The present study was carried out to investigate the possible antioxidant effects of raisin extract “Karkni” on
biochemical parameters in alloxan-induced diabetic rats. Three doses of extract, 125, 250 and 375 mg/kg were orally
administered daily to alloxan-diabetic rats for 4 weeks. Alloxan-induced diabetic rats showed significant increases
in the levels of blood glucose, triglycerides, cholesterol, low density lipoprotein LDL-cholesterol, creatinine, uric acid
, total protein, glycosylated hemoglobin (HbA1c), Aspartate aminotransferase (AST) and alanine aminotransferase
(ALT) while high density lipoprotein HDL cholesterol, total hemoglobin and lactate dehydrogenase (LDH) levels were
significantly decreased compared to normal rats (P<0.05). The changes of the above parameters to their normal
levels after 4 weeks of treatment were observed mainly with the second dose 250 mg/kg. The overall results suggest
that the “Karkni” extract possesses potential hypoglycemic and hypolipidemic activity in alloxan-induced diabetic
rats.
Diabetes is a major degenerative disease in the world today [1],
affecting at least 15 million people and having complications which
include hypertension, atherosclerosis and microcirculatory disorders
[2]. It is also associated with long-term complications, including
retinopathy, nephropathy, neuropathy and angiopathy [3-5].
Controlling blood sugar levels and keeping these closer to normal values
is able to reduce the risk of diabetes and death by these complications.
Since dietary carbohydrates had the most direct impact on blood
sugar levels, controlling the amount of carbohydrate consumed per
meal is the focus of diabetes nutrition management [6]. Raisins, like
all commonly consumed fruits, provide carbohydrates as the only
caloric macronutrient but it is considered as a great source of many
polyphenolic compounds (flavonoids, tannins, phenolic acid) playing
important roles in nutrition and health care [7,8]. Spiller et al. [9] first
studied the hypocholesterolemic effect of plant-based diets, which
included more than one serving of sun-dried raisins daily. Keen and
schram showed that a diet providing 2, 3.5 and 5.5 ounces of raisins per
day for 4 weeks lowered ox-LDL levels [10]. Besides, research suggest
that addition of raisins to the daily diet has been shown to lower total
and LDL cholesterol levels, reduce markers of inflammation, increase
plasma antioxidant capacity and lower circulating levels of oxidized
LDL, a marker of coronary heart disease risk [11,12]. Also browning
reaction products (BRPs) in raisin have been reported to prevent or
retard oxidation reactions in lipid systems [13,14], because they have
similar chemical properties as phenolic acids [15]. Experimental
diabetes in animals has the advantage that it allows the analysis of the
biochemical events that take place not only during the induction of a
diabetic state but also after it has become established and during its
evolution to a severe insulin deficiency or even death [16]. Alloxan is
a specific toxin that destroys the pancreatic β cells, provoking a state
of primary deficiency of insulin without affecting other islet types
[17,18]. Hence, it was selected to induce diabetes in the present study
and the principal objective was to determine the potential beneficial
antidiabetic effects of three concentrations of raisin extract (125, 250
and 375 mg/kg) during 4 weeks on alloxan-induced diabetic rats.
Materials and Methods
Plant material
Dried fruit was extracted with distilled water by grinding with
a mortar and pestle. It was incubated for 24 h and filtered using a
Buckner funnel and Whatman No. 1 filter paper. It was the filtrate that
was administered to the animals in the course of this study, fresh for a
maximum of two days after which fresh extract was prepared.
Experimental animals
Male Wistar rats (Société des Industries Pharmaceutiques
Tunis, Tunisia), weighing 200-250 g were housed under standard
environmental conditions (23°C, 55 ± 5% humidity and a 12 h light/dark
cycle) and maintained with free access to water and a standard diet ad
libitum. Experimental diabetes was induced in rats by intraperitoneal
injection of alloxan monohydrate at a dose of 120 mg/kg. After 2
weeks, animals with blood glucose levels of 200 mg/dl and above were
considered diabetic. The rats were divided into 5 groups of 8 animals
per group. Group 1: Non-diabetic untreated rats (Control), Group 2:
Diabetic untreated rats, Group 3: Diabetic rats treated with 125 mg/kg
“Karkni” extract (D+C1), Group 4: Diabetic rats treated with 250 mg/
kg of the “Karkni” extract (D+C2) and Group 5: Diabetic rats treated with 375 mg/kg of the “Karkni” extract (D+C3). After confirming
diabetes, “Karkni” extract was administered orally, in the morning,
once every 24 hours for 4 weeks. At the end of this treatment period,
the animals were fasted overnight for 12 hours. Blood samples from
rats were collected under mild ether anesthesia in heparinized tubes
and centrifiguted at 3000 g for 15 min at 4°C. Plasma samples were
stored at -20°C in aliquots until analysis. All experimental protocols
used were in accordance with the guidelines of the local Committee on
Use of Laboratory Animals.
Experimental procedure
TC and HDL cholesterol, Creatinine, Urea, Uric acid, Total
protein in plasma were determined by colorimetric method, whereas
LDL cholesterol was calculated by the method of Friedewald et
al. [19]. Coronary Risk Index (CRI) was calculated by dividing
the Total Cholesterol (TC) by High density lipoprotein (HDL).
Glycosylated haemoglobin (HbA1c) was estimated using whole blood.
Hemoglobin, LDH, γ-GT, Alanine Aminotransferase, and Aspartate
Aminotransferase activity levels in the plasma were estimated by using
standard kits purchased from Biomaghreb [20-23].
Statistical analysis
Data were subjected to one-way analysis of variance for means of
comparison, and significant differences were calculated according to
Duncan multiple range test. Data are reported as means ± standard
error of the means. Differences at P<0.05 were considered statistically
significant. SPSS (version 11.0) was used to perform the statistical
analysis.
Results
Effects of “Karkni” extract on blood glucose levels
The effects of “Karkni” extract on blood glucose levels in diabetic
rats were shown in figure 1. The blood glucose levels of rats in the
control group did not change during the experimental period. By
contrast the blood glucose levels in diabetic group were significantly
higher (P<0.05) than those of the treated rats in the other groups; D+C1, D+C2 and D+C3. Of the 3 doses of raisin tested, the 250 mg/kg
dose was found to be the most effective in reducing blood glucose levels
during the 4 weeks.
Figure 1: Effect of “Karkni” extract treatment on blood glucose levels
in diabetic rats. Values with different superscript letters (a,b,c) indicate
significant differences among groups at P< 0.05 by Duncan’s multiple
range test.
C: Control; D: Rats with Alloxane-induced diabetes; D+C1: rats treated with 125
mg/kg/day; D+C2: rats treated with 250 mg/kg/day; D+C3: rats treated with 375
mg/kg/day
Effects of “Karkni” extract on plasma lipid profile like TC,
TG, LDL and HDL
In our study, induction of diabetes significantly altered the normal
lipid profile levels compared to control rat (Table 1). Administration
of three doses of “Karkni” extract significantly decreased (P<0.05)
plasma TC, TG and LDL cholesterol levels compared to diabetic
group. Moreover, the LDL cholesterol in the (D+C2) group was lower
than that in the other groups (D+C1) and (D+C3). HDL cholesterol
concentrations were increased in the (D+C2) and (D+C3) groups
than in diabetic group. Consequently, the ratio of LDL/HDL ratio
was decreased significantly in (D+C2) group than in the diabetic,
(D+C1) and (D+C3) groups (p<0.05), whereas, the coronary risk
was significantly lower in the (D+C2) and (D+C3) groups than in the
others groups.
Table 1: Effect of “Karkni” extract on lipid profile of alloxan-induced diabetic of rats.
Effects of “Karkni” extract on creatinine, Urea, Uric acid,
Total protein, Hemoglobin and HbA1c levels
Plasma levels of blood uric acid were increased following diabetes
induction (Table 2). There were significant difference between the
diabetic and the control group, 187.42 ± 56.34 and 152.5 ± 61.51
μmol/l respectively. But the lowest levels were observed in (D+C2)
group, (136.8 ± 21.01μmol/l). Also the level of creatinine was increased
in the diabetic group but the difference was not significant. Moreover,
plasma protein levels were significantly increased in the diabetic rats
(p<0.05). But after 4 weeks of treatment with “Karkni” extract, the
levels of protein were decreased in (D+C1), (D+C2) and (D+C3)
groups. Increased HbA1c levels were noticed in diabetic rats compared
to control rats whereas hemoglobin and urea levels were significantly
lowered in diabetic rats. But they were restored after four weeks of
treatment by “Karkni” extract.
Table 2: Effect of “Karkni” extract on creatinine, Urea, Uric acid, Total protein and Hemoglobin levels of alloxan-diabetic rats.
Effects of “Karkni” extract on enzymes activity
Induction of diabetes led to increased ALAT activity (Figure 2).
After four weeks of treatment, activity of this enzyme was decreased
in (D+C1) and (D+C2) groups but it still remained higher in the
(D+C3) group. Concerning ASAT activity, it was also found that
alloxan-induced diabetes increased, but it was significantly decreased
in (D+C2) and (D+C3) group (Figure 3). Also a significant (P<0.001)
reduction of LDH activity was observed in diabetic group compared to
control rats. By contrast, γ-GT activity was increased significantly in
diabetic rats (p<0.05). After 4 weeks, enzyme activity was restored in
(D+C1), (D+C2) and (D+C3) groups.
Figure 2: Effect of Karkni extract treatment on ALAT and ASAT activity
in diabetic rats. Values with different superscript letters (a,b,c) indicate
significant differences among groups at P< 0.05 by Duncan’s multiple
range test.
C: Control; D: Rats with Alloxane-induced diabetes; D+C1: Rats treated with
125 mg/kg/day; D+C2: Rats treated with 250 mg/kg/day; D+C3: Rats treated
with 375 mg/kg/day
Figure 3: Effect of Karkni extract treatment on LDH and γ -GT activity
in diabetic rats. Values with different superscript letters (a,b,c) indicate
significant differences among groups at P<0.05 by Duncan’s multiple
range test.
C: Control; D: Rats with alloxane-induced diabetes; D+C1: Rats treated with
125 mg/kg/day; D+C2: Rats treated with 250 mg/kg/day; D+C3: Rats treated
with 375 mg/kg/day
Discussion
Raisin polyphénols have demonstrated significant antioxidant
anticarcinogenic, anti-inflammatory, thermogenic, probiotic, and
antimicrobial properties in numerous human, animal, and in vitro studies [6]. But the effect of raisin polyphenols in diabetes is still
unknown. The present study was conducted to determine the effect
of feeding 3 doses of raisin extract “Karkni” on the lipid parameters
in alloxan rats. Alloxan, at a dose of 120 mg/kg body weight, caused
sufficient damage to pancreatic β cells so that secreted insulin was not
enough to regulate blood glucose and resulted in a significant increase
in blood glucose levels [24-26]. In our experiment, we had shown that
raisin extract decreased plasma glucose for 4 weeks compared with
those of the diabetic’s controls. Both doses (125 and 250 mg/kg) of
the extract showed a significant hypoglycemic effect but rats treated
with 375 mg/kg of “Karkni” extract showed significant increases
in blood glucose after three weeks. These results can be related with
the carbohydrates content in raisin. Previous studies had shown that
hypoglycemic effect of many herbs was related to flavonoids content
and these constituents can preserve the insulin-secreting capacity and
viability of pancreatic β cells [27]. Therefore, the presence of these
constituents in our variety of raisin may explain the hypoglycemic
activity, and we can conclude that the antioxidative activities of raisin extract in alloxan-induced diabetes, at least in part, may be related
to antihyperglycemic capability. The present study found that the
intake of “Karkni” extract significantly lower plasma total cholesterol,
triglycerides and LDL cholesterol concentrations but increased
serum HDL cholesterol in (D+C1), (D+C2) and (D+C3) groups
when compared with those of diabetic control. Also decreased level
of coronary risk was observed in the same group but it was especially
interesting in (D+C2) group, (2.82 mg/dl). These results were similar
to those found by Bruce et al. [11] and Gardner et al. [12]. High levels
of circulating cardiac dysfunction/damage markers such as, LDH
and uric acid, represent a sensitive predictor of increased cardiac
complications [28]. In our study, “Karkni” extract showed a significant
decrease in levels of uric acid in (D+C1) and (D+C2) groups when
compared with those of diabetic control. But it was still remaining
higher in (D+C3) group. In diabetic rats the elevation observed in
uric acid level could be due to the increased abundance and activity
of xanthine oxidase. It was responsible for the formation of uric acid
and also serves as an important biological source of ROS (superoxide)
that contribute to oxidative damage involved in many pathological
processes such as diabetes, atherosclerosis, cancer and aging [29-31].
Moreover, decreasing levels of HbA1c observed in (D+C2) group can
reduce the risk of lipid peroxidation [32]. These explain the important
role of raisin to decrease cardiac damage markers in diabetic person
with 250 mg/kg.
Also results presented in table 2 clearly revealed that the
administration of the three concentrations of “Karkni” extract to the
diabetic rats could restore the changes in the levels of hemoglobin,
urea, creatinine and total protein after 4 weeks of treatment to their
normal levels. Lal et al. proved that in diabetes, elevated levels of serum
urea, uric acid and creatinine are observed which may be due to renal
damage caused by abnormal glucose regulation or elevated glucose
and glycosylated protein tissue levels [33]. So it could be concluded
that the above action of raisin may be due to its potential to control
hyperglycemia and renal damage in diabetic rats. Alloxan induces
increase of AST, and ALT activity in rats (Figure 2). This results
were similar than that observed with streptozotocin induced diabetic
rats [34]. Restoration of the normal level of ALAT may indicate the
normalizing effect of both 125 and 250 mg/kg of “Karkni” extract.
As for ASAT activity, restoration of the normal level was observed
only with 250 and 375 mg/kg of “Karkni” extract. A change in
enzyme activity is presumed to be due to the decreased blood insulin
concentration [35], but is also related to energy metabolism, as these
enzymes play a role in gluconeogenesis [36]. As for LDH activity,
results from our experiment show that diabetes decreases LDH activity
(Figure 3). These result are similar than Tormo et al. [37], but not than
Melinkeri et al. [38]. A highly significant elevation in the activity of
γ-GT was observed in plasma of alloxan-induced diabetic rats. But after
treatment of raisin, increased activity of this enzyme was lowered to
near normal that indicates the possible prevention of necrosis in the
liver by “Karkni” extract. The same result was observed by McLennan
et al. [39]. Also, the increment of the activities of AST, ALT and LDH
in plasma is mainly due to the leakage of these enzymes from the liver
cytosol into the blood stream [40], which gives an indication on the
hepatotoxic effect of alloxan, but raisin extract reduced the activity of
these enzymes in plasma so the oral administration of “Karkni” extract
had a beneficial effect on the diabetic state reducing the hyperglycemia
as well as hyperlipidemia.
Conclusion
In conclusion, our study demonstrates that “Karkni” extract corrects
hyperglycemia and dyslipidemia, thus improving the atherogenic index.
The 250 mg/kg dose was found to be the most effective in restoring
changes during the 4 weeks. This implies that raisin can prevent or be
helpful in reducing the complications of diabetes. However, further
investigations to fully identify the biologically active ingredients and to
define the precise molecular mechanism(s) of these effects are required.
Meng J, Fang Y, Zhang A, Chen S, Xu T, et al. (2011) Phenolic content and antioxidant capacity of Chinese raisins produced in 407 Xinjiang Province. Food Res 44: 2830-2836.
Spiller GA, Schultz L, Spiller M, Ou B (2002) Sun-dried raisins help prevent oxidative DNA damage during intense athletic activity. J Am Coll Nutr21:487.
Keen CL, Schram DD (Unpublished data) The effect of raisins on antioxidant capacity and cholesterol concentration in human subjects.
Gogus F, Bozkurt H, Eren S (1998) Kinetics of Maillard reactions between the major sugars and amino acids of boiled grape juice. Lebensmittel-Wissenschaftund-Tech 31: 196-200.
OMICS Publishing Group is the member of / publishing partner of/source content provider to
OMICS Publishing Group, An Open Access Publisher and Scientific Events Organizer for the Advancement of Science & Technology. All Published content, except where otherwise noted, is licensed under a Creative Commons Attribution License
Please ensure that you are using the latest version of Adobe reader. If you do not have this software installed on your system, you can download the free Adobe Reader by simply clicking on the following link: http://www.adobe.com/products/acrobat/readstep2.html
