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Pharmacologia
Year: 2015  |  Volume: 6  |  Issue: 3  |  Page No.: 97 - 105

Protective Effects of Saponin-Rich Aqueous Leaf Extract of Gymnema sylvestre R. Br. against Cafeteria and High-Fat Diet-Induced Obesity and Oxidative Stress in Rats

Indireddy Rama Manohar Reddy, Pasupuleti Visweswara Rao, Bobbu Pushpa Latha and Tartte Vijaya    

Abstract: Background and Objectives: The present study was aimed to investigate the protective effect of saponin-rich aqueous leaf extract of Gymnema sylvestre (SGE) on obesity and oxidative stress in rats fed with cafeteria (CA) and high-fat (HF) diets. Methodology: Male Wistar rats were fed CA and HF diets for 8 weeks to induce obesity and oxidative stress. The SGE (100 mg kg-1) was administered orally to CA and HF fed rats for 8 weeks. Changes in body weight gain, food consumption, fat storage, serum parameters, fecal lipid excretion and liver oxidative stress parameters were measured. Results: SGE showed protective effect on body weight gain, food consumption and visceral fat accumulation. The SGE treatment elicited a significant reduction in serum levels of glucose, atherogenic index, total cholesterol, triglycerides, Low-Density Lipoproteins-Cholesterol (LDL-C), Very Low Density Lipoproteins-Cholesterol (VLDL-C) and while High-Density Lipoproteins-Cholesterol (HDL-C) level increased. Furthermore, SGE treatment decreased the liver lipids, lipid peroxidation and increased antioxidant levels and fecal lipid excretion in obese rats. Conclusion: In conclusion, intake of SGE may be a feasible therapeutic strategy for prevention of cafeteria and high-fat diet- induced obesity and oxidative stress.

1. In the experimental setting, animals provided with cafeteria and high fat diets associated with over obesity were likewise documented with elevated body weight gain, lipid profiles and oxidative stress. Furthermore, high fat diet leads to an increase in oxidative stress levels2. Oxidative stress has been shown to be involved in the process of atherogenesis3, ischemic heart disease4 and obesity5.

Oxidative stress is highly correlated with a wide variety of inflammatory and metabolic disease states, including obesity6. It is highly correlated with cumulative damage in the body done by free radicals inadequately neutralized by antioxidants6. It has been shown that free radicals may adversely affect cell survival because of membrane damage through the oxidative damage of lipid, protein and irreversible DNA modification7. Lipid peroxidation such as thiobarbituric acid reactive substances and hydroperoxide levels as well as markers of protein oxidation such as carbonyl proteins are markers of oxidative damage of Reactive Oxygen Species (ROS)8.

Furthermore, oxidative damage is aggravated by the decrease in antioxidant enzymes activities such as superoxide dismutase, catalase, glutathione S-transferase and glutathione peroxidase which acts as free radical scavengers in conditions associated with oxidative stress9. Evidence suggests that a clustering of sources of oxidative stress exists in obesity, hyperglycemia, increased tissue lipid levels, inadequate antioxidant defenses, increased rates of free radical formation and chronic inflammation10. Obesity affects several organs in the body such as liver, heart and kidney. Fatty liver and nephropathy are commons complication of obesity11. Atherosclerosis and cardiac complications are more common among obese individuals12. Obesity therapies include reduction of nutrient absorption and administration of drugs that affects lipid mobilization and utilization. Owing to the adverse side effects associated with many anti-obesity drugs, more recent drug trials have focused on screening for natural sources that have been reported to reduce body weight and that generally have minimal side effects13.

Gymnema sylvestre R.Br is a wild plant classified in the Asclepiadaceae family and is a woody perennial that grows in the tropical forests of central and southern India and has a deep history rooted in Ayurvedic medicine. Leaves of G. sylvestre have been used as stomachic, diuretic and antidiabetic remedy. The total saponin fraction of the leaves commonly known as gymnemic acid, has an anti-sweetening effect14, hypolipidemic activity15 and hepatoprotective activity16. As for the active substances involved in G. sylvestre, the triterpenoid saponin and its derivatives have been identified. These are glycosides where gymnemagenin is formed by attachment of glucuronic acid to the triterpenoid structure as aglycone. This glycoside and its derivatives are referred to as gymnemic acids17. It has been reported that the saponin of Platycodi radix showed strong inhibitory effect of body weight, calorie intake, fat storage, hypolipidemic and lipase inhibition and increased fecal lipid excretion18. Therefore, the present study aimed to investigate the protective effect of saponin-rich G. sylvestre aqueous leaf extract in rats fed with cafeteria and high fat diets induced obesity and oxidative stress.

MATERIALS AND METHODS

Plant material: Leaves of G. sylvestre R. Br were collected from Tirumala hills, A. P., India and identified by taxonomist of the Department of Botany, Sri Venkateswara University, Tirupati, A. P., India. A voucher specimen was deposited for future reference.

Extraction of saponin fraction: The aqueous extraction was carried out as described earlier19. Five hundred grams of shade dried G. sylvestre leaves were powdered and extracted twice with 4 L of distilled water in Soxhlet extractor at 60°C for 5 h. After filtration, extracts were combined and acidified with 1 M sulfuric acid to pH 2.0. The precipitate was filtered, dried and then extracted with ethanol and acetone. The insoluble matter was eliminated by filtration and solvents were evaporated. The dark green powder (7.1 g) was designated as SGE and used in the feeding experiment. The fraction was tested for saponins using Froth test and Libermann-Burchard test20.

Acute toxicity studies: The acute toxicity of SGE was determined according to guideline No. 420 of the Organization for European Economic Cooperation (OECD) using male Wistar rats (120±10 g). Initial doses of 100, 400, 800, 1200, 1600 and 2000 mg kg-1 b.wt. of SGE were administered to the respective six groups of four rats each and monitored for three weeks for mortality and general behaviour. Toxic symptoms of mortality were observed till the end of the study with doses of 800-2000 mg kg-1 b.wt. The lethal dose i.e., LD50 was determined as 400 mg kg-1 b.wt. Hence, the experimental dose was selected as one-fourth (100 mg kg-1 b.wt.) of the LD50.

Animal diets: The cafeteria diet consisted of three diets: (a) 10 g condensed milk+10 g bread+5 g pellet chow (4:4:2), (b) 3.75 g chocolate+7.5 g biscuits+7.5 g dried coconut+6.25 g pellet chow (1.5:3:3:2.5) and (c) 10 g cheese+12.5 g boiled potatoes+2.5 g pellet chow (4:5:1). The three variants were presented to the individual rats on days one, two and three, respectively and then repeated for eight weeks in the same succession21. High fat diet (39% carbohydrate, 21.5% fat, 34.5% protein and 5% mineral and vitamin mixture AIN 93) was purchased from National Institute of Nutrition, Hyderabad, A.P., India.

Animals: Male wistar rats (130-140 g) were used in the present study were purchased from Sri Venkateswara Animal Traders, Bangalore. The rats were fed and water ad libitum and maintained under controlled laboratory conditions (12 h light/dark, temp. 26±2°C; relative humidity 50±10%). The animals were divided into six groups of six animals each as follows: Group 1 control for normal diet (N diet), (2) Normal diet+saponins rich G. sylvestre aqueous leaf extract (N diet+SGE), (3) control for cafeteria diet (CA diet), (4) Cafeteria diet+SGE (CA diet+SGE), (5) Control for high fat diet (HF diet) and (6) High fat diet+SGE (HF+SGE). SGE at a dose of 100 mg kg-1 b.wt. was administered for 8 weeks once a day (between 8 am to 10 am) to the respective treatment groups. The dose was suspended in distilled water and given orally using gastric gavage method. The food consumption rate was calculated daily by subtracting the amount of food left over in each cage barrier per each rat from the measured amount of food provided on the previous day (g/rat/day). The animals were weighed at the start of the experiment and then every week thereafter. During the last week of the experiment, rat feces were collected for 3 consecutive days to determine fecal lipid excretion. At the end of experimental period, blood samples were collected from the retro-orbital plexus into centrifuge tubes. The blood samples were allowed to clot for 30 min at room temperature and then centrifuged at 1200 x g for 15 min at 4°C for biochemical analysis of serum parameters. The serum was stored at -40°C until analysis. After the blood collection, the rats were killed under anesthesia using 85 mg kg-1 b.wt. ketamine and 95 mg kg-1 b.wt. xylazine (ip). The liver, peritoneal and perirenal fat mass were excised immediately, rinsed, weighed and stored at -40°C until analysis. The animal experimentation was carried out according to Institutional Animal Ethical Committee Guidelines (CPCSEA), Sri Venkateswara University, Tirupati, India.

Biochemical analysis: The blood glucose was determined using glucometer, Accu Chek Sensor set, Roche Diagnostics (Mannheim, Germany). Serum lipids such as Total Cholesterol (TC), triglycerides (TG) and High-Density Lipoproteins (HDL-C) levels were measured by enzymatic colorimetric methods using biochemical kits purchased from Kamineni Life Sciences, Pvt (Hyderabad, India). Total cholesterol and triglyceride concentrations in the liver and feces were determined using the same kits. The liver and feces lipids were extracted by the method of Folch et al.22 Serum Low-Density Lipoprotein (LDL-C) and very Low-Density Lipoprotein (VLDL-C) cholesterol levels were calculated using Friedewalds formula23. The Atherogenic Index (AI) was calculated by using the equation:

Determination of lipid peroxidation and the activity of liver antioxidant enzymes: Liver homogenate was prepared by homogenizing the tissues in 50 mM phosphate buffer pH (7.0). The homogenate was then centrifuged at 4,000 rpm at 4°C for 20 min. The supernatant fraction was collected and further centrifuged at 10,500 x g at 4°C for 60 min. The final supernatant was then analyzed for estimation of activities of antioxidant enzymes using the procedures described by Superoxide dismutase24, Glutathione peroxidase25, Catalase26 and Glutathione27. Lipid peroxidation in the liver was measured in terms of malondialdehyde content and determined by using the Thiobarbituric Acid (TBA) reagent. The reactivity of the TBA is determined with minor modification of the method adopted by Ohkawa et al.28.

Statistical analysis: Data are expressed as the Mean±SD. The results were analyzed by one way ANOVA. Duncan Multiple Range Test (DMRT) was performed to determine statistical significance among groups by using SPSS software version 11.5. Significant difference was accepted at p<0.05.

RESULTS Body weight and food consumption: The effects of cafeteria and high-fat diets on the body weight of rats are shown in Fig. 1. During the 8 weeks of treatment, the weight of the cafeteria and high-fat diets group increased significantly from the first week until the end of treatment when compared with the normal diet group. The gain of body weight was prevented in the rats treated with SGE compared with the rats that were fed the cafeteria and high fat diets. The daily food consumption was significantly different between the N diet and CA and HF diet groups but the daily food intake of the SGE treated group continued to diverge from the CA and HF diets group rats. The CA and HF diets groups with or without treatment of SGE did not cause diarrhoea during the experiment.




The total food consumption during the whole experimental period was significantly different among the groups (Fig. 2).

Weight of liver and fat storage: The effects of SGE on liver weight and body fat accumulation are shown in Fig. 3. Parallel to the body weight change, the weights of the liver and regional fat mass were higher in the CA and HF diets group than in the N diet group. The relative weight of liver, peritoneal and perirenal fat masses of the CA and HF diet+SGE group were significantly reduced in comparison with those of the CA and HF diets group.

Biochemical parameters: The CA and HF diets increased the serum levels of TG, TC, VLDL-C, LDL-C, AI, blood glucose and decreased HDL-C level as compared with rats fed on N diet. Additional administration of SGE significantly prevented these changes (Table 1).

Liver and fecal lipids: Feeding CA and HF diets developed fatty liver with increase of the liver weight and an accumulation of liver TG and TC content was significantly increased when compared with the N diet group. Treatment with SGE significantly reduced TG and TC content compared with CA and HF diets group (Fig. 4). Fecal lipid analysis revealed that there was significant elevation in TG and TC in the feces of SGE treated groups when compared to CA and HF diets group (Fig. 5).

Liver lipid Peroxidation and antioxidant activities: Liver lipid peroxidation was significantly increased, whereas SOD, CAT, GPx activities and GSH content was decreased in CA and HF diet rats compared to the rats fed on N diet. Treatment with SGE significantly depressed the high level of liver lipid peroxidation and increased the low levels of SOD, CAT, GPx activities and GSH content to the normalized (Table 2).

DISCUSSION The present investigation demonstrates that the SGE used in this study is effective in obesity and oxidative stress in cafeteria and high fat diet induced rats. Feeding cafeteria and high fat diets not only promotes weight gain, hyperglycemia and hyperlipidemia but also accumulation of fat in liver occur and furthermore, lead to oxidative stress. In this sense, cafeteria diet feeding has been broadly applied in animal studies due to its similarities with human obesity29.



This model shares common Western diet features (high-fat intake and hyperphagia), which are thought to lead to hyperinsulinemia, type 2 diabetes and metabolic syndrome conditions30.

In the present study, it was revealed that the CA and HF diets induced increase in body weight, peritoneal and perirenal fat masses. The SGE treatment prevented these alterations, suggesting that the saponin rich fraction of G. sylvestre aqueous leaf extract exhibits pharmacological antiobesity effects. These results show that the CA and HF diets increased body weight in rats, even though food consumption was high compared with the normal diet. The CA and HF diets provide more calories than normal diet, resulting in high level of fat storage in the peritoneal and perirenal region. SGE administration prevented weight gain and reduced the consumption of food in CA and HF diet fed rats. However, SGE alone reduced food consumption and caused weight loss in rats.

The results from the present study reveals that the CA and HF diet caused dyslipidemic changes as illustrated by increasing TG, TC, LDL and VLDL levels, AI and decrease in HDL levels. These finding are in accordance with that of Kamalakkannan et al.31 SGE treatment produced significant decrease in serum TG, TC, LDL, VLDL levels and AI, while there was significant increase in HDL cholesterol in cafeteria and high fat diet fed rats. Bishayee and Chatterjee32 reported that the G. sylvestre leaf extract reduced hyperlipidaemia and antiatherosclerotic condition in albino rats fed high fat diet. The possible explanation for the antihyperlipidemic activity of the G. sylvestre leaf extract is an increased excretion of cholesterol, neutral steroids, bile acids in the feces and lipid lowering effects, resulting in depression of lipid accumulation. It consequently has antiatherosclerotic properties33. The CA and HF diets, respectively, produced significantly increase in blood glucose level. Diminished hepatic and muscular uptake of glucose produces hyperlipidemia due to increased fat mobilization from adipose tissue and resistance to the antilipolytic actions of insulin. Impaired insulin action is associated with an oversupply of lipids. This increased availability leads to either elevated lipid storage in insulin target tissues (e.g. muscle, liver adipose) or increased plasma-free fatty acids or triglycerides34. A significant decrease of serum glucose in rats fed on CA and HF diets along with SGE was observed in agreement with Gholap and Kar35.

The liver is the primary organ responsible for maintaining TC and TG homeostasis. A number of lines of evidence show that energy rich diet raises hepatic TC and TG content resulting in the increase of triglyceride synthesis36. The hepatic lipid content and fecal lipid excretion was evaluated in the present study to investigate the basic mechanism of hepatic lipid-lowering activity of SGE. The reduction of hepatic lipid content and the elevation of fecal lipid excretion in CA and HF diet rats treated with SGE, indicate that hepatic lipid lowering effect of SGE was probably related to lower intestinal lipid absorption, resulting in an increase of hepatic bile acids biosynthesis using cholesterol as the precursor and finally leading to the decrease of hepatic cholesterol and triglyceride accumulation. The same result is shown by study of Asai and Miyazawa37. It is well known that dietary lipid is directly not absorbed from the intestine unless it has been subjected to the action of the pancreatic lipase enzyme which are fatty acids and 2-monoacylglycerides, which are absorbed38, thus the inhibition of this enzyme is beneficial in the treatment obesity. Saponins are also capable of precipitating cholesterol from micelles and interfering with enterohepatic circulation of bile acids making it unavailable for intestinal absorption and hence reduce plasma cholesterol levels39. It is noteworthy that in the fecal excretion of the cholesterol, triglycerides was significantly higher with administration of SGE when compared to that of CA and HF diet rats. Furthermore studies demonstrate that the liver weight was significantly reduced, whereas hepatic cholesterol and triglyceride contents were lowered, respectively36.

Additionally, lipid alterations have been considered as contributory factors to oxidative stress in obesity40. Increased production of reactive oxygen species as well as reduced oxidant and antioxidant defense mechanisms have been suggested to play a role in both humans and animal models of obesity41.

Lipid peroxidation is thought to be a component of obesity induced pathology42. The results obtained in this study showed that obesity increased lipid peroxidation in hepatic tissues as expressed by increased tissue levels of MDA. These results are in agreement with the results of Milagro et al.2 who showed that obesity is an independent risk factor for increasing lipid peroxidation and decreased activity of cytoprotective enzymes. Obesity can cause increased lipid peroxidation by progressive and cumulative cell injury resulting from pressure of the large body mass. Cell injury causes the release of cytokines, especially Tumor Necrosis Factor alpha (TNF-α) which generates ROS from the tissues which in turn cause lipid peroxidation43,44. The hypertriglyceridemia seen in obese rats may contribute to the alteration in the antioxidant balance, suggesting that an increase in the bioavailability of free fatty acids can increase lipidperoxidation42. Hepatic lipid peroxidation, as shown by the malondialdehyde (MDA) level was increased in CA and HF diet rats, while SGE significantly decreased levels of hepatic MDA compared with CA and HF diet fed groups as result of SGE that inhibit ATP citrate lyase which catalyse extra mitochondrial cleavage of citrate to oxaloacetate giving acetyl CO-A which used in fatty acid synthesis, suggesting that SGE has hypolipidemic action and reduces MDA in liver.

Antioxidant enzymes constituted a mutually supportive team of defense against reactive oxygen species. In the present study hepatic antioxidant activities of SOD, CAT, GPx and GSH content was significantly decreased in CA and HF diet rats compared to N diet rats. Another interesting point is that SGE normalized the activities of SOD, CAT, GPx and GSH content in hepatic tissue. These results reveal that eight weeks of CA and HF diet feeding induces oxidative stress to impair the liver tissue. Recent findings indicate that some therapeutic herbs have both lipid lowering ability and antioxidative activities to suppress lipid peroxide production and then eventually may contribute to their effectiveness in preventing atherosclerosis and in protecting various organs at risk from hyperlipidemia45.

In conclusion, saponin rich extract of G. sylvestre leaves was able to decrease the body weight, food intake and their fat storage. They were also effective in high blood glucose, lipid profile and AI levels in rats fed with CA and HF diets for eight weeks. In addition, SGE decreased hepatic lipid content whereas increased fecal lipid excretion undergo significant change. It also suppressed the high level of lipid peroxidation in the liver tissue. Furthermore, SGE significantly enhanced the activities of the antioxidant enzymes in the liver tissue. The present study clearly indicated that the saponin rich extract of G. sylvestre leaves had significant antiobesity action and supports the traditional usage as a substitute drug for G. sylvestre leaves. Hence, it might be helpful in preventing obesity complications and oxidative stress.

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