction of Cys or GSH with dithiophosphoric alk(en)yl
2
.20 (qr, 7.5)
2.56 (tr, 7.5)
.58 (tr, 7.5)
4
C-3
31.6
2
5
6
7
175.1 or 172.9
53.0
1
6
disulfides; however, the multistep chemical synthesis was
difficult to carry out. Here we prepared CySSR/GSSR (R =
methyl, ethyl, propyl, and allyl) by the reaction of prepared
pure TS with Cys or GSH in water. Without any
chromatography purification, >95% purity of CySSR and
4.72 (dd, 4.5, 9.5)
3.01 (dd, 9.5, 14.5)
3.27 (dd, 4.5, 14.5)
C-6
38.4
8
175.1 or 172.9
42.0
9
3.97 (s)
1
GSSR was obtained; H NMR analysis showed the only
10
11
12
13
175.1 or 172.9
133.5
impurity in the reaction product was unreacted Cys or GSH.
Using TS to prepare the disulfide conjugates has the following
advantages: (1) the reaction requires only the TS and the thiol
compound (no catalyst is needed); (2) the reaction is rapid and
almost complete; (3) TS are well soluble in many solvents from
water to CH Cl , so they can be used to react with hydrophilic
6.12 (m)
C-13
C-11
6.12 (m)
C-13
C-12
123.5
1.78 (br, 5.0)
17.7
a
J in hertz in parentheses.
2
2
or lipophilic thiol compounds; and (4) an extra amount of TS
is used in the reaction, and the residue TS can be easily
removed by evaporation or solvent washing (no chromatog-
raphy purification is required).
In a typical tissue reaction described above, one heat-
inactivated onion bulb (∼300 g) and fresh garlic (∼15 g)
generate ∼0.2 g of enriched 1-Pe TS mixture, which can be
used to prepare 0.5−1.0 g of Cys or GSH conjugate mixture,
which has defined compositions containing ∼20% CySSPe or
GSSPe. Compared with a previous multiple-step chemical
synthesis of CySSPe, this vegetable blending strategy is a very
simple and cost-effective method to prepare mixed or pure 1-
propenyl disulfide conjugates.
Preparation of CySSPe and GSSPe. 1-Propenyl TS are
3
major species in fresh onion tissues; thus, the biological effects
19
of the 1-propenyl mixed disulfide conjugate derivatives
(
CySSPe and GSSPe) may be key to understanding the health
benefits of onion. However, our previous method could not be
used to prepare CySSPe and GSSPe because 1-propene-1-thiol
is not available to prepare the synthetic intermediate
dithiophosphoric 1-propenyl disulfide using the synthetic
CySSPe and GSSPe Have the Most Potent Biological
Activities among Allium CySSR/GSSR Conjugates. Che-
moprevention refers to the use of agents to suppress tumor
16
27
strategy in our previous study. Using a modified synthesis
strategy, we have synthesized the dithiophosphoric 1-propenyl
disulfide (Supporting Information, Scheme S1). However, the
reaction of dithiophosphoric 1-propenyl disulfide with Cys or
GSH failed to generate CySSPe or GSSPe (data not shown).
This result suggests that a new method is needed to prepare
CySSPe and GSSPe.
progression, leading to reduced risk of cancers. Two signaling
pathways have been shown to play a key role in the action
mechanisms of many chemopreventive agents: (1) induction of
phase II detoxification enzymes such as QR and glutathione S-
transferase, which are involved in the detoxification of
environmental carcinogens to inhibit the formation of ultimate
electrophilic and carcinogenic species; and (2) inhibition of
inflammation, including the production of inflammatory
mediators and cytokines, such as inducible nitric oxide synthase
(iNOS) and cyclooxygenase-2. Chronic inflammation is widely
Here we prepared CySSPe and GSSPe by the reaction of Cys
or GSH with the enriched 1-propenyl TS prepared from the
above mixture. Figure 4A shows a typical HPLC profile of the
reaction product. For the reaction with Cys or GSH, four peaks
were observed on HPLC, and they were confirmed to be
unreacted starting material Cys or GSH and the methyl, allyl,
and 1-propenyl disulfide conjugates with Cys or GSH. The
identities of peaks 1−3 and 5−7 were confirmed by coelution
with synthesized standards. Peaks 4 and 8 were purified by
semipreparative HPLC and confirmed to be CySSPe and
GSSPe, respectively, by NMR and MS analysis. The structure of
28
believed to be correlated with increased cancer risks.
Our previous study has shown that CySSR and GSSR (R =
methyl, ethyl, propyl, and allyl) have potent effects to induce
QR activities in Hepa 1c1c7 cells and inhibit LPS-induced NO
16
formation in RAW 264.7 cells. Here we further found that
CySSA and GSSA induced the expression of QR protein in
Hepa 1c1c7 cells and inhibited LPS-induced iNOS expression
in RAW 264.7 cells (Supporting Information, Figure S1),
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Journal of Agricultural and Food Chemistry
Article
supporting the effects of CySSR and GSSR are at the protein
level. However, the effects of CySSPe and GSSPe are unknown.
Here we tested the biological activities of the newly prepared 1-
propenyl conjugates CySSPe and GSSPe (Figure 5A−D).
Previous study has shown that many cancer preventive
compounds activate phase II enzymes or inhibit inflammation
via covalent modification of important signaling proteins such
29
as Keap1 for phase II enzyme induction or p65 for NF-κB-
30
mediated inflammation signaling. CySSA has been shown to
3
1,32
covalently interact with protein thiols.
A recent study
showed that it disrupted microtubule polymerization via8
covalent modifications of cysteine residues of microtubule.
Therefore, it is likely that CySSR and GSSR exert their
biological activities via direct interactions with cysteine residues
of Keap1 or p65 proteins. The relative potencies of CySSR and
GSSR may be due to varied reactivity of the conjugates with
protein thiols. More studies are required to characterize the
mode of actions of CySSR and GSSR.
β-Lyase-Dependent Mechanism May Not Be Involved
in Biological Activities of CySSR. A previous study has
shown that CySSA is an efficient substrate of β-lyase, which
cleaves CySSA to generate highly redox-active persulfide
species (RSS), leading to modulation of redox signaling
33
(
proposed mechanism is shown in Figure 6A). To test
Figure 5. Effects of CySSR and GSSR on induction of QR and
inhibition of LPS-induced NO formation: (A) chemical structures of
CySSPe and GSSPe; (B) LPS-induced NO assay of CySSPe and
GSSPe in RAW 274.7 cells; (C) QR assay of CySSPe; (D) QR assay of
GSSPe; (E) structure−activity relationships (SAR) of GSSR for QR
induction; (F) SAR of GSSR conjugates for LPS-induced NO
formation. The results are expressed as the mean ± SD of three
independent experiments with four duplicates in each experiment.
Figure 6. β-Lyase inhibitor propargylglycine (PAG) has no effect on
activity of CySSA in vitro. (A) The previously proposed mechanism of
β-lyase in the activation of CySSR to form redox active RSS species
shows that propargylglycine (PAG) is a chemical inhibitor of β-lyase.
(B) Hepa 1c1c7 cells were treated with a combination of CySSA (0.17
mM) and varied doses of PAG. After 48 h of treatment, QR activities
were assayed. (C) RAW 264.7 cells were treated with a combination of
CySSA (0.09 mM) and varied doses of PAG and then stimulated with
Among all of the Allium CySSR and GSSR conjugates, GSSPe
showed the most potent effects to inhibit LPS-induced NO
formation in RAW 264.7 cells with an IC50 value = 12.6 ± 1.1
μM (Figure 5B) and induce QR specific activity with a CD
value = 14.9 ± 0.8 μM (Figure 5D). CySSPe had a potent effect
with a CD value = 31.1 ± 8 μM (Figure 5C); however, it had a
weak NO inhibitory effect with IC = 120.8 ± 1.8 μM (Figure
1
μg/mL LPS. After 24 h of treatment, the concentration of nitrite in
the medium was analyzed. The results are expressed as the mean ± SD
of three independent experiments with four duplicates in each
experiment.
5
0
5
B). For QR induction, the CD values of CySSPe and GSSPe
were comparable to those of 1-propenyl TS, which have CD
17
values of ∼10 μM. These result suggest that CySSPe and
GSSPe had potential cancer preventive and anti-inflammatory
effects.
whether the β-lyase-dependent mechanism was involved in the
QR inducing and NO inhibitory effects of CySSA, the cells
were treated with a combination of CySSA and varied doses of
propargylglycine (PAG), a chemical inhibitor of β-lyase. As
shown in Figure 6B,C, co-addition of PAG (0.37−7 mM) had
no effect on CySSA-induced QR induction (Figure 6B) or NO
inhibition (Figure 6C), suggesting the β-lyase pathway was not
involved in the QR inducing and NO inhibitory effects of
CySSA.
In summary, here we demonstrate a simple enzymatic
method for gram-scale preparation of Allium TS, as well as their
putative metabolites CySSR and GSSR. To our best knowledge,
this is the first study to use alliinase enzyme for gram-scale
preparation of TS. The prepared CySSR and GSSR conjugates
have potent effects to induce phase II enzymes and inhibit
34
Structure−Activity Relationships (SAR) of CySSR and
GSSR Conjugates. The CD and IC values of CySSR and
5
0
GSSR are shown in Table S1 in the Supporting Information.
Comparison of the biological activities of CySSR and GSSR (R
=
methyl, ethyl, propyl, allyl, and 1-propenyl) revealed that the
R- group plays a key role in the potency of the conjugates. For
GSSR conjugates, an order of potency of GSSPe > GSSA >
GSSP ≈ GSSE ≈ GSSM was observed for both QR and NO
assays (the dose responses of all GSSR conjugates for QR and
NO assays are shown in Figure 5E,F). For CySSR conjugates, a
similar trend was also observed except CySSPe showed a weak
NO inhibitory effect.
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Journal of Agricultural and Food Chemistry
Article
inflammation in vitro, which may contribute to the health-
promoting effects of Allium vegetables.
(6) Germain, E.; Chevalier, J.; Siess, M. H.; Teyssier, C. Hepatic
metabolism of diallyl disulphide in rat and man. Xenobiotica 2003, 33,
1
(
185−1199.
7) Miron, T.; Rabinkov, A.; Mirelman, D.; Wilchek, M.; Weiner, L.
ASSOCIATED CONTENT
■
The mode of action of allicin: its ready permeability through
phospholipid membranes may contribute to its biological activity.
Biochim. Biophys. Acta−Biomembranes 2000, 1463, 20−30.
(8) Xiao, D.; Pinto, J. T.; Soh, J.; Deguchi, A.; Gundersen, G. G.;
Palazzo, A. F.; Yoon, J.; Shirin, H.; Weinstein, I. B. Induction of
apoptosis by the garlic-derived compound 3S-allylmercaptocysteine
*
S
Supporting Information
Additional scheme, table, and figure. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
*
(
SAMC) is associated with microtubule depolymerization and c-Jun
Corresponding Author
E-mail: (K.L.P.) klparkin@wisc.edu or (G.Z.) gdzhang@
ucdavis.edu.
NH -terminal kinase 1 activation. Cancer Res. 2003, 63, 6825−6837.
2
(9) Shirin, H.; Pinto, J. T.; Kawabata, Y.; Soh, J.; Delohery, T.; Moss,
S. F.; Murty, V.; Rivlin, R. S.; Holt, P. R.; Weinstein, I. B.
Antiproliferative effects of S-allylmercaptocysteine on colon cancer
cells when tested alone or in combination with sulindac sulfide. Cancer
Res. 2001, 61, 725−731.
Present Address
†
Department of Entomology and Comprehensive Cancer
Center, University of California, Davis, One Shield Avenue,
Davis, CA, 95616, USA.
(10) Howard, E. W.; Ling, M.; Chua, C. W.; Cheung, H. W.; Wang,
X.; Wong, Y. C. Garlic-derived S-allylmercaptocysteine is a novel in
vivo antimetastatic agent for androgen-independent prostate cancer.
Clin. Cancer Res. 2007, 13, 1847−1856.
Funding
This work was supported by the University of Wisconsin,
College of Agricultural and Life Sciences, in part through an
Agricultural Research Station Hatch grant (WIS01184) and the
American Institute for Cancer Research (project 10A050).
(11) Lawson, L. D.; Wang, Z. J. Low allicin release from garlic
supplements: a major problem due to the sensitivities of alliinase
activity. J. Agric. Food Chem. 2001, 49, 2592−2599.
(12) Block, E.; Bayer, T.; Naganathan, S.; Zhao, S. Allium chemistry:
Notes
synthesis and sigmatropic rearrangements of alk(en)yl 1-propenyl
disulfide S-oxides from cut onion and garlic. J. Am. Chem. Soc. 1996,
The authors declare the following competing financial
interest(s): K.P. and G.Z. have applied for a U.S. patent for
the tissue homogenate method: Process of preparing conjugates
of Allium organosulfur compound with amino acids, peptides
and proteins, US 20110082282, assigned to Wisconsin Alumni
Research Foundation.
1
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18, 2799−2810.
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Wilchek, M. A method for continuous production of allicin using
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sulfoxide lyase and partially competitive inhibition by S-alkyl-l-
cysteines. J. Biol. Chem. 1964, 239, 777−782.
ABBREVIATIONS USED
■
ACSO, (±)-S-alk(en)yl-L-cysteine sulfoxide; Cys, cysteine;
CySR, S-alk(en)yl-L-cysteine; CySSR, S-alk(en)ylmercapto-L-
cysteine; CySSM, S-methylmercapto-L-cysteine; CySSE, S-
ethylmercapto-L-cysteine; CySSP, S-propylmercapto-L-cysteine;
CySSA, S-2-propenylmercapto-L-cysteine; CySSPe, S-1-prope-
nylmercapto-L-cysteine; EtOAc, ethyl acetate; GSH, gluta-
thione; GSSR, S-alk(en)ylmercapto-L-glutathione; GSSM, S-
methylmercapto-L-glutathione; GSSE, S-ethylmercapto-L-gluta-
thione; GSSP, S-propylmercapto-L-glutathione; GSSA, S-2-
propenylmercapto-L-glutathione; GSSPe, S-1-propenylmercap-
to-L-glutathione; iNOS, inducible nitric oxide synthase; NO,
nitric oxide; PTSO, propanethial S-oxide; QR, quinone
reductase or NAD(P)H:quinone oxidoreductase; SAR, struc-
ture and activity relationships; TS, thiosulfinates
(16) Zhang, G.; Li, B.; Lee, C.; Parkin, K. L. Cysteine and glutathione
mixed-disulfide conjugates of thiosulfinates: chemical synthesis and
biological activities. J. Agric. Food Chem. 2010, 58, 1564−1571.
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Revelle, L. K.; Wang, D.; Zhang, X. Allium chemistry: microwave
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