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Anti-obesity effect of standardized ethanol extract of Embelia ribes in murine model of high fat diet-induced obesity
PharmaNutrition 1 (2013) 50–57
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Anti-obesity effect of standardized ethanol extract of Embelia ribes in murine model of high fat diet-induced obesity Uma Bhandari a,*, Hemantkumar Somabhai Chaudhari a, Ajay Narayan Bisnoi a, Vinay Kumar a, Geetika Khanna b, Kalim Javed c a b c
Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi 110062, India Department of Pathology, Vardhman Mahavir Medical College, Safdarjung Hospital, New Delhi 110029, India Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi 110062, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 December 2012 Received in revised form 13 January 2013 Accepted 15 January 2013
Overweight and obesity are the most common nutritional disorder in Western countries. The objective of this study was to evaluate anti-obesity potential of standardized Embelia ribes ethanol extract (ERE) in murine model of high fat diet (HFD)-induced obesity. ERE was standardized by high performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC). Male Wistar rats were fed HFD for 28 days to induce obesity. ERE (100 mg/kg) administered orally to HFD fed rats for 21 days. Changes in body weight gain, body mass index (BMI), blood pressure, serum parameters, and myocardial oxidative stress parameters were measured. ERE showed a preventive effect on body weight gain, visceral fat accumulation and elevated blood pressure. The extract treatment elicited a significant reduction in serum levels of leptin by 45%, insulin by 37%, glucose by 28%, total cholesterol by 18%, and triglycerides by 24% while HDL-C level increased by 31%. Furthermore, ERE treatment decreased the myocardial lipid peroxidation and increased antioxidant levels in obese rats. These findings demonstrated the anti-obesity potential of ERE, possibly through suppression of body weight gain, lipid lowering action, improvement in insulin and leptin sensitivity and increased antioxidant defense. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Embelia ribes Obesity High fat diet Leptin Insulin Oxidative stress
1. Introduction Obesity is set to be the world’s major cause of morbidity and mortality in the 21st Century [1]. In 2008, more than 1.4 billion adults were overweight in the world, and at least 200 million men and nearly 300 million women from them were obese [2]. Obesity significantly increases the risk of developing various lifethreatening diseases, including type II diabetes, hypertension, coronary heart disease, stroke and certain cancers [1]. Many factors affect the onset of obesity including satiety control, reduced levels of physical exercise, and hormonal and genetic parameters which influence the metabolic pathways leading to increase in stored fat [3]. The pharmacotherapy of obesity has recently undergone unprecedented expansion. However, the substantial barriers that undermine long-term obesity management strategies include lack of specific obesity training of health professionals, attitudes, and beliefs, as well as coverage and availability of obesity treatments [4]. The situation as it currently stands, the
* Corresponding author. Tel.: +91 11 26059688; fax: +91 11 26059663. E-mail address: [email protected] (U. Bhandari). 2213-4344/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phanu.2013.01.001
market for anti-obesity drugs is expanding rapidly but the available drugs are extremely sparse in relation to need. Hence, there is need to search for novel anti-obesity drug to tackle the obesity problem. Current therapeutic strategy for treating obesity is mainly focused on diet control and physical exercise. Natural products/ dietary photochemicals have aroused considerable interest in recent years as potential therapeutic agents to counteract obesity. These compounds exert the anti-obesity effects mainly through regulation of various pathways, including lipid absorption, intake and expenditure of energy, increasing lipolysis, and decrease lipogenesis, and differentiation and proliferation of preadipocytes [5]. Embelia ribes Burm (Myrsinaceae) known commonly as vidanga is widely distributed throughout India. Ayurveda describes vidanga as pungent and cures colic and flatulence i.e. it has potential in medoroga (obesity). One ayurvedic formulation, vidangadya cˆurna (powder of vidanga), containing vidanga as main ingredient is taken with honey to alleviate obesity [6]. E. ribes fruits contain a quinone derivative (embelin), an alkaloid (christembine) and a volatile oil (vilangin). Embelin (2,5dihydroxy-3-undecyl-1,4-benzoquinone) is considered one of the major bioactive constituents and has a wide spectrum of
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biological activities including antitumor, antibacterial, antioxidant, analgesic, antifertility, wound healing, anticonvulsant and antidiabetic [7]. Tripathi [8] reported the anti-hyperglycemic activity of the decoction of E. ribes in glucose-fed albino rabbits. Further, Bhandari et al. [9] reported the potential of E. ribes ethanol extract (ERE) in diabetic dyslipidemia and protection from lipid peroxidation in tissues in streptozotocin induced diabetes in rats. The effect of oral treatment with ERE on the basal level of some key serum, tissue antioxidants, blood pressure and glycosylated haemoglobin in streptozotocin-induced oxidative damage in rats has also been reported [10,11]. However, E. ribes has not been investigated so far for its antiobesity potential. We have selected a rat model of diet induced obesity based on numerous experimental studies which indicated that diets high in fat are known to increased body weight and fat mass, induce alterations in carbohydrate and lipid metabolism, lead to insulin resistance, and increase production and release of leptin in humans, rodents, and other animals [12,13]. Hence, the objective of this study was to evaluate anti-obesity effect of E. ribes ethanol extract (ERE) in murine model of high fat diet (HFD)induced obesity. 2. Materials and methods 2.1. Plant material The berries of E. ribes were purchased from North Eastern India Ayurveda Research Institute, Barsjoi,Guwahati, Assam, India, and authenticated by Dr. H. B. Singh, Head, Raw Materials and Herbarium, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi, India, where a voucher specimen is preserved for further reference (Reference No. NISCAIR/RHMD/Consult/-2010-11/1500/98, dated 26 August, 2010). 2.2. Preparation of E. ribes ethanol extract The dried and powdered fruits of E. ribes were extracted with 90% ethanol in a Soxhlet extraction system for 72 h. The solvent was removed under reduced pressure to give a dry extract, 7% yield (w/w) with respect to the crude material and stored at 20 8C until the experiment. This extract was tested for phytochemical analysis and high performance thin layer chromatography (HPTLC) finger printing for standardization [14,15]. 2.3. High performance liquid chromatography (HPLC) analysis The HPLC analysis of ERE was carried out by the methodology of Shelar et al. [16] on HPLC system (Waters Corporation, USA) equipped with Millennium Chromatography software version 2.15 and the chromatographic separations were performed using a Hyperchrome Column (250 mm 4.6 mm), with a flow rate of 1 ml/min and a sample size of 10 ml. The mobile phase used was methanol (A) and 0.1% trifluoroacetic acid (B) with a ratio of 88:12 (A:B (v/v)). The sample analysis was performed at wavelength 288 nm using photodiode array (PDA) detector at ambient temperature. The chromatographic peaks of the ERE were confirmed by comparing its retention time and UV spectra with corresponding reference standard embelin. The result was obtained by comparison of peak area (at 288 nm) of ERE with that of embelin. 2.4. High fat diet-induced obesity The male Wistar rats (150–200 g) were procured from central animal house facility of Hamdard University, New Delhi, India. The
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Table 1 Compositions of high fat diet (g/kg diet). Ingredients
Quantity
Casein L-cystine Starch Sucrose Cellulose Ground nut oil Tallow AIN salt mix AIN vitamin mix
342.0 3.0 172.0 172.0 50.0 25.0 190.0 35.0 10.0
Total (g)
999.0
animals were housed in standard polypropylene cages and maintained under controlled room temperature (22 2 8C) and humidity (55 5%) with 12 h light and 12 h dark cycle. All the rats were provided with commercially available rodent chow diet (Amrut rat feed, Nav Maharastra Chakan Oil Mills Ltd., Delhi, India) and tap water ad libitum. After 1 week of acclimatization with free access to rodent chow diet and water, animals were used in the study. The guidelines of committee for the purpose of control and supervision of experiments on animals (CPCSEA), Government of India were followed and prior permission was sought from the Institutional Animal Ethics Committee, Jamia Hamdard, New Delhi for conducting the study. Rats were fed a HFD in a pellet form and water ad libitum for the period of 4 weeks. HFD was purchased from National Centre for Laboratory Animal Science (NCLAS), National Institute of Nutrition (NIN), Hyderabad, India. Composition of HFD is shown in Table 1. 2.5. Experimental design In this study, a total of 50 rats were used and divided into five groups of 10 rats each. Group I: normal healthy control rats fed with rodent chow diet and administered 1% gum acacia [1 ml/kg, body weight (bw)] for the period of 28 days (NC). Group II: rats fed with HFD for the period of 28 days (HFD). Group III: rats fed with HFD for the period of 28 days + from 8th day, treated with ERE (100 mg/kg, bw) for period of 21 days (HFD + ERE). Group IV: rats fed with HFD for the period of 28 days + from 8th day, treated with Orlistat (10 mg/kg, bw) for period of 21 days (HFD + ORL). Group V: rats fed with rodent chow diet for the period of 28 days + from 8th day, treated with ERE (100 mg/kg, bw) for period of 21 days (ERE per se).
All the drugs were administered by oral gavage once a day. Food and water intakes were measured daily for the period of 28 days at the same time. Food intake and water intake was measured on per cage basis and the average food and water consumed were calculated. At the end of the experimental period (on 29th day), the animals were anesthetized with ether, following overnight fasting. Blood was drawn by retro-orbital method into a tube and the serum was obtained by centrifugation. After collection of blood, rats were sacrificed; heart, and perirenal white adipose tissue (WAT), epididymal WAT, mesenteric WAT were excised immediately, rinsed with phosphate buffer saline and weighed. The serum and heart samples were stored at 70 8C until analysis.
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2.6. Anthropometric measurements The body weights were determined once a week. Body mass index (BMI) was calculated from formula: BMI = body weight (g)/ length2 (cm2) [17]. 2.7. Measurement of hemodynamic parameters Hemodynamic parameters (systolic, diastolic, mean arterial blood pressure and heart rate) were measured by Non-Invasive Blood Pressure Recorder using rat tail-cuff method (Kent Scientific Corporation, USA). 2.8. Biochemical estimation Serum glucose, triglyceride (TG) and total cholesterol (TC) levels were determined by enzymatic methods using commercial assay kits (SPAN Diagnostics Ltd., Surat, India) according to the manufacturer’s protocols. Serum high-density lipoprotein cholesterol (HDL-C) levels were determined using HDL Cholesterol Test Kit (Reckon Diagnostics Pvt. Ltd,. Baroda, India). Serum leptin and insulin levels were measured by immunoassays using a commercially available ELISA assay kit from Bio Vendor (Modrice, Czech Republic) and Alpco Diagnostics (Salem, USA), respectively. Low-density lipoprotein cholesterol (LDL-C) levels were calculated using Friedewald formula [17]. Serum very low density lipoprotein cholesterol fraction (VLDL-C) concentration was calculated by deduction of the sum of HDL-C and LDL-C concentrations from that of TC. 2.9. Determination of lipid peroxidation and free radical scavenging activity in cardiac tissue
[21], glutathione peroxidase (GPx) [22], and glutathione-Stransferase (GST) [23] activities. 2.10. Histopathological analysis For histological examination, the heart tissue was collected and fixed in 10% neutral buffered formalin, embedded in paraffin. Standard sections of 5 mm thickness were cut, which were then stained with hematoxylin and eosin (H&E), and examined by light microscopy. 2.11. Statistical analysis Data are expressed as the mean standard error of the mean (SEM). The statistical significance of difference between the mean values for the treatment groups was analyzed by ANOVA (analysis of variance) followed by Dunnett’s t-test using Graph pad InStat1 version 3.06 (Graph Pad Software, San Diego, CA, USA). Values of p < 0.05 were considered significant. 3. Results 3.1. Preliminary phytochemical screening, HPTLC and HPLC analysis Phytochemical screening of ERE showed positive results for alkaloids, carbohydrates, phenolics, and saponins. HPTLC finger prints of ERE showed the presence of 7 spots with their retention factor (Rf) value 0.06, 0.19, 0.25, 0.41, 0.45, 0.51 and 0.61 in the solvent system ethyl acetate:methanol (9:1) at 365 nm wavelength. Out of these compounds, band with max Rf 0.06 matched exactly with that of embelin (max Rf 0.06). The presence of embelin in ERE was established by HPLC and quantified as 3.2803% (Fig. 1). 3.2. Effect on food and water intake
A portion of heart was minced and homogenized (10%, w/v) for determination of lipid peroxidation (LPO) [18], reduced glutathione (GSH) [19], superoxide dismutase (SOD) [20], catalase (CAT)
There was no significant (p > 0.05) change in food and water intake was observed in HFD-fed group as compared to the NC
Fig. 1. HPLC chromatograms of Embelia ribes ethanol extract (A) and the standard embelin (B; retention time: 3.088).
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Table 2 Effect of ethanol E. ribes extract on food intake, water intake, body weight gain and BMI in HFD-induced obesity in Wistar rats. Groups
Food intake (gm/rat/day)
Water intake (ml/rat/day)
Body weight gain (g)
BMI (g/cm2)
NC HFD HFD + ERE HFD + ORL ERE per se
12.78 2.31 15.65 3.67 15.40 0.67 15.27 0.83 14.89 0.48
40.34 3.81 45.60 4.71 43.50 1.32 43.80 1.41 42.80 1.38
53.13 5.02 124.35 9.26** 94.60 7.29## 87.80 4.22## 47.80 1.49
4.26 0.04 5.87 0.18* 4.61 0.35# 4.15 0.26# 4.03 0.80
All values were expressed as Mean SEM (n = 10). * p < 0.05 as compared to the NC group. ** p < 0.01 as compared to the NC group. # p < 0.05 as compared to HFD group. ## p < 0.01 as compared to HFD group.
Table 3 Effect of ethanol E. ribes extract on heart rate and blood pressure in HFD-induced obesity in Wistar rats. Groups
Heart rate (beats/min)
Systolic B.P. (mmHg)
Diastolic B.P. (mmHg)
Mean arterial B.P (mmHg)
NC HFD HFD + ERE HFD + ORL ERE per se
421.83 11.26 539.20 15.33** 470.89 14.11## 464.83 11.57## 436.21 8.58
127.83 3.60 145.16 2.58** 130.00 1.88## 129.23 4.84## 132.73 3.29
96 3.65 109.00 2.56** 96.66 2.38## 94.53 1.77## 94.21 7.39
106.61 1.22 121.05 2.09** 107.77 1.84## 106.09 1.77## 107.05 2.19
All values were expressed as Mean SEM, (n = 10). * **
p < 0.05 as compared to the NC group. p < 0.01 as compared to the NC group.
# ##
p < 0.05 as compared to HFD group. p < 0.01 as compared to HFD group.
group. Administration of HFD + ERE or HFD + ORL did not produce significant change in the food and water intake as compared to HFD group (Table 2).
1.46-fold, perirenal WAT by 1.25-fold, and mesenteric WAT by 1.3fold when compared to HFD group (Fig. 2). 3.6. Effect on serum lipid levels
3.3. Effect on anthropometric parameters The body weight gain of rats in the HFD-fed group was greater than the values for the NC. A 2.34-fold increase in body weight gain was observed in HFD group compared to NC group. Treatment with HFD + ERE and HFD + ORL significantly (p < 0.01) reduced the body weight gain by 1.31- and 1.42-fold respectively. BMI of the rats in HFD-fed group, which was significantly greater than the value for NC (p < 0.05), was reduced significantly (p < 0.05) when the rats were administered HFD + ERE or HFD + ORL. However, there was no significant difference in body weight gain and BMI among the NC and ERE per se was observed (Table 2). 3.4. Effect on hemodynamic parameters A statistically significant increase (p < 0.01) in heart rate, systolic, diastolic and mean arterial blood pressure was observed in HFD-fed group as compared to NC. ERE in 100 mg/kg dose (i.e. HFD + ERE group) produced a statistically significant (p < 0.01) decrease in heart rate, systolic, diastolic and mean arterial blood pressure was observed. However, the reference drug, orlistat (i.e. HFD + ORL group) exhibited better activity than HFD + ERE group in all the above hemodynamic parameters (p < 0.01; Table 3).
The results of the serum lipid profile are shown in Fig. 3. HFD-fed group showed a significant (p < 0.01) increase in serum TC, TG, LDLC and VLDL-C by 1.64-,2.4-, 1.96-, and 2.4-fold respectively against NC group and a significant (p < 0.01) decrease in HDL-C levels by 1.28-fold as compared to NC group. HFD + ERE administration caused a significant (p < 0.01) decrease in serum TC, TG, LDL-C and VLDL-C levels by 1.21-, 1.32-, 1.37- and 1.32-fold, and HFD + ORL 1.28-, 1.38-, 1.55- and 1.38-fold, respectively, as compared with those in HFD-fed group. There was a significant (p < 0.01) increase in serum HDL-C by 1.31- and 1.36-fold in HFD + ERE and HFD + ORL group, respectively, as compared to HFD-fed group. However, there was no significant (p > 0.05) difference in serum lipid profile among the NC and ERE per se was observed. 3.7. Effect on serum glucose, insulin, and leptin levels HFD-fed rats exhibited significant (p < 0.01) increase in serum glucose, insulin and leptin by 1.56-, 2.13- and 1.52-fold
3.5. Effect on white adipose tissue weights The weights of fat pads from HFD-fed, NC, drug treated groups were assessed after the treatment period. The weight of the WAT tissue were significantly (p < 0.01) higher (epididymal WAT by 1.69fold, perirenal WAT by 1.74-fold, and mesenteric WAT by 1.93-fold higher) by feeding HFD than the value for NC. Conversely, epididymal WAT, perirenal WAT, and mesenteric WAT weights were decreased by 1.19-, 1.13-, 1.19-fold, respectively, in ERE treated rats (i.e. HFD + ERE group) then in their HFD-fed counterparts. HFD + ORL administration decreased epididymal WAT by
Fig. 2. Effect of ethanolic extract of Embelia ribes on white adipose tissue (WAT) weights in HFD-induced obesity in Wistar rats. All values were expressed as Mean SEM, (n = 10); *p < 0.05, **p < 0.01 as compared to NC; #p < 0.05, ##p < 0.01 as compared to HFD.
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Fig. 3. Effect of ethanolic extract of Embelia ribes on serum lipid levels in HFD-induced obesity in Wistar rats. All values were expressed as Mean SEM, (n = 10); * p < 0.05, ** p < 0.01 as compared to NC; #p < 0.05, ##p < 0.01 as compared to HFD.
glucose, insulin and leptin levels by 1.33-, 1.74- and 1.94-fold respectively. However, there was no significant (p > 0.05) difference in serum glucose, insulin and leptin levels among the NC and ERE per se was observed (Figs. 4, 5 and 6). 3.8. Effect on lipid peroxidation and free radical scavenging activity in cardiac tissue
Fig. 4. Effect of ethanolic extract of Embelia ribes on serum glucose levels in HFDinduced obesity in Wistar rats. All values were expressed as Mean SEM, (n = 10); * p < 0.05, **p < 0.01 as compared to NC; #p < 0.05, ##p < 0.01 as compared to HFD.
HFD-fed rats showed a significant (p < 0.01) increase in cardiac TBARS content by 3.48-fold as compared with those in NC group. HFD + ERE treatment caused a significant (p < 0.01) decrease in cardiac TBARS content by 2.28-fold while HFD + ORL showed 2.7fold reduction as compared to HFD group. . As shown in Table 4, the HFD-fed group showed a significant (p < 0.01) depletion in cardiac SOD, CAT, GSH, GPx and GST contents by 1.81-, 1.95-, 2.8-, 1.39- and 2-fold respectively as compared to NC group. HFD + ERE treatment caused a significant (p < 0.01) enhancement in cardiac SOD, CAT, GSH, GPx and GST contents by 1.44-, 1.77-, 2-, 1.32- and 1.76-fold while HFD + ORL showed 1.61-, 1.89-, 2.83-, 1.45- and 1.88-fold elevation respectively as compared to HFD-fed group. However, there was no significant (p > 0.05) difference in cardiac TBARS, SOD, CAT, GSH, GPx and GST contents among the NC and ERE per se was observed. 3.9. Effect on histopathological changes in cardiac tissue
Fig. 5. Effect of ethanolic extract of Embelia ribes on serum insulin levels in HFDinduced obesity in Wistar rats. All values were expressed as Mean SEM, (n = 10); * p < 0.05, **p < 0.01 as compared to NC; #p < 0.05, #p < 0.01 as compared to HFD.
In the histopathological studies, heart sections in NC and ERE per se group showed normal morphology of myocardium. The heart sections of HFD-fed obese rat showed deposition of fat globules in myocardial cells. However, HFD + ERE and HFD + ORL treated rats heart showed normal architecture of myocardium (Fig. 7). 4. Discussion
Fig. 6. Effect of ethanolic extract of Embelia ribes on serum leptin levels in HFDinduced obesity in Wistar rats. All values were expressed as Mean SEM, (n = 10); * p < 0.05, **p < 0.01 as compared to NC; #p < 0.05, ##p < 0.01 as compared to HFD.
respectively against NC group. In contrast, treatment with HFD + ERE significantly (p < 0.01) decreased serum glucose, insulin and leptin levels by 1.39-, 1.58- and 1.81-fold respectively. HFD + ORL treatment significantly (p < 0.01) decreased serum
In the present study, anti-obesity effect of ERE was investigated using a HFD-induced obese rat model. Reduction in body weight gain of HFD-fed rats was accompanied by a depletion of body fat stores, since treatment with ERE also significantly reduced the weight of the visceral WAT compared with that of HFD-fed rats. Excessive growth of adipose tissue results in obesity which involves two growth mechanisms: hyperplasia (cell number increase) and hypertrophy (cell size increase) [24]. In epidemiological studies, BMI is widely used as a measure of fatness because it is highly correlated with body fat and is nearly independent of height [25]. In our study, significant reduction in BMI of ERE treated group was observed. These results indicate that ERE suppresses the HFD-induced increase in fat mass and body weight gain.
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Table 4 Effect of ethanol E. ribes extract on myocardial TBARS, SOD, CAT, GSH, GPx and GST levels in HFD-induced obesity in Wistar rats. Groups
TBARS (nmol MDA/mg protein)
SOD (U/mg protein)
CAT (nmol H2O2 consumed/min/mg protein)
GSH (mmol P liberated/min/mg protein)
GPx (nmol NADPH oxidized/min/mg protein)
GST (nmol CDNB conjugate/formed/min/mg protein)
NC HFD HFD + ERE HFD + ORL ERE per se
0.21 0.01 0.73 0.03** 0.32 0.01## 0.27 0.03## 0.20 0.05
3.85 0.22 2.13 0.13** 3.07 0.09## 3.42 0.06## 3.74 0.12
54.63 1.45 28.03 0.64** 49.55 1.70## 52.95 1.09## 56.46 2.14
26.75 0.97 9.54 0.47** 19.12 0.79## 27.01 0.18## 26.52 1.3
184.81 7.54 132.66 3.14** 174.55 5.70## 192.50 9.18## 188.72 2.04
492.31 4.01 246.43 2.06** 434.38 6.37## 463.21 5.26## 488.21 3.09
All values were expressed as Mean SEM, (n = 10). * **
p < 0.05 as compared to the NC group. p < 0.01 as compared to the NC group.
# ##
p < 0.05 as compared to HFD group. p < 0.01 as compared to HFD group.
Fig. 7. Histopathological changes in heart of experimental rats. Group I (A): normal heart show normal morphology of myocardium; Group II (B): obesity control, HFD treated heart shows deposition of fat globules in myocardial cells; Group III (C): HFD + E. ribes extract (100 mg/kg b.w.) shows no fatty changes; Group IV (D): HFD + Orlistat (10 mg/ kg b.w.) shows no fatty changes and normal architecture of myocardium; Group V (E): E. ribes extract (100 mg/kg) per se shows normal architecture of heart.
Though there was a significant difference in the body weights between the HFD-fed and NC groups, no significant difference in the daily food intake and water intake between groups was observed. This observation provides us with the fact that dietary caloric content was independent of the amount of food
consumed by the animals. The HFD-fed rats continuously consumed similar quantities of food, regardless of the higher calories content in the diet. As a result, the caloric intake was raised in the HFD-fed rats compared to the NC rats. The unchanged food intake in spite of the higher caloric content in
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the diet is proportional to the increment of the body weight, hence, resulting in the obese state. In addition to its weight reducing effect, ERE was also able to improve some lipid metabolites including TC, TG, HDL-, LDL- and VLDL-cholesterol levels in obese rats. It is reported that obesity, especially abdominal obesity, is associated with dyslipidemia, characterized by elevated TG and reduced HDL-C concentrations. TGs are involved in the ectopic accumulation of lipid stores in the liver and are associated with a number of diseases such as metabolic syndrome. VLDL-C transports cholesterol and TG to the tissues, while high HDL-C is helpful in transporting excess cholesterol to the liver for excretion in the bile. High TC and LDL-C levels, and lower HDL-C level are risk factors for coronary heart disease [26]. Thus, this result suggested that ERE would be helpful in the prevention of obesity complications through improving hyperlipidemia. Further, ERE treatment showed improvements in the histological features of heart induced by HFD in rats. These results seemed to correspond to the white adipose tissue weights (Fig. 2) and serum lipid profiles (Fig. 3). Obesity is associated with an impaired ability of tissue to respond to insulin and effectively store glucose. Further, in response to insulin resistance production is increased, leading to hyperinsulinemia, hyperglycemia and ultimately type 2 diabetes [27]. In our study, significant reduction in the serum glucose and insulin levels after the treatment with ERE in HFD-fed rats was observed. Epidemiological studies have found a progressive increase in the prevalence of elevated blood pressure with increasing adipose tissue [25]. Moreover, hypertension and hyperlipidemia often manifest concomitantly in the clinical context of obesity and insulin resistance. In consistent with improvement of lipid profile and insulin resistance by ERE, it is also able to attenuate the development of hypertension in HFD-fed obese rats. Leptin is fat-derived key regulators of food intake and energy expenditure, where its concentration in the plasma is associated with general adiposity [28]. The reductions of leptin level reflect a decreased in the fat mass. Hypothalamus receives direct input from leptin, which cross the blood–brain barrier and provide information on the levels of peripheral adipose mass. The results from the present studies suggest that ERE caused significant adipocytes loss, indicated by reduced leptin levels. Obesity and hyperlipidemia synergistically promote systemic oxidative stress-imbalance between tissue free radicals, reactive oxygen species (ROS) and antioxidants [29]. ROS could react with polyunsaturated fatty acids, which lead to lipid peroxidation [30]. Malondialdehyde is a by-product of lipid peroxidation and reflect the degree of oxidation in the body [18]. Possible mechanisms that generate oxidative stress in obesity include hyperglycemia, elevated lipid levels, inadequate antioxidant defenses and hyperleptinemia [31]. In the present study, cardiac TBARS levels, determined by evaluating malondialdehyde content were decreased after ERE treatment in HFD-fed rats. Furthermore, in our study, the activities of GSH (a major endogenous antioxidant), GPx, GST, SOD and CAT (scavenger of free radicals) decreased in HFD-fed rats as reported earlier [32]. Treatment of HFD-fed rats with ERE had reversed the activities of these enzymatic antioxidants. It is therefore reasonable to assume that ERE treatment improves cardiac oxidative balance in HFD-fed obese rats, because ERE was able to reduce the level of TBARS and free radical generation. Conclusively, observed reduction in body weight gain, BMI, heart rate, blood pressure, serum lipids, glucose, insulin, leptin levels, myocardial tissue lipid peroxidation, and improvement in myocardial lipid accumulation and antioxidant enzyme levels suggests that ERE possesses significant anti-obesity potential.
Layperson’s summary Overweight and obesity are the most common nutritional disorder in Western countries. The objective of this study was to evaluate anti-obesity potential of standardized E. ribes ethanol extract in high fat diet (HFD)-induced obesity in Wistar rats. E. ribes extract treatment for 21 days to obese rats resulted in significant reduction on body weight gain, visceral fat accumulation and elevated blood pressure. The extract treatment also produced a significant reduction in serum levels of lipids, insulin, glucose and elevation in antioxidant levels. These results suggested that E. ribes extract has anti-obesity potential.
Acknowledgements This study was supported by major research grant to Dr. Uma Bhandari from Central Council for Research in Ayurvedic Sciences (CCRAS), Department of AYUSH, Ministry of Health, Government of India, New Delhi, India. References [1] Cheetham SC, Jackson HC, Vickers SP, Dickinson K, Jones RB, Heal DJ. Novel targets for the treatment of obesity: a review of progress. Drug Discovery Today 2004;1:227–35. [2] World Health Organization. Obesity and overweight. WHO; 2012, http:// www.who.int/mediacentre/factsheets/fs311/en/index.html. [3] Jeffcoat R. Obesity - A perspective based on the biochemical interrelationship of lipids and carbohydrates. Medical Hypotheses 2007;68:1159–71. [4] Mauro M, Taylor V, Wharton S, Sharma AM. Barriers to obesity treatment. European Journal of International Medicine 2008;19:173–80. [5] Gonza´lez-Castejo´n M, Rodriguez-Casado A. Dietary phytochemicals and their potential effects on obesity: a review. Pharmacological Research 2011;64:438–55. [6] Vaidya Arya MR. Sthaulya Cikitsa (Treatment of obesity). In: A compendium of ayurvedic medicine: principles and practice; 1999.p. 335–9. [7] Mahendran S, Maithili V, Badami S. Evaluation of antidiabetic effect of embelin from Embelia ribes in alloxan induced diabetes in rats. Biomedicine and Preventive Nutrition 2011;1:25–31. [8] Tripathi SN, Tiwari CM, Upadhyay BN, Singh RS. Screening of hypoglycemic action in certain indigenous drugs. Journal of Research in Indian Medicine Yoga and Homeopathy 1979;14:159–69. [9] Bhandari U, Kanojia R, Pillai KK. Effect of ethanolic extract of Embelia ribes on dyslipidemia in diabetic rats. International Journal of Experimental Diabetes Research 2002;3:159–62. [10] Bhandari U, Jain N, Pillai KK. Further studies on antioxidant potential and protection of pancreatic b-cells by Embelia ribes in experimental diabetes. Experimental Diabetes Research 2007;2007:1–6. [11] Bhandari U, Jain N, Ansari MN, Pillai KK. Beneficial effect of Embelia ribes ethanolic extract on blood pressure and glycosylated hemoglobin in streptozotocin-induced diabetes in rats. Fitoterapia 2008;79:351–5. [12] Mooradian AD, Chehade J, Hurd R, Haas MJ. Monosaccharide-enriched diets cause hyperleptinemia without hypophagia. Nutrition 2000;16:439–41. [13] Brown JL, Spicer MT, Spicer LJ. Effect of high-fat diet on body composition and hormone responses to glucose tolerance tests. Endocrine 2002;19:327–32. [14] Ansari MN, Bhandari U. High performance thin layer chromatographic method for quantification of embelin from Embelia Ribes Burm fruits. Indian Drugs 2008;45:908–10. [15] Bhandari U, Ansari MN. Protective effect of aqueous extract of Embelia ribes Burm fruits in middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Indian Journal of Pharmacology 2008;40:215–20. [16] Shelar R, Maurya C, Tekale P, Katkar K, Naik V, Suthar A, et al. Embelin An HPLC Method for Quantitative Estimation in Embelia ribes Burm. International Journal of Pharmaceutical and Clinical Research 2009;1:146–9. [17] Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clinical Chemistry 1972;18:499–502. [18] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid. Analytical Biochemistry 1979;95:359–64. [19] Sedlak J, Lindsay RH. Estimation of total, protein bound and non protein sulfhydryl group in tissue with Ellman’s reagent. Analytical Biochemistry 1968;25:192–205. [20] Marklund S, Marklund G. Involvement of the superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry 1974;47:469–74.
U. Bhandari et al. / PharmaNutrition 1 (2013) 50–57 [21] Caliborne A. Catalase activity. In: Greenwald RA, editor. Handbook of methods of oxygen radical research. London, UK: CRC Press; 1985 . p. 283–5. [22] Mohandas J, Marshall JJ, Duggin GG, Horvath JS, Tiller DJ. Low activities of glutathione-related enzymes as factors in the genesis of urinary bladder cancer. Cancer Research 1984;44:5086–91. [23] Habig WH, Pabst MJ, Jakoby WB. Glutthione-S-transferase. The first enzymatic step in mercaptouric acid formation. Journal of Biological Chemistry 1974;249:7130–9. [24] Mancini FP, Lanni A, Sabatino L, Moreno M, Giannino A, Contaldo F, et al. Fenofibrate prevents and reduces bodyweight gain and adiposity in dietinduced obese rats. FEBS Letters 2001;491:154–8. [25] Yalcin BM, Sahin EM, Yalcin E. Which anthropometric measurements is most closely related to elevated blood pressure. Family Practice 2005;22:541–7. [26] Chang CJ, Tzeng TF, Liou SS, Chang YS, Liu IM. Kaempferol regulates the lipidprofile in high-fat diet-fed rats through an increase in hepatic PPARa levels. Planta Medica 2011;77:1876–82.
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[27] Knight SF, Imig JD. Obesity, insulin resistance, and renal function. Microcirculation 2007;14:349–62. [28] Staiger H, Haring HU. Adipocytokines: fat-derived humoral mediators of metabolic homeostasis. Experimental and Clinical Endocrinology and Diabetes 2005;113:67–79. [29] Hasty HA, Gruen ML, Terry ES, Surmi BK, Atkinson RD, Gao L, et al. Effects of vitamin E on oxidative stress and atherosclerosis in an obese hyperlipidemic mouse model. Journal of Nutritional Biochemistry 2007;18:127–33. [30] Memisogulları R, Bakan E. Levels of ceruloplasmin, transferin, and lipid peroxidation in the serum of patients with type 2 diabetes mellitus. Journal of Diabetes and its Complications 2004;18:193–207. [31] Vincent HK, Innes KE, Vincent KR. Oxidative stress and potential interventions to reduce oxidative stress in overweight and obesity. Diabetes Obesity and Metabolism 2007;9:813–39. [32] Ansari JA, Bhandari U, Haque SE, Pillai KK. Enhancement of antioxidant defense mechanism by pitavastatin and rosuvastatin on obesity-induced oxidative stress in Wistar rats. Toxicology Mechanisms and Methods 2012;22:67–73.
Contents lists available at SciVerse ScienceDirect
PharmaNutrition journal homepage: www.elsevier.com/locate/phanu
Anti-obesity effect of standardized ethanol extract of Embelia ribes in murine model of high fat diet-induced obesity Uma Bhandari a,*, Hemantkumar Somabhai Chaudhari a, Ajay Narayan Bisnoi a, Vinay Kumar a, Geetika Khanna b, Kalim Javed c a b c
Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi 110062, India Department of Pathology, Vardhman Mahavir Medical College, Safdarjung Hospital, New Delhi 110029, India Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi 110062, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 December 2012 Received in revised form 13 January 2013 Accepted 15 January 2013
Overweight and obesity are the most common nutritional disorder in Western countries. The objective of this study was to evaluate anti-obesity potential of standardized Embelia ribes ethanol extract (ERE) in murine model of high fat diet (HFD)-induced obesity. ERE was standardized by high performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC). Male Wistar rats were fed HFD for 28 days to induce obesity. ERE (100 mg/kg) administered orally to HFD fed rats for 21 days. Changes in body weight gain, body mass index (BMI), blood pressure, serum parameters, and myocardial oxidative stress parameters were measured. ERE showed a preventive effect on body weight gain, visceral fat accumulation and elevated blood pressure. The extract treatment elicited a significant reduction in serum levels of leptin by 45%, insulin by 37%, glucose by 28%, total cholesterol by 18%, and triglycerides by 24% while HDL-C level increased by 31%. Furthermore, ERE treatment decreased the myocardial lipid peroxidation and increased antioxidant levels in obese rats. These findings demonstrated the anti-obesity potential of ERE, possibly through suppression of body weight gain, lipid lowering action, improvement in insulin and leptin sensitivity and increased antioxidant defense. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Embelia ribes Obesity High fat diet Leptin Insulin Oxidative stress
1. Introduction Obesity is set to be the world’s major cause of morbidity and mortality in the 21st Century [1]. In 2008, more than 1.4 billion adults were overweight in the world, and at least 200 million men and nearly 300 million women from them were obese [2]. Obesity significantly increases the risk of developing various lifethreatening diseases, including type II diabetes, hypertension, coronary heart disease, stroke and certain cancers [1]. Many factors affect the onset of obesity including satiety control, reduced levels of physical exercise, and hormonal and genetic parameters which influence the metabolic pathways leading to increase in stored fat [3]. The pharmacotherapy of obesity has recently undergone unprecedented expansion. However, the substantial barriers that undermine long-term obesity management strategies include lack of specific obesity training of health professionals, attitudes, and beliefs, as well as coverage and availability of obesity treatments [4]. The situation as it currently stands, the
* Corresponding author. Tel.: +91 11 26059688; fax: +91 11 26059663. E-mail address: [email protected] (U. Bhandari). 2213-4344/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phanu.2013.01.001
market for anti-obesity drugs is expanding rapidly but the available drugs are extremely sparse in relation to need. Hence, there is need to search for novel anti-obesity drug to tackle the obesity problem. Current therapeutic strategy for treating obesity is mainly focused on diet control and physical exercise. Natural products/ dietary photochemicals have aroused considerable interest in recent years as potential therapeutic agents to counteract obesity. These compounds exert the anti-obesity effects mainly through regulation of various pathways, including lipid absorption, intake and expenditure of energy, increasing lipolysis, and decrease lipogenesis, and differentiation and proliferation of preadipocytes [5]. Embelia ribes Burm (Myrsinaceae) known commonly as vidanga is widely distributed throughout India. Ayurveda describes vidanga as pungent and cures colic and flatulence i.e. it has potential in medoroga (obesity). One ayurvedic formulation, vidangadya cˆurna (powder of vidanga), containing vidanga as main ingredient is taken with honey to alleviate obesity [6]. E. ribes fruits contain a quinone derivative (embelin), an alkaloid (christembine) and a volatile oil (vilangin). Embelin (2,5dihydroxy-3-undecyl-1,4-benzoquinone) is considered one of the major bioactive constituents and has a wide spectrum of
U. Bhandari et al. / PharmaNutrition 1 (2013) 50–57
biological activities including antitumor, antibacterial, antioxidant, analgesic, antifertility, wound healing, anticonvulsant and antidiabetic [7]. Tripathi [8] reported the anti-hyperglycemic activity of the decoction of E. ribes in glucose-fed albino rabbits. Further, Bhandari et al. [9] reported the potential of E. ribes ethanol extract (ERE) in diabetic dyslipidemia and protection from lipid peroxidation in tissues in streptozotocin induced diabetes in rats. The effect of oral treatment with ERE on the basal level of some key serum, tissue antioxidants, blood pressure and glycosylated haemoglobin in streptozotocin-induced oxidative damage in rats has also been reported [10,11]. However, E. ribes has not been investigated so far for its antiobesity potential. We have selected a rat model of diet induced obesity based on numerous experimental studies which indicated that diets high in fat are known to increased body weight and fat mass, induce alterations in carbohydrate and lipid metabolism, lead to insulin resistance, and increase production and release of leptin in humans, rodents, and other animals [12,13]. Hence, the objective of this study was to evaluate anti-obesity effect of E. ribes ethanol extract (ERE) in murine model of high fat diet (HFD)induced obesity. 2. Materials and methods 2.1. Plant material The berries of E. ribes were purchased from North Eastern India Ayurveda Research Institute, Barsjoi,Guwahati, Assam, India, and authenticated by Dr. H. B. Singh, Head, Raw Materials and Herbarium, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi, India, where a voucher specimen is preserved for further reference (Reference No. NISCAIR/RHMD/Consult/-2010-11/1500/98, dated 26 August, 2010). 2.2. Preparation of E. ribes ethanol extract The dried and powdered fruits of E. ribes were extracted with 90% ethanol in a Soxhlet extraction system for 72 h. The solvent was removed under reduced pressure to give a dry extract, 7% yield (w/w) with respect to the crude material and stored at 20 8C until the experiment. This extract was tested for phytochemical analysis and high performance thin layer chromatography (HPTLC) finger printing for standardization [14,15]. 2.3. High performance liquid chromatography (HPLC) analysis The HPLC analysis of ERE was carried out by the methodology of Shelar et al. [16] on HPLC system (Waters Corporation, USA) equipped with Millennium Chromatography software version 2.15 and the chromatographic separations were performed using a Hyperchrome Column (250 mm 4.6 mm), with a flow rate of 1 ml/min and a sample size of 10 ml. The mobile phase used was methanol (A) and 0.1% trifluoroacetic acid (B) with a ratio of 88:12 (A:B (v/v)). The sample analysis was performed at wavelength 288 nm using photodiode array (PDA) detector at ambient temperature. The chromatographic peaks of the ERE were confirmed by comparing its retention time and UV spectra with corresponding reference standard embelin. The result was obtained by comparison of peak area (at 288 nm) of ERE with that of embelin. 2.4. High fat diet-induced obesity The male Wistar rats (150–200 g) were procured from central animal house facility of Hamdard University, New Delhi, India. The
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Table 1 Compositions of high fat diet (g/kg diet). Ingredients
Quantity
Casein L-cystine Starch Sucrose Cellulose Ground nut oil Tallow AIN salt mix AIN vitamin mix
342.0 3.0 172.0 172.0 50.0 25.0 190.0 35.0 10.0
Total (g)
999.0
animals were housed in standard polypropylene cages and maintained under controlled room temperature (22 2 8C) and humidity (55 5%) with 12 h light and 12 h dark cycle. All the rats were provided with commercially available rodent chow diet (Amrut rat feed, Nav Maharastra Chakan Oil Mills Ltd., Delhi, India) and tap water ad libitum. After 1 week of acclimatization with free access to rodent chow diet and water, animals were used in the study. The guidelines of committee for the purpose of control and supervision of experiments on animals (CPCSEA), Government of India were followed and prior permission was sought from the Institutional Animal Ethics Committee, Jamia Hamdard, New Delhi for conducting the study. Rats were fed a HFD in a pellet form and water ad libitum for the period of 4 weeks. HFD was purchased from National Centre for Laboratory Animal Science (NCLAS), National Institute of Nutrition (NIN), Hyderabad, India. Composition of HFD is shown in Table 1. 2.5. Experimental design In this study, a total of 50 rats were used and divided into five groups of 10 rats each. Group I: normal healthy control rats fed with rodent chow diet and administered 1% gum acacia [1 ml/kg, body weight (bw)] for the period of 28 days (NC). Group II: rats fed with HFD for the period of 28 days (HFD). Group III: rats fed with HFD for the period of 28 days + from 8th day, treated with ERE (100 mg/kg, bw) for period of 21 days (HFD + ERE). Group IV: rats fed with HFD for the period of 28 days + from 8th day, treated with Orlistat (10 mg/kg, bw) for period of 21 days (HFD + ORL). Group V: rats fed with rodent chow diet for the period of 28 days + from 8th day, treated with ERE (100 mg/kg, bw) for period of 21 days (ERE per se).
All the drugs were administered by oral gavage once a day. Food and water intakes were measured daily for the period of 28 days at the same time. Food intake and water intake was measured on per cage basis and the average food and water consumed were calculated. At the end of the experimental period (on 29th day), the animals were anesthetized with ether, following overnight fasting. Blood was drawn by retro-orbital method into a tube and the serum was obtained by centrifugation. After collection of blood, rats were sacrificed; heart, and perirenal white adipose tissue (WAT), epididymal WAT, mesenteric WAT were excised immediately, rinsed with phosphate buffer saline and weighed. The serum and heart samples were stored at 70 8C until analysis.
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2.6. Anthropometric measurements The body weights were determined once a week. Body mass index (BMI) was calculated from formula: BMI = body weight (g)/ length2 (cm2) [17]. 2.7. Measurement of hemodynamic parameters Hemodynamic parameters (systolic, diastolic, mean arterial blood pressure and heart rate) were measured by Non-Invasive Blood Pressure Recorder using rat tail-cuff method (Kent Scientific Corporation, USA). 2.8. Biochemical estimation Serum glucose, triglyceride (TG) and total cholesterol (TC) levels were determined by enzymatic methods using commercial assay kits (SPAN Diagnostics Ltd., Surat, India) according to the manufacturer’s protocols. Serum high-density lipoprotein cholesterol (HDL-C) levels were determined using HDL Cholesterol Test Kit (Reckon Diagnostics Pvt. Ltd,. Baroda, India). Serum leptin and insulin levels were measured by immunoassays using a commercially available ELISA assay kit from Bio Vendor (Modrice, Czech Republic) and Alpco Diagnostics (Salem, USA), respectively. Low-density lipoprotein cholesterol (LDL-C) levels were calculated using Friedewald formula [17]. Serum very low density lipoprotein cholesterol fraction (VLDL-C) concentration was calculated by deduction of the sum of HDL-C and LDL-C concentrations from that of TC. 2.9. Determination of lipid peroxidation and free radical scavenging activity in cardiac tissue
[21], glutathione peroxidase (GPx) [22], and glutathione-Stransferase (GST) [23] activities. 2.10. Histopathological analysis For histological examination, the heart tissue was collected and fixed in 10% neutral buffered formalin, embedded in paraffin. Standard sections of 5 mm thickness were cut, which were then stained with hematoxylin and eosin (H&E), and examined by light microscopy. 2.11. Statistical analysis Data are expressed as the mean standard error of the mean (SEM). The statistical significance of difference between the mean values for the treatment groups was analyzed by ANOVA (analysis of variance) followed by Dunnett’s t-test using Graph pad InStat1 version 3.06 (Graph Pad Software, San Diego, CA, USA). Values of p < 0.05 were considered significant. 3. Results 3.1. Preliminary phytochemical screening, HPTLC and HPLC analysis Phytochemical screening of ERE showed positive results for alkaloids, carbohydrates, phenolics, and saponins. HPTLC finger prints of ERE showed the presence of 7 spots with their retention factor (Rf) value 0.06, 0.19, 0.25, 0.41, 0.45, 0.51 and 0.61 in the solvent system ethyl acetate:methanol (9:1) at 365 nm wavelength. Out of these compounds, band with max Rf 0.06 matched exactly with that of embelin (max Rf 0.06). The presence of embelin in ERE was established by HPLC and quantified as 3.2803% (Fig. 1). 3.2. Effect on food and water intake
A portion of heart was minced and homogenized (10%, w/v) for determination of lipid peroxidation (LPO) [18], reduced glutathione (GSH) [19], superoxide dismutase (SOD) [20], catalase (CAT)
There was no significant (p > 0.05) change in food and water intake was observed in HFD-fed group as compared to the NC
Fig. 1. HPLC chromatograms of Embelia ribes ethanol extract (A) and the standard embelin (B; retention time: 3.088).
U. Bhandari et al. / PharmaNutrition 1 (2013) 50–57
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Table 2 Effect of ethanol E. ribes extract on food intake, water intake, body weight gain and BMI in HFD-induced obesity in Wistar rats. Groups
Food intake (gm/rat/day)
Water intake (ml/rat/day)
Body weight gain (g)
BMI (g/cm2)
NC HFD HFD + ERE HFD + ORL ERE per se
12.78 2.31 15.65 3.67 15.40 0.67 15.27 0.83 14.89 0.48
40.34 3.81 45.60 4.71 43.50 1.32 43.80 1.41 42.80 1.38
53.13 5.02 124.35 9.26** 94.60 7.29## 87.80 4.22## 47.80 1.49
4.26 0.04 5.87 0.18* 4.61 0.35# 4.15 0.26# 4.03 0.80
All values were expressed as Mean SEM (n = 10). * p < 0.05 as compared to the NC group. ** p < 0.01 as compared to the NC group. # p < 0.05 as compared to HFD group. ## p < 0.01 as compared to HFD group.
Table 3 Effect of ethanol E. ribes extract on heart rate and blood pressure in HFD-induced obesity in Wistar rats. Groups
Heart rate (beats/min)
Systolic B.P. (mmHg)
Diastolic B.P. (mmHg)
Mean arterial B.P (mmHg)
NC HFD HFD + ERE HFD + ORL ERE per se
421.83 11.26 539.20 15.33** 470.89 14.11## 464.83 11.57## 436.21 8.58
127.83 3.60 145.16 2.58** 130.00 1.88## 129.23 4.84## 132.73 3.29
96 3.65 109.00 2.56** 96.66 2.38## 94.53 1.77## 94.21 7.39
106.61 1.22 121.05 2.09** 107.77 1.84## 106.09 1.77## 107.05 2.19
All values were expressed as Mean SEM, (n = 10). * **
p < 0.05 as compared to the NC group. p < 0.01 as compared to the NC group.
# ##
p < 0.05 as compared to HFD group. p < 0.01 as compared to HFD group.
group. Administration of HFD + ERE or HFD + ORL did not produce significant change in the food and water intake as compared to HFD group (Table 2).
1.46-fold, perirenal WAT by 1.25-fold, and mesenteric WAT by 1.3fold when compared to HFD group (Fig. 2). 3.6. Effect on serum lipid levels
3.3. Effect on anthropometric parameters The body weight gain of rats in the HFD-fed group was greater than the values for the NC. A 2.34-fold increase in body weight gain was observed in HFD group compared to NC group. Treatment with HFD + ERE and HFD + ORL significantly (p < 0.01) reduced the body weight gain by 1.31- and 1.42-fold respectively. BMI of the rats in HFD-fed group, which was significantly greater than the value for NC (p < 0.05), was reduced significantly (p < 0.05) when the rats were administered HFD + ERE or HFD + ORL. However, there was no significant difference in body weight gain and BMI among the NC and ERE per se was observed (Table 2). 3.4. Effect on hemodynamic parameters A statistically significant increase (p < 0.01) in heart rate, systolic, diastolic and mean arterial blood pressure was observed in HFD-fed group as compared to NC. ERE in 100 mg/kg dose (i.e. HFD + ERE group) produced a statistically significant (p < 0.01) decrease in heart rate, systolic, diastolic and mean arterial blood pressure was observed. However, the reference drug, orlistat (i.e. HFD + ORL group) exhibited better activity than HFD + ERE group in all the above hemodynamic parameters (p < 0.01; Table 3).
The results of the serum lipid profile are shown in Fig. 3. HFD-fed group showed a significant (p < 0.01) increase in serum TC, TG, LDLC and VLDL-C by 1.64-,2.4-, 1.96-, and 2.4-fold respectively against NC group and a significant (p < 0.01) decrease in HDL-C levels by 1.28-fold as compared to NC group. HFD + ERE administration caused a significant (p < 0.01) decrease in serum TC, TG, LDL-C and VLDL-C levels by 1.21-, 1.32-, 1.37- and 1.32-fold, and HFD + ORL 1.28-, 1.38-, 1.55- and 1.38-fold, respectively, as compared with those in HFD-fed group. There was a significant (p < 0.01) increase in serum HDL-C by 1.31- and 1.36-fold in HFD + ERE and HFD + ORL group, respectively, as compared to HFD-fed group. However, there was no significant (p > 0.05) difference in serum lipid profile among the NC and ERE per se was observed. 3.7. Effect on serum glucose, insulin, and leptin levels HFD-fed rats exhibited significant (p < 0.01) increase in serum glucose, insulin and leptin by 1.56-, 2.13- and 1.52-fold
3.5. Effect on white adipose tissue weights The weights of fat pads from HFD-fed, NC, drug treated groups were assessed after the treatment period. The weight of the WAT tissue were significantly (p < 0.01) higher (epididymal WAT by 1.69fold, perirenal WAT by 1.74-fold, and mesenteric WAT by 1.93-fold higher) by feeding HFD than the value for NC. Conversely, epididymal WAT, perirenal WAT, and mesenteric WAT weights were decreased by 1.19-, 1.13-, 1.19-fold, respectively, in ERE treated rats (i.e. HFD + ERE group) then in their HFD-fed counterparts. HFD + ORL administration decreased epididymal WAT by
Fig. 2. Effect of ethanolic extract of Embelia ribes on white adipose tissue (WAT) weights in HFD-induced obesity in Wistar rats. All values were expressed as Mean SEM, (n = 10); *p < 0.05, **p < 0.01 as compared to NC; #p < 0.05, ##p < 0.01 as compared to HFD.
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Fig. 3. Effect of ethanolic extract of Embelia ribes on serum lipid levels in HFD-induced obesity in Wistar rats. All values were expressed as Mean SEM, (n = 10); * p < 0.05, ** p < 0.01 as compared to NC; #p < 0.05, ##p < 0.01 as compared to HFD.
glucose, insulin and leptin levels by 1.33-, 1.74- and 1.94-fold respectively. However, there was no significant (p > 0.05) difference in serum glucose, insulin and leptin levels among the NC and ERE per se was observed (Figs. 4, 5 and 6). 3.8. Effect on lipid peroxidation and free radical scavenging activity in cardiac tissue
Fig. 4. Effect of ethanolic extract of Embelia ribes on serum glucose levels in HFDinduced obesity in Wistar rats. All values were expressed as Mean SEM, (n = 10); * p < 0.05, **p < 0.01 as compared to NC; #p < 0.05, ##p < 0.01 as compared to HFD.
HFD-fed rats showed a significant (p < 0.01) increase in cardiac TBARS content by 3.48-fold as compared with those in NC group. HFD + ERE treatment caused a significant (p < 0.01) decrease in cardiac TBARS content by 2.28-fold while HFD + ORL showed 2.7fold reduction as compared to HFD group. . As shown in Table 4, the HFD-fed group showed a significant (p < 0.01) depletion in cardiac SOD, CAT, GSH, GPx and GST contents by 1.81-, 1.95-, 2.8-, 1.39- and 2-fold respectively as compared to NC group. HFD + ERE treatment caused a significant (p < 0.01) enhancement in cardiac SOD, CAT, GSH, GPx and GST contents by 1.44-, 1.77-, 2-, 1.32- and 1.76-fold while HFD + ORL showed 1.61-, 1.89-, 2.83-, 1.45- and 1.88-fold elevation respectively as compared to HFD-fed group. However, there was no significant (p > 0.05) difference in cardiac TBARS, SOD, CAT, GSH, GPx and GST contents among the NC and ERE per se was observed. 3.9. Effect on histopathological changes in cardiac tissue
Fig. 5. Effect of ethanolic extract of Embelia ribes on serum insulin levels in HFDinduced obesity in Wistar rats. All values were expressed as Mean SEM, (n = 10); * p < 0.05, **p < 0.01 as compared to NC; #p < 0.05, #p < 0.01 as compared to HFD.
In the histopathological studies, heart sections in NC and ERE per se group showed normal morphology of myocardium. The heart sections of HFD-fed obese rat showed deposition of fat globules in myocardial cells. However, HFD + ERE and HFD + ORL treated rats heart showed normal architecture of myocardium (Fig. 7). 4. Discussion
Fig. 6. Effect of ethanolic extract of Embelia ribes on serum leptin levels in HFDinduced obesity in Wistar rats. All values were expressed as Mean SEM, (n = 10); * p < 0.05, **p < 0.01 as compared to NC; #p < 0.05, ##p < 0.01 as compared to HFD.
respectively against NC group. In contrast, treatment with HFD + ERE significantly (p < 0.01) decreased serum glucose, insulin and leptin levels by 1.39-, 1.58- and 1.81-fold respectively. HFD + ORL treatment significantly (p < 0.01) decreased serum
In the present study, anti-obesity effect of ERE was investigated using a HFD-induced obese rat model. Reduction in body weight gain of HFD-fed rats was accompanied by a depletion of body fat stores, since treatment with ERE also significantly reduced the weight of the visceral WAT compared with that of HFD-fed rats. Excessive growth of adipose tissue results in obesity which involves two growth mechanisms: hyperplasia (cell number increase) and hypertrophy (cell size increase) [24]. In epidemiological studies, BMI is widely used as a measure of fatness because it is highly correlated with body fat and is nearly independent of height [25]. In our study, significant reduction in BMI of ERE treated group was observed. These results indicate that ERE suppresses the HFD-induced increase in fat mass and body weight gain.
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Table 4 Effect of ethanol E. ribes extract on myocardial TBARS, SOD, CAT, GSH, GPx and GST levels in HFD-induced obesity in Wistar rats. Groups
TBARS (nmol MDA/mg protein)
SOD (U/mg protein)
CAT (nmol H2O2 consumed/min/mg protein)
GSH (mmol P liberated/min/mg protein)
GPx (nmol NADPH oxidized/min/mg protein)
GST (nmol CDNB conjugate/formed/min/mg protein)
NC HFD HFD + ERE HFD + ORL ERE per se
0.21 0.01 0.73 0.03** 0.32 0.01## 0.27 0.03## 0.20 0.05
3.85 0.22 2.13 0.13** 3.07 0.09## 3.42 0.06## 3.74 0.12
54.63 1.45 28.03 0.64** 49.55 1.70## 52.95 1.09## 56.46 2.14
26.75 0.97 9.54 0.47** 19.12 0.79## 27.01 0.18## 26.52 1.3
184.81 7.54 132.66 3.14** 174.55 5.70## 192.50 9.18## 188.72 2.04
492.31 4.01 246.43 2.06** 434.38 6.37## 463.21 5.26## 488.21 3.09
All values were expressed as Mean SEM, (n = 10). * **
p < 0.05 as compared to the NC group. p < 0.01 as compared to the NC group.
# ##
p < 0.05 as compared to HFD group. p < 0.01 as compared to HFD group.
Fig. 7. Histopathological changes in heart of experimental rats. Group I (A): normal heart show normal morphology of myocardium; Group II (B): obesity control, HFD treated heart shows deposition of fat globules in myocardial cells; Group III (C): HFD + E. ribes extract (100 mg/kg b.w.) shows no fatty changes; Group IV (D): HFD + Orlistat (10 mg/ kg b.w.) shows no fatty changes and normal architecture of myocardium; Group V (E): E. ribes extract (100 mg/kg) per se shows normal architecture of heart.
Though there was a significant difference in the body weights between the HFD-fed and NC groups, no significant difference in the daily food intake and water intake between groups was observed. This observation provides us with the fact that dietary caloric content was independent of the amount of food
consumed by the animals. The HFD-fed rats continuously consumed similar quantities of food, regardless of the higher calories content in the diet. As a result, the caloric intake was raised in the HFD-fed rats compared to the NC rats. The unchanged food intake in spite of the higher caloric content in
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the diet is proportional to the increment of the body weight, hence, resulting in the obese state. In addition to its weight reducing effect, ERE was also able to improve some lipid metabolites including TC, TG, HDL-, LDL- and VLDL-cholesterol levels in obese rats. It is reported that obesity, especially abdominal obesity, is associated with dyslipidemia, characterized by elevated TG and reduced HDL-C concentrations. TGs are involved in the ectopic accumulation of lipid stores in the liver and are associated with a number of diseases such as metabolic syndrome. VLDL-C transports cholesterol and TG to the tissues, while high HDL-C is helpful in transporting excess cholesterol to the liver for excretion in the bile. High TC and LDL-C levels, and lower HDL-C level are risk factors for coronary heart disease [26]. Thus, this result suggested that ERE would be helpful in the prevention of obesity complications through improving hyperlipidemia. Further, ERE treatment showed improvements in the histological features of heart induced by HFD in rats. These results seemed to correspond to the white adipose tissue weights (Fig. 2) and serum lipid profiles (Fig. 3). Obesity is associated with an impaired ability of tissue to respond to insulin and effectively store glucose. Further, in response to insulin resistance production is increased, leading to hyperinsulinemia, hyperglycemia and ultimately type 2 diabetes [27]. In our study, significant reduction in the serum glucose and insulin levels after the treatment with ERE in HFD-fed rats was observed. Epidemiological studies have found a progressive increase in the prevalence of elevated blood pressure with increasing adipose tissue [25]. Moreover, hypertension and hyperlipidemia often manifest concomitantly in the clinical context of obesity and insulin resistance. In consistent with improvement of lipid profile and insulin resistance by ERE, it is also able to attenuate the development of hypertension in HFD-fed obese rats. Leptin is fat-derived key regulators of food intake and energy expenditure, where its concentration in the plasma is associated with general adiposity [28]. The reductions of leptin level reflect a decreased in the fat mass. Hypothalamus receives direct input from leptin, which cross the blood–brain barrier and provide information on the levels of peripheral adipose mass. The results from the present studies suggest that ERE caused significant adipocytes loss, indicated by reduced leptin levels. Obesity and hyperlipidemia synergistically promote systemic oxidative stress-imbalance between tissue free radicals, reactive oxygen species (ROS) and antioxidants [29]. ROS could react with polyunsaturated fatty acids, which lead to lipid peroxidation [30]. Malondialdehyde is a by-product of lipid peroxidation and reflect the degree of oxidation in the body [18]. Possible mechanisms that generate oxidative stress in obesity include hyperglycemia, elevated lipid levels, inadequate antioxidant defenses and hyperleptinemia [31]. In the present study, cardiac TBARS levels, determined by evaluating malondialdehyde content were decreased after ERE treatment in HFD-fed rats. Furthermore, in our study, the activities of GSH (a major endogenous antioxidant), GPx, GST, SOD and CAT (scavenger of free radicals) decreased in HFD-fed rats as reported earlier [32]. Treatment of HFD-fed rats with ERE had reversed the activities of these enzymatic antioxidants. It is therefore reasonable to assume that ERE treatment improves cardiac oxidative balance in HFD-fed obese rats, because ERE was able to reduce the level of TBARS and free radical generation. Conclusively, observed reduction in body weight gain, BMI, heart rate, blood pressure, serum lipids, glucose, insulin, leptin levels, myocardial tissue lipid peroxidation, and improvement in myocardial lipid accumulation and antioxidant enzyme levels suggests that ERE possesses significant anti-obesity potential.
Layperson’s summary Overweight and obesity are the most common nutritional disorder in Western countries. The objective of this study was to evaluate anti-obesity potential of standardized E. ribes ethanol extract in high fat diet (HFD)-induced obesity in Wistar rats. E. ribes extract treatment for 21 days to obese rats resulted in significant reduction on body weight gain, visceral fat accumulation and elevated blood pressure. The extract treatment also produced a significant reduction in serum levels of lipids, insulin, glucose and elevation in antioxidant levels. These results suggested that E. ribes extract has anti-obesity potential.
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