Doklady Biological Sciences, Vol. 389, 2003, pp. 188191. Translated from Doklady Akademii Nauk, Vol.

389, No. 4, 2003, pp. 547551.

Original Russian Text Copyright 2003 by Astashkin, Khokhlova, Tilkunova, Zalepugin, Glezer, Grachev.

CELL BIOLOGY

Allicin Suppresses the Production of Oxygen Radicals by Human

Neutrophils Induced by Various Stimulating Agents
E. I. Astashkin*, O. A. Khokhlova*, N. A. Tilkunova**, D. Yu. Zalepugin**,
M. G. Glezer*, and S. V. Grachev*
Presented by Academician M.A. Paltsev November 27, 2002
Received November 27, 2002

Human blood phagocytes, including neutrophils,

are components of the innate immunity system. They
are cells of the first line of defense and are responsible
for the removal of foreign cells (predominantly microbial) and altered self cells via phagocytosis. Phagocytes
efficiently fulfill protective functions due to their ability
to produce significant amounts of oxygen radicals,
which are generated by the multi-component NADPH
oxidase enzyme complex in the plasma membrane. As
a result of the presence of one unpaired electron, oxygen radicals actively interact with all species of
macromolecules in the cell, leading to their denaturation and destruction. An inadequately intense production of oxygen radicals often results in the development of pathologies or their aggravation. For this
reason, the search for compounds that suppress oxygen-radical production or enable the elimination of
already formed radicals is a topical biomedical
problem.
Allicin (diallylthiosulfinate) is one of the main components of garlic determining its characteristic smell [1].
This compound is produced by the enzyme allinase
from the amino acid alliine as a result of mechanical
destruction of garlic cloves [2].
Allicin displays a broad spectrum of biological
activities. It exhibits a bactericidal effect [3], decreases
the level of cholesterol and triglycerides in the blood
plasma [4], suppresses the platelet aggregation [5],
decreases intraocular pressure [6], and has an antitumor
effect [7, 8].
Recently, it was shown using the ESR and spin trap
methods that garlic extracts and allicin had a pronounced antioxidant effect on hydroxyl radicals formed
in Fentons reaction from hydrogen peroxide in the
presence of Fe2+ [2, 9]. In addition, sulfur-containing

*Sechenov Medical Academy, ul. Bolshaya Pirogovskaya

2/6, Moscow, 119881 Russia
**State Research Institute of Organic Chemistry and
Technology, Moscow, Russia

components of the garlic suppressed lipid peroxidation [9]. Based on these facts, it was proposed to use
garlic preparations for prevention of cardiovascular diseases, atherosclerosis, and aging and suppression of
neoplastic growth [10].
The purpose of this work was to study the effect of
allicin on the production of superoxide anion radicals
by human neutrophils induced by various stimulating
agents. The stimulants used in this study were a bacterial tripeptide (N-formyl-Met-Leu-Phe, FMLP) and
a phorbol ester (phorbol 12-myristate 13-acetate,
PMA).
MATERIALS AND METHODS
Reagents. FicollHepaque gradient, formylpeptide
FMLP, PMA, HEPES, lucigenin, superoxide dismutase
(SOD), and reduced glutathione were obtained from
Sigma (United States).
Obtaining allicin. The extract of garlic homogenate
was obtained using a SFChem device for overcritical
extraction equipped with a 2.5-ml Keystone Scientific
flow extractor. Carbon dioxide in overcritical state was
used as a solvent. The extraction was performed at
40C and 280 atm for 120 min. The extract was a dense
yellow-green substance. Allicin was isolated from garlic extracts by semipreparative HPLC using a Gilson
115 chromatograph equipped with an Ultrasil column
(250 10 mm; diameter of particles, 5 m). Allicin was
eluted using the hexanisopropanol system (95 : 5) at a
rate of 3.0 ml/min and UV detection at 254 nm. The
fraction isolated was at least 97.5% allicin. Allicin was
identified using IR and NMR spectroscopy.
Isolation of neutrophils. Neutrophils were isolated
from peripheral blood of donors. Plasma enriched in
cells was layered on one-step FicollHepaque gradient
(density, 1.077 g/ml) and centrifuged at 800 g for
30 min. As a result of centrifugation, neutrophils were
pelleted above erythrocytes. The latter were removed
by osmotic lysis induced by addition of distilled water
for 20 s. Thereafter, neutrophils were washed twice by
centrifugation in the Hanks medium. The number of

0012-4966/03/0304-0188$25.00 2003 MAIK Nauka /Interperiodica

ALLICIN SUPPRESSES THE PRODUCTION OF OXYGEN RADICALS

Chemiluminescence intensity, cps

189

2
(a)

2500

2000

1500

1000

500

0
700

400

800

1200

1600

2000

800

960
Time, s

(b)
2

600
500
400
300
200
100
0

160

320

480

640

Fig. 1. Allicin decreases the level of superoxide-anion radical generated in human neutrophil suspension in the presence of (a) phorbol ester and (b) formylpeptide. (a) 1, 1 M PMA; 2, 30 M allicin; (b) 1, 1 M FMLP; 2, 30 M allicin.

cells was determined in a hemocytometer. The viability

of the cells estimated by the absorption of trypan blue,
a vital dye, was at least 95%. Lucigenin (20 M) was
added to an aliquot of the cell suspension (100 l,
106 cells/ml) as a luminophore. The production of
.
superoxide anion radicals ( O 2 ) was monitored using
a Biotoks-7 chemiluminometer connected through an
interface to a PC [11]. The results were expressed in
counts per second (cps).
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RESULTS AND DISCUSSION

Neutrophils were weakly activated due to adhesion
to the plastic wall of the cuvette. As a result, the basal
level of chemiluminescence slowly increased (Fig. 1a).
To cause a respiratory burst, neutrophils were treated
with standard agents, namely, phorbol ester (1 M) or
the formylpeptide FMLP (1 M). It is known that the
formylpeptide interacts with specific receptors located
on the surface of neutrophils. As a result, membrane

190

ASTASHKIN et al.
Chemiluminescence intensity, cps
(a)

800

600

400

200

0
(b)
600

500
400
1
300
200
100

160

320

480

640

800
Time, s

Fig. 2. A decrease in the level of phorbol ester-induced production of superoxide-anion radicals in the presence of (a) SOD and
(b) reduced glutathione. (a) 1, 1 M PMA; 2, SOD (5IU); (b) 1, 1 M PMA; 2, 0.5 mM reduced glutathione (GSH).

inositol phosphates are hydrolyzed, yielding two secondary messengers, diacylglycerol (DAG) and inositol1,4,5-trisphosphate (IP3). The latter induces Ca2+
release from the intracellular Ca2+ stores into the cytoplasm. DAG selectively interacts with protein kinase C,
which in the presence of Ca2+ is translocated from the
cytoplasm to the inner surface of the plasma membrane
and activates this enzyme [12]. Phorbol ester stimulates
protein kinase C as a result of its binding with the site
of the enzyme that interacts with DAG. An increase in
the intracellular concentration of free Ca2+ and activation of protein kinase C are required for self-assembling of all components of the NADPH-oxidase

enzyme complex, which reduces molecular oxygen to

superoxide anion radical, using NADHP as an electron
donor [13].
As is seen from Fig. 1a, PMA induced intense production of superoxide anion radical (judging by an
increase in the intensity of chemiluminescence of the
cell suspension). Subsequent treatment of the cells with
30 M allicin caused a decrease in the content of oxygen radicals in the suspension. This decrease had a
complex pattern: the chemiluminescence level sharply
dropped during the first several seconds and then gradually decreased. This complex kinetics assumes a complex nature of the allicin effect. It is known that allicin
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ALLICIN SUPPRESSES THE PRODUCTION OF OXYGEN RADICALS

can effectively interact with SH groups in proteins and

low-molecular-weight compounds [9]. In addition, it
has been shown recently that allicin has an antioxidant
effect on hydroxyl radicals [2]. We assumed that the

rapid stage of the decrease in the O 2 level is related to

the antioxidant effect of allicin, whereas the slow stage
of the decrease is caused by allicin influx into the cell
and its subsequent interaction with the SH-containing
intracellular agents, which often serve as endogenous
antioxidants [1, 9]. The ability of allicin to easily penetrate across the membrane is suggested by the data
obtained in [2] using phospholipid membranes, as well
as the results of our experiments showing a rapid Ca2+
release from intracellular stores of lymphocytes treated
with allicin in Ca2+-free medium [14].
When the neutrophil suspensions were treated with
FMLP, we also observed respiratory burst, whose
amplitude and kinetics were different from those
observed in the case of PMA treatment (Fig. 1b). In
these experiments, 30 M allicin also caused a twophase decrease in the oxygen-radical level in the cell
suspension.

Interestingly, the content of O 2 generated in the

presence of both PMA and FMLP decreased after the
allicin treatment below the basal level (Fig. 1a, 1b). For
comparison, we studied the effect of a widely known
antioxidant, SOD (5 IU), which was added after 1 M
phorbol ester (Fig. 2a). At this SOD activity, the level
of oxygen radicals decreased to stationary values. At a

greater SOD activity, the O 2 level decreased to the

basal values. It is known that exogenous SOD does not
penetrate across the plasma membrane to the cytoplasm
and has an antioxidant effect on extracellular radicals.
Apparently, this account for the absence of the second,
slow stage in the SOD effect (Fig. 2a), which is characteristic of allicin (Figs. 1a, 1b). Similar results were
obtained when exogenous reduced glutathione
(0.5 mM) was used as an antioxidant (Fig. 2b).

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It should be noted that the inhibitory effect of allicin

was concentration-dependent and, in our experiment,
expressed within the concentration range from 30 to
150 M.
Thus, the results of this study suggest that allicin has
an antioxidant effect on human cell suspensions. This
effect is complex and is exerted by different mechanisms.
REFERENCES
1. Miron, T. and Rabinkov, A., Mirelman, D., et al., Biochim. Biophys. Acta, 2000, vol. 1463, pp. 2030.
2. Rabinkov, A., Miron, T., Konstantinovski, L., et al., Biochim. Biophys. Acta, 1998, vol. 1379, pp. 233244.
3. Ankri, S. and Mirelman, D., Microb. Infect., 1999,
vol. 1, no. 2, pp. 125129.
4. Kannar, D., Wattanapenpaiboon, N., Savige, G.S., and
Wahlqvist, M.L., J. Amer. Coll. Nutr., 2001, vol. 20,
no. 3, pp. 225231.
5. Qi, R., Liao, F., Inoue, K., et al., Biochem. Pharmacol.,
2000, vol. 60, no. 10, pp. 14751483.
6. Teh-Ching Chu, Burch, J.L., De Paula Brotto, M.A.,
et al., Comp. Biochem. Physiol., 1996, vol. 115, no. 1,
pp. 8994.
7. Sunaram, S. and Milner, J.A., Biochim. Biophys. Acta,
1996, vol. 1315, pp. 1520.
8. Knowles, L.M. and Milner, J.A., Drug Metabol. Drug
Interact., 2000, vol. 17, no. 1/4, pp. 81107.
9. Rabinkov, A., Miron, T., Mirelman, D., et al., Biochim.
Biophys. Acta, 2000, vol. 1499, no. 1/2, pp. 144153.
10. Ames, B.N., Shigenaga, M.K., and Hagen, T.M., Proc.
Natl. Acad. Sci. USA, 1993, vol. 90, pp. 79157922.
11. Astashkin, E.I., Petrov, E.A., Glezer, M.G., et al.,
Voprosy Biol. Med. Farm. Khim., 2001, no. 3, pp. 2529.
12. Rukovodstvo po immunofarmakologii (Handbook of
Immunopharmacology),
Deila,
M.M.
and
Formena, Dzh.K., Eds., Moscow: Meditsyna, 1998,
pp. 3248.
13. Yarilin, A.A., Osnovy immunologii (Principles of Immunology), Moscow: Meditsyna, 1999, pp. 122123.
14. Astashkin, E.I., Belikova, N.A., Tilkunova, N.A., et al.,
Dokl. Akad. Nauk, 2002, vol. 386, no. 6, pp. 822824.