Food and Chemical Toxicology 48 (2010) 24072412

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Food and Chemical Toxicology

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Flax and Pumpkin seeds mixture ameliorates diabetic nephropathy in rats

Mohamed Makni a,b, Mediha Se a,1, Hamadi Fetoui a,1, El Mouldi Garoui a, Nabil K. Gargouri b,
Tahia Boudawara c, Najiba Zeghal a,*
a
Animal Physiology Laboratory, Faculty of Sciences, BP 1171, 3000 Sfax, Tunisia
b
Food Processing Department, ISET, BP 377, 9100 Sidi Bouzid, Tunisia
c
Histopathology Laboratory, CHU Habib Bourguiba, 3029 Sfax, Tunisia

a r t i c l e i n f o a b s t r a c t

Article history: This study investigated the hypoglycemic and antioxidant effects of Flax and Pumpkin seeds mixture on
Received 3 April 2010 the kidney of alloxan-induced diabetic rats. Animals were allocated into three groups of six rats each: a
Accepted 28 May 2010 control group (CD), a diabetic group (DD) and diabetic rats fed with Flax and Pumpkin seeds mixture
(DMS) group. The DD rats showed a signicant increase of glycemia and lipid parameters such as total
lipid, total cholesterol and triglycerides levels compared to those of the control group (CD). In addition,
Keywords: plasma and kidney malonaldialdehyde levels (MDA) were signicantly increased compared to (CD)
Diabetes
group. Antioxidant enzyme activities such as catalase (CAT), superoxide dismutase (SOD) and non-enzy-
Oxidative stress
Antioxidant enzymes
matic levels of reduced glutathione (GSH) signicantly decreased in the plasma and kidney of diabetic
Lipid peroxidation rats compared to those of controls. Diet supplemented with Flax and Pumpkin seeds mixture ameliorated
Flax and Pumpkin seeds the antioxidant enzymes activities observed in diabetic rats and signicantly decreased MDA levels. Kid-
Nephropathy ney histological sections, showed glomerular hypertrophy and tubular dilatation. In DMS rats, these his-
topathological changes were less prominent. Our results suggest that Flax and Pumpkin seeds mixture
supplemented in diet of diabetic rats may be helpful to prevent diabetes and its complications.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
Diabetes mellitus (DM) is a health problem affecting millions of
individuals worldwide. The World Health Organization (WHO) pre-
dicts that 300 millions of people will have diabetes mellitus by the
year 2025 (Pradeepa and Mohan, 2002). DM is a prevalent systemic
disease with well documented devastating effects (Duckworth,
2001).
Hyperglycemia has been found to play a key role in reactive
oxygen species (ROS) generated damage (Ha and Kim, 1999;
Greenberg and Sacks, 2002; Ugochukwu and Babady, 2002;
Abbreviations: AI, atherogenic index; CAT, catalase; CD, control rats; DD,
diabetic rats; DM, diabetes mellitus; DMS, diabetic rats fed diet containing seeds
mixture; DTNB, 5,5-dithiobis-2 nitro benzoic acid; FCE, food conversion efciency;
GSH, reduced glutathione; GTT, glucose tolerance test; HDL-C, high-density
lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; MDA,
malondialdehyde; NBT, nitroblue tetrazolium; PUFAs, polyunsaturated fatty acids;
ROS, reactive oxygen species; SD, standard deviation; SOD, superoxide dismutase;
TBA, thiobarbituric acid; TBARS, thiobarbituric acid-reactive substance; TC,
total cholesterol; TG, triacylglycerol; WHO, World Health Organization.
* Corresponding author. Address: 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: najiba.zeghal@tunet.tn (N. Zeghal).
1
Authors contributed equally to this work.
Ugochukwu et al., 2003; Maritim et al., 2003). Oxygen free radicals
are formed disproportionately in diabetes by glucose oxidation,
non-enzymatic protein glycation and the subsequent oxidative
degradation of glycated proteins (Maritim et al., 2003).
Many scientic reports indicate that diabetic complications are
associated with overproduction of ROS and accumulation of lipid
peroxidation by-products (Palanduz et al., 2001). In diabetes, major
damage occurs in tissues such as kidney where the entry of glucose
is not regulated by insulin (Limaye et al., 2003).
Free radicals generated in diabetes may lead to several kinds of
diabetic complications including nephropathy, neuropathy, cardi-
opathy and many others diseases. Many herbal extracts used as
single agents or in different oral formulations have been recom-
mended to prevent diabetes mellitus due to the fact that they are
less toxic than oral hypoglycemic agents such as sulfonylureas,
metformin, etc. (Ponnachan et al., 1993; Chattopadhyay, 1993).
Antioxidants play a major role in protection against molecular oxi-
dative damage (Evans, 2007).
Traditional medicines, derived mainly from plants, play a major
role in the management of diabetes mellitus (Karunanayake and
Tennekoon, 1993; Patel and Srinivasan, 1997; Ahmed et al., 2004).
World Health Organization (WHO) recommended the evalua-
tion of traditional plant treatments for diabetes as they are effec-
tive, non-toxic, with less or no side effects and are considered to
be excellent candidates for oral therapy (Day, 1998). Recently,

0278-6915/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.05.079
2408 M. Makni et al. / Food and Chemical Toxicology 48 (2010) 24072412
Mankil et al. (2006) have recommended many medicinal plants
used already in traditional medicine, experimental and clinical
antidiabetic. To our knowledge, this is the rst biochemical inves-
tigation undertaken to explore in alloxan diabetic animals, the
antidiabetic and nephroprotective effects of Flax and Pumpkin
seeds mixture rich in PUFAs and antioxidant compounds, in allox-
an diabetic animals.
2. Materials and methods
2.1. Plant material
Flax (Linum usitatissimum L.) and Pumpkin (Cucurbita pepo L.) seeds were pur-
chased from a local market (Sfax city, Tunisia), crushed at ambient temperature
and stored at 4 C prior to use. The seeds mixture of Flax and Pumpkin rich in Ome-
ga 3 and Omega 6 was prepared. The ratio of Omega 6/Omega 3 fatty acids was 5/1
as recommended by the WHO and according to Blandeau and Schneider (2006) and
Grigg (2004).
2.2. Experimental design
Male Wistar rats (190210 g) aged 3 months, were obtained from the Central
Pharmacy of Tunisia (SIPHAT, Tunisia). They were maintained under standard lab-
oratory conditions (22 3 C, 12-h light/dark cycle), with pellated food (Industrial
Company of Rodent Diet, Sfax, Tunisia) and tap water given ad libitum during
30 days of experimental period. The animals were cared in accordance with the
principles of the Guide for the Care and Use of Experimental Animals (Council of
European Communities, 1986). The local Committee of Ethics in Animal Experimen-
tation approved all the experimental procedures of this study.
The present study was conducted in three groups of six rats each: control rats
(CD), diabetic rats (DD) and diabetic rats fed diet containing Flax and Pumpkin
seeds mixture supplemented at 33% (DMS).
2.3. Induction of diabetes
After 2 weeks of acclimatization, diabetes was induced in male rats which re-
ceived by intraperitoneal way, 120 mg kg1 BW of alloxan monohydrate freshly
prepared in normal saline, according to Mansour et al. (2002) and Sheweita et al.
(2002). Rats were orally treated with 20% glucose solution (510 ml) after 6 h, be-
cause alloxan is able to produce fatal hypoglycemia as a result of massive pancreatic
insulin release. The rats received for the next 24 h, 5% glucose water solution to pre-
vent hypoglycemia. Those with moderate diabetes and where blood glucose con-
centration was about 200300 mg dl1 were taken for the experimental tests.
2.4. Biochemical assays
2.4.1. Determination of blood glucose and glucose tolerance test (GTT)
Plasma glucose levels were assayed by enzymatic methods, using commercial
reagent kits purchased from Biomaghreb (Ref. 20121, Ariana Tunis, Tunisia).
The glucose tolerance test (GTT) evaluates the ability to respond appropriately
to a glucose challenge (Matteucci and Giampietro, 2008). GTT was conducted in
control and treated rats, 24 h before sacrice day. Blood samples were collected
from the rats tail veins which were fasted overnight to obtain baseline blood glu-
cose levels. Subsequently, rats of both control and treated groups were injected
intraperitoneally with glucose (2 g kg1 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 insulin concentration
Plasma insulin was determined using rat insulin enzyme-linked immunosor-
bent assay (ELISA) kit Ref. RIT-461 No. AKRIN-010T (Shibayagi Co., Ltd., Japan).
2.4.3. Estimation of urea, uric acid and creatinine
The levels of urea, uric acid and creatinine in plasma were estimated spectro-
photometrically using commercial diagnostic kits, respectively (Refs. 20151,
20143, and 20091) purchased from Biomagreb (Ariana, Tunisia).
2.4.4. Analysis of plasma lipids
Plasma lipids were extracted with chloroform/methanol mixture (2v/1v)
according to the method of Folch et al. (1957). The contents of total lipids in plasma
extracts were quantied gravimetrically by evaporating off the solvents using a ro-
tary evaporator (Heidolph, Laborota 4010 digital, Germany). Plasma lipid parame-
ters 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 (Refs. 20112, 20131, 20113, Ariana, Tuni-
sia). The low-density lipoprotein-cholesterol (LDL-C) fraction and atherogenic index
(AI) were determined according to the Friedewald equations (Friedewald et al.,
1972):
LDL-C Total cholesterol  Triglycerides=5 HDL-C;
AI TC  HDL-C=HDL-C:
2.4.5. Measurement of malonaldialdehyde (MDA) in tissues
Concentrations of MDA in tissues, an index of lipid peroxidation, were deter-
mined spectrophotometrically according to Draper and Hadley (1990). An amount
of 0.5 ml of each plasma sample or kidney extract supernatant (tissue homogenates
in potassium phosphate buffer pH 7.8), was mixed with 1 ml of trichloroacetic acid
solution and centrifuged at 2500g for 10 min. One milliliter solution containing
0.67% thiobarbituric acid (TBA) and 0.5 ml of supernatant were incubated for
15 min at 90 C and cooled. Absorbance of TBA-MDA complex was determined at
532 nm using a spectrophotometer (Jenway UV-6305, Essex, England). Lipid perox-
idation was expressed as nmol of thiobarbituric acid-reactive substances (TBARS),
using 1,1,3,3-tetraethoxypropane as standard.
2.4.6. Antioxidant enzymes and glutathione assays in plasma and kidney
2.4.6.1. Total superoxide dismutase activity (SOD). Superoxide dismutase (SOD) activ-
ity was estimated according to Beauchamp and Fridovich (1971). The reaction mix-
ture contained 50 mM of tissue homogenates in potassium phosphate buffer (pH
7.8), 0.1 mM EDTA, 13 mM L-methionine, 2 lM riboavin and 75 lM Nitroblue tet-
razolium (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 mg of
protein.
2.4.6.2. Catalase activity (CAT). Catalase (CAT) activity was assayed by the method of
Aebi (1984). Enzymatic reaction was initiated by adding an aliquot of 20 ll of the
homogenized tissue and the substrate (H2O2) to a concentration of 0.5 M in a med-
ium containing 100 mM phosphate buffer, pH 7.4. Changes in absorbance were re-
corded at 240 nm. CAT activity was calculated in terms of nmol H2O2 consumed/
min/mg of protein.
2.4.6.3. Glutathione levels (GSH). GSH in tissues was determined by the method of
Ellman (1959) and modied by Jollow et al. (1974) based on the development of
a yellow color when DTNB (5,5-dithiobis-2 nitro benzoic acid) was added to com-
pounds containing sulfhydryl groups. A 500 ll of tissue homogenate in phosphate
buffer were added to 3 ml of 4% sulfosalicylic acid. The mixture was centrifuged
at 1600g for 15 min. Supernatant (500 ll) were taken and added to Ellmans re-
agent. The absorbance was measured at 412 nm after 10 min. Total GSH content
was expressed as mg/ml in plasma and as mg/mg of protein in kidney.
2.5. Histopathological examination
Kidneys removed from the control and tested rats were cleaned and xed in 10%
buffered formalin solution. Then they were embedded in parafn and stained with
hematoxylineosin for histopathological studies. All sections were evaluated for the
degree of tubular and glomerular injury and necrosis.
2.6. Statistical analysis
The data were analyzed using the statistical package program Stat view 5 Soft-
ware for Windows (SAS Institute, Berkley, CA). Statistical analysis between CD and
DD, DMS and CD and DMS and DD groups was performed with one-way ANOVA fol-
lowed by student t-test. All data were expressed as means SD. The results were
considered signicant if p 6 0.05.
3. Results
3.1. Body weight gain, digestibility and feed conversion efciency (FCE)
Table 1 summarizes body weights, digestibility and feed con-
version efciency (FCE) of experimental animals. A signicant de-
crease of body weight gain was observed in DD rats, seeds
mixture seems to exert a protective effect against weight loss in
DMS as compared to DD group. The digestibility of macronutrients
and the food conversion efciency (FCE) ratio were presented in
Table 1. No signicant difference was observed in digestibility
and FCE between groups.
 M. Makni et al. / Food and Chemical Toxicology 48 (2010) 24072412 2409

Table 1 1.4
Body weight gain, feed intake, digestibility, average feces weights, and feed conversion +++
efciency (FCE) in rats fed diets for 4 weeks.

Plasma insulin levels (ng/ml)

1.2
Parameters and treatments CD DD DMS
1
Body weight gaina (g) 99.00 72.00* 87.00
Average feed intake (g/d) 20.00 17.43 22.54+ ***
0.8
FCE ratio 00.20 00.24 0.26
Average feces weight (g/d) 03.09 02.89 3.34
0.6
Apparent digestibility (%) 84.55 83.42 85.18

Signicant differences between the DD and CD groups: *p < 0.05. 0.4

Signicant differences between the DMS and DD groups: +p < 0.05.
a
Values are given as means standard deviation (mean of six determinations). 0.2

3.2. Blood glucose and glucose tolerance test (GTT)
The plasma glucose concentration in the diabetic group (DD)
signicantly increased in comparison to the normoglycemic group
(CD) (Fig. 1). The administration of Flax and Pumpkin seeds mix-
ture to rats with hyperglycemia resulted in the signicant decrease
of plasma glucose level in comparison to the result obtained from
the DD group.
Plasma insulin level (Fig. 2) of DD rats decreased by 42% in com-
parison to the CD group. Flax and Pumpkin seeds mixture supple-
mented to the diet of DMS group increased plasma insulin
concentration by 63% in comparison to the DD group.
Flax and Pumpkin seeds mixture signicantly increased the tol-
erance for glucose (Fig. 3). The maximum glucose tolerance was
noticed 120 min after glucose injection.
3.3. Estimation of urea, uric acid and creatinine
In diabetic rats, levels of urea, creatinine and uric acid were sig-
nicantly higher in plasma (29%, 66% and 16%) than those of con-
trols (Table 2). Administration of Flax and Pumpkin seeds mixture
to diabetic rats signicantly reversed these changes to near normal
values.
0
CD DD DMS
Fig. 2. Plasma insulin levels in CD, DD and DMS groups. Values are given as means
standard deviation [Mean of six determinations]. Signicant differences between
the DD and CD groups ***p < 0.001. Signicant differences between the DMS and DD
groups +++p < 0.001.
els were observed in DD group, LDL/HDL ratio and AI also signi-
cantly increased in the plasma of the last group. In DMS group
plasma TC and TG levels were decreased by 47% and 47%, respec-
tively, compared to those of DD group. The HTR ratio signicantly
increased, while LDL/HDL ratio and AI signicantly decreased in
DMS group as compared to those of DD group.
3.5. Lipid peroxidation in plasma and kidney homogenates
MDA levels in plasma and kidney are presented in Table 4. A
signicant increase in MDA levels in plasma (132%) and in kidney
(102%) was observed in DD group compared to those of control
400
Blood glucose level (mg/dl)

350
3.4. Effect of seeds mixture supplemented to diet on plasma lipid
300
parameters
250
While the DD group recorded an increase in plasma lipids by 200
108%, compared to the CD group, plasma lipids in the DMS group
150
decreased by 21% compared to the DD group (Table 3).
TC, TG, HDL-C, LDL-C levels, HTR ratio and AI are represented in 100
Table 3. Signicant increases in plasma TC (137%) and TG (85%) lev- 50

0
400 0 30 60 90 120
Time (min)
glucose 2g/l
Blood Glucose Levels (mg/dl)

350

300 CD DD DMS

250 Fig. 3. Glucose tolerance test (GTT) in control and diabetic rats. Values are given as
means of six determinations.
200

150
Table 2
100
Plasma levels of creatinine, urea and uric acid of CD, DD and DMS experimental rats.
50
Parameters and CD DD DMS
0 treatments
J+0 J+2 J+7 J+14 J+21 J+30 Creatinine (lmol/l) 112.76 6.98 186.85 7.61*** 100.2 5.95+++
Days Uric acid (lmol/l) 302.55 10.34 350.2 11.76*** 297.6 7.86+++
Urea (mmol/l) 10.13 2.12 13.09 2.83* 10.66 1.78+
CD Group DD Group DMS Group
Values are given as means standard deviation (mean of six determinations).
Fig. 1. Blood glucose (mg/dl) levels of CD, DD and DMS groups. Values are given as Signicant differences between the DD and CD groups: *p < 0.05 and ***p < 0.001.
means standard deviation [mean of six determinations]. Signicant differences between the DMS and DD groups: +p < 0.05 and +++p < 0.001.
2410 M. Makni et al. / Food and Chemical Toxicology 48 (2010) 24072412

Table 3 signicant morphological changes in renal DD rats with severe in-

Plasma lipid prole in CD, DD and DMS groups. jury of tubular and glomeruli (Fig. 4AC).
Parameters and treatments CD DD DMS
Total plasma lipid (mg/ml) 9.77 0.38 20.30 1.45*** 16.07 1.66++ 4. Discussion
Total plasma cholesterol 0.67 0.08 1.59 0.20*** 0.85 0.07+++
(TC) (g/l)
Diabetes mellitus is possibly the worlds largest growing meta-
Plasma triacylglycerol (TG) 0.67 0.05 1.24 0.13*** 0.66 0.05+++
(g/l) bolic disease. The need for more appropriate therapy increases
LDL-cholesterol (g/l) 0.24 0.07 1.10 0.12*** 0.40 0.18+++ (Bailey and Day, 1989). A large number of hypoglycemic/antidia-
HDL-cholesterol (g/l) 0.30 0.02 0.24 0.06* 0.32 0.04+ betic plants and herbs are known in traditional medicine but their
HTR (%) 44.55 3.37 15.32 5.65*** 36.93 4.18+++ introduction into modern therapy waits pharmacological testing
Atherogenic index (AI) 1.24 0.18 5.53 2.21*** 1.71 0.31+++
by recent methods. Thus offer a natural key to unlock a diabetolog-
LDL/HDL ratio 0.80 0.04 4.50 1.10*** 1.28 0.20+++
ists pharmacy for the future.
AI = (TC  HDL)/HDL. The present results suggest that Flax and Pumpkin seeds mix-
HTR (%) = HDL-C/TC ratio.
ture exhibit signicant hypoglycemic, hypolipidemic and nephro-
Values are given as means standard deviation (mean of six determinations).
Signicant differences between the DD and CD groups: *p < 0.05; **p < 0.01; and protector effects in alloxan-induced diabetic rats. Fasting blood
***p < 0.001. glucose level in diabetic rats is an important basal parameter for
Signicant differences between the DMS and DD groups: +p < 0.05; ++p < 0.01; and
+++
p < 0.001.

group CD. Diet supplemented with seeds mixture induced a signif-

icant decrease of MDA levels in plasma (28%) and in kidney (34%)
compared to DD group.

3.6. Antioxidant enzyme activities and glutathione levels in plasma

and kidney

GSH levels and antioxidant enzyme activities (CAT and SOD) in

the plasma and kidney of control and tested groups are shown in
Table 4. In DD group, a signicant decrease of GSH levels, CAT
and SOD activities was observed in plasma (57%, 27% and 60%)
and in kidney (34%, 34% and 25%), respectively, as compared to
those of CD group. Diet supplemented with seeds mixture in
DMS group, improved GSH levels, CAT and SOD activities in plasma
(42%, 11% and 130%) and kidney (44%, 53% and 35%), respectively,
as compared to those of DD group.

3.7. Light microscopy study of kidney tissue

The kidney histological examination of control and DMS groups

showed normal cells architecture. Alloxan treatment elicited

Table 4
MDA, glutathione levels (GSH) and enzymes activities (SOD, CAT) in plasma and
kidney of CD, DD and DMS rats.

Parameters and treatments CD DD DMS

MDAa
Plasma 3.94 0.29 9.16 0.65*** 6.60 0.45++
Kidney 4.02 0.45 8.12 0.95*** 5.33 0.76++
GSHb
Plasma 8.67 1.23 3.70 0.92*** 5.26 1.40+
Kidney 6.22 0.39 4.11 0.19** 5.92 0.25+++
SODc
Plasma 15.99 3.26 6.35 1.21*** 14.65 2.66+++
Kidney 18.03 2.61 13.53 3.71** 18.28 2.64++
CATd
Plasma 6.42 0.91 4.70 0.41** 5.21 0.57+
Kidney 14.49 1.37 9.55 1.06*** 14.61 1.11+++

Values are given as means standard deviation (mean of six determinations).

Signicant differences between the DD and CD groups: *p < 0.05; **p < 0.01; and
***p < 0.001.
Signicant differences between the DMS and DD groups: +p < 0.05; ++p < 0.01; and
+++
p < 0.001.
a
MDA = nmol/ml in plasma and nmol/100 mg in liver.
b
GSH = mg/ml in plasma and mg/mg protein in liver. Fig. 4. Kidney histological sections of CD (A), DD (B) and DMS (C) groups
c
SOD = units/mg protein. (hematoxylin and Eosin, 400). Arrows indicate: : Tubular dilatation, :
d
CAT = lmol H2O2 degraded/min/mg protein. Glomerular space reduction; ?: Necrosis.
 M. Makni et al. / Food and Chemical Toxicology 48 (2010) 24072412
monitoring diabetes (Rajkumar et al., 2005). It has shown that Flax
and Pumpkin seeds mixture supplemented in the diet of DMS
group causes the hypoglycemic and antihyperglycemic effects by
reducing the fasting blood glucose level. The signicant decrease
in the levels of fasting blood glucose in the last group might be ex-
plained by the stimulation of the residual pancreatic mechanism,
regeneration or protection of pancreatic cells that were partially
destroyed by alloxan, potentiating of insulin secretion from pro-
tected b-cells of the islets of Langerhans (Suba et al., 2004), and
probably by increasing peripheral utilization of glucose (Erah
et al., 1996).
Alloxan-induced diabetes, characterized by a severe loss in
body weight (Odetola et al., 2006), induced by the degradation of
structural proteins (Rajkumar et al., 1991), were associated with
classic symptoms of diabetes including polydipsia and polyuria
(Oi et al., 1997). In our study, the decrease of body weight gain
in diabetic rats supports these ndings.
Diabetes mellitus is usually associated with an increase in plas-
ma lipids levels, the risk factor for coronary heart diseases
(Davidson, 1981; Al-Shamaony et al., 1994). A decrease of serum
lipid concentration through drug therapy or dietary measures
seems to decrease the risk of vascular diseases (Rhoads et al.,
1976). Increase in lipids, TG and TC levels in alloxan diabetic rats
observed in the present study may be a result of increased break-
down of lipids and mobilization of free fatty acids from the periph-
eral stores. Administration of Flax and Pumpkin seeds mixture for
30 days normalized lipid prole in diabetic animals. Seed mixtures
not only lowered the TC, TG and LDL but also enhanced the
HDL-cholesterol which is known to play an important role in the
transport of cholesterol from peripheral cells to the liver by a
pathway termed reverse cholesterol transport, and is considered
to be a cardio protective lipid. Thus seeds mixture has a signicant
impact to improve the imbalance in lipoprotein metabolism.
Kidneys remove metabolic wastes such as urea, uric acid, creat-
inine and ions. So optimum chemical composition of body uids is
maintained. The concentrations of the metabolites increase in
blood during renal diseases or renal damage associated with
uncontrolled diabetes mellitus (Almdal and Vilstrup, 1988). This
may be due to metabolic disturbance in diabetes reected in high
activities of xanthine oxidase, lipid peroxidation, and increased tri-
acylglycerol and cholesterol levels (Madinov et al., 2000; Anwar
and Meki, 2003). In the present study, there was an elevation in
urea, uric acid and creatinine in the DD group indicating renal
damage, while signicant decrease in these parameters was ob-
served in the animals of DMS group.
In the present study, diabetes was induced by alloxan. Alloxan
produces hyperglycemia by selective cytotoxic effects on pancre-
atic b-cells. One of the intracellular phenomenons for its cytotoxic-
ity is through generation of free radicals, which has been
demonstrated both in vivo and in vitro (Grandy et al., 1982;
Papaccio et al., 1986). Free radicals and associated reactive oxygen
species have been implicated in diabetes and its complications
(Baynes and Thorpe, 1999). It has been suggested that a variety
of antioxidants that scavenge reactive oxygen species might im-
prove hyperglycemia and prevent diabetic complications. In our
previous study, we have demonstrated that Flax and Pumpkin
seeds mixture possesses free radical scavenging activity in hyper-
cholesterolemic rats (Makni et al., 2008). Antidiabetic activity of
seeds mixture may be due to its protective role in quenching free
radicals (which destroy pancreatic cells) to ensure benecial role
in survival of pancreatic b-cells.
Flax and Pumpkin seeds mixture caused signicant increase in
insulin levels of diabetic treated rats (DMS group) compared to
the DD animals. Enhanced plasma insulin levels in the DMS
animals ensured the regeneration or the protection of pancreatic
b-cells. This effect has been reported by others with some other
2411
medicinal plants like Terminalia chebula, Momordica charantia and
Eugenia jambolana (Sharma et al., 2003; Ahmed et al., 2004; Murali
et al., 2007).
The biochemical parameters were correlated with the renal his-
tological studies. In fact, we revealed that alloxan caused a signif-
icant damage in renal structure showing marked glomeruli and
tubular damages, due probably to the generation of reactive radi-
cals and to subsequent lipid peroxidation induced by alloxan. So,
hydroperoxides accumulated in kidney could cause cytotoxicity
associated with membrane phospholipids peroxidation, the basis
for renal cellular damage and necrotic renal cells. The administra-
tion of Flax and Pumpkin seeds mixture through the diet of dia-
betic rats improved the histological alterations induced by
alloxan, which could be attributed to its antiradical/antioxidant
activities.
Conict of Interest
The authors declare that there are no conicts of interest.
Acknowledgements
The authors thank the skillful technical assistance of the Food
Processing Department of Sidi Bouzid Institute (ISET) Tunisia and
of Histopathology Laboratory of CHU Habib Bourguiba, Sfax, Tuni-
sia. The present work was supported by the DGRST grants (Appui
la Recherche Universitaire de Base ARUB 99/UR/08-73), Tunisia.
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