Abstract
Background
Obesity is the most prevalent metabolic disorder globally, characterized by oxidative stress and inflammation that play a key role in its pathogenesis. Current anti-obesity drugs have adverse effects and fail to address oxidative stress and inflammation. Therefore, there is a growing need for safe and effective therapies. Ayurveda recommends Trimad for obesity management. Trimad is a polyherbal formulation consisting of Cyperus rotundus tubers (Musta), Embelia ribes fruits (Vidang), and Plumbago zeylanica roots (Chitraka) in an equal 1:1:1 ratio.
Objective
This study evaluated the effect of Trimad on ameliorating pathophysiological changes in a rat model of high-fat diet (HFD)-induced obesity.
Material and methods
Male wistar rats were divided into six groups (n = 6; IAEC approval no. BVDUMC/3293/2016/007/002). Group 1 served as the Normal control (normal chow diet), Group 2 as Disease control (HFD), Group 3 as Positive control (HFD + Atorvastatin, 1.2 mg/kg), and Groups 4–6 received HFD with Trimad extract at 50, 100, and 200 mg/kg, respectively for 48 days with treatment starting from day 24. Body weight, feed intake, biochemical parameters, oxidative stress markers, inflammatory cytokines, adipokines, adipocyte morphology, brain monoamines and hepatic lipid-metabolizing gene expression were evaluated.
Results
HFD caused lipid abnormalities, inflammation, oxidative stress, hepatic steatosis, and weight gain. Trimad significantly reduced weight gain, improved lipid metabolism by modulating hepatic lipid-metabolizing genes, and normalized biochemical parameters, oxidative stress, inflammatory markers, adipokines, and adipocyte hypertrophy. There was normalization of brain monoamines in Trimad treated groups.
Conclusion
Trimad effectively ameliorated HFD-induced obesity and demonstrated strong anti-obesity potential.
Keywords: Adipokines, Brain monoamines, Inflammation, Lipid metabolism, Oxidative stress
1. Introduction
Obesity results from an imbalance between energy intake and expenditure [1] and is marked by increased adipose tissue mass [2]. This expansion involves adipocyte hypertrophy and hyperplasia, accompanied by macrophage infiltration that releases pro-inflammatory cytokines and adipokines [3]. The imbalance between pro- and anti-inflammatory cytokines contributes to obesity-related complications such as dyslipidemia, hepatic steatosis, insulin resistance, and type 2 diabetes [4]. With the global rise in obesity, the search for safe anti-obesity therapeutics has intensified. Pharmacological management of obesity has evolved from modestly effective agents like Orlistat to highly efficacious therapies like GLP-1 receptor agonists and metabolism targeting agents like Resmetirom [5]. However, majority of these drugs have been limited by adverse effects [6]. Ayurveda offers a wide range of anti-obesity drugs that can be evaluated for their efficacy. One such formulation is Trimad, traditionally recommended for obesity management [7]. It contains Cyperus rotundus (Musta), Embelia ribes (Vidang), and Plumbago zeylanica (Chitraka) in equal proportion. Each of this constituent is individually reported for weight-reducing potential [[8], [9], [10]], and the formulation has shown clinical efficacy in a randomized placebo-controlled trial [11]. Hence, the present study investigated the effects of Trimad in a high-fat diet (HFD)-induced obese rat model with focus on preliminary pathophysiological alteration viz. inflammation, oxidative stress and hunger-satiety [12].
2. Materials and methods
2.1. Materials
Trimad as a standardized aqueous extract was provided by Pharmanza Herbals Pvt. Ltd., Gujarat (Lot No. TMEP/RAD/005; Product Code: 00000EPTR1). The details of raw material authentication, aqueous extract preparation procedure, and phyto-constituent detection have been provided in Supplementary material- Fig. 1. Atorvastatin (Cipla Ltd.) was procured locally and used as the anti-hyperlipidemic Positive control. The normal chow diet (3% fat) was procured from Nutrivet Life Sciences, Pune. The high-fat diet (35% fat) used for obesity induction was obtained from VRK Nutritional Solutions, Pune. HFD was prepared using soybean oil, lard, and LR-grade cholesterol. Proximate analysis of normal chow diet and HFD is provided in Supplementary material- Table 1.
2.2. Animals and treatments
The work was conducted in accordance with the internationally accepted principles and guidelines for laboratory animal use and care. Institutional animal ethics approval (BVDUMC/3293/2016/007/002) was obtained before initiating the study. The study was carried out in 4-5 week old male Wistar rats weighing up to 125-150 g. The rats were purchased from National Toxicology Center, Pune and were housed in standard conditions (25 ± 1°C/45-55% relative humidity and 12 h light-dark cycle). Throughout the study, the animals had free access to feed and water.
2.3. Experimental design
The animals were randomly divided into 6 groups with n = 6 in each group (Table 1). Based on literature review for usage of herbal drugs in HFD induced animal models, doses for the treatment groups were selected [13]. The dose of Atorvastatin was extrapolated from human anti-hyperlipidaemic dose [14]. Group 1 (Normal control, NC) received only normal diet throughout the study period (i.e. 48 days) while groups 2 to 6 received HFD. Group 2 received only high fat diet throughout the study period (Disease control, DC). In addition to HFD, animals in groups 3 to 6 received treatments as per the assigned groups from day 24 to day 48 (last 24 days). Group 3 was Positive control (PC) and received Atorvastatin. Group 4, 5 and 6 (TM50, TM100 and TM200) received Trimad at doses 50, 100 and 200 mg/kg b.w. respectively in addition to HFD.
Table 1.
Group description with dosage details.
| Group | Description |
|---|---|
| 1 | Normal control (NC), received normal diet during entire study period |
| 2 | Disease control (DC), received only high fat diet (HFD) |
| 3 | Positive control (PC), received HFD + Atorvastatin (1.2 mg/kg b.w.) |
| 4 | TM50 received HFD + Trimad (50.0 mg/kg b.w.) |
| 5 | TM100 received HFD + Trimad (100.0 mg/kg b.w.) |
| 6 | TM200 received HFD + Trimad (200.0 mg/kg b.w.) |
To adhere to the 3Rs principles for animal welfare, the evaluation of Trimad was carried out by sharing control groups with a concurrent study on Triphala. Both polyherbal formulations utilized the same normal, disease, and positive control groups, with only the specific experimental groups added independently. The comparative data for the control arms and Triphala have been published previously [15].
2.4. Study parameters
Throughout the study duration, feed intake and body weight were recorded (once a week) using digital weighing balance.
2.4.1. Serum biochemical parameters
Blood was collected from all animals through retro-orbital puncture under anesthesia on day 0, 24, 48 after overnight fasting. Serum lipid profile (Total Cholesterol; TC, Triglycerides; TG, HDL-C) and glucose were determined using commercially available colorimetric kits from Coral Clinical Systems, India. Adipokines (Adiponectin and Leptin) and inflammatory markers (IL-6 and hs-CRP) were estimated by ELISA kits procured from My Bioresource, USA. Oxidative stress in serum was evaluated as Malondialdehyde (MDA; marker of lipid peroxidation) using TBARS method [16]. Oxygen Radical Absorbance Capacity (ORAC) was measured by fluorescence method described by Choudhari et al. [17].
2.4.2. Changes in key organs
On day 48, all the animals were sacrificed and key organs (liver, brain and adipose tissue) were harvested. The weights of the liver and the brain were recorded. Liver section was divided into two parts. The first part was stored in Trichloroacetic acid for determination of oxidative stress. Oxidative stress (lipid peroxidation) was measured in tissue homogenates using the Thiobarbituric Acid Reactive Substances (TBARS) method [15]. The second part of the liver tissue was stored in Liquid nitrogen and was used for molecular studies. Superoxide dismutase (anti-oxidant enzyme, SOD) was measured using a method described by Zhang et al. (2017) [18]. The details of the molecular studies have been provided in the section ‘Expression of genes involved in lipid metabolism in liver tissue’. Determination of adipocyte diameters was done for 10% formalin fixed adipose tissue by a reported method [15]. Briefly, representative images of adipose tissue sections (5–7 μm) were analyzed using ImageJ software. Measurements of adipocyte diameter were recorded at four different points across each cell and averaged. The average cell diameter value was used as an indicator of adipocyte hypertrophy. Additionally, brain monoamines (dopamine, serotonin and noradrenaline) were determined by HPLC method following a method earlier reported by Lakshmana et al. (1997) [19].
2.4.3. Expression of genes involved in lipid metabolism in liver tissue
Liquid Nitrogen frozen liver tissue was used for total RNA extraction using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Quality and quantity of the extracted RNA was determined using agarose gel electrophoresis (Bio-Rad, Hercules, California, USA) and ND-1000 CV spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) respectively. The genes selected were SREBP-1c (Sterol Regulatory Element Binding Protein-1c), PPAR-γ (Peroxisome Proliferator–Activated Receptor Gamma), ACC (Acetyl-CoA Carboxylase Alpha), ACSS2 (Acyl-CoA Synthetase) and FASN (Fatty Acid Synthase). cDNA was synthesized from extracted RNA (2 μg RNA from each sample) using Invitrogen's reverse transcriptase kit. Using TB Green Premix SYBR green expression assays (TAKARA, Japan), qRT-PCR was performed on Applied Biosystems' (USA) StepOne real-time PCR system. The following conditions were applied for the PCR protocol: an initial denaturation step at 95°C for 10 min, followed by 40 cycles of (1) denaturation at 95°C for 3 s, (2) annealing at 60°C for 30 s, and (3) extension at 72°C for 15 s. A final extension step was carried out at 72°C for 5 min. GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) was used as the housekeeping gene.
2.5. Statistical analysis
Data is presented as Mean ± SD. Treatment groups were compared with Normal control (NC) and Disease control (DC) group by applying Two-way ANOVA followed by Bonferroni posttests. GraphPad Version 5.03 was used for statistical analysis. Level of significance (p < 0.05) was considered significant.
3. Results
As mentioned in methodology, the anti-obesity effect of Trimad was evaluated with another polyherbal formulation (Triphala) to minimize usage of animals owing to 3Rs guidelines. Keeping 3 groups viz. Normal control, Disease control and Positive control same, three dose levels of both formulations were evaluated in the same experiment. The data of Triphala along with the control groups have been already published [15].
3.1. Effect of Trimad on body weight and feed intake
Effect of Trimad on body weight and feed intake is presented in Fig. 1. DC group showed significantly more weight than NC group on day 24 (p < 0.01) (Fig. 1 (A)). Additionally, on day 24, there were no statistically significant difference in the weights for groups 2 to 6. On day 48 (at the end of study), compared with DC, all drug treated groups (3 to 6), demonstrated significant lower body weights (p < 0.001) even though fed on HFD throughout the study period. As shown in Fig. 1 (B), on day 24 and day 48, compared to NC group, DC group showed significantly higher feed intake but all the drug treatment groups (3 to 6) demonstrated lower feed intake when compared to DC group. On day 48, feed intakes in the treatment groups 3 to 6 were similar to NC.
Fig. 1.
Effect of Trimad on body weight and feed intake
Rats were divided in six experimental groups (n = 6). Except Normal control (NC), all the groups; Disease control group (DC), Positive control (PC, treated with Atorvastatin 1.2 mg/kg b.w.) and Trimad treated groups (TM 50, TM 100 and TM 200, groups treated with Trimad 50, 100 and 200 mg/kg b.w. treatment resp. received high fat diet throughout the study period (i.e. for 48 days). Treatment with Atorvastatin or Trimad was started from day 24 to day 48. Weights of the animals (A) and feed intake (B) were measured periodically at day 24 and day 48 and are presented as Mean ± SD (n = 6). Two way ANOVA and Bonferroni posttests were applied for the statistical analysis. ##; p < 0.01 vs NC, ###; p < 0.001 vs NC and ***; p < 0.001 vs DC.
3.2. Effect of Trimad on serum biochemical parameters
In DC group, on day 48, significant increase in TG, TC and glucose levels was observed as compared to NC group (Fig. 2 (A) and 2 (B), and 2 (D)) (p < 0.001). TG levels were found to raise from day 24 to day 48 in DC group. Such increase was not demonstrated by the treatment groups. Similarly, on day 48, TC levels in Trimad treated groups were significantly lower when compared with DC (p < 0.001). HDL-c levels (Fig. 2 (C)) were significantly lower in DC group than NC group on both day 24 and 48 (p < 0.001). Though there was significant lowering of HDL-c in treatment groups compared to NC, in Trimad treated groups these levels were significantly higher than DC.
Fig. 2.
Effect of Trimad on serum biochemical parameters
Rats were divided in six experimental groups (n = 6). Except Normal control (NC), all the groups; Disease control group (DC), Positive control (PC, treated with Atorvastatin 1.2 mg/kg b.w.) and Trimad treated groups (TM 50, TM 100 and TM 200, groups treated with Trimad 50, 100 and 200 mg/kg b.w. treatment resp. received high fat diet throughout the study period (i.e. for 48 days). Treatment with Atorvastatin or Trimad was started from day 24 to day 48. After overnight fasting, serum was collected for the analysis on specified days. Effect of Trimad on Triglycerides (A), Total cholesterol (B), HDL-c (C), Glucose (D), Leptin (E), Adiponectin (F), Interluekin-6 (G) and hs-C-reactive protein (H) was studied. Data is presented as Mean ± SD (n = 6). Two way ANOVA and Bonferroni posttests were applied for the statistical analysis. #; p < 0.05, ##; p < 0.01, ###; p < 0.001 vs NC, *; p < 0.05, **; p < 0.01 and ***; p < 0.001 vs DC.
Similar to observation for TC, Glucose levels were significantly higher in DC when compared with NC (p < 0.001) on day 24 and 48. Such rise in the glucose levels was attenuated by all the treatments (p < 0.001) on day 48.
Effect of Trimad on adipokines (Leptin and Adiponectin) and inflammatory markers (IL-6 and hs-CRP) was also determined. As shown in Fig. 2 (E), on day 24 and 48, DC group showed higher levels of Leptin as compared to NC (p < 0.001). When comparison was done between day 24 and 48 for respective treatments, it was observed that unlike DC, in all treatments, Leptin levels on day 48 were lower than those on day 24. Opposite to Leptin levels (pro-inflammatory mediator), levels of Adiponectin (anti-inflammatory adipokine) (Fig. 2 (F)) were found to be lower in DC group when compared with NC group on day 48 (p < 0.001 vs NC). All Trimad treated groups and PC reversed these changes (p < 0.01 vs DC).
DC group showed significantly higher levels of IL-6 on day 48 (p < 0.001) compared to NC. In all the treatment groups, IL-6 levels were almost normalized (p < 0.001 vs DC) (Fig. 2 (G)). HFD treatment in DC resulted in significant rise in hs-CRP (Fig. 2 (H)) (p < 0.001 vs NC) on day 48. Though the treatment groups did not exhibit any decrease, they prevented further rise in the levels compared to day 24 levels as observed for DC.
3.3. Effect of Trimad on oxidative stress
Effect of Trimad on oxidative stress in serum measured as lipid peroxidation (malondialdehyde; MDA) and oxygen radical absorbance capacity (ORAC) is presented in Fig. 3. Compared to NC group, lipid peroxidation measured as MDA from serum was higher in DC group (Fig. 3 (A)). Trimad (all doses) and Atorvastatin (PC group) reduced these levels significantly (p < 0.001 vs DC). Anti-oxidant capacity of the serum from all the groups was determined in terms of ORAC. On day 24, ORAC of all the groups was similar to NC group (Fig. 3 (B)). On day 48, there was further lowering of ORAC in DC group (p < 0.05) but for Trimad treated groups, there was statistically significant dose-dependent rise in ORAC.
Fig. 3.
Effect of Trimad on oxidative stress
Rats were divided in six experimental groups (n = 6). Except Normal control (NC), all the groups; Disease control group (DC), Positive control (PC, treated with Atorvastatin 1.2 mg/kg b.w.) and Trimad treated groups (TM 50, TM 100 and TM 200, groups treated with Trimad 50, 100 and 200 mg/kg b.w. treatment resp. received high fat diet throughout the study period (i.e. for 48 days). Treatment with Atorvastatin or Trimad was started from day 24 to day 48. After overnight fasting, serum was collected for the analysis on specified days. Effect of Trimad on serum oxidative stress was determined as Malondialdehyde (MDA) (A) and ORAC (Oxygen radical absorbance capacity) (B). Oxidative stress in liver tissue was measured in terms of MDA (C) and SOD (D). Data is presented as Mean ± SD (n = 6). Two way ANOVA and Bonferroni posttests were applied for the statistical analysis. #; p < 0.05 & ###; p < 0.001 vs NC and *; p < 0.05, **; p < 0.01& ***; p < 0.001 vs DC.
When oxidative stress was measured in the liver tissue (Fig. 3 (C)), MDA level were higher in DC and all the treatment groups compared with NC group. Higher doses of Trimad (100 and 200 mg/kg) were able to significantly lower the MDA levels (p < 0.001). In contrast, all the groups had significantly lower liver SOD levels when compared with the NC group (p < 0.001) (Fig. 3 (D)). But when compared with DC, these levels were significantly (p < 0.001) higher in all treatment groups including PC.
3.4. Effect of Trimad on changes in key organs
Effect of Trimad on key organs was studied in terms of organ weights (liver and brain), adipocyte hypertrophy, and brain monoamines. As presented in Table 2 (A), DC group had highest liver weight (16.63 ± 2.90 g) in comparison to NC group (11.10 ± 1.41 g) (p < 0.001). Though the weights of liver in treated groups were lower than DC group, the differences were not statistically significant. In case of brain weights, across all the groups including DC, no such change was seen.
Table 2 A.
Effect of Trimad on organ weights (g).
| Groups | Liver | Brain |
|---|---|---|
| NC | 11.10 ± 1.41 | 1.79 ± 0.25 |
| DC | 16.63 ± 2.90### | 1.80 ± 0.30 |
| PC | 14.46 ± 3.93## | 1.77 ± 0.31 |
| TM 50 | 12.15 ± 2.59 | 1.80 ± 0.36 |
| TM 100 | 13.58 ± 2.89 | 1.90 ± 0.30 |
| TM 200 | 14.25 ± 2.69# | 1.89 ± 0.20 |
Adipocyte hypertrophy is presented as adipocyte diameter (in μm) in Table 2 (B). As shown in the table, DC group adipocytes had significantly larger diameter (118 ± 6.34 μm) when compared with NC group adipocytes (57.93 ± 4.49 μm) (p < 0.001). Adipocytes from Trimad treated groups showed significantly smaller diameter compared to DC group adipocytes (p < 0.001). This effect of Trimad on adipocyte hypertrophy was dose-dependent. For NC group, mean adipocyte diameter was recorded to be 57.93 ± 4.49 μm. It was increased significantly for DC group (118 ± 6.34 μm, p < 0.001 vs NC). For Trimad (200 mg/kg), the effect was comparable to Atorvastatin (PC), 74.9 ± 1.60 μm and 73.25 ± 1.89 μm respectively.
Table 2 B.
Effect of Trimad on adipocyte diameter in rats (μm).
| Groups | Adipocyte diameter |
|---|---|
| NC | 57.93 ± 4.49 |
| DC | 118 ± 6.34### |
| PC | 73.25 ± 1.89*** |
| TM 50 | 87.03 ± 2.65*** |
| TM 100 | 81.51 ± 0.60*** |
| TM 200 | 74.9 ± 1.60*** |
Rats were divided in six experimental groups (n = 6). Except Normal control (NC), all the groups; Disease control group (DC), Positive control (PC, treated with Atorvastatin 1.2 mg/kg b.w.) and Trimad treated groups (TM 50, TM 100 and TM 200, groups treated with Trimad 50, 100 and 200 mg/kg b.w. treatment resp. received high fat diet throughout the study period (i.e. for 48 days). Treatment with Atorvastatin or Trimad was started from day 24 to day 48. At the end of study period, organs were harvested. Weights of the organs were measured and are presented as Mean ± SD (A). Adipocyte diameters were determined from the 5-7 μm thick sections (n = 8). Data is presented as Mean ± SD (B). Two way ANOVA and Bonferroni posttests were applied for the statistical analysis. #; p < 0.05 vs NC, ##; p < 0.01 vs NC, ###; p < 0.001 vs NC and ***; p < 0.001 vs DC.
Effect of Trimad treatment on brain functionality was determined in terms of brain monoamine levels (Fig. 4). In DC, levels of serotonin and dopamine were found to be lower as compared to NC (Fig. 4 (A) and 4 (B) respectively). Levels of these monoamines in all treatment groups were statistically higher when compared with DC levels (p < 0.001). On the other hand, noradrenaline was seen higher in DC group. Trimad treated groups along with PC showed normalization of these levels and all the changes were statistically significant (p < 0.001) (Fig. 4 (C)).
Fig. 4.
Effect of Trimad on brain monoamines
Rats were divided in six experimental groups (n = 6). Except Normal control (NC), all the groups; Disease control group (DC), Positive control (PC, treated with Atorvastatin 1.2 mg/kg b.w.) and Trimad treated groups (TM 50, TM 100 and TM 200, groups treated with Trimad 50, 100 and 200 mg/kg b.w. treatment resp. received high fat diet throughout the study period (i.e. for 48 days). Treatment with Atorvastatin or Trimad was started from day 24 to day 48. At the end of study period, brain tissues were harvested. Brain monoamines determined were Serotonin (A), Dopamine (B) and Noradrenaline (C). Data is presented as Mean ± SD (n = 3). One way ANOVA Tukey's multiple comparison posttest was applied for the statistical analysis. ##; p < 0.01& ###; p < 0.001vs NC and ***; p < 0.001 vs DC.
3.5. Effect of Trimad on expression of genes involved in lipid metabolism in liver tissue
Effect of Trimad on expression of genes involved in liver lipid metabolism was determined using qRT-PCR (Fig. 5). The genes studied were PPARλ, SREBP-1c, ACC, ACSS2 and FASN. The expression of PPARλ was found to be up-regulated by ∼1.5 times in DC group (p < 0.01) in comparison to the NC group (Fig. 5 (A)). Lower dose of Trimad (TM 50) along with PC had lower expression levels for this gene though the effects were significant only for TM 50 (p < 0.05). TM 50 down-regulated PPARλ expression as compared to DC. But highest Trimad dose (i.e. TM 200) had statistically significant higher level of PPARλ (p < 0.001 vs DC). SREBP-1c expression was up-regulated in DC group by ∼4.7 folds as compared to the NC group (p < 0.001) (Fig. 5 (B)). TM 50 and TM 100 showed ∼2.0 and ∼1.8 fold down regulation of SREBP-1c as compared to the DC group (p < 0.001 and p < 0.05 respectively). Such down regulation was not detected in TM 200. Similar to SREBP-1c expression pattern, there was up-regulation of ACC gene expression by ∼1.7 fold in DC in comparison to NC (p < 0.001) (Fig. 5 (C)). In Trimad treated groups, TM 50 significantly (p < 0.001) down-regulated ACC gene expression by ∼1.6 times as compared to DC. But in TM 200 group, instead of down regulation, up-regulation in the expression of this gene was observed. The ACSS2 gene expression in the DC was up-regulated (p < 0.001) by ∼2.2 times in comparison to NC rats (Fig. 5 (D)). There was a statistically significant decrease in the expression of ACSS2 gene in all treated groups (p < 0.001) compared to the DC group. There was upregulation of FASN gene expression in DC group by ∼1.3 times as compared to NC group (Fig. 5 (E)). All treated groups except TM 200, showed down-regulation (p < 0.001) of FASN gene.
Fig. 5.
Effect of Trimad on expression of hepatic genes involved in lipid metabolism
Rats were divided in six experimental groups (n = 6). Except Normal control (NC), all the groups; Disease control group (DC), Positive control (PC, treated with Atorvastatin 1.2 mg/kg b.w.) and Trimad treated groups (TM 50, TM 100 and TM 200, groups treated with Trimad 50, 100 and 200 mg/kg b.w. treatment resp. received high fat diet throughout the study period (i.e. for 48 days). Treatment with Atorvastatin or Trimad was started from day 24 to day 48. At the end of study period, liver tissues were harvested and flash frozen. Expression levels of genes involved in lipid metabolism were determined by qRT-PCR. Results are represented as Mean ± SD (n = 3 and the reactions were carried out in duplicates). Genes studied were PPAR-γ (A), SREBP-1c (B), ACC (C), ACSS2 (D) and FASN (E). Two way ANOVA and Bonferroni posttests were applied for the statistical analysis. #; p < 0.05, ##; p < 0.01 & ###; p < 0.001 vs NC and *; p < 0.05, **; p < 0.01 & ***; p < 0.001 vs DC PPAR-γ: Peroxisome Proliferator-Activated Receptor, SREBP-1c: Sterol Regulatory Element Binding Proteins-1c, ACC: Acetyl-CoA Carboxylase, ACSS2: Acyl-CoA Synthetase Short Chain Family Member 2, FASN: Fatty Acid Synthase.
4. Discussion
In this study, in an HFD-induced obese rat model, Trimad demonstrated significant anti-obesity effects, evaluated alongside Triphala under 3Rs guidelines to minimize animal use. Trimad treatment led to reduced body weight and feed intake, improved serum lipid profiles, and lowered glucose levels compared to the Disease control group. It also attenuated leptin and CRP levels while enhancing adiponectin and antioxidant capacity (ORAC), and significantly reduced oxidative stress markers (MDA) in both serum and liver. Adipocyte hypertrophy was significantly reversed in a dose-dependent manner. Brain monoamine analysis showed normalization of serotonin, dopamine, and noradrenaline levels. Moreover, in liver, Trimad modulated expression of genes involved in lipid metabolism (significant down-regulating SREBP-1c, ACC, ACSS2, and FASN) especially at lower doses. These findings suggest that Trimad effectively ameliorates metabolic, inflammatory, oxidative, and molecular alterations associated with diet-induced obesity.
The effects of HFD on various metabolic pathways are well documented. HFD is reported to induce transient insulin resistance, glucose intolerance and hypertriglyceridemia with impact on total cholesterol [20]. We observed significantly elevated glucose and triglyceride levels along with significantly increased cholesterol levels in Disease control group which had received only HFD throughout the study period. Treatment with Trimad was found to reverse these perturbations. In liver tissue, compared to Disease control, MDA levels were lower and SOD levels were higher. These observations suggest that the beneficial effects by Trimad are probably due to improved fatty acid oxidation in liver [21]. Long term energy balance regulation is mediated by Leptin. It is mainly expressed in adipose tissue and lowers food intake, enhances energy expenditure with simultaneous induction of weight loss [[22], [23]] Inability to induce satiety even in the presence of high leptin levels and energy stores indicates reduction in the leptin sensitivity (leptin resistance). In our study, we observed increased levels of leptin along with increased feed intake in Disease control group indicating leptin resistance. All Trimad treated groups showed decrease in leptin levels along with decrease in feed intake as compared to Disease control group indicating improved leptin sensitivity. Adiponectin, another important adipokine, has both the metabolic and anti-inflammatory activity [24]. Higher adiponectin levels are associated with enhanced insulin sensitivity, fatty acid oxidation in liver & skeletal muscle and energy dissipation [[25], [26]]. In our study, after obesity induction by feeding HFD in Disease control group, adiponectin levels were lowered. This effect on adiponectin was reversed by Trimad treatment. We observed increase in adiponectin levels and corresponding decrease in serum triglyceride after treatment with Trimad. It can be concluded from the above observations that reduced leptin and enhanced adiponectin levels might have restored insulin sensitivity after Trimad treatment and ultimately have resulted in reduced lipid levels and weight.
IL-6 is involved in triglyceride release, lipoprotein lipase down-regulation, insulin sensitivity, and so on [27]. IL-6 receptors are abundantly expressed in hepatocytes (liver), they induce expression of C-reactive protein (acute phase protein, CRP) in liver. Additionally, there is positive co-relation between CRP levels and mass of adipose tissue [[28], [29]]. Elevated IL-6 further induces release of CRP [30]. CRP is not only a marker of inflammation, but it also participates in tissue damage. Trimad treatment showed marked decrease in IL-6 and prevented increase in CRP levels, which indicates that Trimad acts on inflammatory processes mediated through obesity and adipose tissue.
In obesity, elevated circulating glucose and lipid levels (excessive energy supply) can result in elevated metabolic pathway activities in adipose and non-adipose tissues which may result in elevated production of reactive oxygen species (ROS) [31]. When these ROS attack polyunsaturated fatty acids, fatty acids undergo lipid peroxidation. HFD treatment for 48 days increased levels of lipid peroxidation that was measured as MDA in serum. A significant decline in the MDA levels was seen in Trimad administered groups. Further, while HFD treatment reduced the levels of anti-oxidants measured as ORAC, these levels were significantly increased at all dose levels of Trimad. Similarly, in liver tissue of Disease control group, significantly higher MDA levels were detected in comparison to Normal control. But the treatment groups (except Trimad 50 mg/kg) showed significantly lower levels of MDA as compared to Disease control group. At the same time, the levels of Superoxide Dismutase (SOD), an anti-oxidative enzyme was found significantly higher in Trimad treated groups. These findings confirm our earlier observation on anti-oxidant capacity of Trimad [32]. Considering the anti-oxidant, anti-inflammatory and anti-hyperlipidaemic effects of Trimad, it may prove as a potential agent for preventing hepatic steatosis.
Brain monoamines play a vital role in obesity. Though some reports mention that the alterations in monoamines lead to obesity [33], there are reports stating that development of obesity occurs before any monoamine alterations [34]. The Disease control rats exhibited decrease in serotonin levels, which was improved by Trimad treatment. Serotonin (5-HT) is a monoaminergic neurotransmitter that modulates central and peripheral functions. It is known to have anorectic properties [35]. It controls feeding behavior, energy expenditure by restoring the caloric homeostasis within the body [36] and is connected to fat burning mechanism [37]. Opposite to serotonin, norepinephrine levels were found elevated in the Disease control group. Trimad treatment was found to decrease these levels. Norepinephrine contributes to stimulation of hunger and satiety. Additionally, it is also connected to thermogenesis [38]. Central nervous system controls energy balance through norepinephrine. It needs to be noted here that involvement of serotonin and norepinephrine in neural pathways has been targeted for anti-obesity drug development [39]. The levels of dopamine as compared to Disease control group were found significantly increased in all treatment groups. It is well reported that Dopamine is a major contributor in brain reward circuit [40]. It therefore contributes in food intake and eating behaviors. Engagement of dopamine in eating behavior has been proved by brain imaging studies in both animals and humans. Thus, targeting improvement in dopamine function is a new therapeutic option for the prevention and obesity management [41].
It is well known that in obesity, adipocytes store surplus energy as triacylglycerols that leads to expansion in their size (hypertrophy) and number (hyperplasia). Adipocyte hypertrophy results from a relative increase in lipid deposition versus reduced lipolysis. In our study, as compared to Normal control group, adipocyte diameters (adipocyte size) in Disease control group were significantly higher while in treatment groups adipocyte sizes were reduced.
In liver steatosis, lipogenic gene (PPARλ and SREBP1-c) expression is up-regulated [42] which leads to lipid accumulation in the form of droplets in liver. As a result of this, the liver becomes fatty [43]. We observed down regulated PPARλ expression with lower dose of Trimad implying decrease in fatty infiltration in liver. The histopathology of liver also confirmed the reduction in fatty changes in liver. Abundant expression level of SREBP-1c is observed in liver [44]. Over expression of SREBP-1c is associated with elevated triglyceride levels [45]. Various lipogenic genes (ACC, ACSS2 and FASN) are regulated by transcription factor SREBP-1c. All these genes are up-regulated in obesity [[46], [47]] In our study, elevated levels of SREBP-1c further leading to elevation of ACC, ACSS2 and FASN expression resulted in higher cholesterol and triglyceride levels in serum of Disease control group. As compared to the Disease control animals, in Trimad treated animals, these genes were down-regulated. Though, mechanistic investigations were limited to selected gene expression analyses, overall, these modulations in transcription factor and its targeted genes have resulted in the normalization of abnormal lipid profiles along with lowering of body weight in the Trimad treated animals.
It is important to note here that Trimad is a polyherbal formulation with complex phytochemical composition. These phytochemicals can act through distinct molecular pathways and can produce differential effects at different dose levels. Thus, for polyherbal formulations due to multicomponent nature, biological responses may not always be linear across the doses [[48], [49], [50]]. Investigations like receptor saturation studies would better explain the observed dose-dependent responses. Additionally, future in-depth studies assessing the modulation of hepatic steatosis and the molecular pathways associated with obesity and metabolic dysfunction-associated steatotic liver disease (MASLD) are warranted to further elucidate the mechanisms underlying the observed effects of Trimad polyherbal formulation.
5. Conclusion
Obesity involves multiple pathways like hunger-satiety mechanism, insulin signaling, lipid metabolism, oxidative stress and chronic inflammation. To conclude, in our study, Trimad has shown promising results on almost each of these mechanisms though in varying degrees. Of the 3 doses studied, we found the maximum response with 50 mg/kg in terms of body weight, feed intake, leptin, lipid peroxidation, and liver weight. The response at higher doses viz. 100 and 200 mg/kg was found either diminished or blunted.
CRediT author contributions
Conceptualization: SSB. Methodology: SSB, AS. Software: Nil. Validation: SSB, AAJ.
Formal analysis: SSB, AAJ. Investigation: MPS, SPK, AS. Resources: SSB, AS. Data curation: SSB. Writing – Original Draft: SPK. Writing – Review & Editing: SSB, AAJ. Visualization: MPS, SPK, AAJ. Supervision: SSB. Project administration: SSB, AS. Funding acquisition: SSB.
Data availability statement
All data generated or analyzed during this study are included in this article. It can be obtained from the corresponding author upon request.
Ethical statement
This study was approved by Institutional Animal Ethics Committee of Medical college, Bharati Vidyapeeth (Deemed to be university) (Approval number: BVDUMC/3293/2016/007/002).
Declaration of generative AI in scientific writing
The authors declare that they have not used generative AI and AI-assisted technologies in the writing process of this paper.
Funding source
Central Council for Research in Ayurvedic Sciences (CCRAS), Ministry of Ayush, Govt. of India had funded this research work (Grant ID: Z-28015/234/2015-HPC (EMR)-AYUSH-A).
Conflict of interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Supriya Bhalerao reports financial support was provided by Central Council for Research in Ayurvedic Sciences. Dr. Supriya Bhalerao, Corresponding Author, is on the Editorial Board of JAIM. The author has not been involved in the peer review process of this submission. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
Authors acknowledge Central Council for Research in Ayurvedic Sciences (CCRAS), Ministry of Ayush, Govt. of India for the research funding. The authors are thankful to M/s Pharmanza Herbals Ltd. for supplying Trimad (aqueous extract), students from Dept. of Pharmacology, Poona College of Pharmacy for their technical help in monoamine estimation and Cancer Research Laboratory, BVDU-IRSHA for the ORAC study.
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.jaim.2026.101400.
Contributor Information
Asavari Anirudha Joshi, Email: asavari.joshi@bharatividyapeeth.edu.
Megha Pradip Salunke, Email: meghasalunke5@gmail.com.
Suresh Purushottam Khadke, Email: spkhadke@gmail.com.
Arulmozhi Sathiyanarayanan, Email: arulmozhi.s@bharatividyapeeth.edu.
Supriya Sudhakar Bhalerao, Email: supriya.bhalerao@gmail.com.
Appendix A. Supplementary data
The following is the Supplementary data to this article:
References
- 1.Löffler M.C., Betz M.J., Blondin D.P., Augustin R., Sharma A.K., Tseng Y.H., Scheele C., Zimdahl H., Mark M., Hennige A.M. Challenges in tackling energy expenditure as obesity therapy: from preclinical models to clinical application. Mol Metabol. 2021;51 doi: 10.1016/j.molmet.2021.101237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Longo M., Zatterale F., Naderi J., Parrillo L., Formisano P., Raciti G.A., Beguinot F., Miele C. Adipose tissue dysfunction as determinant of obesity-associated metabolic complications. Int J Mol Sci. 2019;20(9):2358. doi: 10.3390/ijms20092358. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Banjare J., Bhalerao S. Obesity associated noncommunicable disease burden. Int J Health Allied Sci. 2016;5:81–87. doi: 10.4103/2278-344X.180429. [DOI] [Google Scholar]
- 4.Chait A., Hartigh L.J.D. Adipose tissue distribution, inflammation and its metabolic consequences, including diabetes and cardiovascular disease. Front Cardiovasc Med. 2020;7:22. doi: 10.3389/fcvm.2020.00022. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Kokkorakis M., Chakhtoura M., Rhayem C., Al Rifai J., Ghezzawi M., Valenzuela-Vallejo L., Mantzoros C.S. Emerging pharmacotherapies for obesity: a systematic review. Pharmacol Rev. 2025;77(1) doi: 10.1124/pharmrev.123.001045. [DOI] [PubMed] [Google Scholar]
- 6.Tak Y.J., Lee S.Y. Long-term efficacy and safety of anti-obesity treatment: where do we stand? Curr Obes Rep. 2021;10(1):14–30. doi: 10.1007/s13679-020-00422-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Salunke M., Deshpande M., Bhalerao S. Experiential documentation of Trimad for its anti-obesity potential: a survey of Ayurvedic physicians from Pune city. J Ayurveda Integr Med. 2017;8(3):190–193. doi: 10.10156/j.jaim.2017.03.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Majeed M., Nagabhushanam K., Bhat B., Ansari M., Pandey A., Bani S., Mundkur L. The anti-obesity potential of Cyperus rotundus extract containing piceatannol, scirpusin A and scirpusin B from rhizomes: preclinical and clinical evaluations. Diabetes Metab Syndr Obes. 2022;15:369–382. doi: 10.2147/DMSO.S348412. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Sharma V., Gautam D.N., Radu A.F., Behl T., Bungau S.G., Vesa C.M. Reviewing the traditional/modern uses, phytochemistry, essential oils/extracts and pharmacology of Embelia ribes Burm. Antioxidants. 2022;11(7):1359. doi: 10.3390/antiox11071359. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Shukla B., Saxena S., Usmani S., Kushwaha P. Phytochemistry and pharmacological studies of Plumbago zeylanica L.: a medicinal plant review. Clin Phytosci. 2021;7(1):34. doi: 10.1186/s40816-021-00271-7. [DOI] [Google Scholar]
- 11.Salunke M., Banjare J., Bhalerao S. Effect of selected herbal formulations on anthropometry and body composition in overweight and Obese individuals: a randomized, double blind, placebo-controlled study. J Herb Med. 2019;17–18 doi: 10.1016/j.hermed.2019.100298. [DOI] [Google Scholar]
- 12.Rodríguez-Correa E., González-Pérez I., Clavel-Pérez P.I., Contreras-Vargas Y., Carvajal K. Biochemical and nutritional overview of diet-induced metabolic syndrome models in rats: what is the best choice? Nutr Diabetes. 2020;10(1):24. doi: 10.1038/s41387-020-0127-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Singh D., Mani D. Protective effect of Triphala Rasayana against paracetamol-induced hepato–renal toxicity in mice. J Ayurveda Integr Med. 2015;6(3):181–186. doi: 10.4103/0975-9476.146553. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Jain P.G., Surana S.J. Isolation, characterization and hypolipidemic activity of ferulic acid in high-fat-diet-induced hyperlipidemia in laboratory rats. EXCLI J. 2016;15:599–613. doi: 10.17179/excli2016-394. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Bhalerao S.S., Joshi A.A., Khadke S., Sathiyanarayan A. Anti-obesity effects of triphala at biochemical and molecular level in high-fat diet-induced Obese rats. Pharmacogn Mag. 2024;20(1):30–42. doi: 10.1177/09731296231198316. [DOI] [Google Scholar]
- 16.Banjare J., Salunke M., Indapurkar K., Ghate U., Bhalerao S. Estimation of serum malondialdehyde as a marker of lipid peroxidation in medical students undergoing examination-induced psychological stress. J Sci Soc. 2017;44(3):137–139. doi: 10.4103/jss.JSS_13_17. [DOI] [Google Scholar]
- 17.Choudhari A.S., Raina P., Deshpande M.M., Wali A.G., Zanwar A., Bodhankar S.L., Kaul-Ghanekar R. Evaluating the anti-inflammatory potential of Tectaria cicutaria L. rhizome extract in vitro as well as in vivo. J Ethnopharmacol. 2013;150(1):215–222. doi: 10.1016/j.jep.2013.08.025. [DOI] [PubMed] [Google Scholar]
- 18.Zhang J., Chen R., Yu Z., Xue L. Superoxide dismutase (SOD) and catalase (CAT) activity assay protocols for Caenorhabditis elegans. Bio-protocol. 2017;7(16) doi: 10.21769/BioProtoc.2505. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Lakshmana M.K., Raju T.R. An isocratic assay for norepinephrine, dopamine, and 5-Hydroxytryptamine using their native fluorescence by high-performance liquid chromatography with fluorescence detection in discrete brain areas of rat. Anal Biochem. 1997;246(2):166–170. doi: 10.1006/abio.1996.9997. [DOI] [PubMed] [Google Scholar]
- 20.Taweesap P., Potue P., Khamseekaew J., Iampanichakul M., Jan O.B., Pakdeechote P., Maneesai P. Luteolin relieves metabolic dysfunction-associated fatty liver disease caused by a high-fat diet in rats through modulating the AdipoR1/AMPK/PPARγ signaling pathway. Int J Mol Sci. 2025;26(8):3804. doi: 10.3390/ijms26083804. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Ghoshal K., Bhattacharyya M. Adiponectin: probe of the molecular paradigm associating diabetes and obesity. World J Diabetes. 2015;6(1):151–166. doi: 10.4239/wjd.v6.i1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Lozano I., Van der Werf R., Bietiger W., Seyfritz E., Peronet C., Pinget M., Jeandidier N., Maillard E., Marchioni E., Sigrist S., Dal S. High-fructose and high-fat diet-induced disorders in rats: impact on diabetes risk, hepatic and vascular complications. Nutr Metab (Lond) 2016;13:15. doi: 10.1186/s12986-016-0074-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Martínez-Sánchez N. There and back again: Leptin actions in white adipose tissue. Int J Mol Sci. 2020;21(17):6039. doi: 10.3390/ijms21176039. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Obradovic M., Sudar-Milovanovic E., Soskic S., Essack M., Arya S., Stewart A.J., Gojobori T., Isenovic E.R. Leptin and obesity: role and clinical implication. Front Endocrinol (Lausanne) 2021;12 doi: 10.3389/fendo.2021.585887. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Li X., Zhang D., Vatner D.F., Goedeke L., Hirabara S.M., Zhang Y., Perry R.J., Shulman G.I. Mechanisms by which adiponectin reverses high fat diet-induced insulin resistance in mice. Proc Natl Acad Sci USA. 2020;117(51):32584–32593. doi: 10.1073/pnas.1922169117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Achari A.E., Jain S.K. Adiponectin, a therapeutic target for obesity, diabetes, and endothelial dysfunction. Int J Mol Sci. 2017;18(6):1321. doi: 10.3390/ijms18061321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Alves-Bezerra M., Cohen D.E. Triglyceride metabolism in the liver. Compr Physiol. 2017;8(1):1–8. doi: 10.1002/cphy.c170012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Ngwa D.N., Pathak A., Agrawal A. IL-6 regulates induction of C-reactive protein gene expression by activating STAT3 isoforms. Mol Immunol. 2022;146:50–56. doi: 10.1016/j.molimm.2022.04.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Faber D.R., Van Der Graaf Y., Westerink J., Visseren F.L.J. Increased visceral adipose tissue mass is associated with increased C-reactive protein in patients with manifest vascular diseases. Atherosclerosis. 2010;212(1):274–280. doi: 10.1016/j.atherosclerosis.2010.04.029. [DOI] [PubMed] [Google Scholar]
- 30.Mouliou D.S. C-reactive protein: pathophysiology, diagnosis, false test results and a novel diagnostic algorithm for clinicians. Diseases. 2023;11(4):132. doi: 10.3390/diseases11040132. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.McMurray F., Patten D.A., Harper M. Reactive oxygen species and oxidative stress in obesity—recent findings and empirical approaches. Obesity. 2016;24(11):2301–2310. doi: 10.1002/oby.21654. [DOI] [PubMed] [Google Scholar]
- 32.Salunke M., Giramkar S., Bhalerao S. In vitro free radical scavenging potential of trimad (A polyherbal formulation) Int J Res Ayurveda Pharm. 2017;8(3):127–131. doi: 10.7897/2277-4343.083183. [DOI] [Google Scholar]
- 33.van Galen K.A., Ter Horst K.W., Booij J., la Fleur S.E., Serlie M.J. The role of central dopamine and serotonin in human obesity: lessons learned from molecular neuroimaging studies. Metabolism. 2018;85:325–339. doi: 10.1016/j.metabol.2017.09.007. [DOI] [PubMed] [Google Scholar]
- 34.Janssen L.K., Herzog N., Waltmann M., Breuer N., Wiencke K., Rausch F., Hartmann H., Poessel M., Horstmann A. Lost in translation? On the need for convergence in animal and human studies on the role of dopamine in diet-induced obesity. Curr Addict Rep. 2019;6(3):229–257. doi: 10.1007/s40429-019-00268-w. [DOI] [Google Scholar]
- 35.Conde K., Fang S., Xu Y. Unraveling the serotonin saga: from discovery to weight regulation and beyond-a comprehensive scientific review. Cell Biosci. 2023;13(1):143. doi: 10.1186/s13578-023-01091-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Cîmpeanu R.C., Caragea E.M., Mustață L.M., Forțofoiu D., Dragne I.G., Alexa R.E., Balta A., Ceasovschih A., Șorodoc L., Săndulescu L.D. The involvement of serotonin in the obesity pathway—A last decade systematic review of the literature. Int J Mol Sci. 2025;26(7):3081. doi: 10.3390/ijms26073081. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Yabut J.M., Crane J.D., Green A.E., Keating D.J., Khan W.I., Steinberg G.R. Emerging roles for serotonin in regulating metabolism: new implications for an ancient molecule. Endocr Rev. 2019;40(4):1092–1107. doi: 10.1210/er.2018-00283. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Tabuchi C., Sul H.S. Signaling pathways regulating thermogenesis. Front Endocrinol. 2021;12 doi: 10.3389/fendo.2021.595020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Caron A., Jane Michael N. New horizons: is obesity a disorder of neurotransmission? J Clin Endocrinol Metab. 2021;106(12):e4872–e4886. doi: 10.1210/clinem/dgab421. [DOI] [PubMed] [Google Scholar]
- 40.Wallace C.W., Fordahl S.C. Obesity and dietary fat influence dopamine neurotransmission: exploring the convergence of metabolic state, physiological stress, and inflammation on dopaminergic control of food intake. Nutr Res Rev. 2022;35(2):236–251. doi: 10.1017/S0954422421000196. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Watanabe H., Nakano T., Saito R., Akasaka D., Saito K., Ogasawara H., Minashima T., Miyazawa K., Kanaya T., Takakura I., Inoue N., Ikeda I., Chen X., Miyake M., Kitazawa H., Shirakawa H., Sato K., Tahara K., Nagasawa Y., Rose M.T., Ohwada S., Watanabe K., Aso H. Serotonin improves high fat diet induced obesity in mice. PLoS One. 2016;11(1) doi: 10.1371/journal.pone.0147143. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Aibara D., Sakaguchi A., Matsusue K. Transcriptional regulation of adipogenin expression in liver steatosis by hepatic peroxisome proliferator‐activated receptor gamma. Genes Cells. 2023;28(8):585–594. doi: 10.1111/gtc.13052. [DOI] [PubMed] [Google Scholar]
- 43.Videla L.A., Pettinelli P. Misregulation of PPAR functioning and its pathogenic consequences associated with nonalcoholic fatty liver disease in human obesity. PPAR Res. 2012;2012(1) doi: 10.1155/2012/107434. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Li N., Li X., Ding Y., Liu X., Diggle K., Kisseleva T., Brenner D.A. SREBP regulation of lipid metabolism in liver disease, and therapeutic strategies. Biomedicines. 2023;11(12):3280. doi: 10.3390/biomedicines11123280. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Li X., Li Y., Yang W., Xiao C., Fu S., Deng Q., Ding H., Wang Z., Liu G., Li X. SREBP-1c overexpression induces triglycerides accumulation through increasing lipid synthesis and decreasing lipid oxidation and VLDL assembly in bovine hepatocytes. J Steroid Biochem Mol Biol. 2014;143:174–182. doi: 10.1016/j.jsbmb.2014.02.009. [DOI] [PubMed] [Google Scholar]
- 46.Moslehi A., Hamidi-Zad Z. Role of SREBPs in liver diseases: a mini-review. J Clin Transl Hepatol. 2018;6(3):332. doi: 10.14218/JCTH.2017.00061. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Yao Q., Li S., Cheng X., Zou Y., Shen Y., Zhang S. Yin Zhi Huang, a traditional Chinese herbal formula, ameliorates diet-induced obesity and hepatic steatosis by activating the AMPK/SREBP-1 and the AMPK/ACC/CPT1A pathways. Ann Transl Med. 2020;8(5):231. doi: 10.21037/atm.2020.01.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Park M.N. Distributed pharmacodynamic architecture in multi-component herbal formulations: a flux-based framework for redox-heterogeneous diseases. Pharmaceutics. 2026;18(3):339. doi: 10.3390/pharmaceutics18030339. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Parasuraman S., Thing G.S., Dhanaraj S.A. Polyherbal formulation: concept of ayurveda. Pharmacogn Rev. 2014;8(16):73. doi: 10.4103/0973-7847.134229. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Zhou X., Seto S.W., Chang D., Kiat H., Razmovski-Naumovski V., Chan K., Bensoussan A. Synergistic effects of Chinese herbal medicine: a comprehensive review of methodology and current research. Front Pharmacol. 2016;7:201. doi: 10.3389/fphar.2016.00201. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Data Availability Statement
All data generated or analyzed during this study are included in this article. It can be obtained from the corresponding author upon request.