Changes in antioxidant defense status in hypercholesterolemic rats treated with Ajuga iva.

Abstract

The aim of the study was to investigate the effect of aqueous extract of Ajuga iva (Ai) on serum and tissues lipid peroxidation as well as antioxidant enzymes activities in red blood cells (RBC) and tissues, in high hypereholesteroclemie rats (HC). Male Wistar rats (n = 12) were fed on 1% cholesterol-enriched diet for 15 d. After this adaptation phase, hypercholesterolemic rats (total cholesterol = 6.5 + 0.6mol/l) were divided into two groups fed the same diet and treated or not with Ai for 15 d. Thiobarbituric acid reactive substances (TBARS) concentrations in serum, LDL-[HDL.sub.1], [HDL.sub.2] and [HDL.sub.3] were respectively, 5-, 7.8-, 2.3- and 5-fold lower in Ai treated than untreated hypercholesterolemic groups. TBARS concentrations were 1.4-fold lower in heart and 2.8-fold higher in kidney in Ai-HC treated than untreated HC group. Superoxide dismutase activity was respectively, 1.2- and 1.4-fold higher in RBC and muscle in Ai treated than untreated group. In RBC, Ajuga iva treatment enhanced glutathione peroxidase (GSH-Px) ( + 9%) and glutathione reductase (GSSH-Red) (+ 12%) in Ai-HC treated than untreated HC group. GSSH-Red activity was 1.4- and 1.5-fold higher in adipose tissue and heart, respectively and 3.7-fold lower in kidney in Ai treated than untreated group. Liver catalase activity was 1.6-fold higher in Ai treated than untreated group. Adipose tissue and muscle total glutathione content represented in Ai treated group 35% and 36% of the value noted in untreated group. Nitric oxide values of liver, adipose tissue and heart were 3.3-, 2.5- and 3.4-fold higher in Ai-HC than HC group. Ajuga iva treatment enhanced [delta]-tocopherol contents ( + 25%) in Ai treated than untreated group. In conclusion, Ajuga iva treatment is more effective to improve the antioxidant capacity of RBC than that of tissues. Indeed, At is able to reduce the oxidative stress in hypercholesterolemic rats by increasing the antioxidant enzymes activity. [c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Hypercholesterolemic rat; Ajuga iva; TBARS; Antioxidant enzyme

Introduction

The beneficial effect of lowering hypercholesterolemia in the prevention of coronary heart diseases is well established (Simons, 2002). Epidemiological studies have clearly shown that diets rich in plant foods protect human against degenerative diseases such as cancer and cardio-vascular diseases (Manach et al., 2005). Several lines of evidence indicate that the oxidative modification of LDL and their accelerated uptake by artery wall macrophages contribute to the formation of atherosclerotic plaque (Chisolm and Steinberg, 2000). In vitro studies suggested that oxidized high density lipoproteins (HDL) have a diminished role in reverse cholesterol transport (Francis, 2000).

A number of epidemiological studies conducted during recent years have clearly demonstrated a link between stress and the development and the course of many diseases (Gumsulu et al., 2002). Antioxidants are important aspect of health maintenance based on their modulation of the antioxidative process in the body (Lee et al., 2002).

Feeding antioxidants attenuate the atherogenic process in animal models, mainly due to their free radical scavenging capabilities (Paul et al., 2001). In addition to food, medicinal plants are relied upon by 80% of the world population for their basic health care needs (Zaidi and Crow, 2005). Various medicinal properties have been ascribed to natural herbs and constitute the main source of new pharmaceuticals and healthcare products (Monsef et al., 2004). As plant could represent a source of natural compounds with antioxidant activities, many studies have been conducted searching for the antioxidant activities of many plant extracts and their constituents (Zhu et al., 2004). Measurements of the glutathione peroxidase (GSH-Px) and the superoxide dismutase (SOD) activities of liver and red blood cells (RBC) of aqueous extract from Rumex patientia (Polygonaceae) administered to rats showed that there was an increase in GSH-Px and SOD activities when compared to the control (Cetinkaya et al., 2002). Many studies were carried out on the effect of medicinal plant on lipid peroxidation and antioxidant status in animal fed a diet without cholesterol supplementation. The effects of a leaf lyophilized aqueous extract of the traditional medicinal plant Rhazya stricta (0.25, 1.0 and 4.0g/kg/day for 3 days) on reduced glutathione (GSH) and lipid peroxidation were studied in rats. The plant aqueous extract, at a dose of 4.0g/kg increased significantly the concentration of GSH and decreased lipid peroxidation in the liver (Ali et al., 2000).

Several scientific studies have been conducted on many species of the genus Ajuga, which are ecologically related and some of their active compounds have been identified (Takasaki et al., 1999). Ajuga iva (L.) Schreber (Labiatae) is used as an anthelmintic, against intestinal disorders (Bellakhdar et al., 1991), and as a diuretic agent (Alliotta and Pollio, 1994). According to ethnobotanical data collected in oriental Morocco by Ziyyat et al. (1997), Ajuga iva, locally known as 'Chendgoura', is also alleged to possess hypoglycaemic activity and it is believed by many Moroccan diabetics that the decoction of this plant consumed over a long time removes the cause of diabetes.

Our previous study (Chenni et al., 2007) using two groups of normocholesterolemic rats (3.0 [+ or -] 0.3mmol/l)submitted to 1% cholesterol-supplemented diet for 28 days have shown that Ajuga iva (0.5% in the diet during the experiment duration) decreased lipid peroxidation and increased the antioxidant enzyme activities.

In this study rats were submitted to 1% cholesterolsupplemented diet during 15 days involving hypercholesterolemia (2.4-> 6.5mmol/l). After this time, Ajuga iva treatment was delivered (0.5% in the diet) for 15 days.

Therefore, the purpose of this study was to examine the influence of aqueous extract of Ajuga iva on serum and tissues lipid peroxidation as well as on the antioxidant enzymes activities of RBC and tissues, in hypercholesterolemic rats.

Material and methods

Preparation of the aqueous extract of Ajuga iva

Mature whole Ajuga iva (L.) Schreber plants were collected in November 2004 from Bechar, South-West of Algeria and stored at room temperature in a dry place prior to use. The arial parts of Ajuga iva plant were dried at ambiant temperature. Then, 500ml of distilled water was added to 50 g of arial plant finely powered and the mixture heated under reflux for 60min and then the decoction was filtered. The filtrate was frozen at-20[degrees]C and lyophilised. The crude yield of the lyophilized material was approximately 18% (w/w), it was stored at ambiant temperature until further use (Chenni et al., 2007).

Animals and diets

Male Wistar rats (n = 12) (Iffa Credo, l'Arbresle, Lyon, France) weighing 120 [+ or -] 5g were used in this study. Experimental hypercholesterolemia was induced by feeding normocholesterolemic rats (with total cholesterol (TC) value of 2.40 [+ or -] 0.62mmol/l) a 1% cholesterol-enriched diet (casein, 200 (95% purity) (Prolabo, Paris, France) combined with Isio 4 oil, 50; sucrose, 40; cornstarch, 585; cellulose, 50; vitamins, 20; minerals 40; cholesterol, 10; cholic acid 5 (Merck, Darmstadt, Germany) (to facilitate cholesterol absorption) for 15 days. After this phase, serum cholesterol level was measured and the value was 6.5 [+ or -] 0.6mmol/l. At the beginning of the treatment (d0), hypercholesterolemic (HC) rats were divided into two groups fed for 15 days (d15) the same diet with or without Ajuga iva (Ai) lyophilised aqueous extract (0.5%). The composition of minerals, vitamins and Isio 4 oil was previously reported by Frenoux et al. (2001).

Diets and tap water were freely available. Animals were kept in wire bottom cages at temperature of 24[degrees]C, relative humidity of 60% and light were automatically turned on from 07:00 to 19:00. We followed the general Guidelines on the Use of Living Animals in Scientific Investigations (Council of European Communities, 1987).

To evaluate the digestibility of lipid in hypercholesterolemic diet, six rats from control and Ai groups were placed individually into metabolism cages. Food intake was measured daily. Faeces were collected from d7 to d15 of the experiment. Total lipids were extracted according to the method of Delsal (1944). The fecal cholesterol content was determined by enzymatic colorimetric method (kit Human, GmbH, Wiesbaden, Germany).

Blood samples

At d15, rats were food deprived for 12 h and anaesthetized with sodium pentobarbital (60mg/kg body weight). Blood was collected from abdominal aorta into dried tubes and centrifuged at 4[degrees]C, 6000g for 15min. Serum was taken and separated RBC were then washed three 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, muscle, heart and kidney were also quickly excised in ice-cold saline, blotted on filter paper and weighed.

Isolation and characterisation of serum LDL-[HDL.sub.1], [HDL.sub.2] and [HDL.sub.3]

Serum LDl-[HDL.sub.1] were isolated by precipitation using [MgCl.sub.2] and phosphotungstate (Sigma Chemical Company, France) by the method of Burstein et al. (1970). [HDL.sub.2] and [HDL.sub.3] were performed by differential dextran sulphate magnesium chloride precipitation according to Burstein et al. (1989). To estimate the validity of this method, ultracentrifugation was performed according to Havel et al. (1955). Total cholesterol (TC) of serum and each fraction was determined by enzymatic colorimetric method (kit Human, GmbH, Wiesbaden, Germany).

Lipid peroxidation

As a marker of the lipid peroxidation, thiobarbituric acid reactive substance (TBARS) concentrations of serum and each fraction were measured according to the method of Quintanilha et al. (1982) using tetramethoxypropane (Prolabo) as precursor of malondialdehyde. One milliliter of diluted sample (protein concentration about 2mg/ml) was added to 2 ml of thiobarbituric acid (final concentration, 0.017 mmol/l), plus butylated hydroxytoluene (concentration, 3.36[micro].mol/1) and incubated for 15min at 100[degrees]C. After cooling and centrifugation, the absorbance of supernatant was measured at 535 nm. Data were expressed as mmol of TBARS produced/ml of serum.

Tissues lipid peroxidation was determined by the method of Ohkawa et al. (1979). Briefly, liver, adipose tissue, heart, muscle or kidney (0.5 g) was homogenized with 4.5 ml of KC1 (1.15%). The homogenate (100[micro]l) was mixed with 0.1 ml SDS (8.1%), 750[micro]l acetic acid (20%) and 750[micro]l TBA reagent (0.8%). The reaction mixture was heated at 95[degrees]C for 60min. After heating, the tubes were cooled and 2.5 ml of n-butanol-pyridine was added. After mixing and centrifugation at 4000g for 10min, the upper phase was taken for measurement at 532 nm. Results were expressed as [mu]mol of TBARS/mg of proteins.

Antioxidant enzyme measurements

All enzymes activities were adapted to micro-plate titration with the micro-plate titrator IEMS reader MF (KIRIAL SA Couternon, France). Superoxide dismutase (SOD; EC 1.15.1.1) activity was measured at 412 nm by the NADH oxidation procedure (Elstner et al., 1983). SOD activity was compared with those of standard solution of known activity (SOD, Sigma). Glutathione peroxidase (GSH-Px; EC 1.11.1.9) was determined by the method of Paglia and Valentine (1967) using cumene hydroperoxide as substrate. One unit of GSH-Px was defined as the oxidation by [H.sub.2][O.sub.2] of 1[micro]mol of reduced glutathione per min at pH 7 at 25[degrees]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 (Goldberg and Spooner, 1992). One unit of enzyme reduces 1[micro] mol oxidized glutathione per min at pH 7 at 25[degrees]C. Catalase activity was determined by the method of Aebi (1974) by measuring the rate of decomposition of [H.sub.2][O.sub.2] at 240 nm. Glutathione was measured according to the procedure of Anderson (1985) using reduced glutathione as standard.

Tissues proteins were estimated by BCA method using bovine serum albumin as a standard.

Nitric oxide determination in serum and tissues and vitamin measurements in serum

Nitric oxide determination was performed by using the Griess reagent (sulphanilamide and n-naphtyl-ethylene diamine) (Cortas and Wakid, 1990). Serum and tissue extracts were clarified by zinc sulphate solution and [NO.sub.3] was then reduced to [NO.sub.2] by cadmium overnight at 20[degrees]C under shaking. Samples were added to the Griess reagent and incubated for 20 min at room temperature. Absorbance was measured at 540 nm. Sodium nitrite was used for a standard curve.

Serum retinol and [alpha]-tocopherol were analyzed by high-performance liquid chromatography with a Varian system (Varian, Les Ulis, France) after hexane extraction. Tocol (Lara Spiral, Couternon, France) was added to samples as an internal standard (Jezequel-Cuer et al., 1995).

Statistical analysis

Results were expressed as means [+ or -] SEM. Statistical evaluation of the data was carried out by the parametric Student 't' test. The limit of statistical significance was set at p<0.05 between the both groups treated and untreated with Ajuga iva extract.

Results

Body weight, food and cholesterol intake

At d15, a similar body weight was noted in the both hypercholesterolemic (HC) rats treated or not with Ai. Food and cholesterol intakes were increased by 11% but lipid digestibility and fecal cholesterol values were similar in Ai treated compared with untreated group (Table 1).

Serum total cholesterol (TC), LDL-[HDL.sub.1]-C, [HDL.sub.2]-C, [HDL.sub.3]-C and atherogenic ratios

At d15 of feeding the hypercholesterolemic diet, rats treated or not with Ajuga iva showed no significant difference in serum TC (4.0 [+ or -] 0.8 mmol/1, the average of HC and Ai HC) but this value was lowered by about --38% compared to that noted at d0 (Table 1).

Table 1. Body weight, lipid digestibility and serum total cholesterol

Rats                            HC                  Ai-HC

Body weight (g)           196 [+ or -] 22     204 [+ or -] 24

Food intake (g/d/rat)    16.0 [+ or -]0.2    18.0 [+ or -]0.5 *

Lipids intake             960 [+ or -] 54    1080 [+ or -]
(mg/d/rat)

Fecal lipids (mg/d/rat)    70 [+ or -] 8       80 [+ or -] 11

Cholesterol intake        160 [+ or -] 5      180 [+ or -] 10 *
(mg/d/rat)

Fecal cholesterol          32 [+ or -] 7       33 [+ or -] 2
(mg/d/rat)

Lipid digestibility (%)    93 [+ or -] 2       93 [+ or -] 4

Serum total cholesterol   6.5 [+ or -] 0.6     .5 [+ or -] 0.6
(mmol/1) (d.sub.0)

Serum total cholesterol  3.70 [+ or -] 0.90  4.30 [+ or -] 0.80
(mmol/1) (d.sub.15)

Values are means [+ or -] SEM of 6 rats per group. Both groups were fed
the cholesterol-enriched diet and treated(Ai-HC) or not (HC) with
Ajuga iva.

Lipid digestibility (%) = [(ingested lipids-excretedlipids)/ingested
lipids] x 100.

* p < 0.05, Ai-HC treated vs untreated HC group.

LDL-[HDL.sub.1]-C contents were similar in the both groups (2.60 [+ or -] 0.06 mmol/1, the average of HC and Ai-HC) (Table 2). [HDL.sub.2]-C and [HDL.sub.3]-C amounts enhanced respectively by 40% and 74% in Ai-HC group compared to HC group. TC/HDL-C and LDL-[HDL.sub.1]-C/HDL-C ratios were respectively, 1.8- and 4-fold lower in Ai treated than untreated hypercholesterolemic group.

Table 2. LDL-[HDL.sub.1]- cholesterol (C), [HDL.sub.2]-C, [HDL.sub.3]-C
and atherogenic ratios

Rats                                HC                Ai-HC

LDL-[HDL.sub.1]-C (mmol/1)  2.50 [+ or -] 0.09  2.70 [+ or -] 0.04
[HDL.sub.2]-C (mmol/1)      0.30 [+ or -] 0.07  0.50 [+ or -] 0.02 *
[HDL.sub.3]-C (mmol/1)      0.20 [+ or -] 0.06  1.00 [+ or -] 0.04 *
TC/HDL-C                    7.00 [+ or -] 1.00  4.00 [+ or -] 0.40 *
LDL-[HDL.sub.1]-C/HDL-C     5.00 [+ or -] 0.80  1.20 [+ or -] 0.40 *

Values are means [= or -] SEM of 6 rats per group.Both groups were fed
the cholesterol-enriched dietand treated (Ai-HC) or not (HC) with
Ajuga iva.

HDL-C = [HDL.sub.2]-C + [HDL.sub.3]-C.

* p<0.05, Ai-HC treated vs untreated HC group.

Serum, LDL-[HDL.sub.1], [HDL.sub.2], [HDL.sub.3] and tissues lipid peroxidation

Lipid peroxidation was significantly decreased in Ai group. Serum, LDL-[HDL.sub.1], [HDL.sub.2] and [HDL.sub.3] TBARS contents were respectively, 5-, 7.8-, 2.3- and 5-fold lower in Ai treated than untreated hypercholesterolemic groups (Table 3).

Table 3. Thiobarbituric acid reactive substances(TBARS) contents in
serum, LDL-[HDL.sub.1], [HDL.sub.2], [HDL.sub.3] (mmol/ml) and in
tissues ([mu]mol/mg protein)

TBARS                          HC                  Ai-HC

Serum                  0.80 [+ or -] 0.20    0.14 [+ or -] 0.04 *
LDL-[HDL.sub.1]       0.230 [+ or -] 0.050  0.030 [+ or -] 0.007 *
[HDL.sub.2]           0.070 [+ or -] 0.007  0.030 [+ or -] 0.005 *
[HDL.sub.3]            0.10 [+ or -] 0.03    0.02 [+ or -] 0.01 *
Liver                  3.00 [+ or -] 0.70    2.00 [+ or -] 0.80
Adipose tissue         5.00 [+ or -] 1.00    6.00 [+ or -] 1.00
Gastrocnemius muscle   2.00 [+ or -] 0.60    2.00 [+ or -] 0.80
Heart                   1.0 [+ or -] 0.1      0.8 [+ or -] 0.3 *
Kidney                 1.40 [+ or -] 0.20    4.00 [+ or -] 0.90 *

Values are means [+ or -] SEM of 6 rats per group.Both groups were fed
the cholesterol-enriched dietand treated (Ai-HC) or not (HC) with
Ajuga iva.

* p<0.05, Ai-HC treated vs untreated HC group.

Heart TBARS concentrations were 1.4-fold lower, whereas those of kidney were 2.8-fold higher in Ai-HC than HC group (Table 3).

Antioxidant enzymes activity in RBC and different tissues

The RBC superoxide dismutase (SOD) activity was 1.2-fold higher in Ai treated than untreated hyperchlesterolemic group. Ai treatment enhanced glutathione peroxidase (GSH-Px) (+ 9%) and glutathione reductase (GSSH-Red) (+ 12%) in RBC.

In heart and muscle, Ajuga iva treatment enhanced SOD activity ( + 28% and + 50%, respectively) in Ai treated than untreated group, whereas, that of kidney was decreased (-80%). Adipose tissue and heart GSSH-Red activity was respectively, 1.4- and 1.5-fold higher in Ai treated than untreated hypercholesterolemic group. Kidney GSSH-Red activity was 3.7-fold lower in Ai treated than untreated group. Liver catalase activity was 1.6-fold higher in Ai treated than untreated hypercholesterolemic group, whereas those of adipose tissue, muscle, heart and kidney were not affected by Ajuga iva treatment (Table 4).

Table 4. Antioxidant enzymes activity in red blood cells and different
tissues

Enzymes                          HC                  Ai-HC

RBC (U/g Hb)

  GSH-Px                  206 [+ or -] 9         228 [+ or -] 5 *
  GSSH-Red               39.0 [+ or -] 1.7      44.0 [+ or -] 1.5 *
  SOD                    5.16 [+ or -] 0.20     6.33 [+ or -] 0.50 *
  Catalase                205 [+ or -] 15        257 [+ or -] 41

Liver (U/mg protein)
  GSH-Px                 66.6 [+ or -] 6.3      64.8 [+ or -] 9.4
  GSSH-Red              139.8 [+ or -] 19.5    135.5 [+ or -] 19.6
  SOD                   17.00 [+ or -] 2.52    12.77 [+ or -] 2.71
  Catalase              13.26 [+ or -] 3.10    20.81 [+ or -] 1.70 *

Adipose tissue (U/mg protein)
  GSH-Px                142.4 [+ or -] 25.0    182.1 [+ or -] 22.4
  GSSH-Red            N 251.2 [+ or -] 35.6    353.1 [+ or -] 36.2 *
  SOD                  254.80 [+ or -] 61.08  280.37 [+ or -] 54.16
  Catalase              59.44 [+ or -] 12.03   78.11 [+ or -] 15.03

Gastrocnemius muscle (U/mg protein)
  GSH-Px                 72.8 [+ or -] 16.0     82.9 [+ or -] 19.0
  GSSH-Red              147.1 [+ or -] 27.0    144.8 [+ or -] 27.0
  SOD                   99.01 [+ or -] 14.05  136.49 [+ or -]21.60 *
  Catalase              52.81 [+ or -] 4.60    57.66 [+ or -] 8.09

Heart (U/mg protein
  GSH-Px                 89.4 [+ or -] 12.6     93.9 [+ or -] 17.0
  GSSH-Red             179.20 [+ or -] 28.30  276.30 [+ or -] 40.88 *
  SOD                  103.48 [+ or -] 26.93  208.75 [+ or -] 53.69 *
  Catalase              32.01 [+ or -] 4.20    32.05 [+ or -] 2.78

Kidney (U/mg protein)
  GSH-Px                69.96 [+ or -] 16.80   55.40 [+ or -] 8.00
  GSSH-Red               74.5 [+ or -] 12.0    20.28 [+ or -] 5.50 *
  SOD                  142.40 [+ or -] 36.70   28.86 [+ or -] 8.53 *
  Catalase              54.17 [+ or -] 6.27    63.01 [+ or -] 5.34

Values are means [+ or -] SEM of 6 rats per group.Both groups were fed
the cholesterol-enriched diet and treated (Ai-HC) or not (HC) with
Ajuga iva.

* p< 0.05, Ai-HC treated vs untreated HC group

Total glutathione content in RBC and tissues

The total glutathione (GSH) contents of RBC, liver and heart were similar in both hypercholesterolemic groups, whereas those of adipose tissue and muscle were 1.5-fold higher in Ai-HC than HC group (Table 5).

Table 5. Total glutathion content (GSH) in RBC (mmol/1) and tissues
(mmol/mg protein)

GSH                        HC             Ai-HC

RBC                   11 [+ or -] 1    11 [+ or -] 1
Liver                 32 [+ or -] 8    33 [+ or -] 5
Adipose tissue        68 [+ or -] 12  104 [+ or -] 12 *
Gastrocnemius muscle  19 [+ or -] 4    30 [+ or -] 6 *
Heart                 39 [+ or -] 14   40 [+ or -] 8
Kidney                12 [+ or -] 2    10 [+ or -] 2

Values are means [+ or -] SEM of 6 rats per group. Both groups were fed
the cholesterol-enriched diet and treated (Ai-HC) or not (HC) with
Ajuga iva.

* p<0.05, Ai-HC treated vs untreated HC group.

Nitric oxide levels in serum and tissues and vitamin contents in serum

Nitric oxide values of serum, muscle and kidney were similar in both hypercholesterolemic groups, whereas those of liver, adipose tissue and heart were 3.3-, 2.5- and 3.4-fold higher in Ai-HC than HC group.

No significant difference in serum retinol contents were found between both groups, whereas, Ajuga iva treatment enhanced [alpha]-tocopherol contents (+ 25%) (Table 6).

Table 6. Nitric oxide levels in serum (mmol/ml) and tissues
([mu]mol/mg protein) and vitamins contents in serum ([mu]g/ml)

                               HC                 Ai-HC

Serum                  0.15 [+ or -] 0.01   0.18 [+ or -] 0.02
Liver                 17.31 [+ or -] 2.68  58.66 [+ or -]16.12 *
Adipose tissue         7.11 [+ or -] 2.19  17.75 [+ or -] 2.35
Gastrocnemius muscle  15.26 [+ or -] 4.32  17.70 [+ or -] 1.78
Heart                  5.73 [+ or -] 0.82  19.20 [+ or -] 7.83 *
Kidney                16.15 [+ or -] 4.38  21.78 [+ or -] 3.12
Retinol               38.78 [+ or -] 3.51  39.65 [+ or -] 7.79
[alpha]-Tocopherol    33.71 [+ or -] 3.11  44.51 [+ or -] 5.73 *

Values are means [+ or -] SEM of 6 rats per group. Both groups were fed
the cholesterol-enriched diet and treated (Ai-HC) or not (HC) with
Ajuga iva.

* p < 0.05, Ai-tiC treated vs untreated HC group.

Discussion

In this investigation, the effect of lyophilised aqucous extract from Ajuga iva on serum and tissues lipid peroxidation as well as its influence on the activities of antioxidant enzymes in RBC and tissues, were reported in hypercholesterolemic rats.

Previous study showed that Ajuga iva lowered plasma total cholesterol by about --18% in rat fed high-cholesterol diet (HCD) for 28 days (Chenni et al., 2007). In addition, feeding of atherogenic diet (0.5% cholic acid and 1% cholesterol) for 3 weeks resulted in an increase of total cholesterol (Minhajuddin et al., 2005). These data confirmed and extended those reported by other finding diets enriched by different amounts of cholesterol, ranging between 0.5% and 4% (Tanaka et al., 2005) for different period of times, supplied or not with cholic acid.

In this investigation, cholesterol enriched diet (1%) for 15 days induced hypercholesterolemia in rats. A decline of --38% was noted in serum cholesterol values with or without Ajuga iva treatment from dO to d15 and the values were similar in both groups. This might indicate that Ajuga iva had no effect on hypercholesterolemia. This could be explained, in part, by the similar values of lipid digestibility and fecal cholesterol excretion. In addition, Ai treated group are more cholesterol (20 mg/d) compared to untreated group, which could be shown by enhanced HDL-C or/and in cholesterol used. Whereas, E1-Hilaly et al. (2006) showed that in normal rats, 6th after a single oral dose of Ajuga iva-extract (10mg/kg BW), a significant reduction in plasma cholesterol was noted. In addition, rats have a strong capability to maintain their serum cholesterol (Fujioka et al., 1995) and are particularly resistant to the development of hypercholesterolemia and atherosclerosis (Giricz et al., 2003).

Hyperlipidemia (mainly high total cholesterol or low LDL-C) is an important risk factor in the initiation and progression of atherosclerotic lesions (Harrison et al., 2003). Several studies showed that increased HDL-C value is associated with a decrease in coronary risk (Harrison et al., 2003). In the present study, there was no significant difference in serum total cholesterol and LDL-C between hypercholesterolemic groups treated or not with Ajuga iva. However, [HDL.sub.2]-C and [HDL.sub.3]-3C values were respectively, 1.7- and 5-fold higher, in Ai-HC than HC group. This was an important advantage in the treatment of hypercholesterolemia, especially among people where low HDL-C was the prevalence of lipoprotein abnormalities. TC/HDL-C or LDL-C/ HDL-C ratios were markedly reduced in Ai-HC than HC groups. These decreased ratios are predictors of coronary risk (National Cholesterol Education program Expert Panel, 1994). Ajuga iva treatment enhanced dietary cholesterol intake but serum cholesterol was similar in the both groups treated or not. This could be explained by a lowered 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA-reductase) activity, the rate-limiting enzyme in biosynthesis of cholesterol (data not published). The reduced cholesterol 7[alpha]-hydroxylase activity (data not published) on the one hand, and the high C-HDL (substrate of this enzyme) on the other hand, might suggest a lower synthesis of bile acids.

Lipid peroxidation is a free radical chain reaction, which is triggered by hydroxyl radical and leads to membrane break down producing more free radicals (Ji et al., 2003). Amold et al. (1992) showed that feeding 1% cholesterol to rats for 8 weeks did not alter their serum susceptibility to lipid peroxidation compared with rats fed a cholesterol-free diet. Our results showed that thiobarbituric acid-reactive substance (TBARS) contents were lower in serum, LDL-[HDL.sub.1], [HDL.sub.2] and [HDL.sub.3] of Ai treated compared to untreated hypercho-lesterolemic group. These decreases, observed with Ajuga iva treatment, could be good indicators of lowered lipid peroxidation. In hypercholesterolemic rats treated with Ajuga iva, there was no effect on liver, adipose tissue and muscle lipid peroxidation. Indeed, Mahfouz and Kummerow (2000) observed no difference in liver TBARS in rat fed a diet containing 1% cholesterol, but Lu and Chiang (2001) noted that cholesterol feeding (1%) to rats for 6 weeks led to markedly decreased levels of hepatic TBARS substances when compared to cholesterol free.

Hypercholesterolemic is a dominant risk factor of atherosclerosis (Deepa and Veralakshmi, 2005) and oxidative stress is one of the causative factors that link hypercholesterolemia (Lee et al., 2002). This stress results from an imbalance between the production of free radicals and effectiveness of antioxidant defence system (Lum and Roebuck, 2001). RBC and hepatic tissue contain enzymes that contribute to the antioxidant defense mechanism (Lee et al., 2002). Red blood cells are susceptible to oxidation by oxygen radical because they are very rich in [Fe.sup.++] containing molecules, primarily hemoglobin (Subah et al., 2004). Disorders in RBC antioxidant parameters have also been reported in subjects with cardiovascular diseases (Akkus et al., 1996).

Mahfouz and Kummerow (2000) did not observe any significant difference in GSH-Px, catalase or SOD activities of RBC between rats fed 1% cholesterol or basal diets, at any time. In the present study, RBC superoxide dismutase activity was higher in the Ai treated than untreated hypercholesterolemic group. This increase might be due to enzyme activation by Ajuga iva treatment. In RBC, in spite of higher glutathione peroxidase (GSH-Px) and glutathione reductase (GSSG-Red) activities, total glutathione (GSH) content was not sensitive to Ajuga iva treatment. Therefore, in the current study, the higher SOD activity appeared to contribute to reduced reactive oxygen species level in the RBC with Ajuga iva treatment. This increase might constitute a protection against superoxide anion elevation. Because SOD catalyses the decomposition of superoxide anions to hydrogen peroxide ([H.sub.2][O.sub.2], this enzyme prevents the further generation of free radicals (Yu, 1994). Superoxide radicals are converted by SOD to [H.sub.2][O.sub.2], which is broken down by catalase and GSH-Px. However, this process can cause lipid peroxidation if [H.sub.2][O.sub.2] is not decomposed immediately (Gumsulu et al., 2002). In addition, GSH serves as a substrate for the enzyme GSH-Px, and it has been suggested that it is through the activity of this enzyme that GSH protects the RBC against oxidative damage. In this study, the similar values of GSH in Ai treated and untreated hypercholesterolemic groups showed that GSH content is sufficient for GSH-Px activity. Vasu et al. (2005) showed that Enicostemma littorale (a small herb of family Gentianaceae) aqueous extract decreased the SOD activity and the lipid peroxidation with increased RBC glutathion contents, in rats fed cholesterol-enriched diet (1%) as compared to untreated rats.

In our experiment, at d15, liver SOD activity was similar in both HC groups treated or not by Ai, whereas Chenni et al. (2007) showed that Ajuga iva treatment combined to high cholesterol diet during 28 days, exhibited higher SOD activity in liver and kidney of rats. This result might indicate that the activation of this enzyme could be observed after d15 of Ai treatment. Ajuga iva treatment of HC rats showed similar values of GSH-Px and GSSG-Red activities and GSH in liver. The same value of GSH-Px activity in liver between both groups treated or not with Ajuga iva could be dependent on the unchanged GSH concentration. In addition, Lu and Chiang (2001) noted that 1% cholesterol feeding to rats for 6 weeks led to markedly decreased hepatic SOD and GSH-Px activities when compared to cholesterol free. Additionally, liver catalase activity was higher in Ai-treated than untreated HC group. The stimulation of the liver catalase activity can protect their tissues against oxidation, thus preventing the accumulation of lipid peroxidation and their secretion within the lipoproteins from the liver into the circulation, in hypercholesterolemic rats (Panasenko et al., 1992). GSSH-Red and SOD activities were lowered in kidneys of Ai treated compared to untreated HC group, suggesting no accumulation of superoxide anion, leading to increased TBARS contents in this tissue. In muscle, SOD activity was higher in Ai treated compared to untreated which would lead to a decrease in superoxide radical numbers. Enhanced SOD activity in heart of Ai treated compared to untreated hypercholesterolemic group was due to higher values of nitric oxide which might be the result of a reduced production of superoxide anion radical. Onody et al. (2003) showed that cardiac NO was significantly decreased in heart of cholesterol-fed rats. However, the mechanism of reduced NO level was not known and the authors hypothesized, that the decrease in cardiac NO level is secondary to increased production of superoxide and formation of peroxinitrite. In this study, higher nitric oxide content in liver, adipose tissue and heart of Ai treated compared to untreated group might be due to enhanced NO biosynthesis or to decreased NO elimination. In addition, in heart, enhanced NO content might be the consequence of decreased formation of superoxide anion. Effectively, SOD activity was higher in Ai treated than untreated group. Increased serum [alpha]-tocopherol of Ai treated compared to untreated hypercholesterolemic group could result from a high endogenous synthesis in response to oxidative stress in these rats.

In conclusion, when compared the antioxidative parameters in RBC and tissues between the hypercholesterolemic groups treated or not with Ajuga iva, it can be suggested that the antioxidative system of the RBC is more effective than that of the studied tissues. The lyophilized aqueous extract of Ajuga iva is able to reduce the oxidative stress which may prevent lipid peroxidation in hypercholesterolemic rats. Flavonoids and iridoids were isolated from Ajuga iva (Ghedira et al., 1991). Since flavonoids are well known for their antioxydant activity (Wagner and Lacaille-Dubois, 1995), it may be suggested that the antioxidant defense status in hypercholesterolemic rats treated with Ajuga iva might be correlated to these compounds.

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S. Bounderbala (a), M.Lamri-Senhadji (a), J. Prost (b), M.A. Lacaille-Dubois (c), *, M. Bouchenak (a)

(a) Laboratorie de Nutrition Clinique et Metabolique, Departement de Biologie, Faculte des Sciences, Universite d'Oran Es-Senia, 31000 Oran, Algeria

(b) UPRES Lipides & Nutrition, Faculte des Sciences Gabriel, Universite de Bourgogne, 21100 Dijon, France

(c) Laboratoire de Pharmacognosie, Faculte de Pharmacie, Unite de Molecules d'Interet Biologique, UMIB, UPRES EA 3660, Universite de Bourgogne, 21100 Dijon, France

* Corresponding author.

E-amil address: [e-mail](M.A. Lacaille-Dubois).

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