Indian Journal of Clinical Biochemistry, 2009 / 24 (4) 419-425
ATTENUATION OF OXIDATIVE STRESS IN STREPTOZOTOCIN-INDUCED DIABETIC RATS
BY EUCALYPTUS GLOBULUS
Alireza Nakhaee, Mohammad Bokaeian*, Mohsen Saravani, Ali Farhangi** and Azim Akbarzadeh**
Department of Biochemistry, Faculty of Medicine and *Department of Laboratory Sciences,
Faculty of Paramedical sciences, Zahedan University of Medical Sciences, Zahedan, Iran
**Pilot Biotechnology Department of Pasteur institute of Iran, Tehran, Iran
In traditional medicine, Eucalyptus globulus (eucalyptus) was used for the treatment of diabetes mellitus.
Hyperglycemia in diabetes has been associated with increased formation of reactive oxygen species (ROS)
and oxidative damage to tissue compounds. The aim of this study was to evaluate the effects of eucalyptus in
the diet (20 g/Kg) and drinking water (2.5 g/L) on lipid peroxidation, protein oxidation and antioxidant power in
plasma and liver homogenate, as well as glycated-Hb (HbA1C) of blood in streptozotocin-induced diabetic rats
for a period of 4 weeks. Diabetes induced in rats by a single intraperitoneal injection of streptozotocin (STZ, 65
mg/Kg). At the end of the treatment period, the level of plasma glucose, plasma and liver malondialdehyde
(MDA, the main product of lipid peroxidation), protein carbonyl (PC, one of the protein oxidation products) and
HbA1C increased and ferric reducing antioxidant power (FRAP) decreased in diabetic rats compared to normal
rats. Eucalyptus administration for 4 weeks caused a significant decrease in the plasma glucose levels, plasma
and liver MDA, PC and HbA1C, also a concomitant increase in the levels of FRAP in diabetic treated rats. In
conclusion, the present study showed that eucalyptus posses antioxidant activities. Eucalyptus probably restores
antioxidant power, due to the improved hyperglycemia in streptozotocin-induced diabetic rats.
Diabetes mellitus, Eucalyptus globulus, Ferric reducing antioxidant power, Malondialdehyde, Protein Carbonyl,
Address for Correspondence :
Dr. Azim Akbarzadeh
Pilot Biotechnology Department
Pasteur Institute of Iran
No. 358, 12 Farvardin Street, Jomhoori Avenue,
Tehran- Iran, 13169- 43551
Tel: + 98 21 6646 5406
Diabetes mellitus is a group of metabolic disorders of glucose
characterized by hyperglycemia resulting from defects in
insulin secretion, insulin action, or both (1). The prevalence of
diabetes for all age-groups worldwide was estimated to be
2.8% in 2000 and 4.4% in 2030. The total number of people
with diabetes is projected to rise from 171 million in 2000 to
366 million in 2030 (2). In spite of the introduction of
hypoglycemic drugs, diabetes and related complications
continue to be a major medical problem (3). The chronic
hyperglycemia was found to increase the production of free
radicals that is associated with long-term damage, dysfunction,
and failure of various organs, especially the eyes, kidneys,
nerves, heart, and blood vessels (4, 5). Several hypotheses
have been reported to explain the genesis of free radicals in
diabetes. These include oxidation of glucose, the non-
enzymatic and progressive glycation of proteins with
consequently increased formation of glucose-derived
advanced glycation end products (AGEs) (6, 7). Evidences
indicate that free radicals, membrane lipid peroxidation and
protein oxidation are significantly increased in diabetic patients
and in experimental diabetic animals (8-10). The increased
production and/or ineffective scavenging of reactive oxygen
Indian Journal of Clinical Biochemistry, 2009 / 24 (4)
species (ROS), resulting in tissue damage that in the most
instances is assessed by the measurement of lipid peroxides
and protein carbonyl (11, 12).
In recent years, there has been renewed interest in plant
medicine for the treatment of diseases such as diabetes (13,
14). Furthermore, evidences have indicated that various plants
including eucalyptus exert antidiabetic effects (15, 16).
Eucalyptus globulus (Myrtaceae) grows in wide range of
climatic conditions and is widely distributed throughout the
Sistan-Balouchestan province of Iran. Moreover, the leaves
of eucalyptus plant were traditionally used to treat diabetes
mellitus (17). Furthermore, recent studies in streptozotocin-
induced diabetic mice were confirmed the anti-hyperglycemic
efficacy of eucalyptus (17, 18). The antioxidant activity of
Eucalyptus globulus leaves has not been previously
investigated. Thus, the purpose of the present study was to
evaluate the effects of eucalyptus on oxidative stress in
streptozotocin-induced diabetic rats.
MATERIALS AND METHODS
Chemicals: TPTZ (2, 4, 6-tri (2-pyridyl)-1, 3, 5-triazine), TBA
(2-thiobarbituric acid: 4, 6- dihydroxy-2-mercaptopyrimidine),
n-butanol, sodium phosphate, sodium hydroxide, sodium
acetate, phosphoric acid, ferric chloride, ferrous sulfate, glacial
acetic acid, TCA (trichloroacetic acid), EDTA (ethylene diamine
tetra acetic acid), guanidine-HCl, DNPH (2,4-
dinitrophenylhydrazine), KCl, NaCl, digitonin, Coomasie blue
brilliant G250 and bovine serum albumin were purchased from
Merck. Streptozotocin and malondialdehyde standard
(malondialdehyde bis dimethyl acetate) were obtained from
Plant material: Fresh eucalyptus leaves were collected from
Mellat garden of Zahedan, Iran. The leaves were washed with
distilled water and dried in 45°C oven. Dried leaves were
crushed and then powdered in an electrical grinder. The
powder was stored at room temperatures (25 ± 2°C) until use.
For animal foods, plant material was prepared according to
method of Gray et al (16). Eucalyptus powder was incorporated
into powdered rat diet (20 g/Kg), thoroughly mixed, distilled
water added and mixed to a stiff paste. The mixture was then
pelleted and dried at 45°C. Control diet was prepared by the
same manner to ensure there was no difference in vitamin
and mineral content as a result of the drying process. Aqueous
extract of eucalyptus was prepared by 15 min decoction of
the powdered leaves at 25 g/L. In brief, 2.5 g powdered
material was placed in 100 ml distilled water, brought to boiling
point, then was released from boiling and allowed to infuse
for 15 min. This suspension was filtered by Whatman paper
No. 1 and the volume readjusted with distilled water to 100 ml
and 10 ml aliquots of the extract were stored at -20°C until
use when they were diluted 10-fold with tap water (2.5 g/L).
Preparation of animals: The study was performed on
matured normoglycemic male Wistar rats, weighing 200-220
g, which were separately housed in cages (one rat per cage).
Animals were maintained in a room at 23°C ± 2, humidity 45%
to 55% with a fixed 12 h artificial light period and allowed to
eat and drink adlib. Rats were fed with standard rodent diet
until initiation of treatment. All animals received humane care,
as outlined in the guide for the care and use of laboratory
Induction of diabetes in animals: Diabetes was induced by
a single intraperitoneal administration of streptozotocin (65
mg/Kg of body weight) in 0.15 M NaCl with 100 mM sodium
citrate buffer (pH 4.5). Control rats received the vehicle alone.
After 5 days of development of diabetes, the rats with plasma
glucose more than 200 mg/dl were considered as diabetic
rats and used for experiment.
Experimental design and treatment: Thirty rats were divided
in to three groups: (1) Non-diabetic control group: Rats of this
group (n=10) received normal saline containing 100mM
sodium citrate and fed with standard rodent diet and tap water
throughout study; (2) Diabetic group: Rats of this group (n=10)
received a single intraperitoneal administration of
streptozotocin and fed with standard rodent diet and tap water
throughout study; (3) Treated-diabetic group: rats of this group
(n=10) received a single intraperitoneal administration of
streptozotocin and fed standard rodent diet and tap water
supplemented with 2.5g /L aqueous extract of eucalyptus 5
day after administration of streptozotocin. The experiment was
carried out for 4 weeks after the initiation of treatment. Body
weight, food and fluid intake of all groups were measured at
the end of experiment period. At the end of the treatment period
overnight-fasted rats were anesthetized under light ether and
blood samples were collected from tip of the tail vein. Blood
sample was collected in EDTA for determination of HbA1C
and preparation of plasma.
Isolation of liver tissue: After blood collection, animals were
killed by cervical decapitation and livers removed and rinsed
of any adhering blood. Then, livers were quickly sliced, and
fragments were homogenated in appropriate buffers.
Liver homogenization for evaluation of oxidative stress:
For TBARS and FRAP assay, a fraction of liver was
homogenized (1:10, w/v) in cold 1.15% KCl and 0.05 M sodium
phosphate buffer pH 7.4, respectively with a Silent Crusher S
homogenizer (Heidolph homogenizer, Germany).
Homogenates were centrifuged at 6000g for 20 min at 4°C.
Tissue homogenates for protein carbonyl assay were prepared
by the method of Evans et al (19). Briefly, 200mg of tissue
was homogenized in 2ml of phosphate buffer containing 0.1%
digitonin. After 15 min, streptomycin sulfate was added to final
concentration of 1% to the homogenized tissue and mixture
was centrifuged at 2800g for 10 min at room temperature.
The resulting supernatants were used for biochemical assays.
Assays: For evaluation of hyperglycemia, glucose levels were
measured by glucose oxidase standard method. HbAlC was
estimated by a commercial Kit (BioSystem, Spain) according
to manufacturer method (20). Concentration of lipid
peroxidation product (MDA) or thiobarbituric acid reactive
substances (TBARS) was determined spectrophotometrically
by the method of Uchiyama and Mihara (21). 3 ml of 1%
phosphoric acid and 1ml 0.6% w/v thiobarbituric acid aqueous
solution was added to 0.5 ml of supernatant or plasma. The
mixture was heated for 45 min in a boiling water bath. After
cooling, 4 ml n-butanol was added, shaken and upper layer
was separated by centrifugation at 1000 xg for 10 min. The
light absorbance of the sample was determined at 535 nm
and TBARS concentration was calculated using MDA standard
curve. Concentration of thiobarbituric acid reactive substances
(TBARS) in plasma and tissue was expressed in nmol/ml and
nmol/mg of protein, respectively. Antioxidant power of plasma
and liver were measured by ferric reducing/antioxidant power
(FRAP). FRAP assay was performed according to the method
as described (22, 23). The method is based on the reduction
of the Fe3+-TPTZ (2, 4, 6-tri-(2-pyridyl)-s-triazin) complex to
the ferrous form at low pH. This reduction is monitored by
measuring the absorption change at 593 nm. Briefly, 3 mL of
working FRAP reagent (25 ml 0.3 M sodium acetate buffer,
pH 3.6; 2.5 ml 0.01 M TPTZ in 0.04 M HCl; 2.5 ml 0.02 M
FeCl3. 6H2O; preheated to 37°C) was mixed with 100 μL of
supernatant or plasma and the absorbance at 593 nm was
recorded after a 5 min incubation at 37°C. The absorption of
the blue FeII-complex was measured at 593 nm using a WPA
biowave II spectrophotometer. FeSO4. 7H2O solutions from
0.2 to 1 mM were used for calibration. FRAP value in plasma
and tissue was expressed as μmoles/L and μmoles/milligram
of protein, respectively. Protein concentration was determined
by Bradford’s method using bovine serum albumin as
Protein carbonyls were measured according to procedure
described by Reznick and Packer (25) using dinitro-
phenylhydrazine (DNPH) reagent and spectrophotometric
method. Four ml of 10 mM DNPH in 2.5 M HCl was added to
1 ml sample (tissue homogenate or 1/5 diluted plasma). In
other tube as blank, 4 ml 2.5 M HCl was added to 1 ml sample.
Tubes were incubated in room temperature and darkness for
1 hr and swirled every 15 min. Protein was precipitated with 5
ml of 20 % (w/v) trichloroacetic acid and the pellets were
washed once with 4 ml of 10 % (w/v) trichloroacetic acid and
three times with 4 ml of an ethanol/ethyl acetate mixture (1:1)
to remove free DNPH and lipid contaminants. Washings were
achieved by mechanical disruption of the pellets in the washing
solution using a small spatula and re-pelleting was done by
centrifugation at 6000 g for 5 min. Finally, the precipitates were
dissolved in 1 ml 6 M guanidine-HCl solution and the
absorbance was measured at 370 nm. The results were
expressed as nanomoles of carbonyl groups per milligram of
protein using a molar extinction coefficient of 22,000 M-1
cm-1. Protein contents were determined on the HCl blank
pellets using a bovine serum albumin standard curve in
guanidine-HCl and reading the absorbance at 280 nm.
Statistical data analysis: Results were expressed as
mean ± SE for ten rats in each experimental group. Statistical
analysis was performed using SPSS 11 software. One-way
analysis of variance (ANOVA) followed by turkey’s post hoc
test was used to compare differences between experimental
groups. The criterion for statistical significance considered
Table 1 shows body weight, food and fluid intake, plasma
glucose concentration and HbA1C levels in different
experimental groups. Diabetic rats showed significant (P<0.05)
weight loss, polyphagia, and polydipsia compared with control
Table 1: Effect of eucalyptus supplementation on food and fluid
intake, body weight, plasma glucose and %HbA1C of rats in
different experimental groups
Food intake (mg/day)23.4 ± 3.252.4 ± 5.6 *
27.7 ± 6.6 **
58.8± 8.65 **
280 ± 25.6**
117.7 ± 5.25 **
7.1 ± 0.85 **
Fluid intake (ml/day)50.4±7.5
298 ± 27.4 254± 20.5*
Body weight (g)
Plasma glucose (mg/dl)89.5 ± 2.6 268± 8.5*
12.8 ± 9.4 *
HbA1C (%)6.4 ± 0.54
The values represent the mean ±SE for ten rats per group. Comparisons
were made by one-way ANOVA test. * P<0.01 compared to control group;
** P<0.01 compared to diabetic group.
Attenuation of Oxidative Stress by Eucalyptus globulus
Indian Journal of Clinical Biochemistry, 2009 / 24 (4)
at the end of the treatment period. Administration of eucalyptus
significantly (P<0.01) reduced the body weight loss, polyphagia
and polydipsia in treated-diabetic rats compared with
untreated-diabetic rats. Plasma glucose concentration and
HbA1C level in diabetic rats receiving eucalyptus were
significantly lower (P<0.01) than untreated-diabetic rats and
were not different from those of control animals.
Figure 1 shows concentration of TBARS in plasma and liver
of control and diabetic animals. Diabetes caused a significant
increase in TBARS concentration in plasma and liver
compared to the corresponding treated-diabetic rats
(P<0.001). Treatment with eucalyptus ameliorated lipid
peroxidation. The changes in antioxidant power (FRAP) were
shown in Figure 2. FRAP was found to be lower (P<0.001) in
plasma and liver of diabetic rats compared to control ones.
Antioxidant power was significantly decreased in treated-
diabetic rats (P<0.001). Treatment with eucalyptus improved
Protein carbonyl concentration in plasma and liver of all animal
groups are indicated in Figure 3. The level of PC was increased
in untreated-diabetic rats compared to control ones (P<0.01).
The results shown in Figure 3 indicate that eucalyptus
treatment significantly reduced protein carbonyl concentration
in treated-diabetic group (P<0.01).
Streptozotocin (STZ, 2-deoxy-2-(3-methyl-3-nitrosoureldo)-D-
glucopyranose), is synthesized by Streptoinycetes
achroniogenes and has long been used to generate animal
models of diabetes (26). Streptozotocin-induced diabetes
animals exhibit most of the diabetic complication (27). During
diabetes, advanced glycation end products form when glucose
reacts with various proteins such as hemoglobin, albumin,
collagen, LDL, or crystalline proteins to form labile Schiff bases,
which then undergo further modification to form Amadori
products (28, 29). Additional rearrangements or modifications
Fig 2: FRAP level in plasma and liver of rats in different experimental
groups. The values represent the mean ±SE for ten rats per group.
Statistical analysis was performed by One-way ANOVA test (*P<0.001
compared to control group; ** P<0.001 compared to diabetic group)
Fig 3: Protein carbonyl (PC) level in plasma and liver of rats in
different experimental groups. The values represent the mean ± SE
for ten rats per group. Statistical analysis was performed by One-
way ANOVA test (*P<0.01 compared to control group; **P<0.01
compared to diabetic group)
Fig 1: TBARS level in plasma and liver of rats in different
experimental groups. The values represent the mean ±SE for ten
rats per group. Statistical analysis was performed by One-way
ANOVA test (*P<0.001 compared to control group; **P<0.001
compared to diabetic group)
may give rise to advanced glycation end products seen in
prolonged hyperglycemia. A common modification is generated
by oxidative cleavage of Amadori intermediates to form epsilon-
(carboxymethyl) lysine structures (30). The rate of glycation
is proportional to the blood glucose concentration (31).
Glycated hemoglobin (HbA1C) was found to increase in the
patients with uncontrolled or poorly controlled diabetes mellitus
and amount of increase is directly proportional to the
hyperglycemic state (32). Evidence showed that glycation itself
may induce the formation of oxygen-derived free radicals in
diabetic condition, and the level of HbA1C is considered as
one of the markers of degree of oxidative stress in diabetes
mellitus (33). Therefore, the measurement of HbA1C is
supposed to be a very sensitive index for glycemic control
(34). In the present investigation, diabetic animals showed
higher levels of HbA1C, compared with control rats. Treatment
with eucalyptus reduced significantly (P<0.01) levels of HbA1C,
in treated-diabetic rats that could be due to an improvement
in hyperglycemia. On the other hand, streptozotocin-induced
diabetes is characterized by loss in body weight, polyphagia
and polydipsia and these were also seen in our study. The
decrease in body weight observed in uncontrolled diabetic
might be the result of protein wasting due to unavailability of
carbohydrate for utilization as an energy source. Body weight
enhanced significantly (P<0.01) in eucalyptus treated diabetic
rats when compared with untreated-diabetic ones. Likewise,
eucalyptus decreased significantly (P<0.01) polyphagia and
polydipsia in treated-diabetic rats when compared with diabetic
Chronic hyperglycemia induces carbonyl stress, which in turn
can lead to increased lipid peroxidation (35). The increased
concentration of lipid peroxidation induces oxidative damage
by increasing peroxy radicals and hydroxyl radicals (36). Thus,
lipid peroxidation is one of the characteristics features of
chronic uncontrolled diabetes. The most commonly used
indicator of lipid peroxidation is TBARS (37). The increased
lipid peroxidation in the plasma and tissues of diabetic animals
may be due to the observed remarkable increase in the
concentration of TBARS and MDA as a main product of lipid
peroxidation in the plasma and liver (38). In the present study,
significant (P<0.001) elevations of TBARS levels were
observed in the plasma and liver homogenate of diabetic rats
compared to the corresponding control rats. Administration of
eucalyptus decreased significantly (P<0.001) TBARS in treated
diabetic when compared with diabetic rats. Several studies
have been shown that some constitutes isolated from
Eucalyptus globulus significantly reduces free radicals and
inhibited lipid peroxidation (39, 40). Oxidative stress in diabetes
coexists with a reduction in the antioxidant power (41). Cakatay
et al also indicated that total antioxidant capacity (FRAP) levels
in plasma of chronic diabetic animals were decreased
significantly as compared to those of control animals (42).
The present work, indicated the same results and showed
significant (P<0.001) reduction in plasma and liver
homogenate FRAP of diabetic rats when compared with control
animals. Treatment with eucalyptus improved significantly
(P<0.001) antioxidant power in treated-diabetic rats when
compared with untreated-diabetic group. Elevated protein
carbonyl levels have been detected in diabetes (10, 43). High
plasma PC levels in diabetic children and adolescents without
complications compared with control subjects indicate that
oxidative protein damage occurs at the onset of disease and
tends to increase in the later stages. Furthermore, decreased
antioxidant defenses might increase the susceptibility of
diabetic patients to oxidative injury (44). The results of the
present study also showed that tissue and plasma PC levels
were increased in untreated diabetic rats compared to controls
(P<0.01) and Eucalyptus causes improvement of protein
In conclusion, the results of this study suggest that Eucalyptus
globulus possess antidiabetic and antioxidant activity. Previous
studies have shown antihyperglycemic and improving effects
of eucalyptus on loss of body weight and polydipsia in
streptozotocin-induced diabetic animals (17, 18). Also, data
of this study indicates that eucalyptus can either increase
antioxidant power or reduce the oxidative stress or both.
Eucalyptus globulus probably improved oxidative stress, due
to reduction in plasma glucose level in diabetic rats, which
prevents excessive production of free radicals through
glycation of the proteins.
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