1

Cissus quadrangularis stem alleviates insulin resistance, oxidative injury
and fatty liver disease in rats fed high fat plus fructose diet
Chidambaram Jaya, Carani Venkatraman Anuradha
*
Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India
article info
Article history:
Received 4 December 2009
Accepted 28 April 2010
Keywords:
Insulin resistance
Liver steatosis
High fat–high fructose diet
Oxidative stress
Free-radical scavenger
Cissus quadrangularis
abstract
The study evaluated the protective effects of Cissus quadrangularis stem extract (CQEt) on oxidant–anti-
oxidant balance and insulin resistance (IR) in rats fed high fat–high fructose diet (HFFD) and also tested
its free-radical scavenging property in vitro. Rats were fed either control diet or HFFD for 15 days, follow-
ing which the diet was fortified with CQEt at a dose of 10 g/100 g diet. After 60 days, HFFD caused dele-
terious metabolic effects, including hyperglycemia, IR and liver dysfunction. Rats fed HFFD alone showed
increased activities of hepatocellular enzymes in plasma, lipid deposition, significant decline in antioxi-
dants, and elevated lipid peroxidation indices and protein carbonyl in liver. CQEt addition significantly
improved insulin sensitivity, reduced liver damage and oxidative changes, and brought back the antiox-
idants and lipids towards normal. Histopathology of the liver confirmed the changes induced by HFFD
and the heptoprotective effect of CQEt. The effects of CQEt in vivo were comparable with that of standard
drug, metformin. Through in vitro assays, CQEt was found to contain large quantities of polyphenols, vita-
mins C and E. CQEt exhibited radical scavenging ability in a dose-dependent manner. These data suggest
that CQEt affords hepatoprotection by its antioxidant and insulin-sensitizing activities.
Ó2010 Elsevier Ltd. All rights reserved.
1. Introduction
Nonalcoholic fatty liver disease (NAFLD) is one of the most com-
mon causes of chronic liver injury in many countries around the
world. It has a broad pathologic spectrum which ranges from sim-
ple fatty infiltration of the liver or steatosis, to nonalcoholic steato-
hepatitis (NASH), fibrosis, cirrhosis and to liver failure.
Studies have shown that intake of high fat diet or fructose diet
results in insulin resistance (IR), hepatic steatosis, excessive gener-
ation of reactive oxygen species (ROS), malfunctioning of the liver
and depletion of the hepatocyte population (Assy et al., 2000). A
combination of high fat–high fructose diet (HFFD) reduces the
intrinsic antioxidant defense system, creates an unbalanced
oxidative status and fatty infiltration in rats (Aragno et al., 2009).
There is evidence that oxidative stress contributes to the develop-
ment of steatohepatitis from steatosis induced by high-energy
diet (Barbuio et al., 2007). The development of NAFLD has been
associated with a ‘‘two-hit hypothesis”, the first hit being triglycer-
ide and free fatty acid accumulation and second hit involving oxi-
dative stress and inflammation of the liver (Demori et al., 2006).
Studies indicate that feeding HFFD can trigger all these pathologi-
cal events and this model closely resembles human NAFLD.
The most common ROS are superoxide anion (O
2
), hydroxyl
radicals (OH
) and hydrogen peroxide (H
2
O
2
). These species are
capable of initiating and promoting oxidative damage to the major
biomolecules (Kovacic and Cooksy, 2005). Uncontrolled generation
of ROS readily attack membrane lipids, protein and DNA and is be-
lieved to be involved in many metabolic, degenerative and inflam-
matory health disorders and in conditions like IR and dyslipidemia
(Pryor and Ann, 1982).
Based on growing interest in free-radical biology and lack of
effective therapies for most chronic diseases, the usefulness of nat-
ural antioxidants from plant materials have been evaluated for
therapeutic efficiency against diseases related to oxidative stress.
Plants are rich source of bioactive components, the most important
of these are flavonoids and polyphenolic compounds. They exhibit
high antioxidant properties that terminate free-radical mediated
reactions by donating hydrogen atom or an electron to the radicals
(Shariff, 2001; Havsteen, 2002; Snoog and Barlow, 2004). This as-
pect reinforces the idea that the dietary inclusion of natural antiox-
idants present in plant foods is an important health-protecting and
disease-preventing factor in humans.
0278-6915/$ - see front matter Ó2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.04.044
Abbreviations: CQEt,Cissus quadrangularis stem extract; IR, insulin resistance;
HFFD, high fat–high fructose diet; HOMA, homeostatic model assessment; QUICKI,
quantitative insulin sensitivity check index; TG, triglycerides; GSH, glutathione;
GPx, glutathione peroxidase; SOD, superoxide dismutase; CAT, catalase; GST,
glutathione S-transferase; H and E, hematoxylin and eosin; DPPH
, 2,2
0
-diphenyl-1-
hydrazine; ABTS
+
, 2,2
0
-azino-di[3-ethylbenzthiazoline sulphonate].
*Corresponding author. Tel.: [phone]; fax: [phone].
E-mail address: [email protected] (A. Carani Venkatraman).
Food and Chemical Toxicology 48 (2010) 2021–2029
Contents lists available at ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox

Cissus quadrangularis Linn. belongs to the family Vitaceae and is
indigenous to India, Sri Lanka, Malaysia, Thailand and Africa. The
plant has a great medicinal value and the stem part of C. quadrang-
ularis is traditionally used for the treatment of skin infections, con-
stipation, piles, anemia, asthma, irregular menstruation, burns and
wounds (Kritikar and Basu, 2000). Studies have reported that
methanolic extract from C. quadrangularis (CQEt) have antimicro-
bial, antiulcer and antioxidant properties (Subbu, 1970; Anoop
and Jagdeesan, 2002; Chidambara Murthy et al., 2003). The potent
fracture-healing property of this plant was the first report in 1963
documenting its benefits. Since then, a number of animal studies
have claimed its utility in a wide range of disease conditions like
gastric ulcer, bone fracture and hepatic toxicity (Prasad and Udupa,
1963; Chopra et al., 1975). A recent report shows that CQEt effec-
tively reduces the body weight by inhibiting the oxidation of LDL
cholesterol and by lowering the blood glucose in obese patients
(Oben et al., 2006). It has also been proved that CQEt has therapeu-
tic action against hepatic damage induced by carbon tetrachloride
(CCl
4
)(Jainu and Devi, 2005). However, there is a lack of data on
the protective effects of CQEt on the pathological development of
IR-associated liver disease induced by HFFD.
Metformin, an insulin sensitizer, has become the established
treatment for type 2 diabetes mellitus. Proposed mechanisms for
the antihyperglycaemic effect include enhanced insulin-stimulated
glucose uptake from the blood into the tissues, and decreased glu-
cose production in the liver by suppression of hepatic gluconeo-
genesis (Srividhya and Anuradha, 2002). This study aims to
evaluate the effect of CQEt on HFFD-induced fatty liver and IR
and compare the data with that of metformin. Additionally, a sys-
tematic evaluation of its free-radical scavenging property of CQEt is
carried out in vitro.
2. Materials and methods
2.1. Preparation of plant extract
The fresh stem of C. quadrangularis was purchased from the local market, at
Chidambaram, washed, cut into pieces and sun-dried. The dried stems were finely
powdered. One kilogram of the dried plant material was exhaustively extracted
with 2 L methanol. The extract was filtered and distilled on a water bath. The crude
extract was vacuum dried and powdered (Jainu and Devi, 2005). The yield of the ex-
tract was 4.8 g% .The powder was mixed in the diet for in vivo studies and dissolved
in saline for in vitro studies.
2.2. Animals and treatment protocol
Adult male Wistar albino rats weighing 150–170 g were obtained from the Cen-
tral Animal House, Rajah Muthiah Medical College, Annamalai University. The ani-
mals were housed in large polypropylene cages in a temperature-controlled room
and provided with standardized pelleted feed (Amrut rat and mice feed, Bangalore,
India) and clean drinking water ad libitum. All the experimental procedures were
carried out in accordance with the guidelines of the Institutional Animal Ethics
Committee (IAEC).
After acclimatization a period of one week, the animals were divided into two
groups and fed the control or HFFD. After 15 days, IR was confirmed by measuring
fasting glucose and insulin and by computing fasting insulin sensitivity indices. On
day 16, the rats in each group were divided treated with CQEt, metformin or left un-
treated. Accordingly five groups of rats were maintained for 60 days as follows:
Group I – CON – normal diet-fed rats.
Group II – HFFD-fed rats.
Group III – HFFD + CQEt – HFFD-fed rats treated with CQEt (10 g/100 g diet).
Group IV – HFFD + MET – HFFD-fed rats treated with metformin (50 mg/kg/
day).
Group V – CON + CQEt – normal diet-fed rats treated with CQEt (10 g/100 g
diet).
Diet and water were provided ad libitium and body weight was recorded at reg-
ular intervals. At the end of 60 days, the animals were deprived of food overnight,
administrated ketamine hydrochloride (35 mg/kg) and sacrificed by decapitation.
Blood was collected and plasma was separated by centrifugation (1500 g, 15 min,
room temperature). Plasma glucose and insulin levels were determined. Insulin
sensitivity was assayed by calculating homeostatic model assessment (HOMA) val-
ues. Liver was immediately dissected out and washed in ice-cold saline. Liver tissue
homogenate (10%) was prepared using 0.025 M Tris–HCl buffer, pH 7.5 or phos-
phate buffer, pH 7.
2.3. In vivo assays
2.3.1. Glucose, insulin and insulin sensitivity indices
Plasma glucose and insulin were measured using kits obtained from Agappe
Diagnostic Pvt. Ltd., Kerala and Accubind microwells, Monobind Inc., CA, USA,
respectively. IR was assessed by computing HOMA, quantitative insulin sensitivity
check index (QUICKI) and insulin resistance indices FIRI (Shalam et al., 2006). The
formulae used are given below:
HOMA = Insulin (
l U/mL) glucose (mM)/22.5
QUICKI ¼1
logðglucose mg=dLÞþlogðinsulin mU=LÞ
FIRI ¼ðfasting insulin ð
l U=mLÞfasting glucoseðmg=dLÞÞ=25:
2.3.2. Marker enzymes
To assess the liver injury, activities of aspartate transaminase (AST), alanine
transaminase (ALT), alkaline phospatase (ALP) and gamma glutamyl transferase
(GGT) were assayed using kits obtained from Agappe Diagnostics, Kerala, India.
2.3.3. Liver lipid levels
Lipids were extracted from liver by the method of Folch et al. (1957). Total lipid,
triglycerides (TG) (Foster and Dunn, 1973), cholesterol (Zlatkis et al., 1953), free
fatty acids (FFA) (Falholt et al., 1973) and phospholipids (Zilversmit and Davis,
1950) were determined.
2.3.4. Lipid and protein damage
Lipid peroxidation was evidenced by measuring the formation of thiobarbitutric
acid reactive substance (TBARS) and lipid hydroperoxide (LHP) in liver samples fol-
lowing the method Niehaus and Samuelson (1968) and Jiang et al. (1992), respec-
tively. The protein carbonyl content in the liver was measured by the method of
Levine et al. (1990).
2.3.5. Antioxidant status
The antioxidant status in plasma and liver was evaluated by estimating the lev-
els of non-enzymatic antioxidants such as reduced glutathione (GSH) by the meth-
od of Ellman (1959), vitamin E was estimated in the lipid extract by the method of
Baker et al. (1980) and vitamin C by that of Roe and Kuether (1942). The activities of
enzymatic antioxidants such as glutathione peroxidase (GPx), superoxide dismu-
tase (SOD) and catalase (CAT) and glutathione S-transferase (GST) were assayed
in both hemolysate and liver by the methods outlined elsewhere (Anitha Nandhini
et al., 2002).
2.3.6. Histopathological analysis
Histology of liver was studied using hematoxylin and eosin (H and E) and oil red
O staining. A portion of the liver was fixed in 10% buffered formalin, dehydrated in
graded (50–100%) alcohol embedded in paraffin. Thin sections (4–5
l m) were cut
and stained with H and E. For oil red O staining, frozen liver sample was processed
using cryostat and then fixed and stained.
2.4. In vitro assays
2.4.1. Estimation of total phenolic content and vitamins C and E
Total phenolic content in CQEt was determined using Folins–Ciocalteau reagent
by the method of Singleton and Rossi (1965). The values were expressed as mg of
gallic acid equivalents (GAE)/100 g. Potential antioxidant status of the plant extract
was evaluated by estimating the levels vitamin C by that of Roe and Kuether (1942)
and vitamin E by the method of Baker et al. (1980).
2.4.2. Hydroxyl radical scavenging activity
Hydroxyl radical is the most reactive of the free radicals that can cause damage
to proteins, lipids and DNA (Spencer et al., 1994). The hydroxyl radical scavenging
activity of CQEt was determined by the method of Halliwell et al. (1987). The incu-
bation mixture in the total volume of 1 mL contained 0.2 mL of 100 mM potassium
dihydrogen phosphate–potassium hydroxide buffer pH 7.4, varying volumes of
CQEt (10, 20, 30, 40 and 50
l g/mL), 0.2 mL of 500 l M ferric chloride, 0.1 mL of
1 mM ascorbic acid, 0.1 mL of 1 mM ethylenediamine tetra acetate (EDTA), 0.1 mL
of 10 mM hydrogen peroxide and 0.2 mL of 2-deoxyribose. The contents were
mixed thoroughly and incubated at room temperature for 60 min. Then 1 mL of
1% thiobarbitutric acid (TBA) (1 g in 100 mL of 0.05 N NaOH) and 1 mL of 28% tri-
chloro acetic acid (TCA) were added. All the tubes were kept in boiling water bathy
for 30 min. Gallic acid and vitamin C were used as a positive control for comparison.
The absorbance was read in a spectrophotometer at 532 nm with reagent blank
2022 J. Chidambaram, A. Carani Venkatraman / Food and Chemical Toxicology 48 (2010) 2021–2029

containing water in the place of extract. Decreased absorbance of the reaction mix-
ture indicated increased hydroxyl radical scavenging activity. The percentage scav-
enging was calculated as per the formula given below.
Scavenging ð%Þ¼Absorbance of control Absorbance of test
Absorbance of control
2.4.3. Superoxide anion-scavenging activity
Superoxide anion radical scavenging activity of CQEt was determined by the
method of Nishimiki et al. (1972) with modifications. The assay was based on the
oxidation of nicotinamide adenine dinucleotide (NADH) by phenazine methosulfate
(PMS) to liberate a PMS
red
. PMS
red
converted oxidized nitroblue tetrazolium
(NBT
oxi
) to NBT
red,
which forms a violet colour. The reaction mixture in a final vol-
ume of 2.5 mL contained, 1 mL of NBT (100
l mol NBT in 100 mM phosphate buffer,
pH 7.4), 1 mL of NADH solution (468
l mol in 100 mM phosphate buffer, pH 7.4) and
varying volumes of CQEt (10, 20, 30, 40 and 50
l g/mL). The reaction was started by
the addition of 100
l L PMS (60 l mol/100 mM phosphate buffer, pH 7.4). The reac-
tion mixture was incubated at 30 °C for 15 min after which the absorbance was
measured at 560 nm. Blank contained the all the solutions and water in place of
CQEt. Gallic acid and vitamin C were used as a positive control for comparison. De-
creased absorbance of the reaction mixture indicated increased superoxide anion-
scavenging activity. The percentage scavenging was calculated as per the formula
given above.
2.4.4. Reducing power
The reducing ability of a compound generally depends on the presence of reduc-
tants which can exert antioxidative potential by breaking the free-radical chain,
donating a hydrogen atom .The reducing power of CQEt was determined by the
method of Oyaizu (1986). Substances, which have reduction potential, react with
potassium ferricyanide (Fe
3+
) to form potassium ferrocyanide (Fe
2+
), which then re-
acts with ferric chloride to form ferric–ferrous complex that has as absorption max-
imum at 700 nm. Varying volumes of CQEt (10, 20, 30, 40 and 50
l g/mL) taken in
test tubes, mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of
potassium ferricyanide (1% w/v). The mixture was incubated at 50 °C for 20 min. La-
ter, 1.5 mL of 10% TCA was added and centrifuged at 3000 g for 10 min. From all the
tubes, 0.5 mL of supernatant was mixed with 1 mL of distilled water and 0.5 mL of
FeCl
3
(0.1 w/v). The absorbance was measured at 700 nm in a spectrophotometer
against a blank that contained water in the place of CQEt. Increased absorbance
of the reaction mixture indicated increasing reducing power. Gallic acid was used
as positive control for comparison.
2.4.5. Nitric oxide radical scavenging activity
Scavenging of nitric oxide radical was determined by the method of Garrat
(1964). In this method, 2 mL of sodium nitroprusside (10 mM in water), and differ-
ent concentrations of CQEt [20–100
l g] were incubated at 25 °C for 15 min. After
15 min, 0.5 mL of incubation solution containing nitrite was mixed with 1 mL of
sulfanilic acid (0.33% in 20% glacial acetic acid) reagent and allowed to stand for
5 min. Then 1 mL of naphthylethylenediamine hydrochloride (0.1% NEDD in 0.1 M
HCl) was added, mixed and allowed to stand for 30 min at 25 °C. The absorbance
of the chromophore formed during the diazotization of nitrite with sulphanilamide
and subsequent coupling with NEDD was read at 540 nm. The amount of nitrite was
calculated from standard curved constructed with sodium nitrite.
2.4.6. DPPH
radical scavenging assay
The radical scavenging activity of CQEt against 2,2
0
-diphenyl-1-hydrazine
(DPPH
) was determined by spectrophotometrically by the method of Brand-Wil-
liams et al. (1995). The reaction mixture in a total volume of 3 mL contained
1 mL of 100
l M DPPH in methanol, equal volumes of CQEt [10–50 l g] and 1 mL
of phosphate buffer pH 7.4. The tubes were incubated for 10 min at 37 °C in the
dark. The absorbance was monitored at 517 nm. The control tube contained DPPH
alone. The percentage scavenging was calculated as shown above.
2.4.7. Antiradical activity against ABTS
+
The total antioxidant capacity was assessed based on the ability of a compound
to scavenge the stable 2,2
0
-azino-di[3-ethylbenzthiazoline sulphonate] (ABTS
+
)
radical (Woifenden and Willson, 1982). The experiments were carried out using
an improved ABTS
+
decolorisation assay (Re et al., 1999), which involves the gen-
eration of the ABTS
+
chromophore by the oxidation of ABTS
+
(1.8 mM) with potas-
sium persulphate (2.45 mM). The mixture was allowed to stand in the dark at room
temperature for 12–16 h. About 0.54 mL of ABTS
+
and 0.5 mL of phosphate buffer
were added and mixed well. To this, different concentrations of CQEt [20–100
l g]
was added and made up to 5 mL with distilled water. The absorbance was measured
at 734 nm. The percentage inhibition of CQEt was calculated and compared with
trolox, the water soluble analogue of vitamin E, was used as a reference standard.
2.5. Statistical analysis
For in vivo assays, the results of animal experiments are given as mean ± SD of
six rats from each group and statistically evaluated by Student’s t-test for unpaired
comparisons. For in vitro assays, the results given are the average of five determina-
tions and were analyzed by Students t-test for unpaired comparisons. The level of
statistical significance was set at P< 0.05.
3. Results
3.1. In vivo assays
Table 1 shows a significant elevation in the circulating levels of
glucose, insulin, HOMA, QUICKI and FIRI for 15 days HFFD
treatment.
Table 2 shows final body weight, liver weight, liver index (Li, li-
ver weight/body weight 100) glucose, insulin and HOMA values
in the different groups. Final body weight, Li, glucose, insulin
HOMA, QUICKI and FIRI were significantly increased in rats fed
the HFFD-diet compared with all other groups, which did not differ
significantly from each other.
Table 1
Glucose, insulin and insulin sensitivity indices in control and HFFD-fed rats at the end
of 15 days.
Parameters CON HFFD
Glucose (mg/dL) 81.03 ± 5.16
c
130.20 ± 9.42
a
Insulin ( l U/mL) 45.39 ± 5.68
c
64.16 ± 6.12
a
HOMA 8.97 ± 0.43
c
20.40 ± 0.96
a
QUICKI 0.280 ± 0.001
c
0.255 ± 0.002
a
FIRI 147.5 ± 7.51
c
334.17 ± 24.23
a
Values are the mean ± SD (n= 6 per group).
HOMA, homeostatic model assessment.
FIRI, fasting insulin resistance indices.
Values not sharing a common superscript were significantly differ from each other
(P< 0.05, ANOVA followed by Duncan’s multiply range test (DMRT)).
Table 2
Body weight, liver weight and liver index, glucose, insulin and homeostatic model assessment, insulin resistance indices in the different groups at the end of 60 days.
Parameters CON HFFD HFFD + CQEt HFFD + MET CON + CQEt
Body weight(g) 184.6 ± 12.7
c
214.3 ± 15.1
a
204.2 ± 14.5
b
199.8 ± 13.9
c
180.4 ± 13.8
c
Liver weight(g) 5.58 ± 0.57
c
8.93 ± 0.79
a
7.64 ± 0.62
b
6.01 ± 0.50
c
5.5 ± 0.52
c
Liver index 3.02 ± 0.23
c
4.16 ± 0.24
a
3.74 ± 0.32
b
3.00 ± 0.36
c
3.05 ± 0.28
c
Glucose (mg/dL) 83.72 ± 5.92
c
151.11 ± 8.65
a
134.16 ± 6.98
b
120.14 ± 4.58
c
80.23 ± 5.91
c
Insulin ( l U/mL) 49.45 ± 4.13
c
75.14 ± 6.23
a
56.12 ± 5.84
b
51.33 ± 4.34
c
46.97 ± 5.33
c
HOMA 10.10 ± 0.79
c
27.73 ± 1.4
a
18.39 ± 0.64
b
15.05 ± 0.70
c
9.20 ± 0.37
c
QUICKI 0.27 ± 0.001
c
0.24 ± 0.003
a
0.25 ± 0.002
b
0.26 ± 0.001
c
0.27 ± 0.002
c
FIRI 165.5 ± 7.51
c
454.17 ± 24.23
a
301.16 ± 16.02
b
246.67 ± 12.11
c
150.73 ± 7.22
c
Values are the mean ± SD (n= 6 per group).
The liver index was calculated as liver weight / body weight x 100.
HOMA, Homeostatic model assessment.
FIRI, Fasting insulin resistance indices.
Values not sharing a common superscript were significantly differ from each other (P< 0.05, ANOVA followed by Duncan’s multiply range test (DMRT)).
J. Chidambaram, A. Carani Venkatraman / Food and Chemical Toxicology 48 (2010) 2021–2029 2023

Increased plasma activities of AST, ALT, ALP and GGT were
found in HFFD-fed rats, indicating damage to liver cells (Table 3).
Treatment of HFFD-fed rats with CQEt or metformin resulted in sig-
nificantly (P< 0.05) lower AST, ALT, ALP and GGT activity compared
with the untreated HFFD-fed rats.
Plasma lipid concentrations in the different groups are shown in
Table 4. HFFD-fed rats exhibited a twofold increase in total choles-
terol compared with control rats. In addition, significant increases
were observed in TG and FFA in the HFFD-fed rats (by 63%, 24%,
and 38%, respectively). CQEt treatment of HFFD-fed rats resulted
in a significant reduction in TG, phospholipid, FFA, and cholesterol
levels to near-normal values. Metformin also lowered the hepatic
lipid levels when administered to HFFD-fed rats.
Table 5 lists TBARS, LHP, and protein carbonyl levels in the dif-
ferent groups. Significantly higher TBARS, LHP, and protein car-
bonyl levels were found in HFFD-fed rats compared with control
rats. In CQEt-treated HFFD-fed rats, TBARS, LHP, and protein car-
bonyl levels were significantly (P< 0.05) lower compared with
the untreated HFFD-fed group. This result was also comparable
with metformin.
Tables 6 and 7 list the activities of enzymatic and non-enzy-
matic antioxidants in the plasma and liver of the different groups.
The SOD, CAT, GPx and GSH activities, as well as vitamin C and E
levels, were significantly (P< 0.05) decreased in HFFD-fed rats by
43%, 42%, 55%, 37%, 36% and 44%, respectively compared with con-
trol. In CQEt and metformin-treated HFFD-fed rats, these parame-
Table 3
Levels of liver marker enzymes in plasma (IU/L).
Parameters CON HFFD HFFD + CQEt HFFD + MET CON + CQEt
ALP 41.82 ± 1.90
c
78.70 ± 5.64
a
52.69 ± 3.59
b
43.55 ± 2.41
c
40.55 ± 2.04
c
GGT 19.84 ± 0.94
c
41.15 ± 3.25
a
29.45 ± 2.16
b
21.61 ± 0.99
c
20.77 ± 1.35
c
SGPT 51.97 ± 2.23
c
88.96 ± 6.79
a
63.27 ± 3.88
b
53.15 ± 3.68
c
52.04 ± 1.76
c
SGOT 41.82 ± 2.18
c
88.81 ± 6.05
a
54.98 ± 3.68
b
44.46 ± 2.93
c
43.77 ± 1.92
c
Values are means ± SD of six rats from each group. CON – control rats; HFFD-high fat–high fructose-fed rats;
HFFD + CQEt – high fat–high fructose-fed rats treated with Cissus quadrangularis extract (10 g/100 g diet);
HFFD + MET – high fat–high fructose-fed rats treated with metformin (50 mg/kg b.w).
CON +CQEt – control rats treated with Cissus quadrangularis extract (10 g/100 g diet).
Values not sharing a common superscript were significantly differ from each other (P< 0.05, ANOVA followed by Duncan’s multiply range test (DMRT)).
Table 4
Levels of total cholesterol, triglyceride (TG), free fatty acid (FFA) and phospholipids in liver (mg/g tissue).
Parameters CON HFFD HFFD + CQEt HFFD + MET CON + CQEt
Cholesterol 3.06 ± 0.0.21
c
8.05 ± 0.42
a
6.33 ± 0.53
b
5.37 ± 0.40
c
3.08 ± 0.25
c
TG 4.16 ± 0.31
c
7.04 ± 0.32
a
6.12 ± 0.33
b
5.09 ± 0.29
c
4.21 ± 0.33
c
FFA 4.13 ± 0.31
c
7.09 ± 0.51
a
6..05 ± 0.33
b
5.10 ± 0.40
c
4.15 ± 0.34
c
Phospholipid 16.10 ± 0.55
c
12.28 ± 0.57
a
14.16 ± 0.59
b
15.16 ± 0.50
c
16.08 ± 0.56
c
Values are means ± SD of six rats in each group. CON – control rats; HFFD – high fat–high fructose-fed rats;
HFFD – CQEt – high fat–high fructose-fed rats treated with Cissus quadrangularis extract (10 g/100 g diet);
HFFD + MET – high fat–high fructose-fed rats treated with metformin (50 mg/kg b.w).
CON +CQEt – control rats treated with Cissus quadrangularis extract (10 g/100 g diet).
Values not sharing a common superscript were significantly differ from each other (P< 0.05, ANOVA followed by Duncan’s multiply range test (DMRT)).
Table 5
Levels of lipid peroxidation indices such as thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LHP) in liver.
Parameters CON HFFD HFFD + CQEt HFFD + MET CON + CQEt
TBARS (
g mol/mg protein) 2.89 ± 0.15
c
4.51 ± 0.45
a
3.52 ± 0.23
b
3.01 ± 0.25
c
2.93 ± 0.19
c
LHP ( g mol/mg protein) 1.49 ± 0.07
c
4.92 ± 0.44
a
2.49 ± 0.17
b
1.64 ± 0.12
c
1.50 ± 0.07
c
Protein carbonyl ( l g/mg protein) 1.08 ± 0.07
c
2.42 ± 0.18
a
1.66 ± 0.13
b
1.29 ± 0.13
c
1.12 ± 0.09
c
Values are means ± SD of six rats in each group. CON – control rats; HFFD – high fat–high fructose-fed rats;
HFFD – CQEt – high fat–high fructose-fed rats treated with Cissus quadrangularis extract (10 g/100 g diet);
HFFD + MET – high fat–high fructose-fed rats treated with metformin (50 mg/kg b.w).
CON +CQEt – control rats treated with Cissus quadrangularis extract (10 g/100 g diet).
Values not sharing a common superscript were significantly differ from each other (P< 0.05, ANOVA followed by Duncan’s multiply range test (DMRT)).
Table 6
Activities of enzymatic antioxidants in liver.
Parameters CON HFFD HFFD + CQEt HFFD + MET CON + CQEt
SOD (
l mol/min/mg protein) 8.85 ± 0.25
c
5.03 ± 0.33
a
7.05 ± 0.49
b
8.64 ± 0.19
c
8.74 ± 0.54
c
CAT ( l mol/min/mg protein) 72.23 ± 2.78
c
42.19 ± 3.74
a
59.08±.28
b
68.34 ± 4.22
c
71.60 ± 2.93
c
GPx ( l g/min/mg protein) 12.73 ± 0.61
c
5.69 ± 0.52
a
9.80 ± 0.67
b
11.86 ± 0.78
c
12.40 ± 0.65
c
Values are means ± SD of six rats in each group. CON – control rats; HFFD – high fat–high fructose-fed rats;
HFFD + CQEt – high fat–high fructose-fed rats treated with Cissus quadrangularis extract (10 g/100 g diet);
HFFD + MET – high fat–high fructose-fed rats treated with metformin (50 mg/kg b.w).
CON +CQEt – control rats treated with Cissus quadrangularis extract (10 g/100 g diet).
Values not sharing a common superscript were significantly differ from each other (P< 0.05, ANOVA followed by Duncan’s multiply range test (DMRT)).
2024 J. Chidambaram, A. Carani Venkatraman / Food and Chemical Toxicology 48 (2010) 2021–2029

ters returned to normal levels. There were no significant differ-
ences in these parameters between the CQEt-treated and untreated
control groups.
Histopathological examinations also demonstrated that CQEt
effectively stabilizes the hepatocyte against HFFD as seen by re-
duced severity of hepatic lesions. Fig. 1 shows photomicrographs
of H and E stained liver tissues. The histology of the liver appears
normal in the control group (Fig. 1A), while liver from HFFD group,
shows predominant microvesicular fatty change with focal macr-
ovasicular fatty change (Fig. 1B). Treatment with CQEt shows lesser
microvesicular fatty changes when compared with HFFD group
(Fig. 1C). Treatment with metformin preserved the hepatic archi-
Table 7
Activities of non-enzymatic antioxidants in liver.
Parameters CON HFFD HFFD + CQEt HFFD + MET CON + CQEt
GSH 159.65 ± 0.18
c
100.98 ± 6.82
a
130.47 ± 9.52
b
169.34 ± 10.74
c
160.75 ± 9.25
c
Vitamin C 0.78 ± 0.04
c
0.50 ± 0.02
a
0.63 ± 0.05
b
0.72 ± 0.05
c
0.76 ± 0.04
c
Vitamin E 6.49 ± 0.43
c
3.64 ± 0.32
a
5.21 ± 0.34
b
6.15 ± 0.38
c
6.28 ± 0.38
c
Values are means ± SD of six rats in each group. CON – control rats; HFFD – high fat–high fructose-fed rats;
HFFD + CQEt – high fat–high fructose-fed rats treated with Cissus quadrangularis extract (10 g/100 g diet);
HFFD + MET – high fat–high fructose-fed rats treated with metformin (50 mg/kg b.w).
CON +CQEt – control rats treated with Cissus quadrangularis extract (10 g/100 g diet).
Values not sharing a common superscript were significantly differ from each other (P< 0.05, ANOVA followed by Duncan’s multiply range test (DMRT)).
Fig. 1. Photomicrograhs of liver sections (H and E, 20X). (A) CON: normal hepatocytes showing normal architecture. (B) HFFD: micro and macro vesicular fatty change were
seen. (C) HFFD + CQEt (10 g/100 g diet): microvesicular fatty changes are mild (D) HFFD + MET: retains normal hepatic architecture. (E) CON + CQEt: shows normal
appearance.
J. Chidambaram, A. Carani Venkatraman / Food and Chemical Toxicology 48 (2010) 2021–2029 2025