Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : August 12, 2022
Accepted : August 29, 2022
Published online : September 30, 2022
doi:10.24659/gsr.9.3_146

Original article

S-allyl cysteine increases blood flow in NO-dependent and-independent manners
Shigekazu Takemura 1, 4), Hideshi Ihara 2), Kanako Nakagawa 1), Yukiko Minamiyama 3)1) Department of Frontier Life-science, Graduate School of Medicine,
Osaka Metropolitan University, Osaka, Japan
2) Department of Biological Chemistry, Graduate School of Science,
Osaka Metropolitan University, Sakai, Osaka, Japan.
3) Food Hygiene and Environmental Health Division of Applied Life Science,
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan
4) Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine,
Osaka Metropolitan University, Osaka, Japan

Abstract

S-allylcysteine (SAC) is a bioactive substance found in aged garlic and is known to improve liver function, inhibit organ
fibrosis, and have anticancer activity. A single oral administration of SAC (10 mg/kg) increased tail vein blood flow in rats
by up to 10 % over one hour, and a 0.3% SAC mixed diet increased tail vein blood flow compared to the normal diet after
2 months. To investigate the relationship between these increases in blood flow and nitric oxide (NO), a single oral dose ofSAC (100 mg/kg) followed by a 3-day administration of L-nitroarginine (LNA, 2 mM), an NO synthase inhibitor, resulted
in the disappearance of SAC-induced increases in blood flow. Whereas, when LNA and excess L-arginine were mixed withdrinking water, the SAC-induced increase in blood flow was restored. This phenomenon was also observed in plantar blood
flow. These results indicate that the increase in blood flow induced by SAC is NO-dependent. To investigate the relationshipbetween these increases in blood flow and NO, a single oral dose of SAC (100 mg/kg) followed by a 3-day administrationof LNA (2 mM), resulted in the disappearance of SAC-induced increases in blood flow. Whereas, when LNA and excess
L-arginine were mixed with drinking water, the SAC-induced increase in blood flow was restored. This phenomenon was also
observed in plantar blood flow. These results indicate that the increase in blood flow by SAC is NO-dependent. Next, Western
blotting of vasodilator-stimulated phosphoprotein (VASP) was performed to confirm this in a vascular model. Phosphorylated
VASP (pVASP) is characterized as a substrate for both cGMP- dependent and cAMP-dependent kinases. LNA abolished
the aortic pVASP band, whereas LNA plus SAC partially recovered. Focusing on the NO-independent vasodilator H 2S,
measurements of plasma reactive persulfide showed that LNA treatment significantly reduced thiols, i.e., cysteine, cystine,
glutathione (GSH), and H2S. Of the thiols reduced by LNA treatment, the concurrent SAC application significantly restored
the reduction of cysteine and cystine, but not H2S. These results suggest that SAC may maintain nitrosothiols (compoundsof nitric oxide and thiols) that act as NO donors and indirectly increase the bioavailability of NO, resulting in blood flow
augmentation through its antioxidant capacity by free radical scavenging.

KEY WORDS: S-allyl cysteine, blood flow, nitric oxide (NO), vasodilator-stimulated phosphoprotein (VASP),

		nitrosothiol, L-nitro-arginine

Correspondent to: Associate Professor Shigekazu Takemura M.D., PhD
Department of Frontier Life-science, Graduate School of Medicine,
Osaka Metropolitan University,
Abeno-ku, Osaka 545-8585, Japan
TEL & FAX: +81-6-6645-3692 e-mail: take@omu.ac.jp
Co-authors; Ihara H, iharah@omu.ac.jp;
Nakagawa K, k-nakagawa@nichinichi-phar.co.jp;
Minamiyama Y, yukiko-m@kpu.ac.jp

Glycative Stress Research 2022; 9 (3) : 146 -157
(c) Society for Glycative Stress Research_ 14 6 _Glycative Stress Research

Introduction
S-allyl cysteine (SAC) is a biological active organosulfur
component of aged garlic extracts. SAC has been extensively
studied about tumoricidal effects, anti-inflammatory effects,
etc. We have also reported about the improvement of SAC
on lung injury 1) and fibrosis 2), liver injury 3) and fibrosis 4, 5),
and sperm dysfunction 6). Thus, SAC has a potential of
multifunctional and disease-preventing drug.
SAC decreased H 2 O 2 -induced ROS production, restoredcellular nitric oxide (NO) and cGMP level in human placenta
trophoblast cells 7). In the study, it has been reported that
SAC also possesses the ability to increase NO and cGMP
level at non-oxidative stress status in the cells. However,
it is unknown whether SAC can directly increase bloodflow by NO in vivo. Furthermore, a gas mediator other thanNO, H2S is produced from L-cysteine by cystathionine
β-synthase (CBS), cystathionine γ-lyase (CSE), and3-mercaptopyruvate sulfurtransferase (3MST) coupled withcysteine aminotransferase (CAT). It has been confirmed thatH 2 S activates endothelial NO synthase via Akt to increase
NO production 8), and this effect is thought to be one of thereasons for the synergistic effect.
Therefore, in the present study, the effects of SAC
treatment for blood flow and the mechanisms are studied inrats.

Materials and Methods
Animals

All the rats were taken care of according to thespecifications outlined in the Guiding Principles for the Careand Use of Laboratory Animals, and the study was approvedby the authorities of the local committee on experimental
animal research of Osaka City University Medical School( No.18018).

Single administration of SAC
Male Wistar rats (8 weeks age, 220 ~ 240 g; SLC, Co.,
Shizuoka) were anesthetized with 1 g/kg urethane. Then, therats were kept at 37 ℃ on the heating mat for more than 30
min, and the tail vein or pedal blood flow was measured
with MoorLDI (Moor instruments LTD, Devon, UK). The
measurement conditions were as follows: Scan Speed: 4 ms/
pixel, Scan Distance: 30 cm, Image Resolution: 126 x 54.
Changes in blood flow rate were expressed as changes insignal intensity.
To evaluate the effect of single administration of SAC,
SAC was orally administered at 10 mg to 200 mg/kg bodyweight using a gastric tube, and blood flow was measured at
15, 30, 45, 60, 75, and 90 min.

Consecutive administration of SAC
Eight-week-old male Wistar rats were intraperitoneallyanesthetized with the following mixed anesthetic agents(2. 5 mL/kg) every 4 weeks for 24 weeks. The mixed
anesthetic agents were made with total volume 2.5 mL withmedetomidine [0.15 mg/0.15 mL], midazolam [ 2 mg/0.4 mL],butorphanol [2.5 mg/0.5 mL], and saline [1.45 mL]. Rats
were maintained on a 37 ℃ warming mat for at least 30 min
to measure tail vein blood flow as described above.
To evaluate NO-dependent blood flow by SAC, 8-weekold male Wistar rats were fed with drinking water containing
L-nitro-arginine (LNA; Sigma, N5501) 40 µmol/kg/day for
3 days. In addition, to cancel the effect of LNA, excess
L-arginine (Arg; Wako, 019- 04611) at 2.4 mmol/kg/day
was mixed with LNA solution. SAC (200 mg/ kg) was
administered orally as a single dose, and then tail vein bloodflow was measured over time under urethane anesthesia. The
same experiment was performed in rats fed ad libitum with
LNA 10 µmol/kg/day and L-Arg 1 mmol/kg/day in drinking
water for 3 days. For the measurement of plantar blood flow,rats were placed face down on a plate at 37 ℃ and the heels
were fixed with tape with the soles facing upward.

Western blotting
For western blot analysis, animals were dissected 60
min after administration of 0.5 % CMC or SAC (200 mg/kg,
p.o.). Blood was drawn from the abdominal aorta into aheparinized syringe and then into ice-cold saline (50 mL).
Blood was drawn from the abdominal aorta into a heparinizedsyringe and then ice-cold saline (50 mL) was perfusedthrough the abdominal aorta. Thoracic aorta was immediately
isolated and frozen in liquid nitrogen. The samples werestored at −80 ℃ until ready for measurement.
Thoracic aorta was homogenized in RIPA buffer andWestern blotting was performed. Antibodies used were VASP
(Calbiochem, 676600, 1 : 5,000) and eNOS (endothelial nitricoxide synthase) (BD Biosciences, 610296, 1 : 5,000).

Analysis for Reactive Persulfide Species in plasma
Reactive persulfide species were analyzed by LCESIMS/MS (Liquid Chromatograph-Electrospray Ionization
Tandem Mass Spectrometer) with β-(4-hydroxyphenyl) ethyl
iodoacetamide (HPE-IAM, Molecular Bioscience, Boulder,
CO, USA) as a trapping agent for persulfide species 9, 10).
Plasma was diluted 5 times with ice-cold methanol containing
1.25 mM HPE-IAM, and incubated at 37 ℃ for one hour.
The mixture was centrifuged at 15,000 × g at 4℃ for 5min, and the resultant supernatant was diluted 10 times with
0.1% formic acid containing known amounts of isotopelabeled internal standards, which were synthesized as in ourprevious work 9, 10). HPE-IAM adducts with various reactive
sulfide species were analyzed with a Waters Alliance HPLC
system coupled to a Waters Xevo TQD ESI triple-stage
quadrupole mass spectrometer. Separation was achieved byusing a Mightysil RP-18 column (50 mm long × 2 mm innerdiameter, Kanto Chemical) with a linear 1 ~ 95 % methanol
gradient for 10 min in 0.1% formic acid at 40 ℃. The total
flow rate was 0.3 mL/min. Mass spectrometric analyses were
performed on line using ESI-MS/MS in the positive ion
mode with multiple reaction monitoring (MRM) mode. The
MRM parameters were summarized in Table 1.

Statistical Analysis
Unless otherwise stated, data are presented as means± standard error (SE). Results were considered significant_ 147 _

S-allyl Cysteine and Blood Flow: Nitric Oxide

Table 1. Parameters of Multiple reaction monitoring (MRM) mode.
AnalytePrecursor (m/z)

Product (m/z)

Cone potential (ev)Collision energy (eV)CysS-HPE-AM[ 13 C]CysS-HPE-AM
CysS-S-HPE-AM
CysS- 34 S-HPE-AM
Bis-S-HPE-AM
Bis- 34 S-HPE-AM
Bis-S 2 -HPE-AM
Bis- 34 S 2 -HPE-AM
GS-HPE-AM[ 13 C]GS-HPE-AM
GSS-HPE-AM
GS 34 S-HPE-AM

299.1
300.1
330.8
332.8
388.9
390.9
420.9
424.9
484.9
487.9
516.9
518.9

121.0
121.0
121.0
121.0
121.0
121.0
121.0
121.0
356.3
359.1
388.3
390.3

30
30
35
35
35
35
40
40
35
35
40
40

29
29
29
29
30
30
23
23
18
18
18
18

at p < 0.05. Statistical analysis was performed by using
unpaired Student’s t-tests or analysis of variance (ANOVA)
for repeated measures followed by Fisher's test for post hoc
analysis.

Results

Blood flow after a single administration of SAC

Fig. 1-a shows a representative figure of tail vein bloodflow changes. The blue to red color indicates higher blood
flow. The results of tail vein blood flow measurement are
shown in Fig. 1-b, compared with the control group thatreceived 0.5 % carboxymethyl cellulose (CMC) at 2 mL/kg
body weight, the blood flow increased in the group thatreceived SAC from 15 min after administration, and theeffect was sustained even after 75 min.

Blood flow of tail vein after long-term SAC
administration

The results of blood flow measurement are shown in
Fig. 2. The blood flow after SAC administration increased
significantly at 8, 20, and 24 weeks compared to that of rats
fed with normal diet.

Relationship with NO
Fig. 3 shows the results of the study using rats pretreatedwith LNA, an inhibitor of NO synthase; blood flow in the
LNA-treated group was not increased by SAC. Furthermore,
the LNA + Arg group did not recover to the SAC alone group(Fig. 4 ). This finding suggests that the increase in blood flowby SAC is due to NO-dependent and NO-independent effects.
Similarly, plantar blood flow was also decreased by LNA
administration, and the increase in blood flow increased
more slowly when SAC was administered than when SAC
was administered alone (Fig. 5 ).
The expression of vasodilator-stimulated phosphoprotein(pVASP/VASP), which is associated with the vasorelaxant
response, was decreased by LNA treatment and the decrease

was ameliorated by SAC treatment (Fig. 6 ). eNOS levels were
unchanged.

Involvement of H 2 S-related substances
We investigated the involvement of H2 S-related substances
as NO-independent vasodilators. The concentrations ofcysteine (Cys-SH), cystine, GSH, H 2 S, oxidized GSH (GSSH),
and H2 S2 in plasma were measured at 1 hour after a single
administration of SAC under various conditions (Fig. 7 ). In
the normal rats treated with SAC, only GSSH concentration
increased; in the LNA-treated group, Cys-SH, cystine, GSH,
and H 2 S were all significantly decreased; only Cys-SH andcystine were ameliorated by SAC.

Discussion
The present study showed that SAC treatment increased
peripheral blood flow in normal rats in both NO - dependent
and NO -independent ways. Fig. 8 shows the estimatedpathway of SAC in improving blood flow.
This aspect of the increase in blood flow was foundto be consistent with the kinetics 11), which remained highfor several hours after oral administration in proportion to
the amount of SAC in plasma (Suppl. 2). SAC ameliorates
ischemic injury in retina by inhibiting excessive elevation of
HIF-1α, VEGF, and matrix metalloprotease (MMP)-9 12).
Inhibition of VEGF inhibits metastasis of hepatocellular
carcinoma 13). However, it has not been reported that SAC
increases peripheral blood flow under normal conditions. In
humans, 600 mg of garlic was administered for one week,
and plasma NOx and IL-6 were measured. As a result, there
was a correlation between IL-6 and blood flow, so there is areport that blood flow may be increased via IL-6, but it is notclear 14).
The results of Figs. 3, 4, and 5 suggest that vasodilation
by SAC involves at least the effect of NO. However, SAC had
no effect on plasma NOx, urinary NOx, or plasma renin activity(Suppl. 1 ), nor did it affect blood pressure (data not shown).
These suggest that the changes are due to local, or perivascular,_ 14 8 _Glycative Stress Researcha)0.5 % CMC

(min)

SAC

0

15

30

45

60

0

b)

100

200

300

400

500

600

700

Relative signal intensity

1.2

1.1

**

1.0

0

15

30

45

60

75

90

Time after administration (min)Fig. 1. Blood flow in the rat tail vein.

a) Representative figures of blood flow (left vein) after SAC administration. It shows increased blood flow as
the color changes from blue to red. b) Changes in blood flow. SAC was suspended in 5 mg/mL 0.5 % CMC andadministered orally at 2 mL/kg. Blood flow in the tail vein of rats was measured under anesthesia. The blood flow
signal before treatment was set as one and the change over time was shown. Open circle ( ), 0.5 % CMC in control
rats; closed circle ( ), SAC (100 mg/kg) in control rats. Results are expressed as mean ± SE, ** p < 0.01 between
SAC (n = 6) and control (CMC, n = 17). SAC, S-allylcysteine; CMC, carboxymethyl cellulose; SE, standard error.

_ 14 9 _Changes in signal intensity

S-allyl Cysteine and Blood Flow: Nitric Oxide

1.2
0.8

*

0.4
0
- 0.4
- 0.8
0

4

8

12

16

20

Time after administration (weeks)

Fig. 2. Blood flow changes in long-term administration of SAC.

Blood flow was measured every 4 weeks under triple mixed anesthesia in rats fed a 0.3% SAC mixed diet. * p < 0.05,
SAC vs. Control, n = 6 each. Red line, SAC; Blue dotted line, Control; SAC, S-allylcysteine; SD, standard deviation.

Relative intensity of blood flow

1.4
1.3
1.2

*

1.1
1.0
0.9
0

0

15

30

45

60

75

Time after treatment (min)Fig. 3. Effects of inhibition of NO synthesis on tail vein blood flow.

*

1.2

*

1.0
0.9
0

0

SAC

1.1

LNA+Arg+SAC* *LNA+SAC

Relative intensity of blood flow

LNA (2 mM) was administered to rats as a drinking water ad libitum for 3 days. Figure indicates % changes in tail blood flow after SAC(100 mg/kg, p.o.) administration. Open circle ( ), 0. 5 % CMC in control rats; closed circle ( ), SAC (100 mg/kg) in control rats; open
square ( ), 0.5 % CMC in LNA treated rats; closed square ( ), SAC in LNA treated rats. Results are expressed as mean ± SE, n = 6 ~18, * p < 0.05. SAC vs. LNA+ SAC. SAC, S-allylcysteine; CMC, carboxymethyl cellulose; LNA, L-nitro-arginine; SE, standard error.

15

Fig. 4. Effects of inhibition of NO synthesis on tail vein blood flow.

30

(min)

LNA (2 mM) and Arg (100 mM) were administered to rats as a drinking water ad libitum for 3 days. Figure indicates changes in relative
intensity of tail blood flow after SAC (100 mg/kg, p.o.) administration. Open circle ( ), 0.5% CMC in control rats; closed circle ( ),SAC (100 mg/kg); open square ( ), 0. 5 % CMC in LNA treated rats; closed square ( ), SAC in LNA treated rats. Results are expressed
as mean ± SE, n = 3 ~ 4, * p < 0.05 vs. LNA + SAC. SAC, S-allylcysteine; CMC, carboxymethyl cellulose; LNA, L-nitro-arginine; Arg,
L-arginine; SE, standard error.

_ 15 0 _Glycative Stress Researcha)Change in signal intensity*

SAC (200 mg/kg)

*

0.04
0.00

Control (CMC)- 0.04

LNA + SAC200- 0.08- 0.12- 0.16

0

15

30

45

60

75

90

Time after treatment (min)

b)
(min)

CMC

SAC200

LNA + SAC200

0

15

30

45

0

100

200

300

400

Fig. 5. Effects of SAC on plantar blood flow.

500

600

700

a) Changes in blood flow of foot pad after a single oral administration of 200 mg/kg of SAC to rats that received LNA (0.5 mM) for 3
days. Open circle ( ), 0. 5 % CMC in control rats (n = 17); closed circle ( ), SAC (200 mg/kg) (n = 7); closed square ( ), SAC in LNA
treated rats (n = 5). Results are expressed as mean ± SE, * p < 0.05, vs LNA + SAC. b) Representative changes in plantar blood flow.
SAC, S-allylcysteine; CMC, carboxymethyl cellulose; L NA, L-nitro -arginine; Arg, L-arginine; SE, standard error.

_ 151 _

S-allyl Cysteine and Blood Flow: Nitric Oxide

Cont SAC

Cont

SAC

LNA
NOS3 140 kDa

a)Cont SAC

Cont

4KO( LNA

SAC- actin 42 kDa

NOS3 140 kDa

NOS3
NOS3
Relative Intensity
Relative Intensity

2.5

4KO(

2.0

- actin 42 kDa

2.5

1.5

2.0

1.0

1.5

0.5

1.0

0.0

0.5
0.0

Cont

SAC

Cont

Cont

SAC

Cont

SAC
LNA
SAC
LNA

b)

Cont SAC

Cont
LNA

1.2
Cont SACpVASP/ VASP
pVASP/ VASP
Relative Intensity
Relative Intensity

1.0

Cont

P-VASP 50 kDa
VASP 46 kDa
SAC

LNA

1.2
0.8

- actin
42 kDaP-VASP
50 kDaVASP 46 kDa

*

1.0
0.6

- actin 42 kDa

＃＃

0.8
0.4

*

0.6
0.2

＃＃

0.4
0.0
0.2

SAC

Cont

SAC

Cont

Cont

SAC

Cont

SAC
LNA

0.0

Fig. 6. Western blot analysis of thoracic aorta.

SAC
LNA

a) Expression of eNOS protein, b) Expression of pVASP. Rats were treated with 0. 5 mM LNA for 3 days and

0.5% CMC (Control) or 200 mg/kg SAC orally. Results are expressed as mean ± SE, n = 3 ~ 4, ## p < 0.01
vs. vehicle control, * p < 0.05 vs. LNA. eNOS, endothelial nitric oxide synthas; VASP, vasodilator-stimulated
phosphoprotein; pVASP, phosphorylated VASP; SAC, S-allylcysteine; CMC, carboxymethyl cellulose; LNA,
L-nitro-arginine; SE, standard error.

_ 152 _Glycative Stress Research

Cystine (nM)

Cys-SH (nM)
350
300
a)
350
250
300
350
200
250
300
150
200
250
100
150
200
50
100
1500
50
100
0
50
0
a)

8000

Cys-SH (nM)*

Cys-SH (nM)

*

##

*

##
##
Cont

SAC

Cont

SAC

Cont

SAC

Cont

LNA
SAC

Cont

SAC

Cont LNASAC

a)

7000

6000
7000
5000
8000
6000
4000
7000
5000
3000
6000
4000
2000
5000
3000
1000
4000
2000
30000
1000
2000
0
1000

b)0

GSH (nM)

1000
1200
800
1000
1200

c)

700
800
600
700
500
800
600
400
700
500
300
600
400
200
500
300
100
400
200
3000
100
200
0
100
0

Cont

SAC

Cont

SAC

Cont

SAC

Cont

LNA
SAC

Cont

SAC

Cont LNA SAC
LNA

GSSH (nM)

9070

GSSH (nM)

Cont

SAC

Cont

SAC

Cont

SAC

Cont

LNA
SAC

80
60
90
70
50
80
60
40
70
50
30
60
40
20
50
30
10
40
200
30
10
20
0
10

SAC

0

d)

Cont LNASAC

#

#

#

Cont

SAC

Cont

Cont

SAC

Cont

Cont

SAC

Cont LNA SAC

d)

LNA

25

SAC

H 2 S 2 (nM)2025

H 2 S (nM)#LNA

H 2 S 2 (nM)d)

H 2 S (nM)

SAC

LNA

f)

H 2 S (nM)

800

*

##
GSH (nM)
##

c)
e)
c)

##

GSSH (nM)

##

Cont

*

##

80

200
400
600

0

##

GSH (nM)

400
600
800

2000

*

b)
d)90

600
800
1000

0
200
400

Cystine (nM)

b)

LNA

a)
1200
c)

Cystine (nM)

b)
8000

H 2 S 2 (nM)

25
15
20

#

20
10
15

#

15
105

Cont
Cont
Cont

SAC
SAC
SAC

Cont
Cont

1050

SAC

50

LNA
SAC

0

ContLNASAC

Cont

SAC

Cont

Cont

SAC

Cont

Cont

SAC

Cont LNA SAC

f)Fig. 7.e)Analysis for reactive persulfide species by LC-ESI-MS/MS
in plasma.

SAC
LNA

SAC

LNA
a) Cys-SH, b) Cystine, c) GSH,LNAd) GSSH, e) H 2 S, f ) H 2 S2 . Rats were treated with 0. 5 mM LNA
for 3 days ande)0.5 % CMC (Control) or 200 mg/kg SAC orally. Results aref) expressed as mean ± SE, n = 3 ~ 4, # p < 0.05, ## p<
0.01 vs.
Fig.
7 vehicle control, * p < 0.05 vs. LNA. Cys-SH, cysteine; GSH, glutathione; GSSH, oxidized glutathione;e)SAC, S-allylcysteine; CMC, carboxymethyl cellulose; LNA,f) L-nitro-arginine; SE, standard error.
Fig. 7
Fig. 7_ 153 _

S-allyl Cysteine and Blood Flow: Nitric Oxide

SAC
Cystathionine

LNA

ROS
CSE

LNA

L-cysteine

GSH

cystine

CBS
CSE

LNA

CBS
CSE

H2S
GSH

GS-SH

H2S

VASP

Cys-SH

PKG-I

GSSNO?
Vasorelaxation

pVASP

cGMP
SAC

sGC
NO

LNA

Fig. 8. Estimated pathway of SAC in improving blood flow.

In addition to improving blood flow by increasing the bioavailability of NO through free radical scavenging, SAC
may also be a source of cysteine, which increases SH compounds and increases NO releaser by increasing nitroso
compounds, resulting in increased blood flow. It is also thought that the increase in NO releaser due to the increasein nitroso compounds may cause increased blood flow. Cys-SH, cysteine; GSH, glutathione; SAC, S-allylcysteine;
LNA, L-nitro-arginine; ROS, reactive oxygen species; GSH, glutathione; CSE, cystathionine γ-lyase; CBS,cystathionine β-synthase; VASP, vasodilator-stimulated phosphoprotein; pVASP, phosphorylated VASP; PKG-1,
protein kinase G-1; cGMP, cyclic guanosine monophosphate; sGC, soluble guanylate cyclase.

Fig. 8_ 15 4 _

4

2
0

a)
0.8

2

2W

4W

6W

0

plasma Nox (µM)

0.6
12

6W

0.7
0.6
0.5
0.4
0.3

4
0.1

0

4W

Urine NOx (µmol/mg creatinine)

0.8

0.5
10
0.4
8
0.3
6
0.2
2
0

2W

b)

Urine NOx (µmol/mg creatinine)

0.7
14

Glycative Stress Research

0.2

2W

4W

6W

2W

4W

6W

0.1
0

2W

4W

6W

c)
90
80
0.8
70
0.7
60
0.6
50
0.5
40

Plasma Renin (ng /mL/hr)
Urine NOx (µmol/mg creatinine)80
70

Suppl.
1.
60
Levelsof plasma NOx, urine NOx, and plasma renin.
50a)40 Plasma NOx, b) Urine NOx, c) plasma renin. Plasma

0.430

concentrations were measured after a control diet or a 0.3 % SAC
30
mixed
diet at the indicated time. Open column, Control; Stripe
column,SAC. Values are means ± SD, n = 6. SAC, S-allylcysteine;20
SD, standard deviation.

20
0.3
10
0.2
0
0.1
0

Plasma Renin (ng /mL/hr)

90

2W

4W

6W

2W

4W

6W

10
0

2W

4W

6W

(µM)
900

Plasma Renin(ng /mL/hr)
800

90

700

80
70

600

60
50

500

40

400

30

300

20

200

10
0

2W

100

4W

0

0

6W
30

60

90

120

Time after SAC administration (min)Suppl. 2. Effects of SAC concentration after the oral administration.

SAC (200 mg/kg) was orally treated to rats. At the indicated times, heparinized blood was collected via thefemoral vein. Values are means ± SE, n = 3 ~ 6. SAC, S-allylcysteine; SE, standard error.

_ 155 _

S-allyl Cysteine and Blood Flow: Nitric Oxide

physiological fluctuations rather than systemic changes.
H2 S has recently attracted much interest as a potent
vaso-relaxative substance that may establish itself alongsideanother gaseous signal molecule, NO. In contrast to NO, themajor source of H2 S in blood may be production by red blood
cells 15) or by vascular smooth muscle cells 16). H2 S is produced
from Cys-SH, involving the enzymes cystathionine betasynthase and cystathionine gamma-lyase (CSE). The
importance of CSE was recently demonstrated in a mouselacking CSE which showed reduced H 2 S levels anddeveloped hypertension and reduced endothelium-mediatedvasorelaxation. These data establish H2 S as a new andimportant biological signal molecule and as a new regulatorof vascular blood flow and blood pressure 17). Furthermore,
H 2 S up-regulated the effect of NO/cGMP/sGC/PKG
signaling pathways. Partially, the dilating effect of H2 S isrealized via activation of ATP-sensitive K(+) channels inplasmalemma of smooth muscle cells. In the brain, ischemia/
reperfusion injury degrades the ability of pial arteries todilate via inhibition of NO-mediated signaling pathway 18).
N-Nitro-L-arginine methylester (L-NAME) inhibitedCSE activity and H 2 S generation. Dysfunction of thevascular H 2 S synthase/H 2 S pathway was found in L-NAMEinduced hypertensive rats. Exogenous H 2 S effectively
prevented the development of hypertension induced by
L-NAME. These findings suggest that the H2 S synthase/H2 S
pathway participates in hypertension 19).

bioavailability of NO was reduced by O 2 −, but the enzymes
that scavenge it, SOD1 and SOD2, were unchanged (datanot shown). Whereas, it has been reported that S-propargylcysteine (SPRC), an H2 S donor, is a structural analogue of
SAC. It was investigated for its potential anti-cancer effecton gastric cancer cells 20). Therefore, the possibility that non
detectable H 2 S is produced around the microvasculature of
SAC administration cannot be denied, but it is not clear.
Nitrosothiols are compounds produced by the reactionof NO 2 or NO 2 − with thiols (compounds indicated byR-SH) and have vasorelaxant activity in vivo. The S-nitroso
forms of Cys-SH and GSH, as nitrosothiols, act as NOproviders (donors) in vivo to relax vascular smooth muscle
and exert vasodilation. In the present experiment, LNA
administration decreased Cys-SH, cystine, GSH, and H2 S,
while SAC improved Cys-SH and cystine; it is possible that
SAC maintains Cys-SH and cystine, which react with NO toproduce nitrosothiols.
In addition, in a double blind random clinical trial of
SAC in humans, it was reported that administration of 2
mg capsules for 4 weeks improved fatigue 21). Therefore, wewould like to study the effect of the capsules on improving
blood flow in humans.

Conclusion

However, SAC had little effect on H2 S concentrations,suggesting that LNA inhibits the synthesis of CysSH from cystathionine and that NO is not synthesized,
which may lead to a decrease in Cys-SH by increasing
ROS. The bioavailability of NO is decreased by O 2 −. The

In this study, we examined the mechanism of actionof SAC to increase blood flow, suggesting that SAC may
increase blood flow by indirectly increasing the bioavailabilityof NO through the maintenance of nitrosothiols, which act
as NO donors, and antioxidant capacity through free radical
scavenging activity.

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