Antihyperglycemic, antihyperlipidemic and ... - Pathophysiology

Pathophysiology 19 (2012) 35–42

Antihyperglycemic, antihyperlipidemic and antioxidant activities of traditional aqueous extract of Zygophyllum album in streptozotocin diabetic mice Jamel El Ghoul a,b,∗,1 , Moêz Smiri b,1 , Saad Ghrab c , Naceur A. Boughattas d , Mossadok Ben-Attia a a

Unité de Toxicométrie & Chronobiométrie, Laboratoire de Biosurveillance de l’Environnement (LR01/ES14), Faculté des Sciences de Bizerte, Université de Carthage, 7021 Zarzouna, Tunisia b Département des Sciences de la Vie, Faculté des Sciences de Bizerte, Université de Carthage, 7021 Zarzouna, Tunisia c Laboratoire de Chimie Structurale Organique, Synthèses et études physicochimique, Faculté des Sciences de Tunis, Département de Chimie, Campus Universitaire El Manar I, 2092 Tunis, Tunisia d Laboratoire de Pharmacologie (04/UR/09-01), Faculté de Médecine de Monastir, Université de Monastir, 5019 Monastir, Tunisia Received 11 October 2011; received in revised form 20 November 2011; accepted 12 December 2011

Abstract Objective: The aim of this work was to investigate the antihyperglycemic, antioxidant and antihyperlipidemic effects of the aqueous extract of Zygophyllum album on streptozotocin (STZ)-induced diabetic mice. Methods: Diabetes was induced in Swiss albino mice by the administration of STZ (45 mg/kg b.w.) intraperitoneally. Aqueous extract of Z. album (100 and 300 mg/kg b.w.) was administered by oral gavage once a day for a period of 15 days. The effect of the extract on blood glucose, lipids, cholesterol levels in plasma, and also on enzymatic and non enzymatic antioxidants of defence systems such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) enzyme activities, and vitamin C, vitamin E and glutathione reductase (GSH) levels in liver and pancreas were studied. Results: Our results showed that Z. album extract reduced the blood glucose, total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) levels in STZ-diabetic mice. It also significantly abolished the increase in MDA level, and GPx, SOD and CAT activities in both liver and pancreas. The levels of GSH, vitamin C and high-density lipoprotein (HDL) were significantly augmented in Z. album treated diabetic mice in comparison with control group. Our findings suggest that Z. album aqueous extract prevented the diabetic induced MDA levels via the enhancement of the tissue GSH and blood vitamin C levels. Conclusions: These results suggest that Z. album extract exerts the anti-diabetic and antihypercholesterolemic activities through its antioxidant properties. © 2011 Elsevier Ireland Ltd. All rights reserved. Keywords: Diabetes mellitus; Zygophyllum album; Streptozotocin; Antioxidants; Plasma lipid; Liver; Pancreas; Mice

1. Introduction Diabetes mellitus is increasingly common metabolic disorder and one of the five leading causes of death in the world. It is projected that 300 million people will have the ∗ Corresponding author at: Unité de Toxicométrie & Chronobiométrie, Laboratoire de Biosurveillance de l’Environnement (LR01/ES14), Faculté des Sciences de Bizerte, Université de Carthage, 7021 Zarzouna, Tunisia. E-mail address: [email protected] (J.E. Ghoul). 1 These authors contributed equally to this work.

0928-4680/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2011.12.001

disease by the year 2025 [1]. It is a chronic metabolic disorder of multiple aetiologies; and is mainly diagnosed by blood glucose rise subsequently to insulin deficiency. Type I, insulin-dependent diabetes mellitus, is characterized when the body is totally short of the production of insulin. Diabetes patients of type I take daily doses of insulin. Type II, non insulin-dependent diabetes mellitus, is defined by loosing accuracy to produce enough quantity or appropriately use insulin. This type constitutes the epidemic form of the disease [2]. Such disorders lead to various profound

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J.E. Ghoul et al. / Pathophysiology 19 (2012) 35–42

secondary complications like as, atherosclerosis, hypertension, hypercholesterolemia, myocardial infarction, ischemic attacks, retinopathy and nephropathy [3]. Hyperglycemia can increase the oxidative stress by several mechanisms, including glucose auto-oxidation, non-enzymatic protein glycation and activation of polyol pathway. The rise in free radical activity is suggested to play an important role in lipid peroxidation and protein oxidation of cellular structures resulting in cell injury and is implicated in the pathogenesis of vascular disease which are the mainly cause of morbidity and mortality in both type I and type II diabetes [4]. Traditional medicines derived mainly from plants have played and still plays major role in the management of diabetes mellitus [5–7]. In recent years, the role of alternative therapeutic approaches has become very popular [8] and since a single plant may have many pharmacological activities (anti-diabetic, anti-oxidant and anti-stress activity) they can be effectively utilized to delay or counter diabetic complications. Various plant extracts had been evaluated as treatment against diabetes such as Gongronema latifolium [9], Ricinus communis [10], Teucrium polium [11], Zygophyllum gaetulum [12] and Zygophyllum coccineum [13]. The decoction of Zygophyllum album (Zygophyllacae), leaves is widely used to treat hyperglycemia in folk medicine, in Tunisia; but few studies had investigated its effects and mechanism of action. Streptozotocin is frequently used to induce diabetes mellitus in experimental animals through its toxic effects on pancreatic ␤-cells [14–17]. STZ has drawn attention as a potential inducer of oxidative stress and several evidences indicate that the reactive oxygen species (ROS) generation is responsible for its wide spectrum of genotoxic effects in vitro [18]. Although the genotoxic effects of STZ are fairly well established in vitro, data on its potential to induce mutagenic alterations in vivo are limited [19]. Disturbances of antioxidant defence systems in diabetes have been demonstrated, including alteration in the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and impaired glutathione (GSH) metabolism [20]. Implication of oxidative stress in the pathogenesis of diabetes is suggested not only by oxygen free-radical generation but also due to nonenzymatic protein glycosylation, auto-oxidation of glucose [21–23], impaired glutathione metabolism [24], alteration in antioxidant enzymes [25], lipid peroxides formation [26] and decreased ascorbic acid levels [27]. In addition to GSH [28] there are other defence mechanisms against free radicals like the enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) whose activities contribute to eliminate superoxide, hydrogen peroxide and hydroxyl radicals [29]. Since that, the goal of this study was to evaluate the effects of aqueous extracts of Z. album on the blood glucose level in streptozotocin-induced diabetic mice. The effect of Z. album extracts on the levels of malondialdehyde (MDA) and some oxidative stress scavengers in blood and liver and pancreas were also determined.

2. Materials and methods 2.1. Preparation of the aqueous extract Fresh whole Z. album plants were collected from Southern Tunisia (Douz/Gbéli) between May and July 2007. The plant was identified by Mr. EL-OUNI botanist and Professor Emeritus at the University of Carthage (Bizerte, Tunisia). A voucher specimen was deposited in the herbarium of our laboratory. The plant material was dried at ambient temperature and stored in a dry place prior to use. The plant was washed well with water, dried at room temperature in the dark, and then ground in an electric grinder to give a coarse powder. A 100 g of the powdered aerial parts were suspended in 1000 ml distilled water, heated and boiled under reflux for 30 min. The decoction obtained was filtered, and the filtrate frozen at −20 ◦ C and then lyophilised. The average yield of the lyophylised material (Za-extract) was approximately 18.3%. It was stored at ambient temperature until further use. 2.2. Chemicals and reagents Reagents were obtained from Sigma Aldrich (Milano, Italy) and Merck (Darmstadt, Germany), and were of the highest commercial grade available. 2.3. Experimental induction of type 2 diabetes in mice The experiments were carried out with 8–10 weeks-old adult male Swiss albino mice weighing about 25 (±2) g. The animals were kept in polyacrylic cages (22.5 cm × 37.5 cm) with 5 mice per cage and maintained under standard housing conditions (room temperature 24–27 ◦ C and humidity 60–65%) with a 12-h light and dark cycle. Food, in the form of dry pellets, and water were available ad libitum. For the experimental induction of diabetes, streptozotocin (STZ) (Sigma–Aldrich Corp, St. Louis, MO, USA) was freshly prepared by dissolving in citrate buffer (0.01 M, pH 4.5) and ice cooled, before administration [30]. A single dose of 45 mg/kg STZ was intraperitoneally injected to mice. One week after STZ injection, only mice with blood glucose concentration higher than 250 mg/dl (at fasting state) were considered as diabetic and included in the present study. 2.4. Experimental design A total of 40 mice were divided into five groups in as follows: Group I: normal control mice, received only saline solution. Group II: diabetic control mice, received STZ in single dose (45 mg/kg, i.p.). Group III: treated diabetic mice received the aqueous extract of Z. album extract (100 mg/kg, p.o.). Group IV: treated diabetic mice received the aqueous extract of Z. album extract (300 mg/kg, p.o.).


Group V: STZ-diabetic mice received gliclazide (25 mg/kg, i.p.). Animals were treated by oral gavage once a day for a period of 15 days. 2.5. Blood and tissue collection After an overnight fasting, mice were decapitated, and total arterio-venous blood was collected for glucose and vitamins analysis. Thereafter, an abdominal incision was performed, in order to harvest livers and pancreas. The whole organs were cleaned with chilled normal saline on the ice. A 10% (w/v) homogenate of organ samples (0.03 M sodium phosphate buffer, pH −7.4) was prepared by using an Ultra-Turrax homogenizer at a speed of 9500 rpm. The homogenized tissue preparation was used to measure MDA, CAT, SOD, GPx and GSH levels.

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2.9. Lipid peroxidation MDA, as a measure of lipid peroxidation, was measured spectrophotometrically by the method of Ohkawa et al. [34], using 1,1,3,3-tetraethoxypropane as standard. MDA is expressed as n moles per mg protein. To 500 ␮l of liver or pancreatic homogenates in phosphate buffer (pH 7.4), 300 ␮l of 30% trichloroacetic acid (TCA), 150 ␮l of 5 N HCl and 300 ␮l of 2% (w/v) 2-thiobarbituric acid (TBA) were added and then the mixture was heated for 15 min at 90 ◦ C. The mixture was centrifuged at 12,000 × g for 10 min. Pink colored supernatant was obtained, which was measured spectrophotometrically at 532 nm. 2.10. Reduced glutathione (GSH)

Blood glucose was measured by O-toluidine method of Sasaki et al. [31]. Briefly, 0.1 ml of the blood was mixed 1.9 ml of 10% trichloroacetic acid (TCA) solution to precipitate proteins and then centrifuged. 1.0 ml of supernatant was mixed with 4.0 ml of O-toluidine reagent and was 6 kept in boiling water bath for 15 min and cooled. The absorbance was read at 620 nm. Data were expressed as mg/dl.

GSH content was estimated by method of Sedlak and Lindsay [35]. The tissues were homogenized in 0.02 M with ethylenediaminetetraacetic acid (EDTA). Aliquots of 5 ml of the homogenates were mixed in test tube with 4 ml of cold distilled water and 1 ml of 50% TCA. The tubes were shaken for 10 min using vortex mixer and the centrifuged at 1200 × g for 15 min. Following centrifugation 2 ml of supernatant was mixed with 4 ml of 0.4 M Tris–buffer (pH 8.9). The whole solution was mixed and 0.1 ml of 0.01 M DTNB {5,5 -dithiobis (2-nitrobenzoic acid)} was added to it. The absorbance was read within 5 min of the addition of DTNB at 412 nm against a reagent blank with no homogenate.

2.7. Vitamin E determination

2.11. Glutathione peroxidase (GPx)

Vitamin E was measured by the method of Desai [32]. To 0.25 ml of plasma, 0.25 ml of ethanol was added and thoroughly mixed. Then 0.75 ml of petroleum ether was added, shaken rapidly and centrifuged. 0.5 ml of supernatant was taken and evaporated to drought. To this 0.05 ml of 0.2% bathophenanthoraline was added. The assay mixture was protected from light and 0.05 ml of 0.001 M ferric chloride was added followed by 0.05 ml of 0.001 M phosphoric acid. The total volume was made up to 0.75 ml with ethanol. The color developed was read at 530 nm. The level of vitamin E was expressed as mg/dl of plasma.

GPx was assayed by the method of Rotruck et al. [36]. The reaction mixture consisted of 0.2 ml of 0.8 mM EDTA, 0.1 ml of 10 mM sodium azide, 0.1 ml of 2.5 mM hydrogen peroxide (H2 O2 ), 0.2 ml of 4 mM reduced glutathione, 0.4 ml of 0.4 M phosphate buffer (pH 7.0), and 0.2 ml homogenate was incubated at 37.8 ◦ C for 10 min. The reaction was arrested by the addition of 0.5 ml of 10% TCA and the tubes were centrifuged at 2000 rpm. To the supernatant 3 ml of 0.3 M disodium hydrogen phosphate and 1.0 ml of 0.04% DTNB were added and the color developed was read at 420 nm immediately. The activity of GPx was expressed as ␮moles of glutathione oxidized/min/mg protein.

2.6. Blood glucose measurement

2.8. Vitamin C determination 2.12. Catalase (CAT) This assay was based on the method described by Omaye et al. [33]. To 0.25 ml of plasma, 0.25 ml of water and 0.5 ml of 5% TCA were added, mixed thoroughly and centrifuged. To 0.5 ml of the supernatant, 0.1 ml of, 4-dinitrophenyl hydrazine–thiourea–CuSO4 (DTC) reagent was added and incubated at 37.8 ◦ C for 3 h. Then 0.75 ml of 65% sulfuric acid was added mixed well and the solution was allowed to stand at room temperature for another 30 min. The color developed was read at 520 nm. The level of vitamin C was expressed as mg/dl of plasma.

CAT was assayed by the method of Aebi [37]. To 1.2 ml of 50 mM phosphate buffer (pH 7.0), 0.2 ml of the tissue homogenate was added and the enzyme reaction was started by the addition of 1.0 ml of 30 mM H2 O2 solution. The decrease in absorbance was measured at 240 nm at 30 s intervals for 3 min. The enzyme blank was run simultaneously with 1.0 ml of distilled water instead of hydrogen peroxide. The enzyme activity was expressed as ␮moles of H2 O2 decomposed/min/mg protein.

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2.13. Superoxide dismutase (SOD) SOD was assayed by the method of Misra and Fridovich [38]. 0.1 ml of tissue homogenate was added to tubes containing 0.75 ml ethanol and 0.15 ml chloroform (chilled in ice) and centrifuged. To 0.5 ml of supernatant, added 0.5 ml of 0.6 mM EDTA solution and 1 ml of 0.1 M carbonate–bicarbonate buffer (pH 10.2). The reaction was initiated by the addition of 0.5 ml of 1.8 mM epinephrine and the increase in absorbance at 480 nm was measured. The enzyme activity is expressed as 50% inhibition of epinephrine autooxidation/min. 2.14. Measurement of lipid profile Lipid profiles were estimated by the method of Yadav et al. [39] and were obtained by using commercially available kits. Total cholesterol, high density lipoprotein (HDL) cholesterol and triglyceride (TG) levels in serum were determined according to the instructions of the manufacturer (Sigma Diagnostics). For the determination of very low density lipoprotein (VLDL) and low density lipoprotein (LDL), Friedwald’s (1972) formula was used which states: VLDL cholesterol = Triglyceride/5 and LDL cholesterol = Total cholesterol − (VLDL + HDL cholesterol). 2.15. Protein determination Protein content was determined by the method of Bradford using assay kit (Sigma Diagnostics). 2.16. Statistical analysis Results are expressed as the means ± SD. Student’s t-test for unpaired samples was performed using GraphPad Prism (GraphPad Software, Version 4.0). A value of p < 0.05 was considered significant.

3. Results 3.1. Effect of aqueous extract of Z. album on blood glucose (Fig. 1) Induction of diabetes in the experimental mice was confirmed of a high fasting blood glucose level. A significant (p < 0.05) increase in the level of blood glucose (group II) was observed in diabetic mice when compared to controls (group I). Administration of aqueous extract of Z. album at a dose of 100 mg/kg (group III) and 300 mg/kg (group IV) to diabetic mice significantly (p < 0.05) decreased the level of blood glucose again to near normal level (Fig. 1).

Fig. 1. Effect of aqueous extract of Zygophyllum album on blood glucose level in STZ-induced diabetic mice. The data are expressed as means ± S.D. (n = 8). Values are statistically significant at p < 0.05. a Diabetic control vs. control. b Diabetic + Zygophyllum album extract 100 mg/kg vs. c diabetic control. Diabetic + Zygophyllum album extract 300 mg/kg vs. diabetic control. d Diabetic + gliclazide vs. diabetic control.

3.2. Effect of aqueous extract of Z. album on lipid peroxidation (Fig. 2) Thiobarbituric acid reactive substances, as quantified by MDA, were significantly augmented in diabetic control group, in comparison with the other groups of mice (I, III, IV and V), both in liver and pancreas (p < 0.05). MDA level was also greater in group III in comparison with groups I, IV and V (p < 0.05), in both pancreas and liver (Fig. 2). 3.3. Effect of aqueous extract of Z. album on serum vitamin C and E (Fig. 3) The levels on serum vitamin E and vitamin C level in STZ-induced diabetic mice are shown in Fig. 3. Diabetic mice (group II) had low level of vitamin C and the elevation of vitamin E in serum when compared with control mice (group I). Diabetic mice (group II) treated with aqueous extract (groups III and IV) and with glicazide (group V) showed a significant increase (p < 0.05) in the level of vitamin C and a decrease (p < 0.05) in the level of vitamin E. 3.4. Effect of aqueous extract of Z. album in liver and pancreatic activities of GSH, GPx, SOD and CAT (Tables 1 and 2) Tables 1 and 2 show the levels of the oxidative stress enzymes’ activities, respectively, in liver and pancreas. GPx, SOD and CAT activity levels were significantly increased in diabetic control mice than in other groups (p < 0.05). However, the GSH level was significantly diminished in diabetic mice when compared to shame, and diabetic treated mice with either Z. album extracts or glicazide. 3.5. Effect of ethanolic extract of Z. album on serum lipid profiles Serum TC, TG, LDL, VLDL and HDL levels of the experimental groups of animals are shown in Table 3. Serum TC,


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Table 1 Effect of traditional aqueous extract of Zygophyllum album on anti-oxidative enzymes activities in the liver in STZ-induced diabetic mice. Groups GSH GPx SOD CAT

I 6.83 11.94 4.02 18.69

II ± ± ± ±

0.23 0.17 0.3 0.48

1.94 18.74 8.91 27.33

III ± ± 0.27a ± 0.15a ± 0.39a

0.19a

3.24 15.04 6.39 24.1

IV ± ± 0.13b ± 0.2b ± 0.09b 0.16b

6.01 13.56 3.98 21.05

V ± ± 0.6c ± 0.24c ± 0.27c 0.09c

6.02 9.85 3.88 16.89

± ± ± ±

0.14d 1.32d 0.36d 1.2d

The data are expressed as means ± S.D. (n = 8). Values are statistically significant at p < 0.05. Nanomoles per milligram of protein for GSH. Micromoles of glutathione oxidized per minute per milligram of protein for GPx. Activity is expressed as: 50% of inhibition of epinephrine auto-oxidation per minute for SOD; Micromoles of hydrogen peroxide decomposed per minute per milligram of protein for CAT. a Diabetic control vs. control. b Diabetic + Zygophyllum album extract 100 mg/kg vs. diabetic control. c Diabetic + Zygophyllum album extract 300 mg/kg vs. diabetic control. d Diabetic + gliclazide vs. diabetic control.

Table 2 Effect of aqueous extract of Zygophyllum album on anti-oxidative enzymes activities in pancreas in STZ-induced diabetic mice. Groups

I

GSH GPx SOD CAT

2.23 7.03 2.35 15.63

± ± ± ±

0.08 0.19 0.2 0.53

II

III

IV

V

0.63 ± 0.06a 13.01 ± 0.2a 6.2 ± 0.12a 26.22 ± 0.48a

0.97 ± 0.025b 11.23 ± 0.21b 4.31 ± 0.06b 22.07 ± 0.31b

2.08 ± 0.012c 7.13 ± 0.3c 2.95 ± 0.07c 17.63 ± 0.43c

2.02 ± 0.006d 8.05 ± 0.17d 2.65 ± 0.17d 16.02 ± 0.35d

The data are expressed as means ± S.D. (n = 8). Values are statistically significant at p < 0.05. Nanomoles per milligram of protein for GSH. Micromoles of glutathione oxidized per minute per milligram of protein for GPx. Activity is expressed as: 50% of inhibition of epinephrine auto-oxidation per minute for SOD; Micromoles of hydrogen peroxide decomposed per minute per milligram of protein for CAT. a Diabetic control vs. control. b Diabetic +Zygophyllum album extract 100 mg/kg vs. diabetic control. c Diabetic + Zygophyllum album extract 300 mg/kg vs. diabetic control. d Diabetic + gliclazide vs. diabetic control.

TG, LDL and VLDL were significantly higher (p < 0.05) in STZ-induced diabetic mice (group II) than those in normal controls (group I). Decreased levels of HDL were observed in STZ induced diabetic mice compared to normal untreated mice. Treatment with aqueous extract of Z. album and gliclazide resulted in a significant decrease (p < 0.05) in TC, TG, LDL and VLDL levels compared to those in STZ-induced diabetic mice. Serum HDL levels were significantly increased (p < 0.05) in the diabetic treated group.

4. Discussion Recently, a growing concern has brought back to traditional and alternative medicines, both for their pharmacological properties and their economical interest. As mentioned above, various phyto-therapeutic products are already used; and convey satisfactory results. In essence, Zygophyllum sp., Mediterranean Zygophyllacea plants, are widely used in traditional medicine for their anti-diabetic, antiseptic, antispasmodic, anti eczema [40] anti-diarrheal

Table 3 Effects of aqueous extract of Zygophyllum album on serum lipids content in STZ-induced diabetic mice. Groups

I

II

III

IV

V

TC HDL VLDL LDL TG

2.18 ± 0.19 0.96 ± 0.09 0.326 ± 0.035 0.894 ± 0.1 1.63 ± 0.13

4.88 ± 0.13 0.51 ± 0.08a 0.916 ± 0.09a 3.73 ± 0.21a 3.02 ± 0.15a

3.26 ± 0.09 0.73 ± 0.06b 0.64 ± 0.015b 1.614 ± 0.35b 2.75 ± 0.18b

2.65 ± 0.08 1.05 ± 0.1c 0.376 ± 0.1c 1.224 ± 0.12c 1.88 ± 0.21c

2.14 0.88 0.35 0.91 1.75

± ± ± ± ±

0.18 0.12d 0.081d 0.13d 0.19d

Plasma total cholesterol (TC), high-density lipoprotein (HDL), very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and triglycerides (TG) were measured by kits. Values are expressed as mmol/l and are given as mean ± SD for five groups of 8 animals each. Values are statistically significant at p < 0.05. a Diabetic control vs. control. b Diabetic + Zygophyllum album extract 100 mg/kg vs. diabetic control. c Diabetic + Zygophyllum album extract 300 mg/kg vs. diabetic control. d Diabetic + gliclazide vs. diabetic control.

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Fig. 2. Effect of aqueous extract of Zygophyllum album on lipid peroxidation (MDA) in liver (A) and pancreas (B) in STZ-induced diabetic mice. The data are expressed as means ± S.D. (n = 8). Values are statistically significant at p < 0.05. a Diabetic control vs. control. b Diabetic + Zygophyllum album extract 100 mg/kg vs. c diabetic control. Diabetic + Zygophyllum album extract 300 mg/kg vs. diabetic control. d Diabetic + gliclazide vs. diabetic control.

[41] and anti inflammatory [42] effects. In this paper, we had experimentally investigated the anti-diabetic effect of Z. album aqueous extracts, with special focus on their impact on the oxidative/anti-oxidative system. Our results showed that the daily injection of the aqueous extract of Z. album during 15 days abolished the blood sugar increase in the STZ induced diabetic mice. This effect was dose dependent. These results were well corroborated those reported by other authors using various Zygophyllacea plants [40,43]. The mainly proposed mechanism of such glycaemia decrease may be bypassed through enhancement of the peripheral sugar uptake and metabolism and insulin secretion [44]. Because of the misbalanced oxidative/anti oxidative system in diabetes mellitus and its involvement in the mechanism of various pathological complications, the effect of the aqueous extract of this plant on the oxidative stress, was investigated. In accordance to results reported by Kakkar

Fig. 3. Effect of aqueous extract of Zygophyllum album on serum vitamin C (A) and vitamin E (B) level in STZ-induced diabetic mice. The data are expressed as means ± S.D. (n = 8). Values are statistically significant at p < 0.05. a Diabetic control vs. control. b Diabetic + Zygophyllum album extract 100 mg/kg vs. diabetic control. c Diabetic + Zygophyllum album extract 300 mg/kg vs. diabetic control. d Diabetic + gliclazide vs. diabetic control.

et al. [45], we had found that streptozotocin increased both the lipid peroxidation (MDA) and the anti-oxidant enzyme (SOD, GPx and CAT) activities, in diabetic control mice. The authors delineated that the increase in antioxidant enzyme activities could primarily be a response to the diabetic state; whereas STZ appears to not exert any direct effects on these activities [45]. Interestingly, the administered Z. album aqueous extract potentially prevented the liver and pancreatic MDA increase observed in diabetic mice, in a dose dependent manner. Such diminution in the lipid peroxidation may be a consequence of the observed enhancement of the enzymatic (GSH) and non enzymatic (vitamin C) anti oxidative scavengers. According to Evans and Halliwell [46] the antioxidant defences consist of low molecular mass antioxidants, intracellular enzymes, sequestration of transition metal ions and repair mechanisms. Particularly important low molecular mass antioxidants are glutathione, vitamin E, ubiquinone, ␤-carotene, vitamins C and A. GSH


and vitamin C strongly prevent the lipid peroxidation and DNA damages induced by the hydroxyl radical [47]. Currently used drugs against diabetes are essentially focused on controlling and lowering blood glucose to a normal level, via different pathways, while diabetic complications require other treatments. Somewhat, undesirable side-effect such as hypoglyceamia, lactic acid intoxication and gastrointestinal upset appear after patients took these medicines [2]. To disclose the wonder, alternative medicines are looked as an adequate solution. The reported results, herein, suggest that Z. album aqueous extract is able to re-establish both the blood glucose level and the oxidative stress status, which are determinants of diabetic secondary complications. The actions of the extract need to be further studied to clarify which compounds are active and to establish their mechanisms of action in the therapeutic effects. The levels of serum lipids are usually elevated in diabetes mellitus and such an elevation represents a risk factor for coronary heart disease [48]. Marked increase in total cholesterol, triglycerides, VLDL, LDL and decreased HDL cholesterol are observed in untreated diabetic mice. Several authors have reported anti-hyperlipedimic activity of medicinal plants [49–51]. Diabetes mellitus is related to a hyperlipidemia and leads to serious anomalies in lipids composition and concentration [52]. These anomalies can induce cardiovascular diseases [53]. It is known that the increase in serum lipids in STZ-induced diabetic rats played an important role in such pathology [54]. In our study, we recorded a significant increase in the serum concentration of total cholesterol and triglyceride levels at STZ-induced diabetic mice (p < 0.001). The high level of total cholesterol in the blood could be seen as a major risk factor generating coronary heart disease [55]. These results agree with those found by Eddouks et al. [56], Ravi et al. [57] who suggest that the abnormal high concentration of serum lipids observed in diabetic subjects is mainly due to the increase in the mobilization of fatty acids from fat tissue [57]. Indeed, Betterridge [58] have indicated that the deficiency in insulin or the insulin resistance may be responsible for hyperlipidaemia due to the insulin inhibiting action on the 3-hydroxy-3-methyl-methylglutaryl coenzyme-A – a key enzyme in the cholesterol biosynthesis. During diabetes, the hyperlipidemia may be regarded as a result of the noninhibiting action of the lipolytic hormones on adipose tissues [59].

5. Conclusion The present study showed that increased oxidative stress is apparent in STZ-induced diabetic animals. The traditional aqueous extract of Z. album exhibited a significant antihyperglycemic as well as antioxidant activity in experimentally diabetic mice.

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Conflict of interest statement No conflicts of interest.

Acknowledgment We are grateful to the Tunisian Ministry of Superior Education and Scientific Research for financial support.

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