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. 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