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Nutrition Research 30 (2010) 358 – 365

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Iridoid extracts from Ajuga iva increase the antioxidant enzyme activities
in red blood cells of rats fed a cholesterol-rich diet
Sherazede Bouderbala a,⁎, Josiane Prost b , Marie Aleth Lacaille-Dubois c , Malika Bouchenak a
a
Laboratoire de Nutrition Clinique et Métabolique, Département de Biologie, Faculté des Sciences, Université d'Oran Es-Sénia, 31000 Oran, Algeria
b
UPRES Lipides & Nutrition, Faculté des Sciences Gabriel, Université de Bourgogne, 21100 Dijon, France
c
Laboratoire de Pharmacognosie, Faculté de Pharmacie, Unité de Molécules d'Intérêt Biologique, UMIB, UPRES EA 3660, Université de Bourgogne,
21100 Dijon, France
Received 11 March 2010; revised 23 April 2010; accepted 5 May 2010

Abstract

The lyophilized aqueous extract of Ajuga iva (Ai) is able to reduce oxidative stress, which may
prevent lipid peroxidation in hypercholesterolemic rats. Iridoids (I) were isolated from Ai. We
hypothesized that the antioxidant defense status in red blood cells (RBC) and tissues in rats fed a
cholesterol-rich diet and treated with Ai may be correlated to these compounds. Male Wistar rats
(n = 32) weighing 120 ± 5 g were fed a diet containing 1% cholesterol for 15 days. After this phase,
hypercholesterolemic (HC) rats were divided into groups, fed the same diet, and received either the
same or different doses (5, 10, or 15 mg/kg body weight by intraperitoneal injection) of I for 15 days.
Compared with the HC group, total cholesterol value was 1.4- and 1.2-fold lower in the I5-HC and
I10-HC groups. Serum thiobarbituric acid reactive substance content was 2.3-, 2.9-, and 3-fold lower
in the I5-HC, I10-HC, and I15-HC groups compared with the HC group. In RBC, glutathione
peroxidase, glutathione reductase, and superoxide dismutase activities were significantly higher in
the I5-HC, I10-HC, and I15-HC groups than the HC group. Liver, heart, and muscle glutathione
peroxidase and superoxide dismutase activities were significantly higher in the groups treated with I
than the HC group. Muscle glutathione reductase activity was increased 1.4-fold in the I5-HC, 1.5-
fold in the I10-HC, and 1.5-fold in the I15-HC group. In HC rats, different doses of I increase the
antioxidant enzyme activities in RBC and act differently in tissues. Treatment with I may play an
important role in suppressing oxidative stress caused by dietary cholesterol and, thus, may be useful
for the prevention and/or early treatment of hypercholesterolemia.
© 2010 Elsevier Inc. All rights reserved.
Keywords: Hypercholesterolemic rat; Ajuga iva; Iridoids; TBARS; Antioxidant enzymes
Abbreviations: Ai, Ajuga iva; BW, body weight; CAT, catalase; GSH, glutathione; GSH-Px, glutathione peroxidase; GSSH-Red,
glutathione reductase; HC, hypercholesterolemic; HDL, high-density lipoproteins; I, iridoids; LDL, low-density
lipoproteins; RBC, red blood cells; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances;
TC, total cholesterol.

1. Introduction hypercholesterolemia-induced vasculopathies [2]. To protect

tissues from these damaging effects, the organism possesses
At high concentrations, reactive oxygen species can be
enzymatic and nonenzymatic antioxidant systems [3].
important mediators of damage to cell structures, nucleic
Enzymatic antioxidant defenses include superoxide dismu-
acids, lipids, and proteins [1], which could be involved in
tase (SOD), glutathione peroxidase (GSH-Px), and catalase
(CAT). Nonenzymatic antioxidants are represented by
⁎ Corresponding author. ascorbic acid (vitamin C), α-tocopherol (vitamin E), gluta-
E-mail address: bsherazede@yahoo.fr (S. Bouderbala). thione (GSH), carotenoids, flavonoids, and other antioxidants.
0271-5317/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.nutres.2010.05.004
 S. Bouderbala et al. / Nutrition Research 30 (2010) 358–365
Under normal conditions, there is a balance between both
the activities and the intracellular levels of these antiox-
idants. This balance is essential for the survival of organisms
and their health [4].
Hypercholesterolemia, high-cholesterol diet, and oxida-
tive stress increase serum total cholesterol (TC) and low-
density lipoprotein (LDL) cholesterol levels, resulting in
increased risk for atherosclerosis development [5]. In such
conditions, antioxidants play an important role in inhibiting
and scavenging radicals, thus providing protection to
humans against infectious and degenerative diseases.
Hypercholesterolemia is among the most common health
problems treated with traditional remedies [6]. However,
literature shows that medicinal plants have high antioxidant
capacity [5] and also the potential to reduce lipid and
cholesterol in body due to their bioactive compounds [7,8].
Iridoids (I) represent a group of natural constituents with a
monoterpene cyclic ring. They exist usually as glycosidic
forms in nature, but are also found on rare occasion as
aglycone [9].
Iridoids manifest dual facets of biological activities: one
is to act as a defense substance for certain plant species
[10], and other is to produce a variety of pharmacologic
actions for animals. Iridoids are found in many medicinal
plants and may be responsible for some of their phar-
maceutical activities. Isolated and purified, I exhibit a wide
range of bioactivities including cardiovascular, hypoglyce-
mic, anti-inflammatory [11], antioxidant, and hypolipi-
demic [12] activities.
Our previous study showed that treatment by Ajuga iva
(Ai) aqueous extract, which contains sugar, tanins, and I, was
more effective in improving the antioxidant capacity of red
blood cells (RBC) than that of several tissues. Indeed, this
extract was able to reduce the oxidative stress in hypercho-
lesterolemic (HC) rats by increasing the antioxidant enzymes
activities [13].
The hypothesis that the antioxidant defense status in
RBC and tissues in rats fed a cholesterol-rich diet and
treated with Ai may be correlated to I was tested following
2 objectives. The first was to study the effect of different
doses of I from Ai aqueous extract on the lipid peroxidation
in RBC, serum, serum lipoprotein, and tissues. The second
was to measure the antioxidant enzymes activity in RBC
and different tissues in rats.
2. Methods and materials
2.1. Preparation of the aqueous extract of Ai
Mature whole Ai (L) Schreber plants were collected in
November 2004 from Béchar, southwest of Algeria, and
stored at room temperature in a dry place before use. The
arial parts of Ai plant were dried at ambient temperature.
Afterward, 500 mL of distilled water was added to 50 g of
arial plant finely powered, the mixture was heated under
reflux for 60 minutes, and then the decoction was filtered.
359
The filtrate was frozen at −20°C and lyophilized. The
crude yield of the lyophilized material was approximately
18% (wt/wt); it was stored at ambient temperature until
further use [13].
2.2. Extraction and isolation of I from Ai
Aqueous extract of Ai was added to H2O-saturated
n-butanol. After mixing, the supernatant was recuperated
and evaporated to dryness in vacuum. The extract was
submitted to a vacuum liquid chromatography on RP-C18
silica gel to eliminate the free sugar. Samples were subjected
to flash chromatography to give different fractions. After
evaporation of the solvent in vacuum, the fractions were
diluted with H2O; and the crude yield of the lyophilized
material was approximately 2% (wt/wt). The compound in
the aqueous extract of Ai was identified as I and especially
8-O-acetylharpagide according to the literature data [14].
2.3. Animals and diets
Male Wistar rats (n = 32) (Iffa Credo, l'Arbresle, Lyon,
France) weighing 120 ± 5 g were used in this study.
Experimental hypercholesterolemia was induced by feeding
normocholesterolemic rats (with TC value of 2.40 ± 0.62
mmol/L) 1% cholesterol-enriched diet for 15 days (Table 1).
After this phase, serum TC concentration was measured;
and the value was 2.7-fold higher than that in the beginning
of the study (6.5 ± 0.6 mmol/L).
Hypercholesterolemic rats were then divided into 4
groups fed for 15 days with the same diet: 3 groups were
treated by different doses (5, 10, or 15 mg/kg body weight
Table 1
Ingredient composition of the cholesterol-enriched diet fed to rats a
Ingredient g/kg
Casein b
200
Corn starch c 542.5
Sucrose c 40
Sunflower oil c 50
Cellulose b 50
Vitamin mix d 20
Mineral mix e 20
Cholesterol f 10
Cholic acid f 5
Methionine f 3
a
The diet contained 17 780 J/kg and was given in powdered form.
b
Prolabo, Paris, France.
c
Commercial products, Oran, Algeria.
d
UAR 200 Villemoisson, Epinay, S/Orge, France. Vitamin mix
provides (in milligrams per kilogram diet) the following: thiamin, 40;
riboflavin, 30; nicotinic acid, 140; pyridoxine, 20; pyridoxal, 300;
cyanocobalamin, 0.1; ascorbic acid, 1600; tocopherol, 340; calcium
panthotenate, 200; choline, 2720; pteroylmonoglutaric acid, 10; p-
aminobenzoic acid, 100; biotin, 0.6; retinol, 12; and cholecalciferol, 0.125.
e
UAR 205. Mineral mix provides (in grams per kilogram diet) the
following: Ca, 4; K, 2.4; Na, 1.6; Mg, 0.4; Fe, 0.12; Mn, 0.032; Cu, 0.005;
Zn, 0.018; Co, 0.00004; and I, 0.00002.
f
Merck, Darmstadt, Germany.
360 S. Bouderbala et al. / Nutrition Research 30 (2010) 358–365
[BW] by intraperitoneal injection) of I from lyophilized
aqueous extract of Ai, and 1 group was untreated. Diets
and tap water were freely available. Animals were kept in
wire bottom cages at a temperature of 24°C and relative
humidity of 60%, and lights were automatically turned on
from 7:00 AM to 7:00 PM. The General Guidelines on the Use
of Living Animals in Scientific Investigations [15] were
followed, and the protocol and use of rats were approved
by the institutional committee on animal care and use.
2.4. Blood and tissues samples
At day 15, rats were food deprived for 12 hours,
anesthetized with sodium pentobarbital (60 mg/kg BW),
and euthanized with overdose. Blood was collected from
abdominal aorta into dried tubes and centrifuged at 4°C,
1000g for 15 minutes. Serum was taken, and separated RBC
were then washed 3 times by resuspending in 0.9% NaCl
solution and repeating the centrifugation. The washed cells
were lysed in an equal volume of water and mixed
thoroughly. Liver, adipose tissue, gastrocnemius muscle,
heart, and kidney were also excised in ice-cold saline, blotted
on filter paper, and weighed.
2.4.1. Isolation and characterization of serum
LDL–high-density lipoprotein1, high-density lipoprotein2,
and high-density lipoprotein3
Serum LDL–high-density lipoprotein (HDL)1 was iso-
lated by precipitation using MgCl2 and phosphotungstate
(Sigma Chemical Company, Paris, France) by the method of
Burstein et al (1970) [16]. High-density lipoprotein2 and
HDL3 were determined by differential dextran sulfate
magnesium chloride precipitation according to Burstein et
al (1989) [17]. Total cholesterol of serum and each
lipoprotein fraction were determined by enzymatic colori-
metric method (kit Human, GmbH, Wiesbaden, Germany).
2.4.2. Lipid peroxidation
As a marker of the lipid peroxidation, thiobarbituric
acid reactive substances (TBARS) concentrations of serum
and lipoproteins were measured according to the method of
Quintanilha et al (1982) [18] and those of tissues by the
Table 2
Body weight, food intake, and relative organs weights of rats given the different treatments
HC
BW (g) 196 ± 22
Food intake (g/d per rat) 16.00 ± 1.00
Relative weight (g/100 g BW)
Liver 4.63 ± 0.76
Adipose tissue 1.14 ± 0.34
Muscle 0.47 ± 0.07
Heart 0.34 ± 0.04
Kidney 0.72 ± 0.11
Rats were fed a diet containing 1% cholesterol for 15 days. After this phase, rats were fed the cholesterol-enriched diet and treated with I from Ai at doses of 5 mg/
kg BW (I5-HC), 10 mg/kg BW (I10-HC), and 15 mg/kg BW (I15-HC) or not (HC). Statistical evaluation of the data was carried out by the parametric Student
t test. Values are means ± SEM of 6 rats per group.
* P b .05, I-HC treated vs untreated HC group.
method of Ohkawa et al (1979) [19], as previously
described [13].
2.4.3. Total antioxidant capacity of whole blood and RBC
Total antioxidant capacity of whole blood was measured
by monitoring the rat hemolysis (KRL TM test), using a
microplate titrator according to the method described by
Prost (1989) [20] and Blach and Prost (1992) [21]. Whole
blood and washed RBC were diluted (1:25 and 1:50, vol/
vol) with KRL buffer (300 mOsm/L), and 50 μL of whole
blood or RBC suspension was assayed using a 96-well
microplate coated with a free radical generator (GRL, 400,
Kirial SA, Couternon, France). The kinetics of sample
resistance to hemolysis was determined by monitoring the
changes at 620-nm absorbance at 37°C.
2.4.4. Antioxidant enzyme measurements
All enzymes activities were adapted to microplate
titration with the microplate titrator IEMS reader MF (Kirial
SA). Superoxide dismutase (EC 1.15.1.1) activity was
measured at 412 nm by the NADH oxidation procedure
[22]. Glutathione peroxidase (EC 1.11.1.9) was determined
by the method of Paglia and Valentine (1967) [23] using
cumene hydroperoxide as substrate. One unit of GSH-Px
was defined as the oxidation by H2O2 of 1 mmol of reduced
glutathione per minute at pH 7 at 25°C. Glutathione
reductase (GSSH-Red; EC 1.6.4.2) activity was evaluated
at 340 nm by measuring the decrease in NADPH absorbance
in the presence of oxidized glutathione [24]. One unit of
enzyme reduces 1 mmol oxidized glutathione per minute at
pH 7 at 25°C. Catalase activity was determined by the
method of Aebi (1974) [25] by measuring the rate of
decomposition of H2O2 at 240 nm. Glutathione was
measured according to the procedure of Anderson (1985)
[26] using reduced glutathione as standard. Protein concen-
trations were measured according to the method of Lowry
et al (1951) [27] using bovine serum albumin as a standard.
2.5. Statistical analyses
Results were expressed as means ± SEM for 6 rats per
group. Statistical evaluation of the data was carried out by
i5-HC i10-HC i15-HC
265 ± 10 ⁎ 290 ± 15 ⁎ 277 ± 15 ⁎
19.00 ± 0.10 ⁎ 19.50 ± 0.20 ⁎ 19.00 ± 0.10 ⁎
3.56 ± 0.33 3.86 ± 0.26 3.91 ± 0.15
0.99 ± 0.17 0.91 ± 0.20 0.98 ± 0.25
0.36 ± 0.03 0.39 ± 0.01 0.37 ± 0.04
0.34 ± 0.02 0.35 ± 0.02 0.33 ± 0.05
0.73 ± 0.03 0.73 ± 0.04 0.77 ± 0.02
 S. Bouderbala et al. / Nutrition Research 30 (2010) 358–365 361

Table 3
Serum TC, C-LDL-HDL1, C-HDL2, and C-HDL3 and atherogenic ratios of rats given the different treatments
HC i5-HC i10-HC i15-HC
TC (mmol/L) 3.70 ± 0.90 2.68 ± 0.09 ⁎ 2.98 ± 0.33 ⁎ 4.18 ± 1.31
C-LDL-HDL1 (mmol/L) 2.50 ± 0.09 0.32 ± 0.03 ⁎ 0.60 ± 0.09 ⁎ 0.58 ± 0.11 ⁎
C-HDL2 (mmol/L) 0.30 ± 0.07 0.96 ± 0.10 ⁎ 0.67 ± 0.07 ⁎ 0.97 ± 0.20 ⁎
C-HDL3 (mmol/L) 0.20 ± 0.06 0.80 ± 0.07 ⁎ 0.85 ± 0.01 ⁎ 1.75 ± 0.07 ⁎
TC/C-HDL 7.00 ± 1.00 1.52 ± 0.03 ⁎ 1.96 ± 0.01 ⁎ 1.53 ± 0.03 ⁎
C-LDL-HDL1/C-HDL 5.00 ± 0.80 0.18 ± 0.02 ⁎ 0.39 ± 0.05 ⁎ 0.34 ± 0.03 ⁎
Rats were fed a diet containing 1% cholesterol for 15 days. After this phase, rats were fed the cholesterol-enriched diet and treated with I from Ai at doses of
5 mg/kg BW (I5-HC), 10 mg/kg BW (I10-HC), and 15 mg/kg BW (I15-HC) or not (HC). Statistical evaluation of the data was carried out by the parametric
Student t test. Values are means ± SEM of 6 rats per group. C-HDL = C-HDL2 + C-HDL3.
* P b .05, I-HC treated vs untreated HC group.

the parametric Student t test. The calculations were
performed using STATISTICA 6.0 (for Windows, StatSoft
Inc software, Tulsa, OK). The limit of statistical significance
was set at P b .05 between the different groups treated or not
with I from Ai aqueous extract.
3. Results
3.1. Body weight, food intake, and relative tissues weight
At day 15, BW and food intake were increased,
respectively, by 26% and 34%, 30% and 16%, and 18%
and 16% in the I5-HC, I10-HC, and I15-HC groups compared
with the untreated HC group. The relative tissues weights
(liver, adipose tissue, gastrocnemius muscle, heart, and
kidney) were similar in all the groups (Table 2).
3.2. Serum TC, C-LDL-HDL1, C-HDL2, C-HDL3, and
atherogenic ratios
Serum TC concentration was 1.4- and 1.2-fold lower in
the I5-HC and I10-HC groups compared with the HC group.
In all treated groups, C-LDL-HDL1 contents were 7.8-, 4.2-,
and 4.3-fold lower in the I5-HC, I10-HC, and I15-HC groups
than the untreated group. In contrast, C-HDL2 and C-HDL3
amounts were higher, respectively, 3.2- and 4-fold in the I5-
HC group, 2.2- and 4.2-fold in the I10-HC group, and 3.2-
and 8.7-fold in the I15-HC group than in the HC group.
The TC/C-HDL and C-LDL-HDL1/C-HDL ratios repre-
sented 78% and 96% in the I5-HC group, 72% and 92% in
the I10-HC group, and 78% and 93% in the I15-HC group of
the value noted in the HC group (Table 3).
3.3. RBC, serum, LDL-HDL1, HDL2, and HDL3
lipid peroxidation
When compared with the HC untreated rats, the
cholesterol-enriched diet with I treatment for 15 days
showed that RBC TBARS contents were 1.4- and 1.3-fold
lower in the I5-HC and I10-HC groups, respectively. In
serum, the concentrations were, respectively, 2.3-, 2.9-, and
3.-fold lower in the I5-HC, I10-HC, and I15-HC groups; those
of LDL + HDL1 and HDL3 were, respectively, 2- and 2-fold
decreased in the I5-HC than the HC group (Table 4).
3.4. Tissues lipid peroxidation
Liver TBARS values were decreased by 36% and 47% in
the I10-HC and I15-HC groups, respectively. Muscle and
heart TBARS were lowered by 50% and 64% in the I5-HC
group, 73% and 72% in the I10-HC group, and 68% and 20%

Table 4
Thiobarbituric acid reactive substances in RBC, serum LDL-HDL1, HDL2, HDL3, and tissues of rats given the different treatments
HC i5-HC i10-HC i15-HC
RBC (nmol/L) 21.60 ± 3.00 15.11 ± 2.00 ⁎ 16.22 ± 1.5 ⁎ 20.85 ± 2.00
Serum (mmol/L) 0.74 ± 0.25 0.32 ± 0.07 ⁎ 0.25 ± 0.04 ⁎ 0.24 ± 0.05 ⁎
LDL-HDL1 (mmol/L) 0.23 ± 0.05 0.11 ± 0.03 ⁎ 0.13 ± 0.05 0.19 ± 0.03
HDL2 (mmol/L) 0.07 ± 0.007 0.069 ± 0.03 0.065 ± 0.01 0.065 ± 0.05
HDL3 (mmol/L) 0.10 ± 0.03 0.05 ± 0.01 ⁎ 0.06 ± 0.01 0.09 ± 0.02
Liver (mmol/mg protein) 3.32 ± 0.75 2.17 ± 0.50 1.50 ± 0.11 ⁎ 1.56 ± 0.26 ⁎
Adipose tissue (mmol/mg protein) 5.44 ± 0.93 6.11 ± 1.20 5.70 ± 0.65 6.70 ± 1.08
Muscle (mmol/mg protein) 2.21 ± 0.65 1.11 ± 0.25 ⁎ 0.59 ± 0.09 ⁎ 0.70 ± 0.05 ⁎
Heart (mmol/mg protein) 1.49 ± 0.07 0.53 ± 0.13 ⁎ 0.42 ± 0.10 ⁎ 1.20 ± 0.09 ⁎
Kidney (mmol/mg protein) 1.40 ± 0.24 1.35 ± 0.09 0.50 ± 0.03 ⁎ 1.20 ± 0.30
Rats were fed a diet containing 1% cholesterol for 15 days. After this phase, rats were fed the cholesterol-enriched diet and treated with I from Ai at doses of
5 mg/kg BW (I5-HC), 10 mg/kg BW (I10-HC), and 15 mg/kg BW (I15-HC) or not (HC). Statistical evaluation of the data was carried out by the parametric
Student t test. Values are means ± SEM of 6 rats per group.
* P b .05, I-HC treated vs untreated HC group.
362 S. Bouderbala et al. / Nutrition Research 30 (2010) 358–365

Table 5
Total antioxidant status of RBC, antioxidant enzymes activity, and GSH content in RBC and tissues of rats given the different treatments
HC i5-HC i10-HC i15-HC
Blood (T1/2 hemolysis) (min) 71.89 ± 2.40 85.93 ± 0.29 ⁎ 82.63 ± 2.60 ⁎ 81.23 ± 1.53 ⁎
RBC (T1/2 hemolysis) (min) 54.80 ± 3.18 65.96 ± 0.43 ⁎ 60.29 ± 0.97 ⁎ 64.00 ± 1.26 ⁎
RBC (U/g Hb)
GSH-Px 206 ± 09 420 ± 10 ⁎ 511 ± 50 ⁎ 560 ± 25 ⁎
GSSH-Red 39 ± 2 70 ± 11 ⁎ 85 ± 5⁎ 82 ± 6 ⁎
SOD 5.16 ± 0.2 10.16 ± 1.00 ⁎ 11.25 ± 2.50 ⁎ 10.30 ± 1.70 ⁎
CAT 205 ± 15 210 ± 10 225 ± 16 209 ± 1
Liver (U/mg protein)
GSH-Px 66.60 ± 6.30 170.11 ± 2.80 ⁎ 165.00 ± 5.20 ⁎ 168.00 ± 11.00 ⁎
GSSH-Red 139.80 ± 19.50 150.00 ± 11.00 155.00 ± 2.30 140.00 ± 13.20
SOD 17.00 ± 2.52 40.25 ± 3.02 ⁎ 45.00 ± 0.25 ⁎ 48.00 ± 2.10 ⁎
CAT 13.26 ± 3.10 12.11 ± 5.30 24.00 ± 0.70 ⁎ 30.00 ± 1.85 ⁎
Adipose tissue (U/mg protein)
GSH-Px 132.40 ± 25.00 130.00 ± 11.00 129.10 ± 12.10 135.00 ± 25.00
GSSH-Red 251.20 ± 35.60 240.00 ± 20.17 245.00 ± 40.10 260.00 ± 23.40
SOD 254.80 ± 61.08 270.00 ± 25.00 280.00 ± 12.11 275.11 ± 17.13
CAT 59.44 ± 12.03 101.14 ± 20.50 112.00 ± 15.17 113.00 ± 10.25
Muscle (U/mg protein)
GSH-Px 72.86 ± 15.90 102.11 ± 10.2 120 ± 9.45 195.70 ± 1.75
GSSH-Red 147.10 ± 26.90 200.12 ± 9.54 215 ± 10.25 220 ± 9.27
SOD 99.01 ± 14.05 150.11 ± 10.2 197 ± 13 130.2 ± 9.10
CAT 52.81 ± 4.60 50.70 ± 1.17 53 ± 0.97 41.11 ± 0.81
Heart (U/mg protein)
GSH-Px 89.40 ± 12.60 120.00 ± 10.00 ⁎ 140.51 ± 0.60 ⁎ 135.00 ± 9.17 ⁎
GSSH-Red 179.20 ± 28.30 180.17 ± 10.00 160.35 ± 17.10 169.19 ± 9.65
SOD 103.48 ± 26.93 320.00 ± 17.00 ⁎ 330.17 ± 11.65 ⁎ 411.17 ± 20.97 ⁎
CAT 32.02 ± 4.20 60.67 ± 1.30 ⁎ 45.00 ± 5.17 ⁎ 42.00 ± 03.00 ⁎
Kidney (U/mg protein)
GSH-Px 69.96 ± 16.80 70.17 ± 9.11 65.00 ± 6.07 42.17 ± 1.13
GSSH-Red 142.40 ± 36.70 140.00 ± 11.00 136.00 ± 8.17 141.00 ± 2.23
SOD 28.86 ± 8.53 12.52 ± 0.30 ⁎ 20.17 ± 2.25 15.00 ± 0.30 ⁎
CAT 54.17 ± 6.27 60.20 ± 7.11 58.10 ± 1.20 59.35 ± 2.11
GSH
RBC (mmol/L) 11.00 ± 1.00 12.00 ± 1.00 15.40 ± 2.30 ⁎ 15.70 ± 2.11 ⁎
Liver (mmol/mg protein) 31.86 ± 8.50 53.10 ± 0.25 ⁎ 65.15 ± 2.10 ⁎ 65.70 ± 3.10 ⁎
Adipose tissue (mmol/mg protein) 67.57 ± 12.02 70.62 ± 10.25 68 ± 9.15 72.00 ± 10.42
Muscle (mmol/mg protein) 18.94 ± 3.53 30.24 ± 0.13 ⁎ 28.70 ± 1.25 ⁎ 26.00 ± 2.61 ⁎
Heart (mmol/mg protein) 38.73 ± 14.89 67 ± 11 68.25 ± 05 55.17 ± 7.91
Kidney (mmol/mg protein) 12.12 ± 1.98 26.30 ± 1.00 ⁎ 24.20 ± 0.25 ⁎ 32.17 ± 5.11 ⁎
Rats were fed a diet containing 1% cholesterol for 15 days. After this phase, rats were fed the cholesterol-enriched diet and treated with I from Ai at doses of
5 mg/kg BW (I5-HC), 10 mg/kg BW (I10-HC), and 15 mg/kg BW (I15-HC) or not (HC). Statistical evaluation of the data was carried out by the parametric
Student t test. Values are means ± SEM of 6 rats per group.
* P b .05, I-HC treated vs untreated HC group.

in the I15-HC group. In kidney, the values were decreased
by 36% in the I10-HC group (Table 4).
3.5. Total antioxidant capacity of whole blood and RBC
The total antioxidant capacity of whole blood and RBC
was, respectively, higher 1.19- and 1.2-fold in the I5-HC
group, 1.5- and 1.10-fold in the I10-HC group, and 1.13- and
1.17- fold in the I15-HC group compared with the untreated
group (Table 5).
3.6. Antioxidant enzymes activity in RBC and
different tissues
Glutathione peroxidase, GSSH-Red, and SOD activities
in RBC represented, respectively, 40%, 56%, and 51% in the
I5-HC group; 40%, 46%, and 46% in the I10-HC group; and
36%, 47%, and 50% in the I15-HC group of the values noted
in the HC group.
Liver GSH-Px and SOD activities were, respectively,
higher 2.6- and 2.4-fold in the I5-HC group, 2.5- and 2.6-fold
in the I10-HC group, and 2.5- and 2.8-fold in the I15-HC
group than the untreated group. Liver CAT activity was 1.8-
and 2.3- fold higher in the I10-HC and I15-HC groups.
Adipose tissue CAT activity was, respectively, increased
1.7-, 1.9-, and 1.9-fold in the I5-HC, I10-HC, and I15-HC
groups compared with the HC group. Muscle GSH-Px,
GSSH-Red, and SOD activities were, respectively, higher
1.4-, 1.4-, and 1.5-fold in the I5-HC group; 1.6-, 1.5-, and
1.5-fold in the I10-HC group; and 2.7-, 1.5-, and 1.3-fold in
the I15-HC group than the HC group. In the heart, I treatment
 S. Bouderbala et al. / Nutrition Research 30 (2010) 358–365
enhanced GSH-Px, SOD, and CAT activities; and the values
were higher 1.3-, 3.1-, and 1.9-fold in the I5-HC group; 1.6-,
3.2-, and 1.4-fold in the I10-HC group; and 1.5-, 1.4-, and
1.3-fold in the I15-HC group than in the untreated HC group.
In the kidney, SOD activity was significantly decreased in
the I5-HC and I15-HC groups than in the HC group (P b .05)
(Table 5).
3.7. Total glutathione content in RBC and tissues
The RBC GSH content was similar in the I5-HC group,
but was 1.4-fold higher in both the I10-HC and I15-HC
groups than the untreated HC group.
In the liver, muscle, and kidney, I treatment enhanced
GSH values by 40%, 37%, and 54% in the I5-HC group;
50%, 34%, and 50% in the I10-HC group; and 51%, 27%,
and 62% in the I15-HC group compared with the untreated
HC group. In the heart and adipose tissue, the values were
similar in all the I-treated groups compared with the
untreated HC group (Table 5).
4. Discussion
In the present study, we have demonstrated that the I from
lyophilized aqueous extract of Ai have significant antioxi-
dant defense status in RBC and tissues in rats fed a
cholesterol-rich diet. The I extracted from aqueous Ai at
different doses (5, 10, or 15 mg/kg BW) enhanced the BW as
compared with HC untreated rats; and this was probably due
to the increased food intake.
Our previous results showed that cholesterol-enriched
diet (1%) for 15 days induced hypercholesterolemia in rats,
with a decline of 38% in serum TC values with or without
Ai treatment from the beginning to day 15 of the
experiment [13]. However, in the present study, compared
with the HC group, the I treatment at doses 5 or 10 mg/kg
BW significantly decreased TC; but there was no sustained
hypocholesterolemia in rats receiving 15 mg/kg BW dose.
The possible underlying mechanism by which I could exert
their cholesterol-lowering activities was not elucidated. At
this stage of the study, several fundamental mechanisms
could be proposed to explain our results. Iridoids can act
by decreasing the cholesterol biosynthesis especially by
decreasing the 3-hydroxy-3-methyl-glutaryl-coenzyme A
reductase activity and/or by reducing the NADPH required
for fatty acids and cholesterol synthesis. On the other
hand, it could be suggested that I led to a decrease in
cholesterol absorption from the intestine and could have
the ability to inhibit the intestinal absorption of bile acids
and neutral steroids and to enhance hepatic cholesterol 7α-
hydroxylase activity.
It was notable that the reduced TC levels observed with
the I5-HC and I10-HC groups were accompanied by a low
value of C-LDL-HDL1. Iridoids may improve hypercholes-
terolemia by modifying lipoproteins metabolism and
enhance uptake of LDL by increasing LDL receptors and/
363
or by increasing the lecithin: cholesterol acyltransferase
activity, which may contribute to the regulation of blood
lipids. Effectively, our study showed an increase in lecithin:
cholesterol acyltransferase activity (data not shown). In
addition, in these groups and compared with the untreated
HC rats, the TC/C-HDL and C-LDL-HDL1/C-HDL ratios
were decreased. In humans, at least, these low ratios are
considered to reduce the risk of coronary heart disease, even
when TC level is elevated. In addition, several studies
showed that increased C-HDL value is associated with a
decrease in coronary risk [28].
It has been suggested that lipids and proteins oxidation in
lipoproteins and cells membranes leads to impaired lipid
transport and cell injury and thereby contributes to the
development of various diseases [29]. It was suggested also
that hypercholesterolemia increased the level of lipid
peroxidation in the serum of HC rabbits and guinea pigs [30].
Our results showed that RBC TBARS were lower in
rats treated with I, in particular at doses 5 and 10 mg/kg
BW. In addition, I treatment at doses 10 and 15 mg/kg
BW involved a decrease in serum and liver TBARS levels.
Indeed, I treatment reduced TBARS contents by inhibit-
ing the oxidation of LDL, which would seem to imply
that I maintained an antioxidant efficacy as well as an
anti-HC effect.
In HC rats treated with Ai aqueous extract, there was no
significant effect on liver, adipose tissue, and muscle lipid
peroxidation [13], whereas, in the present study, I treatment
decreased TBARS concentrations in the liver, muscle, and
heart, in particular at 10 and 15 mg/kg BW. This result
showed that 5 mg of I per kilogram BW was not sufficient to
lower lipid peroxidation in HC rat.
Enhanced production of superoxide has been demonstrat-
ed in the arterial vessels of HC animals [31]. Erythrocyte is
especially susceptible to oxidative damage resulting from a
high concentration of oxygen and hemoglobin, and the latter
would appear to be a particularly powerful promoter of
oxidative processes. The antioxidant capacity of blood and
RBC was higher in HC rats treated with different doses of
I than in untreated HC rats. These bioactive molecules
are known to play an antioxidant role [12].
Glutathione peroxidase scavenges H2O2 and lipid
peroxides, whereas CAT is responsible for the scavenging
or detoxification of H2O2. Our results showed that RBC
enzymes activities (GSH-Px, GSSH-Red, and SOD) were
higher in all groups treated with I compared with the
untreated HC group. In addition, the high values of GSH-
Px activity and GSH in rats treated with 10 and 15 mg/kg
BW of I increased the protection against lipid peroxidation,
whereas the CAT activity was not sensitive to I treatment.
Schull et al (1991) [32] showed that the decrease of GSH-
Px activity may lower the erythrocyte capacity to deal with
H2O2 and probably lead to an increase in CAT activity as
an adaptation process.
There was no change in liver SOD and GSH-Px activities
in rat fed the cholesterol-enriched diet and treated with Ai
364 S. Bouderbala et al. / Nutrition Research 30 (2010) 358–365
compared with untreated rat [13], whereas, in this study,
SOD activity of the liver, muscle, and heart increased in
HC rats treated with different doses of I. This could be
explained by the Ai other compounds that could inhibit I
activity. The higher SOD activity is believed to be due to
increased dismutation of superoxide anions due to their
high production [33]. In the kidney, SOD activity was
decreased in the I5-HC and I15-HC groups, suggesting
that, in this tissue, 10 mg/kg BW of I was more effective
than 5 and 15 mg/kg BW to lower the accumulation of
superoxide anion.
Liver CAT activity was also higher in the I10-HC and I15-
HC groups compared with the HC group; these doses may
prevent the accumulation of TBARS and their secretion
within the lipoproteins from the liver into the circulation. The
same effect observed in the diabetic rats treated with aqueous
extract of Gongronema latifolium compared with untreated
rats may be caused by induction of CAT activity for
increased protection against H2O2, rather than an increase in
reactive oxygen species concentrations [34].
In conclusion, the observed cholesterol-reducing actions
of the I from Ai at doses of 5 and 10 mg/kg BW indicate
that these bioactive molecules possess some potential
medicinal value and can explain their ethnomedical use.
In addition, I extracts may decrease the lipid peroxidation
in RBC and tissues associated with an increase in the
antioxidant enzymes activities in rats fed a cholesterol-
enriched diet. In future studies, it is necessary to confirm
these results in human subjects and to adjust the dose for
better therapeutic effects.
Acknowledgment
This research was supported by the Algerian Ministry of
Higher Education and Scientific Research and by the French
Foreign Office with International Research Extension Grant
04 MDU 629.
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