Trends Sci. 2025; 22(9): 10407

Obesity is one of the significant public health problems worldwide due to its continuously increasing prevalence across all age groups. According to the World Obesity Atlas 2023 report, the prevalence of obesity (BMI ≥ 30 kg/m²) is estimated to rise from 14 to 24%, or approximately 2 billion adults, children, and adolescents, by 2035. Furthermore, the double prevalence in adults, from 4 to 10% in men and 8 to 16% among women, will occur in this period. Indonesia was the country with the most rapid growth, at the rate of 5.8% for adults and 7.9% for children [1]. Obesity is now a major risk factor for metabolic and degenerative diseases such as cardiovascular diseases, diabetes, dyslipidemia, sleep apnea, and various cancers [2]. Diabetes has become one of the leading causes of premature death and disability for people of all ages and sexes in the world [3]. By 2045, the International Diabetes Federation estimates that 260 million people in the Western Pacific region, including Indonesia, will be afflicted with diabetes. Available data indicate that in 2021, the prevalence of diabetes in adults in Indonesia was 10.8% [4].

Obesity results from a constant imbalance between food intake and energy expenditure, tightly regulated by the central nervous system and by peripheral hormones such as T cell protein tyrosine phosphatase (TCPTP), leptin, adiponectin, and insulin. Leptin is an appetite-regulating hormone released by adipocytes. In obese people, leptin secretion is overzealous, causing leptin resistance. Currently, leptin resistance has become a key factor in obesity pathophysiology, which reduces anorexic response to leptin stimulus, thus causing hyperphagia [2]. In this condition, uncontrolled appetite leads to overnutrition and fat accumulation in the adipose tissues and impairs insulin action to further uptake blood glucose. As a result, it continues insulin secretion and insulin resistance, characterized by raised blood insulin levels (hyperinsulinemia). In addition, overnutrition leads to fat deposits that can elevate blood triglycerides, total cholesterol, and LDL-C levels, paired with a simultaneous fall in HDL-C levels. Perilipin-1 proteins will increase to engage elevated triglycerides for lipolysis, releasing free fatty acids in the bloodstream. Elevated fat accumulation promotes subcutaneous fat development, increases body weight and obesity index, and induces inflammation, marked by higher ROS and MDA levels in the blood circulation. This inflammatory process is followed by reductions of anti-inflammatory cytokines, including adiponectin [5,6].

Obesity diagnosis in clinical and community settings traditionally relies on anthropometric measurements like body weight, body mass index, waist circumference, and waist circumference to height ratio. In animals, various measurements were used, such as Rohrer, Lee, and TM indexes, body fat content, and visceral fat measurement [7]. However, combining anthropometric measurements with radiological approaches like dual-energy X-ray absorptiometry (DXA) and computed tomography (CT) scans is crucial to assess fat accumulation effectively. The DXA allows for a precise evaluation of fat mass, fat-free mass, and bone density with minimal error rates, while CT scans aid in cardio-metabolic risk analysis by examining different fat types [8,9]. Despite their effectiveness, both DXA and CT involve ionizing radiation that can pose risks like gene mutations and cancer [10]. Ultrasound imaging is an alternative technique to observe tissue morphology and blood flow without radiation exposure [11].

There are several treatment modalities for managing obesity, but the current treatments are either ineffective or have several side effects [12]. Lifestyle changes and dietary modifications have weight loss effects, but many obese individuals do not adhere to dietary rules, leading to weight regain [13]. The use of medication for obesity is only prescribed for obese patients with moderate to high risks. Some of the anti-obesity drugs currently used include orlistat, lorcaserin, and naltrexone. However, the long-term use of those drugs remains unknown, and it has been reported to have several side effects, such as dizziness, nausea, hypertension, insomnia, and hypoglycaemia [14]. Therefore, it requires an alternative therapy to control body weight and modulate adipose tissues in obese people.

Medicinal therapy using plant-based drugs is a growing trend in current obesity management. In silico studies have shown that a rutacridone phytochemical in the Rue (Ruta graveolens) plant can interact with the TCPTP, thus potentially reducing leptin resistance and addressing obesity [15]. Rue leaves are one of the medicinal plants known in Indonesia, containing over 231 active compounds with various pharmacological effects [16]. For example, quercetin, an important flavonoid, is reported to decrease obesity by increasing adiponectin and decreasing leptin levels [17]. A previous research study states that the administration of 240 mg/kg/day quercetin for 11 weeks in obese rats can control obesity by reducing intracellular oxidative stress, and chronic low-grade inflammation, inhibiting adipogenesis and lipogenesis, and suppressing the differentiation of preadipocytes into mature adipocytes [18]. It seems that natural supplements obtained from traditional medicinal plants are a rational approach to treating obesity, and recent experiments have revealed many useful herbal products for treating obesity [19]. Therefore, the aim of this study was to investigate the methanol extract of Rue leaves (MERL) in addressing obesity through the TCPTP inhibition mechanism in an obese animal model.

Materials and methods

Preparation of methanol extract of rue leaves (MERL)

Fresh rue leaves were obtained from the Center for Research and Development of Medicinal Plants and Traditional Medicine, Tawangmangu, Karanganyar, Central Java, Indonesia. The leaves were dried in an oven at 60 °C for 24 h and then ground using a blender to produce a powder. The extraction of Rue powder was soaked in 70% methanol at room temperature for 3 days. The filtrate of rue powder was then concentrated using a rotary evaporator at 60 °C, 80 rpm, and 175 mbar [18]. MERL was stored at 4 °C before further analysis.

Phytochemical analysis using liquid chromatography high-resolution mass spectrometry (LC-HRMS)

The methanol extract of rue leaves was chemically analyzed using an LC-HRMS (Dionex™ Ultimate 3000 RSLC nano, Thermo Scientific™, USA) at the Integrated Research and Testing Laboratory, Universitas Gadjah Mada (UGM), Yogyakarta, Indonesia. The MERL sample was dissolved in 1.000 µL of methanol and then vortexed for 3 min. The homogenized sample was filtered using a 0.2 µM Millex filter, and 5 µL MERL was injected into the analytical column: Phenyl Hexyl 100 mm × 2.1. The LC-HRMS consisted of mobile phase A (water + 0.1% Formic Acid) and B (Acetonitrile + 0.1% Formic acid) with a flow rate of 0.20 mL/min for 30 min. Identification of bioactive compounds was performed using Thermo Scientific™ Compound Discoverer Software.

Development of obese rat model

Male white rats (Rattus norvegicus), the Sprague Dawley strain, were adapted for 7 days in a cage with 12 h of light, 12 h of darkness at 25 °C, and controlled humidity. The rats were given Comfeed Br-2 feed and aquadest. After adaptation, all male rats were evaluated for their body weight, body length, and subcutaneous fat thickness. A total of 40 rats were developed to become obese through the administration of a high-fat high-fructose (HFHFr) diet for 30 days. The HFHFr consisted of beef fat, duck egg yolk, chicken liver, and butter, along with 10% fructose in their drinking water, which had nutrient values of 100 g and consisted of 610 kcal of total energy and 16.6% of total carbohydrates. 13.11% of total protein and 54.64% of total fat. The HFHFr was given 10% of the rats’ body weight every day [20]. BW, BL, and SFT were regularly monitored every week. The male rats with obesity were established using Lee’s obesity index ≥ 30 g/cm³ [7]. The rat’s SFT was measured using a portable ultrasonography with ≥ 1.54 mm obese value [39].

Study design

This study was animal experiment research with a pre-posttest control group design for body weight, obesity index, subcutaneous fat thickness, fasting blood glucose, lipid profile, leptin, adiponectin, malondialdehyde levels, and a posttest only group design for protein expression of TCPTP and Perilipin-1. Male white rats (Rattus norvegicus), Sprague Dawley strain, were used in this study, and they were aged 8 - 10 weeks and weighed approximately 300 g. The sample size was calculated using the formula: (n×t) – t, where n = the number of rats and p = the number of rat groups, establishing a minimum of 4 rats per treatment group. In anticipation, if there are rats that drop out, an estimate of up to 50% is added [21]. The normal group or non-obese rats (N) was given BR-2 comfeed and aquadest drinking water for 30 days. Meanwhile, obese rats were divided into 5 groups. The negative (NC) and positive control (PC) groups were given a standard diet and 1 mL 0.9% NaCl and 0.6 mg/kg BW/day rutacridone solution (Sigma, USA), respectively, for 30 days. Treatment groups (T1, T2 and T3) were given a standard diet and 80, 100 and 200 mg/kg BW/day MERL, respectively, for 30 days. The protocol of this research study was approved by the Health Research Ethics Committee, Dr. Moewardi, General Hospital, Surakarta, Indonesia, number 100/I/HREC/2023, on January 30, 2023.

Measurement of BW, BL, OI and SFT

BW and BL measurements were conducted weekly in the morning using a body weight scale (Sonic, Jakarta) and a stadiometer (Measure King, Jakarta). The OI was calculated using the Rohrer Index formula = {BW (g)/Naso − anal length (cm)³} × 10³, where rats were considered obese if they had an index ≥ 30 g/cm³. Before measuring SFT, the rats’ hair in the abdominal area was shaved to approximately 5 cm in diameter. The SFT measurement was conducted weekly in the morning before giving the standard feed. The shaved abdomen was evenly covered with ultrasound gel (Onemed, Sidoarjo). Rats were considered obese if they had an SFT ≥ 1.54 mm.

Measurement of FBG, lipid profiles, LEP and ADP levels

Blood samples were collected thrice on days 0, 15 and 30 in the morning before intervention. All collected blood samples were centrifuged at 4.000 rpm for 10 min to obtain serum for measuring fasting blood glucose (FBG) levels, lipid profiles: Triglyceride (TG), total cholesterol (TC), Low-Density Lipoprotein Cholesterol (LDL-C), High Density Lipoprotein Cholesterol (HDL-C) levels, leptin (LEP), and adiponectin (ADP) levels. FBG levels were determined using the GOD-PAP method, TG levels using the GPO-PAP method, and TC, LDL-C, and HDL-C levels using the CHOD-PAP method. The measurements of LEP and ADP levels used ELISA assays provided by Elabscience®, China (E-EL-R0582 and E-OSEL-R0006, respectively). The ELISA protocols for measuring LEP and ADP levels were based on the manufacturer.

Western blotting analysis of TCPTP and perilipin-1

The examination of TCPTP and Perilipin-1 protein expression used rats’ brains and adipose tissues, which were collected on day 30 of intervention and lysed using RIPA buffer (Thermo Scientific, USA) [22]. Before SDS PAGE Electrophoresis, total protein concentrations of the cell lysate of all rats were measured using a BCA Protein Assay Kit (Thermo Scientific, USA catalog number: 23227). The following antibodies were used: Primary Antibody (Perilipin1 Recombinant Rabbit Monoclonal Antibody (Invitrogen, ARC1122; 1: 3.000), beta Actin Antibody (Abcam a68227; 1: 10.0000), and PTPN2 antibody (Sigma-Aldrich SAB4200249; 1: 3.000)) and Secondary antibody (Goat Anti-Rabbit IgG H&L (HRP), Abcam ab6721; 1:3.000). The SDS-PAGE process began with the preparation of reagents and samples, electrophoresis using the Mini Protean Tetra System-Biorad, loading the samples and protein marker (Precision Plus Protein Dual Color Standards, Biorad cat #1610734) into the wells, and running the gel at 150 V for 45 - 60 min. The proteins were then transferred to wells, 50 μg for the brain sample and 200 μg for the lipids sample, each well (Mini Transblot-cell), followed by incubation with primary and secondary antibodies. Visualization was conducted by transferring the PVDF membrane, adding ECL, incubating for 5 min, and then drying the membrane before placing it on a sample tray and visualizing using the ChemiDoc Touch Imaging System (Biorad).

Statistical analysis

The obtained data were then analyzed using IBM SPSS (Statistical Package for the Social Sciences) version 25 and presented as mean ± standard deviation. Parametric test of BW, OI, SFT, TG, TC, LDL-C, HDL-C, FBG, LEP, ADP levels, expression of TCPTP and PLIN-1 using 1-way ANOVA followed by the LSD post hoc test. To compare means within the same group on different days, we used the dependent T-test, and the data were considered significantly different if the p-value was < 0.05.

Results and discussion

The results of the data analysis indicated that the administration of 80, 100 and 200 mg/kg BW MERL declines SFT, BW, OI, TG, TC, LDL-C, FBG, and leptin levels, and low relative expression of TCPTP and Perilipin-1 in obese male rats in a dose-dependent manner, except for Leptin, Perilipin-1, TG, TC, and LDL-C. Therefore, our findings suggest that MERL will become a potential anti-obesity drug in the future.

Phytochemical screening of methanol extract of rue leaf

Based on the results of the phytochemical screening of the MERL, we selected the top 20 compounds based on their peak areas. Of the top 20 compounds, several appeared more than once. The data of the phytochemical screening results of the MERL are presented in Table 1.

From Table 1, we found 13 compounds that have various health-related activities. Based on their activities, these compounds are divided into antidiabetics such as 4,7,8-trimethoxyfuro[2,3-b] quinoline, Rutamarin, Osthol, Psoralen, Isoimperatorin, 4-Hydroxycoumarin, Scoparone, L-Tyrosine, Bis(2-ethylhexyl) phthalate, and Quercetin. Antioxidants such as Osthol, Rutin, 4-Hydroxycoumarin, L-Tyrosine, and Quercetin. Antihyperlipidemic such as Quercetin and anti-obesity such as Osthol, Psoralen, Rutin, Scoparone, and Quercetin.

The results of the MERL phytochemical test revealed 121 compounds contained in the extract. We targeted one compound, rutacridone, but the results of the phytochemical test did not show the presence of this compound, because rutacridone is only slightly soluble in water. In our study, the solvent used when making rue leaf extract was methanol, so it is suspected that rutacridone cannot dissolve in this solvent. Based on previous research, it was stated that rutacridone dissolves very well in ethyl lactate solvent. Meanwhile, rutacridone dissolved in ethanol cannot be dissolved like ethyl lactate [23]. Although we did not find the rutacridone compound, we found several compounds with the largest area that had good activity in overcoming obesity. There are 13 compounds (Table 1) with various activities, such as 4,7,8-trimethoxyfuro[2,3-b] quinoline, rutamarin, osthol, psoralen, isoimperatorin, 4-Hydroxycoumarin, scoparone, L-tryosine, Bis(2-ethylhexyl) phthalate, asparagin, quercetin which have antidiabetic activity. In addition, there are compounds that have antiobesity activity, such as scoparone, quercetin, rutin, psoralen, and osthol, so these 5 compounds are suspected to play a role in overcoming obesity in our study. Figures 1(A) - 1(E) is the LC-HRMS result of MERL compounds with anti-obesity activity. Osthol compound (Figure 1(A)) is a compound with anti-obesity activity that has the largest area compared to other compounds, which is 7.4×10⁹ (Table 1). However, the compound with the highest intensity is Psoralen compound at RT 12.012 (Figure 1(B)).

Effect of MERL on body weight, obesity index, and subcutaneous fat thickness

Obesity is characterized by an increase in body weight (BW) due to food consumption exceeding the body’s required nutrient intake. Excess energy is then converted into fat, leading to the accumulation of fat cells under the skin. Figure 2(A) shows that after 30 days of intervention, the PC, T1, T2, and T3 groups showed a significant decrease in BW (p = 0.001, 0.036, 0.010 and 0.023). The largest decrease in BW was observed in the T3 group (42.75 ± −5.06 g), while the smallest decrease in BW was observed in the NC group (12.5 ± −2.44 g). The mean BW levels among rat groups after 30 days of intervention were significantly different (p = 0.001). The highest mean BW levels were found in the NC group (378.75 ± 8.54 g), while the lowest mean BW levels in obese rats were found in the T3 group (348.88 ± 18.09 g). In our study, we observed a significant reduction in BW in the PC, T1, T2 and T3 groups, although the T3 group (treated with 200 mg/kg BW of MERL) showed the greatest average BW reduction among the obese rat groups (42.75 ± 5.06 g). However, the 200 mg/kg BW dose of MERL was unable to restore the BW of obese rats to normal levels within the 30-day intervention period. The decreased BW in our study was in accordance with a previous study that used methanol extract of R. graveolens and a high-fat diet, although the largest decrease in our study (42.75 ± 5.06 g) was lower than the previous study (558 g). This difference is related to the use of the animal model, which we used male Sprague Dawley rats, while the previous study used rabbits [24].

There was a significant decrease in OI in the PC, T1, T2, and T3 groups (p = 0.012). The largest and the smallest decrease in OI was observed in the T3 and NC groups (−9.07 ± 0.8 and −3.43 ± −0.3 g/cm³). The highest and the lowest mean OI levels were found in the NC and T3 groups (30.64 ± 0.53 and 24.16 ± 1.52 g/cm³) (Figure 2(B)). The OI is one of the methods used to detect obesity. A significant reduction was recorded for the obesity index in the PC, T1, T2, and T3 groups after the intervention period of our study, thus proving that MERL can reduce OI by lowering their body weight and length ratio. A previous study reported a significant reduction of OI in obese rats receiving 1 g/kg/day of Safoof Mohazzil, a mixture of Ptychotis ajowan, Origanum majorana, Foeniculum vulgare, Carum carvi, Coccus lacca, and Ruta graveolens, for 2 weeks, compared to the obese control group. However, our results showed a greater reduction in OI (−9.07 ± 0.8 g/cm³). The previous research study used Mohazil, which consists of a mixture of several plant extracts, and one of them is Ruta graveolens. In addition, the animal models were Female Sprague Dawley rats with 100 - 150 g BW, while our study used Male Sprague Dawley rats with ± 300 g BW [25].

Table 1 Results of LC-HRMS testing of methanol extract of rue leaf.

Figures 3(A) - 3(F) shows the SFT measurements representing each treatment group after treatment. It can be seen that the SFT with the highest thickness is in NC (Figure 3(B)), and the thinnest SFT is in PC (Figure 3(C)). A significant decrease in SFT was observed in the NC, PC, T1, T2, and T3 groups (p = 0.012, 0.011, 0.012, 0.011, and 0.012, respectively). The largest decrease in SFT was observed in the PC group (0.71 ± −0.01 mm). Meanwhile, the smallest decrease in SFT in obese rats was observed in the NC group (0.33 ± 0.01 mm). The highest mean SFT levels were found in the NC group (1.36 ± 0.10 mm), while the lowest mean SFT levels were found in the PC group (0.97 ± 0.05 mm) (Figure 3). Continuously increasing BW leads to the distribution of excess lipids to various parts of the body. Subcutaneous adipose tissue stores the majority of these lipids. Based on the research findings, administration of 80, 100, and 200 mg/kg BW MERL significantly reduced SFT (1.15 ± 0.06, 1.06 ± 0.08, and 1.04 ± 0.02 mm, respectively), although it did not match the reduction observed in the positive control group (0.97 ± 0.05 mm). This indicates that the pure compound rutacridone is more effective in reducing subcutaneous fat thickness. Until now, there is no similar study that uses ultrasound to measure SFT in obese rats, so this parameter is an update on our study.

The mean TG, TC, LDL-C, HDL-C, and FBG levels among obese rat groups on day 0 showed no significant differences (p = 0.908, 0.906, 0.102, 0.988, and 0.906). Figure 4(A) shows that after 30 days of intervention, there was a decrease in mean TG levels in all rat groups. A significant decrease in TG was observed in the PC, T1, T2, and T3 groups (p = 0.012). The smallest and the highest decrease in TG levels in obese rats was observed in the NC and T2 groups (−13.69 ± 0.18 and −59.86 ± 9.38 mg/dL). The mean TG levels among rat groups after 30 days of intervention were significantly different (p = 0.001). The highest mean TG levels in obese rats on day 30 were found in the NC group (104.02 ± 25.95 mg/dL), while the lowest mean TG levels were found in the T3 group (61.63 ± 14.34 mg/dL).

Figure 1 Results of LC-HRMS testing of compounds (a) osthol, (b) psoralen, (c) rutin, (d) scoparone, and (e) quercetin in the MERL.

The triglycerides are a group of lipids that are absorbed in the intestinal tract. Blood vessels, muscle tissues, and adipose tissues then utilize these lipids. Following digestion by the lipases, triglycerides are conveyed to the liver, which then catalyzes conversion into LDL cholesterol. The level of triglycerides increases because of intake that exceeds what the body can utilize, which causes an increase in the level of carbohydrates in the body. High levels of triglycerides and cholesterol cause the deposition of these compounds on blood vessel walls, forming plaques. The study’s results showed that leaf extract of ginkgo, at doses of 80, 100, and 200 mg/kg BW, significantly reduced the triglyceride levels, with the best dose being T2 (100 mg/kg BW). A previous research study reported that the administration of 0.25 mg/kg R. graveolens and flavonoids extract was able to reduce TG levels among all groups in rats with a diabetic model. The results of TG levels in that study were even lower (42.3 ± 1.9 mg/dL) than the TG levels in our study (61.63 ± 14.34 mg/dL). This different result may be related to the use of an animal model, which used male Wistar rats with 200 ± 20 g BW, while our study used male Sprague Dawley with 300 ± g BW [26]. A previous study reported that administration of 50 mg/kg rutin and induction of hyperlipidemia resulted in lower TG levels compared to rats with a hyperlipidemia model by reducing the HMG-CoA reductase activity as well as increasing fecal sterols. The results of TG levels in that study were higher (3,495.5 ± 874.7 mg/dL) than TG levels in our study (61.63 ± 14.34 mg/dL). This difference may suggest that the previous study used pure quercetin, while the MERL in our study contained several bioactive compounds [27].

Figure 2 Mean (A) BW and (B) OI in male obese rat models on day 0 and day 30, presented as mean ± SD. ^ns) Independent t-test not significant (p > 0.05) *) Independent t-test significant (p < 0.05) **) Post hoc test significant compared to the negative control (NC). ***) The post hoc test was significant compared to the positive control (PC). ^#) Post hoc test was significant compared to treatment 1 (T1). ^##) The post hoc test was significant compared to treatment 2 (T2). Statistical analysis was performed using Repeated-Measures ANOVA (for normally distributed data) and the Friedman test (for non-normally distributed data). N = Normal group (non-obese rats), NC = Negative control group (OR + standard diet + 0.9% NaCl), PC = Positive control group (OR + BR-2 + 0.6 mg/kg BW/day pure rutacridone), T1, T2 and T3 were OR + BR-2 + 80, 100 and 200 mg/kg BW/day MERL).

A significant decrease in TC (Figure 4(B)) was observed only in the PC and T3 groups (p = 0.012). The smallest and the highest decrease in TC was observed in the T1 and T2 groups (19.64 ± −3.9 and 23.86 ± −2.43 mg/dL). Although T2 had the highest decrease in TC, this decrease was not significant (p = 0.05). The highest mean TC levels in obese rats on day 30 were found in the NC group (93.59 ± 14.47 mg/dL), while the lowest mean TC levels were found in the T2 group (68.84 ± 16.07 mg/dL). Cholesterol is a highly influential factor that is an amphipathic compound from fat. In our study, 200 mg/kg BW MERL significantly reduced total cholesterol level by 23.15 ± 11.29 mg/dL in the study on obese rats. In a previous study, it was reported that the decrease in TC levels was 72.1% in the 40 mg/kg of Ruta chalepensis ethanolic extract group compared to the high cholesterol diet control. The results of TC levels in the study were still higher than the TC levels in our study (68.84 ± 16.07 mg/dL). This different result is that the previous study made a hyperlipidemia rat model by administering Triton WR-1339 intraperitoneally, while the rats in our study were made obese models by administering HFHFr [28].

Figure 3 Results of 30-day SFT intervention period using USG (A) N (B) NC (C) PC (D) T1 (E) T2 and (F) T3, each image shown is a SFT representing each group, (G) mean SFT in male obese rat models on day 0 and day 30, presented as mean ± SD. ^ns) Independent t-test not significant (p > 0.05) *) Independent t-test significant (p < 0.05) **) Post hoc test significant compared to the negative control (NC). ***) The post hoc test was significant compared to the positive control (PC). ^#) The post hoc test was significant compared to the T1 group. ^##) The post hoc test was significant compared to the T2 group. Statistical analysis was performed using Repeated-Measures ANOVA. N = Normal group (non-obese rats), NC = Negative control group (OR + standard diet + 0.9% NaCl), PC = Positive control group (OR + BR-2 + 0.6 mg/kg BW/day pure rutacridone), T1, T2 and T3 were OR + BR-2 + 80, 100 and 200 mg/kg BW/day MERL).

Effect of MERL on lipid profiles and FBG levels

Figure 4 Mean levels of (A) TG, (B) TC, (C) LDL-C, (D) HDL-C, and (D) FBG in male obese rat models on day 0 and day 30, presented as mean ± SD. ^ns) Independent t-test not significant (p > 0.05) *) Independent t-test significant (p < 0.05) **) Post hoc test significant compared to the negative control (NC). ***) The post hoc test was significant compared to the positive control (PC). ^#) Post hoc test was significant compared to treatment 1 (T1). ^##) Post hoc test was significant compared to treatment 2 (T2). Statistical analysis was performed using Repeated-Measures ANOVA. N = Normal group (non-obese rats), NC = Negative control group (OR + standard diet + 0.9% NaCl), PC = Positive control group (OR + BR-2 + 0.6 mg/kg BW/day pure rutacridone), T1, T2 and T3 were OR + BR-2 + 80, 100 and 200 mg/kg BW/day MERL).