journal of functional foods 17 (2015) 792801

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Effect of dietary supplementation of ginger and

turmeric rhizomes on angiotensin-1 converting
enzyme (ACE) and arginase activities in L-NAME
induced hypertensive rats
Ayodele Jacob Akinyemi a,b,c, Gustavo Roberto Thome c,
Vera Maria Morsch c, Naiara Stefanello c, Jeferson Ferraz Goularte d,
Adriane Bell-Klein d, Ganiyu Oboh a,*,
Maria Rosa Chitolina Schetinger c,**
a

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

Received 15 March 2015

but the mechanism remains unclear. This study evaluated the effects of ginger and tur-

Received in revised form 29 May

meric rhizomes on angiotensin 1 converting enzyme (ACE) and arginase activities in

2015

hypertensive rats. The animals were divided into seven groups (n = 10): normotensive control

Accepted 2 June 2015

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

tension by oral administration of N-nitro-L-arginine methyl ester hydrochloride (L-

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.

journal of functional foods 17 (2015) 792801

1.

Introduction

Hypertension is a well-defined risk factor for cardiovascular

diseases such as strokes and coronary heart disease (Touyz &
Schiffrin, 2004). Nitric oxide (NO) has been demonstrated and
proven to have an important role in the maintenance of normal
blood pressure and body fluid homeostasis (Krier & Romero,
1998). Several disease conditions, including essential hypertension, have been linked to impaired synthesis or action of
NO (Cardoso et al., 2012, 2014), which makes chronic inhibition of basal NO with an orally active nitric oxide synthase (NOS)
inhibitor, N-nitro-L-arginine methyl ester hydrochloride (LNAME), a particularly interesting model of hypertension. The
chronic administration of L-NAME causes significant increase in blood pressure in experimental animals and its
physiological and pathological characteristics resemble essential or primary hypertension because it raises high blood
pressure with no obvious underlying cause (Cardoso et al., 2012,
2014). Therefore, reducing blood pressure values or preventing its rise will reduce target organ damage (Touyz & Schiffrin,
2004).
The reninangiotensin aldosterone system (RAS) plays a
major physiologic role in blood pressure control and sodium
volume homeostasis (Akinyemi, Ademiluyi, & Oboh, 2013). This
system has been suggested to be of importance in pathologic
conditions such as high blood pressure and cardiac complications (Udenigwe, Lin, Hou, & Aluko, 2009). Angiotensin
converting enzyme (ACE) is a zinc metallopeptidase which plays
a major role in regulation of vascular tone by converting the
inactive peptide, angiotensin I (Ang I) into vasoconstrictor and
trophic angiotensin II (Ang II) (Udenigwe, Lin, Hou, & Aluko,
2009). The octapeptide Ang II is the active component of the
reninangiotensin system (RAS) and has a major role in the
regulation of cardiovascular, renal and endocrine functions
(Udenigwe, Lin, Hou, & Aluko, 2009). ACE exists both as a
membrane-bound enzyme in various organs such as heart,
blood vessels, kidney and in a freely soluble form in plasma
(Johnston, 1992). There are some reports suggesting that RAS
is altered in this model of chronic administration of L-NAME
(Ackermann, Fernandez-Alfonso, & Gonzalez, 1998; Tekemoto,
Egashira, Tomita, & Takeshita, 1997). It has been shown that
blockade of the RAS with angiotensin converting enzyme inhibitor or with an angiotensin II (Ang II) receptor antagonist
prevents the elevation of blood pressure in L-NAME-treated
animals (Ackermann et al., 1998; Tekemoto et al., 1997).
Angiotensin II (Ang II) actions in endothelial cells are
mostly associated with endothelial NOS (eNOS) dysfunction
and uncoupling, which lead to decreased levels of NO and
increased superoxide production (Satoh et al., 2008). NO is produced by conversion of L-arginine (L-Arg) and oxygen to
L-citrulline and NO by nitric oxide synthase (NOS). Depletion
of endogenous L-arginine substrate has been associated
with systemic hypertension via elevated arginase activity, a
hydrolytic enzyme responsible for conversion of L-arginine
into urea and L-ornithine (Demougeot, Prigent-Tessier, Marie,
& Berthelot, 2005). Because NOS and arginase use L-arginine
as a common substrate, arginase may down-regulate nitric
oxide (NO) biosynthesis by competing with NOS for L-arginine
degradation. Consistent with this hypothesis, NO production

793

has been inversely correlated to arginase activity in

vessels at both physiological and pathological conditions
such as hypertension (Demougeot et al., 2005). A few
studies have demonstrated the benefits of a chronic treatment with an arginase inhibitor for treating endothelial
dysfunction associated with hypertension (Bagnost et al., 2008,
2010).
Although many classes of antihypertensive drugs are
available in modern medicine to achieve this purpose, the
treatment of arterial hypertension in developing countries is
still facing important problems because pharmaceuticals
are unaffordable for almost the entire population, which
then refer to herbal therapy. During the last two decades
many studies have focused on the dietary prevention of
hypertension development, with particular interest in plant
foods.
Zingiber officinale Roscoe (Zingiberaceae), commonly
known as ginger, is known and used universally for its medicinal and culinary properties (Abdullahi, 2011; Chan, Lim,
& Wong, 2009). It contains several valuable compounds
and new constituents are still being found (Abdullahi, 2011).
Recently, Akinyemi, Ademiluyi, and Oboh (2014) reported
the hypocholesterolaemic effect of ginger rhizomes in vivo.
Also, the arginase and ACE activity inhibitory effects of
ginger rhizomes have been reported (Akinyemi et al., 2013;
Akinyemi, Oboh, Ademiluyi, Boligon, & Athayde, 2015). In traditional medicine, it is used as therapy against hypertension
and several cardiovascular diseases (Duke, 2002). The hypotensive effect of aqueous extract of ginger rhizomes has been
reported by Ghayur and Gilani (2005). Another notable member
of this plant family (Zingiberaceae) is turmeric rhizomes
(Curcuma longa), which is a rhizomatous herbaceous perennial plant in the ginger family, employed as a dye source and
food colourant due to its characteristic yellow colour (Chan
et al., 2009). Turmeric is one of the main ingredients in curry
powder, and can be made into a drink to treat cold and
stomach complaints (Chan et al., 2009). Curcuminoids
are the major phytochemicals of the turmeric responsible for
the characteristic yellow colour and has been reported
to have several medicinal values (Chan et al., 2009). Turmeric
as well as curcumin has been reported to reduce the
uptake of cholesterol from the gut (Arafa, 2005). Arun
and Nalini (2002) have reported the hypoglycaemic
properties of turmeric in diabetic albino rats. In addition,
curcumin from turmeric rhizomes have been shown to
possess anti-inflammatory properties (Kowluru & Kanwar,
2007).
Both rhizomes are useful in herbal medicine
against hypertension and are considered safe herbal medicines because no significant side effect has yet been described
(Ghayur, Gilani, & Afridi, 2005; Weidner & Sigwart, 2000) and
their effect on NO production has been published (Bird & Shah,
2013). However, their effects in an in vivo model in which endothelial dysfunction and hypertension are induced by nitric
oxide synthesis blockade remain unexplored. Hence, the
aim of the present study was to evaluate the effect of dietary
supplementation of the two ginger varieties on activities of
some key enzymes currently linked with hypertension management (ACE and arginase) in L-NAME-induced hypertensive
rats.

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journal of functional foods 17 (2015) 792801

2.

Materials and methods

2.1.

Chemicals

The substrates Hippuryl-histidyl-leucine and L-arginine, as well

as urea, N-(l-naphthyl) ethylenediamine dihydrochloride, TrisHCl buffer, HEPES, L-NAME, and Coomassie brilliant blue G were
obtained from Sigma Chemical Co (St. Louis, MO, USA) and
bovine serum albumin, nitrate, and VCl3 were obtained from
Reagen (Colombo, Paran, Brazil). All the other chemicals used
in this experiment were of the highest purity, while the water
was glass distilled. All the kits used for the bioassay were
sourced from Randox Laboratories Ltd. (Crumlin, Dublin, Northern Ireland, UK).

2.2.

Plant material

The fresh samples of ginger (Zingiber officinale) and turmeric

(Curcuma longa) rhizomes were obtained from a farmland at
Akure metropolis, Nigeria. Authentication of the plants was
carried out at the Department of Biology, Federal University of
Technology, Akure, Nigeria. A voucher specimen of the rhizomes has been deposited in the Universitys Botany
Departmental Herbarium.

2.3.

Analysis of phenolic compounds by HPLC-DAD

Reverse phase chromatographic analyses were carried out under

gradient conditions using C18 column (4.6 mm 150 mm) packed
with 5 m diameter particles; the mobile phase was water containing 2% acetic acid (A) and methanol (B), and the composition
gradient was: 5% of B until 2 min and changed to obtain 25,
40, 50, 60, 70 and 100% B at 10, 20, 30, 40, 50 and 80 min, respectively, following the method described by Barbosa-Filho
et al. (2014) with slight modifications. Ginger and turmeric
aqueous extracts were analysed at a concentration of 15 mg/
ml. The presence of some antioxidant compounds was
investigated. Identification of these compounds was performed by comparing their retention time and UV absorption
spectrum with those of the commercial standards. The flow
rate was 0.6 ml/min, injection volume 50 l and the wavelength were 270 nm for gallic acid, 281 nm for catechin and
epicatechin, 327 nm for caffeic acid, 365 nm for quercetin,
quercitrin, kaempferol, rutin and luteolin, and 240 nm for
curcumin. The samples and mobile phase were filtered through
0.45 m membrane filter (Millipore) and then degassed by ultrasonic bath prior to use. Stock solutions of standard references
were prepared in the HPLC mobile phase at a concentration
range of 0.0300.250 mg/ml for catechin, epicatechin, quercetin, quercitrin, rutin, kaempferol, curcumin and luteolin, and
0.0500.300 mg/ml for caffeic and gallic acids. The chromatography peaks were confirmed by comparing their retention time
with those of reference standards and by DAD spectra (200
500 nm). All chromatography operations were carried out at
ambient temperature and in triplicate.

2.4.

Animals

Adult male Wistar rats (200300 g) from the Central Animal

House of the Federal University of Santa Maria, Rio Grande Sul

(RS), Brazil, were used in this experiment. The animals were

maintained at a constant temperature (22 2 C) on a 12 h light/
dark cycle with free access to food and water. The animals were
used according to the guidelines of the National Council for
Animal Experiments Control (CONCEA) and are in accordance with international guidelines. The research project was
approved by the ethics committee of the Federal University of
Santa Maria Brazil, with project number 23/2011.

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

The diets were freshly formulated according to a modified

method of Akinyemi et al. (2014) and in consultation with the
Department of Animal Production and Health, Federal University of Technology, Akure (Table 1).

2.7.

Haemodynamic parameter determination

In all rats, systolic blood pressure (SBP) was measured in awaken

animals, by tail-cuff plethysmography (Kent Scientific; RTBP1001

795

journal of functional foods 17 (2015) 792801

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.

Determination of ACE activity

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.

Determination of arginase activity

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,

-isonitrosopropiophenone (25 l, 9% in absolute ethanol) was

then added and the mixture was heated at 100 C for 45 min.
After placing the sample in the dark for 10 minutes at room
temperature, the urea concentration was determined spectrophotometrically by the absorbance at 550 nm measured with
a microplate reader (Bio-Tek Instrument). The amount of urea
produced, after normalization with protein, was used as an
index for arginase activity.

2.10.

Measurement of nitric oxide (NO) level

NO content in serum and kidney homogenate was estimated

in a medium containing 400 ml of 2% vanadium chloride (VCl3)
in 5% HCl, 200 ml of 0.1% N-(l-naphthyl) ethylenediamine
dihydrochloride, 200 ml of 2% sulphanilamide (in 5% HCl). After
incubating at 37 C for 60 min, nitrite levels, which correspond to an estimative level of NO, were determined
spectrophotometrically at 540 nm, based on the reduction of
nitrate to nitrite by VCl3 (Miranda, Espay, & Wink, 2001). Serum
and kidney nitrite and nitrate levels were expressed as
nanomole of NO/milligram of protein.

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

The statistical analysis used was one-way ANOVA, followed by

Duncans multiple range tests; p < 0.05 was considered to represent a significant difference in both analyses used. All data
were expressed as mean SEM.

796

journal of functional foods 17 (2015) 792801

Table 2 Composition of turmeric (Curcuma longa) and

ginger (Zingiber officinale) aqueous extracts.
Ginger
compounds

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.

sure (SBP) when compared with the normotensive control rats,

validating the hypertensive model. However, we could observe
that dietary supplementation of both rhizomes as well as antihypertensive drug (atenolol) caused a significant reduction of
SBP when compared with the hypertensive L-NAME group
(Fig. 2).

Systolic blood Pressure

250

SBP (mm Hg)

Fig. 1 Representative high performance liquid

chromatography profile of (A) turmeric and (B) ginger
aqueous extract. Gallic acid (peak 1), catechin (peak 2),
caffeic acid (peak 3), epicatechin (peak 4), rutin (peak 5),
quercitrin (peak 6), quercetin (peak 7), kaempferol (peak 8),
luteolin (peak 9) and curcumin (peak 10). (Source:
Akinyemi, Oboh, Ademiluyi, Boligon, & Athayde, 2015).

200

150
a

100
50

3.1.

Characterization of phenolic compounds

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.

Fig. 2 Final systolic blood pressure (SBP) measurements

from L-NAME induced hypertensive rats treated with
dietary supplementation with ginger and turmeric
rhizomes. Data are presented as mean SEM (n = 10). Bars
with different letters are statistically different (p < 0.05).
Control: Normotensive control rats placed on basal diet
only; Induced: Hypertensive (L-NAME) rats placed on basal
diet + L-NAME (40 mg/kg/day); L-NAME + AT: Hypertensive
(L-NAME) rats placed on basal diet + L-NAME (40 mg/kg/
day) + atenolol (10 mg/kg/day); RG Normal: Normotensive
rats placed on basal diet supplemented with 4% turmeric
rhizomes only; RG + L-NAME: Hypertensive (L-NAME) rats
placed on basal diet supplemented with 4% turmeric
rhizomes + L-NAME (40 mg/kg/day); WG Normal:
Normotensive rats placed on basal diet supplemented with
4% ginger rhizomes only; WG + L-NAME: Hypertensive (LNAME) rats placed on basal diet supplemented with 4%
ginger rhizomes + L-NAME (40 mg/kg/day).

797

journal of functional foods 17 (2015) 792801

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

Fig. 4 Arginase activity from L-NAME induced

hypertensive rats treated with dietary supplementation
with ginger and turmeric rhizomes. Data are presented as
mean SEM (n = 10). *Statistically different from control
(p < 0.05). For details see the legend of Fig. 2.

Serum ACE activity

0.08

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.

Kidney ACE activity

0.08

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

Fig. 3 Angiotensin 1 converting enzyme (ACE) activity in

serum (A) and kidney (B) from L-NAME induced
hypertensive rats treated with dietary supplementation
with ginger and turmeric rhizomes. Data are presented as
mean SEM (n = 10). Bars with different letters are
statistically different (p < 0.05). For details see the legend of
Fig. 2.

The results obtained for serum and kidney arginase activity

are presented in Fig. 4. As can be observed, serum and kidney
arginase activity with L-arginine as substrate was significantly (p < 0.05) increased in the induced (hypertensive L-NAME)
group (23.4% for serum arginase and 65.3% for kidney arginase) when compared to the control (normotensive) group. Pretreatment with both rhizomes as well as the positive control
treated with atenolol (10 mg/kg/day) caused a significant
(p < 0.05) decrease of 35.1%, 34.3% and 23.8% for serum arginase and 39.3%, 61.6% and 58.2% for kidney arginase in
L-NAME + AT, RG + L-NAME and WG + L-NAME groups respectively when compared to the induced (hypertensive L-NAME)
group.

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

journal of functional foods 17 (2015) 792801

nmol NOx/mg protein

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

Fig. 5 Nitric oxide (NO) level from L-NAME induced

hypertensive rats treated with dietary supplementation
with ginger and turmeric rhizomes. Data are presented as
mean SEM (n = 10). *Statistically different from control
(p < 0.05). For details see the legend of Fig. 2.

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

have been employed after L-NAME treatment when SBP is found

to be increased, but our approach is more appropriate to show
the preventive effect of the ginger varieties.
The reninangiotensin system (RAS) is one of the main
targets for the regulation of blood pressure. Its inhibition at
three possible levels, angiotensin I-converting enzyme (ACE),
upstream renin activity or downstream angiotensin receptors, is the pharmacological basis for commonly used
antihypertensive drugs (Udenigwe et al., 2009). ACE inhibition is also the most aimed target for antihypertensive foodderived phenolics developed as an alternative to drugs
(Martnez-Maqueda, Miralles, Recio, & Hernndez-Ledesma,
2012; Udenigwe et al., 2009). Oral administration of L-NAME
resulted in the activation of reninangiotensin system via increased ACE activity (Fig. 3). Several studies have shown that
L-NAME given orally produced a sustained increase in arterial blood pressure due to the inhibition of the production of
NO and also by persistent activation of reninangiotensin
system (Nguelefack et al., 2008; Nguelefack-Mbuyo et al., 2008).
In the present study, our results revealed that dietary supplementation with both rhizomes caused a significant reduction
in serum and kidney ACE activity. The decrease in ACE activity as a result of introduction of the dietary rhizomes could
be linked to the synergistic effect of the phenolic compounds
such as caffeic acid, gallic acid, quercetin, curcumin, etc., present
in the extract (Fig. 1 and Table 1). Phenolic compounds such
as curcumin, quercetin, gallic acid and caffeic acids have been
reported to inhibit ACE activity either as a single compound
or in synergy with other compounds (Bhullar et al., 2014; Kang
et al., 2015; Pang et al., 2015). This approach has been used in
various practices of traditional medicine where mixture of plant
constituents is commonly prescribed for the treatment/
management of several cardiovascular diseases (Luo, Xu, &
Chen, 2013; Xiong & Wang, 2014). Also, Akinyemi et al. (2013)
have reported that aqueous extracts of ginger rhizomes possess
inhibitory property on ACE activity.
Angiotensin II produced in RAS from angiotensin I by the
action of ACE is a vasoconstrictor in renal vessels and has been
implicated in hypertension (Satoh et al., 2008). However, its
effect under pathological conditions is counteracted by NO
which serves as a potent vasodilator and plays an important
role in maintaining vascular tone (Satoh et al., 2008). In the
kidney, NO is synthesized primarily by eNOS and neuronal NOS
(Patzak et al., 2008), while iNOS seems to have a less important role. Because of its fundamental contribution to the
maintenance of vasodilator tone, arginine is primarily viewed
as a substrate for NO formation, but arginine is involved in multiple biochemical pathways, and the availability of arginine for
NO formation depends upon the concentration of plasma
L-arginine and the relative activity of competing intracellular
pathways (Morris, 2009). Arginase converts L-arginine to form
urea and ornithine, and studies suggest that there is intracellular competition between eNOS and arginase enzymes for their
common substrate L-arginine (Morris, 2009). Our recent study
shows that there is an increase in arginase activity in hypertensive rats treated with L-NAME (Fig. 4). However, pretreatment with both rhizomes prevented the increase of
arginase activity in hypertensive rats. Previous studies have reported up-regulation of arginase activity and decreased NO in
hypertension (Bagnost et al., 2008, 2010; Maquiaveli et al., 2014).

journal of functional foods 17 (2015) 792801

Table 3 Effect of dietary supplementation with ginger

and turmeric rhizomes on renal function biomarkers in
L-NAME induced hypertensive rats.
Groups

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.

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