Fd Chem, 2012

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Dose-dependent effect in the inhibition of oxidative stress and anticlastogenic
potential of ginger in STZ induced diabetic rats
Nirmala Kota ⇑
, Virendra Vasant Panpatil, Rajakumar Kaleb, Bhaskar Varanasi, Kalpagam Polasa
Food and Drug Toxicology Research Centre, National Institute of Nutrition (ICMR), PO – Jamai Osmania, Hyderabad 500 007, India
a r t i c l e i n f o
Article history:
Received 1 March 2012
Received in revised form 29 May 2012
Accepted 26 June 2012
Available online 14 July 2012
Keywords:
Ginger
Antioxidant enzymes
Genotoxicity
Dose–response
a b s t r a c t
Ginger is an important medicinal herb has numerous bioactive components and is used in the manage-
ment, control and/or treatment of diseases including diabetes mellitus. The present study was under-
taken to see the dose–response effect of ginger and evaluate the possible protective effects of dietary
ginger on oxidative stress and genotoxicity induced by streptozotocin (STZ) diabetic rats. Inbred male
Wistar/NIN rats of 8–9 weeks old were treated with 30 mg/kg of STZ. Rats were divided into different
groups of control, diabetic non-treated, and diabetic treated with ginger powder at 0.5%, 1% and 5%
respectively. After feeding for a month, blood and tissues were collected to see the effect of ginger on
antioxidant status, DNA damage and bone marrow genotoxicity. In this study ginger exerted a protective
effect against STZ-induced diabetes by modulating antioxidant enzymes and glutathione and down reg-
ulating lipid and protein oxidation and inhibition in genotoxicity in a dose–response manner.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Plant phenols play an important role in modifying the antioxi-
dant status and significantly reduce the oxidative state. Cancer
chemopreventive potential of naturally occurring phytochemicals
in spices possesses antioxidant, antimutagenic and anticarcino-
genic properties (Vinda-Martos, Riuz-Navajas, Fernandez- Lopez,
& Perez-Alvarez, 2011). Oxidative damage is involved in the path-
ogenesis of cancer, diabetes, CVD, cataract, infection, inflammation
and other diseases. Since diabetes is known for its complications
like nephropathy, retinopathy, prevention and control of complica-
tions associated with it is one of the important factors in the man-
agement of diabetes. Chemoprotection by diet derived
antioxidants has emerged as a cost effective approach in prevent-
ing genotoxicity and carcinogenicity. As a part of the dietary treat-
ment of diabetes, there has been continuous search for novel
antidiabetic drugs from plant sources (Srinivasan, 2005).
Ginger is well known as an important medicinal herb and is also
a component of human diet containing some important com-
pounds like gingerols, shogaols and paradols (Ali et al., 2008) have
been found to possess potential chemopreventive activities. Anti-
oxidants play an important role to protect against damage by reac-
tive oxygen species. The suppression of lipid peroxidation and
oxidative damage in rats was observed when they were given gin-
ger which is an indication that ginger has antioxidant effect in vivo
which could be related to the prevention of carcinogenesis (Ippou-
shi, Takenchi, Ito, Horie, & Azuma, 2007). Earlier in vivo studies on
rats showed that ginger feeding to rats improved the antioxidant
status (Nirmala, Prasanna, & Polasa, 2008) and by virtue of its anti-
oxidant property, also influenced the xenobiotic metabolism by
inducing drug metabolizing enzymes such as glutathione-s-trans-
ferase, quinone reductase and glutathione peroxidase (Nirmala
et al., 2010). The present study was undertaken to evaluate the
possible protective effects of dietary ginger under oxidative stress
using streptozotocin (STZ) induced diabetic rats.
2. Materials and methods
Male Wistar NIN (WNIN) rats were obtained from the National
Centre for Laboratory Animal Science (NCLAS) and housed in the
animal facility where the temperature was maintained at 24–
25 °C with 12-h dark/light cycle. The experimental protocol was
approved by the Institutional Animal Ethics Committee (IAEC) un-
der Committee for Purpose of Control and Supervision on Experi-
ments on Animals (CPCSEA), Ministry of Environment and
Forests, Government of India. Ginger powder of standard grade
(AGMARK) was procured from local market, Hyderabad, India.
2.1. Study design
Inbred male WNIN rats of 8–9 weeks old and weighing 175 g
were randomly divided into four groups of 12 rats per group and
were given standard laboratory diet containing ginger powder at
0.5%, 1% and 5% and a normal group (control) without ginger.
The standard diet contained wheat flour 15%, roasted Bengal gram
0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.06.116
⇑ Corresponding author. Tel.: +91 40 27197329; fax: +91 40 27019074.
E-mail addresses: knimy7@yahoo.com, nirmala.nin@gmail.com (N. Kota).
Food Chemistry 135 (2012) 2954–2959
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

flour 58%, groundnut flour 10%, skimmed milk powder 5%, casein
4%, refined oil 4%, salt mixture 4% and vitamin mixture 0.2%. The
rats had free access to food and drinking water. After 1 month of
feeding, half the number of animals from each group was given
intraperitoneal administration of a single dose of 30 mg/kg of STZ
dissolved in 0.1 M sodium citrate buffer after an overnight fasting
.The control group was given equal volume of citrate buffer. The
animals were monitored for 4 weeks after the administration of
STZ by measuring the blood glucose levels; above 200 mg/dl they
were considered as diabetic. Following the induction of diabetes
after 4 weeks, blood samples of overnight fasted rats were col-
lected from retro-orbital venous plexus using heparinised glass
capillary. A small aliquot of blood was used for comet assay. The
plasma was separated for the analysis of biochemical parameters
such as total cholesterol, triglycerides and high density lipoprotein
using respective diagnostic kits procured from Biosystems (Spain).
Animals were sacrificed by euthanisation and tissues such as liver
and kidney were dissected, rinsed with normal saline and were fro-
zen immediately in liquid nitrogen and stored at À80 °C for further
use. Body weights were taken and blood glucose levels were deter-
mined before and after the experiment. The tissues were minced
and a 20% homogenate was prepared in cold buffer using a poly-
tron homogenizer (kinematica) and processed as per the standard
procedures for estimation of antioxidant enzymes and other anti-
oxidant parameters. Femur bones were collected for the analysis
by rodent bone marrow micronucleus test.
2.2. Measurement of antioxidant parameters
Superoxide dismutase (SOD), catalase, glutathione peroxidase
(GSHPx), malonaldehyde (MDA) and protein carbonyl levels were
estimated as described in our earlier study (Nirmala et al., 2008).
2.3. Reduced glutathione(GSH)
GSH was estimated according to the method of Hissin and Hilf
(1976). GSH reacts with a fluorescent reagent orthophthalaldehyde
(OPT) to yield a fluorescent complex at pH 8. The fluorescence of
the complex formed was measured in a spectrofluorimeter at exci-
tation and emission wavelength of 350 and 420 nm, respectively. A
portion of liver tissue was homogenized in phosphate (0.1 M) EDTA
(0.005 M) buffer and 1 ml of 25% phosphoric acid and the homog-
enate was centrifuged in a refrigerated centrifuge at 25,000g. An
aliquot of 100 ll of the diluted supernatant was taken for the assay
and was mixed with phosphate EDTA buffer and OPT (100 ll of
1 mg/ml) and the fluorescence was measured. Glutathione stan-
dards were also run simultaneously and the concentration of
GSH in the samples was read from the graph. Values were ex-
pressed as lg/g liver.
The protein was estimated by the method of Lowry, Rosen-
brongh, Farr, and Rendall (1951).
2.4. Genotoxicity assays
2.4.1. Comet assay
The use of Comet assay which is relatively simple and rapid
method to examine DNA damage and repair is an important bio-
marker for the study of the effects of nutrition and cancer. This as-
say was taken up using the method of Singh, McCoy, Tice, and
Schneider (1988). Briefly, 40 ll of whole blood was minced with
0.5% of low melting point agarose (LMP) and placed on a frosted
microscope slide that has already been prelayered with 1% normal
melting point agarose (NMP). After cooling, the slides were covered
with a third layer of LMP. Later they were immersed in lysing solu-
tion (1% Sodium sarcosinate, 2.5 M NaCl, 100 mM sodium EDTA,
10 mM Tris–HCl of pH-10 and 1% Triton X-100) for 1 h at 4 °C.
The slides were then kept in alkaline electrophoresis buffer for
20 min at 25 mV and 300 mA. The slides were then rinsed with
0.4 M Tris (pH-7.4) and finally with 70% and 100% ethanol respec-
tively for 2 min. They were allowed to dry and then stained with
ethidium bromide. Fluorescent microscope (Leica) that was
equipped with an excitation filter of 516–560 nm and a barrier fil-
ter of 590 nm was used to examine the slides. The extent of DNA
damage was quantified by measuring the width of the head and
length of the tail of the Comet images using visual scoring system
with an ocular micrometre. About 50 cells per slide were counted
in duplicates.
2.4.2. Rodent bone marrow micronucleus test
The most commonly examined target organ is the rodent bone
marrow and the micronucleus is sensitive to many aneuploidy
inducing agents and is one of the most widely used short term as-
say for identification of genotoxic effects associated with mutagens
and carcinogens (Hayes, Doherty, Adkins, Oldman, & O’Donovan,
2009).
The femur bones were cut at the ends and the contents of bone
marrow was gently flushed out in a beaker containing foetal calf
serum and made a fine colloid with a syringe and centrifuged at
800 rpm for 5 min. The supernatant was removed and the sedi-
ment was overlayered with two drops of calf serum. The cell sus-
pension was smeared on glass slide and air dried. They were
stained successively with May-Gruenwald and Giemsa stain for
detecting micronucleated polychromatic erythrocytes (MNPCEs)
(Salamone & Mavourin, 2005 and Celikler et al., 2009). The normo-
chromatic erythrocytes (NCEs) were also scored and the frequency
of PCE among the first 200 NCEs were counted using Leica micro-
scope with plain objective at 100Â/1.25 oil mount magnification,
to calculate the PCE/NCE ratio. The % reduction in the frequency
of MNPCEs was also calculated.
2.4.3. Statistical analysis
The Statistical Package for Social Sciences (SPSS) windows ver-
sion 15.0 was used for the analysis of the data. Mean and SD values
were calculated for all the variables and the mean values were
compared by oneway ANOVA with post hoc test of Least Significant
Differences (LSD) among groups. Non-parametric tests of Kruskal–
Wallis Wilcoxn signed rank test was performed whenever the
assumptions of parametric tests varied. For the analysis of micro-
nuclei, 2000 PCEs were scored to calculate the MN frequencies
and 200 NCEs were examined to determine the ratio of PCE to
NCE. Differences in the incidence of MNPCE per group and of PCE
per 2000 erythrocytes (PCE + NCE) were compared between nor-
mal, diabetic and diabetic + ginger fed groups using the Mann–
Whitney U-test (two-tailed).
3. Results
3.1. Effect of ginger on body weights and glucose levels
Body weights, blood glucose levels and serum lipid profiles
were monitored weekly in all the groups before the commence-
ment and till the end of the experiment. Prior to STZ administra-
tion, the fasting blood glucose levels did not differ between the
normal and diabetic group. One week after the administration of
STZ, glucose levels were significantly higher in STZ treated groups
and remained elevated over a period of 4 weeks. The control group,
treated with citrate buffer, maintained a normal blood glucose le-
vel throughout the experimental period. The results revealed a sig-
nificant increase in serum triglycerides and total cholesterol in
diabetic rats compared to the normal control rats.
N. Kota et al. / Food Chemistry 135 (2012) 2954–2959 2955

The body weights of the rats at the beginning of the study were
similar in all groups. At the end of experiment, diabetic rats and
ginger fed diabetic rats showed a significant reduction in the body
weights compared to the normal control and ginger fed non-dia-
betic rats (p < 0.01). However, the weight loss in ginger fed diabetic
rats was lower than the diabetic rats and the increase in body
weight was comparable to the increase in body weight of normal
controls (Table 1). There was a slight trend in the increase of body
weight within the diabetic groups compared to its control but was
not significant.
A dose dependent decrease in glucose levels was observed in
ginger fed diabetic rats compared to the diabetic control. Signifi-
cant decrease was seen within the treatment groups in diabetic
rats between diabetic control + 1% ginger (p < 0.05) and between
diabetic control + 5% ginger (p < 0.01) (Table 2).
3.2. Lipid profile
There were no significant differences in the serum cholesterol
and HDL levels in non-diabetic rats fed with ginger compared to
the normal control. The serum cholesterol level of the control dia-
betic rats increased compared to the basal values. However, in gin-
ger fed diabetic rats a significant decrease was seen compared to
the diabetic control and the maximum reduction was at 5% level
(p < 0.05).The serum triglyceride levels also showed a significant
decrease in their levels with the maximum reduction being at 1%
(p < 0.05) and at 5% (p < 0.01) in the diabetic groups compared to
its diabetic control. HDL was found to be lower in diabetic groups
compared to the normal control but there was an increase in HDL
at 5% level (p < 0.05) in the diabetic groups. Dose dependent reduc-
tion was observed in the level of triglycerides and cholesterol in
the diabetic rats fed with ginger (Table 3).
3.3. Antioxidant parameters
A dose–response increase in the activity of SOD, catalase and
GSHPx was observed in liver of rats fed with ginger at 0.5%, 1%
and 5% levels (Table 4). A significant increase in liver SOD activity
was seen in both non-diabetic (p < 0.01 at all the levels) and dia-
betic groups (p < 0.01 at 5% level) compared to their respective
controls. Stimulation in catalase activity was also observed in both
non-diabetic (p < 0.01 at 1% and 5%) and diabetic (p < 0.01 at 5%)
against their respective controls. A significant increase in GSHPx
activity was observed in non-diabetic (p < 0.05 at 1% and p < 0.01
at 5% level) and diabetic groups (p < 0.01 at 5%) compared to their
respective controls. There was an increase in the GSH content in
non-diabetic groups, but was not significant. In diabetic groups a
significant increase was seen at 1% and 5% levels (p < 0.01) com-
pared to the diabetic control.
A dose-related effect was seen in the inhibition of MDA levels in
liver homogenates of rats fed with ginger at 0.5%, 1% and 5%,
respectively, in both non-diabetic (p < 0.05) and diabetic groups
(p < 0.01) compared to their respective control groups. An inhibi-
tion of 30%, 54% and 59% was seen in the non-diabetic rats and a
7%, 33% and 46% inhibition in the diabetic rats was observed (Ta-
ble 4). A reduction in the carbonyl levels was also observed in liver
cytosol at all the levels of ginger feeding in both non-diabetic and
diabetic rats compared to their respective controls, and a dose–re-
sponse relation was seen. An inhibition of 18%, 27% and 40% in the
non-diabetic groups and 8%, 19% and 29% in the diabetic groups
was observed (Table 4).
There was an increase in the SOD activity in the kidney of both
the non-diabetic (p < 0.01 at 1% and 5%) and the diabetic groups fed
with ginger (p < 0.01 at 5%) compared to their respective controls
The levels of MDA formed in kidney homogenates was also
Table 1
Initial and final body weights of normal and STZ-induced diabetic rats.
Groups Initial Final
Normal STZ induced Normal STZ induced
Control 175.8 ± 23.99 175.0 ± 24.29 280.3 ± 24.09 223.3 ± 19.18**
0.5%
Ginger
177.2 ± 32.13 175.1 ± 31.05 278.5 ± 27.65 225.6 ± 23.89*
1% Ginger 176.5 ± 33.71 174.5 ± 30.82 282.8 ± 22.34 234.1 ± 25.03*
5% Ginger 175.7 ± 25.98 175.6 ± 23.09 288.2 ± 28.79 237.8 ± 31.61*
All values are mean ± SD.
*
p < 0.01, Significant differences in the final body weights between non-diabetic
and diabetic.
**
p < 0.001, Significant differences in the final body weights between non-diabetic
and diabetic.
Table 2
Serum glucose levels (mg/dl) in normal and STZ-induced diabetic rats fed with ginger.
Groups Basal After 72 h After 4 weeks
Normal
Control 81.7 ± 7.41 90.1 ± 18.32 93.6 ± 16.86
0.5% Ginger 82.3 ± 6.31 83.3 ± 6.19 87.4 ± 2.23
1.0% Ginger 87.1 ± 12.31 86.5 ± 3.10 92.8 ± 13.13
5% Ginger 85.8 ± 2.17 87.9 ± 2.12 87.7 ± 2.20
STZ induced
Control 83.0 ± 3.10 243.6 ± 30.25 222.9 ± 28.99
0.5% Ginger 84.7 ± 5.75 227.9 ± 22.44 208.0 ± 31.38
1.0% Ginger 86.3 ± 9.09 218.0 ± 32.27 185.8 ± 29.52*
5% Ginger 81.4 ± 4.72 222.7 ± 11.63 169.8 ± 12.42**
All values are mean ± SD of 6 rats/group.
*
p < 0.05, Diabetic control Vs 1% G + STZ.
**
p < 0.01, Diabetic control Vs 5% G + STZ
Table 3
Serum cholesterol, triglycerides and HDL levels (mg/dl) in normal and STZ-induced diabetic rats fed with ginger.
Groups Cholesterol (mg/dl) Triglycerides (mg/dl) HDL-Cholesterol (mg/dl)
Initial Final Initial Final Initial Final
Normal
Control 78.9 ± 7.91 94.1 ± 30.12 114.8 ± 15.89 126.5 ± 13.77 71.5 ± 8.55 68.6 ± 10.21
0.5% Ginger 81.5 ± 8.78 88.3 ± 9.60 113.5 ± 14.33 122.1 ± 9.37 66.7 ± 9.48 70.8 ± 9.12
1% Ginger 80.7 ± 10.14 85.1 ± 10.02 109.5 ± 14.73 120.5 ± 17.94 69.3 ± 10.59 71.1 ± 9.55
5% Ginger 80.3 ± 19.13 82.3 ± 9.42 113.9 ± 8.82 121.9 ± 10.87 68.2 ± 12.08 76.4 ± 14.05
STZ induced
Control 79.5 ± 10.95 106.6 ± 17.62a
108.7 ± 22.30 159.6 ± 28.90a
69.0 ± 19.30 56.3 ± 6.96a
0.5% Ginger 82.6 ± 10.16 98.2 ± 11.23a
115.3 ± 13.97 144.4 ± 11.57a
70.0 ± 12.40 58.1 ± 6.79a
1% Ginger 78.3 ± 26.28 94.2 ± 10.31a
106.5 ± 21.21 132.7 ± 16.22b
66.9 ± 11.14 59.8 ± 13.40a
5% Ginger 77.7 ± 10.47 88.5 ± 11.65b
111.3 ± 22.24 122.3 ± 20.97c
68.1 ± 18.55 67.6 ± 7.71b
All values are mean ± SD of 6 rats/group. Different superscripts are significant at p < 0.05 (ab)
and p < 0.01 (bc)
in STZ + ginger fed groups compared to their respective diabetic
controls for all the parameters.
2956 N. Kota et al. / Food Chemistry 135 (2012) 2954–2959

reduced significantly in a dose–response manner in both non-dia-
betic (p < 0.05) and diabetic groups (p < 0.01) compared to their
respective controls. An inhibition of 25%, 34% and 59% in the
non-diabetic group and of 6%, 31% and 38% in the diabetic rats
was observed (Table 5).
3.4. Genotoxicity
3.4.1. Comet assay in erythrocytes
The length of the Comet (L) and cell diameter (D) was measured
in 50 cells/slide and the group means ± SD were calculated. A sig-
nificant difference in the Comet ratios (y/x i.e. the width of head/
length of the tail) was seen between the normal control compared
to the diabetic control. The diabetic control showed a decrease in
the Comet ratio (0.768 ± 0.047) as compared to the normal control
(0.938 ± 0.022). The DNA damage in diabetic rats fed with ginger
decreased in a dose–response manner compared to the diabetic
control. An increase in the Comet ratios was observed in the dia-
betic groups with the increase in concentration of ginger showing
a dose-dependent inhibitory action on DNA damage (Table 6).
3.4.2. Rodent bone marrow micronucleus test
An increase in the frequency of micronuclei (MN) formation
was observed in diabetic control group (11.3 ± 2.86) compared to
the normal control group (3.7 ± 0.94), which was approximately
threefold higher than the normal group. A significant dose–re-
sponse reduction in MN formation was observed in the diabetic
groups fed with ginger at all levels (p < 0.05). The extent of reduc-
tion seen was 43.4%, 69.7% and 93.4% at 0.5%, 1% and 5%, respec-
tively, compared to the diabetic control. The PCE/NCE ratio was
also determined to evaluate the cytotoxic effect on bone marrow
cells, and ginger by itself did not show any cytotoxic effect and
the results clearly demonstrated the anticlastogenic potential
(Table 6).
4. Discussion
The present study was undertaken to see the dose–response
effect of ginger in the inhibition of oxidative stress and clastogenic-
ity in STZ induced diabetic rats. Several studies revealed the
benefits of medicinal plants like ginger which showed a hypoglyce-
mic effect and also a delay in the development of diabetes mellitus
(Al-Attar & Zari, 2007). Diet has been recognized as an important
factor in the management of diabetes mellitus. Among spices,
ginger and clove oil have been proved to possess antidiabetic
potential (Srinivasan, 2005). An aqueous extract of raw ginger at a
dose of 500 mg/kg lowered serum glucose, increased the insulin
levels in ginger-treated diabetic rats compared to the positive
control (Al-Amin, Thomson, Al-qattan, Peltonen-Shalaby, & Ali,
2006) in type 1 diabetes possibly involving 5-HT receptors. An
aqueous extract of ginger was studied to evaluate the hypoglycemic
and anti hyperglycemic effects on normoglycemic and STZ-diabetic
Table 4
Enzymatic and non-enzymatic antioxidant effect of ginger in liver of normal and STZ induced diabetic rats.
Groups SOD
(U/mg protein)
Catalase
(U/mg protein)
GSHPx cytosol
(oxidized/mg
protein/min)
GSH (lg/g) MDA (nmol/mg
protein)
%
Inhibition
Protein carbonyl
(nmol/mg protein)
% Inhibition
Normal
Control 3.15 ± 0.293 37.45 ± 4.052 290.63 ± 42.02 340.9 ± 39.27 3.73 ± 0.449 – 2.61 ± 0.425 –
0.5% Ginger 4.32 ± 0.366**
46.83 ± 8.347 320.0 ± 30.22 353.4 ± 40.94 2.61 ± 0.622*
30 2.15 ± 0.381*
18
1% Ginger 5.54 ± 0.412**
59.31 ± 7.940**
354.1 ± 36.08*
365.3 ± 39.85 1.72 ± 0.244*
54 1.91 ± 0.229*
27
5% Ginger 6.38 ± 0.427**
71.32 ± 5.044**
384.7 ± 29.06**
390.1 ± 41.64 1.52 ± 0.195*
59 1.56 ± 0.243*
40
STZ induced
Control 2.23 ± 0.305 9.24 ± 1.049 210.8 ± 35.80 192.2 ± 30.56 8.13 ± 0.991 – 3.86 ± 0.457 –
0.5% Ginger 2.54 ± 0.354 11.52 ± 1.035 237.6 ± 35.40 207.12 ± 33.27 7.59 ± 0.808**
7 3.54 ± 0.311 8
1% Ginger 2.77 ± 0.392 22.88 ± 3.971 257.3 ± 36.38 245.6 ± 39.34*
6.31 ± 1.110**
33 3.12 ± 0.272**
19
5% Ginger 3.22 ± 0.418*
40.39 ± 6.06**
278.9 ± 30.94**
312.5 ± 30.49*
4.39 ± 0.903**
46 2.76 ± 0.439**
29
*
p < 0.05, Significance between non-diabetic and diabetic groups compared to their respective controls
**
p < 0.01, Significance between non-diabetic and diabetic groups compared to their respective controls
Table 6
Influence of ginger on micronucleated polychromatic erythrocytes (MNPCE) and DNA damage in erythrocytes of STZ-induced diabetic rats.
Groups Comet ratio (y/x) MNPCE/2000 PCE % Reduction PCE/NCE ratio
Normal STZ induced Normal STZ induced Normal STZ induced
Control 0.938 ± 0.022 0.768 ± 0.047a
3.7 ± 0.94 11.3 ± 2.86a
0.77 ± 0.077 0.76 ± 0.145
0.5% Ginger 0.928 ± 0.023 0.860 ± 0.043b
4.3 ± 1.99 8.0 ± 1.41b
43.4 0.67 ± 0.170 0.64 ± 0.217
1% Ginger 0.935 ± 0.013 0.914 ± 0.019c
3.7 ± 1.47 6.0 ± 2.80c
69.7 0.65 ± 0.178 0.67 ± 0.154
5% Ginger 0.958 ± 0.025 0.927 ± 0.004c
4.6 ± 2.10 4.2 ± 0.98d
93.4 0.69 ± 0.143 0.64 ± 0.127
Values are mean ± SD of six animals per group. (y = Width of the head and x = length of the tail.)
abcd
p < 0.01, Different superscripts are significantly different between diabetic groups and positive control.
Table 5
Antioxidant effect of ginger in the kidney of normal and STZ-induced diabetic rats.
Groups SOD (U/mg protein) MDA (nmol/mg protein) % inhibition
Normal
Control 2.11 ± 0.174 2.63 ± 0.475 –
0.5% Ginger 2.39 ± 0.320 1.98 ± 0.435*
25
1% Ginger 3.24 ± 0.313**
1.73 ± 0.299**
34
5% Ginger 4.76 ± 0.444**
1.07 ± 0.232**
59
STZ induced
Control 1.59 ± 0.230 7.31 ± 1.002 –
0.5% Ginger 1.70 ± 0.208 6.88 ± 0.748 6
1% Ginger 1.88±.0211 5.06 ± 0.621**
31
5% Ginger 1.94 ± 0.219**
4.56 ± 0.595**
38
*
p < 0.05, Significance between non-diabetic and diabetic groups compared to
their respective controls.
**
p < 0.01, Significance between non-diabetic and diabetic groups compared to
their respective controls.

rats and to assess the possible herb–drug interactions with
glibenclamide and insulin. The interaction of ginger extract with
these two was found to be effective in lowering blood glucose levels
(Ihsan, Fatima, & Abdulazim, 2012). The results of our in vivo study
showed that by feeding ginger at different concentrations such as
0.5%, 1% and 5% effectively lowered glucose, cholesterol, triglycer-
ides in STZ-induced diabetic rats in a dose–response manner
(Table 3). However, it should be noted that the serum glucose levels
in ginger fed diabetic rats did not reach the normal levels though a
dose-dependent reduction was observed. Similar results were
reported in a study on the effect of ginger juice in STZ-induced
diabetic rats (Akhani, Vishwakarma, & Goyal, 2004; Saraswat
et al., 2010). There was a significant decrease in glucose levels
within the treated diabetic groups, and the maximum decrease
was at 5% level.
Earlier reports showed that compounds of ginger such as 6-
gingerol possess hypoglycemic and other pharmacological proper-
ties (Jiang et al., 2006). In a recent study by Saraswat, Reddy, Muth-
enna, and Reddy (2009), aqueous extracts of some herbs
particularly in ginger, cumin and cinnamon extracts and several
biochemical tests were done to measure the cross-linking of lens
proteins that occur during AGE formation with fructose. Ginger
was found to be more effective in protecting the lens proteins from
attack by fructose. Besides, ginger exhibited hypolipidemic effect
both in non-diabetic and diabetic rats fed with ginger as a result
of synergistic action of bioactive components present in it. In this
study a significant decrease in the levels of cholesterol and triglyc-
erides was seen and the lipid profile appeared to be markedly al-
tered favourably by ginger feeding and the abnormalities
developed in STZ-induced diabetic rats were effectively countered
by ginger. From the experimental data it is evident that ginger effi-
ciently regulated blood glucose in diabetic rats and also amelio-
rated lipid abnormalities associated with diabetes by virtue of its
antioxidant and antidiabetic compounds like gingerols and shoga-
ols present in ginger. During recent years, spices such as onion, tur-
meric, fenugreek and cumin and their active principles were
studied for their antidiabetic potential and as possible ameliorative
or preventive agents in addition to experimentally induced animal
diabetic models. Diallyl sulfide, an active principle of garlic, re-
duced adenosine induced platelet aggregation in women with type
2 diabetes (Abhay kumar et al., 2011).
Diabetes mellitus may be associated with increased lipid perox-
idation caused by oxidative stress and may also affect the progress
of diabetic complications. Therefore lipid and protein oxidation
and antioxidant status may be one mechanism by which dietary
treatment like ginger contributes to the prevention of diabetic
complications. The determination of carbonyl levels was used as
an index of the extent of oxidative damage of the protein, and
MDA was used as a marker of lipid oxidation. The MDA and car-
bonyl levels increased in the liver and kidney of the STZ induced
diabetic rats suggesting an increase in the free radical mediated
damage of the cell membrane (Qujeq, Habibinudeh, Daylmkatol,
& Rezvani, 2005). The mechanism of lipid and protein metabolism
is impaired in the tissues of diabetic rats.
The balance between oxidative stress and antioxidant defence
mechanism may be impaired by the depletion of enzymatic antiox-
idants and increased levels of MDA and carbonyl content in dia-
betic rats (Bhor, Raghuram, & Sivakami, 2004). Dose–response
reduction in MDA and protein carbonyl levels was demonstrated
in this study. The maximum reduction was noted at 5% level in li-
ver (Table 4) and the maximum reduction in MDA levels in kidney
was also at 5% (Table 5). Impaired glucose metabolism leads to oxi-
dative stress, protein glycation and formation of free radicals and
thus an augmentation of plasma antioxidant capacity decreases
plasma free radicals by consuming ginger, which is a rich source
of antioxidants (Sanjay, Santosh, & Ramesh, 2010).
The antioxidant effect of ginger was determined in our earlier
study which exhibited enhancement in the activities of SOD, cata-
lase and GSHPx (Nirmala et al., 2008). It was necessary to deter-
mine the antioxidant effect in vivo in STZ-induced diabetic rats
and also study the dose–response effect of ginger, which could
be useful in preventing diabetic complications. This study showed
a dose–response effect in the activities of SOD, catalase, GSHPx and
GSH (Tables 4 and 5). Glutathione (GSH) known as body’s master
antioxidant is a very important nutrient chemical which protects
tissues and organs from ageing and oxidative related diseases like
atherosclerosis, coronary artery disease, diabetes, cancer etc. The
largest amount of GSH is found in liver which is a key to detoxifi-
cation. The GSH content also showed a significant dose–response
increase with increasing the ginger concentrations. In one study,
ginger supplementation for 30 days to diabetic rats exerted a ther-
apeutic protective effect in diabetes by decreasing oxidative stress,
hepatic and renal damage (Shanmugam, Mallikarjuna, Nishanth, &
Satyavelu Reddy, 2011), whereas another study showed a decrease
in lipid peroxidation, increased plasma antioxidant capacity and
also reduced renal nephropathy in STZ-induced diabetic rats (Afs-
hari et al., 2007). Peroxy-nitrate induced nitration of protein tyro-
sine residues which is considered as one of the major pathological
causes of several human diseases, such as CVD and diabetes, was
suppressed by ginger, and it had more scavenging ability compared
to other spices (Ho, Tang, Lin, & Liew, 2010). Oxidative stress can
be modulated by suggesting a diet containing naturally occurring
compounds such as ginger which is found to be effective in exert-
ing protective effects (Rafat et al., 2008). Ginger extract inhibited
the hydroxyl radicals by 79.6% at 37 °C and 74.8% at 80 °C, which
showed a higher antioxidant activity than quercetin (Stoilova,
Krastanov, Stoyanova, & Gargova, 2007). The ginger extract che-
lated Fe+3
in the solution. In this study, decreased activities of anti-
oxidant enzymes, MDA and protein carbonyl levels were
augmented in normal and diabetic rats fed with ginger by acceler-
ating the antioxidant defence mechanisms and downregulating the
MDA and carbonyl levels. Thus, ginger may be used as therapeutic
agent in preventing complications in diabetic patients (Shanmu-
gam et al., 2011).
The Comet assay is sensitive to many aneuploidy agents and is
one of the most widely used short term assays for identification of
genotoxic effects associated with carcinogens. Diabetes mellitus is
a group of heterogenous, hormonal, metabolic and chronic disorder
associated with many complications and is also considered to be a
major factor for CVD. It is hypothesized that the diabetogenic ac-
tion of STZ-treated animals is mediated through a reduction of
NAD in pancreatic cells. The DNA caused by STZ-mediated alkyl-
ation is repaired by an excision repair process which requires the
activation of NAD-dependent enzyme polysynthetase thus deplet-
ing the cells of NAD and eventually leading to cell death (Weiss,
1982). In a study, ginger extract reduced the incidence of micronu-
cleated cell formation induced by Ehrlich ascites cells inoculation,
to almost normal or less than the control values; there was also a
decrease in DNA fragmentation (Hanafy, 2009). Ginger extract did
not inhibit the development of mouse bladder tumors induced by
N-butyl-N(4-hydroxy butyl) nitosamine BBN/N-methyl nitrosurea
(MNU) in male Swiss mice fed with diets containing 1.2% and 2%
ginger extract, respectively, and ginger by itself was not genotoxic
(Bidinolto, Spinardi-Barbesan, Rocha, Favero Salvadori, et al.,
2006). Earlier in vitro studies proved ginger to be antigenotoxic
(Nirmala, Prasanna, & Polasa, 2007a, 2007b; Nirmala et al., 2008).
In this study, a dose-dependent reduction in the frequencies of
STZ-mediated bone marrow micronuclei was observed which
may be mediated by the antioxidant-enhancing effects of natural
dietary agent such as ginger. Inhibition in the DNA damage in
the erythrocytes of rats fed with ginger also showed a dose–re-
sponse effect (Table 6).
2958 N. Kota et al. / Food Chemistry 135 (2012) 2954–2959

Ginger exerted a protective effect against STZ-induced diabetes
by modulating antioxidant enzymes, lipid and protein oxidation
and genotoxicity. The results of the study may suggest that a diet
containing even low levels of different naturally occurring com-
pounds such as ginger is effective in exerting antigenotoxic and
antioxidant effects by inhibiting the STZ-induced clastogenic activ-
ity. Further studies on the role of oxidative stress in the regulation
of gene expression by ginger and the post transcriptional activation
of enzymes involved in the detoxification, where gene expression
changes are a function of redox-sensitive transcription factor, are
undertaken to understand the mechanism of its action.
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