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j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j ff
Functional Foods and Nutraceuticals Unit, Department of Biochemistry, Federal University of Technology
Akure, Private Mail Bag 704, Akure 340001, Nigeria
b
Department of Biochemistry, Afe Babalola University Ado-Ekiti, Private Mail Bag 5454, Nigeria
c
Programa de Ps Graduao em Cincias Biolgicas, Bioqumica Toxicolgica, Centro de Cincias Naturais e
Exatas, Universidade Federal de Santa Maria, Campus Universitrio, Camobi, Santa Maria, RS CEP 97105-900,
Brazil
d
Health Basic Sciences Institute, Department of Physiology, Federal University of Rio Grande do Sul, Porto
Alegre, RS, Brazil
A R T I C L E
I N F O
A B S T R A C T
Article history:
Ginger and turmeric rhizomes are used in folk medicine for the treatment of hypertension
but the mechanism remains unclear. This study evaluated the effects of ginger and tur-
2015
hypertensive rats. The animals were divided into seven groups (n = 10): normotensive control
rats; hypertensive rats; hypertensive rats treated with atenolol; normotensive diet group
Available online
supplemented with turmeric rhizomes; hypertensive rats supplemented with turmeric rhizomes; normotensive diet group supplemented with ginger rhizomes; and hypertensive diet
Keywords:
group supplemented with ginger rhizomes respectively. After 14 days of pre-treatment with
Ginger
ginger and turmeric rhizomes-supplemented diet, the animals were induced with hyper-
Hypertension
L-NAME
NAME). The results revealed a significant increase in ACE and arginase activities in hypertensive
ACE
rats when compared with the control. However, pre-treatment with both rhizomes respec-
Arginase
tively caused a significant decrease in ACE and arginase activities with a concomitant increase
NO
in nitric oxide (NO) level. These activities could further buttress their antihypertensive benefits in folk medicine.
2015 Published by Elsevier Ltd.
* Corresponding author. Functional Foods and Nutraceuticals Unit, Department of Biochemistry, Federal University of Technology Akure,
Private Mail Bag 704, Akure 34000157. ia.
E-mail address: goboh2001@yahoo.com (G. Oboh).
** Corresponding author. Departamento de Bioqumica e Biologia Molecular, Centro de Cincias Naturais e Exatas, Universidade Federal
de Santa Maria (UFSM), CEP 97105-900, Santa Maria/RS, Brazil. Tel.: +55 55 3220 9557; fax: +55 55 3220 9557.
E-mail address: mariachitolina@gmail.com (M.R.C. Schetinger).
http://dx.doi.org/10.1016/j.jff.2015.06.011
1756-4646/ 2015 Published by Elsevier Ltd.
1.
Introduction
793
794
2.
2.1.
Chemicals
2.2.
Plant material
2.3.
2.4.
Animals
2.5.
Experimental protocol
The rats were acclimatized for two weeks and randomly divided
into seven groups of ten animals each (n = 10). Group 1: (Control)
served as the normotensive control group placed on a basal
diet; Group 2: (Induced) served as the hypertensive (L-NAME)
group placed on a basal diet plus L-NAME; Group 3: (LNAME + AT) served as the positive control placed on a basal
diet plus L-NAME plus atenolol (10 mg/kg/day); Group 4: (RG
Normal) served as the normotensive diet group placed on a
diet supplemented with turmeric rhizomes (4%); Group 5:
(RG + L-NAME) served as the hypertensive group placed on a
diet supplemented with turmeric rhizomes (4%) plus L-NAME;
Group 6: (WG Normal) served as the normotensive diet group
placed on a diet supplemented with ginger rhizomes (4%); and
Group 7: (WG + L-NAME) served as the hypertensive group
placed on a diet supplemented with ginger rhizomes (4%) plus
L-NAME. The rats were placed on their respective diet for two
weeks before induction of hypertension (Table 1). Daily feed
intake was monitored and body weight was taken both at the
beginning and at the end of the experiment. In the hypertensive groups, hypertension was induced by the oral
administration of the nitric oxide synthase (NOS) inhibitor
L-NAME (40 mg/kg/day) by gavage for ten days (Furstenau et al.,
2008). In the normotensive groups, the animals received water
by gavage throughout the entire experiment to be submitted
to the same stress (normotensive groups). These rats were euthanized 24 h after the last treatment session. The experiment
lasted for 24 days, after which the animals were submitted to
euthanasia, being previously anaesthetized with isoflurane, and
their blood collected for serum preparation and determination of enzyme activities and testing of renal function. The
kidneys were removed and rinsed in ice-cold 1.15% KCl, after
which they were blotted and weighed. The kidneys were minced
with scissors in three volumes of ice-cold 100 mM potassium
phosphate buffer (pH 7.4) and homogenized in a Teflon glass
homogenizer. The homogenates were centrifuged for 10 min
at 12,000 g to yield a pellet that was discarded. A low-speed
supernatant (S1) was used for subsequent analysis.
2.6.
Diet formulation
2.7.
795
Table 1 Diet formulation for basal and supplemented diets for control and test groups (g/kg).
Component
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Skimmed milk
Oil
Vitamin mixture
Corn starch
Ginger
394
100
40
466
394
100
40
466
394
100
40
466
394
100
40
426
40
394
100
40
426
40
394
100
40
426
40
394
100
40
426
40
Notes: Skimmed milk = 32% protein; the vitamin mixture (mg or IU/g) h was the following composition: 3200 IU vitamin A, 600 IU vitamin D3,
2.8 mg vitamin E, 0.6 mg vitamin K3, 0.8 mg vitamin B1, 1 mg vitamin B2, 6 mg niacin, 2.2 mg pantothenic acid, 0.8 mg vitamin B6, 0.004 mg
vitamin B12, 0.2 mg folic acid, 0.1 mg biotin H2, 70 mg choline chloride, 0.08 mg cobalt, 1.2 mg copper, 0.4 mg iodine, 8.4 mg iron, 16 mg manganese, 0.08 mg selenium, 12.4 mg zinc, 0.5 mg antioxidant.
Group 1 (Control): the normotensive control group placed on a basal diet only;
Group 2 (Induced): the hypertensive (L-NAME) group placed on a basal diet + L-NAME;
Group 3 (L-NAME + AT): the positive control placed on a basal diet + L-NAME + atenolol (antihypertensive drug);
Group 4 (RG Normal): the normotensive diet group placed on a diet-supplemented with turmeric rhizomes (4%) only;
Group 5 (RG + L-NAME): the hypertensive group placed on a diet-supplemented with turmeric rhizomes (4%) + L-NAME;
Group 6 (WG Normal): the normotensive diet group placed on a diet-supplemented with ginger rhizomes (4%) only; and
Group 7 (WG + L-NAME): the hypertensive group placed on a diet-supplemented with ginger rhizomes (4%) + L-NAME.
Rat Tail Blood Pressure System for rats and mice, Litchfield, MN,
USA). Rats were conditioned with the apparatus before measurements were taken. SBP was recorded at the end of
experiment (last treatment week). The measurements were
done blindly by the same person in quadruplicate per animal.
2.8.
The kidney and serum ACE activity was determined as described by Cushman and Cheung (1971). The substrate [hippurylhistidyl-leucine (Bz-Gly-His-Leu)] was purchased from SigmaAldrich Chemie GmbH, Steinheim, North Rhine-Westphalia,
Germany. The amount of cleaved hippuric acid from hippurylhistidyl-leucine was measured by the enzymatic method. Fifty
microlitres of the samples and 150 l of 8.33 mM of hippurylhistidylleucine (Bz-Gly-His-Leu) in 125 mM Tris-HCl buffer
(pH 8.3) were incubated at 37 C for 30 min. After incubation,
the reaction was arrested by adding 250 l of 1M HCl. The
GlyHis bond was then cleaved, and the hippuric acid produced by the reaction was extracted with 1.5 ml ethyl acetate.
Next, the mixture was centrifuged to separate the ethyl acetate
layer; then, 1 ml of the ethyl acetate layer was transferred to
a clean test tube and evaporated. The residue was re-dissolved
in distilled water, and its absorbance was measured at 228 nm.
The ACE activity was expressed as mmol/min/mg protein.
2.9.
Arginase activity in the serum and kidney cortex was determined by measuring the rate of urea production using
-isonitrosopropiophenone (9% in absolute ethanol) as previously described by Zhang, Hein, Wang, Chang, and Kuo (2001).
Briefly, 50 l of the samples was added into 75 l of Tris-HCl
(50 mmol/l, pH 7.5) containing 10 mmol/l MnCl2 and was preincubated at 37 C for 10 minutes to activate the enzyme. The
hydrolysis reaction of L-arginine by arginase was performed
by incubating the mixture containing activated arginase with
50 l of L-arginine (0.5 mol/l, pH 9.7) at 37 C for 1 hour and was
stopped by adding 400 l of the acid solution mixture [H2SO4/
H3PO4/H2O = 1:3:7 (v/v/v)]. For calorimetric determination of urea,
2.10.
2.11.
Renal function
Renal function test was carried out by measuring serum creatinine and urea levels using commercial colorimetric enzymatic
diagnostic kits purchased from Randox Laboratories Ltd.
(Crumlin, Dublin, Northern Ireland, UK).
2.12.
Protein determination
Protein was measured by the Coomassie blue method according to Bradford (1976) using serum albumin as a standard.
2.13.
Statistical analysis
796
Turmeric
(mg/g)
Ginger
(mg/g)
Gallic acid
Catechin
Caffeic acid
Epicatechin
Rutin
Quercitrin
Quercetin
Kaempferol
Luteolin
Curcumin
3.27 0.02a
5.08 0.01b
2.15 0.03c
3.31 0.02a
3.19 0.01a
10.52 0.03d
3.28 0.02a
5.11 0.01b
4.06 0.02e
12.75 0.01f
1.83 0.03a
4.95 0.02b
2.93 0.01c
3.05 0.03c
1.87 0.01a
5.01 0.02b
6.78 0.03d
1.80 0.02a
3.09 0.01c
6.93 0.01d
Notes: The results are expressed as mean SEM of three determinations. Averages followed by different letters differ by Tukey test
at p < 0.05. The units are expressed as mg/g weight of sample.
200
150
a
100
50
3.1.
In an attempt to identify the major constituents of the two rhizomes, we performed reversed-phase HPLC analysis using
standard flavonoids and phenolic acids [Sigma-Aldrich Chemie
GmbH, Steinheim, North Rhine-Westphalia, Germany]. The compounds gallic acid (tR = 9.97 min; peak 1), catechin (tR = 16.81 min;
peak 2), caffeic acid (t R = 24.79 min; peak 3), epicatechin
(tR = 32.56 min; peak 4), rutin (tR = 38.07 min; peak 5), quercitrin
(tR = 46.51 min; peak 6), quercetin (tR = 50.43 min; peak 7),
kaempferol (tR = 53.97 min; peak 8), luteolin (tR = 58.62 min; peak
9) and curcumin (tR = 65.19 min; peak 10) were identified by RT
and UV/VIS spectra that matched with the standards (Fig. 1
and Table 2). The observed result has already been characterized in this extract according to our previous work by Akinyemi,
Oboh, Ademiluyi, Boligon, & Athayde (2015).
3.2.
Effect of supplemented diet on systolic blood pressure
in L-NAME induced hypertensive rats
In this study, the oral administration of L-NAME by gavage was
associated with a significant rise in the final systolic blood pres-
0
on
tr
ol
In
du
Lce
N
d
A
M
E
+
A
R
T
G
N
or
R
G
m
al
+
LN
A
M
W
E
G
N
W
or
G
m
al
+
LN
A
M
E
Results
3.
797
Serum
Kidney
80
60
40
*
20
0
The results as shown in Fig. 3 revealed that there was a significant increase (p < .05) in the serum (40.5%) and kidney (56.2%)
ACE activity in hypertensive rats when compared with the normotensive control group (without L-NAME). However, there was
a significant (p < 0.05) inhibitory effect on ACE activity as a result
of supplementation with ginger and turmeric rhizomes respectively when compared with the induced group [RG + L-
A.
mmol./min/mg protein
NAME = serum (45.2%) and kidney (51.4%), and WG + LNAME = serum (51.9%) and kidney (50.1%) ACE activity].
0.06
a
0.04
3.5.
Effect of supplemented diet on arginase enzyme
activity in L-NAME induced hypertensive rats
c
c
0.02
LN
or
m
al
In
du
ce
d
A
M
E
+
A
R
T
G
N
or
R
G
m
al
+
LN
A
M
W
E
G
N
W
or
G
m
al
+
LN
A
M
E
0.00
Groups
B.
mmol./min/mg protein
on
t
3.4.
Effect of supplemented diet on angiotensin 1
converting enzyme (ACE) activity in L-NAME induced
hypertensive rats
ro
l
In
d
Luc
N
ed
A
M
E
+
A
R
T
G
N
o
R
r
G
m
al
+
LN
A
M
W
E
G
N
W
or
G
m
al
+
LN
A
M
E
Regarding body weight (BW) and feed intake, there was no significant (p < 0.05) difference observed after the administration
of L-NAME (40 mg/kg/day) by gavage among the experimental groups (data not shown).
100
mol./min/mg protein
3.3.
Effect of supplemented diet on body weight and feed
intake in L-NAME induced hypertensive rats
0.06
a
0.04
a
c
0.02
a
c
LN
on
t
ro
l
In
du
ce
d
A
M
E
+
A
R
T
G
N
or
R
G
m
al
+
LN
A
M
W
E
G
N
W
or
G
m
al
+
LN
A
M
E
0.00
Groups
3.6.
Effect of supplemented diet on nitric oxide (NO) level
in L-NAME induced hypertensive rats
Nitric oxide (NO) level in the serum was decreased in induced
group (hypertensive rats) when compared with the control (normotensive) group. In the diet-supplemented hypertensive group
the levels of NO were clearly elevated compared to the induced
group (hypertensive rats) but were not significantly different
from the control (normotensive animals) as presented in Fig. 5.
3.7.
Effect of supplemented diet on renal function
biomarkers in L-NAME induced hypertensive rats
As presented in Table 2, there was a significant (p < 0.05) increase in serum creatinine and urea levels of hypertensive group
when compared with the control. However, pre-treatment with
ginger and turmeric rhizomes supplemented diet caused a
798
50
Serum
Kidney
40
30
20
10
*
*
on
tr
ol
In
d
Luc
N
ed
A
M
E
+
A
R
T
G
N
or
R
G
m
al
+
LN
A
M
W
E
G
N
W
or
G
m
al
+
LN
A
M
E
decrease in the levels of serum creatinine and urea when compared with the hypertensive (L-NAME) group.
4.
Discussion
It has been obviously demonstrated that chronic administration of L-NAME in rats induced arterial hypertension associated
with the deficiency of nitric oxide (NO) (Cardoso et al., 2012,
2014; Furstenau et al., 2008). The present study analysed the
effect of ginger and turmeric rhizomes on arterial hypertension associated with the chronic deficiency of NO. As presented
in Fig. 2, we observed a significant rise in systolic blood pressure after treatment with L-NAME by oral gavage. This result
is in agreement with previously described studies where
L-NAME given in the drinking water to animals produces a prolonged increase in blood pressure over many hours (Cardoso
et al., 2012, 2014; Furstenau et al., 2008). This effect occurs due
to the capacity of L-NAME to inhibit the production of nitric
oxide, a well known vasodilator molecule, by blocking nitric
oxide synthase (NOS) activity (Cardoso et al., 2012, 2014).
However, dietary supplementation with the two ginger varieties and treatment with a positive control drug (atenolol)
caused a significant reduction of SBP in the hypertensive rats
(Fig. 2). This is in agreement with Ghayur and Gilani (2005),
where they reported hypotensive effect of aqueous extract of
ginger in normotensive rats under anaesthesia. The observed
fall in SBP in hypertensive rats is in line with the traditional
use of ginger as an antihypertensive and vasodilator agent
(Ghayur et al., 2005). Nevertheless, the synergy of the phenolic compounds present in the ginger extract could be responsible
for the reduction in blood pressure (Fig. 1 and Table 1). Phenolic compounds such as curcumin, quercetin, gallic acid and
caffeic acid have been reported to exhibit blood pressure lowering effect in hypertensive rats (Bhullar, Lassalle-Claux, Touaibi,
& Rupasinghe, 2014; Kang et al., 2015; Pang et al., 2015). However,
in a clinical perspective, treatment with both rhizomes should
Creatinine
(mg/dl)
Urea
(mg/dl)
Control
Induced
L-NAME + AT
RG Normal
RG + L-NAME
WG Normal
WG + L-NAME
0.33 0.01a
0.73 0.09b
0.36 0.07a
0.40 0.02a
0.43 0.03a
0.37 0.05a
0.50 0.09a
35.4 3.5a
47.8 3.1b
37.6 2.8a
37.3 2.1a
36.6 1.7a
39.0 3.3a
37.3 3.1a
Notes: Values are presented as mean SEM (n = 10). Values with different superscript letters along the columns are statistically different
(p < 0.05). The units are expressed as mg/dl of serum. For details see
the legend of groups in Table 1.
This result is in agreement with recent studies that inhibition of arginase activity is crucial for the management of
hypertension (Bagnost et al., 2008, 2010; Maquiaveli et al., 2014).
However, several authors have reported dietary plant phenolics exhibited inhibitory effect on arginase activity (Kim et al.,
2013; Manjolin, dos Reis, Maquiaveli, Santos-Filho, & Da Silva,
2013).
Enhanced arginase activity can impair endotheliumdependent vasorelaxation by decreasing L-arginine availability
to endothelial nitric oxide synthase (eNOS), thereby reducing
NO production and uncoupling eNOS function. Nitric oxide (NO)
is essential to normal cardiovascular function and blood pressure control. We observed a significant decrease in level of NO
in L-NAME induced hypertensive rats (Fig. 5). The result is in
agreement with previous studies where L-NAME has been
shown to be a chronic inhibitor of NOS (Cardoso et al., 2012,
2014; Furstenau et al., 2008). Also, it is in line with our earlier
results in Fig. 4, where we observed an increase in arginase
activity which can deplete NO production. Nevertheless, dietary
supplementation with ginger rhizomes restores the level of NO
in hypertensive rats. This increase in NO could be a result of
the fact that ginger rhizomes exhibited inhibitory effect on arginase activity or by increasing endogenous L-arginine level
through dietary means as reported by Ajayi, Akomolafe, and
Akinyemi (2013).
It is well known that NO is an important regulator of renal
haemodynamics and sodium handling (Bech, Nielsen, Ivarsen,
Jensen, & Pedersen, 1998). Inhibition of nitric oxide synthesis
by the administration of L-NAME leads to increase in serum
creatinine and urea levels as shown in Table 3. This indicates
a reduction in renal function due to hypertensive state. Several
studies have reported a diminished renal function such as decrease in urinary sodium excretion along with the decreased
renal blood flow, urine flow rates, glomerular filtration rates
(GFR), and free-water clearance in L-NAME hypertensive rats
(Bech et al., 1998).
5.
Conclusions
Dietary supplementation of ginger and turmeric rhizomes inhibited ACE and arginase activities as well as increased NO
799
production in L-NAME induced hypertensive rats. These activities could suggest possible mechanism of action for their
antihypertensive benefits in traditional medicine. However, the
observed effect could be attributed to the phenolic compounds acting either synergistically or additively. Moreover,
further work to isolate the bioactive principle is in progress
in our laboratory.
Conflict of interest
The authors declare no conflicting interest.
Acknowledgments
One of the authors (Ayodele Jacob Akinyemi) is a beneficiary
of 2013 CNPq/TWAS sandwich postgraduate fellowship.
Therefore, we wish to thank the Conselho Nacional de
Desenvolvimento Cientfico e Tecnolgico (CNPq), Fundao de
Amparo Pesquisa do Estado do Rio Grande do Sul (FAPERGS),
Fundao Coordenao de Aperfeioamento de Pessoal de Nvel
Superior (CAPES) and the Academy of Sciences for the Developing World (TWAS) for their support towards this study.
REFERENCES
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