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Philadelphia Biomedical Research Institute, King of Prussia, PA 19406
3To whom correspondence should be addressed. E-mail: stohnishi{at}aol.com.
ABSTRACT
The efficacy of S-allylcysteine (SAC) as a free radical
scavenger was studied using rat brain ischemia models. In a middle
cerebral artery occlusion model, preischemic administration of SAC had
the following effects: it improved motor performance and memory
impairment and reduced water content and the infarct size. In a
transient global ischemia model, the time course of free radical
(alkoxyl radical) formation as studied by electron paramagnetic
resonance (EPR) spectroscopy and
-phenyl-N-tert-butylnitrone (PBN) was
biphasic; the first peak occurred at 5 min and the second at 20 min
after reperfusion. Although SAC did not attenuate the first peak, it
did affect the second peak, which is related to lipid peroxidation. The
lipid peroxidation as estimated by thiobarbituric acid reactive
substances (TBARS) increased significantly at 20 min after reperfusion.
SAC decreased TBARS to the levels found without ischemia. These results
suggest that SAC could have beneficial effects in brain ischemia and
that the major protective mechanism may be the inhibition of free
radical–mediated lipid peroxidation.
KEY WORDS: • S-allylcysteine • brain ischemia • motor performance • memory impairment • free radicals • lipid peroxidation
Reactive oxygen species
(ROS)4are involved in cerebral ischemia, particularly in
ischemia-reperfusion (Demopoulos et al. 1977
,
Flamm et al. 1978
, Sakamoto et al. 1991
,
Siesjö 1981
). Transgenic mice with enhanced
superoxide dismutase (SOD) activity were less seriously affected in
brain ischemia-reperfusion (Chan et al. 1991
and 1993
, Kinouchi et al. 1991
).
Lipid peroxidation inhibitors were reported to have beneficial effects
against the brain subjected to ischemia-reperfusion injury
(Braughler et al. 1987
, Hall et al. 1988
,
Hall and Yonkers 1988
, Young et al. 1988
). Several free radical scavengers were reported to prevent
brain ischemic damage effectively (Abe et al. 1988
,
Carney and Floyd 1991
, Floyd and Carney 1992
, Knuckey et al. 1995
, Kurata et al. 1991
, Ohnishi et al. 1989
, Oishi et al. 1989
).
Using a spin trap, -phenyl-N-tert-butylnitrone
(PBN), Sakamoto et al. (1991)
demonstrated that oxygen
free radicals were produced during ischemia-reperfusion. Several
studies have identified the free radicals produced as primary
oxygen-centered (superoxide, hydroxyl) free radical spin adducts
(Dugan et al. 1995
, Phillis et al. 1994
,
Sakamoto et al. 1991
, Sen and Phyllis
1993
).
We focused on thiol-containing compounds found in garlic
(Allium sativum) (Forman et al. 1983
,
Haugaard et al. 1969
) because various thiol compounds
are known to prevent lipid peroxidation (Horie et al. 1989
and 1992
, Kagawa et al. 1986
, Lewin and Popov 1994
). We reported earlier that aged garlic extract (AGE) also
inhibited edema formation after rat brain ischemia (Numagami et al. 1996
). In this paper, we will characterize the
pharmacologic features of S-allylcysteine (SAC, a
water-soluble component of AGE) as a free radical scavenger in
brain ischemia.
MATERIALS AND METHODS
Drugs and administration.
Drugs and saline (control) were administered intraperitoneally 30 min before the onset of ischemia. The control comprised a saline solution (injection volume, 1.5 mL/kg body); SAC (Wakunaga, Mission Viejo, CA) was administered as a saline solution at doses of 100, 300 and 600 mg/kg body.
Two water-insoluble sulfuric compounds, diallyl sulfide (DAS) and diallyl disulfide (DADS), were purchased from Aldrich (Milwaukee, WI), dissolved in polyethylene glycol-400 and diluted in a 1% gum arabic/saline solution before intraperitoneal injection.
Animal preparation.
Adult male Sprague-Dawley rats weighing 250–300 g were purchased from Ace Animals (Boyertown, PA). They were housed in grid-floor cages (similar to the passive-avoidance test apparatus we used) for >1 wk before experiment. All animal experimental procedures were approved by the institutional animal care and use committee.
After 0.04 mg of atropine sulfate intraperitoneal injection, anesthesia was induced with 4% and maintained with 1% isoflurane in a mixture of 70% N2O and 30% O2. Throughout the experiment, head temperature was measured in temporal muscles to maintain the level between 37 and 37.5°C with the use of a heat lamp.
Focal ischemia study.
Focal cerebral ischemia was produced by middle cerebral artery
occlusion (Chen et al. 1986
) as follows: the right
common carotid artery (CCA) and the right middle cerebral artery (MCA)
were ligated permanently. The left CCA was then occluded for 1 h
with a clip. Sham-operated rats were prepared as above but did not
undergo MCA ligation and CCA clipping.
The water content of both right and left hemispheres was measured using a dry-weight method 3 d after the ischemic insult. The values were calculated as (wet weight - dried weight)/(wet weight).
We examined motor performance with an inclined plane test, a balance
beam test and a prehensile test (Combs and D’Alecy 1987
, Tomigaga and Ohnishi 1989
). The
total motor score was the sum of each score and therefore ranges from 0
to 12.
Rats were tested in a step-through type passive avoidance test
(Ader et al. 1972
). The apparatus consists of two
compartments, one that is illuminated and one that is dark; each is
equipped with a grid floor and separated from the outside by a
guillotine door. This apparatus was placed in a dark room. All tests
were conducted between 4 and 8 h after the start of the dark cycle
in the caging facility. The acquisition trial was conducted 4 h
before surgery (d 0) as follows: a rat was placed in the illuminated
compartment and allowed to enter the dark compartment; 3 s after
the rat entered the dark compartment, foot shock (50 mA, 3 s) was
delivered through the grid floor. The retention test was conducted
24 h after the acquisition trial (d 1). The rat was placed again
in the illuminated compartment and the latency to enter the dark
compartment was measured. If the rat avoided entry for >900 s, a
ceiling value of 900 was assigned.
The area of infarction was determined using the 2,3,5-triphenyltetrazolium chloride (TTC) staining method for viable mitochondrial dehydrogenase activity. Three days after surgery, the brain was removed under pentobarbital anesthesia. Coronal brain slices (2 mm thick) were then stained in a 2% TTC/100 mmol/L phosphate buffer (pH 7.4). The percentage of the infarct area (no TTC staining area) with respect to the total area was calculated with an appropriate correction in accordance with the degree of edema.
Global ischemia study.
Forebrain ischemia was produced by the combination of bilateral CCA
occlusion and hemorrhagic hypotension (Smith et al. 1984
). In brief, the bilateral femoral arteries were canulated
with two lines, one for blood withdrawal and the other for continuous
monitoring of mean arterial blood pressure (MABP) and blood gas
analysis. Heparin (150 IU/kg) was injected to prevent
coagulation. Bilateral CCA were then occluded by clips, and systemic
hypotension (MABP = 30 mm Hg) was produced by the withdrawal of
arterial blood. A syringe was placed at a height equivalent to 30 mm Hg
to store the blood and at the same time to maintain a constant MABP.
Isoflurane was discontinued after a flat electroencephalogram (EEG) was
observed. After 20 min of ischemia, the clips were detached and the
blood in the syringe was reinfused. Then, the rat was maintained for
various reperfusion periods (0, 1, 3, 5, 10, 20, 40 and 60 min).
Sham-operated (no ischemia) rats were prepared as above but did not
undergo CCA occlusion, blood withdrawal and reperfusion.
Spin trapping.
PBN was purchased from OMRF Spin Trap Source (Oklahoma City, OK), and
the ex vivo spin trapping was performed according to Sakamoto et al. (1991)
with some modifications. Brain samples were
collected into 100 mmol/L PBN as follows: at the beginning of surgery,
a 3-mm diameter ring-shaped groove was made on the rat skull with a
dental drill under saline irrigation (the dura was not exposed at this
point). After various reperfusion periods, the bone inside the groove
was removed using a dental hook. The brain tissue was then collected
into a dounce-type homogenizer containing 2.5 mL ice-cold 100
mmol/L PBN using negative pressure suction (from a vacuum pump) for
4 s. The brain suction began at the portion under the parietal
bone and terminated at the cranial base. The tissue was homogenized
immediately. Thus, the entire procedure was performed in a very short
time; the time between the brain suction and the brain homogenization
was 6–8 s. The homogenate was processed following the method of
Mergner et al. (1991)
, with some modification provided
by Dr. J. H. Kramer (personal communication, George Washington
University). In brief, the homogenate was mixed immediately
with 2.0 mL deoxygenated HPLC-grade toluene and centrifuged at 7000
x g for 10 min at 5°C. Then, the sample was
frozen at -80°C. The sample was thawed and centrifuged again at 7000
x g for 10 min at 5°C to reproducibly separate
the toluene layer from tissue residuals. The residuals were dried and
their dry weight was measured.
The supernatant (1.5 mL toluene) was concentrated to 0.5 mL under nitrogen gas flow at 0°C and transferred into an electron paramagnetic resonance (EPR) spectroscopy quartz tube for analysis. EPR measurements were performed at room temperature using a Varian E-4 spectrometer at the following settings: gain, 2.0 x 104; microwave power 20 mW; modulation amplitude 0.2 mT; time constant 3.0 s; scan range 10 mT; scan time 8 min. Two scans were made and the signals were averaged. Spin adduct formation was calculated as follows: at the midfield doublet signals, the peak-to-peak heights of both signals were added and regarded as the intensity of free radical signals. The signal intensity divided by the tissue dry weight was considered to represent the specific radical intensity.
Lipid peroxidation.
The formation of thiobarbituric acid reactive substance (TBARS) was
determined as described by Aust (1985)
.
Statistical analysis.
Results are given as means ± (SEM), except for the motor scores, which are displayed as median scores. For the analysis of three or more groups, one-way ANOVA with Fisher’s protected least significant difference as post-hoc test was used, except for the motor performance test, for which the Kruskal-Wallis test followed by Mann-Whitney’s U test was used. For the analysis between two groups, Student’s t test was used. A P-value of < 0.05 was considered significant.
RESULTS
The effects of SAC on the water content were studied at three
dosages, i.e., 100, 300 and 600 mg/kg. The water content of the control
group (saline-treated) was 81.50 ± 0.07%. As shown in
Figure 1
, a significant effect was observed at the doses of 300 and 600 mg/kg,
suggesting that the effect reached a plateau at the lower dose.
|
).
|
)
|
)
|
). The percentage of the infarct volume in reference to the entire brain
volume (calculated from the values for all slices) was 13.5 ± 1.0% for the control group and 8.6 ± 0.9% for the
SAC-treated group, a 36% reduction (P < 0.01;
Fig. 4B
).
|
shows typical ERR spectra of spin adducts obtained from brain cortex
extracts. They are from the brain of (panel A)
the control (saline-treated) rat; (panel B) the SAC (300
mg/kg)-treated rat, before ischemia, at 20 min ischemia/5 min
reperfusion and at 20 min ischemia/20 min reperfusion. In panel
C, alkoxyl radicals were chemically generated from cumene
hydroperoxide and Fe2+, which was trapped by PBN
and extracted by toluene. The spectra obtained from the brain exposed
to ischemia reperfusion (Fig. 5A
and B) had
parameters of aN = 13.5–13.8 G and
aH = 2.0–2.2 G. The PBN adduct signal from the
chemically produced alkoxyl radical (aN = 13.7 G
and aH = 2.2 G, Fig. 5A
) was
essentially identical to that obtained from the brain exposed to
ischemia reperfusion. We assumed that the sum of both peaks of the
doublet at the midfield divided by the dried tissue weight was
proportional to the amount of free radicals produced in the brain
tissue, and plotted the time course of the spin-adduct formation
(Fig. 6
). The figure shows that upon reperfusion, there occurred a burst of
spin-adduct formation, which reached a peak at 5 min (P
< 0.01 compared with the data for no ischemia). The
spin-adduct formation decreased once at 10 min, but increased again
to form the second peak at 20 min (P < 0.001). The
pretreatment with SAC (300 mg/kg) significantly reduced the
spin-adduct formation not at 5 min of reperfusion but at 20 min of
reperfusion (P < 0.01), whereas it did not change the
baseline (before ischemia) of the spin-adduct formation. There was
no significant difference among the weights of all brain specimens.
|
|
, in the control group, TBARS increased significantly after 20 min of
reperfusion compared with the group without ischemia. The preischemic
administration of SAC (300 mg/kg) prevented the increase in TBARS,
representing a decrease of 22% (P < 0.05).
|
DISCUSSION
Thiol group–containing agents such as cysteine (Deneke et al. 1983
, Forman et al. 1983
, Landolt et al. 1992
, Lyrer et al. 1991
, Uemura et al. 1991
), thioproline (Fuente et al. 1993
),
2-mercaptopropionylglycine, captopril (Ayene et al. 1993
, Suzuki et al. 1993
) and
N-acetylcysteine (Aruoma et al. 1989
,
Ferrari et al. 1991
, Knuckey et al. 1995
)
are reported to have free radical–scavenging effects. These
investigators suggested that the scavenging mechanisms are based on an
increased amount of reduced glutathione (GSH). We confirmed through
this study that SAC, a cysteine-derived compound and one of the
components of Allium sativum (garlic), had therapeutic value
in the rat focal ischemia because it reduced edema formation, the
infarction area, motor dysfunction and memory impairment.
In an attempt to demonstrate the effects of SAC as a free radical scavenger, its effect on the production of free radicals was measured in brain ischemia-reperfusion in a transient global ischemia model. This model was chosen because it produces a greater amount of free radicals; it can provide well-defined ischemia-reperfusion and expose a wider area to ischemia-reperfusion.
It is interesting to note that not all sulfur-containing components in garlic are protective. Two lipid-soluble compounds, DAS and DADS, exacerbated ischemic brain injury in contrast to SAC, which is water soluble.
Recently, Kramer et al. (1994)
used toluene for
spin-adduct extraction. They found that lipid peroxides in venous
blood formed PBN spin-adducts after heart ischemia-reperfusion
and that the time course had two peaks, i.e., the first, a few minutes
after the onset of reperfusion, and the second, after 
20 min. They
reported that the signal of the spin adducts was consistent with
PBN/alkoxyl radicals (Kramer et al. 1994
, Mergner et al. 1991
, Tortolani et al. 1993
).
Our results show that, like the reperfused ischemic heart, the
PBN/alkoxyl adducts were the dominant signal and that the time course
of adduct production had two peaks after the onset of reperfusion,
first 5 min and then 20 min after reperfusion (Fig. 5A
, B
C
). A possible explanation for a phenomenon of
transient suppression between the two peaks (at 10 min reperfusion) is
that the propagation of lipid peroxidation was temporarily inhibited by
endogenous free radical scavengers, such as ascorbate and
-tocopherol. However, the capacity of endogenous scavengers is not
sufficient to terminate the reaction completely because of their
limited amounts, thereby allowing the production of the second peak.
Thus, SAC seems to work as a free radical scavenger.
Primary free radicals, such as superoxide and hydroxyl radical, are so
unstable that the extent to which they could cause injury may be
limited. However, the appearance of secondarily formed lipid free
radicals, such as alkoxyl radicals, may result in extensive oxidative
damage in the cells because alkoxyl radicals can abstract hydrogen
atoms from lipids and lead to further lipid peroxidation
(Halliwell 1992
). Thus, the appearance of alkoxyl
radicals strongly suggests that lipid peroxidation took place.
Therefore, this method seems to permit us to achieve the following:
1) detect and partially identify PBN adducts as alkoxyl
radicals; 2) establish the time course of PBN adduct
production; and 3) demonstrate the role of free radical
scavengers in the cascade reaction leading to the formation of alkoxyl
radicals.
Because free radicals are unstable, a suction method, which removes and homogenizes the brain tissue in the ice-cold PBN solution within several seconds, was devised. This new method enabled us to trap free radicals in a reproducible fashion in the rat brain exposed to ischemia-reperfusion.
The data demonstrated that the administration of SAC inhibited only the
second peak. We hypothesized that the first peak is the result of a
direct response to primary free radical attack, which initiates the
lipid peroxidation during the subsequent reperfusion periods, but that
the second peak represents the propagation of lipid peroxidation after
the burst of primary free radical production has ceased. To examine
this hypothesis, the effects of SAC on the lipid peroxidation during
ischemia reperfusion were studied. The amount of TBARS was measured as
an index of lipid peroxidation. We found that TBARS increased at 20 min
but not at 5 min after reperfusion. This result agreed with results of
previous investigators (Sakamoto et al. 1991
,
Yoshida et al. 1980
). SAC inhibited TBARS formation at
20 min after reperfusion. Therefore, the results again suggest that SAC
works as a free radical scavenger, namely, a propagation inhibitor of
lipid peroxidation.
FOOTNOTES
1 Presented at the conference "Recent Advances
on the Nutritional Benefits Accompanying the Use of Garlic as a
Supplement" held November 15–17, 1998 in Newport Beach, CA. The
conference was supported by educational grants from Pennsylvania State
University, Wakunaga of America, Ltd. and the National Cancer
Institute. The proceedings of this conference are published as a
supplement to The Journal of Nutrition. Guest editors:
John Milner, The Pennsylvania State University, University Park, PA and
Richard Rivlin, Weill Medical College of Cornell University and
Memorial Sloan-Kettering Cancer Center, New York, NY. 
2 Present address: Division of Neurosurgery,
Institute of Brain Diseases, Tohoku University School of Medicine,
Sendai, Japan.
4 Abbreviations: CCA, common carotid artery; DADS,
diallyl disulfide; DAS, diallyl sulfide; EPR, electron paramagnetic
resonance; GSH, reduced glutathione; MABP, mean arterial blood
pressure; MCA, middle cerebral artery; PBN,
-phenyl-N-tert-butylnitrone; ROS,
reactive oxygen species; SAC, S-allylcysteine; SOD,
superoxide dismutase; TBARS, thiobarbituric acid reactive substances;
TTC, 2,3,5-triphenyltetrazolium chloride.
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