ga
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mm
ra P
dia
Diabetes mellitus
Oral glucose tolerance test
Medicinal plants
Antioxidants
ose
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nce
triglycerides by 54.12% and glucose–insulin index by 59.8% of the fructose fed rats. Regarding liver anti-
a mark
r in the
aride
actually or potentially deleterious, e.g., hyperlipidemia, hyperinsu-
linemia, insulin resistance, hyperuricemia, hypertension, glucose
intolerance and non-enzymatic fructosylation of proteins (Thor-
sagayam and Sainis, 2002). It is known to have hepatoprotective
(Nagarkatti et al., 1994), immunostimulatory (Kapil and Sharma,
1997) and hyperlipidemic properties (Prince and Menon, 1999). A
variety of constituents have been isolated from this plant belong
to different classes such as alkaloids, diterpenoid lactones, glyco-
sides, steroids, sesquiterpenoid, phenolics, aliphatic compounds
and polysaccharides (Singh et al., 2003). Although, the antidiabetic
and antioxidant activities of this plant in experimentally diabetic
rats has been well documented in scientific literature (Prince and
Menon, 1999; Grover et al., 2001), studies regarding its efficacy in
Abbreviations: AUC, area under the curve; CAT, catalase; GPx, glutathione
peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GST, glutathione-
S-transferase; HFCS, high-fructose corn syrup; OGTT, oral glucose tolerance test;
ROS, reactive oxygen species; SOD, superoxide dismutase; TC, Tinospora cordifolia;
TCAE, aqueous extract of Tinospora cordifolia stem.
* Corresponding author. Tel.: +91 08554 255879; fax: +91 08554 255805.
Food and Chemical Toxicology 47 (2009) 2224–2229
Contents lists availab
Food and Chemi
journal homepage: www.elsevE-mail address: skumari1@yahoo.co.in (D. Saralakumari).tose) and high-fructose corn syrup (HFCS; 55–90% fructose
content) (Bray et al., 2004). Processed-food manufacturers often
prefer HFCS to sucrose because it is inexpensive, sweeter and
mixes well in many foods. The increase in HFCS consumption far
exceeds the increases in intake of any other food or food group.
The disturbing fact is fructose consumption (excluding that which
occurs naturally in fruits and vegetables) increased from less than
0.5 g/day in 1970 to more than 40 g/day in 1997 (more than an
80-fold increase) (Gaby, 2005).
Concern has arisen because of the realization that fructose, at
elevated concentrations, can promote metabolic changes that are
Rats fed with a high-fructose diet form a model of diet-induced
insulin resistance, associated with hyperinsulinemia, hypertriglycer-
idemia and glucose intolerance (Thorburnet al., 1989). Recently, anti-
oxidants are found to be effective in preventing a majority of the
abnormalities inducedbyhigh-fructosediet (Faure et al., 1997, 1999).
Tinospora cordifolia (Menispermaceae) is a glabrous, succulent,
climbing shrub distributed throughout tropical Indian subcontinent.
The plant is commonly known as Guduchi, Giloy or Amritha. This
plant has beenwidely used in the Indian SystemofMedicine (Ayurv-
eda) as Rasayana for the treatment of diabetes, jaundice, rheumatoid
arthritis, gout, generalweakness, skin diseases and infections (Deva-Sweetener
Metabolic syndrome
1. Introduction
The last 25 years have witnessed
capita fructose intake as a sweetene
ily in the form of sucrose (a disacch0278-6915/$ - see front matter  2009 Elsevier Ltd. A
doi:10.1016/j.fct.2009.06.008oxidant status, fructose fed rats showed higher values of lipid peroxidation (91.3%), protein carbonyl
groups (44%) and lowered GSH levels (42.1%) and, lowered activities of enzymatic antioxidants, while
TCAE treatment prevented all these observed abnormalities. In conclusion, our data indicate the preven-
tive role of T. cordifolia against fructose-induced insulin resistance and oxidative stress; hence this plant
could be used as an adjuvant therapy for the prevention and/or management of chronic diseases charac-
terized by hyperinsulinemia, hypertriglyceridemia, insulin resistance and aggravated antioxidant status.
 2009 Elsevier Ltd. All rights reserved.
ed increase in total per
food industry, primar-
consisting of 50% fruc-
burn et al., 1989; Hwang et al., 1987; Reddy et al., 2008; Dills,
1993). In addition, excessive fructose consumption may be respon-
sible in part for the increasing prevalence of obesity, diabetes mel-
litus, non-alcoholic fatty liver disease and cardiovascular diseases
(Jurgens et al., 2005; Reaven, 1988; Reiser, 1985).Keywords:
(400 mg/kg/day) were given simultaneously for a period of 60 days. Fructose fed rats showed hypergly-
cemia, hyperinsulinemia, hypertriglyceridemia, impaired glucose tolerance and impaired insulin sensi-
tivity (P < 0.05). TCAE treatment prevented the rise in glucose levels by 21.3%, insulin by 51.5%,Preventive effect of Tinospora cordifolia a
insulin resistance and oxidative stress in
Singareddy Sreenivasa Reddy a, Pasurla Ramatholisa
aDepartment of Biochemistry, Sri Krishnadevaraya University, Anantapur 515 003, Andh
bDepartment of Biochemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh, In
a r t i c l e i n f o
Article history:
Received 6 February 2009
Accepted 5 June 2009
a b s t r a c t
High intake of dietary fruct
was to investigate whethe
diet-induced insulin resistall rights reserved.inst high-fructose diet-induced
ale Wistar rats
a b, Rasineni Karuna a, Desireddy Saralakumari a,*
radesh, India
exerts a number of adverse metabolic effects. The aim of the present study
ueous extract of Tinospora cordifolia stem (TCAE) alleviates high-fructose
and oxidative stress in rats. High-fructose diet (66% of fructose) and TCAE
le at ScienceDirect
cal Toxicology
ier .com/locate/ foodchemtox

After the experimental period the animals were fasted overnight and killed by
cervical decapitation. The body was cut open and liver was dissected out into ice-
cal Tpreventing insulin resistance which plays a role in pathophysiology
of type 2 diabetes mellitus have not been undertaken.
The anti-hyperglycemic, anti-hyperlipidemic and antioxidant
properties of T. cordifolia (TC) (in diabetic rat model) prompted
us to design the present study to investigate whether management
with TC has any preventive effect on plasma glucose, insulin, tri-
glycerides, hepatic lipid peroxidation and activities of both enzy-
matic and non-enzymatic antioxidant status in fructose fed rat
model of insulin resistance.
2. Materials and methods
2.1. Chemicals
Thiobarbituric acid and pyrogallol were obtained from the Sigma Chemical Co.,
St. Louis, MO, USA. All other chemicals and solvents were of analytical grade and
were from Sisco Research Laboratories (P) Ltd., Mumbai, India.
2.2. Plant extract
An aqueous extract of T. cordifolia stem (TCAE; brown, dry powder with Lot No.
L5111031) was procured from the manufacturers and exporters of herbal extracts,
Ms. Plantex Pvt. Ltd., Vijayawada, Andhra Pradesh, India. Procedure followed by the
firm for the preparation of extract is as follows: The plant was identified by Dr. K.
Narasimha Reddy, Taxonomist, Laila Impex R&D Center, Vijayawada. The collected
plant sample (stem) was washed thoroughly with tap water, dried at room temper-
ature away from sun light, cut into small pieces and then powdered. The aqueous
extract was prepared by cold maceration of stem powder in drinking water for
7 days. The extract was filtered, concentrated under reduced pressure and finally
dried in a vacuum desiccator. Herb-to-product ratio was 10:1. A voucher specimen
has been deposited in the Department of Biochemistry, Sri Krishnadevaraya Univer-
sity, Anantapur, under number SK-TC-08.
2.3. Control and fructose diet
The control diet for the rats contained 66% starch, 15% protein, 8% fat, 4% cellu-
lose, 1% of each mineral and vitamin mix. The fructose diet contained 66% of fruc-
tose instead of starch and remaining composition is same as that of the control diet.
Both the diets were obtained from National Centre for Laboratory Animal Sciences,
National Institute of Nutrition (Hyderabad, India).
2.4. Animals
Male Albino Wistar rats (140–160 g) used for the present study were procured
from Sri Venkateswara Enterprises (Bangalore, India). The animals were acclima-
tized for 7 days in our animal house (Regd. No. 470/01/a/CPCSEA) before dietary
manipulation. They were housed two per cage in an air-conditioned room
(22 ± 2 C) with 12 h light/dark cycle and had free access to standard pellet diet
and water. All the procedures were performed in accordance with the Institutional
Animal Ethics Committee.
2.5. Experimental design
All the animals were 6 weeks of age, weighing around 200 g at the time of die-
tary manipulation. Animals were randomly assigned into four groups of eight each
as given below:
 Group-C: normal control rats, received tap water and control diet,
 Group-F: fructose fed rats, received tap water and fructose diet,
 Group-F+TC: TCAE treated fructose fed rats, received TCAE (400 mg/kg/day) and
fructose diet,
 Group-C+TC: TCAE treated normal rats, received TCAE (400 mg/kg/day) and con-
trol diet.
Vehicle (tap water for group-C and -F) and TCAE (dissolved in tap water) were
administered orally by gastric intubation. The animals were maintained in their
respective groups for 60 days. The dose of TCAE used in the current study was based
on the earlier report on the anti-hyperglycemic effect of this plant in experimental
diabetic rats (Grover et al., 2000) and our previous dose fixation studies for anti-
hyperglycemic effect of TCAE in alloxan-induced diabetic rats (data not shown).
The body weight, fasting plasma glucose, insulin and triglycerides of all animals
were measured on initial, 15, 30, 45 and 60th day of experiment.
2.6. Oral glucose tolerance test (OGTT)
S.S. Reddy et al. / Food and ChemiAt the end of experimental period (60 days), the 12-h fasted animals were sub-
jected to oral glucose tolerance test. For this, a 40% glucose solution was introducedcold saline and then thoroughly rinsed.
2.9. Biochemical measurements
The concentration of plasma glucose was measured by the glucose oxidase
method, using Span Diagnostic Kit (Surath, India). Plasma triglyceride level was
estimated by GPO–POD enzymatic method using the Monozyme Diagnostic kit
(Secunderabad, India). Insulin was determined by radioimmunoassay kit (RIAK-1)
provided by Bhabha Atomic Research Center (Mumbai, India) according to the
method of Yalow and Berson (1961). Human insulin was used for the preparation
of standard curve of insulin.
The concentration of lipid peroxidation intermediates, liver thiobarbituric acid
reactive substances (TBARS) was measured by following the method of Utley et al.
(1967), using 10% liver homogenate in 0.15 M KCl and expressed as nmol MDA
formed/15 min/mg protein. The extent of protein carbonyl groups (Levine et al.,
1990) and reduced glutathione (GSH) levels (Ellman, 1959) in liver were deter-
mined. Protein content in the liver homogenate was measured by the method of
Lowry et al. (1951).
2.10. Enzyme assays
Ten percent liver homogenate was prepared in ice-cold 0.15 M KCl, centrifuged
at 12,000 rpm for 45 min in Sigma Laboratory centrifuge 3K18 model, rotor No.
12150. The clear supernatant thus obtained was used for the assay of superoxide
dismutase (SOD; E.C.1.15.1.1; Soon and Tan, 2002), catalase (CAT; E.C.1.11.1.6;
Beers and Sizer, 1952), glutathione peroxidase (GPx; E.C.1.15.1.9; Rotsruck et al.,
1973), glutathione-S-transferase (GST; E.C.2.5.1.14; Habig et al., 1974) and glutathi-
one reductase (GR; E.C.1.8.1.7; Pinto and Bartley, 1969).
2.11. Statistical analysis
All results were expressed as means ± SEM for the number, n = 8 of animals in
the group as indicated in the figures and table. To determine the statistical signifi-
cance of clinical and laboratory findings Duncan Multiple Range test (DMRT) was
used. P values of less than 0.05 were regarded as significant.
3. Results
3.1. Effect of TCAE on body weight
The body weights of four groups of animals during experimen-
tal period are represented in Fig. 1A. No significant variation in
body weights of groups C+TC and F+TC was observed when com-
pared with group-C. Whereas, group-F also showed no significant
variation up to 15 days but a significant (P < 0.05) increase was
observed from 30 days onwards till the end of experimental period
when compared with group-C.
3.2. Fasting plasma glucose
There was no significant variation in the plasma glucose con-directly into the stomach through a fine gastric catheter at a dose of 2 g/kg body
weight to conscious rats. Plasma glucose and insulin levels were determined at 0
(before glucose administration), 30, 60 and 120 min after glucose administration.
2.7. Measurement of glucose–insulin index
The action of insulin on glucose disposal rate was measured using the glucose–
insulin index, which is the product of the areas under the curve (AUC) of glucose
and insulin during the glucose tolerance test.
2.8. Sample collection
Blood was collected from 12-h fasted rats with capillary tube from retino-orbi-
tal plexus of the animals in fresh vials containing EDTA (10 mg/ml) as anticoagu-
lant. The samples were centrifuged at 3000 rpm for 5 min (MSE Micro Centaur,
UK) and the plasma obtained was aliquoted and frozen for insulin assay. Plasma
glucose and triglycerides were determined immediately. The blood sample col-
lected during oral glucose tolerance test was from the tail vein of animals.
oxicology 47 (2009) 2224–2229 2225centrations of group-C and C+TC throughout the experimental per-
iod (Fig. 1B). Group-F showed a gradual and significant increase in
plasma glucose levels from 30 days onwards till the end of exper-