Telechargé par m-elkabbaoui

1

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 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
a r t i c l e
i n f o
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
a b s t r a c t
The study evaluated the protective effects of Cissus quadrangularis stem extract (CQEt) on oxidant–antioxidant 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, following which the diet was fortiﬁed with CQEt at a dose of 10 g/100 g diet. After 60 days, HFFD caused deleterious 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 antioxidants, 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 antioxidants 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, vitamins 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.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver injury in many countries around the
world. It has a broad pathologic spectrum which ranges from simple fatty inﬁltration of the liver or steatosis, to nonalcoholic steatohepatitis (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 generation 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 development of steatohepatitis from steatosis induced by high-energy
diet (Barbuio et al., 2007). The development of NAFLD has been
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, 2,20 -diphenyl-1hydrazine; ABTS+, 2,20 -azino-di[3-ethylbenzthiazoline sulphonate].
* Corresponding author. Tel.: +91 (0) 4144 239141; fax: +91 (0) 4144 238080.
E-mail address: [email protected] (A. Carani Venkatraman).
0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.04.044
associated with a ‘‘two-hit hypothesis”, the ﬁrst hit being triglyceride and free fatty acid accumulation and second hit involving oxidative stress and inﬂammation of the liver (Demori et al., 2006).
Studies indicate that feeding HFFD can trigger all these pathological events and this model closely resembles human NAFLD.
The most common ROS are superoxide anion (O2), hydroxyl
radicals (OH) and hydrogen peroxide (H2O2). 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 believed to be involved in many metabolic, degenerative and inﬂammatory 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 natural 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 aspect reinforces the idea that the dietary inclusion of natural antioxidants present in plant foods is an important health-protecting and
disease-preventing factor in humans.
2022
J. Chidambaram, A. Carani Venkatraman / Food and Chemical Toxicology 48 (2010) 2021–2029
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. quadrangularis is traditionally used for the treatment of skin infections, constipation, piles, anemia, asthma, irregular menstruation, burns and
wounds (Kritikar and Basu, 2000). Studies have reported that
methanolic extract from C. quadrangularis (CQEt) have antimicrobial, 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 effectively 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 therapeutic action against hepatic damage induced by carbon tetrachloride
(CCl4) (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 glucose production in the liver by suppression of hepatic gluconeogenesis (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 systematic 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 extract 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 Central Animal House, Rajah Muthiah Medical College, Annamalai University. The animals 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 untreated. Accordingly ﬁve groups of rats were maintained for 60 days as follows:
Group
Group
Group
Group
day).
Group
diet).
I – CON – normal diet-fed rats.
II – HFFD-fed rats.
III – HFFD + CQEt – HFFD-fed rats treated with CQEt (10 g/100 g diet).
IV – HFFD + MET – HFFD-fed rats treated with metformin (50 mg/kg/
V – CON + CQEt – normal diet-fed rats treated with CQEt (10 g/100 g
Diet and water were provided ad libitium and body weight was recorded at regular 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) values. 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 phosphate 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 (lU/mL) glucose (mM)/22.5
1
logðglucose mg=dLÞ þ logðinsulin mU=LÞ
FIRI ¼ ðfasting insulin ðlU=mLÞ fasting glucoseðmg=dLÞÞ=25:
QUICKI ¼
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 following the method Niehaus and Samuelson (1968) and Jiang et al. (1992), respectively. 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 levels of non-enzymatic antioxidants such as reduced glutathione (GSH) by the method 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 dismutase (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 lm) 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 incubation 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 lg/mL), 0.2 mL of 500 lM 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% trichloro 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
2023
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 mixture indicated increased hydroxyl radical scavenging activity. The percentage scavenging was calculated as per the formula given below.
Scavenging ð%Þ ¼
Absorbance 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 PMSred. PMSred converted oxidized nitroblue tetrazolium
(NBToxi) to NBTred, which forms a violet colour. The reaction mixture in a ﬁnal volume of 2.5 mL contained, 1 mL of NBT (100 lmol NBT in 100 mM phosphate buffer,
pH 7.4), 1 mL of NADH solution (468 lmol in 100 mM phosphate buffer, pH 7.4) and
varying volumes of CQEt (10, 20, 30, 40 and 50 lg/mL). The reaction was started by
the addition of 100 lL PMS (60 lmol/100 mM phosphate buffer, pH 7.4). The reaction 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. Decreased absorbance of the reaction mixture indicated increased superoxide anionscavenging 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 reductants 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 (Fe3+) to form potassium ferrocyanide (Fe2+), which then reacts with ferric chloride to form ferric–ferrous complex that has as absorption maximum at 700 nm. Varying volumes of CQEt (10, 20, 30, 40 and 50 lg/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. Later, 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
FeCl3 (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.
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)
Insulin (lU/mL)
HOMA
QUICKI
FIRI
81.03 ± 5.16c
45.39 ± 5.68c
8.97 ± 0.43c
0.280 ± 0.001c
147.5 ± 7.51c
130.20 ± 9.42a
64.16 ± 6.12a
20.40 ± 0.96a
0.255 ± 0.002a
334.17 ± 24.23a
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)).
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 different concentrations of CQEt [20–100 lg] 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 2,20 -diphenyl-1-hydrazine
(DPPH) was determined by spectrophotometrically by the method of Brand-Williams et al. (1995). The reaction mixture in a total volume of 3 mL contained
1 mL of 100 lM DPPH in methanol, equal volumes of CQEt [10–50 lg] 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 2,20 -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 generation of the ABTS+ chromophore by the oxidation of ABTS+ (1.8 mM) with potassium 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 lg]
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 determinations 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, liver 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 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)
Liver weight(g)
Liver index
Glucose (mg/dL)
Insulin (lU/mL)
HOMA
QUICKI
FIRI
184.6 ± 12.7c
5.58 ± 0.57c
3.02 ± 0.23c
83.72 ± 5.92c
49.45 ± 4.13c
10.10 ± 0.79c
0.27 ± 0.001c
165.5 ± 7.51c
214.3 ± 15.1a
8.93 ± 0.79a
4.16 ± 0.24a
151.11 ± 8.65a
75.14 ± 6.23a
27.73 ± 1.4a
0.24 ± 0.003a
454.17 ± 24.23a
204.2 ± 14.5b
7.64 ± 0.62b
3.74 ± 0.32b
134.16 ± 6.98b
56.12 ± 5.84b
18.39 ± 0.64b
0.25 ± 0.002b
301.16 ± 16.02b
199.8 ± 13.9c
6.01 ± 0.50c
3.00 ± 0.36c
120.14 ± 4.58c
51.33 ± 4.34c
15.05 ± 0.70c
0.26 ± 0.001c
246.67 ± 12.11c
180.4 ± 13.8c
5.5 ± 0.52c
3.05 ± 0.28c
80.23 ± 5.91c
46.97 ± 5.33c
9.20 ± 0.37c
0.27 ± 0.002c
150.73 ± 7.22c
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)).
2024
J. Chidambaram, A. Carani Venkatraman / Food and Chemical Toxicology 48 (2010) 2021–2029
Table 3
Levels of liver marker enzymes in plasma (IU/L).
Parameters
ALP
GGT
SGPT
SGOT
CON
HFFD
c
HFFD + CQEt
a
41.82 ± 1.90
19.84 ± 0.94c
51.97 ± 2.23c
41.82 ± 2.18c
b
78.70 ± 5.64
41.15 ± 3.25a
88.96 ± 6.79a
88.81 ± 6.05a
52.69 ± 3.59
29.45 ± 2.16b
63.27 ± 3.88b
54.98 ± 3.68b
HFFD + MET
CON + CQEt
c
43.55 ± 2.41
21.61 ± 0.99c
53.15 ± 3.68c
44.46 ± 2.93c
40.55 ± 2.04c
20.77 ± 1.35c
52.04 ± 1.76c
43.77 ± 1.92c
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)).
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 signiﬁcantly (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 cholesterol 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 different groups. Signiﬁcantly higher TBARS, LHP, and protein carbonyl levels were found in HFFD-fed rats compared with control
rats. In CQEt-treated HFFD-fed rats, TBARS, LHP, and protein carbonyl 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-enzymatic 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 control. In CQEt and metformin-treated HFFD-fed rats, these parame-
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
TG
FFA
Phospholipid
3.06 ± 0.0.21c
4.16 ± 0.31c
4.13 ± 0.31c
16.10 ± 0.55c
8.05 ± 0.42a
7.04 ± 0.32a
7.09 ± 0.51a
12.28 ± 0.57a
6.33 ± 0.53b
6.12 ± 0.33b
6..05 ± 0.33b
14.16 ± 0.59b
5.37 ± 0.40c
5.09 ± 0.29c
5.10 ± 0.40c
15.16 ± 0.50c
3.08 ± 0.25c
4.21 ± 0.33c
4.15 ± 0.34c
16.08 ± 0.56c
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 (gmol/mg protein)
LHP (gmol/mg protein)
Protein carbonyl (lg/mg protein)
2.89 ± 0.15c
1.49 ± 0.07c
1.08 ± 0.07c
4.51 ± 0.45a
4.92 ± 0.44a
2.42 ± 0.18a
3.52 ± 0.23b
2.49 ± 0.17b
1.66 ± 0.13b
3.01 ± 0.25c
1.64 ± 0.12c
1.29 ± 0.13c
2.93 ± 0.19c
1.50 ± 0.07c
1.12 ± 0.09c
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 (lmol/min/mg protein)
CAT (lmol/min/mg protein)
GPx (lg/min/mg protein)
8.85 ± 0.25c
72.23 ± 2.78c
12.73 ± 0.61c
5.03 ± 0.33a
42.19 ± 3.74 a
5.69 ± 0.52a
7.05 ± 0.49b
59.08±.28 b
9.80 ± 0.67 b
8.64 ± 0.19c
68.34 ± 4.22c
11.86 ± 0.78c
8.74 ± 0.54c
71.60 ± 2.93c
12.40 ± 0.65c
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)).
2025
J. Chidambaram, A. Carani Venkatraman / Food and Chemical Toxicology 48 (2010) 2021–2029
Table 7
Activities of non-enzymatic antioxidants in liver.
Parameters
GSH
Vitamin C
Vitamin E
CON
HFFD
c
159.65 ± 0.18
0.78 ± 0.04c
6.49 ± 0.43c
HFFD + CQEt
a
100.98 ± 6.82
0.50 ± 0.02a
3.64 ± 0.32a
HFFD + MET
b
130.47 ± 9.52
0.63 ± 0.05b
5.21 ± 0.34b
169.34 ± 10.74
0.72 ± 0.05c
6.15 ± 0.38c
CON + CQEt
c
160.75 ± 9.25c
0.76 ± 0.04c
6.28 ± 0.38c
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)).
ters returned to normal levels. There were no signiﬁcant differences 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 reduced 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 macrovasicular 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-
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.
2026
J. Chidambaram, A. Carani Venkatraman / Food and Chemical Toxicology 48 (2010) 2021–2029
tecture (Fig. 1D). CQEt treatment of rats fed the control diet had no
signiﬁcant effect, with liver sections appearing normal (Fig. 1E).
Fig. 2 shows photomicrographs of oil red O stained liver specimens. The histology of liver is normal in the control group (Fig. 2A).
In HFFD group, liver shows widespread deposition of lipid droplets
inside the parenchyma cells (Fig. 2B). Liver from the CQEt group
showed scattered droplets of fat when compared with HFFD group
(Fig. 2C). Fat staining is negligible in metformin treated rats
(Fig. 2D). The liver from CQEt-treated group shows normal appearance (Fig. 2E).
The results of biochemical tests together with histological
observations suggest that CQEt treatment improves IR, lowers steatosis and prevents peroxidative damage and the effects comparable with that of metformin.
3.2. In vitro assays
3.2.1. Total phenolic content, vitamins C and E
The total polyphenolic content of CQEt was found to be
585.40 ± 0.16 mg GAE/100 g and the levels of vitamins C and E
were estimated to be 327 mg/100 g and 696 mg/100 g of powdered
extract, respectively.
3.2.2. Hydroxyl radical scavenging activity
Fig. 3 shows the percentage hydroxyl radical scavenging activity
of CQEt, gallic acid and vitamin C at various concentrations. Addition of CQEt scavenged hydroxyl radical in a concentration-dependent manner. CQEt was a better scavenger when compared with
the reference compounds. At 50 lg/mL concentration, the extract
signiﬁcantly inhibited (83%) degradation of deoxyribose mediated
by the hydroxyl radicals compared to gallic acid (69%) and vitamin
C (81%).
3.2.3. Superoxide anion-scavenging activity
Superoxide anion, is an important primary free radical since the
biological system converts it into more reactive OH radical and
singlet oxygen. Fig. 4 shows that scavenging of superoxide anion
by CQEt was proportional to concentration of the extract added
and was comparable to that of gallic acid and vitamin C. The scavenging capacity of the CQEt was 42%, gallic acid and vitamin C
showed 76% and 48% at 40 lg/mL.
Fig. 2. Lipid staining of rat liver sections (oil red O, 40); (A) CON: normal. (B) HFFD: widespread deposition of lipid droplets inside the parenchymal cells. (C) HFFD + CQEt
(10 g/100 g diet): scattered droplets of fat are seen. (D) HFFD + MET: retains normal structure. (E) CON + CQEt: shows normal appearance.
2027
J. Chidambaram, A. Carani Venkatraman / Food and Chemical Toxicology 48 (2010) 2021–2029
70
90
#
*
80
60
#
60
% Scavenging
% Scavenging
70
50
40
30
20
10
50
40
#
#
# *
*
*
*
30
20
10
0
10
20
30
40
0
50
20
Concentration (µg/ml)
40
60
80
100
Concentration (µg/ml)
Fig. 3. Hydroxyl radical scavenging action of CQEt and standard antioxidants (N –
CQEt, – gallic acid, j – vitamin C).
90
80
*#
70
#
60
*#
60
50
*
*
40
*
30
– CQEt,
80
% Inhibition
% Scavenging
70
#
#
#
#
Fig. 6. Nitric oxide scavenging effect of CQEt and standard antioxidants (
– gallic acid,
– vitamin C).
20
10
*#
*
50
*
40
30
20
0
10
20
30
40
10
50
Concentration (µg/ml)
0
10
Fig. 4. Superoxide anion-scavenging action of CQEt and standard antioxidants (N –
CQEt, – gallic acid, j – vitamin C).
3.2.4. Reducing power
The reduction potential of CQEt at various concentrations is presented in Fig. 5. There was a concentration-dependent increase in
the reducing power of the extract as determined by the colour formation due to Fe2+–Fe3+ transition. However, CQEt showed a less
reducing power as compared with gallic acid and vitamin C.
3.2.5. Nitric oxide radical scavenging activity
Fig. 6 shows that the scavenging of nitric oxide by CQEt increases with increasing concentration. CQEt scavenged nitric oxide
20
30
40
50
Concentration (µg/ml)
Fig. 7. DPPH scavenging capacity of CQEt and standard antioxidants. (N – CQEt, –
gallic acid, j – vitamin C).
in a concentration-dependent manner. Maximum scavenging
activity was observed at 50 lg/mL among the doses tested and this
effect was comparable to gallic acid and vitamin C.
3.2.6. DPPH radical scavenging assay
Fig. 7 shows that the inhibition of DPPH radical formation was
proportional to increasing concentrations of the CQEt. At a concentration of 50 lg/mL, CQEt shows maximum inhibition capacity
0.5
#
#
0.3
#
*
45
*
*
40
*
*
% Inhibition
Absorbance at 700nm
0.4
50
#
#
0.2
0.1
35
30
25
20
15
10
5
0
10
20
30
40
0
50
20
Concentration (µg/ml)
40
60
80
100
Concentration (µg/ml)
Fig. 5. Reducing power of CQEt and standard antioxidants (
acid,
– vitamin C).
– CQEt,
– gallic
+
Fig. 8. ABTS radical scavenging ability of CQEt (
– CQEt).
2028
J. Chidambaram, A. Carani Venkatraman / Food and Chemical Toxicology 48 (2010) 2021–2029
(40%) and this effect was comparable to the effect of gallic acid and
vitamin C.
3.2.7. Antiradical activity against ABTS+
Fig. 8 depicts a decrease in the absorbance at 734 nm in the
presence of the CQEt at concentrations 20–100 lg/mL. The percentage inhibition was concentration-dependent with maximum inhibition of 43% at 100 lg/mL. The standard drug trolox showed 92%
inhibition of colour formation at 100 lg/mL (data not shown in
ﬁgure).
4. Discussion
Administration of HFFD diet induces the development of metabolic syndrome characterized by obesity, IR and liver steatosis
(Angulo, 2002). A body of evidence indicates that accumulation
of fat in the liver increases the susceptibility to other insults such
as oxidative stress and subsequent inﬂammation that results in the
progression of steatosis to steatohepatitis, ﬁbrosis and cirrhosis
(Koteish and Diehl, 2002). Considering the recently recognized
association between IR, oxidative stress and inﬂammation, the
present experiment conﬁrms that the combination of fructose
and fat could result in oxidative liver injury. Induction of oxidative
stress is evident from the increased peroxidation markers and
inadequate antioxidant status in the blood and liver of rats fed
HFFD. The markers of oxidative injury (TBARS, LHP and protein carbonyl) were signiﬁcantly elevated. CQEt could effectively protect
against the hepatic oxidative stress induced by HFFD. These ﬁndings are concordant with those of other investigators (Oben et al.,
2006).
Moderate but signiﬁcant elevations in blood glucose in the presence of hyperinsulinemia in rats fed HFFD indicate IR. The presence
of IR is further indicated by higher values of HOMA-IR, QUICKI and
FIRI. These ﬁndings are consistent with our early reports (Thirunavukkarasu and Anuradha, 2004) and those of other investigators (Aragno et al., 2009).
HFFD administration was associated with hepatocellular damage and microvesicular steatosis. The increased activities of marker
enzymes, AST, ALT, ALP and GGT are suggestive of liver injury.
Treatment with CQEt notably prevented the elevation of these enzymes to an extent that was comparable to the standard drug metformin. CQEt prevents liver cell damage and preserves cell integrity
possibly leading to survival of the functionally active cells. These
results are in line with the ﬁndings reported by Jainu and Devi
(2005) who observed the hepatoprotective action of CQEt in CCl4treated rats.
Levels of total lipid and those of cholesterol, TG and FFA were
signiﬁcantly elevated in liver. Fatty liver is a serious risk factor
for the development of liver injury. Results of the histological
changes in HFFD rats, such as widespread deposition of lipid droplets inside the parenchymal cells are consistent with the result of
the biochemical analysis. Evidence of lipid accumulation in liver
exposed to HFFD (Aragno et al., 2009) and in rats drinking fructose-sweetened beverages (Jurgens et al., 2005) has been reported.
Fructose is highly lipogeneic and the HFFD diet used in this study
may have resulted in the increased delivery of fatty acids through
the portal circulation resulting in fatty liver. Treatment of HFFDfed rats with CQEt showed considerable restoration of lipid levels
to that of control. Lipid dysregulation in fructose-fed rat model
has been associated to the activation of oxidative stress and
inﬂammatory pathways in the liver which favours the progression
to NAFLD (Basciano et al., 2005). An evolving hypothesis is that
metabolic disease, ROS formation and inﬂammation create a progressive cycle leading to disease progression and NAFLD (Raval
et al., 2006).
The derangement in enzymatic antioxidant potential indicates
that HFFD-fed rats are unable to cope up with excess free-radical
formation and oxidative damage. The reduction in the activities
of enzymatic antioxidants results in the accumulation of free radicals, which lead to tissue damage (Peterhans, 1997). Our results
show that HFFD caused signiﬁcant decreases in SOD, CAT, GPx
and GST activities. CQEt supplementation improved the antioxidant defense mechanisms and suppressed oxidative damage in
HFFD-fed rats.
Non-enzymatic antioxidants such as GSH, vitamins C and E are
closely interlinked to each other and participate to form an antioxidant network. This helps to regenerate one another from their oxidized forms there by playing an excellent role in protecting the
cells from oxidative damage. One of the most prominent antioxidant in the liver is GSH which is present in high concentration than
any other antioxidant. Liver GSH concentration reﬂects the detoxiﬁcation potential in the liver. Our results show decreased hepatic
GSH in rats receiving HFFD which indicates enhanced oxidation of
GSH to the oxidized form (GSSG). Consequent to this, vitamins C
and E level also reduced in HFFD-fed groups. These ﬁndings are
in agreement with our previous data in fructose-fed rats demonstrating a decrease of antioxidant defense with histological signs
of hepatic steatosis and liver malfunction (Sumiyabanu et al.,
2009). In this regard, Aragno et al. (2009) have also observed that
HFFD induces oxidative stress in the liver, impairing the activities
of antioxidants such as GSH and SOD. The administration of CQEt
improved GSH, vitamins E and C status in HFFD-fed rat liver.
Plant phenolics constitute one of the major groups of compounds acting as primary antioxidants or free-radical terminators
(Cao et al., 1997). They possess a wide spectrum of biochemical
activities such as antioxidant, antimutageneic, anticarcinogenic
as well as ability to modify the gene expression. In our study, the
methanolic extract contained substantially high amount of polyphenolic compounds which is comparable to that present in common food products and other plant sources reported in the
literature by other investigators (Gupta and Nair, 1997). The study
observed signiﬁcant free-radical scavenging activity of CQEt
against a wide variety of free radicals in a concentration-dependent manner among the tested doses which was comparable with
standard antioxidants. Speciﬁcally, CQEt was more effective in neutralizing OH radical when compared to the gallic acid and vitamin
C. Other investigators have evidenced the free-radical scavenging
activity of CQEt against superoxide radical (Chidambara Murthy
et al., 2003) and ABTS+ (Jainu and Devi, 2005). Also, CQEt reduced
tetra butyl hydroperoxide-induced erythrocyte lipid peroxidation
in vivo (Jainu and Devi 2005).
The scavenging potential of CQEt might be due to the presence
of phenolics and antioxidant vitamins. The active compounds identiﬁed in C. quadrangularis include vitamin C, b-carotene, tritepenoids, b-sitosterols, ﬂavonoids and quadrangularis A, B, and C
(Mehta et al., 2001; Chidambara Murthy et al., 2003), most of
which are potent antioxidants. Di and monohydroxyl substitutions
in the aromatic ring of ﬂavonoid structure confer potent hydrogen
donating ability and thus provide protection against oxidative
damage induced by the free radicals.
In vitro assays provide direct evidence that CQEt possesses potent free-radical scavenging effect while studies in vivo demonstrate hepatoprotective value by inhibiting liver steatosis and by
mitigating oxidative damage. Plasma glucose, insulin and IR were
favourably modiﬁed by CQEt, suggesting that changes in insulin-related parameters in the liver might contribute to the beneﬁcial effects of CQEt on steatohepatitis. Reports concerning the role of CQEt
in connection with insulin sensitivity are not available in the literature and this study is the ﬁrst to suggest that CQEt may enhance
insulin sensitivity. Although many synthetic antioxidants are
promising compounds for various human ailments, their pro-oxi-
J. Chidambaram, A. Carani Venkatraman / Food and Chemical Toxicology 48 (2010) 2021–2029
dant or cytotoxic nature at higher concentrations prevents them
from long term use (Barlow, 1990). Plant extracts are generally less
expensive to obtain, more accessible to the population and are
wholesome. Since, IR is widely prevalent in the population, the
inclusion of C. quadrangularis in daily diet may be worthy in fatty
liver disease associated with IR.
5. Conclusion
Our data clearly indicates that CQEt has considerable antioxidant activity in vitro and exerts its protective effect on HFFD-induced toxicity through interrelated mechanisms such as
improvement of insulin sensitivity, lowering of lipid levels, scavenging free radicals and enhancement of antioxidant protection.
This preliminary study indicates a need for further studies on the
chemical analysis of CQEt and molecular mechanisms of its protective action in vivo. Whether CQEt interrupts the inﬂammatory cascade and redox-sensitive cell signaling are being investigated in
our laboratory.
Conﬂict of Interest
The authors declare that there are no conﬂicts of interest.
Acknowledgements
The authors thank Dr. Pradeepa, Professor, Department of
Pathology, Sri Ramachandra Medical College and Hospital, Porur,
Chennai for her help in histopathological studies.
References
Angulo, P., 2002. Non alcoholic fatty liver disease. N. Engl. J. Med. 18 (346), 1221–
1231.
Anitha Nandhini, A.T., Balakrishnan, S.D., Anuradha, C.V., 2002. Taurine modulates
antioxidant potential and controls lipid peroxidation in the aorta of high
fructose-fed rats. J. Biochem. Mol. Biol. Biophys. 6, 129–133.
Anoop, A., Jagdeesan, M., 2002. Gastric and duodenal antiulcer and cytoprotective
effect of Cissus quadrangularis Linn. variant II in rats. Nigerian J. Nat. Prod. Med.
6, 1–7.
Aragno, M., Tomasinelli, C.E., Vercellinatto, I., Catalano, M.G., Collino, M., Fantozzi,
R., Danni, O., Boccuzzi, G., 2009. SREBP-1c in NAFDL induced by western-type
high-fat diet plus fructose in rats. Free Radical Biol. Med. 47, 1067–1074.
Assy, N., Kaita, K., Mymin, D., Levy, C., Rosser, B., Minuk, G., 2000. Fatty inﬁltration of
liver in hyperlipidemic patients. Dig. Dis. Sci. 45, 1929–1934.
Baker, H.O., Fuanko, B., De Angelis, S., Feingold, F., 1980. Plasma tocopherol in man
at various times after ingesting free or acetylated tocopherol. Nutr. Rep. Int. 21,
531–536.
Barbuio, R., Milanski, M., Bertolo, M.B., Saad, M.J., Vellosa, L.A., 2007. Inﬂiximab
reverses steatosis and improves insulin signal transduction in liver of rats fed a
high-fat diet. J. Endocrinol. 194, 539–550.
Barlow, S.M., 1990. Toxicological aspects of antioxidants used as food additives. In:
Hudson, B.J.F. (Ed.), Food Antioxidants. Elsevier, Amsterdam, p. 23.
Basciano, H., Federico, L., Adeli, K., 2005. Fructose, insulin resistance, and metabolic
dyslipidemia. Nutr. Metab. (Lond.) 2, 5–19.
Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of free radical method to
evaluate antioxidant activity. Lebensm.-Wiss. Technol. 28, 25–30.
Cao, G., Soﬁc, E., Prior, R.L., 1997. Antioxidant and prooxidant behavior of
ﬂavonoids: structure–activity relationships. Free Radical Biol. Med. 22 (5),
749–756.
Chidambara Murthy, K.N., Vanitha, A., Mahadeva Swamy, M., Ravishanker, G.A.,
2003. Antioxidant and antimicrobial activity of Cissus quadrangularis Linn.. J.
Med. Foods 6, 99–105.
Chopra, S.S., Patel, M.R., Gupta, L.P., Datta, I.C., 1975. Studies on Cissus qudrangularis
in experimental fracture repair: effect on chemical parameters in blood. Indian
J. Med. Res. 63, 824–828.
Demori, I., Voci, A., Fugassa, E., Burlando, B., 2006. Combined effects of high-fat and
ethanol induce oxidative stress in rat liver. Alcohol 40, 185–191.
Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77.
Falholt, K., Lund, B., Falholt, W., 1973. An easy colorimetric micromethod for routine
determination of free fatty acids in plasma. Clin. Chim. Acta 46, 105–111.
Folch, J., Lees, M., Sloane Stanley, G.H., 1957. A simple method for the isolation and
puriﬁcation of total lipids from animal tissues. J. Biol. Chem. 226, 497–509.
Foster, L.B., Dunn, R.T., 1973. Stable reagents for determination of serum
triglycerides by a colorimetric Hantzsch condensation method. Clin. Chem.
19, 338–340.
2029
Garrat, D.C., 1964. The Quantitative Analysis of Drugs, vol. 3. Chapman and Hall,
Japan. pp. 456–458.
Gupta, R., Nair, S., 1997. Antioxidant ﬂavonoids in common Indian diet. South Asian
J. Prevent. Cardiol. 3, 83–94.
Halliwell, B., Gutteridge, J.M., Aruoma, O.I., 1987. The deoxyribose method: a simple
test-tube assay for determination of rate constants for reactions of hydroxyl
radicals. Anal. Biochem. 165 (1), 215–219.
Havsteen, B.H., 2002. The biochemistry and medical signiﬁcance of the ﬂavonoids.
Pharmacol. Ther. 96, 67–202.
Jainu, M., Devi, C.S.S., 2005. In vitro and in vivo evaluation of free radical scavenging
potential of Cissus quadrangularis. Afr. J. Biomed. Res. 8, 95–99.
Jiang, Z.Y., Hunt, J.V., Wolf, S.P., 1992. Detection of lipid hydroperoxides using the
FOX method. Anal. Biochem. 202, 384–389.
Jurgens, H., Haas, W., Castanedana, T.R., 2005. Consuming fructose-sweetened
beverages increases body adiposity in mice. Obes. Res. 13, 1146–1156.
Koteish, A., Diehl, A.M., 2002. Animal models of steatohepatitis. Semin. Liver Dis. 21,
89–104.
Kovacic, P., Cooksy, A., 2005. Iminium metabolite mechanism for nicotine toxicity
and addiction: oxidative stress and electron transfer. Med. Hypotheses 64 (1),
104–111.
Kritikar, K.R., Basu, B.D., 2000. In: Basu, L.M. (Ed.), Indian Medicinal Plants. Lalit
Mohan Basu Publisher, Allahabad, India, pp. 841–843.
Levine, R.L., Garland, D., Oliver, C.N., Amici, A., Climent, I., Lenz, A.G., Ahn, B.W.,
Shaltiel, S., Stadtman, E.R., 1990. Determination of carbonyl content in
oxidatively modiﬁed proteins. Methods Enzymol. 186, 464–478.
Mehta, M., Kaur, N., Bhutani, K.K., 2001. Determination of marker constituents from
Cissus quadrangularis Linn. and their quantitation by HPTLC and HPLC.
Phytochem. Anal. 12 (2), 91–95.
Niehaus, W.G., Samuelson, S., 1968. Formation of malondialdehyde from
phospholipids arachidonate during microsomal lipid peroxidation. Eur. J.
Biochem. 6, 126–130.
Nishimiki, M., Rao, N.A., Yagi, K., 1972. The occurrence of superoxide anion in the
reaction of reduced phenazine methosulphate and molecular oxygen. Biochem.
Biophys. Res. Commun. 46, 849.
Oben, J., Kuate1, D., Agbor, G., Momo, C., Talla, X., 2006. The use of a Cissus
quadrangularis formulation in the management of weight loss and metabolic
syndrome. Lipids Health Dis. 5, 24.
Oyaizu, M., 1986. Studies on product of browning reaction prepared from glucose
amine. Jpn. J. Nutr. 44, 307–315.
Peterhans, E., 1997. Oxidants and antioxidants in viral diseases: disease
mechanisms and metabolic regulation. J. Nutr. 127, 962–965.
Prasad, G.C., Udupa, K.N., 1963. Effect of Cissus quadrangularis on the healing of
cortisone treated fractures. Indian J. Med. Res. 51, 667–676.
Pryor, W.A., Ann, N.Y., 1982. Free radical biology: xenobiotics, cancer, and aging.
Acad. Sci. 393, 1–22.
Raval, J., Lyman, S., Nitta, T., Mohuczy, D., Lemasters, J.J., Kim, J.S., 2006. Basal
reactive oxygen species determine the susceptibility to apoptosis in cirrhotic
hepatocytes. Free Radical Biol. Med. 41, 1645–1654.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999.
Antioxidant activity applying an improved ABTS radical cation decolorisation
assay. Free Radical Biol. Med. 26 (9/10), 1231–1237.
Roe, J.H., Kuether, C.A., 1942. A color reaction for dehydroascorbic acid useful in the
determination of vitamin C. Science 95, 77.
Shariff, Z.U., 2001. Modern Herbal Therapy for Common Ailments. Nature Pharmacy
Series, vol. 1. Spectrum Books Ltd., Ibadan, Nigeria in Association with Safari
Books (export) Ltd., UK, pp. 9–84.
Shalam, M.D., Harish, M.S., Farhana, S.A., 2006. Prevention of dexamethasone- and
fructose-induced insulin resistance in rats by SH-01D, a herbal preparation.
Indian J. Pharmacol. 38, 419–422.
Singleton, S.L., Rossi, J.A., 1965. Colorimetry of total phenolics with
phosphomolybdic phosphotungstic acid reagents. Am. J. Enol. Viticult. 16, 44–
158.
Snoog, Y.Y., Barlow, P.J., 2004. Antioxidant activity and phenolic of selected fruit
seeds. Food Chem. 88, 411–417.
Spencer, J.P.E., Jenner, A., Aruoma, O.I., Evans, P.J., Kaur, H., Dexter, D.T., 1994.
Intense oxidative DNA damage promoted by L-DOPA and its metabolites,
implications for neurodegenerative disease. FEBS Lett. 353, 246–250.
Srividhya, S., Anuradha, C.V., 2002. Metformin improves liver antioxidant potential
in rats fed a high-fructose diet. Asia Pac. J. Clin. Nutr. 11 (4), 319–322.
Subbu, V.S.V., 1970. Mechanism of action of Vitis glucoside on myocardial tissue.
Indian J. Med. Sci. 25, 400–403.
Sumiyabanu, M., Palanisamy, N., Pooranaperundevi, M., Viswanathan, P.,
Anuradha, C.V., 2009. Genistein improves liver function and attenuates
non-alcoholic fatty liver disease in a rat model of insulin resistance. J.
Diabetes 1, 278–287.
Thirunavukkarasu, V., Anuradha, C.V., 2004. Inﬂuence of lipoic acid on lipid
peroxidation and antioxidant defence system in blood of insulin resistant
rats. Diabetes Obes. Metab. 6 (3), 200–207.
Woifenden, B.S., Willson, R.L., 1982. Radical cations as reference chromogens in
kinetic studies of one-electron transfer reactions: pulse radiolysis studies of
2,20 -azinobis-(3-ethylbenzthiazoline-6-sulphonate). J. Chem. Soc. Perkin Trans.
2, 805–812.
Zilversmit, D.B., Davis, A.K., 1950. Microdetermination of plasma phospholipids by
means of precipitation with trichloroacetic acid. Laboratorio 10, 127–135.
Zlatkis, A., Zak, B., Boyle, A.J., 1953. A new method for the direct determination of
serum cholesterol. J. Lab. Clin. Med. 41, 486–492.