Beruflich Dokumente
Kultur Dokumente
DOI: 10.1007/s11010-006-9244-1
cgSpringer
2006
Abstract
Rutin, a polyphenolic flavonoid, was investigated for its antioxidant potential in streptozotocin (STZ)-induced diabetic rats.
Rats were rendered diabetic by a single intraperitoneal injection of streptozotocin (50 mg/kg). The levels of fasting plasma
glucose and insulin were estimated. Lipid peroxidative products and antioxidants were estimated in liver, kidney and brain.
Histopathological studies were carried out in these tissues. A significant (p < 0.05) increase in the levels of fasting plasma
glucose, lipid peroxidative products (thiobarbituric acid reactive substances [TBARS] and lipid hydroperoxides [HP]) and a
significant (p < 0.05) decrease in plasma insulin, enzymic antioxidants (superoxide dismutase [SOD], catalase, glutathione
peroxidase [GPx] and glutathione reductase [GRx]) and nonenzymic antioxidants (reduced glutathione [GSH], vitamin C and E)
in diabetic liver, kidney and brain were observed. Oral administration of rutin (100 mg/kg) for a period of 45 days significantly
(p < 0.05) decreased fasting plasma glucose, increased insulin levels and improved the antioxidant status of diabetic rats by
decreasing lipid peroxidative products and increasing enzymic and nonenzymic antioxidants. Normal rats treated with rutin (100
mg/kg) showed no significant (p < 0.05) effect on any of the parameters studied. Histopathological studies of the liver, kidney
and brain showed the protective role of rutin. Thus, our study clearly shows that rutin has antioxidant effect in STZ-induced
experimental diabetes. (Mol Cell Biochem 293: 211219, 2006)
Key words: rutin, diabetes mellitus, streptozotocin, lipid peroxidation, antioxidants
Introduction
Diabetes mellitus is a serious health problem affecting millions of individuals worldwide. By the year 2025, the World
Health Organization (WHO) predicts that 300 million people
will have diabetes mellitus [1]. Treatment of diabetes mellitus and its complications in the recent context have focused
on the usage of plant extracts and their constituents. Much
interest has grown in the role and usage of natural antioxidants as a means to prevent oxidative damage in diabetes with
high oxidative stress. The WHO had estimated that
= 80% of
the earths inhabitants rely on traditional medicine for their
primary health care needs, and most of this therapy involves
the use of plant extracts or their active components [2].
Address for offprints: P. Stanely Mainzen Prince, Senior Lecturer, Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar-608
002, Tamilnadu, India (E-mail: ps mainzenprince@yahoo.co.in)
212
as glycosylated and sulfated derivatives [8]. Flavonoid glycosides are much more readily absorbed by humans than the
aglycones [9].
Quercetin (3,3 ,4 ,5,7-pentahydroxy flavone) is one of the
most common native flavonoids occurring mainly in glycosidic forms such as rutin (5,7,3 ,4 -OH, 3-rutinose) [10].
Quercetin and rutin are the flavonoids most abundantly consumed in foods [11]. In Western diets, the richest sources
of quercetin glycosides are onions (347 mg/kg), apples
(36 mg/kg), tea (20 mg/kg) and red wine (11 mg/kg) [12].
Quercetin and rutin are widely used in many countries as vasoprotectants and are ingredients of numerous multivitamin
preparations and herbal remedies [13].
A wide variety of pharmacological activities of rutin were
reported- antitumor [14], anti-inflammatory [15], antidiarrhoeal [16], antimutagenic [17], myocardial protecting [18],
immunomodulator [19] and hepatoprotective activities [20].
A study by Gao et al. [21] showed that rutin increased
the antioxidant status in the kidney of normal mouse liver.
Nagasawa et al. [22] have shown that, rutin and a rutin analogue exhibited significant antioxidant activity in a liposomal
model reaction. Nagasawa et al. [23] have also shown that,
0.2% of G-rutin (a rutin-glucose derivative) supplemented in
20% casein diet for a period of one month to STZ-induced
diabetic rats decreased kidney thiobarbituric acid reactive
substances (TBARS). Previously, we have reported in STZinduced diabetic rats, rutin decreased plasma glucose, glycosylated hemoglobin, TBARS, lipid hydroperoxies (HP) and
increased the levels of insulin, C-peptide, hemoglobin, total
proteins, reduced glutathione, vitamin C, vitamin E and ceruloplasmin [24]. The present study was designed to evaluate
the role of rutin on lipid peroxidation and antioxidant status
in the diabetic liver, kidney and brain. In addition, the effect
of rutin on the histopathological alterations of these tissues
in normal and diabetic rats were also studied.
glucose, 1.8% vitamins and 56.17% carbohydrates. It provided a metabolisable energy of 3600 kcal. They were maintained in a controlled environment (12 h/12 h light/dark cycle)
and temperature (30 2 C). The animals were acclimatized
to the laboratory conditions for one week before starting the
experiment.
Chemicals
Rutin hydrate and STZ were purchased from Sigma Chemical Co., St. Louis, MO, USA. Carboxymethyl cellulose sodium salt, phosphotungstic acid, thiobarbituric acid,
1,1 ,3,3 -tetramethoxy propane, butylated hydroxy toluene,
xylenol orange, 2,2 -dinitro-5,5 -dithiodibenzoic acid, ascorbic acid, 2,2 -dipyridyl, p-phenylene diamine hydrochloride
and sodium azide were obtained from S.D. Fine Chemicals,
Mumbai, India. All the other chemicals used in the present
study are of analytical grade.
Animals
The experimental protocol was approved by the Animal
Ethical Committee of Annamalai University (Reg. No.
160/99/CPCSEA; vide No. 169, 2003). Eight week old male
albino Wistar rats (150180 g) obtained from the Central Animal House, Department of Experimental Medicine, Rajah
Muthiah Medical College and Hospital, Annamalai University were used in this study. The animals were fed on a standard pellet diet (Pranav Agro Industries, Pune, India) and water was freely available. The pellet diet consisted of 22.02%
crude protein, 4.25% crude oil, 3.02% crude fibre, 7.5% ash,
1.38% sand silica, 0.8% calcium, 0.6% phosphorus, 2.46%
Experimental design
Previously, a pilot study was conducted with three doses of
rutin (25, 50 and 100 mg/kg body weight) to determine the
dose dependent effect in STZ-induced diabetic rats. We found
that 25 mg/kg, 50 mg/kg and 100 mg/kg of rutin significantly
(p < 0.05) decreased plasma glucose levels and rutin at a dose
of 100 mg/kg was more effective in reducing plasma glucose
levels significantly (p < 0.05) after 45 days of experimental
study. Hence, we have chosen the dose 100 mg/kg of rutin
for further studies [24].
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To investigate the antioxidant effect of rutin, a total of 32
rats divided into four groups were maintained. Each group
contained 8 rats. Group I, normal control; Group II, normal +
rutin (100 mg/kg) [24]; Group III, diabetic control; Group IV,
diabetic + rutin (100 mg/kg) [24]. Rutin was suspended in
carboxymethyl cellulose (CMC) (0.01 g/ml) and was orally
administered to rats using an intragastric tube for a period
of 45 days. Normal control and diabetic control rats received
CMC alone.
After the last treatment (45 days), rats were fasted
overnight and sacrificed by cervical decapitation. Blood was
collected and plasma was obtained after centrifugation. It
was used for the estimation of glucose and insulin. Liver,
kidney and brain tissues were excised immediately from the
rats and stored in ice-cold containers. They were then homogenized with appropriate buffer, centrifuged at low speed
(3000 rpm/min), and the supernatant was collected. Biochemical estimations were carried out in the homogenates on the
same day of sacrifice.
Biochemical analysis
Plasma glucose was estimated using a commercial kit as
stated above. Plasma insulin was assayed by an enzyme
linked immunosorbent assay (ELISA) method using a commercial kit (Catalog No. SP-401) from United Biotech Inc.,
Mountain View, CA, USA. Thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (HP) were estimated by the methods of Fraga et al. [26] and Jiang et al.
[27] respectively. Reduced glutathione (GSH) [28], vitamin
C [29] and vitamin E [30] were also estimated in the tissues. Further, the activities of antioxidant enzymes such as
superoxide dismutase (SOD) [31], catalase [32], glutathione
peroxidase (GPx) [33] and glutathione reductase (GRx) [34]
were assayed.
Histopathological studies
For histopathological studies, rats from control and experimental groups were perfused with 10% neutral formalin solution. Liver, kidney and brain were removed immediately
from the rats, paraffin sections of 5 m thickness were made
and stained by hematoxylin-eosin (H&E) stain. After staining, the sections were observed under light microscope and
photographs were taken.
Statistical analysis
All the grouped data were analysed by one way analysis of
variance (ANOVA) followed by Duncans multiple range
Table 1. Effect of rutin on fasting plasma glucose and insulin levels in normal
and diabetic rats
Groups
Plasma glucose
(mmol/L)
Plasma insulin
(U/ml)
Normal control
Normal + rutin (100 mg/kg)
Diabetic control
Diabetic + rutin (100 mg/kg)
3.83 0.29a
3.89 0.30a
21.17 1.62b
7.89 0.60a
13.67 1.04a
13.74 1.05a
6.89 0.22b
10.92 0.48c
Each value is mean s.d. for 8 rats in each group. Values that have
a different superscript letter (a,b,c) differ significantly with each other
(p < 0.05, DMRT).
Results
Effect of rutin on fasting plasma glucose and insulin levels
Fasting plasma glucose levels were significantly (p < 0.05)
increased and plasma insulin levels significantly decreased
(p < 0.05) in diabetic control rats. Diabetic rats when treated
with rutin significantly (p < 0.05) decreased the plasma glucose levels and brought the levels to normal. The plasma
insulin levels were significantly (p < 0.05) increased in diabetic rats treated with rutin (Table 1).
Effect of rutin on TBARS and HP
The concentration of TBARS and HP in liver, kidney and
brain of normal and diabetic rats is given in Table 2. In diabetic rats, TBARS in liver, kidney and brain increased significantly (p < 0.05). A significant (p < 0.05) increase of
HP was also observed in diabetic liver, kidney and brain.
Treatment of diabetic rats with rutin significantly (p < 0.05)
decreased the concentration of TBARS and HP in these tissues and the concentration of HP was normalized in the
kidney.
Effect of rutin on nonenzymic antioxidants
The concentration of nonenzymic antioxidants (GSH, vitamin C and vitamin E) in liver and kidney of normal and diabetic groups is depicted in Table 3. In liver of diabetic rats,
the concentration of nonenzymic antioxidants were found to
be significantly (p < 0.05) decreased. Diabetic kidney also
exhibited significantly (p < 0.05) low concentration of GSH,
vitamin C and vitamin E. Diabetic rats when treated with
214
Table 2. Effect of rutin on TBARS and HP in liver, kidney and brain of normal and diabetic rats
TBARS (nmol/g wet tissue)
Groups
Liver
Kidney
Brain
Liver
Kidney
Brain
Normal control
Normal + rutin (100 mg/kg)
Diabetic control
Diabetic + rutin (100 mg/kg)
0.62 0.05a
0.61 0.05a
1.73 0.13b
0.81 0.06c
1.02 0.08a
1.01 0.07a
2.14 0.16b
1.20 0.09c
0.35 0.02a
0.35 0.02a
0.77 0.06b
0.49 0.05c
43.91 3.34a
43.80 3.32a
122.02 9.29b
64.01 4.87c
64.01 4.80a
63.93 4.85a
96.02 7.31b
70.01 5.33a
20.30 2.26a
20.11 2.02a
42.08 5.64b
29.52 2.87c
Each value is mean s.d. for 8 rats in each group. Values that have a different superscript letter (a,b,c) differ significantly with each other (p < 0.05,
DMRT).
Table 3. Effect of rutin on GSH in liver, kidney and brain and vitamin C and vitamin E in liver and kidney of normal and diabetic rats
GSH (mg/100 g tissue)
Groups
Liver
Kidney
Brain
Liver
Kidney
Liver
Kidney
Normal control
Normal + rutin (100 mg/kg)
Diabetic control
Diabetic + rutin (100 mg/kg)
34.11 2.37a
35.45 2.39a
20.00 1.52b
28.00 2.13c
24.00 1.98a
24.92 2.03a
14.60 0.88b
20.40 1.78c
28.55 2.12a
29.06 2.00a
13.66 1.47b
22.80 2.42c
1.38 0.10a
1.79 0.10a
0.92 0.07b
1.28 0.09c
1.20 0.08a
1.21 0.08a
0.82 0.06b
1.12 0.08a
0.69 0.05a
0.69 0.05a
0.44 0.04b
0.62 0.04c
0.55 0.04a
0.56 0.04a
0.36 0.03b
0.49 0.04c
Each value is mean s.d. for 8 rats in each group. Values that have a different superscript letter (a,b,c) differ significantly with each other (p < 0.05,
DMRT).
Discussion
Effect of rutin on plasma glucose and insulin
Administration of streptozotocin resulted in increased levels
of plasma glucose and decreased levels of insulin. Treatment
with rutin decreased plasma glucose and increased insulin
levels in diabetic rats. Rutin by its ability to scavenge free radicals and to inhibit lipid peroxidation, prevents STZ-induced
oxidative stress and protects -cells resulting in increased
insulin secretion and decreased plasma glucose levels [24].
Effect of rutin on lipid peroxidative products
Lipid peroxidation is a free-radical mediated propagation
of oxidative insult to polyunsaturated fatty acids (PUFA)
215
Table 4. Effect of rutin on the activities of SOD and catalase in liver, kidney and brain of normal and diabetic rats
SOD (Units/mg protein)
Groups
Liver
Kidney
Brain
Liver
Kidney
Brain
Normal control
Normal + rutin (100 mg/kg)
Diabetic control
Diabetic + rutin (100 mg/kg)
10.72 0.82a
11.02 0.82a
5.72 0.44b
9.14 0.70c
12.06 0.92a
12.68 0.93a
6.33 0.48b
10.50 0.83c
5.97 0.33a
6.03 0.40a
3.33 0.30b
5.02 0.48c
72.01 5.48a
72.66 5.53a
50.51 3.85b
66.33 5.05a
52.11 3.97a
53.01 4.02a
36.01 2.74b
47.31 3.60c
3.11 0.28a
3.18 0.20a
1.82 0.10b
2.24 0.23c
Each value is mean s.d. for 8 rats in each group. Values that have a different superscript letter (a,b,c) differ significantly with each other (p < 0.05,
DMRT).
SOD Units: enzyme concentration required to inhibit the OD at 560 nm of chromogen production by 50% in 1 min
Catalase Units:moles of H2 O2 consumed/min
Table 5. Effect of rutin on the activities of GPx and GRx in liver, kidney and brain of normal and diabetic rats
GPx (g of GSH consumed/min/mg protein)
Groups
Liver
Kidney
Brain
Liver
Kidney
Brain
Normal control
Normal + rutin (100 mg/kg)
Diabetic control
Diabetic + rutin (100 mg/kg)
11.00 0.84a
11.82 0.85a
5.80 0.44b
9.90 0.75c
7.46 0.57a
7.65 0.57a
4.00 0.23b
6.11 0.51c
7.22 0.43a
7.43 0.38a
3.64 0.20b
5.87 0.41c
25.60 1.90a
24.80 1.92a
18.00 0.91b
23.80 1.68c
22.00 1.68a
22.21 1.40a
16.00 0.76b
20.50 1.51c
3.18 0.21a
3.20 0.16a
2.24 0.18b
2.83 0.23c
Each value is mean s.d. for 8 rats in each group. Values that have a different superscript letter (a,b,c) differ significantly with each other (p < 0.05,
DMRT).
involving several types of free radicals, and termination occurs through enzymatic means or by free radical scavenging
by antioxidants [35]. Increased concentration of TBARS and
HP were noted in the tissues of diabetic rats. This increase was
well mitigated by the treatment of rutin. This could be due to
the ability of rutin to transfer electrons, free radicals, chelate
Fig. 2. Normal + rutin treated liver showing portal triad () and normal
hepatocytes (H&E 100x).
216
Fig. 3. Diabetic liver showing dilatation of hepatic sinusoids () and kupffer cell hyperplasia () (H&E 100x).
Potential causes of increased oxidative stress in diabetes mellitus include increased production of ROS by NADPH oxidase, decreased antioxidant enzyme activity and reduced
levels of glutathione, -tocopherol and ascorbate [39]. SOD
combats oxygen toxicity by catalytically reducing superoxide
radical anions to hydrogen peroxide, which, if not degraded
enzymatically, can be reduced in the presence of transition
metals to highly toxic hydroxyl radicals [40]. Catalase catalyzes the transformation of hydrogen peroxide within the
cell to harmless products, thereby curtailing the quantity of
cellular destruction inflicted by lipid peroxidation byproducts
[41]. Diminished activities of SOD and catalase in diabetic
217
Fig. 12. Diabetic + rutin treated diabetic brain showing near normal histology with reduction of oedema (H&E 100x).
Fig. 10. Normal + rutin treated brain showing normal histology (H&E
100x).
tissues might be linked to increased oxidative stress in diabetes accompanied by hyperglycemia [42].
Glutathione provides a first line of defence against ROS, as
it can scavenge free radicals and reduce H2 O2 . The decreased
concentration of GSH in liver, kidney and brain might be due
to NADPH depletion or GSH consumption in the removal of
peroxides [43]. GSH-dependent enzymes provide a second
line of defence as they primarily detoxify noxious byproducts
generated by ROS and also help to prevent propagation of free
radicals [44]. GPx serves to detoxify peroxides by reacting
them with GSH [45]. Low GPx activity in diabetic tissues
might be due to low GSH content, since GSH is a substrate
and cofactor of this enzyme [46]. In the process of catalyzing
H2 O2 to water, GPx converts GSH to GSSG, which is reduced
218
to GSH by GRx [40]. The activity of GRx was also decreased
in the diabetic tissues in this study.
Glutathione may contribute to antioxidant defence by networking with other major antioxidants such as vitamins E
and C. Vitamin E can transfer its phenolic hydrogen to a peroxyl free radical of a peroxidized PUFA, thereby breaking
the radical chain reaction and preventing the peroxidation of
PUFA in cellular and subcellular membrane phospholipids.
As a reducing agent, vitamin C reacts with a vitamin E radical to yield a vitamin C radical while regenerating vitamin E.
A vitamin C radical is converted back to vitamin C by GSH
[47]. These vitamins also directly scavenge ROS and upregulate the activities of antioxidant enzymes [47]. Treatment
with rutin increased the activities of enzymic antioxidants
and also the concentration of nonenzymic antioxidants in diabetic liver and kidney.
Conclusions
From the results obtained, it could be concluded that rutin improves the antioxidant status in the tissues of diabetic rats by
virtue of its antioxidant property, by possessing the structural
features of an antioxidant, the ability to sequester metal ions
and by forming metal ion chelates. Morphological assessments also show that the damage caused by streptozotocin to
the tissues was also markedly reduced by the administration
of rutin. Hence, rutin could be developed as an antidiabetic
drug for type 2 diabetes mellitus and its complications.
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