Antidiabetic effect of flax and pumpkin seed mixture powder: effect on hyperlipidemia and antioxidant status in alloxan diabetic rats

Journal of Diabetes and Its Complications 25 (2011) 339–345

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Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M

Antidiabetic effect of flax and pumpkin seed mixture powder: effect on hyperlipidemia and antioxidant status in alloxan diabetic rats Mohamed Makni a,b, Hamadi Fetoui a,1, Nabil K. Gargouri b,1, El Mouldi Garouia, Najiba Zeghala,⁎ a b

Animal Physiology Laboratory, Faculty of Sciences, BP 1171, 3000 Sfax, Tunisia Food Processing Department, ISET, BP 377, 9100 Sidi Bouzid. Tunisia

a r t i c l e

i n f o

Article history: Received 1 July 2010 received in revised form 14 August 2010 accepted 8 September 2010 Available online 23 November 2010 Keywords: Diabetes flax and pumpkin seeds Antioxidant enzymes Lipid peroxidation Oxidative stress

a b s t r a c t Reactive oxygen species play a crucial role in the pathogenesis of diabetes and its complications. This study aims to examine the effects of flax and pumpkin powder seed mixture on alloxan induced diabetes in Wistar rats. Animals were allocated into three groups of six rats each: a control group (CD), diabetic group (DD) and diabetic rats fed with flax and pumpkin seed mixture (DMS) group. The diabetic rats (DD) presented a significant increase in glycemia, plasma and liver lipid parameters such as total lipid, total cholesterol and triglycerides compared to the control group (CD). In addition, plasma and liver malonaldialdehyde levels (MDA, an index of lipid peroxidation) significantly increased compared to (CD). Antioxidant enzymes activities such as catalase, superoxide dismutase, and reduced glutathione (GSH) levels significantly decreased in the plasma and liver of diabetic rats compared to controls. Diet supplemented with flax and pumpkin seed mixture in the DMS group ameliorated antioxidant enzymes activities and level of GSH in diabetic rats and significantly decreased MDA levels. The present study revealed a significant increase in the activities of aspartate aminotransferase and alanine aminotransferase on diabetic status, indicating considerable hepatocellular injury. The administration of flax and pumpkin seed mixture attenuated the increased levels of the plasma enzymes produced by the induction of diabetes and caused a subsequent recovery towards normalization comparable to the control group animals. Our results thus suggest that flax and pumpkin seed mixture supplemented to diet may be helpful in preventing diabetic complications in adult rats. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Diabetes mellitus (DM) is a chronic metabolic disorder characterized by high levels of glucose in the blood due to the non-secretion of insulin or insulin insensitivity (American Diabetes Association ADA, 2005). DM affects approximately 4% of the population worldwide and is expected to increase by 5.4% in 2025 (Kim, Hyun, & Choung, 2006). Although the underlying mechanisms of diabetes complications remain unclear, clinical and preclinical evidence suggests that diabetes is associated with oxidative stress, leading to an increased production of reactive oxygen species (ROS), including superoxide radical (O2•−), hydrogen peroxide (H2O2) and hydroxyl radical (OH•) or a reduction in the antioxidant defense system (Ihara et al., 1999; Rahimi, Nikfar, Larijani, & Abdollahi, 2005; Rudge et al., 2007). The oxidant/antioxidant imbalance in favour of

⁎ Corresponding author. Animal Physiology Laboratory, UR 08-73, Sfax Faculty of Sciences, BP 1171, 3000 Sfax, Tunisia. Tel.: +216 98 914 154; fax: +216 74 274 437. E-mail address: [email protected] (N. Zeghal). 1 Authors contributed equally to this work. 1056-8727/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jdiacomp.2010.09.001

oxidants contributes to the pathogenesis of different diabetic complications which are considered to result from enhanced reactive oxygen species generation via nicotinamide adenine dinucleotide phosphate-oxidase (Baynes & Thorpe, 1999; Garg, Ojha, & Bansal, 1996; Ha & Kim, 1999). The pathophysiology of diabetes involves a very complex cascade of several interrelated mechanisms. Elevated blood glucose induces auto-oxidative glycosylation and formation of glycation product, activates protein kinase-C, and increases polyol pathway activity and hexosamine flux, which are the key components of the cascade. These pathways are responsible for the generation of reactive oxygen species (superoxide, hydroxyl radical, hydrogen peroxide) and peroxynitrite, which ultimately contribute to oxidative stress (Ahmed, Adeghate, Cummings, Sharma, & Singh, 2004; Baynes & Thorpe, 1999; Ha & Kim, 1999). Current approaches to diabetes therapy involve mainly drugs enhancing insulin secretion or signalling as well as inhibiting endogenous glucose production (Anuradha & Selvam, 1993), while the role of antioxidants as the important agents to restore the redox balance of the organism is still underestimated. Dietary intervention, particularly the use of traditional food and medicine derived from natural sources, is a mainstay in the

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management of diabetes. In this context, various dietary sources are presently receiving considerable attention across the world for the potential health benefits in relation to many diseases such as diabetic disorders. Among them, flax seeds (Linum usitatissimum L., member of Linaceae family) and pumpkin seeds (Cucurbita pepo L., member of Cucurbitaceae family) are becoming new compounds of the traditional health food in Tunisia and other North African countries. Flax seeds contain high levels of Omega-3 fatty acid (Burdge & Calder, 2005), fiber components and phytochemicals such as lignans bioressource (Vijaimohan et al., 2006). The main physiological benefits of flax seeds are attributed primarily to the high linoleic acid content which contributes to their antioxidant properties (Simopoulos, 1991) against various diseases, including atherosclerosis, diabetes, hypertension, anti-inflammatory, and anticarcinogenic effects (Fukuda, Osawa, Namiki, & Ozaki, 1985). Pumpkin seeds are utilized for human consumption as snacks after salting and roasting, in Arabian countries (Al-Khalifa, 1996). These seeds are excellent sources of protein (25.2–37%), vitamins and oil (37.8–45.4%) (Barbara & Murkovic, 2004; Murkovic, Piironen, Lampi, Kraushofer, & Gerhard, 2004), especially Omega 6 fatty acids which have a number of biological applications along with significant antioxidant activity, in addition to anti-inflammatory and hypolipidemic effects (Suresh & Das, 2003). In our previous study, we demonstrated that flax and pumpkin seed mixture supplemented to diet of hypercholesterolemic rats had a significant anti-atherogenic, hypolipidemic and antioxidant potency. Flax and pumpkin seed mixture had a pronounced antioxidant activity due to their richness in antioxidant components (Makni et al., 2008). Several studies have demonstrated the beneficial effects of dietary seeds. Hence, the protective effects of flax and pumpkin seed mixture on diabetic complications would be worth studying. The present study was carried out in order to evaluate both the problems induced by diabetes and the protective effects of flax and pumpkin seed mixture. 2. Materials and methods 2.1. Plant material Flax (Linum usitatissimum L.) and pumpkin (Cucurbita pepo L.) seeds were purchased from a local market, crushed at ambient temperature and stored at 4°C prior to use. The seed mixture of flax and pumpkin rich in Omega 3 and Omega 6 was prepared. The ratio of Omega 6/Omega 3 fatty acids was 5:1, as recommended by the World Health Organization and according to several reports (Blandeau & Schneider, 2006; Grigg, 2004). 2.2. Experimental design Male Wistar rats (aged 11–12 weeks, weighing 190–210 g) were obtained from the Central Pharmacy of Tunisia (SIPHAT, Tunisia). They were maintained under standard laboratory conditions (22±3°C, 12-h light/dark cycle), with pellated food (Industrial Company of Rodent Diet, Sfax, Tunisia) and tap water ad libitum during 30 days of experimental period. The general guidelines on the use of living animals in scientific investigations (Council of European Communities, 1986) and the guidelines for care and use of laboratory animals controlled by the Tunisian Research Ministry were followed. This experimental study was conducted on three 6-rats groups: control group (CD), diabetic rats (DD) and diabetic rats fed with diet supplemented with flax and pumpkin seed mixture at 33% (DMS). 2.3. Induction of diabetes After 2 weeks of acclimatization, diabetes was induced in male rats with a freshly prepared solution of alloxan monohydrate in normal saline at a dose of 120 mg kg−1 body weight (BW) injected

intraperitoneally (Mansour, Newairy, Youssef, & Sheweita, 2002; Sheweita, Newairy, Mansour, & Youssef, 2002). Because alloxan is capable of producing fatal hypoglycemia as a result of massive pancreatic insulin release, rats were orally treated with 20% glucose solution (5–10 ml) after 6 h. The rats were then kept for the next 24 h on 5% glucose water solution to prevent hypoglycemia. Rats with moderate diabetes that exhibited glycosuria and hyperglycemia (i.e., blood glucose concentration 200–300 mg dl−1) were taken for the experimental tests. 2.4. Biochemical assays 2.4.1. Determination of plasma glucose, hepatic glycogen levels and glucose tolerance test 2.4.1.1. Glucose levels. Plasma glucose levels were assayed by enzymatic methods, using commercial reagent kits purchased from Biomaghreb (Ariana Tunis, Tunisia). 2.4.1.2. Hepatic glycogen. Hepatic glycogen content was determined by treatment with O-toluidine reagent and measured at 620 nm using a spectrophotometer (Geissbuhler, 1974). 2.4.1.3. Glucose tolerance test. The glucose tolerance test (GTT) evaluates the ability to respond appropriately to a glucose challenge (Matteucci & Giampietro, 2008). GTT was conducted in control and treated rats, 24 h before sacrifice day. Blood samples were collected from the rats' tail vein (control and treated groups) which were fasted overnight to obtain baseline blood glucose levels. Subsequently, rats of both control and treated groups were injected intraperitoneally with glucose (2 g kg−1 BW). Blood was collected from the rats' tail vein at interval of 30 min up to 2 h for glucose estimation using a glucometer (Esprit 2, BAYER, France). 2.4.2. Estimation of plasma insulin concentration Plasma insulin level was determined using rat Insulin enzymelinked immunosorbent assay kit ref. RIT-461 No. AKRIN-010T (Shibayagi, Japan). 2.4.3. Analysis of lipids in plasma and liver Tissue lipids were extracted with chloroform/methanol mixture (2v/1v) according to the method of Folch, Lees, and Stanley (1957). The contents of total lipids in liver and plasma extracts were quantified gravimetrically by evaporating off the solvents using a rotary evaporator (Heidolph, Laborota 4010 digital, Germany). Plasma lipid parameters such as total cholesterol (TC), triacylglycerol (TG) and High-density lipoprotein-cholesterol (HDL-C) levels were determined by enzymatic methods, using commercial kits from Biomaghreb (Ariana Tunis, Tunisia). The low-density lipoprotein-cholesterol (LDL-C) fraction and atherogenic index (AI) were determined according to the Friedewald equations (Friedewald, Levy, & Fredrickson, 1972): LDL−C = TC−ðTriglycerides = 5 + HDL−CÞ; AI = ðTC−HDL−C = HDL−CÞ: The dried hepatic lipid residues were dissolved in 1ml absolute ethanol for cholesterol and triacylglycerol assays. Hepatic total cholesterol and triacylglycerol contents were analyzed with the same enzymatic kits used in plasma analysis. 2.4.4. Measurement of malonaldialdehyde in tissues Concentrations of MDA in tissues, an index of lipid peroxidation, was determined spectrophotometrically according to Draper and Hadley (1990). An amount of 0.5 ml of each plasma sample or liver extract supernatant was mixed with 1 ml of trichloroacetic acid

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solution and centrifuged at 2500g for 10 min. A 1-ml solution containing 0.67% thiobarbituric acid (TBA) and 0.5 ml of supernatant were incubated for 15 minutes at 90°C and cooled. Absorbance of TBA-MDA complex was determined at 532 nm using a spectrophotometer (Jenway UV-6305; Essex, England). Lipid peroxidation was expressed as nanomoles of TBA reactive substances using 1,1,3,3tetraethoxypropane as standard. 2.4.5. Antioxidant enzymes and glutathione assays in plasma and liver 2.4.5.1. Total superoxide dismutase (SOD) activity. SOD activity was estimated according to Beauchamp and Fridovich (1971). The reaction mixture contained 50 mM of tissue homogenates in potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM L-methionine, 2 μM riboflavin and 75 μM nitroblue tetrazolium (NBT). The developed blue color in the reaction was measured at 560 nm. Units of SOD activity were expressed as the amount of enzyme required to inhibit the reduction of NBT by 50% and the activity was expressed as units per milligrams of protein. 2.4.5.2. Catalase activity (CAT). CAT activity was assayed by the method of Aebi (1984). Enzymatic reaction was initiated by adding an aliquot of 20 μl of the homogenized tissue and the substrate (H2O2) to a concentration of 0.5 M in a medium containing 100 mM phosphate buffer, pH 7.4. Changes in absorbance were recorded at 240 nm. CAT activity was calculated in terms of nanomoles H2O2 consumed per minute per milligram of protein. 2.4.5.3. Glutathione levels (GSH). GSH in tissues was determined by the method of Ellman (1959) modified by Jollow, Mitchell, Zampaglione, and Gillete (1974) based on the development of a yellow color when DTNB (5, 5-dithiobis-2 nitro benzoic acid) was added to compounds containing sulfhydryl groups; 500 μl of tissue homogenate in phosphate buffer was added to 3 ml of 4% sulfosalicylic acid. The mixture was centrifuged at 1600g for 15 min; 500 μl of supernatant was taken and added to Ellman's reagent. The absorbance was measured at 412 nm after 10 min. Total GSH content was expressed as milligrams per milliliter in plasma and as milligrams per milligram of protein in liver. 2.4.6. Estimation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities AST and ALT activities in plasma, used as biochemical markers for hepatic damage, were determined by enzymatic methods using commercial reagent kits from Biomaghreb (Ariana Tunis, Tunisia).

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2.5. Histopathological examination The pancreas, intended for histological examination by light microscopy, was removed and immediately fixed in formalin solution, embedded in paraffin, serially sectioned at 5 μm and stained with hematoxylin-eosin. 2.6. Statistical analysis The data were analyzed using the statistical package program StatView 5 Software for Windows (SAS Institute, Berkley, CA, USA). Statistical analysis between CD and DD, DMS and CD, and DMS and DD groups was performed with one-way analysis of variance followed by Student t test. All data were expressed as means±S.D. The results were considered significant if P≤.05. 3. Results 3.1. Blood glucose, hepatic glycogen concentration and GTT The plasma glucose concentration in the DD group significantly increased in comparison to the normoglycemic group (CD) (Fig. 1). The administration of flax and pumpkin seed mixture to rats with hyperglycemia resulted in the significant decrease of glucose concentration in comparison to the result obtained from the DD group. The concentration of plasma insulin (Fig. 2) of DD rats decreased by −42% in comparison to the CD group. Flax and pumpkin seed mixture supplemented to the diet of DMS group increased the insulin concentration in plasma by 63% in comparison to the DD group. Fig. 1 also shows hepatic glycogen in control and diabetic rats. Treatment with flax and pumpkin seed mixture increased significantly hepatic glycogen levels as compared with the diabetic group. The administration effect of flax and pumpkin seed mixture on glucose tolerance is presented in Fig. 3. flax and pumpkin seed mixture significantly increased the tolerance for glucose. The maximum glucose tolerance was noticed for the tested dose levels 120 min after glucose injection. 3.2. Effect of seed mixture supplemented to diet on plasma and liver lipid parameters Although the DD group recorded an increase in plasma and liver lipids by 108% and 30%, respectively, compared to the CD group, in

Fig. 1. Blood glucose (mg/dl) and hepatic glycogen (mg/g) levels of CD, DD and DMS groups.

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M. Makni et al. / Journal of Diabetes and Its Complications 25 (2011) 339–345 Table 1 Plasma and liver lipid profile in the CD, DD, and DMS groups Parameters and treatments

Fig. 2. Plasma insulin levels in CD, DD and DMS groups.

the DMS group, both plasma and liver lipids decreased by −21% and −19% compared to the DD group (Table 1). TC, TG, HDL-C, LDL-C levels, HTR (HDL-c/TC) ratio and AI are represented in Table 1. Significant increases in plasma and liver TC (137%; 40%) and plasma TG (85%) levels were observed in the DD group; LDL/HDL ratio and AI also significantly increased in the plasma of the last group. In the DMS group, plasma and liver TC and TG levels dropped by −47%, −45% and −47%, and −15%, respectively, compared to those of the DD group. The HTR ratio significantly increased, while the LDL/HDL ratio and AI significantly decreased in the DMS group as compared to those of the DD group. 3.3. Lipid peroxidation in plasma and tissue homogenates MDA levels in plasma and liver are illustrated in Table 2. A significant increase in MDA levels in plasma (132%) and in liver (102%) was observed in the DD group compared to those of the CD. Diet supplemented with seed mixture induced a significant decrease of MDA levels in plasma (−28%) and in liver (−51%) compared to the DD group. 3.4. Antioxidant enzyme activities and glutathione levels in plasma and liver Antioxidant enzyme activities (CAT and SOD) and GSH levels in the plasma and liver of control and tested groups are shown in Table 2. In DD group, a significant decrease of GSH levels and CAT and SOD activities was observed in plasma (−57%, −27%, −60%) and liver (−57%, −66%, −52%), respectively, as compared to the CD group. Diet supplemented with seed mixture improved, in the DMS group, GSH levels, CAT, and SOD activities in plasma (42%, 11%,

Total lipid Plasma (mg/ml) Liver (mg/g) TC Plasma (g/l) Liver (mg/g) TG Plasma (g/l) Liver (mg/g) LDL-cholesterol (g/l) HDL-cholesterol (g/l) HTR (%) AI LDL/HDL ratio

CD

DD

DMS

9.77±0.38 95.35±1.06

20.30±1.45⁎⁎⁎ 123.55±2.22⁎⁎⁎

16.07±1.66++ 100.13±5.64+++

0.67±0.08 11.01±0.06

1.59±0.20⁎⁎⁎ 15.41±0.02⁎

0.85±0.07+++ 8.51±0.01+++

0.67±0.05 13.63±0.05 0.24±0.07 0.30±0.02 44.55±3.37 1.24±0.18 0.80±0.04

1.24±0.13⁎⁎⁎ 13.01±0.04NS 1.10±0.12⁎⁎⁎ 0.24±0.06⁎ 15.32±5.65⁎⁎⁎ 5.53±2.21⁎⁎⁎ 4.50±1.10⁎⁎⁎

0.66±0.05+++ 11.02±0.03NS 0.40±0.18+++ 0.32±0.04+ 36.93±4.18+++ 1.71±0.31+++ 1.28±0.20+++

AI=(TC-HDL)/HDL. HTR (%)=HDL-C/TC ratio. Values are given as means±S.D. (mean of six determinations). Significant differences between the DD and CD groups: ⁎Pb.05; ⁎⁎Pb.01; ⁎⁎⁎Pb.001. Significant differences between the DMS and DD groups: +Pb.05; ++Pb.01; +++Pb.001.

130%) and liver (70%, 185%, 56%), respectively, as compared to those of the DD group. 3.5. ALT and AST activities The activities of AST and ALT significantly increased in diabetic rats compared to controls (Table 3). These activities decreased significantly by (−44% and −26%), respectively, after the supplementation of flax and pumpkin seed mixture in the diet of diabetic rats. 3.6. Light microscopy study of pancreas tissue The pancreas histological examination of the CD and DMS groups showed normal β-cell architecture. Alloxan administration elicited significant morphological changes in DD rats with severe injury of pancreatic β-cells, such as decreasing the islets cell numbers, cell damage, and cell death (Fig. 4A–C). 4. Discussion Our previous investigation (Makni et al., 2008) showing the potent hypolipidemic and antioxidant activity of supplemented flax and Table 2 MDA, GSH levels and enzymes activities (SOD, CAT) in plasma and liver of CD, DD, and DMS rats Parameters and treatments

CD

DD

DMS

a

MDA Plasma Liver GSHb Plasma Liver SODc Plasma Liver CATd Plasma Liver

Fig. 3. GTT in control and diabetic rats.

3.94±0.29 6.96±0.93

9.16±0.65⁎⁎⁎ 14.12±1.42⁎⁎⁎

6.60±0.45++ 6.98±1.09+++

8.67±1.23 6.97±0.84

3.70±0.92⁎⁎⁎ 3.02±0.54⁎⁎⁎

5.26±1.40+ 5.14±0.57+++

15.99±3.26 16.89±2.26

6.35±1.21⁎⁎⁎ 8.02±0.95⁎⁎⁎

14.65±2.66+++ 12.55±2.08++

6.42±0.91 5.26±1.66

4.70±0.41⁎⁎ 1.77±0.62⁎⁎⁎

5.21±0.57+ 5.05±1.10+++

Values are given as means±S.D. (mean of six determinations). Significant differences between the DD and CD groups: ⁎Pb.05; ⁎⁎Pb.01; ⁎⁎⁎Pb.001. Significant differences between the DMS and DD groups: +Pb.05; ++Pb.01; +++Pb.001. a MDA=nmol/ml in plasma and nmol/100 mg in liver. b GSH=mg/ml in plasma and mg/mg protein in liver. c Superoxide dismutase=U/mg protein. d Catalase=μmol H2O2 degraded/min/mg protein.

M. Makni et al. / Journal of Diabetes and Its Complications 25 (2011) 339–345 Table 3 Plasma AST and ALT enzyme activities in CD, DD, and DMS groups Parameters and treatments

CD

DD

DMS

AST (IU/l) ALT(IU/l)

103.67±4.53 40.82±4.88

230.4±6.53⁎⁎⁎ 86.77±4.62⁎⁎⁎

128.77±6.49+++ 64.26±3.09++

Values are given as means±S.D. (mean of six determinations). Significant differences between the DD and CD groups ⁎⁎⁎Pb.001. Significant differences between the DMS and DD groups ++Pb.01;

+++

Pb.001.

pumpkin seed mixture in hypercholesterolemic rats confirmed the ethnomedical use of these seeds against metabolic syndrome. In the present study, we investigated whether the flax and pumpkin seed mixture had any hypoglycemic, hypolipidemic, and antioxidant action in normal and alloxan-diabetic rats. The most important result of the present study was that rats, fed a flax and pumpkin seed-enriched diet, were able to partly recover from alloxan-induced diabetes within a short time compared with rats fed control diet. Interestingly, such an effect could be related to the partial regeneration or preservation of pancreatic β-cell mass after alloxan

Fig. 4. Pancreas histological sections (hematoxylin and eosin, ×400). (A) CD group. (B) DD group. (C) DMS group.

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treatment. Indeed, at the end of the experiment, pancreatic β-cell mass in the DMS group was similar to that of the CD group. We may also hypothesize that flax and pumpkin seed mixture supplementation may have a protective effect against alloxan. However, during the first 2 days after alloxan injection, glycemia rose to the same extent in all treated rat groups, suggesting similar acute alloxan toxic effects on the endocrine pancreas and no protective effect of flax and pumpkin seed mixture supplementation. Nevertheless, when the time course of glycemia was measured from Day 2 to Day 14 following alloxan injection, diabetes in the DMS rats was less developed than in the DD rats. Indeed, by day 14, hyperglycemia was back to near normal value in the DMS group but remained significantly higher in the DD group. At the end of the experiment, hyperglycemia was still elevated in the DD group, thereby underscoring the less protective effect of the control diet compared with flax and pumpkin seed mixture. In order to elucidate the modulatory mechanism of flax and pumpkin seed mixture on glucose metabolism in rats, we also focused on the hepatic glucose metabolism which was reflected by changes in hepatic glycogen. The results obtained showed that flax and pumpkin seed mixture increased hepatic glycogen content. This suggests that the preservation of hepatic glycogen was maintained and the gluconeogenesis rate was depressed. In parallel, plasma insulin level significantly decreased in the DD group compared with the DMS animals. This indicated that changes in insulin may bring about changes in hepatic glycogen content and lead to the regulatory effect of flax and pumpkin seed mixture on glucose metabolism in alloxan-induced diabetic rats and confirmed a defect in pancreatic β-cell function and/ or a decreased β-cell mass, as shown in the histological examination of different diabetes pancreatic sections. DM is also one of the most common human metabolic diseases, and derangements in lipid metabolism in diabetic subjects are often important determinants of the course and status of the disease (Fumelli, Romagnoli, Carlino, Fumelli, & Boemi, 1996). The administration of flax and pumpkin seed mixture extract significantly decreased plasma and liver lipid and cholesterol and plasma triacylglycerol in DMS rats. In continence with the present data, other works reported that the administration of fenugreek lowered both serum triacylglycerol and total cholesterol in diabetic rats (Khosla, Gupta, & Nagpal, 1995) and hypercholesterolemic patients (Prasanna, 2000). The hypolipidemic action of soluble dietary fiber fraction could be the result of the retardation of carbohydrate and fat absorption due to the presence of bioactive fiber in the agent (Hannan et al., 2003). Our results confirmed our previous data (Makni et al., 2008, 2010), which showed that seed mixture rich in polyunsaturated fatty acids (PUFAs) had strong hypotriglyceridemic and hypocholesterolemic effects on rats with a reduction in plasma LDL-C levels and an increase in HDL-C levels. Furthermore, the atherogenic index markedly decreased, causing a significant reduction in LDL/HDL ratio in the DMS group. The increase in HDL-C or HTR ratio is one of the most important criteria of antiatherogenic agents. Moreover, numerous studies have demonstrated that high levels of HDL-C are associated with a lower incidence of cardiovascular diseases (Shali, Kaul, Nilsson, & Cercek, 2001; Young, 2005). The increase in HDL-C levels, observed in our studies, might be due to the stimulation of pre-β HDL-C and reverse cholesterol transport, as demonstrated by previous findings (Gupta, Ross, Myers, & Kashyap, 1993). Besides, epidemiological studies have shown that high HDL-C levels could potentially contribute to its anti-atherogenic properties, including its capacity to inhibit LDL oxidation and protect endothelial cells from the cytotoxic effects of oxidized LDL (Assmann & Nofer, 2003). The antiatherogenic effect of flax and pumpkin seed mixture found in our studies might be due to the presence of PUFAs, phytosterols, tocopherols and β-carotene (ElAdawy &Taha, 2001; Vijaimohan et al., 2006). The

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major total fatty acids present in seed mixture are unsaturated fatty acids such as oleic acid, linolenic acid and linoleic acid, which play a crucial role in reducing blood cholesterol in humans and rats (Makni et al., 2008, 2010; Nettleton, 1991; Takada, Saitoh, & Mori, 1994). Nevertheless, it has been shown in pigs that conjugated linoleic acid and Omega-3 PUFA were important pharmaconutrients for modulating inflammatory bowel disease through the activation of keratinocyte growth factor (KGF) expression (Bassaganya-Riera & Hontecillas, 2006). Interestingly, Movassat and Portha (2007) recently found that the early administration of KGF improved β-cell regeneration in rats with streptozotocin-induced diabetes. Thus, flax and pumpkin seed mixture supplementation could probably lead to increased KGF expression, thus contributing to pancreatic regeneration and/or preservation. It is now well established that fatty acid (FA) composition is crucial to explaining the differential effects of seeds on parameters such as the lipid profile (Dhar, Bhattacharyya, Bhattacharyya, & Ghosh, 2006; Luo et al., 1998), cardiac cell and vascular function (Ghosh, An, Pulinilkunnil, Qi, & Lau, 2004). It has been shown that 2-week treatments with evening primrose oil rich in PUFA lowered lipid and haemostatic risk factors for cardiovascular disease in diabetic rats (Ford, Cotter, Cameron, & Greaves, 2001). The beneficial effects of gamma-linoleic supplementation on nerve conduction velocity, Na+/K+ ATPase activity, and membrane FA composition in the sciatic nerve of diabetic rats have also been demonstrated (Coste et al., 1999). FAs are also clearly identified as insulin secretion modulators, depending on their chain length and saturation degree (Poitout & Robertson, 2008). Thus, linoleic and linolenic acid—the major FA in flax and pumpkin seeds oil as demonstrated in our previous study (Makni et al., 2008)—may be involved in the modulation of pancreatic β-cell function, as recently reported by Feng et al. (Feng et al., 2006). Other authors demonstrated that linoleate reduced the voltage-gated K+ current in rat β-cells through GPR40 and the cAMP-protein kinase A system, leading to an increase in [Ca2+] and insulin secretion (Feng et al., 2006). Similar data were found in vivo in mice in which the dietary supplementation of conjugated linoleic acid and Omega-3 polyunsaturated FA augmented insulin secretion partly because of increased islet glucose oxidation (Winzell, Pacini, & Ahren, 2006). Thus, it may be postulated in our study that the supplementation of diet with flax and pumpkin seed mixture, which provided linoleic and linolenic acid, also had a protective or regenerative effect on the endocrine pancreas. It has been hypothesized that one of the principal causes of diabetes-induced injury is the formation of lipid peroxides by free radical derivatives. Thus, the antioxidant activity or the inhibition of the generation of free radicals is important in the protection against diabetes-induced hepatopathy (Castro et al., 1974). The body has an effective defense mechanism to prevent and neutralize the free radical-induced damage. This is proficient by a set of endogenous antioxidant enzymes such as SOD and CAT. These enzymes constitute a mutually supportive team of defense against ROS (Amresh, Kant, et al., 2007; Amresh, Rao, & Singh, 2007). In diabetes, the balance between ROS production and these antioxidant defenses may be lost, resulting in oxidative stress which, through a series of events, deregulates the cellular functions leading to hepatic necrosis, for example. The reduced activities of SOD and CAT point out the tissues' damage in the diabetic rats. DMS group showed a significant increase in the level of these enzymes as compared to DD group, which indicates the antioxidant activity of the seed mixture. Regarding non enzymic antioxidants, GSH is a critical determinant of tissue susceptibility to oxidative damage and the depletion of GSH has been shown to be associated with an enhanced toxicity to chemicals (Hewawasam, Jayatilaka, Pathirana, & Mudduwa, 2003), including diabetic status. In the present study, a decrease in plasma and hepatic tissue GSH level was observed in diabetic group. The increase in

plasma and hepatic GSH level in the DMS rats may be due to the novo GSH synthesis or GSH regeneration. The level of lipid peroxide (MDA) is a measure of membrane damage and alterations in the structure and function of cellular membranes. In the present study, the elevation of lipid peroxidation in the plasma and liver of diabetic rats was observed. The increase in MDA levels suggests an enhanced lipid peroxidation leading to tissue damage and the failure of antioxidant defense mechanisms to prevent the formation of excessive free radicals (Amresh, Kant, et al., 2007; Amresh, Rao, et al., 2007). Supplementation of flax and pumpkin seed mixture significantly reversed these changes. Hence, it is possible that the mechanism of hepatoprotection may be due to its antioxidant activity. On phytochemical screening, flax and pumpkin seed mixture revealed the presence of flavonoids and phenolics as major compounds. These antioxidant phytochemicals might contribute to the hepatoprotective and antioxidant activities of the whole mixture seeds. Plasma enzymes including AST and ALT are used in the evaluation of hepatic disorders (Achliya, Wadodkar, & Dorle, 2004; Thabrew, Joice, & Rajatissa, 1987). An increase in these enzyme activities reflects active liver damage. Inflammatory hepatocellular disorders result in extremely elevated transaminase levels (Foreston, Tedesco, & Starnes, 1985; Hultcrantz, Glaumann, & Lindberg, 1986). In accordance with these findings, alloxan treatments had a significant role in the alteration of liver functions because the activity of AST and ALT was significantly higher than those of normal values (Sheweita, El-Gabar, & Bastawy, 2001). The present study revealed a significant increase in the activities of AST and ALT on diabetic status, indicating considerable hepatocellular injury. The administration of flax and pumpkin seed mixture attenuated the increased levels of the plasma enzymes produced by induction of diabetes and caused a subsequent recovery towards normalization comparable to the control group animals. The hepatoprotective effect of the mixture was further accomplished by histopathological examinations as demonstrated in our previous study (Makni et al., 2008). In conclusion, our data suggest that supplementing diet with flax and pumpkin seed mixture partly preserved pancreatic function and improved peripheral glucose in alloxan induced diabetic rats. The identification of the active components in such seeds, traditionally used in folk medicine to treat arterial hypertension and/or diabetes in Mediterranean countries, may well contribute to our knowledge of their precise molecular effects. Acknowledgments The authors thank the skilful technical assistance of the Food Processing Department of Sidi Bouzid Institute (ISET) Tunisia. We also extend our thanks to Mr. Bejaoui Hafed, teacher of English at the Sfax Faculty of Science, who helped proofread and edited this manuscript. The present work was supported by the DGRST grants (Appui à la Recherche Universitaire de Base ARUB 99/UR/08-73), Tunisia.

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