The Antihypertensive Effect of Black Garlic

(Allium Sativum) in Spontaneously
Hypertensive Rats via Scavenging of Free
Radicals
Yelian Miao*1, Jieyu Chen2, Guangyong Zhou3, Xiaobian Xu4, Qimei Zhang5, Jining Wang6
*1,3,4,5College of Food Science and Light Industrial Engineering, Nanjing University of Technology, Jiangsu 211816,
China
2 Faculty of Bioresource Science, Akita Prefectural University, Akita 010-0195, Japan
6 School of Economics and Management, Nanjing University of Technology, Jiangsu 211816, China
*1ylmiao@njut.edu.cn
Received 17 Sep, 2013; Accepted 10 Nov, 2013; Published 10 Jan, 2014
© 2014 Science and Engineering Publishing Company
Abstract
Black garlic (Allium sativum) is a new garlic product with
high free-radical-scavenging ability. In the present study, the
antihypertensive effect of black garlic was investigated in
vivo using spontaneously hypertensive rats (SHRs, 185±12
mm Hg) as the test animals. Total antioxidant capacity (TAOC)
and malondialdehyde (MDA) content in both plasma
and hypothalamic paraventricular nucleus (PVN) of the rats
were measured to explore underlying biochemical
mechanism of the antihypertensive effect. The
administration of black garlic for 14 days significantly
lowered the blood pressure of SHRs to 121±10 mm Hg (with
a decline rate of 34.6% in average), while it did not affect the
normal blood pressure of Wistar rats. The black garlic had
more antihypertensive effect on SHRs than fresh garlic. In
the plasma of SHRs receiving the black garlic, the T-AOC
increased from 4.2±1.0 U/mL to 5.4±1.1 U/mL (with an
increase rate of 28.6% in average), and the MDA content
decreased correspondingly from 10.2±2.2 nmol/mL to 7.9±0.7
nmol/mL (with a decrease rate of 22.5% in average). In the
PVN of SHRs receiving the black garlic, the T-AOC
increased from 4.4±0.7 U/mg-protein to 7.2±1.6 U/mg-protein
(with an increase rate of 63.6% in average), and the MDA
content decreased correspondingly from 8.2±1.5 nmol/mgprotein
to 3.9±1.2 nmol/mg-protein (with a decrease rate of
52.4% in average). The findings indicated that the black
garlic exerts a potential antihypertensive effect through
scavenging excessive oxygen free radicals (OFRs) in the
plasma and PVN of SHRs.
Keywords
Black Garli, Antioxidant Activit, Antihypertensive Effect, Oxygen
Free Radical, Hypertension
Abbreviations
CSAR, cardiac sympathetic afferent reflex
FRAP, ferric reducing ability of plasma
MDA, malondialdehyde
OFRs, oxygen free radicals
PVN, paraventricular nucleus
SHRs, spontaneously hypertensive rats
T-AOC, total antioxidant capacity
TBA, thiobarbituric acid
Introduction
Hypertension is a commonly occurring cardiovascular
disease in the world. It is estimated that by 2025, the
incidence of hypertension will increase to 24% in
developed countries and to 80% in developing
countries (Messerli et al., 2007; Quiñones et al., 2013).
Hypertension may be caused by oxygen free radicals
(OFRs) in the human body (Russo et al., 1998; Bogdan
et al., 2000). Antioxidant therapy helps to scavenge
excessive OFRs, and to prevent the pathogenesis of
hypertension and its complications (Vaziri et al., 2000;
Ortiz et al., 2001). Recent research also indicated that
excessive OFRs in hypothalamic paraventricular
nucleus (PVN) contribute to the potentiation of
vasoconstrictor angiotensin II (Ang II) and cardiac
sympathetic afferent reflex (CSAR), which may induce
www.seipub.org/rhn Research in Health and Nutrition (RHN) Volume 2, 2014
6
hypertension (Han et al., 2007; Grassi et al., 1999). In
contrast, microinjection of tempol (i.e. a superoxide
anion scavenger) into the PVN of hypertensive rats
can abolish the potentiation of Ang II and CSAR, and
lower the high blood pressure (Han et al., 2011; Koga
et al., 2008).
Black garlic (Allium sativum) is a new garlic product
processed by the fermentation of fresh garlic in a
temperature- and humidity–controlled room for a
month. It is black in color with a fruit-like sweetness
and non-irritating order. In particular, black garlic has
outstanding antioxidative activity since it is rich in
bioactive compounds such as ajoene, S-allyl-L-cysteine
and polyphenols (Zhou et al., 2010; Wang et al., 2010;
Kim et al., 2012). Experimentation on rats showed that
black garlic has a higher ability to increase antioxidase
activity, and to decrease malondialdehyde (MDA)
content in the blood and the liver tissue, in
comparison with fresh garlic (Apitz-Castro et al., 1992;
Zhu et al., 2008). The heat-extracts of black garlic
enforces anti-tumor activity with a 50% cure rate of
BALB/c mouse fibrosarcoma since it enhances the
cellular immunity by raising the activity of natural
killer cells (Sasaki et al., 2007). Black garlic also has
favourable hepatoprotective, nephroprotective,
hypolipidemic, and antiobesity effects, but no
hypoglycemic effects (Jung et al., 2011). A black garlic
formulation containing 10% black garlic extract is
effective in protecting skin from UVB photodamage
(Kim et al., 2012).
In the present study, black garlic with high freeradical-scavenging
ability was prepared, and its
antihypertensive effect was investigated in vivo using
spontaneously hypertensive rats (SHRs) as the test
animals. Total antioxidant capacity (T-AOC) and MDA
content in both plasma and PVN of the rats were
measured to explore underlying biochemical
mechanism of the antihypertensive effect.
Materials and Methods
Garlics
Fresh garlic (Allium sativum) with purple skin,
produced in Henan province, was obtained from a
local market in Nanjing, China. Black garlic was
prepared by maintaining the fresh garlic in an
incubator with a temperature of 65℃ and a relative
humidity of 70% for 30 days (Miao, 2006).
The content of main compounds and the free-radicalscavenging
ability of fresh and black garlics are shown
in Table 1. Moisture was measured by drying a 5 g
garlic sample at 105℃ for 24 h. Alliin and S-allyl-Lcrysteine
were measured using a high performance
liquid chromatograph (HPLC) system (Ichikawa et al.,
2006; Kodera et al., 2002). Total polyphenols were
measured with the Folin–Ciocalteu method (Li et al.,
2009; Kim et al., 2012). Ajoene was measured using a
LC-MS system and its amount was expressed by the
peak area of LC chromatogram (Iberl et al., 1990; Fu,
2006). The free-radical-scavenging ability was
determined with the ABTS method (Re et al., 1999;
Zulueta et al., 2008). The content of main compounds
and the free-radical-scavenging ability were expressed
on the basis of dry garlic. The black garlic contained
only little alliin and other volatile organic sulfides, so
that it did not have unacceptable odor and sharp taste
of fresh garlic. Moreover, the black garlic had a freeradical-scavenging
ability of 63.4 g-Trolox/kg, which
was about 9 times higher than that of fresh garlic. The
black garlic’s high ability of free-radical-scavenging
resulted from remarkable increases in the content of
bioactive compounds, i.e. total polyphenols, ajoene
and S-allyl-L-cysteine.
TABLE 1 THE CONTENT OF MAIN COMPOUNDS AND THE FREE-RADICALSCAVENGING
ABILITY OF GARLICS
Garlic Fresh garlic Black garlic
Moisture (%,w.b.) 66.2 50.3
Alliin (%,d.b.) 3.8 0.7
Total Poly- phenols (g/kg) 0.8 4.7
Ajoene (mAU•s/ kg) 0.45×106 4.44×106
S-allyl-L- cysteine (mg/kg) 21.0 106.1
Free-radical- scavenging
ability (g-Trolox/kg) 7.1 63.4
Preparation of Animal Model
Normotensive Wistar rats (male: n=18, female: n=18)
and spontaneously hypertensive rats (SHRs, male:
n=18, female: n=18), weighing approximately 150-260 g
(20-week-old), were supplied by Shanghai Laboratory
Animal Center of Chinese Academy of Sciences. The
male Wistar rats, female Wistar rats, male SHRs and
female SHRs were housed separately in polycarbonate
boxes, and had free access to food and tap water. The
animal room was maintained at approximately 22°C
and 50% humidity with a 12-h light/dark cycle. All of
animal experimental design was approved by the
Institutional Animal Care and Ethical Committee of
Nanjing Medical University.
Rat Grouping and Garlic Administration
The Wistar rats, as well as SHRs, were randomly
Research in Health and Nutrition (RHN) Volume 2 Issue 1, January 2014 www.seipub.org/rhn
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divided into three groups (6 male and 6 female rats in
each group), i.e. (1) Group VI: rats receiving the
administration of deionized water; (2) Group FG: rats
receiving the administration of fresh garlic; (3) Group
BG: rats receiving the administration of black garlic. In
addition, the data obtained from Wistar rats and SHRs
before the treatment was used as the control values,
respectively.
The flesh portions of fresh and black garlics were
mashed to puree in a mortar. 100 mL of garlic
homogenate with a concentration of 16% (w/v) were
prepared by mixing 47.4 g of the fresh garlic puree
(with a moisture content of 66.2%,w.b.) or 32.2 g of the
black garlic puree (with a moisture content of
50.3%,w.b.) with deionized water, and stored at -10°C.
The frozen garlic homogenates were thawed by tap
water before being used.
The deionized water, fresh garlic homogenate and
black garlic homogenate were administrated to rats
once a day at 10:00 AM for 14 days, using an injector
equipped with an intragastric administration needle.
The administration volume was 2 mL each time for a
rat weighing 200-260 g, and 1.5 mL each time for a rat
weighing 150-200 g. The average dose of garlic in dry
matter was 1.38 g/kg rat. It corresponded to that an
adult weighing 70 kg receives 10 g of dry garlic, based
on the assumption that the dose for adults is one tenth
of that for rats (Chen, 2006).
Measurement of the Body Weight, Blood Pressure and
Heart Rate of Rats
Body weight was measured every three days using an
electronic balance. Systolic blood pressure and heart
rate were measured for the awake rats at 24 h before
the first administration and 24 h after the last
administration, with the caudal artery method (Li,
2002). The testing apparatus consisted of a tail sleeve,
a pressure sensor, a bridge amplifier (QUAD bridge,
AD Instruments, Australia) and a PowerLab data
analytical processing system (8SP, AD Instruments,
Australia). The measurement for each rat was in
triplicate, and their average was used.
Biochemical Determinations for the Plasma and
Hypothalamic PVN of Rats
The T-AOC and MDA content in plasma and PVN of
rats were determined. In order to determine T-AOC
and MDA content in plasma, 0.2 mL of blood was
collected from each rat by tail venipuncture under the
anesthetized condition with intraperitoneale (i. p.)
injection of chloral hydrate (400 mg/kg), at 24 h before
the first administration and 24 h after the last
administration. Then, the blood samples were
centrifuged at 3000 r/min and 4°C for 10 min. The
plasma (supernatant) was separated and stored at –
80°C until being assayed. In order to determine TAOC
and MDA content in PVN, all rats were
sacrificed under the anesthetized condition with
chloral hydrate (400 mg/kg, i. p.) at 24 h after the last
administration. The brains were taken out quickly,
and coronally cut in 500 μm thick slices using a
cryostat. PVN (0.84 mm anterior and 0.60 mm
posterior to the bregma) regions in the frozen slices
were micro-dissected using a 26-gauge stainless steel
tubing, and then stored at -80°C until being assayed.
Before the assay, the frozen plasma was homogenized
and thawed in ice water. The frozen PVN was
homogenized by sonication with a pH 7.5
homogenization buffer (containing 140 mM NaCl, 5
mM KCl, 0.8 mM MgC12, l.8 mM CaC12, l mM
Na2HPO4, 25 mM HEPES, l% glucose) in ice water,
followed by centrifugation at 3000 r/min and 4°C for
10 min to attain the supernatant for the assay.
For the plasma and PVN, the T-AOC was determined
with the FRAP (ferric reducing ability of plasma)
method (Benzie and Strain, 1996), and the MDA
content was determined with the TBA (thiobarbituric
acid) method (Placer et al., 1966). The soluble protein
contained in PVN was measured with the Bradford
method (Bradford, 1976). Commercial assay kits
(Nanjing Jiancheng Bioengineer Institute, China) were
used for the biochemical determination. T-AOC and
MDA content in the plasma was expressed as U/mL
and nmol/mL, while T-AOC and MDA content in the
PVN was expressed as U/mg-protein and nmol/mgprotein,
respectively. Data were obtained from each
group of twelve rats throughout the experiments.
Statistical Analysis
The group data were expressed as mean ± SD
(standard deviation), and analyzed by one-way
analysis of variance (ANOVA) using State 9.2 software
(STATA Corporation, USA). Differences between the
means were considered to be significant when p<0.05.
Results and Discussion
Body Weight and Heart Rate of Rats
Both the Wistar rats and the SHRs originally had a
body weight of 217±30 g. The body weight did not
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8
change significantly during the administration of
deionized water, fresh and black garlics (Table 2).
The heart rate of rats before and after the
administration is shown in Fig. 1. It was 352±26
times/min in the Wistar rats and 393±33 times/min in
the SHRs before the administration. The heart rate in
both the Wistar rats and the SHRs did not change
significantly after the administration. In general, the
heart rate of adult Wistar rats ranges from 250
times/min to 400 times/min (Zhang and Sannajust,
2000), while that of adult SHRs ranges from 350
times/min to 400 times/min (Li, 2002). It was
confirmed that both the Wistar rats and the SHRs used
in the present study had a normal heart rate during
the administration.
TABLE 2 BODY WEIGHT (MEAN±SD) OF RATS DURING THE
ADMINISTRATION (G)
Time
(d)
Wistar rats SHRs
VI FG BG VI FG BG
0 221±11 217±18 213±24 208±31 229±44 214±54
1 220±10 217±16 214±26 208±36 230±43 214±58
4 219±11 214±18 211±25 204±27 225±46 215±48
7 217±12 211±22 207±23 206±23 229±48 218±47
11 221±12 211±21 209±26 212±25 228±43 219±48
14 221±14 214±17 210±24 211±23 227±43 219±49
VI, FG and BG: rats receiving the administration of deionized water,
fresh garlic and black garlic, respectively.
150
250
350
450
550
Wistar rats SHRs
Heart rate（times/min ）
CR VI FG BG CR VI FG BG
++
FIG. 1 HEART RATE (MEAN±SD) OF RATS BEFORE AND AFTER
THE ADMINISTRATION
CR: control before the administration; VI: after the administration of
deionized water; FG: after the administration of fresh garlic; BG:
after the administration of black garlic; ++: p<0.05 vs. CR of Wistar
rats
Lowering of High Blood Pressure by Black Garlic
The blood pressure of rats before and after the
administration is shown in Fig. 2. Before the
administration, the Wistar rats and the SHRs had a
blood pressure of 115±11 mm Hg and 185±12 mm Hg,
respectively. After the administration, the blood
pressures in Groups VI, FG and BG of Wistar rats, and
in Group VI of SHRs were almost the same as those
before the administration, respectively. However, the
blood pressure of SHRs declined significantly to
164±11 mm Hg (with a decline rate of 11.4% in average)
in Group FG, and to 121±10 mm Hg (with a decline
rate of 34.6% in average) in Group BG. The difference
between the blood pressures in Group BG and Group
FG of SHRs was significant (p<0.05). It should be noted
that the high blood pressure of SHRs was lowered to
that of Wistar rat by administrating of the black garlic.
The black garlic had more antihypertensive effect on
SHRs than the fresh garlic.
50
100
150
200
250
300
Wistar rats SHRs
Blood pressure（mmHg ）
CR VI FG BG CR VI FG BG
££
$$ ££
$$
&&
++
FIG. 2 BLOOD PRESSURE (MEAN±SD) OF RATS BEFORE AND
AFTER THE ADMINISTRATION
CR: control before the administration; VI: after the administration of
deionized water; FG: after the administration of fresh garlic; BG:
after the administration of black garlic; ++: P<0.05 vs. CR of Wistar
rats; ££: P<0.05 vs. CR of SHRs; $$: P<0.05 vs. VI of SHRs; &&: P<0.05
vs. FG of SHRs.
It has been reported that adult normotensive Wistar
rats have a blood pressure of about 120 mm Hg, while
adult SHRs have a blood pressure higher than 170 mm
Hg (Shi, 1989). An agent is considered to be
antihypertensive when the high blood pressure of
hypertensive rats declines to a normal blood pressure
level or by an amount of more than 20 mm Hg after its
administration (Chen, 2006).
Change of Antioxidative Activity in Plasma
Figure 3 shows the plasma T-AOC and MDA content
of rats before and after the administration. Before the
administration, the SHRs had a lower plasma T-AOC
of 4.0±1.0 U/mL and a higher plasma MDA content of
10.2±2.2 nmol/mL, in comparison with those of Wistar
rats (plasma T-AOC: 4.9±0.7 U/mL, plasma MDA
content: 6.0±0.8 nmol/mL), respectively (p<0.05).
Research in Health and Nutrition (RHN) Volume 2 Issue 1, January 2014 www.seipub.org/rhn
9
After the administration, the plasma T-AOC in Group
BG of SHRs increased to 5.4±1.1 U/mL (with an
increase rate of 28.6% in average) (Fig. 3A).
Correspondingly, the plasma MDA content in Group
BG of SHRs decreased to 7.9±0.7 nmol/mL (with a
decrease rate of 22.5% in average) (Fig. 3B). In contrast
to Group BG of SHRs, other groups did not have
significant change in the levels of plasma T-AOC and
plasma MDA content. It was indicated that the
antioxidative activity of plasma in SHRs was increased
by the administration of black garlic.
0
2
4
6
8
10
Wistar rats SHRs
T-AOC（U/mL）
CR VI FG BG CR VI FG BG
++
££
$$
&&
A
0
6
12
18
MDA（nmol/mL）
CR VI FG BG CR VI FG BG
++
B
££
$$
&&
Wistar rats SHRs
FIG. 3 PLASMA T-SOD (A) AND MDA CONTENT (B) (MEAN±SD)
OF RATS BEFORE AND AFTER THE ADMINISTRATION
CR: control before the administration; VI: after the administration of
deionized water; FG: after the administration of fresh garlic; BG:
after the administration of black garlic; ++: P<0.05 vs. CR of Wistar
rats; ££: P<0.05 vs. CR of SHRs; $$: P<0.05 vs. VI of SHRs; &&: P<0.05
vs. FG of SHRs.
Change of Antioxidative Activity in PVN
Figure 4 shows the PVN T-AOC and MDA content of
rats after the administration. The PVN T-AOC was
almost the same at 4.4±0.7 U/mg-protein in Groups VI,
FG and BG of Wistar rats, and Group VI of SHRs. It
increased to 4.8±1.0 U/mg-protein in Group FG of
SHRs, and to 7.2±1.6 U/mg-protein (with an increase
rate of 63.6% in average) in Group BG of SHRs (Fig.
4A). The differences between PVN T-AOC levels in
Group BG of SHRs and other groups were significant
(p<0.05).
The PVN MDA content levels were roughly the same
at 3.7±0.6 nmol/mg-protein in Groups VI, FG and BG
of Wistar rats. Group VI of SHRs had a PVN MDA
content of 8.2±1.5 nmol/mg-protein, which was much
higher than that in Group VI of Wistar rats (p<0.05).
The high PVN MDA content in Group VI of SHRs
decreased to 6.0±1.0 nmol/mg-protein (with a decrease
rate of 26.8% in average) in Group FG, and to 3.9±1.2
nmol/mg-protein (with a decrease rate of 52.4% in
average) in Group BG (Fig. 4B). The difference
between PVN MDA content levels in Group BG and
Group FG of SHRs was significant (p<0.05). It should
be noted that the PVN MDA content of SHRs was
decreased to that of Wistar rats by the administration
of black garlic.
0
4
8
12
Wistar rats SHRs
T-AOC（U/mg-protein ）
VI FG BG VI FG BG
$$
&&
A
0
4
8
12
Wistar rats SHRs
MDA（nmol/mg-protein ）
VI FG BG VI FG BG
$$
$$
&&
##
B
FIG. 4 PVN T-SOD (A) AND MDA CONTENT (B) (MEAN±SD) OF
RATS AFTER THE ADMINISTRATION
VI: administration of deionized water; FG: administration of fresh
garlic; BG: administration of black garlic; ##: P<0.05 vs. VI of Wistar
rats; $$: P<0.05 vs. VI of SHRs; &&: P<0.05 vs. FG of SHRs.
Mechanisms for the Antihypertensive Effect of Black
Garlic
OFRs are metabolites of oxygen in the human body,
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10
including superoxide free radicals (O2-
), superoxide
anions (O2-
), hydroxyl free radicals (OH), hydroxyl
anions (OH-
), hydrogen peroxide (H2O2), singlet
oxygen (1O2), lipidic superoxide free radicals (·ROO)
and alcoxyl free radicals (·RO). In addition, brain is an
organ with the highest metabolism rate in the body.
Most of the energy required for brain activity is
provided by aerobic metabolism. Although the mass
of brain tissue is only 2% of the body, the brain
consumes 20% of total oxygen for the body. Because
50% of the consumed oxygen is reduced to OFRs, the
brain tissue is subjected to more attack of OFRs than
the other organs (Bosnjak et al., 2003).
Based on the experimental results of present study and
the medical evidences reported previously (Han et al.,
2007; Grassi et al., 1999; Han et al., 2011; Koga et al.,
2008), it may be presumed that the existence of
excessive OFRs in the plasma and PVN of SHRs is the
main reason for high blood pressure. When the black
garlic is given to SHRs, its bioactive compounds, such
as polyphenols, ajoene and S-allyl-L-crysteine, pass
through the blood brain barrier into PVN to scavenge
the excessive OFRs, which results in the decline of
high blood pressure via abolishing the potentiation of
Ang II and CSAR. Therefore, the black garlic has great
potential to prevent the pathogenesis of hypertension,
as a health-oriented food product.
In addition to the scavenging of excessive OFRs,
inhibition of plasma angiotensin-converting enzyme
(ACE) may also contribute to the antihypertensive
effect of black garlic (Quiñones et al., 2013). ACE is an
important factor controlling vascular tone by
producing extremely potent Ang II. Pomegranate juice
and several polyphenols such as procyanidins have
been demonstrated to inhibit ACE activity (Aviram
and Dornfeld, 2001; Actis-Goretta et al., 2003;
Kivimäki et al., 2013). Further investigations are
needed.
Conclusions
(1) The administration of black garlic for 14 days
significantly lowered the blood pressure of SHRs to
121±10 mm Hg (with a decline rate of 34.6% in
average), while it did not affect the normal blood
pressure of Wistar rats. The black garlic had more
significant antihypertensive effect on SHRs than fresh
garlic.
(2) In the plasma of SHRs receiving the black garlic,
the T-AOC increased from 4.2±1.0 U/mL to 5.4±1.1
U/mL (with an increase rate of 28.6% in average), and
the MDA content decreased correspondingly from
10.2±2.2 nmol/mL to 7.9±0.7 nmol/mL (with a decrease
rate of 22.5 % in average).
(3) In the PVN of SHRs receiving the black garlic, the
T-AOC increased from 4.4±0.7 U/mg-protein to 7.2±1.6
U/mg-protein (with an increase rate of 63.6% in
average), and the MDA content decreased
correspondingly from 8.2±1.5 nmol/mg-protein to
3.9±1.2 nmol/mg-protein (with a decrease rate of 52.4%
in average).
(4) It was indicated that the black garlic exerted a
potential antihypertensive effect via scavenging
excessive OFRs in the plasma and PVN of SHRs.
ACKNOWLEDGEMENT
The present study was financially supported by the
National Basic Research Program of China (973
Program, 2009CB724700), and the National Nature
Science Fund of China (71173103/G0310). The authors
thank professor Ling Chen of the School of Basic
Medical Science, Nanjing Medical University, China
for giving us advice and chance to use laboratory for
the animal experimentation. The authors declare no
conflict of interest.
REFERENCES
Actis-Goretta, L., Ottaviani, J. I., Keen, C. L., Fraga, C. G.
“Inhibition of angiotensin converting enzyme (ACE)
activity by flavan-3-ols and procyanidins”. FEBS Letters
555(2003): 597–600.
Apitz-Castro, R., Badimon, J. J., Badimon, L. “Effect of ajoene,
the major antiplatelet compound from garlic, on platelet
thrombus formation.” Thrombosis Research 68 (1992):
145-155.
Aviram, M., Dornfeld, L. “Pomegranate juice consumption
inhibits serum angiotensin converting enzyme activity
and reduces systolic blood pressure”. Atherosclerosis 158
(2001): 195–198.
Benzie, I. F., Strain, J. J. “The ferric reducing ability of
plasma (FRAP) as a measure of “antioxidant power”: the
FRAP assay.” Analytical Biochemistry 239 (1996): 70-76.
Bogdan, C., Röllinghoff, M., Diefenbach, A. “Reactive oxygen
and reactive nitrogen intermediates in innate and specific
immunity.” Current Opinion in Immunology 12 (2000):
64-76.
Research in Health and Nutrition (RHN) Volume 2 Issue 1, January 2014 www.seipub.org/rhn
11
Bradford, M. M. “A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding.” Analytical
Biochemistry 72 (1976): 248-254.
Chen Q. “Methodology for the study of pharmacology of
Chinese medicine (2nd ed.) (in Chinese).” Beijing:
People’s Medical Publishing House, 2006.
Fu, X. “Studies on the purification and enzymatic production
of ajoene in garlic (in Chinese).” China Agricultural
University, 2006.
Grassi, G., Giannattasio, C., Seravalle, G. “Sympathetic
activation in the pathogenesis of hypertension and
progression of organ damage.” Hypertension 34 (1999):
724-728.
Han, Y., Shi, Z., Zhang, F., Yu, Y., Zhong, M. K., Gao, X. Y.,
et al. “Reactive oxygen species in the paraventricular
nucleus mediate the cardiac sympathetic afferent reflex
in chronic heart failure rats.” European Journal of Heart
Failure 9/10 (2007): 967-973.
Han, Y., Fan, Z. D., Yuan, N., Xie, G. Q., Gao, J., De, W., et al.
“Superoxide anions in the paraventricular nucleus
mediate the enhanced cardiac sympathetic afferent reflex
and sympathetic activity in renovascular hypertensive
rats.” Journal of Applied Physiology 110/3 (2011): 646-652.
Iberl, B., Winkler, G., Knobloch, K. “Products of Allicin
Transformation: Ajoenes and Dithiins, Characterization
and their Determination by HPLC*.” Planta Medica 56
(1990): 202-211.
Ichikawa, M., Ide, N., Yoshida, J., Yamaguchi, H., Ono, K.
Determination of seven organosulfur compounds in
garlic by high-performance liquid chromatography.
Journal of Agricultural and Food Chemistry 54/5 (2006),
1535-1540.
Jung, Y. M., Lee, S. H., Lee, D. S., You, M. J., Chung, I. K.,
Cheon, W. H., et al. “Fermented garlic protects diabetic,
obese mice when fed a high-fat diet by antioxidant
effects.” Nutrition Research 31 (2011): 387-396.
Kim, S. H., Jung, E. Y., Kang, D. H., Chang, U. J., Hong, Y. H.,
Suh, H. J. “Physical stability, antioxidative properties,
and photoprotective effects of a functionalized
formulation containing black garlic extract.” Journal of
Photochemistry and Photobiology B: Biology 117 (2012):
104-110.
Kivimäki, A. S., Siltari, A., Ehlers, P. I., Korpela, R.,
Vapaatalo, H. “Lingonberry juice lowers blood pressure
of spontaneously hypertensive rats (SHR) “. Journal of
Functional Foods 5(2013): 1432-1440.
Kodera, Y., Suzuki, A., Imada, O., Kasuga, S., Sumioka, I.,
Kanezawa, A., et al. “Physical, chemical, and biological
properties of S-allylcysteine, an amino acid derived from
garlic.” Journal of Agricultural and Food Chemistry 50
(2002): 622-632.
Koga, Y., Hirooka, Y., Araki, S., Nozoe, M., Kishi, T.,
Sunagawa, K. “High salt intake enhances blood pressure
increase during development of hypertension via
oxidative stress in rostral ventrolateral medulla of
spontaneously hypertensive rats.” Hypertension
Research 31 (2008): 2075-2083.
Li, C., Du, H., Wang, L., Shu, Q., Zheng, Y., Xu, Y., et al.
“Flavonoid composition and antioxidant activity of tree
peony (Paeonia section Moutan) yellow flowers.” Journal
of Agricultural and Food Chemistry 57 (2009): 8496-8503.
Li, Y. “Experimental studies of the effect of Huanglian
Qingjiang mixture on spontaneously hypertensive rats
(in Chinese).” Journal of Traditional Chinese Medicine
21/7 (2002): 421-424.
Mancia, G., Grassi, G., Giannattasio, C., Seravalle, G.
“Sympathetic activation in the pathogenesis of
hypertension and progression of organ damage.”
Hypertension 34 (1999): 724-728.
Messerli, F. H., Williams, B., Ritz, E. “Essential
hypertension.” The Lancet, 370/9587 (2007):591–603.
Miao, Y. “A bioprocessing technology for garlic.” China:
ZL200610086040.8.2006.
Miura, H., Bosnjak, J. J., Ning, G., Saito, T., Miura, M.,
Gutterman, D. D. “Role for hydrogen peroxide in flowinduced
dilation of human coronary arterioles.”
Circulation Research 92/2 (2003): e31-e40.
Ortiz, M.C., Manriquez, M.C., Romero, J.C., Juncos, L.A.
“Antioxidants block angiotensin Ⅱ-induced increases in
blood pressure and endothelin.” Hypertension 38 (2001):
655-659.
Placer, Z. A., Cushman, L. L., Johnson, B. C. “Estimation of
product of lipid peroxidation (malonyl dialdehyde) in
biochemical systems.” Analytical Biochemistry 16/2
(1966): 359-364.
www.seipub.org/rhn Research in Health and Nutrition (RHN) Volume 2, 2014
12
Quiñones, M., Guerrero, L., Suarez, M., Pons, Z., Aleixandre,
A., Arola, L., Muguerza, B. “Low-molecular procyanidin
rich grape seed extract exerts antihypertensive effect in
males spontaneously hypertensive rats”. Food Research
International 51 (2013): 587–595.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M.,
Rice-Evans, C. “Antioxidant activity applying an
improved ABTS radical cation decolorization assay.” Free
Radical Biology and Medicine 26 (1999): 1231-1237.
Russo, C., Olivieri, O., Girelli, D., Faccini, G., Zenari, M. L.,
Lombardi, S., et al. “Anti-oxidant status and lipid
peroxidation in patients with essential hypertension.”
Journal of hypertension 16/9 (1998): 1267-1271.
Sasaki, J., Ch, L. U., Machiya, E. “Processed black garlic
(Allium sativum) extracts enhance anti-tumor potency
against mouse tumors.” Medical Aromatic Plant Science
and Biotechnoloy 1/2 (2007): 278-281.
Shi, X. “Medical laboratory animal science (in Chinese).”
Shanxi: Science and Technology Publishing House. 1989.
Vaziri, N. D., Wang, X. Q., Oveisi, F., Rad, B. “Induction of
oxidative stress by glutathione depletion causes severe
hypertension in normal rats.” Hypertension 36 (2000):
142-146.
Wang, D., Feng, Y., Liu, J., Yan, J., Wang, M., Sasaki, J. I., et
al. “Black garlic (Allium sativum) extracts enhance the
immune system.” Medicinal and Aromatic Plant Science
and Biotechnology 4/1 (2010): 37-40.
Zhang, B. L., Sannajust, F. “Diurnal rhythms of blood
pressure, heart rate, and locomotor activity in adult and
old male Wistar rats.” Physiology & Behavior 70 (2000):
375-380.
Zhou, G., Miao, Y., Chen, J., Li, Y., Liu, J. “Changes in the
main components and free radical scavenging ability of
black garlic during storage (in Chinese).” Journal of
Chinese Institute of Food Science and Technology 10/6
(2010): 64-71.
Zhu, B., Wu, H., Liu, Y., Tun, J., Yao, Z. “The antioxidation
of black garlic (in Chinese).” Food Research and
Development 29/10 (2008): 58-60.
Zulueta, A., Esteve, M. J., Frígola, A. “ORAC and TEAC
assays comparison to measure the antioxidant capacity
of food products.” Food Chemistry 114 (2009): 310-316.