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Antioxidant effect of Ajuga iva aqueous extract in streptozotocin-induced diabetic rats.

Abstract

The purpose of this study was to investigate the possible antioxidant effect of an aqueous extract of Ajuga iva (Ai) in streptozotocin (STZ)-induced diabetic rats. Twelve diabetic rats were divided into two groups fed a casein diet supplemented or not with Ai (0.5%), for 4 weeks. In vitro, the Ai extract possessed a very high antioxidant effect (1 mg/ ml was similar to those of trolox 300 mmol/1). The results indicated that plasma thiobarbituric acid reactive substances (TBARS) values were reduced by 41% in Ai-treated compared with untreated diabetic rats. TBARS concentrations were lower 1.5-fold in liver, 1.8-fold in heart, 1.9-fold in muscle and 2.1-fold in brain in Ai-treated than untreated group. In erythrocytes, Ai treatment increased significantly the activities of glutathione peroxidase (GSH-Px) ( + 25%) and glutathione reductase (GSSH-Red) ( + 22%). Superoxide dismutase activity was increased in muscle (+22%), while GSH-Px activity was significantly higher in liver ( + 28%), heart ( + 40%) and kidney ( + 45%) in Ai-treated compared with untreated group. Liver and muscle GSSH-Red activity was, respectively, 1.6- and 1.5-fold higher in Ai-treated than untreated diabetic group. Catalase activity was significantly increased in heart (+36%) and brain ( + 32%) in Ai-treated than untreated group. Ai treatment decreased plasma nitric oxide (-33%), carbonyls (-44%) and carotenoids (-68%) concentrations. In conclusion, this study indicates that Ajuga iva aqueous extract improves the antioxidant status by reducing lipid peroxidation and enhancing the antioxidant enzymes activities in plasma, erythrocytes and tissues of diabetic rats.

[C] 2009 Published by Elsevier GmbH.

Keywords: Ajuga iva; Lamiaceae; Diabetic rats; Lipid peroxidation; Antioxidant status

Introduction

Diabetes is a metabolic disorder characterized by hyperglycaemia resulting from a defect in insulin secretion or action, or both (Limaye et al., 2003). The elevated levels of blood glucose in diabetes are associated with increased lipid peroxidation, which may contribute to long-term tissue damage (Bhor et al. 2004). Hyperglycemia increases oxidative stress through overproduction of reactive oxygen species (ROSs) (Nogueira et al. 2005). The concentrations of ROSs are modulated by antioxidant enzymes and non-enzymatic scavengers (Saxena et al. 1993). Alterations in the antioxidant enzymes activities such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), the three primary scavenging enzymes, have been demonstrated in different tissues of diabetic animals (Kakkar et al. 1995). Traditional medicine, which deals with plants and plant products, uses the active ingredients present in plants for treating disease (Fabricant and Farnsworth 2001) and some of the plants are used by the population, as antidiabetic remedies (Pari and Venkateswaran 2003). Clinical research has confirmed the efficacy of several plants in the modulation of oxidative stress associated with diabetes mellitus (Pari and Latha 2004).

Ajuga iva (L.) Schreiber (Lamiaceae), locally known as "Chendgoura,'" in Algeria is used in phytomedicine around the world for a variety of diseases. Ajuga iva possesses hypoglycaemic (El-Hilaly and Lyoussi 2002), vasorelaxant (El-Hilaly et al. 2004) and hypolipidemic (El-Hilaly et al. 2006; Chenni et al. 2007) effects, which have been experimentally demonstrated.

However, the effect of Ajuga iva on lipid peroxidation and antioxidant enzymes activities in diabetes has never been examined. Thus, the present investigation was carried out in order to study the possible antioxidant effect of an aqueous extract of Ajuga iva in streptozo-tocin (STZ)-induced diabetic rats.

Materials and methods

Plant material and preparation of the aqueous extract of Ajuga iva

Ajuga iva (L.) Schreiber (Lamiaceae) plant was collected in South Algeria (Ain-Sefra) between March and April 2005. The plant material was stored at room temperature in a dry place prior to use.

The preparation of Ajuga iva aqueous extract and the identification of its main chemical groups were realized as previously described (Chenni et al. 2007). The crude yield of the lyophilized Ajuga iva (Ai) extract was approximately 25% (w/w). The identified compounds belonged to three structural types: ecdysone, terpenoid and flavonoid. After isolation by several column chromatographic steps from this aqueous extract and characterisation by spectroscopic methods, two main compounds of Ai from Ain-Sefra were identified as naringenin-7-O-[alpha]-L-rhamnopyranosyl(l- > 2)-[beta]-D-glucopyranoside (flava-noid) and 8-O-acetylharpagide (iridoid) by comparison with literature values (Ghedira et al. 1991).

In vitro test for antioxidant activity of Ai extract

The antioxidant (AOX) activity of the Ai extract was conducted by using KRL test (Biache and Prost 1992). This test evaluates the resistance to free radical aggression by measuring the capacity of total blood to withstand free radical-induced hemolysis. Trolox is a water-soluble synthetic analogue of vitamin E. Trolox was used as standard and the AOX activity of the extract was compared with those of trolox and given as mmol/1 trolox.

Animals and dietary treatment

Male Wistar rats (Iffa Credo, l'Arbresle, Lyon, France), weighing 260 [+ or -] 20 g were housed under standard environmental conditions (23 [+ or -]1[degrees]C, 55 + 5% humidity and a 12 h light/dark cycle) and maintained with free access to water and a standard diet ad libitum. The general guidelines for the care and use of laboratory animals recommended by the Council of European Communities (1987) were followed.

Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) (Sigma, St Louis, Mo, USA) at a dose of 60mg/kg body weight. STZ was dissolved in 0.05 mol/l cold sodium citrate buffer, pH 4.5 immediately before use. After 48 h, hyperglycaemia was confirmed using a Glucometer (Glucotrend, Germany). Only animals with fasting blood glucose levels greater than 2.50 g/1 were considered diabetic and then included in this study. Diabetic rats (n = 12) were randomly divided into two groups. The untreated group received a casein diet and the treated group received the same diet supplemented with lyophilized aqueous extract of Ai (0.5%), for 4 weeks (Table 1). Food intake and body weight were recorded weekly.
Table 1. Composition of the diets (g/kg diet) (a).

   Constituent     Casein diet  Casein diet + Ai extract

Casein (b)             200                200

Corn starch            590                585

Sucrose                 50                 50

Cellulose (b)           50                 50

Sunflower oil           50                 50

Mineral mix (c)         40                 40

Vitamin mix (d)         20                 20

Ai extract               0                  5

(a) Diets were isoenergetic (16.28 MJ/kg diet) and given in powdered
form.
(b) Prolabo, Paris, France.
(c) UAR 205B (Villemoisson, Epinay sur Orge, France). Mineral mixture
provided the following amounts (g/kg diet): Ca, 4; K, 2.4; Na, 1.6; Mg,
0.4; Fe, 0.12; elements (traces): Mn, 0.032; Cu, 0.005; Zn, 0.018; Co,
0.00004; I, 0.00002.
(d) UA.R 200 (Villemoisson, Epinay sur Orge, France). Vitamin mixture
provided the following amounts (mg/kg diet): retinol, 1.8;
cholecalciferol, 0.019; thiamine, 6; riboflavin, 4.5; pantothenic acid,
21; pyridoxine, 3; inositol, 45; cyanocobalamin, 0.015; ascorbic acid,
240; D-L-[alpha]-tocopherol, 51; menadione, 12; nicotinic acid, 30;
para-amino-benzoic acid, 15; folic acid, 1.5; biotin, 0.09.


Blood and tissue samples

After 4-week of experiment and overnight fasting, six rats from each group were anesthetized with sodium pentobarbital (60mg/kg body weight). Blood was collected from the abdominal aorta into tubes containing EDTA, and plasma was prepared by low-speed centrifugation (1000g for 20min, 4[degrees]C). The erythrocyte sediment was washed twice with ice-cold distilled water (1/4, v/v) and centrifuged (1000 g for 10 min, 4[degrees]C). Liver, heart, gastrocnemius muscle, brain and kidney were removed immediately, rinsed with cold saline, and weighed. Plasma, erythrocytes and tissue samples were stored at -70 [degrees]C until use.

Determination of plasma glucose, insulin and lipids concentrations

Fasting glycaemia was determined as described above. Insulin was measured using an enzyme-linked immunosorbent assay (ELISA) (kit LINCO Research, Missouri, USA) with rat insulin as a standard. Plasma total cholesterol and triacylglycerols were determined by enzymatic methods (kits Biomerieux, Lyon, France).

Plasma and tissues lipid peroxidation measurement

Lipid peroxidation was estimated in plasma and tissues as previously described (Chenni et al. 2007).

Antioxidant enzymes activities

In erythrocytes and tissues, superoxide dismutase (SOD; EC 1.15.1.1) activity was measured at 412 nm by testing the inhibition degree of nitrite formation (Elstner et al. 1983). Glutathione peroxidase (GSH-Px; EC 1.11.1.9) activity was determined according to the method of Paglia and Valentine (1967) using cumene hydroperoxide as substrate. Glutathione reductase (GSSH-Red; EC 1.6.4.2) activity was evaluated at 340 nm by measuring the decrease in NADPH absor-bance in the presence of oxidized glutathione (Goldberg and Spooner 1992). One unit of enzyme reduces 1 umol of oxidized glutathione per min at pH 7 at 25 [degrees]C. Catalase (CAT; EC 1.11.1.6) activity was assayed in tissues by measuring the rate of decomposition of hydrogen peroxide at 240 nm (Aebi 1974).

Protein concentrations were measured according to the method of Lowry et al. (1951) using bovine serum albumin as a standard.

Determination of plasma nitric oxide (NO), carbonyls and carotenoids concentrations

NO determination was performed using the Griess reagent (sulfanilamide and n-naphtyl-ethylene diamine) (Cortas and Wakid 1990). Plasma was clarified by zinc sulfate 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.

Carbonyls contents were determined according to the method of Levine et al. (1990) using the 2,4-dinitrophe-nylhydrazine (DNPH) reagent. Briefly, two tubes of 1.0 ml plasma sample were taken; one was marked as "test" and the other as "control". 0.5 ml of 10mmol/l DNPH prepared in 2.5 mol/1 HC1 was added to the test sample and 0.5 ml of 2.5 mol/1 HC1 alone was added to the control sample. The contents were mixed thoroughly and incubated in the dark (room temperature) for 1 h. The tubes were shaken intermittently every 15 min. Then 0.5 ml of 20% TCA (w/v) was added to the both tubes. The tubes were then centrifuged at 11,000g for 3 min to obtain the protein pellet. The supernatant was carefully aspirated and discarded. The precipitates were washed three times with 1 ml of ethanol-ethyl acetate (1/1, v/v) to remove unreacted DNPH and lipid remnants. The final protein pellet was dissolved in 0.6 ml of 6 mol/1 guanidine hydrochloride and incubated at 37 [degrees]C for 15 min. The insoluble materials were removed by centrifugation. The carbonyl content was determined by taking the spectra of the representative samples at 250-300 nm. Each sample was read against the control sample (treated with 2.5 mol/lHCl). The carbonyl content was calculated using an absorption coefficient ([epsilon]) of 22,000 [moll.sup.-1] [cm.sup.-1].

Carotenoids concentrations were determined according to the method of Bortolotti et al. (1996). Twenty [micro]l of plasma were mixed with 180 [micro]l ethanol and after centrifugation; the absorbance of supernatant was measured at 450 nm. Lutein was used for a standard curve.

Statistical analysis

Data were expressed as means [+ or -] SEM for six rats per group. Statistical analysis was carried out by the Student "t" test. The calculations were performed using STATISTIC A 6.0 (for Windows, StatSoft Inc. software, Tulsa, OK, USA). A difference of p < 0.05 was considered significant between the both groups treated and untreated with Ajuga iva extract.

Results

Antioxidant activity of Ai extract

The Ai extract developed an important AOX activity. The AOX activity of lmg/ml of Ai extract was similar to those of 300 mmol/l of trolox. This AOX activity increased with the concentration of the Ai extract (Fig. 1).

[FIGURE 1 OMITTED]

Body weight, food intake and relative organ weight

Body weight and food intake did not differ between Ai-treated and untreated diabetic rats (Table 2). The relative weight of liver and muscle was, respectively, increased by + 12% and + 34% in .Ai-treated than untreated diabetic rats, whereas there was no significant difference in heart, brain and kidney values in the both groups.
Table 2. Body weight, food intake and organ relative weights in
untreated and Ai-treated diabetic rats.

                        Untreated rats       Ai-treated rats

Body weight (g)       212.8 [+ or -] 32.2  220.5 [+ or -] 10.6
Food intake (g/d)     23.31 [+ or -] 0.87  23.23 [+ or -] 1.71

Relative weight
Liver                  3.47 [+ or -] 0.33   3.95 [+ or -] 0.27 *
Heart                  0.35 [+ or -] 0.02   0.33 [+ or -] 0.03
Gastrocnemius muscle   1.02 [+ or -] 0.30   1.54 [+ or -] 0.37 *
Brain                  0.81 [+ or -] 0.11   0.78 [+ or -] 0.05
Kidney                 0.64 [+ or -] 0.12   0.62 [+ or -] 0.02

Values are means [+ or -]SEM of 6 rats per group.
* p < 0.05, Ai-treated vs untreated diabetic rats. Relative weight =
[Organ weight/Body weight] x 100.


Plasma glucose, insulin, total cholesterol and triacylglycerols concentrations

A significant reduction in glycaemia value was observed in Ai-treated rats (2.83 [+ or -] 0.53 g/1) vs untreated diabetic rats (4.82 [+ or -] 0.54 g/1) (Table 3). Moreover, compared with the baseline value (5.14 [+ or -] 0.72 g/1), the plasma glucose concentration was 1.8-fold lower in Ai-treated rats, whereas no significant difference was found in untreated diabetic rats. However, plasma insulin concentrations remained unchanged in the both groups (75.42 [+ or -] 16.49 vs 80.22 [+ or -] 12.63 pmol/1). Ajuga iva treatment lowered significantly plasma total cholesterol (-28%) and triacylglycerols (-50%) contents when compared with untreated diabetic group.
Table 3. Glycemia, insulinemia, total cholesterol and tria-cylglycerols
concentrations in untreated and Ai-treated diabetic rats.

                             Untreated rats       Ai-treated rats

Glycemia (mmol/1)           26.75[+ or -]3.00   15.71[+ or -]2.94 *

Insulinemia (pmol/1)        75.42[+ or -]16.49  80.22[+ or -]12.63

Total cholesterol (mmol/1)   2.72[+ or -]0.10    1.86[+ or -]0.14 **

Triacylglycerols (mmol/1)    1.52[+ or -]0.48    0.66[+ or -]0.13 **

Values are means [+ or -]SEM of 6 rats per group.
* p < 0.05 and ** p < 0.01, A i-treated vs untreated diabetic rats.


Plasma and tissues TBARS concentrations

Ajuga iva treatment decreased significantly TBARS concentrations in plasma (-41%), liver (-34%), heart (-44%), muscle (-48%) and brain (-52%) (Table 4). However, there was no significant difference in kidney TBARS values in the both groups.
Table 4. Plasma and tissue thiobarbituric acid-reactive substances
(TBARS) concentrations in untreated and Ai-treated diabetic rats.

                       Untreated rats          Ai'-treated rats

Plasma (nmol/ml)      9.10 [+ or -] 0.29      5.38 [+ or -] 0.26 *

Liver (nmol/g)      185.6 [+ or -] 6.9      122.5 [+ or -] 14.6 **

Heart (nmol/g)       97.6 [+ or -] 8.2       54.2 [+ or -] 8.1 **

Muscle (nmol/g)     103.8 [+ or -] 16.8      54.3 [+ or -] 8.2 **

Brain (nmol/g)      335.5 [+ or -] 19.3     150.1 [+ or -] 15.0 **

Kidney (nmol/g)     177.5 [+ or -] 7.7      160.1 [+ or -] 8.7

Values are means[+ or -] SEM of 6 rats per group.
* p < 0.05 and ** p < 0.01, Ai-treated vs untreated diabetic rats.


Antioxidant enzymes activities

SOD, GSH-Px and GSSH-Red activities in erythrocytes of Ai-treated and untreated diabetic rats were presented in Fig. 2. In erythrocytes SOD activity was similar in the both groups, whereas an increase was observed in GSH-Px ( + 25%) and GSSH-Red ( + 22%) activities, in Ai-treated compared with untreated diabetic groups.

[FIGURE 2 OMITTED]

Tissue antioxidant enzymes activities of Ai-treated and untreated diabetic rats are presented in Fig. 3. Ajuga iva treatment increased SOD activity in muscle (+ 22%) but there was no effect on the other studied tissues. GSH-Px activity was significantly higher in liver ( + 28%), heart ( + 40%) and kidney ( + 45%), whereas muscle and brain were not sensitive. GSSH-Red activity was higher 1.6-fold in liver and 1.5-fold in muscle by Ajuga iva treatment, but no significant difference was observed in the other tissues. Ajuga iva treatment increased catalase activity in heart ( + 36%) and brain ( + 32%).

[FIGURE 3 OMITTED]

Plasma nitric oxide (NO), carbonyls and carotenoids concentrations

Ajuga iva treatment decreased significantly plasma nitric oxide (-33%), carbonyls (-44%) and carotenoids (-68%) concentrations (Fig. 4).

[FIGURE 4 OMITTED]

Discussion

The aim of the present study was to investigate the effect of Ajuga iva (L.) Schreiber (Lamiaceae) aqueous extract on lipid peroxidation and antioxidant status in STZ-induced diabetic rats, which represented the model for human type 1 diabetes (Tenner et al. 2003). After 4-week of treatment with Ai extract, plasma glucose values decreased significantly in STZ-induced diabetic rats (p < 0.001) (Table 3). However, Ai extract had no effect on plasma insulin concentrations in STZ-diabetic rats. These results confirmed those reported previously by El-Hilaly and Lyoussi (2002). It could be suggested that the observed hypoglycaemic effect in the absence of significant variation in insulin concentration, with Ai treatment may be due to an extrapancreatic mechanism. Some species of plants either in the same family (Lamiaceae) (Jimenz et al. 1986) or in others (Lemhadri et al. 2004; Eddouks et al. 2005; Maghrani et al. 2005) exhibited a hypoglycemic effect via an insulin-independent mechanism. In addition, Ajuga iva treatment reduced plasma cholesterol and triacylglycerol contents. Similar results were reported by El-Hilaly et al. (2006), who showed that daily administration of Ajuga iva extract caused a significant decrease in plasma cholesterol and triglycerides levels in STZ-diabetic rats. In this study, the hypolipidemic action of Ai extract occurs without stimulating insulin secretion. As previously shown, the extract produced a hypoglycaemic activity in STZ-diabetic rats, so its hypolipidemic effect could also be mediated by the improvement of glycaemia.

It is well known that hyperglycaemia increases lipid peroxidation, which may contribute to long-term tissue damage (Bhor et al. 2004). The increase of TBARS that represented the lipid peroxidation products, in tissue and blood of STZ-induced diabetic rats has been previously reported (Miyake et al. 1998). After 4-week of treatment with Ai extract, plasma TBARS concentrations were significantly lower in Ai-treated compared with untreated diabetic rats (Table 4). Moreover, tissues TBARS concentrations were significantly reduced in Ai-treated compared with untreated diabetic rats, excepted in kidneys (Table 4).

These results showed that Ai extract might protect the plasma and tissues against the cytotoxic action and oxidative stress of streptozotocin. Furthermore, the reduction in lipid peroxidation could be due to the improvement of the glycemic control and the increased of antioxidant status, since Ai aqueous extract was highly antioxidant (Fig. 1) and had hypoglycaemic activity in STZ-diabetic rats (Table 3).

Chemical studies on Ajuga iva aqueous extract have revealed the presence of several flavonoids, tannins, terpenes and steroids (Houghton and Raman 1998). In our study, the phytochemical analysis of Ai extract showed the presence of several flavonoids and terpenoids (see Materials and methods). Since flavonoids have been reported to present antioxidant and hypo-cholesterolemic activity (Syrov et al. 1983; Wagner and Lacaille-Dubois 1995), it may be suggested that the hypolipidemic and antioxidant activity of Ai might be related to these compounds. Indeed, it is well established that flavonoids act as free radical scavenger that prevents lipid peroxidation (Soto et al. 2003) and that tannins and triterpenes have antioxidant effects (Larkins and Wynn 2004).

Moreover, the deleterious effects of superoxide anion and hydroxyl radicals can be counteracted by antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase GSH and catalase (CAT). The present study showed that Ai-treated group had increased activities of GSH-Px and GSSH-Red in erythrocytes (p < 0.05) (Fig. 2) and decreased level of lipid peroxidation in plasma (Table 4), indicating the efficacy of the Ai extract to reduce oxidative stress in vivo. The elevated activities of erythrocytes GSH-Px and GSSH-Red noted with Ai extract could be due to the influence of flavonoids, terpenes and tannins present in this plant, since it is well known that flavonoids and other polyphenols are natural antioxidants (Badami et al. 2003; Frei and Higdon 2003; Sudheesh and Vijayalakshmi 2005).

In diabetes, changes in the antioxidant parameters status have been reported in various tissues (Kakkar et al. 1995, 1997) and there are contradictory results in the literature regarding the effect of diabetes-induced hyperglycaemia on antioxidant enzymes activities (Genet et al. 2002; Limaye et al. 2003; Panneerselvam and Govindasamy 2004). Our results showed that Ai treatment increased significantly the superoxide dismutase (Fig. 3) activity in muscle of STZ-diabetic rats, but there was no effect on the other tissues. SOD is the first line of defense against free radical attacks. Its function is to catalyse the conversion of superoxide radicals to hydrogen peroxide ([H.sub.2][O.sub.2]). The observed increase in SOD activity in muscle of Ai-treated rats can lead to an important elimination of superoxide ions, which can then inhibit the formation of hydroxyl radical in this tissue. The [H.sub.2][O.sub.2] produced by SOD is excreted as [H.sub.2]O based on the activity of glutathione peroxidase and catalase, therefore protecting the body from oxygen toxicity. However, this process can cause lipid peroxidation if [H.sub.2][O.sub.2] is not decomposed immediately.

Furthermore, GSH-Px activity was significantly higher in liver, heart and kidney of Ai-treated compared with untreated diabetic rats (Fig. 3). GSH-Px is an enzyme that scavenges [H.sub.2][O.sub.2] and lipid peroxides. This result explained the decrease in TBARS levels in liver and heart since GSH-Px has been known to inactivate lipid peroxidation reactions (Levy et al. 1999). Indeed, in liver and heart, glutathione peroxidase activity is increased along with that of glutathione reductase and catalase, respectively. GSSH-Red regenerates reduced glutathione (GSH) from oxidized glutathione, which has been formed by oxidation while CAT is responsible for the scavenging or detoxification of [H.sub.2][O.sub.2]. These antioxidant enzymes have a complementary catalytic activity leading to reduced TBARS concentrations in these tissues. However, kidney seems to be more sensitive than the other tissues and hence the increase in glutathione peroxidase activity is not sufficient to reduce TBARS concentrations and thus to protect this tissue from lipid peroxidation.

Low glutathione levels in diabetes have been considered to be an indicator of increased oxidative stress (McLennan et al. 1991). GSSH-Red activity which detoxifies oxidized glutathione, was higher in muscle of Ai-treated compared with untreated diabetic group (Fig. 3). This result appears to be responsible for the reduced TBARS concentrations and might lead to an increase in GSH content in this tissue. Reduced glutathione plays an important role in scavenging the toxic intermediates on incomplete oxidation.

The increase in brain catalase activity in Ai-treated compared with untreated diabetic group (Fig. 3) might suggest brain protection against oxidation and accumulation of lipid peroxidation.

Our findings are in agreement with other investigations that reported a significant increase of antioxidant enzymes activities in tissues of diabetic rats treated with extracts from several plants (Ananthan et al. 2004; Sabu and Kuttan 2004; Satheesh and Pari 2004; Ramesh and Pugalendi 2006; Vijayakumar et al. 2006).

Nitric oxide (NO), a potent vasodilator is produced in vascular endothelium (Leclercq et al. 2002), where it plays a central role in modulating endothelial function. However, during hyperglycemia an overproduction of both superoxide and nitric oxide has been reported. This simultaneous overgeneration favours the production of a cytotoxic product, the peroxynitrite anion (Beckman and Koppenol, 1996). Our findings showed that diabetic rats treated with Ajuga iva exhibited lower plasma NO concentrations (Fig. 4). In fact, these rats showed lower glycemia value and hence improvement in oxidative stress (via decreased lipid peroxidation and increased antioxidant enzymes activities) and decreased production of nitric oxide, resulting in unaltered endothelial functions in this group.

Carbonyl groups are the end products of protein oxidation. Their levels in tissues and plasma serve as relatively stable markers of oxidative damage (Chevion et al. 2000). Reactive carbonyl compounds are the result of oxidative stress and could be an active contributor to pathogenesis of diabetic complications. In this study, plasma carbonyl concentrations were significantly lower in Ai-treated compared with untreated diabetic group (Fig. 4). This result suggests that Ajuga iva, by decreasing oxidative stress, may be effective in preventing oxidative protein damage, which may contribute to the development of diabetic complications.

Carotenoids are antioxidant substances, which can neutralize the deleterious effects of reactive oxygen species. In Ajuga iva-treated rats, the low plasma carotenoids levels might be due to an increase in their utilization to neutralize free radicals.

In conclusion, our study demonstrate that the AOX effect of Ajuga iva extract improves oxidative stress in STZ-diabetic rats by reducing lipid peroxidation and increasing antioxidant enzymes activities. In the other hand, the Ai treatment corrects hyperglycemia and dyslipidemia, thus improving the atherogenic index. This implies that Ai treatment can prevent or be helpful in reducing the complications of diabetes. Several secondary metabolites belonging to the terpenoid and flavonoid structural-type were isolated and identified from the aqueous extract of Ai and their presence can explain at least in part the antioxidant activity of Ai in STZ-induced diabetic rats and support its use in North African traditional medicine. However, further investigations to fully identify the biologically active ingredients and to define the precise molecular mechanism(s) of these effects are required.

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D. Taleb-Senouci (a), H. Ghomari (a), D. Krouf (a), S. Bouderbala (a), J. Prost (b), M.A. Lacaiile-Dubois (c), *, M. Bouchenak (a)

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

(b) UP RES Lipides & Nutrition, Faculte des Sciences Gabriel, Universite de Bourgogne, 21000 Dijon, France

(c) Laboratoire de Pharmacognosie Faculte de Pharmacie, Unite de Molecules d'Interet Biologique, UMIB, UPRES EA 3660, Universite de Bourgogne, 21079 Dijon, France

* Corresponding author. Tel: + 33 3 80 39 3229.

E-mail address: malacd@u-bourgogne.fr (M.A. Lacaille-Dubois).

doi: 10.1016/j.phymed.2008.12.004
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Author:Taleb-Senouci, D.; Ghomari, H.; Krouf, D.; Bouderbala, S.; Prost, J.; Lacaiile-Dubois, M.A.; Bouchen
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:6ALGE
Date:Jun 1, 2009
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