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

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Journal of Diabetes and Its Complications

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

Mohamed Maknia,b, Hamadi Fetouia,1, Nabil K. Gargourib,1, El Mouldi Garouia, Najiba Zeghala,⁎a Animal Physiology Laboratory, Faculty of Sciences, BP 1171, 3000 Sfax, Tunisiab Food Processing Department, ISET, BP 377, 9100 Sidi Bouzid. Tunisia

⁎ Corresponding author. Animal Physiology LaboratoSciences, BP 1171, 3000 Sfax, Tunisia. Tel.: +216 98 914

E-mail address: najiba.zeghal@tunet.tn (N. Zeghal).1 Authors contributed equally to this work.

1056-8727/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.jdiacomp.2010.09.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 July 2010received in revised form 14 August 2010accepted 8 September 2010Available online 23 November 2010

Keywords:Diabetesflax and pumpkin seedsAntioxidant enzymesLipid peroxidationOxidative stress

Reactive oxygen species play a crucial role in the pathogenesis of diabetes and its complications. This studyaims to examine the effects of flax and pumpkin powder seed mixture on alloxan induced diabetes in Wistarrats. Animals were allocated into three groups of six rats each: a control group (CD), diabetic group (DD) anddiabetic rats fed with flax and pumpkin seed mixture (DMS) group. The diabetic rats (DD) presented asignificant increase in glycemia, plasma and liver lipid parameters such as total lipid, total cholesterol andtriglycerides 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 enzymesactivities such as catalase, superoxide dismutase, and reduced glutathione (GSH) levels significantlydecreased in the plasma and liver of diabetic rats compared to controls. Diet supplemented with flax andpumpkin seed mixture in the DMS group ameliorated antioxidant enzymes activities and level of GSH indiabetic rats and significantly decreased MDA levels.The present study revealed a significant increase in the activities of aspartate aminotransferase and alanineaminotransferase on diabetic status, indicating considerable hepatocellular injury. The administration offlax and pumpkin seed mixture attenuated the increased levels of the plasma enzymes produced by theinduction of diabetes and caused a subsequent recovery towards normalization comparable to the controlgroup animals.Our results thus suggest that flax and pumpkin seed mixture supplemented to diet may be helpful inpreventing diabetic complications in adult rats.

ry, UR 08-73, Sfax Faculty of154; fax: +216 74 274 437.

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© 2011 Elsevier Inc. All rights reserved.

1. Introduction

Diabetes mellitus (DM) is a chronic metabolic disorder characte-rized by high levels of glucose in the blood due to the non-secretionof insulin or insulin insensitivity (American Diabetes AssociationADA, 2005). DM affects approximately 4% of the populationworldwide and is expected to increase by 5.4% in 2025 (Kim, Hyun,& Choung, 2006). Although the underlying mechanisms of diabetescomplications remain unclear, clinical and preclinical evidencesuggests that diabetes is associated with oxidative stress, leading toan increased production of reactive oxygen species (ROS), includingsuperoxide radical (O2•−), hydrogen peroxide (H2O2) and hydroxylradical (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

oxidants contributes to the pathogenesis of different diabeticcomplications which are considered to result from enhanced reactiveoxygen species generation via nicotinamide adenine dinucleotidephosphate-oxidase (Baynes & Thorpe, 1999; Garg, Ojha, & Bansal,1996; Ha & Kim, 1999).

The pathophysiology of diabetes involves a very complex cascadeof several interrelated mechanisms. Elevated blood glucose inducesauto-oxidative glycosylation and formation of glycation product,activates protein kinase-C, and increases polyol pathway activity andhexosamine flux, which are the key components of the cascade. Thesepathways are responsible for the generation of reactive oxygenspecies (superoxide, hydroxyl radical, hydrogen peroxide) andperoxynitrite, which ultimately contribute to oxidative stress(Ahmed, Adeghate, Cummings, Sharma, & Singh, 2004; Baynes &Thorpe, 1999; Ha & Kim, 1999). Current approaches to diabetestherapy involve mainly drugs enhancing insulin secretion or signal-ling as well as inhibiting endogenous glucose production (Anuradha& Selvam, 1993), while the role of antioxidants as the importantagents to restore the redox balance of the organism is still under-estimated. Dietary intervention, particularly the use of traditionalfood and medicine derived from natural sources, is a mainstay in the

340 M. Makni et al. / Journal of Diabetes and Its Complications 25 (2011) 339–345

management of diabetes. In this context, various dietary sources arepresently receiving considerable attention across the world for thepotential health benefits in relation to many diseases such as diabeticdisorders. Among them, flax seeds (Linum usitatissimum L., member ofLinaceae family) and pumpkin seeds (Cucurbita pepo L., member ofCucurbitaceae family) are becoming new compounds of the traditionalhealth food in Tunisia and other North African countries. Flax seedscontain high levels of Omega-3 fatty acid (Burdge & Calder, 2005),fiber components and phytochemicals such as lignans bioressource(Vijaimohan et al., 2006). Themain physiological benefits of flax seedsare attributed primarily to the high linoleic acid content whichcontributes to their antioxidant properties (Simopoulos, 1991)against various diseases, including atherosclerosis, diabetes, hyper-tension, anti-inflammatory, and anticarcinogenic effects (Fukuda,Osawa, Namiki, & Ozaki, 1985). Pumpkin seeds are utilized for humanconsumption 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 biologicalapplications along with significant antioxidant activity, in additionto anti-inflammatory and hypolipidemic effects (Suresh & Das, 2003).

In our previous study, we demonstrated that flax and pumpkinseedmixture supplemented to diet of hypercholesterolemic rats had asignificant anti-atherogenic, hypolipidemic and antioxidant potency.Flax and pumpkin seedmixture had a pronounced antioxidant activitydue to their richness in antioxidant components (Makni et al., 2008).

Several studies have demonstrated the beneficial effects of dietaryseeds. Hence, the protective effects of flax and pumpkin seed mixtureon diabetic complications would be worth studying.

The present study was carried out in order to evaluate both theproblems induced by diabetes and the protective effects of flax andpumpkin 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 ambienttemperature and stored at 4°C prior to use. The seed mixture of flaxand pumpkin rich in Omega 3 and Omega 6 was prepared. The ratio ofOmega 6/Omega 3 fatty acids was 5:1, as recommended by the WorldHealth 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 (IndustrialCompany of Rodent Diet, Sfax, Tunisia) and tap water ad libitumduring 30 days of experimental period. The general guidelines on theuse of living animals in scientific investigations (Council of EuropeanCommunities, 1986) and the guidelines for care and use of laboratoryanimals 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 dietsupplemented with flax and pumpkin seed mixture at 33% (DMS).

2.3. Induction of diabetes

After 2 weeks of acclimatization, diabetes was induced in male ratswith a freshly prepared solution of alloxan monohydrate in normalsaline 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 iscapable of producing fatal hypoglycemia as a result of massivepancreatic insulin release, rats were orally treated with 20% glucosesolution (5–10 ml) after 6 h. The rats were then kept for the next24 h on 5% glucose water solution to prevent hypoglycemia. Ratswith moderate diabetes that exhibited glycosuria and hyperglycemia(i.e., blood glucose concentration 200–300 mg dl−1) were taken forthe experimental tests.

2.4. Biochemical assays

2.4.1. Determination of plasma glucose, hepatic glycogen levels andglucose tolerance test

2.4.1.1. Glucose levels. Plasma glucose levels were assayed byenzymatic methods, using commercial reagent kits purchased fromBiomaghreb (Ariana Tunis, Tunisia).

2.4.1.2. Hepatic glycogen. Hepatic glycogen content was determined bytreatment with O-toluidine reagent and measured at 620 nm using aspectrophotometer (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 andtreated rats, 24 h before sacrifice day. Blood samples were collectedfrom the rats' tail vein (control and treated groups) which were fastedovernight to obtain baseline blood glucose levels. Subsequently, ratsof both control and treated groups were injected intraperitoneallywith glucose (2 g kg−1 BW). Blood was collected from the rats' tailvein at interval of 30 min up to 2 h for glucose estimation using aglucometer (Esprit 2, BAYER, France).

2.4.2. Estimation of plasma insulin concentrationPlasma insulin level was determined using rat Insulin enzyme-

linked immunosorbent assay kit ref. RIT-461 No. AKRIN-010T(Shibayagi, Japan).

2.4.3. Analysis of lipids in plasma and liverTissue 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 werequantified gravimetrically by evaporating off the solvents using arotary evaporator (Heidolph, Laborota 4010 digital, Germany). Plasmalipid parameters such as total cholesterol (TC), triacylglycerol (TG)and High-density lipoprotein-cholesterol (HDL-C) levels were deter-mined by enzymatic methods, using commercial kits from Biomagh-reb (Ariana Tunis, Tunisia). The low-density lipoprotein-cholesterol(LDL-C) fraction and atherogenic index (AI) were determinedaccording to the Friedewald equations (Friedewald, Levy, & Fredrick-son, 1972):

LDL−C = TC− Triglycerides = 5 + HDL−Cð Þ;AI = TC−HDL−C=HDL−CÞ:ð

The dried hepatic lipid residues were dissolved in 1ml absoluteethanol for cholesterol and triacylglycerol assays. Hepatic totalcholesterol and triacylglycerol contents were analyzed with thesame enzymatic kits used in plasma analysis.

2.4.4. Measurement of malonaldialdehyde in tissuesConcentrations of MDA in tissues, an index of lipid peroxidation,

was determined spectrophotometrically according to Draper andHadley (1990). An amount of 0.5 ml of each plasma sample or liverextract supernatant was mixed with 1 ml of trichloroacetic acid

341M. Makni et al. / Journal of Diabetes and Its Complications 25 (2011) 339–345

solution and centrifuged at 2500g for 10 min. A 1-ml solutioncontaining 0.67% thiobarbituric acid (TBA) and 0.5 ml of supernatantwere incubated for 15 minutes at 90°C and cooled. Absorbance ofTBA-MDA complex was determined at 532 nm using a spectropho-tometer (Jenway UV-6305; Essex, England). Lipid peroxidation wasexpressed as nanomoles of TBA reactive substances using 1,1,3,3-tetraethoxypropane 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 wasestimated according to Beauchamp and Fridovich (1971). Thereaction mixture contained 50 mM of tissue homogenates in potas-sium phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM L-methionine,2 μM riboflavin and 75 μM nitroblue tetrazolium (NBT). Thedeveloped blue color in the reaction was measured at 560 nm. Unitsof SOD activity were expressed as the amount of enzyme requiredto inhibit the reduction of NBT by 50% and the activity was expressedas units per milligrams of protein.

2.4.5.2. Catalase activity (CAT). CAT activity was assayed by themethod of Aebi (1984). Enzymatic reaction was initiated by adding analiquot of 20 μl of the homogenized tissue and the substrate (H2O2) toa concentration of 0.5 M in a medium containing 100 mM phosphatebuffer, pH 7.4. Changes in absorbance were recorded at 240 nm.CAT activity was calculated in terms of nanomoles H2O2 consumedper minute per milligram of protein.

2.4.5.3. Glutathione levels (GSH). GSH in tissues was determined by themethod of Ellman (1959) modified by Jollow, Mitchell, Zampaglione,and Gillete (1974) based on the development of a yellow color whenDTNB (5, 5-dithiobis-2 nitro benzoic acid) was added to compoundscontaining sulfhydryl groups; 500 μl of tissue homogenate inphosphate buffer was added to 3 ml of 4% sulfosalicylic acid. Themixture was centrifuged at 1600g for 15 min; 500 μl of supernatantwas taken and added to Ellman's reagent. The absorbance wasmeasured at 412 nm after 10min. Total GSH content was expressed asmilligrams per milliliter in plasma and as milligrams per milligram ofprotein in liver.

2.4.6. Estimation of aspartate aminotransferase (AST) and alanineaminotransferase (ALT) activities

AST and ALT activities in plasma, used as biochemical markers forhepatic damage, were determined by enzymatic methods usingcommercial reagent kits from Biomaghreb (Ariana Tunis, Tunisia).

Fig. 1. Blood glucose (mg/dl) and hepatic glycoge

2.5. Histopathological examination

The pancreas, intended for histological examination by lightmicroscopy, was removed and immediately fixed in formalin solution,embedded in paraffin, serially sectioned at 5 μm and stained withhematoxylin-eosin.

2.6. Statistical analysis

The data were analyzed using the statistical package programStatView 5 Software for Windows (SAS Institute, Berkley, CA, USA).Statistical analysis between CD and DD, DMS and CD, and DMS and DDgroups was performed with one-way analysis of variance followed byStudent t test. All data were expressed as means±S.D. The resultswere considered significant if P≤.05.

3. Results

3.1. Blood glucose, hepatic glycogen concentration and GTT

The plasma glucose concentration in the DD group significantlyincreased in comparison to the normoglycemic group (CD) (Fig. 1).The administration of flax and pumpkin seed mixture to ratswith hyperglycemia resulted in the significant decrease of glucoseconcentration in comparison to the result obtained from theDD group.

The concentration of plasma insulin (Fig. 2) of DD rats decreasedby −42% in comparison to the CD group. Flax and pumpkin seedmixture supplemented to the diet of DMS group increased the insulinconcentration 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 signifi-cantly hepatic glycogen levels as compared with the diabetic group.

The administration effect of flax and pumpkin seed mixture onglucose tolerance is presented in Fig. 3. flax and pumpkin seedmixture significantly increased the tolerance for glucose. Themaximum glucose tolerance was noticed for the tested dose levels120 min after glucose injection.

3.2. Effect of seed mixture supplemented to diet on plasma and liverlipid parameters

Although the DD group recorded an increase in plasma and liverlipids by 108% and 30%, respectively, compared to the CD group, in

n (mg/g) levels of CD, DD and DMS groups.

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

Table 1Plasma and liver lipid profile in the CD, DD, and DMS groups

Parameters and treatments CD DD DMS

Total lipidPlasma (mg/ml) 9.77±0.38 20.30±1.45⁎⁎⁎ 16.07±1.66++

Liver (mg/g) 95.35±1.06 123.55±2.22⁎⁎⁎ 100.13±5.64+++

TCPlasma (g/l) 0.67±0.08 1.59±0.20⁎⁎⁎ 0.85±0.07+++

Liver (mg/g) 11.01±0.06 15.41±0.02⁎ 8.51±0.01+++

TGPlasma (g/l) 0.67±0.05 1.24±0.13⁎⁎⁎ 0.66±0.05+++

Liver (mg/g) 13.63±0.05 13.01±0.04NS 11.02±0.03NS

LDL-cholesterol (g/l) 0.24±0.07 1.10±0.12⁎⁎⁎ 0.40±0.18+++

HDL-cholesterol (g/l) 0.30±0.02 0.24±0.06⁎ 0.32±0.04+

HTR (%) 44.55±3.37 15.32±5.65⁎⁎⁎ 36.93±4.18+++

AI 1.24±0.18 5.53±2.21⁎⁎⁎ 1.71±0.31+++

LDL/HDL ratio 0.80±0.04 4.50±1.10⁎⁎⁎ 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.

Table 2MDA, GSH levels and enzymes activities (SOD, CAT) in plasma and liver of CD, DD, andDMS rats

342 M. Makni et al. / Journal of Diabetes and Its Complications 25 (2011) 339–345

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 arerepresented in Table 1. Significant increases in plasma and liver TC(137%; 40%) and plasma TG (85%) levels were observed in the DDgroup; LDL/HDL ratio and AI also significantly increased in the plasmaof the last group. In the DMS group, plasma and liver TC and TG levelsdropped by −47%, −45% and −47%, and −15%, respectively,compared to those of the DD group. The HTR ratio significantlyincreased, while the LDL/HDL ratio and AI significantly decreasedin 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. Asignificant 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 decreaseof MDA levels in plasma (−28%) and in liver (−51%) compared tothe DD group.

3.4. Antioxidant enzyme activities and glutathione levels in plasmaand liver

Antioxidant enzyme activities (CAT and SOD) and GSH levels inthe plasma and liver of control and tested groups are shown inTable 2. In DD group, a significant decrease of GSH levels and CATand SOD activities was observed in plasma (−57%, −27%, −60%)and liver (−57%, −66%, −52%), respectively, as compared to theCD group. Diet supplemented with seed mixture improved, in theDMS group, GSH levels, CAT, and SOD activities in plasma (42%, 11%,

Fig. 3. GTT in control and diabetic rats.

130%) and liver (70%, 185%, 56%), respectively, as compared to thoseof the DD group.

3.5. ALT and AST activities

The activities of AST and ALT significantly increased in diabetic ratscompared to controls (Table 3). These activities decreased signifi-cantly by (−44% and −26%), respectively, after the supplementationof 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 groupsshowed normal β-cell architecture. Alloxan administration elicitedsignificant morphological changes in DD rats with severe injury ofpancreatic β-cells, such as decreasing the islets cell numbers, celldamage, and cell death (Fig. 4A–C).

4. Discussion

Our previous investigation (Makni et al., 2008) showing the potenthypolipidemic and antioxidant activity of supplemented flax and

Parameters and treatments CD DD DMS

MDAa

Plasma 3.94±0.29 9.16±0.65⁎⁎⁎ 6.60±0.45++

Liver 6.96±0.93 14.12±1.42⁎⁎⁎ 6.98±1.09+++

GSHb

Plasma 8.67±1.23 3.70±0.92⁎⁎⁎ 5.26±1.40+

Liver 6.97±0.84 3.02±0.54⁎⁎⁎ 5.14±0.57+++

SODc

Plasma 15.99±3.26 6.35±1.21⁎⁎⁎ 14.65±2.66+++

Liver 16.89±2.26 8.02±0.95⁎⁎⁎ 12.55±2.08++

CATd

Plasma 6.42±0.91 4.70±0.41⁎⁎ 5.21±0.57+

Liver 5.26±1.66 1.77±0.62⁎⁎⁎ 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.

Table 3Plasma AST and ALT enzyme activities in CD, DD, and DMS groups

Parameters and treatments CD DD DMS

AST (IU/l) 103.67±4.53 230.4±6.53⁎⁎⁎ 128.77±6.49+++

ALT(IU/l) 40.82±4.88 86.77±4.62⁎⁎⁎ 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.

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pumpkin seed mixture in hypercholesterolemic rats confirmed theethnomedical use of these seeds against metabolic syndrome. In thepresent study, we investigated whether the flax and pumpkin seedmixture had any hypoglycemic, hypolipidemic, and antioxidantaction in normal and alloxan-diabetic rats.

The most important result of the present study was that rats, fed aflax and pumpkin seed-enriched diet, were able to partly recover fromalloxan-induced diabetes within a short time compared with rats fedcontrol diet. Interestingly, such an effect could be related to the partialregeneration 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.

treatment. Indeed, at the end of the experiment, pancreatic β-cellmass in the DMS group was similar to that of the CD group.

We may also hypothesize that flax and pumpkin seed mixturesupplementation may have a protective effect against alloxan.However, during the first 2 days after alloxan injection, glycemiarose to the same extent in all treated rat groups, suggesting similaracute alloxan toxic effects on the endocrine pancreas and noprotective effect of flax and pumpkin seed mixture supplementation.

Nevertheless, when the time course of glycemia was measuredfrom Day 2 to Day 14 following alloxan injection, diabetes in theDMS rats was less developed than in the DD rats. Indeed, by day 14,hyperglycemia was back to near normal value in the DMS group butremained significantly higher in the DD group. At the end of theexperiment, hyperglycemia was still elevated in the DD group,thereby underscoring the less protective effect of the control dietcompared with flax and pumpkin seed mixture.

In order to elucidate the modulatory mechanism of flax andpumpkin seed mixture on glucose metabolism in rats, we alsofocused on the hepatic glucose metabolism which was reflected bychanges in hepatic glycogen. The results obtained showed that flaxand pumpkin seed mixture increased hepatic glycogen content. Thissuggests that the preservation of hepatic glycogen was maintainedand the gluconeogenesis rate was depressed. In parallel, plasmainsulin level significantly decreased in the DD group compared withthe DMS animals.

This indicated that changes in insulin may bring about changes inhepatic glycogen content and lead to the regulatory effect of flax andpumpkin seed mixture on glucose metabolism in alloxan-induceddiabetic rats and confirmed a defect in pancreatic β-cell function and/or a decreased β-cell mass, as shown in the histological examinationof different diabetes pancreatic sections.

DM is also one of the most common human metabolic diseases,and derangements in lipid metabolism in diabetic subjects are oftenimportant determinants of the course and status of the disease(Fumelli, Romagnoli, Carlino, Fumelli, & Boemi, 1996). The adminis-tration of flax and pumpkin seed mixture extract significantlydecreased plasma and liver lipid and cholesterol and plasmatriacylglycerol in DMS rats. In continence with the present data,other works reported that the administration of fenugreek loweredboth serum triacylglycerol and total cholesterol in diabetic rats(Khosla, Gupta, & Nagpal, 1995) and hypercholesterolemic patients(Prasanna, 2000). The hypolipidemic action of soluble dietary fiberfraction could be the result of the retardation of carbohydrate and fatabsorption due to the presence of bioactive fiber in the agent (Hannanet 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 hypocholesterolemiceffects on rats with a reduction in plasma LDL-C levels and an increasein HDL-C levels. Furthermore, the atherogenic index markedlydecreased, causing a significant reduction in LDL/HDL ratio in theDMS group. The increase in HDL-C or HTR ratio is one of the mostimportant criteria of antiatherogenic agents. Moreover, numerousstudies have demonstrated that high levels of HDL-C are associatedwith a lower incidence of cardiovascular diseases (Shali, Kaul, Nilsson,& Cercek, 2001; Young, 2005). The increase in HDL-C levels, observedin our studies, might be due to the stimulation of pre-β HDL-Cand reverse cholesterol transport, as demonstrated by previousfindings (Gupta, Ross, Myers, & Kashyap, 1993). Besides, epidemio-logical studies have shown that high HDL-C levels could potentiallycontribute to its anti-atherogenic properties, including its capacity toinhibit LDL oxidation and protect endothelial cells from the cytotoxiceffects of oxidized LDL (Assmann & Nofer, 2003). The antiatherogeniceffect of flax and pumpkin seed mixture found in our studies mightbe due to the presence of PUFAs, phytosterols, tocopherols andβ-carotene (ElAdawy &Taha, 2001; Vijaimohan et al., 2006). The

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major total fatty acids present in seed mixture are unsaturatedfatty acids such as oleic acid, linolenic acid and linoleic acid, whichplay a crucial role in reducing blood cholesterol in humans andrats (Makni et al., 2008, 2010; Nettleton, 1991; Takada, Saitoh, &Mori, 1994).

Nevertheless, it has been shown in pigs that conjugated linoleicacid and Omega-3 PUFA were important pharmaconutrients formodulating inflammatory bowel disease through the activation ofkeratinocyte growth factor (KGF) expression (Bassaganya-Riera &Hontecillas, 2006). Interestingly, Movassat and Portha (2007)recently found that the early administration of KGF improved β-cellregeneration in rats with streptozotocin-induced diabetes. Thus,flax and pumpkin seed mixture supplementation could probablylead to increased KGF expression, thus contributing to pancreaticregeneration and/or preservation.

It is nowwell established that fatty acid (FA) composition is crucialto explaining the differential effects of seeds on parameters such asthe 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-weektreatments with evening primrose oil rich in PUFA lowered lipid andhaemostatic risk factors for cardiovascular disease in diabetic rats(Ford, Cotter, Cameron, & Greaves, 2001). The beneficial effects ofgamma-linoleic supplementation on nerve conduction velocity,Na+/K+ ATPase activity, and membrane FA composition in the sciaticnerve 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 inflax and pumpkin seeds oil as demonstrated in our previous study(Makni et al., 2008)—may be involved in the modulation of pancreaticβ-cell function, as recently reported by Feng et al. (Feng et al., 2006).Other authors demonstrated that linoleate reduced the voltage-gatedK+ current in rat β-cells through GPR40 and the cAMP-protein kinaseA system, leading to an increase in [Ca2+] and insulin secretion(Feng et al., 2006). Similar data were found in vivo in mice in whichthe dietary supplementation of conjugated linoleic acid and Omega-3polyunsaturated FA augmented insulin secretion partly because ofincreased islet glucose oxidation (Winzell, Pacini, & Ahren, 2006).Thus, it may be postulated in our study that the supplementationof diet with flax and pumpkin seed mixture, which provided linoleicand linolenic acid, also had a protective or regenerative effect onthe endocrine pancreas.

It has been hypothesized that one of the principal causes ofdiabetes-induced injury is the formation of lipid peroxides by freeradical derivatives. Thus, the antioxidant activity or the inhibition ofthe generation of free radicals is important in the protection againstdiabetes-induced hepatopathy (Castro et al., 1974). The body has aneffective defense mechanism to prevent and neutralize the freeradical-induced damage. This is proficient by a set of endogenousantioxidant enzymes such as SOD and CAT. These enzymes constitutea mutually supportive team of defense against ROS (Amresh, Kant,et al., 2007; Amresh, Rao, & Singh, 2007). In diabetes, the balancebetween 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, forexample. The reduced activities of SOD and CAT point out the tissues'damage in the diabetic rats. DMS group showed a significant increasein the level of these enzymes as compared to DD group, whichindicates the antioxidant activity of the seed mixture. Regardingnon enzymic antioxidants, GSH is a critical determinant of tissuesusceptibility to oxidative damage and the depletion of GSH has beenshown to be associated with an enhanced toxicity to chemicals(Hewawasam, Jayatilaka, Pathirana, & Mudduwa, 2003), includingdiabetic status. In the present study, a decrease in plasma and hepatictissue GSH level was observed in diabetic group. The increase in

plasma and hepatic GSH level in the DMS rats may be due to thenovo GSH synthesis or GSH regeneration.

The level of lipid peroxide (MDA) is a measure of membranedamage and alterations in the structure and function of cellularmembranes. In the present study, the elevation of lipid peroxidationin the plasma and liver of diabetic rats was observed. The increasein MDA levels suggests an enhanced lipid peroxidation leading totissue damage and the failure of antioxidant defense mechanisms toprevent the formation of excessive free radicals (Amresh, Kant, et al.,2007; Amresh, Rao, et al., 2007). Supplementation of flax andpumpkin seed mixture significantly reversed these changes. Hence,it is possible that the mechanism of hepatoprotection may be due toits antioxidant activity.

On phytochemical screening, flax and pumpkin seed mixturerevealed the presence of flavonoids and phenolics as major com-pounds. These antioxidant phytochemicals might contribute to thehepatoprotective and antioxidant activities of the wholemixture seeds.

Plasma enzymes including AST and ALT are used in the evaluationof hepatic disorders (Achliya, Wadodkar, & Dorle, 2004; Thabrew,Joice, & Rajatissa, 1987). An increase in these enzyme activitiesreflects active liver damage. Inflammatory hepatocellular disordersresult in extremely elevated transaminase levels (Foreston, Tedesco, &Starnes, 1985; Hultcrantz, Glaumann, & Lindberg, 1986). In accor-dance with these findings, alloxan treatments had a significant role inthe alteration of liver functions because the activity of AST and ALTwas significantly higher than those of normal values (Sheweita,El-Gabar, & Bastawy, 2001).

The present study revealed a significant increase in the activities ofAST and ALT on diabetic status, indicating considerable hepatocellularinjury. The administration of flax and pumpkin seed mixtureattenuated the increased levels of the plasma enzymes produced byinduction of diabetes and caused a subsequent recovery towardsnormalization comparable to the control group animals. The hepato-protective effect of the mixture was further accomplished byhistopathological examinations as demonstrated in our previousstudy (Makni et al., 2008).

In conclusion, our data suggest that supplementing diet with flaxand pumpkin seed mixture partly preserved pancreatic function andimproved peripheral glucose in alloxan induced diabetic rats. Theidentification of the active components in such seeds, traditionallyused in folk medicine to treat arterial hypertension and/or diabetes inMediterranean countries, may well contribute to our knowledge oftheir precise molecular effects.

Acknowledgments

The authors thank the skilful technical assistance of the FoodProcessing Department of Sidi Bouzid Institute (ISET) Tunisia. We alsoextend our thanks to Mr. Bejaoui Hafed, teacher of English at the SfaxFaculty of Science, who helped proofread and edited this manuscript.The present work was supported by the DGRST grants (Appui à laRecherche Universitaire de Base ARUB 99/UR/08-73), Tunisia.

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