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Learn more: PMC Disclaimer | PMC Copyright Notice Mol Nutr Food Res . 2026 Apr 9;70:e70456. doi: 10.1002/mnfr.70456 Search in PMC Search in PubMed View in NLM Catalog Add to search Ellagic Acid Reduces Inulin's Adverse Effects: A Combined Approach to Enhance Therapeutic Potential in Nonalcoholic Steatohepatitis Tharani Senavirathna Tharani Senavirathna 1 Centre For Precision Health, School of Medical and Health, Edith Cowan University, Joondalup, Western Australia Find articles by Tharani Senavirathna 1 , Armaghan Shafaei Armaghan Shafaei 2 School of Science, Edith Cowan University, Joondalup, Western Australia Find articles by Armaghan Shafaei 2 , Ricky R Lareu Ricky R Lareu 3 Curtin Medical School, Faculty of Health Sciences, and Curtin Medical Research Institute, Curtin University, Bentley, Western Australia Find articles by Ricky R Lareu 3, ✉ , Lois Balmer Lois Balmer 1 Centre For Precision Health, School of Medical and Health, Edith Cowan University, Joondalup, Western Australia 4 Centre For Diabetes Research, Harry Perkins Institute For Medical Research, University of Western Australia, Nedlands, Western Australia 5 Dobney Hypertension Centre, School of Medicine, Royal Perth Hospital Unit, Faculty of Medicine, Dentistry & Health Sciences, The University of Western Australia, Perth, Western Australia Find articles by Lois Balmer 1, 4, 5, ✉ Author information Article notes Copyright and License information 1 Centre For Precision Health, School of Medical and Health, Edith Cowan University, Joondalup, Western Australia 2 School of Science, Edith Cowan University, Joondalup, Western Australia 3 Curtin Medical School, Faculty of Health Sciences, and Curtin Medical Research Institute, Curtin University, Bentley, Western Australia 4 Centre For Diabetes Research, Harry Perkins Institute For Medical Research, University of Western Australia, Nedlands, Western Australia 5 Dobney Hypertension Centre, School of Medicine, Royal Perth Hospital Unit, Faculty of Medicine, Dentistry & Health Sciences, The University of Western Australia, Perth, Western Australia ✉ Corresponding author. Revised 2026 Jan 30; Received 2025 May 19; Accepted 2026 Feb 11; Issue date 2026 Apr 15. © 2026 The Author(s). Molecular Nutrition & Food Research published by Wiley‐VCH GmbH. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. PMC Copyright notice PMCID: PMC13066767 PMID: 41958187 ABSTRACT This mouse study investigates the efficacy of ellagic acid (EA), a potent antioxidant in fruits and nuts, and inulin, a prebiotic known for gut microbiota benefits, in nonalcoholic steatohepatitis (NASH). A Western‐style diet (WD) was used to induce NASH in C57BL/6J male mice for 16 weeks. Mice then received EA (100 mg/kg/day, oral gavage, 5 days/week) and/or 5% inulin (w/w in diet) for 12 weeks while continuing WD. Inulin increased energy intake and trended toward higher body weight and fasting glucose. EA reduced liver weights and restored the liver‐to‐body weight ratio. EA and inulin combined significantly reduced steatosis and hepatocyte ballooning, while EA alone reduced liver inflammation and improved biomarkers. In contrast, inulin alone worsened several liver parameters and increased LDL serum levels. EA, alone or combined with inulin, significantly reduced LDL. EA‐treated groups showed elevated urinary 3'‐methylellagic acid. Importantly, the combination of EA and inulin significantly increased urolithin A, C, and D, indicating enhanced EA metabolism and postbiotic production. While inulin alone exacerbated NASH features, EA significantly improved liver health. The combination of EA and inulin offset the adverse effects of inulin but did not further enhance the therapeutic efficacy of EA. Keywords: ellagic acid, inulin, NAFLD, NASH, nutraceuticals, urolithins This study investigated the individual and combined effects of ellagic acid (EA) and inulin on metabolic and liver health outcomes in a mouse model of NASH. Inulin alone worsened metabolic parameters, elevating fasting blood glucose, LDL levels, and steatosis. EA treatment improved liver injury markers and reduced LDL levels. The combination of EA and inulin significantly enhanced metabolites of EA, urolithin A, C, and D, and reduced hepatic steatosis and ballooning, suggesting a synergistic interaction that improves postbiotic metabolism and liver function. Abbreviations ALT alanine aminotransferase AMPK AMP‐activated protein kinase AST aspartate aminotransferase EA ellagic acid FBG fasting blood glucose FDA Food and Drug Administration H&E hematoxylin and eosin LC‐QQQ‐MS liquid chromatography‐triple quadrupole‐mass spectrometry MASH metabolic dysfunction‐associated steatohepatitis MASLD metabolic dysfunction‐associated steatotic liver disease MRM multiple reaction monitoring NAFLD nonalcoholic fatty liver disease NASH nonalcoholic steatohepatitis OGTT oral glucose tolerance test ROI regions of interest SEM standard error of the mean TC total cholesterol TG triglycerides WD Western diet 1. Introduction Nonalcoholic fatty liver disease (NAFLD) has become the most prevalent liver disease globally, affecting approximately 38% of the world's population, with a significantly higher prevalence among overweight and obese individuals [ 1 , 2 , 3 ]. NAFLD represents a wide spectrum of liver conditions, ranging from simple steatosis to its more severe form, nonalcoholic steatohepatitis (NASH). The current global prevalence of NASH is estimated to range from 4% to 6% [ 1 ]. NASH, a progressive form of NAFLD, is characterized by hepatic fat accumulation, lobular inflammation, hepatocellular injury, and ballooning, which can progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma [ 4 , 5 , 6 ]. The progression of NASH is best explained by the multiple‐hit hypothesis, which suggests that factors such as obesity, metabolic dysfunction, and insulin resistance synergistically lead to lipotoxicity, oxidative stress, inflammation, and hepatocellular injury [ 7 , 8 ]. Recently, the Delphi consensus proposed reclassifying NAFLD as metabolic dysfunction‐associated steatotic liver disease (MASLD) and NASH as metabolic dysfunction‐associated steatohepatitis (MASH) [ 9 ]. This redefinition aims to reflect the metabolic dysfunction underlying these conditions more accurately and align with the modern understanding of their pathogenesis [ 10 , 11 ]. However, since the majority of existing literature and clinical trials focus on NAFLD/NASH and due to the limited studies specifically addressing MASLD/MASH, we will use the terms NAFLD/NASH throughout this paper to maintain consistency with available research. Despite recent advances, including the FDA approval of Rezdiffra (resmetirom) for NASH, the absence of broadly effective pharmacological treatments continues to limit therapeutic progress [ 12 , 13 , 14 ]. As such, lifestyle interventions, particularly dietary modifications, remain the first‐line approach for managing NASH. Since oxidative stress and inflammation are key drivers of NASH pathogenesis, dietary interventions rich in antioxidants and anti‐inflammatory compounds are vital in targeting the underlying disease progression and offer a promising approach to complement other potential treatments [ 15 , 16 , 17 ]. Ellagic acid (EA) is a nonflavonoid polyphenolic compound commonly found in fruits such as pomegranate, strawberries, raspberries, and grapes, as well as nuts such as walnuts and pecans [ 18 , 19 , 20 ]. Dietary EA exists either in its free form or as ellagitannins, which are complex polymers that release free EA and gallic acid upon hydrolysis [ 18 , 19 , 21 ]. Free EA is primarily absorbed in the stomach and small intestine, while the remainder is absorbed in the large intestine or metabolized by gut microbiota to produce a microbial‐derived metabolite, urolithins [ 22 , 23 , 24 ]. Microbial metabolism of EA involves an initial ring cleavage and decarboxylation leading to the formation of urolithin D. Subsequent sequential dehydroxylation leads to the production of urolithin C, urolithin A, isourolithin A, and urolithin B [ 22 , 24 , 25 ]. The low bioavailability of EA and its metabolites poses a challenge to the detection and elucidation of its metabolic pathways. In contrast, its microbial metabolites, urolithins, are detectable in human plasma at concentrations ranging from 0.024 to 35 µM, whereas EA is present at substantially lower levels in plasma (∼200 ng/mL) [ 22 , 23 ]. Ellagic acid exerts hepatoprotective effects primarily through its antioxidant activity, driven by its four hydroxyl and two lactone functional groups. These functional groups enable EA to effectively scavenge a broad spectrum of free radical species, including reactive oxygen species and reactive nitrogen species [ 26 , 27 ]. Thereby providing substantial protection against free radical‐induced cellular damage. Additionally, EA demonstrates anti‐inflammatory and antifibrotic properties and contributes to the regulation of lipid synthesis [ 19 , 20 ]. Urolithin A has been shown to reduce LDL cholesterol and increase HDL cholesterol levels while enhancing lipid metabolism through gene regulation [ 28 ]. Urolithin C also contributes to hepatoprotection by activating the hepatic AMP‐activated protein kinase (AMPK) pathway, thereby mitigating the progression of NAFLD [ 29 ]. Unlike EA, which exhibits low bioavailability and limited intestinal epithelium permeability, urolithins possess higher liposolubility, facilitating rapid absorption and systemic distribution [ 22 , 25 , 30 ]. Consequently, urolithins are often regarded as the primary active compounds combating NAFLD/NASH. However, direct comparative studies between EA and urolithins remain limited. In a high‐fat/high‐sucrose‐fed mouse model, one study revealed that only EA, not urolithin A, reduced proton leakage in primary hepatocytes, indicating a distinct role for EA in enhancing mitochondrial respiratory capacity during insulin resistance [ 31 ]. Therefore, a comprehensive assessment of both EA and urolithins is essential to fully elucidate the polyphenol's mechanism of action against NAFLD/NASH. Recent studies have identified prebiotics as promising alternatives for managing chronic diseases, including NAFLD/NASH [ 32 , 33 , 34 , 35 ]. Inulin, a polysaccharide primarily derived from chicory roots, Jerusalem artichoke, and garlic, is a soluble and fermentable dietary fiber that resists hydrolysis in the upper gastrointestinal tract and undergoes fermentation by gut microbiota in the colon [ 36 , 37 ]. During fermentation, inulin produces short‐chain fatty acids (SCFAs) that activate metabolically relevant receptors contributing to improved host health [ 38 , 39 ]. Inulin has gained recognition as a dietary supplement due to its numerous health benefits, including glycaemic regulation, weight reduction, lipid‐lowering effects, and anti‐inflammatory properties [ 37 , 40 , 41 ]. Additionally, inulin promotes the growth of beneficial gut bacteria, suppresses pathogenic intestinal bacteria, and enhances gut health [ 39 , 42 , 43 , 44 ]. However, some studies suggest that high doses of inulin could lead to adverse effects, such as an increased risk of liver cancer in immunodeficient mice, and that long‐term interventions may not significantly alleviate metabolic syndrome in high‐fat diet‐induced obese mice [ 44 , 45 ]. While inulin has demonstrated potential in preventing NAFLD, its efficacy in treating established NAFLD/NASH remains unclear. Despite the benefits, the specific effect of inulin on hepatic lipid metabolism is not well understood. Given its widespread availability as a commercial prebiotic supplement, further research is needed to determine whether the hepatoprotective effects observed in animal models can be effectively translated to human patients. The study aimed to assess the therapeutic efficacy of EA and the impact of inulin supplementation in an established NASH mouse model using physiological, biochemical, and histological parameters. Additionally, we explored the urinary metabolomics of EA to elucidate the mechanisms through which EA may prevent NASH progression. We hypothesized that inulin would enhance the therapeutic effects of EA. However, contrary to expectations, inulin alone exacerbated NASH, leading to increased body weight, elevated food intake, and worsened liver function as evidenced by the levels of aspartate transaminase (AST), alanine aminotransferase (ALT), LDL and triglycerides. Interestingly, coadministration of inulin with EA mitigated these adverse effects. Notably, EA treatment alone significantly improved key liver parameters, including reduced total steatosis, decreased energy consumption, and restoration of the normal liver‐to‐body weight ratio, suggesting a potential role in alleviating diet‐induced chronic liver disease. 2. Materials and Methods 2.1. Animals and Diets Sixty‐one C57BL/6J male mice (6–7 weeks of age) were purchased from the Animal Resource Centre (Perth, Western Australia) and delivered to the Curtin Life Science Facility. Mice underwent 1‐week acclimatization at 23 ± 2°C with a 12‐h day/night cycle with 60% humidity whilst maintained on a standard diet and ad libitum access to water. Initially, mice ( n = 61) were randomly assigned to two dietary groups (Controls and NASH) for 16 weeks. Control mice ( n = 14) were fed normal chow containing 12.8 MJ/Kg digestible energy (meat‐free rat and mouse maintenance diet (Specialty Feeds, Glen Forrest, Western Australia)). To induce the NASH disease model, mice were fed a Western‐style diet (NASH group; n = 47) characterized by high levels of refined sugars in solid as well as liquid form ad libitum. Solid feed (pellets) contained 19.7 MJ/Kg digestible energy with the following constituents: fructose, 20%; sucrose, 22.4%; fats, 23.1%; cholesterol, 0.2% (SF21‐155; Specialty feeds). Pellets were weighed and changed three times per week. Drinking water was a solution of 42 g/L of fructose (55%) and glucose (45%), changed three times per week (Merck Life Science, VIC, Australia). See Table 1a for a comparison of key feed constituents. At the end of the 16 weeks, three Control and three NASH mice were euthanised for histopathological assessment. TABLE 1a. Composition of diets. Percentage composition of key constituents (%) Total digestible energy Fats Refined sugars (sucrose and fructose) Cholesterol Protein Fiber Normal chow 12.8 MJ/Kg 4.8 0 0 14.5 31 Western‐style diet Pellet 19.7 MJ/Kg 21.1 42.4 0.2 18.3 5 Drinking Water 672 kJ/L 0 4.2 (m/v) 0 0 0 Open in a new tab After 16 weeks, the NASH mice ( n = 44) were randomly allocated into four treatment groups for a further 12 weeks: NASH positive disease control treated group; NASH + EA treated group (Merck Life Science, Victoria, Australia); NASH + In (inulin) treated group; NASH + In + EA combination‐treated group. The standard chow group (Controls) remained on the standard diet throughout ( n = 11). All experimental groups were gavaged orally five days per week with either water (Control, NASH, NASH + In) or 100 mg/kg with EA (NASH + EA, NASH + In + EA). Treatment groups NASH + In and NASH + In + EA were fed the same Western‐style diet, additionally containing 5% inulin (SF21‐156; Specialty Feeds) mixed into the solid food pellets. See Table 1b for details. TABLE 1b. Treatment Groups. Experimental groups Solid food Drinking water Gavage Control NC Plain Water NASH WD Refined sugar Water NASH + EA WD Refined sugar EA NASH + In WD Refined sugar Water NASH + In + EA WD Refined sugar EA Open in a new tab 2.2. Sample Collection of Urine, Blood, and Faeces Blood and urine were collected at the beginning of each month. Urine was immediately snap‐frozen in liquid nitrogen. Blood was collected by the submandibular method in tubes containing lithium heparin (MiniCollect; Greiner Bio‐One, Singapore), centrifuged to separate the haematocrit and the plasma, and snap‐frozen. Six mice were euthanised at the end of the first 16 weeks (3 Control and 3 NASH), and the remaining mice ( n = 55) were euthanised after the completion of 12 weeks in the specific diet groups. Blood was collected by cardiac puncture under anaesthesia and processed as above; liver and cecum faeces were collected once the animal was euthanised and snap‐frozen in liquid nitrogen before storing at −80°C. 2.3. Experimental Animals This study was approved by the Animal Ethics Committees of Curtin University (ARE2022‐7) and Edith Cowan University (24079 BALMER). All animal procedures comply with the required National Health and Medical Research Guidelines on the Care and Use of Laboratory Animals in Australia, the Australian Code of Practice for the care and use of animals for scientific purposes (eighth Edition 2013, updated 2021), the Conduct of Ethical Research and Teaching Involving Animals Policy, and the Animal Welfare Act. 2.4. Weight Gain, Energy Intake, and Glucose Tolerance Test Body weights were measured weekly, and dietary intake (solid food) was measured three times per week per cage throughout the study. Energy consumption was calculated using the average feed intake per cage per mouse. The oral glucose tolerance test (OGTT) was performed one week before euthanasia in both the initial 16‐week period and the following 12 weeks, according to the optimized parameters described by Andrikopoulos and colleagues [ 46 ]. Briefly, following 6 h of fasting, a glucose solution (2 g/Kg body weight) was administered by oral gavage. Just prior to glucose administration (time 0) and at 10, 20, 40, 60 and 120 min following administration, blood glucose was measured with a handheld glucometer (OneTouch VerioIQ; LifeScan, Inc., Chesterbrook, PA, USA) from the tail vein. 2.5. Biochemical Analysis Plasma concentration of total cholesterol (TC; mmol/L), LDL (mmol/L), HDL (mmol/L), triglycerides (TG; mmol/L), and plasma activities (U/L) of AST and ALT were quantified commercially by PathWest Laboratories (Fiona Stanley Hospital, Perth). 2.6. Hepatic Cholesterol Liver cholesterol was quantified from frozen liver tissues with the RX Monza CH200 kit (Randox Laboratories, United Kingdom). Briefly, liver tissue was homogenized in isopropanol (1 mL/50 mg tissue) and incubated at 4°C for 1 h. After incubation, the samples were centrifuged at 685 g for 5 min at 4°C and subjected to the kit's enzymatic hydrolysis and oxidation steps according to the Manufacturer's instructions. Colorimetric quantification was carried out on a microplate reader EnSight (PerkinElmer, NSW, Australia). 2.7. Histopathological Stains A portion of the left lateral lobe of the liver was collected and fixed in fresh 4% paraformaldehyde for 24 h at room temperature with gentle shaking, followed by storing in phosphate‐buffered saline (PBS; pH 7.4) at 4°C (Sigma‐Aldrich, St. Louis, MO, USA). Tissues were sequentially dehydrated, embedded in paraffin and sectioned at 4 µm. Sections were stained with hematoxylin and eosin (H&E) (Hurstchem Laboratory Chemicals, WA, Australia), then scanned and digitized at 20x magnification with the Axioscan Z.1 Digital Slide Scanner (Carl Zeiss GmbH, Germany) and analyzed with the ZEN v3.8.3 (Carl Zeiss GmbH, Germany). Liver histopathology was evaluated for total steatosis, inflammation and hepatocyte damage, that is, ballooning. Total liver steatosis was evaluated quantitatively with ZEN v3.8.3 using machine learning with the Zeiss Zen Intellesis module (Carl Zeiss GmbH, Germany). Briefly, multiple regions of interest (ROI) were made to cover >90% of the H&E‐stained section. The ROI was batch‐analyzed with an Intellesis model (Carl Zeiss GmbH, Germany) trained on representative images to identify total steatosis. Inflammation was semiquantitatively evaluated by averaging the number of inflammatory foci in three representative ROI of H&E sections (3×1.5 mm2). Identification of inflammatory foci consisted of ≥ 5 immune cells in a cluster and not in a row. Hepatocyte ballooning was semiquantitatively evaluated by averaging the ballooning score from three representative ROI of H&E sections (3×1.5 mm2). The hepatocyte ballooning criteria were hypertrophic cells (enlarged and rounded) with steatosis and disrupted cytoskeleton, with disrupted nuclear structure and/or Mallory–Denk bodies. Due to the extensive area of liver affected, an extended ballooning score system was developed: ‘0’, none too few ballooned hepatocytes that are seen in healthy animals; ‘1’, low, several scattered individual ballooned hepatocytes; ‘2’, moderate, many scattered individual and/or few small ballooned foci; ‘3’, high, many ballooned hepatocytes, mostly grouped as several to many foci affecting large regions; ‘4’, severe, many interconnected foci of ballooned hepatocytes throughout area with steatosis. For the evaluation of fibrosis, sections were dehydrated and stained with 0.04% (w/v) picrosirius red in saturated picric acid for 30 min, followed by counter staining with 0.1% fast green for 10 min. Sections were then dehydrated and imaged as for the haematoxylin and eosin sections. 2.8. Urine Metabolomics Urinary EA metabolites from each mouse urine sample, collected at euthanasia, were analyzed and quantified using a liquid chromatography‐triple quadrupole‐mass spectrometry (LC‐QQQ‐MS) system. Chromatographic separation was carried out on an UltiMate 3000 Liquid Chromatograph (Thermo Scientific, CA, USA) coupled to a Thermo Scientific TSQ Quantiva Triple Quadrupole Mass Spectrometer (Thermo Scientific, CA, USA) equipped with an ESI source. Separation was performed on an ACE C18‐AR column (100 mm × 2.1 mm ID, 1.7 µm particles; Advanced Chromatography Technologies, Scotland) using a mobile phase of water (A) and acetonitrile (B), each containing 0.1% formic acid. The gradient program lasted 16 min, starting with 96% solvent A and 4% solvent B, and held for 1 min. Solvent B was then increased to 20% over 7 min, 55% over the next 2 min, 60% over 2 min, and 96% over 3 min. The mobile phase was returned to the initial conditions within 1 min, followed by a 3.5‐min re‐equilibration. The flow rate was set at 0.2 mL/min, the column temperature was maintained at 35°C, and the injection volume was 4 µL. Detection was performed in negative mode (−2500 V) with spectra acquired in multiple reaction monitoring (MRM) mode. Argon was used as the collision gas, while nitrogen served as the nebulizer and heater gas. Mass spectrometer (MS) conditions included sheath gas at 35, auxiliary gas at 15, and sweep gas at 0 (arbitrary units), with an ion transfer temperature of 325°C and vaporizer temperature of 275°C. For MS parameter optimization, individual standard solutions (1 µg/mL in methanol) were introduced into the (‐)‐ESI source via direct infusion at a flow rate of 5 µL/min. Table S1 summarizes the optimal MS parameters for each standard and internal standard, including precursor ions, product ion transitions, and collision energies. 2.9. Statistical Analysis Prior to analysis, all assumptions for normality were assessed using the omnibus K2 D'Agostino & Pearson test. Outliers were then identified using the ROUT method ( Q = 1%) in GraphPad Prism and excluded. For normally distributed data, we performed repeated measures ANOVA to assess changes over time and one‐way ANOVA with Dunnett's post hoc test to compare each group against the NASH group as the control. Kruskal–Wallis tests were used for nonparametric data and corrected for multiple comparisons via Dunn's test. All data are presented as mean ± standard error of the mean (SEM) unless otherwise stated. Statistical significance was set at a 2‐sided type‐1 error rate of p < 0.05. Analyses were conducted using GraphPad Prism, version 10 (San Diego, USA). 3. Results 3.1. Establishment of the NASH Model The Western diet group (NASH) had significantly greater body weight gain from week 2 compared to the control group over the induction period (Figure 1a,b ), including significantly higher fasting blood glucose levels and area under the curve (AUC) for the OGTT assay (Figure 1c,d ). Histological examination of liver tissue revealed profound changes between the control and NASH groups. The control liver exhibited normal architecture, whereas the NASH liver showed clear signs of extensive steatosis, hepatocyte ballooning (containing the presence of Mallory–Denk bodies) and inflammation (Figure 1e–g ). These histological features were quantitatively greater for the NASH group (Figure 1h–j ). These findings confirm the successful induction of NASH and highlight the adverse metabolic and hepatic changes associated with this condition prior to commencing the administration of EA and inulin. FIGURE 1. Open in a new tab The induction of the NASH disease model. Mice were fed either a normal rodent chow (Control group) or a Western‐style diet (NASH group) for 16 weeks. Three mice from each group were used to evaluate the establishment of the NASH disease model. (a) Change in body weight. (b) Total body weight gain at the end of treatment. (c) Fasting plasma glucose levels. (d) The area under the curve for the oral glucose tolerance test. H&E‐stained liver sections: (e) Control group with well‐organized hepatocytes, (f) NASH group displaying extensive steatosis, and (g) magnified insert demonstrating hepatocyte ballooning with Mallory–Denk bodies (circle), macrovesicular (green arrowhead), microvesicular (oval), and steatosis and inflammatory foci (black arrow). Quantitative assessment, (h) total steatosis, (i) hepatocyte ballooning, and (j) number of inflammatory foci. All quantitative values are reported as the mean ± SEM per group ( n = 3 biological replicates per group)* p < 0.05;**** p < 0.0001. Statistical analysis was performed with the unpaired t ‐test. 3.2. Ellagic Acid and Inulin Improve Physiological Parameters in NASH Mice Following the 16‐week establishment of the NASH model, treatment with EA, inulin or in combination was carried out for a further 12 weeks on a Western‐style diet. All treatment groups showed a significant difference from the Control group by the end of the 12 weeks, though the onset of these differences varied across groups (Figure 2a,b ). Inulin alone led to a highly significant difference in weight gain as early as week 3 of treatment. Both inulin‐containing groups showed a trend toward the greatest overall weight gain, whereas EA alone was the last group to show a significant difference, starting at week 9, with a lower weight gain compared to the NASH group. The greater weight gain in the inulin‐supplemented groups may be attributed to their significantly higher solid food intake over the 12 weeks (Figure 2c ). Energy consumption in the EA‐alone treated group was lower than in the inulin‐supplemented groups but not significantly different from the positive disease control (NASH). A significant increase in fasting blood glucose was observed only in the inulin‐alone group compared to the Control (Figure 2d ). However, no significant differences in blood glucose response were observed among the treatment groups with OGTT, except when compared to the Control group (Figure 2e ). FIGURE 2. Open in a new tab Physiological changes in response to nutraceutical treatments. Mice with NASH were treated with nutraceuticals for a further 12 weeks. Control mice were maintained on standard rodent chow, while the NASH groups were maintained on the Western‐style diet. (a, left panel) Change in total body weight and (a, right panel) p ‐value matrix showing the statistical differences, range in weeks, between treatment groups. (b) Change in total body weight at the end of treatment. (c) Average energy consumption of solid food during treatment. (d) Week 12 fasting plasma glucose levels. (e) The area under the curve for the plasma glucose response was measured by the oral glucose tolerance test. (f) Total liver weight at the end of treatment. (g) Liver‐to‐body weight ratio. (h) Correlation between liver and body weights. All values are reported as the mean ± SEM of 11 mice per group: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Statistical analysis was performed using one‐way ANOVA with Bonferroni's post hoc test unless otherwise indicated. g, grams; KJ, kilojoules. The Western‐style diet increased liver weight, but the inclusion of EA limited this increase (Figure 2f ). This is more clearly demonstrated when normalized to the weight of the mouse (Figure 2g ). A correlation analysis between the liver and body weights showed interesting results. The inclusion of EA in the NASH disease significantly restored an association between body weight and liver weight ( R 2 = 0.89; p < 0.0001). When inulin was included, this correlation was still present but not as strong ( R 2 = 0.74; p < 0.001). This suggests that while the combination of inulin and EA was beneficial in mitigating the adverse effects of NASH, the addition of inulin did not enhance the effect as significantly as EA alone in terms of restoring the normal liver‐to‐body weight ratio (Figure 1h ). 3.3. Ellagic Acid and Inulin Improve Histopathological Parameters in NASH Mice The histopathology analysis of the liver progressed from the central vein (hepatic acinus zone III) toward the portal triads (zone I). The greater the severity, the greater the area affected, with most samples showing histopathological changes to zones III and I. The exception was the liver samples from the nontreatment Control group that had healthy histology. With severity, steatosis appeared to progress from microvesicular steatosis to greater proportions of macrovesicular steatosis. Representative images are shown in Figure 3a–e . The positive control disease group, NASH, had the largest area affected by steatosis (Figure 3b ). Although all nutraceutical treatments demonstrated a limited reduction in steatosis, the combination treatment of EA and inulin showed a statistically significant reduction (Figure 3f ). A statistically significant reduction was detected when the NASH and NASH + EA groups were discretely analyzed with an unpaired t ‐test, #, p < 0.05. FIGURE 3. Open in a new tab Histopathological changes in response to nutraceutical treatments. Representative images of fixed, hematoxylin and eosin‐stained liver sections following 12 weeks of nutraceutical treatment for (a) Control group, (b) NASH group displaying extensive steatosis, with reduced steatosis for the (c) ellagic acid (EA) group, (d) inulin (In) group, and (e) the combination EA and In group. Quantitative data for (f) total steatosis of area percentage affected, (g) hepatocyte ballooning score, and (h) inflammatory foci number. Representative images with picrosirius red stain to detect collagen in the (i) Control group and (j) inulin group. All values are reported as the mean ± SEM of 11 mice per group: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. # is p < 0.05 with the unpaired t ‐test; otherwise, one‐way ANOVA with Bonferroni's post hoc test was applied. The target of this Western‐style diet was significant liver damage, specifically hepatocyte damage. The damage was so extensive that an extended scoring system was developed (see Section 2 ). Although there was a reduction in the level of hepatocyte ballooning in the EA alone, statistical significance was only seen with the combined nutraceutical treatment (NASH+In+EA) compared to NASH and NASH + In treatments (Figure 3g ). Interestingly, inulin supplementation alone did not reduce hepatocyte damage. These trends were also observed with the level of inflammation, based on the number of inflammatory foci (Figure 3h ). Although NASH + EA and NASH + In + EA combination treatments demonstrated higher inflammation than the Control group, they were not statistically different. To evaluate the level of fibrosis, we used picrosirius red to stain collagen fibers. Interestingly, we only observed increased collagen deposition in five mice: three from the NASH + In treatment and two from the combination treatment (Figure 3j ). All other mice did not have collagen deposition greater than the Control treatment (Figure 3i ). The pattern of increased collagen deposition was restricted to the areas surrounding the central vein. 3.4. EA Supplementation Improves Biochemical Parameters Biochemical analysis revealed markedly elevated AST and ALT levels in both the NASH and inulin‐only groups compared to the control group, indicating liver damage (Figure 4a,b ). EA supplementation significantly reduced both ALT and AST levels in the EA‐treated groups, with or without inulin. LDL levels were similarly elevated in the NASH and inulin‐only groups but were reduced following EA treatment alone and in combination with inulin, showing a statistically significant improvement (Figure 4c ). The HDL levels trended higher for the EA‐treated group compared to the NASH group, but not statistically significantly (Figure 4d ). Plasma triglyceride levels were significantly elevated in the inulin‐only group but showed a favorable trend in both EA‐treated groups (Figure 4e ). Again, EA containing treatments showed a favorable trend toward lower plasma cholesterol levels compared to the NASH group (Figure 4f ). Liver tissue cholesterol levels also trended lower in the groups treated with EA, inulin or their combination, though the changes were not statistically significant (Figure 4g ). These findings highlight EA's beneficial role in improving liver function and lipid metabolism in NASH. FIGURE 4. Open in a new tab Biochemical and lipid changes in response to nutraceutical treatments. Key biochemical and lipid parameters were tested on fasting serum after the 12‐week treatment. (a) Aspartate aminotransferase (AST). (b) Alanine aminotransferase (ALT). (c) Low‐density lipoprotein (LDL). (d) High‐density lipoprotein (HDL). (e) Triglycerides. (f) Total cholesterol. (g) Liver tissue cholesterol. All values are reported as the mean ± SEM of 11 mice per group: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. # is p < 0.05 with the unpaired t ‐test; otherwise, one‐way ANOVA with Bonferroni's post hoc test was applied. 3.5. Urinary Metabolomics of Ellagic Acid and Urolithins Urinary levels of EA, two of its major metabolites and 3 postbiotic metabolites, urolithins A, C, and D, were analyzed across the treatment groups using LC‐QQQ‐MS. As expected, low levels of EA were detected across the groups (Figure 5a ). Levels of the most abundant EA metabolite, 3'‐methylellagic acid, were elevated in both EA‐containing treatment groups compared to the control and NASH groups (Figure 5b ). Similarly, urinary levels of 3,3'‐di‐ O ‐methylellagic acid were higher in both EA‐containing treatment groups, though statistical significance was reached only in the EA‐only group. Interestingly, nonsignificant elevated levels were detected in some of the mice in the control group, likely attributable to dietary sources (Figure 5c ). Notably, EA and in combination with inulin groups, demonstrated significantly higher urinary levels of urolithin A, urolithin C, and urolithin D compared to most other groups. (Figure 5d–f ). Urolithin B was not detected in any group. These findings suggest that the combination treatment of inulin and EA produced the most pronounced increases in urinary EA metabolites and urolithins, highlighting a potential synergistic effect that enhances the production of these metabolites. FIGURE 5. Open in a new tab This panel shows the urinary metabolomic measurements at the end of 12 weeks of the treatment phase with and without ellagic acid (EA) and inulin (In). (a) 3,3‐di‐ O ‐methylellagicacid, (b) 3‐methylellagicacid, (c) ellagic acid, (d) urolithin A, (e) urolithin C, and (f) urolithin D. All values are reported as the mean ± SEM of 11 mice per group: * p < 0.05, ** p < 0.01, *** p < 0.001, and *** p < 0.0001.Statistical analysis was performed using one‐way ANOVA with Bonferroni's post hoc test unless otherwise indicated. 4. Discussion This study provides novel insights into the therapeutic potential of EA and its combination with inulin in alleviating the metabolic and hepatic complications associated with NASH. The early stages of NASH are characterized by excessive lipid accumulation, which progresses to prolonged inflammation and hepatocyte damage. In recent years, plant‐derived metabolites have garnered significant interest as potential therapeutic agents for NASH. EA, a non‐flavonoid polyphenolic compound found in pomegranates, blueberries, and walnuts, has been shown to improve oxidative stress, inflammation, and lipid metabolism [ 18 , 20 , 47 ]. Inulin, a prebiotic with diverse health benefits, has been reported to reduce body weight, attenuate blood glucose levels, and improve lipid regulation [ 37 , 40 , 41 ]. In this study, EA demonstrated remarkable therapeutic potential against Western diet‐induced NASH by significantly reducing energy intake, liver damage, and lipid profiles. It also improved liver health by alleviating total steatosis, hepatocyte ballooning, and inflammation, which are hallmarks of disease progression. Notably, the combination of EA and inulin further amplified these benefits, enhancing liver health during NASH progression. However, inulin alone was associated with adverse effects, including increased body weight, blood glucose levels, energy consumption, and lipid profiles, as well as worsened liver damage, highlighting the importance of combination therapy. In this study, we applied a Western‐style diet to mimic the diets in the real world, which is likely to lead to a higher incidence of NASH [ 48 , 49 , 50 ]. The successful establishment of the NASH model was confirmed by profound histopathological changes, including extensive steatosis, hepatocyte ballooning and inflammation, along with physiologic and metabolic alterations such as increased body weight, fasting blood glucose, and impaired glucose tolerance. These findings align with established literature on the adverse hepatic and metabolic effects of a Western‐style diet, providing a robust platform to evaluate the therapeutic effects of EA and inulin [ 5 , 51 , 52 , 53 ]. Our data suggest that inulin supplementation led to an unexpected increase in body weight gain throughout the treatment phase. This contrasts with previous studies, which have consistently reported that inulin supplementation has a positive impact on weight reduction in obese mice [ 54 , 55 ]. Similarly, EA alone did not show an overall effect on reducing body weight in our study, which is consistent with findings from a previous study [ 56 ] however, it did delay the significant change in weight until week 9 of treatment. The biological variation observed in weight gain and glucose tolerance in our dataset highlights the inherent variability in metabolic responses among the test groups. This suggests that future studies with larger sample sizes are necessary to confirm statistical significance and provide more robust conclusions. Interestingly, energy consumption was observed to increase with inulin supplementation, and even the combination of inulin with EA did not mitigate this effect. In contrast, supplementation with EA alone led to a significant reduction in energy consumption compared to the NASH cohort, indicating potential for weight reduction. While the combination of inulin and EA may provide other beneficial effects, it does not effectively address the adverse impact of inulin on energy intake, highlighting a potential limitation in the combined approach. Previous studies suggest that the effects of inulin on metabolic outcomes are highly dependent on the duration of its use. Short‐term supplementation, such as over the course of a month, has been shown to blunt metabolic syndrome and reduce body weight, demonstrating its potential benefits in improving metabolic health [ 54 , 55 , 57 ]. However, Isken and colleagues noted that long‐term use of inulin in high‐fat diet‐fed mice has been associated with adverse effects, such as increased body weight and elevated triglyceride levels [ 45 ]. These contrasting outcomes highlight the critical role of treatment duration in determining the metabolic impact of inulin and emphasize the need for careful consideration when designing dietary interventions. In our study, treatments with EA significantly improved the liver‐to‐body weight ratios, suggesting its role in reducing hepatic fat accumulation. Although the liver weights were not significantly decreased, the strong correlation between EA treatment and liver‐to‐body weight ratio ( R 2 = 0.89; p < 0.0001) highlights the potential of EA to mitigate liver fat accumulation and enlargement, a critical feature of NASH progression. Combining with inulin, while beneficial ( R 2 = 0.74; p < 0.001), appeared to be less effective in this regard. Interestingly, the combination of EA and inulin did not yield a synergistic improvement, indicating that EA's direct mechanisms of action may dominate in modulating liver weight restoration. Steatosis, lobular inflammation, and hepatocellular ballooning are essential histological features required for diagnosing NASH [ 5 ]. The NASH cohort demonstrated clear signs of advanced liver damage, as evidenced by extensive steatosis, hepatocyte ballooning‐Mallory–Denk bodies, and inflammatory foci. These features are hallmarks of severe NASH pathology and are associated with liver cell injury, inflammation, and progression toward fibrosis [ 5 , 58 ]. The inclusion of fructose in the drinking water substantially increased the level of liver damage. Hepatocyte ballooning was so extensive that we needed to extend the scoring of hepatocyte ballooning levels, based on the NAFLD Activity Score [ 51 ] to accurately evaluate this NASH parameter. Despite its effects on body weight, blood glucose levels, and lipid metabolism, inulin alleviated hepatic steatosis in our model. However, it was associated with increased hepatic inflammation and ballooning, suggesting a complex and potentially adverse impact on liver pathology. Although not consistent across all inulin‐treated mice, five of the 22 mice treated with inulin (in the combination and inulin alone treatments) had extensive fibrosis. This was also observed by Singh and Colleagues in a mouse model when they supplemented a high fat diet with inulin [ 44 ]. Interestingly, combined supplementation with EA provided more robust protective effects on the Western‐style diet‐induced NASH cohort, effectively reducing steatosis, ballooning, and inflammation. Notably, EA alone also demonstrated similar promising effects, highlighting its strong therapeutic potential in mitigating the pathological features of NASH. As expected, dyslipidemia was evident in mice with NASH, characterized by significant alterations in plasma lipid profiles, including elevated TG, increased LDL and reduced HDL. Biochemical data revealed a marked reduction in lipid profiles with EA treatment. Similar results were observed in previous studies [ 25 , 59 , 60 , 61 ]. The combination of EA and inulin resulted in the most pronounced reductions in plasma LDL levels, further supporting the hypothesis of complementarity. Elevated LDL is a major risk factor for cardiovascular diseases, which are commonly associated with NASH. This finding is consistent with the cardioprotective role of EA, as its ability to lower LDL levels could contribute to reducing the overall cardiovascular burden in individuals with metabolic disorders [ 62 , 63 ]. Notably, inulin alone led to elevated plasma lipid levels, which were effectively mitigated by the addition of EA. Elevated levels of ALT and AST, key biomarkers of liver injury, further confirmed hepatic dysfunction in mice with NASH, reflecting the extent of liver damage and inflammation associated with the disease. These markers are widely recognized as indicators of hepatocyte damage, as they are released into circulation when liver cells undergo necrosis or apoptosis [ 64 , 65 , 66 ]. Interestingly, inulin intervention also led to a marked increase in both these biomarkers, suggesting liver damage. However, when combined with EA, these effects were mitigated, demonstrating the therapeutic role of EA. Notably, EA alone demonstrated the most pronounced reduction in ALT and AST levels, highlighting its greater potential in alleviating liver injury in NASH. The beneficial effects of EA are attributed not only to its intact form present in food but also to its microbial metabolites, urolithins [ 20 , 22 , 24 , 25 , 67 ]. EA is known to have poor absorption and undergo extensive metabolism by gut microbiota, resulting in the formation of urolithins, which possess significantly higher bioavailability [ 22 , 24 , 68 , 69 ]. While several studies suggest that urolithins may share metabolic properties with EA, the exact physiological contribution remains challenging due to the complexity of their interactions and metabolism [ 20 , 67 , 70 , 71 ]. Urinary metabolomics revealed that EA treatment, particularly in combination with inulin, significantly increased the levels of EA metabolites and urolithins, indicating enhanced bioavailability and metabolic conversion. The pronounced elevation of urolithins in the combination group suggests that inulin may enhance the microbial metabolism of EA. This finding highlights the potential of dietary fiber to synergize with polyphenols, enhancing their therapeutic efficacy. As a prebiotic, inulin may stimulate gut microbiota activity, particularly the microbial communities involved in the conversion of EA to urolithins, thereby further amplifying its health benefits. While this study demonstrates clear effects of EA and inulin in a NASH model, it did not include direct gut microbiota profiling. Our inferences regarding microbial involvement rely on elevated urinary urolithin levels, which suggest postbiotic production. Previous studies have shown that EA can regulate gut microbiota composition, reducing the relative abundance of pro‐inflammatory taxa such as Faecalibaculum and Ruminococcus , while enriching beneficial genera like Akkermansia . These microbial shifts are associated with improvements in NAFLD/NASH outcomes [ 72 , 73 , 74 , 75 ]. Without direct microbial sequencing in our model, we cannot determine whether these microbial shifts occurred or contributed to the observed effects. Future studies incorporating 16S rRNA or metagenomic sequencing will be important to confirm microbial drivers of urolithin formation, particularly taxa such as Gordonibacter and Ellagibacter , and to clarify whether inulin's adverse effects are linked to SCFA alterations and dysbiosis. In addition, future research should focus on how EA and its metabolites modulate hepatic pathways such as AMPK, nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and sterol regulatory element‐binding protein (SREBP). In conclusion, the present study highlights that EA ameliorates NASH by improving hepatocellular function and enhancing lipid metabolism. The inclusion of inulin may influence these effects by modulating gut microbiota, as suggested by the elevated urinary levels of urolithins. However, inulin alone exhibited adverse effects, which may be attributed to the dysbiotic gut environment associated with NASH. Inulin, as a prebiotic, may further exacerbate this imbalance by amplifying microbial populations that thrive under dysbiosis, thereby complicating its role in NASH management. The findings from this study highlight the potential of nutraceutical interventions to address the growing burden of NASH, with EA emerging as a promising candidate for therapeutic development. While EA significantly improved liver health, the combined treatment primarily neutralized the adverse effects of inulin rather than enhancing the core efficacy of EA. These results underscore the importance of dietary components in modulating treatment outcomes and provide a rationale for integrated dietary and nutraceutical strategies in NASH management. Funding This study was funded by a Higher Degree Research Scholarship of Edith Cowan University, RL, Curtin Medical School, Faculty of Health Sciences, Curtin University, W.A., Australia. LB, School of Medical and Health Science, ECU, WA, Australia; additional financial support was provided to AS by Edith Cowan University through Strategic Research Fellow funding, WA, Australia. Conflicts of Interest The authors declare no conflicts of interest. Supporting information Supporting File : mnfr70456‐sup‐0001‐SuppMat.docx. MNFR-70-e70456-s001.docx (35.1KB, docx) Acknowledgments The authors acknowledge the infrastructure and staff support provided by the Life Science Facility at Curtin University. The authors thank the staff at the Curtin Medical Research Institute, with particular thanks to Mr. Michael Nesbit for his assistance in image analysis. The authors would like to thank the staff at the School of Science, Edith Cowan University, for their invaluable support in conducting this research. Open access publishing facilitated by Edith Cowan University, as part of the Wiley ‐ Edith Cowan University agreement via the Council of Australasian University Librarians Contributor Information Ricky R. Lareu, Email: [email protected]. Lois Balmer, Email: [email protected]. 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