[3H]-allicin: Preparation and Applications
T. Miron, T Bercovici, A. Rabinkov, M. Wilchek and D. Mirelman1
Department of Bilogical Chemistry, The Weizmann Institute of Science Rehovot
76100, Israel.
1
Corresponding author: David Mirelman,
Department of Biological Chemistry,
The Weizmann Institute of Science, Rehovot 76100,
Israel.
Tel: 972-8-343160; Fax: 972-8-468256;
e-mail: david.mirelman@weizmann.ac.il
Key words: Allicin, Alliin, Alliinase, Garlic
Abbreviations: DTNB, 5,5' dithiobis-(2-nitrobenzoic acid); NTB, nitrothiobenzoate,
GSH, reduced glutathione; GSSA, S-allylmercaptoglutathione. PLP, pyridoxal-5’phosphate, DTT, dithiotreitol.
Abstract
Allicin (diallylthiosulfinate), the active substance of garlic has been shown to
possess a variety of biological activities. Mechanistic and pharmacokinetic studies of
allicin and its derivatives raise the need for a labeled compound. However, labeling of
this volatile and unstable liquid requires delicate handling. In the present study we
describe a simple method for the preparation of [3H]-labeled allicin. This was achieved
by applying synthetic [3H]-alliin (2,3-[3H]- allylcysteine sulfoxide) to a column
containing immobilized alliinase [EC 4.1.1.4.] from garlic. Purification of [3H]-allicin
was done by differential adsorbtion of the reaction components on a neutral polystyrene
resin, Porapak Q. Thiol-containing compounds are known to be the main target of
allicin. In this work we demonstrated that [3H]-allicin can be used for the synthesis of
labeled [3H]-allylmercapto derivatives of SH- peptides and proteins. Thus, we prepared
[3H]-S-allylmercaptoglutathione which can be used in metabolic studies. Moreover, we
showed that incubation of alliinase with [3H]-allicin led to modification of 1.4 cysteine
residues per one subunit of the enzyme.
2
Introduction
Allicin (diallylthiosulfinate) a volatile liquid, responsible for the pungent smell
of garlic, is chemically an unstable and high reactive molecule. It represents about 70%
of the overall thiosulfinates present in the cloves upon mechanical crushing [1-3]. Since
its discovery by Cavalitto and Bailey [4] allicin is the best characterized biologically
active compound of garlic and has been shown to possess a variety of biological effects,
including antimicrobial, hypolipidemic, antithrombotic, anticancer and other activities
[1, 2, 5]. Most of these effects can be related to its strong SH-modifying capacity and
antioxidant properties [4, 6-8]. The activated disulfide bond -S(O)-S- of allicin (see
scheme 1) reacts with different thiols containing molecules, including SH-containing
proteins. After its injection into the blood, allicin rapidly disappears from the
circulation, due to its high reactivity, and consequent transformation into secondary
products [1, 9].
The study of the mechanism of action and pharmacokinetics of allicin, both in vitro and
in vivo, could be facilitated by using radioactively labeled allicin. The organ
distribution and clearance rate of 35S-alliin, 35S-allicin (prepared in situ) and 35Svinyldithiine in mice were previously described [10]. However, 35S-allicin preparation
was not purified and its metabolites were not identified. Moreover, 35S-labeled material
is short-lived. Another attempt to study the metabolism of garlic thiol-containing
compounds used deuterated S-allyl cysteine, the early precursor of allicin. However,
the end product, allicin was not further prepared [11, 12].
In the present work we describe a method combining chemical and enzymatic synthesis
of [3H]-allicin. First, [3H]-alliin was synthesized and it was then converted to [3H]allicin by passing it through an immobilized alliinase column [13]. The product, [3H]allicin, was purified by using preparative reverse phase column Porapak Q. The overall
reaction and its catalysis by alliinase is shown in scheme 1 [14].
3
1. Oxidation
O
O
*
*
H2O2
S
-
*
O
*
NH +
3
O
S
+ H2O
-
O
NH +
3
[2,3 3H] Allyl cysteine
[2,3 3H] Allyl cysteine sulfoxide
([3H] Alliin)
2. Enzymatic condensation
O
O
2 H2 C=CH-CH 2-S-CH 2-CH-COO
NH+
3
-
Alliinase
+ H2 O
O
+
H2 C=CH-CH 2 -S-S-CH 2-CH=CH 2 + 2 CH3 -C-COO + 2NH4
[3H] Alliin
[3H] Allicin
Pyruvate
Scheme 1
Material and methods
[2,3 3H]-S-allyl L-cysteine (specific radioactivity: 50 Ci/mmole, mCi/ml) was prepared
by American Radiolabeled Chemicals St. Louis, MO (USA). Hydrogen peroxide
(30%) was obtained from Merck. GSH, PLP and N,N'-Disuccinimidyl Carbonate were
purchased from Sigma-Aldrich Corporation, St. Louis, MO (USA). S-allyl-L-cysteine
and alliin were synthesized as described [14]. CL-Sepharose-4B was obtained from
Pharmacia (Sweden). Porapak Q, (100-120 mesh) was obtained from Waters
Associates, INC, Milford, Mass (USA). Alliinase, isolated from crushed garlic [15] was
coupled to Cl-Sepharose 4B as described [13]. Immobilized alliinase was packed in a
small column and pre-equilibrated with Na phosphate buffer (50 mM, pH 6.5)
containing PLP (0.02 mM) (buffer A). The reaction mixture containing crude [3H]alliin was loaded onto the column and crude [3H]-allicin was collected.
Quantitative analysis of alliin and allicin was performed using LKB HPLC system
equipped with a chromatography station for Windows (DataApex Ltd. 1998, Czech
Republic). The substances were separated on a LiChrosorb RP-18 (7µm) column
(Merck, Darmstadt, Germany), using methanol (60%) in water containing 0.05 %
4
trifluoroacetic acid at a flow rate of 0.55 ml/min., and their absorbance was detected at
210 nm.
Radioactivity (disintegration/min, DPM) of 0.1 ml samples in 4 ml xylene-Lumax (3:1
v/v) (Lumax TM, Lumac, Schaesberg, The Netherlands), was measured using a βcounter liquid scintillation analyzer (Tri-carb 2100TR, Packard Inc. Meriden, CT,
USA).
Synthesis of [3H]-alliin
Alliin was synthesized as described [14] with several modifications.
[3H]-alliin was prepared by oxidation of [2,3 3H]-S-allyl L-cysteine with hydrogen
peroxide. [2,3 3H]-S-allyl L-cysteine (0.1 mCi in 0.1 ml ethanol) was dried in a small
glass tube using a Speed Vac at room temperature. S-allyl-L-cysteine (0.07 ml of 100
mg/ml water solution) and hydrogen peroxide (0.01 ml of 15%) were added over the
dried radioactive precursor. The mixture was incubated in a water bath (40oC) for 30
min and then left at room temperature for another 30 min. Acetone (0.09 ml) was added
to the reaction mixture, left at room temperature for 1 h and centrifuged (12000 rpm 2
min) to remove impurities. The acetone supernatant (52% final concentration) was
collected and more acetone (0.1 ml/tube) was added (70% acetone final concentration)
in order to precipitate [3H]-alliin. The mixture was kept overnight at 4oC. Finally, [3H]alliin was collected after centrifugation, washed with acetone (0.2ml), dried in a Speed
Vac and stored at -20oC.
Preparation and purification of [3H]-allicin
[3H]-Alliin (0.2 mCi) was dissolved in 0.5 ml of Na phosphate buffer (50 mM, pH 6.5)
containing PLP (0.02 mM) (buffer A). It was then loaded onto a mini column (6x15
mm) packed with immobilized alliinase-CL-Sepharose 4B, pre-equilibrated with buffer
A. The column was eluted with 1.0 ml of buffer A, and then spun down to collect the
rest of the supernatant (1.0 ml). [3H]-Allicin obtained was further purified on Porapak
Q column (bed volume about 1 ml). The unbound material was washed away with
water, [3H]-allicin was eluted with ethanol (100%) and stored at -80oC.
Synthesis of [3H]-S-allylmercaptoglutathione and its purification
The reaction between allicin and glutathione (GSH) was performed as described earlier
[16]. GSH at pH 6.0, was added to ethanolic solution of [3H]-allicin. The reaction was
followed by HPLC analysis. Fraction (0.5 ml) were collected and monitored by βcounter for [3H].
5
Results
Preparation of [3H]-alliin
[3H]-alliin (S-allyl-L-cysteine sulfoxide) was obtained by oxidation of [2,3 3H]-S-allylL-cysteine with hydrogen peroxide (oxidation step, Scheme 1). The yield of the
radioactivity in the dry preparation was 60%. Dry alliin (2nd acetone precipitation)
contained both isomers, (+) and (-) S-allyl-L-cysteine sulfoxide, the (+) isomer being
the more active in terms of alliinase substrate.
The specific radioactivity of [3H]-alliin can be modulated by using various ratios of
[3H]-labeled to non-labeled S-allyl -L-cysteine, in the oxidation process.
Preparation and purification of [3H]-allicin
[3H]-alliin (racemic mixture) was loaded onto a mini column of immobilized alliinase.
The effluent containing crude [3H]-allicin was analyzed by HPLC. Fractions were
collected and the radioactivity was measured using a β-counter. The yield of [3H]allicin was 50-70% of the total radioactivity in the various preparations. The specific
radioactivity of allicin in the various preparations ranged between 2-7 µCi/ µmole.
Crude [3H]-allicin, eluted from the immobilized alliinase column, was purified by
adsorption on Porapak Q column [17]. The column was washed with water (to remove
non adherent materials such as [3H]-alliin). [3H]-allicin was eluted with ethanol (100%).
The retention time of [3H]-allicin on the HPLC column was identical to that of its nonradioactive counterpart. The characteristic HPLC pattern of allicin eluted from Porapak
Q column is shown in Fig. 1.
The use of [3H]-allicin for labeling SH-peptide: Preparation of [3H]-Sallylmercaptoglutathione:
Allicin is known to react with free SH group on peptides or proteins. The reactivity of
tritiated allicin was therefore tested on a representative of each group.
A. Preparation of [3H]-S-allylmercaptoglutathione
Reduced glutathione (10 mM, 100 µl), pre-adjusted to pH 6 was added to purified [3H]allicin (12 mM, 2µCi/µmole, 100 µl) in ethanol. The kinetics of the reaction was
followed by HPLC analysis at various time intervals. Fractions (0.5 ml) were collected
and radioactivity monitored. Fig. 2 represents the separation pattern of the reaction
mixture 8 min after the onset of the reaction. The reaction reached completion after 30
min at room temperature. It was then acidified with 1N HCl (100 µl), diluted with
water (1.0 ml) and the product was loaded on Porapak Q column to remove excess of
6
allicin. [3H]-S-allylmercaptoglutathione did not adhere to the Porapak column (Fig. 3),
whereas allicin remained attached to the column. The specific radioactivity of [3H]-Sallylmercaptoglutathione was 0.99 µCi/µmole (nearly 100% yield).
B. Preparation of [3H]-S-allylmercaptoalliinase
Radioactive allicin can be used to determine minute amounts of free SH in proteins.
It was suggested previously that alliinase may contain one or two free SH groups
(unpublished data). In order to verify their existence and quantify them, the protein was
reacted with purified [3H]-allicin. Radioactive allicin (10 µl, 5mM, 5µCi/µmole) was
added to alliinase (1mg/0.3ml in 40 mM Na phosphate buffer pH 6.5 containing 25%
glycerol). The reaction mixture was incubated in an ice bath for 30 min and gel-filtrated
on Sephadex G-50 (Fig. 4). It was found that [3H]-allicin modified 1.4 residues of
alliinase. Therefore we may conclude that there are 2 free SH residues in alliinase. This
is in good correlation with the presence of 2 free SH-groups per one alliinase subunit
previously studied (x-ray unpublished data).
Discussion
During the last few years we established and confirmed that allicin is the most active
molecule in garlic, responsible for its biological activities and health benefits. These
studies were enabled thanks to the availability of isolated pure allicin.
In order to study the mechanism of action, the pharmacokinetics and the organ
distribution of allicin in vivo, radioactive allicin was required. Previously 35S-alliin and
35
S-allicin were prepared for pharmacokinetic studies, but these reagents were far from
being satisfactory due to the fact that allicin was not purified and that 35S has a short
half-life. We prepared a long-lived radioactive [3H]-allicin, labeled in the allyl group,
using a novel chemical and enzymatic combined approach. We started with the
synthetic substrate of alliinase, racemic [3H]-S-allylcysteine sulfoxide, which unlike the
natural (+) S-allylcysteine sulfoxide (alliin) contains both (+) and (-) isomers. Nonradioactive alliin can be separated into its enantiomers by differential precipitation, but
it is not practical when radioactive material is used. Therefore, a method was developed
for efficient purification of allicin prepared from the racemic mixture of alliin, using a
Porapak Q column. Allicin is adsorbed to the column while all the other materials are
not, and can be washed off. Allicin is then eluted with ethanol. This column can also be
used to prepare large quantities of pure allicin. [3H]-allicin was shown to be identical to
7
native allicin in its elution pattern on a reverse-phase column, and in its reactions with
free SH groups containing compounds such as reduced glutathione.
Using [3H]-allicin we also showed that it reacts with two free SH groups on alliinase.
Allicin is known to react with free SH groups of proteins and peptides. The availability
of [3H]-allicin, as well as of non-labeled pure allicin will facilitates quantitative studies
of SH containing proteins using MS and radioactive counting, thus introducing new
strategies into the field of proteomics. Furthermore, the availability of pure 3H-allicin
will enable studies on its mechanism of action, its pharmacokinetics, its organ
distribution and the proteins with which it interacts in the body. Such a study may lead
to the discovery of new target compounds more stable and less volatile than allicin, for
drug intervention.
References
[1]
E. Block, The organosulfur chemistry of the genus Allium Implications for the organic chemistry of sulfur, Angew. Chem. 31
(1992) 1135-1178.
[2]
L. D. Lawson, Garlic: A review of its medicinal effects and indicated
active compounds, in: Lawson, L. D., and Bauer, R. (Eds.)
Phytomedicines of Europe: Their Chemistry and Biological Activity,
American Chemical Society, Washington, 1998, 176-209.
[3]
J. Han, L. Lawson, G. Han, and P. Han, A spectrophotometric method
for quantitative determination of allicin and total garlic thiosulfinates,
Anal. Biochem. 225 (1995) 157-160.
[4]
C. J. Cavallito, and J. H. Bailey, Allicin, the antibacterial principle of
Allium sativum. I. Isolation, physical properties and antibacterial action,
J. Am. Chem. Soc. 66 (1944) 1950-1951.
[5]
H. P. Koch, and L. D. Lawson, Garlic: The Science and therapeutic
application of Allium sativum l. and related species, Williams &
Wilkins, Baltimore, 1996.
[6]
E. D. Wills, Enzyme inhibition by allicin, the active principle of garlic,
Biochem. J. 63 (1956) 514-520.
8
[7]
K. Prasad, V. A. Laxdal, M. Yu, and B. L. Raney, Evaluation of Hydroxyl
Radical-Scavenging Property of Garlic, Mol. Cell. Biochem. 154 (1996) 5563.
[8]
A. Rabinkov, T. Miron, L. Konstantinovski, M. Wilchek, D. Mirelman,
and L. Weiner, The mode of action of allicin: trapping of radicals and
interaction with thiol containing proteins, Biochim. Biophys. Acta 1379
(1998) 233 -244.
[9]
E. Block, S. Naganathan, D. Putnam, and S. H. Zhao, Allium chemistry HPLC analysis of thiosulfinates from onion, garlic, wild garlic
(Ramsoms), Leek, Scallion, Shallot, Elephant (Great-Headed) garlic,
Chive, and Chinese Chive - uniquely high allyl to methyl ratios in
some garlic samples, J. Agric. Food Chem. 40 (1992) 2418-2430.
[10]
G. Lachmann, D. Lorenz, W. Radeck, and M. Steiper, The pharmacokinetics
of the S35 labeled labeled garlic constituents alliin, allicin and vinyldithiine,
Arzneimittelforschung 44 (1994) 734-743.
[11]
R. T. Rosen, R. D. Hiserodt, E. K. Fukuda, R. J. Ruiz, Z. Zhou, J. Lech, S. L.
Rosen, and T. G. Hartman, The determination of metabolites of garlic
preparations in breath and human plasma, Biofactors 13 (2000) 241-249.
[12]
R. T. Rosen, R. D. Hiserodt, E. K. Fukuda, R. J. Ruiz, Z. Zhou, J. Lech, S. L.
Rosen, and T. G. Hartman, Determination of allicin, S-allylcysteine and
volatile metabolites of garlic in breath, plasma or simulated gastrifluids, J
Nutr. 131(3s) (2001) 968S-971S.
[13]
B. Tchernychev, A. Rabinkov, T. Miron, and M. Wilchek, Natural
antibodies against alliinase in human serum and polyclonal antibodies
elicited in rabbit, share the same immunogenic determinants,
Immunol. Letters 71 (2000) 43-47.
[14]
A. Stoll, and E. Seebeck, Chemical investigation on alliin, the specific
principle of garlic, Adv. Enzymol. 11 (1951) 377-400.
[15]
A. Rabinkov, X. Z. Zhu, G. Grafi, G. Galili, and D. Mirelman, Alliin
Lyase (Alliinase) from Garlic (Allium sativum), App. Biochem.
Biotechnol. 48 (1994) 149-171.
[16]
A. Rabinkov, T. Miron, D. Mirelman, M. Wilchek, S. Glozman, E. Yavin, and
L. Weiner, S-Allylmercaptoglutathione: the reaction product of allicin with
glutathione possesses SH-modifying and antioxidant properties, Biochim.
Biophys. Acta 1499 (2000) 144-153.
9
[17]
A. Niederwieser, Adsorption of neutral polystyrene resin. A simple method for
extraction of 2,4-dinitrophenyl derivatives from aqueous solution and for
decoloration of protein hydrolysates, J Chromatogr 54 (1971) 215-223.
10
Legends to figures
Fig. 1. HPLC pattern of [3H]-allicin eluted from Porapak Q column with 100% ethanol.
Sample diluted in HPLC running buffer (1:100) was loaded (20 µl) on a
LiChrosorb RP-18 column.
Fig.2. HPLC-pattern of the reaction mixture containing GSH and [3H]-allicin. A
sample, taken after 8 min, diluted in HPLC running buffer (1:100) was loaded
(50 µl) on a LiChrosorb RP-18 column, fractions were collected (0.5 ml) and
radioactivity of 0.1 ml from each fraction was counted.
Fig. 3. HPLC pattern of [3H]-S-allylmercaptoglutathione that was not adsorbed to the
Porapak Q column. The pass through fraction was diluted in HPLC running
buffer (1:10) and was loaded (20 µl) on a LiChrosorb RP-18 column.
Fig. 4. [3H]-allicin-modified alliinase was subjected to gel-filtration on Sephadex G-50
(18x1.5cm), preequilibrated and run with Na phosphate buffer (50 mM, pH 6.5).
Fractions (1.2 ml) were collected, OD 280nm was recorded and aliquots (50 µl)
were examined for radioactivity as described in Fig. 2.
11
300
Voltage (mV)
200
100
0
0
5
10
Time (min.)
Fig. 1
12
15
20
S- [3H]-allyl mercaptoglutathione
5000
mV (HPLC)
[3H] DPM
3000
[3H]DPM
Voltage (mV)x10
4000
[3H]-allicin
2000
1000
reduced glutathione
0
0
2
4
6
Retention Time (min)
Fig. 2
13
8
10
[3H]-S-Allylmercaptoglutathione
Voltage (mV)
300
200
100
0
0
2
4
6
Time (min.)
Fig. 3
14
8
20.0
[3H]DPM
[3H]-allicin
A280
[3H]DPM (x102)
A280 (x10-1)
15.0
10.0
[3H]-alliinase
5.0
0.0
0
10
20
30
Eluted ml
Fig. 4
15
40
50