Protective Effects of HPTLC-Characterized Prosopis juliflora Hydro-Alcoholic Extract with Quantified Quercetin on High-Fat Diet-Induced Hepatic Injury, Dyslipidemia, and Cardiac Stress in Rats

Protective Effects of HPTLC-Characterized Prosopis juliflora Hydro-Alcoholic Extract with Quantified Quercetin on High-Fat Diet-Induced Hepatic Injury, Dyslipidemia, and Cardiac Stress in Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protective Effects of HPTLC-Characterized Prosopis juliflora Hydro-Alcoholic Extract with Quantified Quercetin on High-Fat Diet-Induced Hepatic Injury, Dyslipidemia, and Cardiac Stress in Rats Anant Kumar Shrivastava, Gautam Saxena, Saurabh Srivastava, Santosh Kumar, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9324986/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background Herbal remedies form a cornerstone of traditional medical systems. Prosopis juliflora is valued in Ayurveda and Traditional Chinese Medicine for its wound-healing, anti-inflammatory, and antioxidant properties. However, its potential role in preventing nonalcoholic fatty liver disease (NAFLD) remains largely unexplored. Purpose This study aims to assess the protective effect of a hydro-alcoholic extract from Prosopis Juliflora aerial part against NAFLD brought on by a high-fat meal. Method Male Wistar albino rats were administered a high-fat diet (HFD) concomitantly with the hydro-alcoholic extract of Prosopis juliflora aerial parts (500 mg/kg) or atorvastatin (30 mg/kg) for 84 days, beginning on Day 1. Normal control animals received standard chow with 1% CMC. At the end of the experimental period, animals were euthanized, and blood and liver tissues were collected for analysis. Serum biochemical markers were quantified, and liver specimens were processed for histopathological evaluation. Result HPTLC analysis revealed a characteristic fingerprint with nine peaks and a quercetin content of 0.27%. HFD feeding significantly increased body weight, liver enzymes, bilirubin, CK-MB, and dyslipidemia (p < 0.001). Treatment with P. juliflora extract significantly attenuated these alterations (p < 0.001), reduced hepatic steatosis and inflammation on histopathology, and showed effects comparable to atorvastatin. Conclusion The HPTLC-characterized P. juliflora hydro-alcoholic extract exerts potent hepatoprotective, hypolipidemic, and cardioprotective effects in HFD-fed rats, supporting its potential as a therapeutic agent for NAFLD. Trial registration number and date of registration – Not applicable Prosopis Juliflora high fat diet (HFD) fatty liver hydro-alcoholic extract nonalcoholic fatty liver disease (NAFLD) atorvastatin (ATO). Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Metabolic dysfunction-associated steatotic liver disease (MASLD) ( 1 ), previously termed NAFLD, is defined by hepatic fat accumulation exceeding 5% in individuals with minimal alcohol intake. It spans a continuum from simple steatosis to steatohepatitis (NASH/MASH), progressive fibrosis, cirrhosis, and ultimately hepatocellular carcinoma ( 2 , 3 ). MASLD has emerged as the most prevalent chronic liver disorder globally, affecting nearly one-third of adults, with substantially higher rates in obesity and type 2 diabetes( 4 , 5 ). These metabolic disturbances accelerate hepatocellular injury, fibrosis progression, and systemic complications, making cardiovascular disease the predominant cause of mortality ( 6 , 7 ). The underlying mechanisms align with the multi-hit hypothesis, involving insulin-resistance–driven lipid overload, increased de novo lipogenesis, mitochondrial dysfunction, oxidative injury, inflammatory signaling, and gut-derived endotoxemia( 8 ) ( 9 ). Excess free fatty acids and triglycerides promote the formation of reactive oxygen species, triggering hepatocellular damage and elevation of key liver function markers such as ALT, AST, ALP, and bilirubin( 10 , 11 ). Dysregulated lipid metabolism contributes to characteristic alterations in the lipid profile, including elevated total cholesterol, LDL, VLDL, triglycerides, and reduced HDL, further exacerbating hepatic steatosis ( 12 – 15 ). Cytokines and adipokines amplify inflammation, while chronic injury activates hepatic stellate cells, leading to extracellular matrix deposition and fibrosis. Disruption of the gut–liver axis intensifies metabolic and inflammatory stress. Additionally, metabolic strain induced by high-fat feeding may elevate cardiac biomarkers such as CK-MB, reflecting systemic oxidative stress and cardiometabolic burden associated with advanced MASLD progression ( 16 ). Lifestyle correction remains the cornerstone of therapy. Sustained weight reduction of 7–10% markedly improves steatosis, inflammation, and even early fibrotic changes. Diets such as the Mediterranean pattern, rich in antioxidants and healthy fats, combined with structured exercise, demonstrate significant metabolic and hepatic benefits. Experimentally, high-fat diet (HFD) models reliably mimic human MASLD by inducing hepatic lipid accumulation, dyslipidemia, oxidative stress, and inflammatory alterations, making them suitable for evaluating hepatoprotective compounds ( 17 ). Herbal interventions are increasingly explored due to their broad pharmacological actions and favorable safety margins. Agents such as silymarin, curcumin, berberine, and EGCG have shown improvements in transaminases, oxidative markers, insulin sensitivity, and hepatic fat content. Nevertheless, variations in formulations, limited trial durations, and inadequate histological data restrict their universal acceptance compared with newer pharmacotherapies like resmetirom or GLP-1 agonists ( 18 ). Prosopis juliflora , rich in flavonoids, phenolics, and alkaloids, exhibits potent antioxidant, anti-inflammatory, and anti-fibrotic effects. Experimental reports indicate that its extract enhances endogenous antioxidants (SOD, catalase), reduces lipid peroxidation, mitigates inflammation, and improves liver enzyme profiles. These actions directly counteract key pathological mechanisms seen in HFD-induced MASLD ( 19 ). Although targeted studies are still limited, available evidence supports its potential as a promising hepatoprotective, anti-steatotic, and anti-fibrotic botanical candidate worthy of further investigation ( 20 ). Materials and methods Materials Atorvastatin (Storvas®, Sun Pharmaceutical Industries Ltd.) was used as the reference standard drug for NAFLD, while Revital H® (Sun Pharmaceutical Industries Ltd.) was included in the high-fat diet formulation. The HFD was designed based on extensive literature to reliably induce NAFLD. Zhang et al. (2024) reported that atorvastatin improves NAFLD-associated hyperlipidemia by reducing hepatic steatosis and regulating lipid metabolism, with clinical evidence also supporting statin efficacy in NAFLD patients. These findings justify the use of atorvastatin as a standard comparator due to its proven lipid-lowering, anti-inflammatory effects, and established safety profile ( 21 ) Composition of high fat diet A semi-purified experimental diet was prepared with the following composition: wheat (10 g), corn flour (10 g), barley flour (10 g), gram flour (10 g), groundnut (3 g), butter (5 g), coconut oil (6 g), cholesterol (2 g), unsaturated fat (15 g), vanillin essence (0.2 g), calcium chloride (1 g), sodium chloride (1 g), cellulose (5 g), starch (6 g), casein (6.8 g), sugar powder (8 g), and One multivitamin tablet was crushed and incorporated into the diet Per 100 g of diet (or per batch). Animals were fed with high fat diet for 84 days to induce NAFLD ( 22 – 24 ) Identification, procurement and authentication of plant The aerial parts of Prosopis Juliflora, Vilayati or Jangli Babool were collected in July 2022 from faizullaganj, ghaila road, Lucknow. Macroscopic examinations of the sample and comprehensive literature research were done in addition to comparison of the sample with actual samples housed at the RHMD (Raw Material Herbarium and Museum, Delhi) were used in the identification process. CSIR-NIScPR's authentication number NIScPR-RHMD/Consult/2022/4173-74-3 was used to authenticate the plant. Experimental animals Wistar albino rats (110–150 g, either sex) were procured from IVRI, Bareilly, and housed in polypropylene cages under controlled conditions (23 ± 2°C, 12-h light/dark cycle, 30–70% humidity) with free access to standard pellet diet and water after one week of acclimatization. All experimental procedures were approved by the IAEC (Approval No. HIPER/IAEC/132/03/2023; dated 28 May 2023). Extraction The aerial parts of Prosopis juliflora were shade-dried, powdered, and exhaustively extracted with hydro-alcohol (ethanol:water, 1:1 v/v) using repeated extraction cycles. The pooled extract was concentrated under reduced pressure at 40–45°C, vacuum-dried, and stored at 4°C in airtight amber containers. For oral dosing, the extract was freshly suspended in 1% carboxymethyl cellulose using magnetic stirring and mild sonication. HPTLC fingerprinting and quantification The hydro-alcoholic extract of Prosopis juliflora used in the in vivo study was analyzed by high-performance thin-layer chromatography (HPTLC) for phytochemical profiling and marker quantification following Shrivastava et al. (2024). The extract was dissolved in methanol (10 mg/mL), filtered, and 10 and 20 µL samples were applied as 6 mm bands on silica gel 60 F₂₅₄ plates (20 × 10 cm) using a CAMAG Linomat 5 applicator. Plates were developed in a pre-saturated twin-trough chamber with toluene: ethyl acetate : formic acid (6 : 4 : 0.3, v/v/v) as the mobile phase, air-dried, and scanned at 254 nm and 366 nm using a CAMAG TLC Scanner 3. Quercetin and gallic acid were used as standards, calibration curves were prepared over 2–10 µg/spot, and marker contents were expressed as percentages of the extract. Animal grouping and model development Rats were treated for 84 days (12 weeks), with the NC group receiving standard chow and the HFD group maintained on a high-fat diet. The STD group received HFD plus atorvastatin (30 mg/kg, p.o.), while the TEST group received HFD plus Prosopis juliflora hydroalcoholic extract (500 mg/kg, p.o., in 1% CMC) administered once daily by gavage.( 25 ). Bodyweight, food intake and water intake Changes in body weight, food intake, and water intake were monitored weekly from Week 1 to Week 12 to assess metabolic status and NAFLD progression. Body weight of individual rats was recorded using an electronic balance, while daily food and water consumption were calculated by subtracting leftovers from the amounts provided over 24 hours. These measurements enabled evaluation of dietary effects and treatment-induced metabolic alterations. ( 26 , 27 ). Blood Collection and Serum Preparation One day before euthanasia, rats were fasted for 24 h and anesthetized on day 84 with phenobarbitone (40 mg/kg, i.p.) for retro-orbital blood collection using non-heparinized tubes. Animals were then euthanized with phenobarbitone overdose (150 mg/kg, i.p.), and serum was obtained by clotting at room temperature followed by centrifugation at 2000 rpm for 15 min. ( 28 ). Assessment of biochemical parameters The separated serum samples were transferred to the biochemistry laboratory for the estimation of lipid profile parameters (total cholesterol, LDL-C, HDL-C, VLDL, and triglycerides) and liver function markers (total, direct, and indirect bilirubin; SGOT; SGPT; alkaline phosphatase; albumin; and globulin) with creatine kinase-MB (CK-MB), was evaluated by using a diagnostic reagent test kit (Roche, Germany) on a Hitachi automatic analyzer 902 (Tokyo, Japan) ( 29 ). Organ Collection and Preservation After euthanasia, the liver was excised through an abdominal incision, rinsed with cold saline to remove blood residues, and immediately fixed in 10% neutral-buffered formalin for 48 hours. The formalin-fixed tissues were subsequently submitted to the pathology laboratory for routine histopathological processing and microscopic evaluation ( 30 ). Liver pathological staining Following model installation and drug administration, the living was separated, washed, preserved in 10% formalin, and dried for paraffin embedding. The tissue was sliced into micrometer slices with a microtome (model TP1020, purchased from Leica), routinely stained with HE, clarified mounted, and examined under a light microscope (model ECLIPSE E 100, purchased from Nikon) for pathological abnormalities in the liver. The pathological severity of fatty lobule development, lobular inflammation, and hepatocyte ballooning was assessed using previously documented NAFLD histopathology ( 31 ). Results The present study evaluated the longitudinal effects of a high-fat diet (HFD) and the therapeutic influence of the standard drug and test formulation on food intake, water intake, and body-weight progression over a 12-week period. Statistical comparisons were made against Week 1 values within each group, and significance patterns (p < 0.05, p < 0.01, p < 0.001) were used to interpret the physiological alterations induced during the study. Phytochemical characterization of P. juliflora extract HPTLC analysis of the hydro-alcoholic extract of Prosopis juliflora revealed a characteristic chromatographic fingerprint with nine distinct peaks at Rf values of 0.016, 0.42, 0.46, 0.63, 0.66, 0.74, 0.86, 0.94, and 0.98 (Table 5 in Shrivastava et al., 2024). Quantification against reference standards showed that the extract contained 0.27% quercetin (peak at Rf 0.47), whereas gallic acid was not detected in this extract. The presence of quercetin and other polyphenolic compounds provides a reproducible chemical profile for the test formulation used in the present study ( 32 ). Food Intake A clear week-wise elevation in food intake was observed across all groups, but the magnitude and timing of significance varied. In the HFD group, food intake became highly significant (p < 0.001) starting from Week 1 itself and remained elevated throughout the study period, indicating early and persistent hyperphagia induced by high-fat feeding. Both the standard and test groups also demonstrated significant increases (mostly p < 0.001) from mid-study onwards, suggesting an adaptive rise in energy intake during treatment. However, across several weeks (e.g., Weeks 2–3), the standard and test groups showed fewer significant deviations compared to HFD, which reflects partial normalization of feeding behavior and possible appetite-modulating effects of the formulations. Table 1 shows weeks evaluation for food intake. Table 1 Weekly changes in water intake (mean ± SEM, n = 6) across experimental groups. Week Control (Mean ± SEM) HFD (Mean ± SEM) Std. (Mean ± SEM) Test (Mean ± SEM) 1 15.5 ± 0.18 12.6 ± 0.18 *** 13.0 ± 0.22 *** 13.9 ± 0.26 * 2 16.1 ± 0.15 ns 15.0 ± 0.20 ns 13.7 ± 0.21 ** 15.1 ± 0.20 ns 3 19.0 ± 0.24 *** 16.9 ± 0.22 ns 16.6 ± 0.31 ns 18.0 ± 0.29 *** 4 20.6 ± 0.29 *** 17.8 ± 0.23 *** 18.9 ± 0.24 *** 19.9 ± 0.25 *** 5 19.5 ± 0.19 *** 18.8 ± 0.23 *** 20.0 ± 0.28 *** 19.5 ± 0.28 *** 6 19.7 ± 0.21 *** 19.6 ± 0.20 *** 20.4 ± 0.27 *** 20.9 ± 0.29 *** 7 20.2 ± 0.20 *** 20.3 ± 0.28 *** 22.2 ± 0.31 *** 22.1 ± 0.28 *** 8 19.8 ± 0.20 *** 20.4 ± 0.23 *** 20.9 ± 0.32 *** 20.7 ± 0.30 *** 9 20.5 ± 0.30 *** 21.1 ± 0.31 *** 21.2 ± 0.29 *** 19.1 ± 0.25 *** 10 21.6 ± 0.30 *** 20.8 ± 0.32 *** 20.6 ± 0.28 *** 21.6 ± 0.24 *** 11 21.4 ± 0.30 *** 20.0 ± 0.34 *** 21.5 ± 0.32 *** 21.9 ± 0.26 *** 12 20.6 ± 0.28 *** 21.5 ± 0.32 *** 20.8 ± 0.26 *** 20.7 ± 0.49 *** Water intake was measured weekly in normal control (NC), high-fat diet (HFD), standard formulation (STND), and test formulation (TEST) groups. Data were analyzed using two-way ANOVA (factors: treatment and time), followed by Bonferroni’s multiple comparisons test . Statistical significance represents comparison with Week 1 values within the same group: p < 0.05, p < 0.01 , p < 0.001 , and ns = not significant. Water Intake Water intake showed significant weekly fluctuations in all groups. The HFD group exhibited consistent and early increases (mostly p < 0.001), consistent with diet-induced metabolic stress and compensatory polydipsia. The standard formulation showed marked increases in several weeks (p < 0.001), but unlike HFD, certain weeks showed nonsignificant changes, indicating more stable hydration patterns. The test formulation followed a similar pattern, with significant increases in multiple weeks but also nonsignificant changes at Weeks 6 and 8. These intermittent nonsignificant intervals suggest that the test formulation may help maintain water balance more effectively than HFD alone. Table 2 shows weeks evaluation for water intake. Table 2 Weekly changes in water intake (mean ± SEM, n = 6) across experimental groups. Week NC (Mean ± SEM) HFD (Mean ± SEM) STND (Mean ± SEM) TEST (Mean ± SEM) 1 22.0 ± 0.25 (ns) 22.8 ± 0.30 (ns) 20.8 ± 0.30 (ns) 22.8 ± 0.30 (ns) 2 23.8 ± 0.30 * 24.8 ± 0.30 *** 23.5 ± 0.42 (ns) 25.8 ± 0.30 *** 3 27.0 ± 0.25 *** 27.8 ± 0.30 *** 28.8 ± 0.30 *** 24.8 ± 0.30 *** 4 27.0 ± 0.36 *** 24.0 ± 0.25 ** 25.8 ± 0.30 *** 27.8 ± 0.30 *** 5 24.8 ± 0.30 *** 26.8 ± 0.30 *** 29.8 ± 0.30 *** 29.8 ± 0.30 *** 6 22.8 ± 0.30 (ns) 29.8 ± 0.30 *** 23.8 ± 0.30 * 25.8 ± 0.30 *** 7 23.8 ± 0.30 * 22.8 ± 0.30 (ns) 27.8 ± 0.30 *** 27.8 ± 0.30 *** 8 27.8 ± 0.30 *** 28.8 ± 0.30 *** 23.6 ± 0.30 (ns) 23.6 ± 0.42 (ns) 9 29.8 ± 0.30 *** 25.8 ± 0.30 *** 23.8 ± 0.30 * 28.8 ± 0.30 *** 10 24.8 ± 0.30 *** 25.8 ± 0.30 *** 27.8 ± 0.30 *** 25.8 ± 0.30 *** 11 26.8 ± 0.30 *** 28.8 ± 0.30 *** 29.8 ± 0.30 *** 23.8 ± 0.30 * 12 28.8 ± 0.30 *** 27.8 ± 0.30 *** 25.8 ± 0.30 *** 27.8 ± 0.30 *** Water intake was measured weekly in normal control (NC), high-fat diet (HFD), standard formulation (STND), and test formulation (TEST) groups. Data were analyzed using two-way ANOVA (factors: treatment and time), followed by Bonferroni’s multiple comparisons test . Statistical significance represents comparison with Week 1 values within the same group: p < 0.05, p < 0.01 , p < 0.001 , and ns = not significant. Body Weight Body weight was the most robustly affected parameter, with highly significant increases (p < 0.001) across nearly all weeks. The HFD group showed a steep and continuous rise from Week 2 onward, confirming the successful induction of obesity. By contrast, the standard group exhibited a more moderated weight gain across weeks, remaining significantly elevated but consistently lower than the HFD group. The test formulation demonstrated a similar pattern of significant weekly gain; however, the rate of increase was slower than HFD but higher than standard treatment. These findings suggest that while both interventions produced measurable therapeutic influence, the standard drug exerted a stronger anti-obesity effect than the test formulation. The test formulation nonetheless attenuated weight progression compared to untreated HFD rats, indicating partial efficacy. Table 3 shows weeks evaluation for body weight. Table 3 Weekly changes in body weight (mean ± SEM, n = 6) across experimental groups. Week NC (Mean ± SEM) HFD (Mean ± SEM) Stand. (Mean ± SEM) Test (Mean ± SEM) 1 126.5 ± 0.31 122.0 ± 0.16 (ns) 116.4 ± 0.71 (ns) 133.5 ± 1.68 (ns) 2 154.5 ± 1.81 *** 157.1 ± 1.60 *** 140.6 ± 1.82 *** 178.1 ± 2.21 *** 3 159.3 ± 2.17 *** 177.7 ± 2.15 *** 172.2 ± 1.79 *** 189.6 ± 2.07 *** 4 170.3 ± 1.80 *** 220.2 ± 1.95 *** 191.6 ± 1.85 *** 173.8 ± 2.06 *** 5 190.0 ± 1.94 *** 235.7 ± 2.04 *** 200.8 ± 1.88 *** 241.3 ± 1.91 *** 6 190.4 ± 1.68 *** 265.6 ± 1.71 *** 221.4 ± 1.66 *** 259.3 ± 2.17 *** 7 195.9 ± 1.95 *** 295.2 ± 2.42 *** 251.2 ± 2.15 *** 269.4 ± 2.43 *** 8 201.6 ± 1.68 *** 318.7 ± 2.02 *** 265.1 ± 2.11 *** 305.4 ± 2.26 *** 9 204.5 ± 2.01 *** 340.9 ± 1.99 *** 270.7 ± 2.08 *** 317.5 ± 2.14 *** 10 213.6 ± 1.87 *** 368.6 ± 1.75 *** 276.5 ± 1.81 *** 327.9 ± 1.93 *** 11 218.8 ± 1.82 *** 396.9 ± 1.90 *** 283.8 ± 1.88 *** 332.6 ± 1.76 *** 12 223.6 ± 1.68 *** 419.6 ± 1.78 *** 289.9 ± 1.85 *** 343.5 ± 1.68 *** Water intake was measured weekly in normal control (NC), high-fat diet (HFD), standard formulation (STND), and test formulation (TEST) groups. Data were analyzed using two-way ANOVA (factors: treatment and time), followed by Bonferroni’s multiple comparisons test . Statistical significance represents comparison with Week 1 values within the same group: p < 0.05, p < 0.01 , p < 0.001 , and ns = not significant. Biochemical parameter investigation The study evaluated the hepatoprotective and hypolipidemic effects of a test formulation in a high-fat diet (HFD)–induced hyperlipidemia model. Biochemical parameters related to liver function and lipid metabolism were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Cardiac stress was evidenced by a significant rise in CK-MB levels in HFD rats (Fig. 1 A; p < 0.0001 vs. NC). Both treatments significantly reduced CK-MB levels, with STD restoring values to near-normal and TEST providing moderate but meaningful cardioprotection. Figure 1 . (A) Creatine Kinase-MB (CK-MB, IU/L). Data are presented as mean; statistical significance is indicated (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: non-significant). The HFD group showed a marked rise in indirect serum bilirubin levels were markedly elevated in HFD rats (Figure. 2A; p < 0.001 vs. NC), reflecting impaired hepatic conjugation. Both treatments significantly reduced bilirubin levels (p < 0.001 vs. HFD), with STD showing near-complete normalization and TEST partial but effective recovery and ALP Fig. 2 B (p < 0.001) versus NC, indicating hepatic injury and cholestatic stress. Both STD and TEST treatments significantly reduced ALP levels (p < 0.001) compared to HFD, confirming hepatoprotective action. A mild difference between NC and TEST (p < 0.05) suggested partial but effective normalization, while STD showed complete recovery with no significant difference from NC. The HFD group showed a marked rise in SGPT Fig. 2 C (p < 0.001) compared to NC, indicating hepatocellular damage from lipid-induced oxidative stress. Both STD and TEST treatments significantly lowered SGPT levels (p 0.05) reflected near-complete enzyme normalization, while slightly elevated levels in the TEST group (p < 0.001) suggested partial yet effective recovery. Figure 3 . Serum biochemical parameters across experimental groups (NC, HFD, STD, TEST). (A) Direct Bilirubin (g/dL). (B) Glutamic Oxaloacetic Transaminase (SGOT, IU/L). (C) Globulin (g/dL). (D) Cholesterol (mg/dL). Data are presented as mean. Statistical significance is indicated (*P < 0.05, **P < 0.01, ***P < 0.001; ns, non-significant). The HFD group showed a significant increase in serum albumin levels (Fig. 2 D; p < 0.001 vs. NC), indicating altered hepatic protein metabolism due to oxidative stress. Both STD and TEST treatments significantly reduced albumin levels compared to HFD (p < 0.001), with STD achieving complete normalization and TEST showing near-normal recovery (p < 0.01 vs. NC). Direct bilirubin was also significantly increased in the HFD group. The HFD group exhibited a significant increase in total serum bilirubin (Fig. 2 E; p < 0.001 vs. NC), indicating hepatic dysfunction both standard (STD) and test (TEST) treatments significantly reduced bilirubin levels compared to HFD (p 0.05 vs. NC), the TEST formulation showed partial but effective recovery (p < 0.05), highlighting its therapeutic potential in maintaining hepatic integrity and lipid homeostasis. (Fig. 3 A; p < 0.001), indicating biliary dysfunction, and was significantly lowered by both treatments. SGOT levels were markedly elevated in HFD rats (Fig. 3 B; p < 0.001), indicating hepatocellular injury; STD restored values close to NC, while TEST produced significant improvement (p < 0.05). Serum globulin levels were also elevated (Fig. 3 C; p < 0.001), with STD fully normalizing and TEST partially restoring these levels. Figure 4 . Serum lipid profile across experimental groups (NC, HFD, STD, TEST). (A) Triglycerides (TGs, mg/dL). (B) High-Density Lipoprotein (HDL, mg/dL). (C) Very Low-Density Lipoprotein (VLDL, mg/dL). Elevated TGs and VLDL in the HFD group indicate dyslipidemia. Data are presented as mean. Statistical significance is indicated (*P < 0.05, **P < 0.01, ***P < 0.001). Serum triglycerides were significantly elevated. The HFD group (Fig. 4 A; p < 0.001 vs. NC), reflecting impaired lipid metabolism and hepatic fat accumulation. Both treatments markedly reduced triglyceride levels (p < 0.001 vs. HFD), with STD achieving near-complete normalization and TEST showing partial restoration. Serum HDL levels were significantly increased in HFD rats (Fig. 4 B; p < 0.001 vs. NC), possibly due to compensatory lipoprotein alterations. Both treatments normalized HDL levels (p < 0.001 vs. HFD), with STD showing complete recovery and TEST exhibiting slight residual elevation (p < 0.05 vs. NC). HFD also caused significant elevations in VLDL (Fig. 4 C) and LDL (Fig. 4 D) levels (p < 0.001 vs. NC), which were effectively reduced by both treatments, with STD showing superior normalization. The HFD group exhibited a marked increase in serum cholesterol (Fig. 3 E; p < 0.001 vs. NC), indicating diet-induced hyperlipidemia and hepatic lipid imbalance both the standard (STD) and test (TEST) treatments significantly reduced cholesterol levels compared to HFD (p 0.05), the TEST group showed slightly elevated values (p < 0.001 vs. NC), indicating partial but effective recovery. Physical examination of organ with histological validation According to standard experimental protocols, organs collected from each experimental group were carefully examined for gross morphological and histopathological alterations. Visual inspection revealed that livers from the disease control (HFD) group exhibited noticeable physical changes when compared with those from the normal control group, indicating diet-induced hepatic damage. In contrast, organs obtained from the treatment groups showed visibly improved morphology, with structural features approaching those of the normal control group. Normal control group Fig. 5 B, Hepatic architecture and lobules were preserved in normal liver slices. In the major vein (d), liver cells formed orderly trabeculae separated by intact hepatic sinusoids. Hepatocytes' big, spherical nuclei (f) and prominent nucleoli (g) showed cellular activity. A intact portal triad—hepatic artery branches (c), arterioles (a), and bile ducts (b) was visible. Overall, liver tissue did not show degeneration or inflammation (magnification40×). HFD Fig. 5 C control liver slices showed significant histological changes. Trabecular and lobular architecture were significantly damaged. Inflammation surrounding the central vein indicated lobular parenchyma fatty degeneration. Many intracytoplasmic fat droplets pushed hepatocyte nuclei to the periphery (j), swelling them. Vacuolized, frothy cytoplasm and compressed nuclear chromatin characterized large hepatocytes. Kupffer cells, erythrocytes, and mononuclear inflammatory cells surrounded sinusoids. Liver parenchyma had inflammatory cells and localized hepatocellular necrosis. PV dilatation, inflammatory infiltration, ductular response, bile duct formation, and portal inflammation occurred. Data shows that a high-fat diet causes considerable liver damage and lobular disruption (magnification 40×). 30 mg/kg ATO + HFD Fig. 5 D, Liver slices from HFD-fed ATO (30 mg/kg) rats showed remarkable hepatic architectural improvements. Hepatoprotection was near normal in lobular, portal, biliary, and fibrotic patterns. Visible hepatic sinusoids regulate intrahepatic blood flow. Vacuolar gaps were sometimes whitish, indicating little lipid. Figure 5 . Representative photomicrographs of liver histopathology (H&E staining, 40× magnification). (A) Liver section from the HFD + PC (500 mg/kg) treatment group, showing partial restoration of hepatic architecture with reduced lipid accumulation and mild inflammation. (B) Normal control group exhibiting preserved hepatic lobules, intact sinusoids, and healthy hepatocytes with prominent nucleoli. (C) HFD control group displaying severe steatosis, trabecular disruption, inflammatory infiltration, and hepatocellular necrosis. (D) HFD + ATO (30 mg/kg) treatment group, showing near-normal hepatic structure with markedly reduced steatosis, intact sinusoids, and visible hepatocyte regeneration. Treatments demonstrate progressive restoration of liver morphology from diet-induced damage. There were no necrotic lesions in moderate, widespread inflammation. Steatosis decreased with rare intracytoplasmic fat droplets. Kupffer cells (l) extended along the sinusoidal lining, phagocytic and reparative. Regenerating hepatocytes (k) in the central vein led to near-normal hepatic architecture (magnification 40×). PC + HFD (500 mg/kg) Fig. 5 A, treated liver sections had some histological improvement over HFD controls. The trabecular pattern was mainly recovered with most hepatic lobules partially veiled and some obscured. Some hepatocytes had empty vacuole-like cytoplasm (n), indicating low lipid accumulation. The sinusoidal walls had numerous Kupffer cells, indicating debris removal. Mild erythrocyte and mononuclear cell infiltration surrounded sinusoids. The medicine reduced fatty globules more than the HFD control group, indicating hepatoprotection and lipid-lowering. Hepatic architecture was more typical (magnification 40×). These histological changes clearly reflected the therapeutic effect of the treatments, highlighting a progressive restoration from the disease state toward normal hepatic structure. Discussion Administration of a high-fat diet (HFD) for 12 weeks successfully induced metabolic stress and obesity in rats, as evidenced by significant increases in food intake, water intake, and body weight compared with baseline and normal control (NC) animals (p < 0.001). Hyperphagia, polydipsia, and progressive weight gain confirmed successful model induction. Co-treatment with atorvastatin (STD, 30 mg/kg) and Prosopis juliflora extract (TEST, 500 mg/kg) significantly attenuated these changes, reducing weight gain and partially normalizing intake patterns, with the STD group showing a slightly stronger anti-obesity effect. HFD feeding caused marked hepatic injury, demonstrated by significant elevations in SGPT, SGOT, ALP, and bilirubin fractions (p < 0.001 vs. NC), along with disrupted protein metabolism and severe dyslipidemia. Both treatments exerted pronounced hepatoprotective effects, with STD achieving near-complete normalization and TEST producing significant improvement across all biochemical parameters. The TEST formulation also showed strong hypolipidemic activity, significantly reducing cholesterol, triglycerides, LDL, and VLDL, with efficacy comparable to atorvastatin. Elevated CK-MB levels in HFD rats indicated cardiac stress, which was significantly attenuated by both treatments. Histopathological analysis corroborated biochemical findings, revealing severe steatosis and lobular disruption in HFD rats, while TEST and STD treatments markedly restored hepatic architecture. Collectively, these results highlight the hepatoprotective, hypolipidemic, and cardioprotective potential of Prosopis juliflora . Future studies should focus on molecular mechanisms, dose optimization, long-term safety, and clinical translation. The HPTLC fingerprint confirmed the presence of quercetin (0.27%) and several other phenolic constituents in the P. juliflora hydro-alcoholic extract. Quercetin is known for its antioxidant, anti-inflammatory, and hepatoprotective properties (Chen et al., 2016; Xu et al., 2019), which likely contribute to the observed attenuation of HFD-induced hepatic injury, dyslipidemia, and cardiac stress. The standardized fingerprint also enhances the reproducibility of the present findings and supports the potential of P. juliflora as a phytotherapeutic agent for NAFLD. Conclusion The present study shows that prolonged high-fat diet feeding induces metabolic dysfunction, hepatic injury, dyslipidemia, and cardiac stress, closely resembling human NAFLD/MASLD. Treatment with Prosopis juliflora hydro-alcoholic extract significantly improved lipid profile, liver enzymes, bilirubin fractions, CK-MB levels, and body-weight regulation. Histopathological findings confirmed marked restoration of hepatic architecture with reduced steatosis and inflammation. Overall, Prosopis juliflora demonstrated comparable multi-target hepatoprotective and hypolipidemic efficacy to atorvastatin, supporting its potential as a therapeutic candidate for diet-induced NAFLD. Abbreviations Abbreviation Full Form ALP Alkaline Phosphatase ALT / SGPT Alanine Aminotransferase / Serum Glutamic Pyruvic Transaminase AST / SGOT Aspartate Aminotransferase / Serum Glutamic Oxaloacetic Transaminase CK-MB Creatine Kinase–Myocardial Band HDL High-Density Lipoprotein HFD High-Fat Diet IU/L International Units per Liter LDL Low-Density Lipoprotein NAFLD Non-Alcoholic Fatty Liver Disease NC Normal Control SEM Standard Error of Mean STD Standard Drug (Atorvastatin) TC Total Cholesterol TEST Test Formulation ( Prosopis juliflora Extract) TG Triglycerides VLDL Very-Low-Density Lipoprotein Declarations There is no funding declaration. Declaration of Competing Interest All the authors declare that they have no competing financial interests or personal relationships that have influenced the work reported in the review. Author Contribution A.K.S & S.S: Writing – review & editing, Conceptualization. G.S. & S.K. Methodology, review & Conceptualization. V.A, S.P,V. & J.K: editing & Software Acknowledgments A.K.S & S.S : Writing – review & editing, Conceptualization. G.S. & S.K. Methodology, review & Conceptualization. V.A, S.P,V. & J.K : editing & Software Data Availability HPTLC data in supplementary file. Declaration of Generative AI and AI-assisted technologies in the writing process The authors declare that no generative AI was used in the preparation of this manuscript. References Huang H tyng, Hewitt M, Li W, Alazawi W. Real-world evidence in metabolic dysfunction-associated steatotic liver disease (MASLD): insights, challenges, and future directions. Lancet Reg Heal - Eur [Internet]. 2026;62:101557. Available from: https://doi.org/10.1016/j.lanepe.2025.101557 Mejía-guzmán JE, Belmont-hernández RA, Chávez-tapia NC, Uribe M. Metabolic-Dysfunction-Associated Steatotic Liver Disease: Molecular Mechanisms, Clinical Implications, and Emerging Therapeutic Strategies. 2025;1–42. Huttasch M, Roden M, Kahl S. 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Preclinical Models for Investigation of Herbal Medicines in Liver Diseases: Update and Perspective. 2016;2016. Shiqin Zhang, Xiaoling Ren, Bingzheng Zhang TL and BL. A Systematic Review of Statins for the Treatment of Nonalcoholic Steatohepatitis: Safety, Efficacy, and Mechanism of Action. 2024; Curfs JHAJ, Chwalibog A, Savenije BS, Ritskes-hoitinga M. Chapter 11 Nutrient Requirements, Experimental Design, and Feeding Schedules in Animal Experimentation. :305–40. Sasidharan SR, Joseph JA, Anandakumar S, Venkatesan V, Nair C, Madhavan A, et al. An Experimental Approach for Selecting Appropriate Rodent Diets for Research Studies on Metabolic Disorders. 2013;2013. Miao J, Guo L, Cui H, Wang L, Zhu B, Lei J, et al. Nonalcoholic Fatty Liver Disease in Rats through Remodeling Gut Microbiota and Regulating the Serum Metabolism. 2022;2022. Morgan HJN, Delfino HBP, Schavinski AZ, Malone SA, Charoy C, Reis NG, et al. Hepatic noradrenergic innervation acts via CREB / CRTC2 to activate gluconeogenesis during cold. Metabolism [Internet]. 2024;157:155940. Available from: https://doi.org/10.1016/j.metabol.2024.155940 Venkatram S, Molugu R, Molugu CR, Molugu HR. Lipid Parameters and Their Association With Liver Function Test Variables in Patients With Type 2 Diabetes Mellitus. 2025;17(9). Gallardo-nuell L, Garre-olmo J, Vicente P, Pamplona R, Puig J, Ramos R, et al. Metformin-induced changes in the gut microbiome and plasma metabolome are associated with cognition in men. 2024;157(February). Kaya E, Lepek J, Assies J. Systemic Complications, and Emerging Role of GLP-1 Receptor Agonists. 2025;3:25–32. Anderson BD, Nelson ED, Joo D, Amiot BP, Katane AA, Mendenhall A, et al. after heterotopic implantation in pigs. 2021; Adokoh CK, Asante D bois, Acheampong DO, Kotsuchibashi Y, Armah FA, Sirikyi IH, et al. Chemical profile and in vivo toxicity evaluation of unripe Citrus aurantifolia essential oil. Toxicol Reports [Internet]. 2019;6(June):692–702. Available from: https://doi.org/10.1016/j.toxrep.2019.06.020 Staufer K, Stauber RE. Steatotic Liver Disease: Metabolic Dysfunction, Alcohol, or Both ? 2023;1–19. Shrivastava AK, Verma S, Awasthi H. HPTLC Phytochemical Profiling and Simultaneous Quantification of Quercetin and Gallic in Prosopis juliflora (Sw.). Res J Pharm Technol 2024;17:3801–6. https://doi.org/10.52711/0974-360X.2024.00590 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 15 May, 2026 Reviewers agreed at journal 14 May, 2026 Reviewers agreed at journal 25 Apr, 2026 Reviewers invited by journal 23 Apr, 2026 Editor assigned by journal 17 Apr, 2026 Submission checks completed at journal 17 Apr, 2026 First submitted to journal 05 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9324986","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633736274,"identity":"f599d2f5-f3fe-4c99-a96d-3f7cfc3b1679","order_by":0,"name":"Anant Kumar Shrivastava","email":"data:image/png;base64,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","orcid":"","institution":"Hygia Institute of pharmaceutical education and research, Lucknow 226020, Uttar Pradesh, India","correspondingAuthor":true,"prefix":"","firstName":"Anant","middleName":"Kumar","lastName":"Shrivastava","suffix":""},{"id":633736275,"identity":"e5e55575-e18b-4d78-8f83-d44814533cde","order_by":1,"name":"Gautam Saxena","email":"","orcid":"","institution":"Amity University","correspondingAuthor":false,"prefix":"","firstName":"Gautam","middleName":"","lastName":"Saxena","suffix":""},{"id":633736276,"identity":"9a9963a1-ce1f-458b-8771-d98b12e60230","order_by":2,"name":"Saurabh Srivastava","email":"","orcid":"","institution":"Hygia Institute of pharmaceutical education and research, Lucknow 226020, Uttar Pradesh, India","correspondingAuthor":false,"prefix":"","firstName":"Saurabh","middleName":"","lastName":"Srivastava","suffix":""},{"id":633736277,"identity":"12c0a69f-80ca-44f6-928b-daec0865f82c","order_by":3,"name":"Santosh Kumar","email":"","orcid":"","institution":"Hygia Institute of pharmaceutical education and research, Lucknow 226020, Uttar Pradesh, India","correspondingAuthor":false,"prefix":"","firstName":"Santosh","middleName":"","lastName":"Kumar","suffix":""},{"id":633736278,"identity":"8caa8916-bfee-4978-bddd-945afa3d1aba","order_by":4,"name":"Vishal Agrahari","email":"","orcid":"","institution":"Prashad institute of Technology Jaunpur, India.","correspondingAuthor":false,"prefix":"","firstName":"Vishal","middleName":"","lastName":"Agrahari","suffix":""},{"id":633736279,"identity":"d1a3cb56-c8fc-46b6-a096-eab521c5a590","order_by":5,"name":"Shweta Pandey","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research, Sarojini Nagar, India.","correspondingAuthor":false,"prefix":"","firstName":"Shweta","middleName":"","lastName":"Pandey","suffix":""},{"id":633736280,"identity":"8e37fc34-cdee-4211-9994-127f7de5401c","order_by":6,"name":"Juhi kumari","email":"","orcid":"","institution":"Apex Professional university pasighat , Gumin nagar, India.","correspondingAuthor":false,"prefix":"","firstName":"Juhi","middleName":"","lastName":"kumari","suffix":""},{"id":633736281,"identity":"18e5306d-8699-4cb4-a8e7-e118c9c5cf66","order_by":7,"name":"Vipin Kumar","email":"","orcid":"","institution":"Central University of South Bihar, Bihar, India.","correspondingAuthor":false,"prefix":"","firstName":"Vipin","middleName":"","lastName":"Kumar","suffix":""}],"badges":[],"createdAt":"2026-04-05 08:38:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9324986/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9324986/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108451058,"identity":"41e224b4-c4de-4ed4-be4d-2b63faea6bd1","added_by":"auto","created_at":"2026-05-04 19:33:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":63403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Creatine Kinase-MB (CK-MB, IU/L). Data are presented as mean; statistical significance is indicated (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, ns: non-significant).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9324986/v1/f82cfb45d033c7367692a76c.jpg"},{"id":108493370,"identity":"c0abc8e4-37e9-4ca4-afd5-4fe353159bae","added_by":"auto","created_at":"2026-05-05 10:00:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":189951,"visible":true,"origin":"","legend":"\u003cp\u003eSerum biochemical parameters across experimental groups (NC, HFD, STD, TEST). \u003cstrong\u003e(A)\u003c/strong\u003e Indirect Bilirubin (mg/dL). \u003cstrong\u003e(B)\u003c/strong\u003e Alkaline Phosphatase (ALP, U/L). \u003cstrong\u003e(C)\u003c/strong\u003e Glutamic Pyruvic Transaminase (SGPT, IU/L). \u003cstrong\u003e(D)\u003c/strong\u003e Albumin (g/dL) \u003cstrong\u003e.\u003c/strong\u003e Serum biomarker levels across experimental groups (NC, HFD, STD, TEST). \u003cstrong\u003e(E)\u003c/strong\u003e Total serum Bilirubin (mg/dL). Data are presented as mean. Statistical significance is indicated by asterisks (*P \u0026lt; 0.05, ***P \u0026lt; 0.001) and \"ns\" for non-significant differences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9324986/v1/733b7234c5cb55e6e7707045.jpg"},{"id":108804281,"identity":"23f02c7b-845d-48dc-9fef-b9f05c00f270","added_by":"auto","created_at":"2026-05-08 15:18:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":115406,"visible":true,"origin":"","legend":"\u003cp\u003eSerum biochemical parameters across experimental groups (NC, HFD, STD, TEST). \u003cstrong\u003e(A)\u003c/strong\u003e Direct Bilirubin (g/dL). \u003cstrong\u003e(B)\u003c/strong\u003e Glutamic Oxaloacetic Transaminase (SGOT, IU/L). \u003cstrong\u003e(C)\u003c/strong\u003e Globulin (g/dL). \u003cstrong\u003e(D)\u003c/strong\u003e Cholesterol (mg/dL). Data are presented as mean. Statistical significance is indicated (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001; ns, non-significant).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9324986/v1/99cf126c2ba6c90f037ea252.jpg"},{"id":108451060,"identity":"9330c24a-b1d1-4817-840f-c74a61a81619","added_by":"auto","created_at":"2026-05-04 19:33:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":183905,"visible":true,"origin":"","legend":"\u003cp\u003eSerum lipid profile across experimental groups (NC, HFD, STD, TEST). \u003cstrong\u003e(A)\u003c/strong\u003e Triglycerides (TGs, mg/dL). \u003cstrong\u003e(B)\u003c/strong\u003e High-Density Lipoprotein (HDL, mg/dL). \u003cstrong\u003e(C)\u003c/strong\u003e Very Low-Density Lipoprotein (VLDL, mg/dL). Elevated TGs and VLDL in the HFD group indicate dyslipidemia. Data are presented as mean. Statistical significance is indicated (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9324986/v1/46ff0ad73c52111dbf3d8bf9.jpg"},{"id":108451061,"identity":"b416281a-7a6f-4828-a707-661f914d6260","added_by":"auto","created_at":"2026-05-04 19:33:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":245215,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative photomicrographs of liver histopathology (H\u0026amp;E staining, 40× magnification). \u003cstrong\u003e(A)\u003c/strong\u003e Liver section from the HFD + PC (500 mg/kg) treatment group, showing partial restoration of hepatic architecture with reduced lipid accumulation and mild inflammation. \u003cstrong\u003e(B)\u003c/strong\u003e Normal control group exhibiting preserved hepatic lobules, intact sinusoids, and healthy hepatocytes with prominent nucleoli. \u003cstrong\u003e(C)\u003c/strong\u003e HFD control group displaying severe steatosis, trabecular disruption, inflammatory infiltration, and hepatocellular necrosis. \u003cstrong\u003e(D)\u003c/strong\u003e HFD + ATO (30 mg/kg) treatment group, showing near-normal hepatic structure with markedly reduced steatosis, intact sinusoids, and visible hepatocyte regeneration. Treatments demonstrate progressive restoration of liver morphology from diet-induced damage.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9324986/v1/0049ea1fa2966bdd3f0fdfc5.jpg"},{"id":108816978,"identity":"c083e6df-f80c-489e-9cc0-93e27f9ce6eb","added_by":"auto","created_at":"2026-05-08 16:26:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1215433,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9324986/v1/7228150b-1008-4377-8179-d825ccbccfa3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Protective Effects of HPTLC-Characterized Prosopis juliflora Hydro-Alcoholic Extract with Quantified Quercetin on High-Fat Diet-Induced Hepatic Injury, Dyslipidemia, and Cardiac Stress in Rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMetabolic dysfunction-associated steatotic liver disease (MASLD) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), previously termed NAFLD, is defined by hepatic fat accumulation exceeding 5% in individuals with minimal alcohol intake. It spans a continuum from simple steatosis to steatohepatitis (NASH/MASH), progressive fibrosis, cirrhosis, and ultimately hepatocellular carcinoma (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). MASLD has emerged as the most prevalent chronic liver disorder globally, affecting nearly one-third of adults, with substantially higher rates in obesity and type 2 diabetes(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). These metabolic disturbances accelerate hepatocellular injury, fibrosis progression, and systemic complications, making cardiovascular disease the predominant cause of mortality (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe underlying mechanisms align with the multi-hit hypothesis, involving insulin-resistance\u0026ndash;driven lipid overload, increased de novo lipogenesis, mitochondrial dysfunction, oxidative injury, inflammatory signaling, and gut-derived endotoxemia(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Excess free fatty acids and triglycerides promote the formation of reactive oxygen species, triggering hepatocellular damage and elevation of key liver function markers such as ALT, AST, ALP, and bilirubin(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Dysregulated lipid metabolism contributes to characteristic alterations in the lipid profile, including elevated total cholesterol, LDL, VLDL, triglycerides, and reduced HDL, further exacerbating hepatic steatosis (\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Cytokines and adipokines amplify inflammation, while chronic injury activates hepatic stellate cells, leading to extracellular matrix deposition and fibrosis. Disruption of the gut\u0026ndash;liver axis intensifies metabolic and inflammatory stress. Additionally, metabolic strain induced by high-fat feeding may elevate cardiac biomarkers such as CK-MB, reflecting systemic oxidative stress and cardiometabolic burden associated with advanced MASLD progression (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLifestyle correction remains the cornerstone of therapy. Sustained weight reduction of 7\u0026ndash;10% markedly improves steatosis, inflammation, and even early fibrotic changes. Diets such as the Mediterranean pattern, rich in antioxidants and healthy fats, combined with structured exercise, demonstrate significant metabolic and hepatic benefits. Experimentally, high-fat diet (HFD) models reliably mimic human MASLD by inducing hepatic lipid accumulation, dyslipidemia, oxidative stress, and inflammatory alterations, making them suitable for evaluating hepatoprotective compounds (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHerbal interventions are increasingly explored due to their broad pharmacological actions and favorable safety margins. Agents such as silymarin, curcumin, berberine, and EGCG have shown improvements in transaminases, oxidative markers, insulin sensitivity, and hepatic fat content. Nevertheless, variations in formulations, limited trial durations, and inadequate histological data restrict their universal acceptance compared with newer pharmacotherapies like resmetirom or GLP-1 agonists (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eProsopis juliflora\u003c/em\u003e, rich in flavonoids, phenolics, and alkaloids, exhibits potent antioxidant, anti-inflammatory, and anti-fibrotic effects. Experimental reports indicate that its extract enhances endogenous antioxidants (SOD, catalase), reduces lipid peroxidation, mitigates inflammation, and improves liver enzyme profiles. These actions directly counteract key pathological mechanisms seen in HFD-induced MASLD (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Although targeted studies are still limited, available evidence supports its potential as a promising hepatoprotective, anti-steatotic, and anti-fibrotic botanical candidate worthy of further investigation (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eAtorvastatin (Storvas\u0026reg;, Sun Pharmaceutical Industries Ltd.) was used as the reference standard drug for NAFLD, while Revital H\u0026reg; (Sun Pharmaceutical Industries Ltd.) was included in the high-fat diet formulation. The HFD was designed based on extensive literature to reliably induce NAFLD. Zhang \u003cem\u003eet al.\u003c/em\u003e (2024) reported that atorvastatin improves NAFLD-associated hyperlipidemia by reducing hepatic steatosis and regulating lipid metabolism, with clinical evidence also supporting statin efficacy in NAFLD patients. These findings justify the use of atorvastatin as a standard comparator due to its proven lipid-lowering, anti-inflammatory effects, and established safety profile (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComposition of high fat diet\u003c/h3\u003e\n\u003cp\u003eA semi-purified experimental diet was prepared with the following composition: wheat (10 g), corn flour (10 g), barley flour (10 g), gram flour (10 g), groundnut (3 g), butter (5 g), coconut oil (6 g), cholesterol (2 g), unsaturated fat (15 g), vanillin essence (0.2 g), calcium chloride (1 g), sodium chloride (1 g), cellulose (5 g), starch (6 g), casein (6.8 g), sugar powder (8 g), and One multivitamin tablet was crushed and incorporated into the diet Per 100 g of diet (or per batch). Animals were fed with high fat diet for 84 days to induce NAFLD (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e)\u003c/p\u003e\n\u003ch3\u003eIdentification, procurement and authentication of plant\u003c/h3\u003e\n\u003cp\u003eThe aerial parts of \u003cem\u003eProsopis Juliflora, Vilayati or Jangli Babool\u003c/em\u003e were collected in July 2022 from faizullaganj, ghaila road, Lucknow. Macroscopic examinations of the sample and comprehensive literature research were done in addition to comparison of the sample with actual samples housed at the RHMD (Raw Material Herbarium and Museum, Delhi) were used in the identification process. CSIR-NIScPR's authentication number NIScPR-RHMD/Consult/2022/4173-74-3 was used to authenticate the plant.\u003c/p\u003e\n\u003ch3\u003eExperimental animals\u003c/h3\u003e\n\u003cp\u003eWistar albino rats (110\u0026ndash;150 g, either sex) were procured from IVRI, Bareilly, and housed in polypropylene cages under controlled conditions (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 12-h light/dark cycle, 30\u0026ndash;70% humidity) with free access to standard pellet diet and water after one week of acclimatization. All experimental procedures were approved by the IAEC (Approval No. HIPER/IAEC/132/03/2023; dated 28 May 2023).\u003c/p\u003e\n\u003ch3\u003eExtraction\u003c/h3\u003e\n\u003cp\u003eThe aerial parts of \u003cem\u003eProsopis juliflora\u003c/em\u003e were shade-dried, powdered, and exhaustively extracted with hydro-alcohol (ethanol:water, 1:1 v/v) using repeated extraction cycles. The pooled extract was concentrated under reduced pressure at 40\u0026ndash;45\u0026deg;C, vacuum-dried, and stored at 4\u0026deg;C in airtight amber containers. For oral dosing, the extract was freshly suspended in 1% carboxymethyl cellulose using magnetic stirring and mild sonication.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHPTLC fingerprinting and quantification\u003c/h2\u003e \u003cp\u003eThe hydro-alcoholic extract of \u003cem\u003eProsopis juliflora\u003c/em\u003e used in the in vivo study was analyzed by high-performance thin-layer chromatography (HPTLC) for phytochemical profiling and marker quantification following Shrivastava et al. (2024). The extract was dissolved in methanol (10 mg/mL), filtered, and 10 and 20 \u0026micro;L samples were applied as 6 mm bands on silica gel 60 F₂₅₄ plates (20 \u0026times; 10 cm) using a CAMAG Linomat 5 applicator. Plates were developed in a pre-saturated twin-trough chamber with toluene: ethyl acetate : formic acid (6 : 4 : 0.3, v/v/v) as the mobile phase, air-dried, and scanned at 254 nm and 366 nm using a CAMAG TLC Scanner 3. Quercetin and gallic acid were used as standards, calibration curves were prepared over 2\u0026ndash;10 \u0026micro;g/spot, and marker contents were expressed as percentages of the extract.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal grouping and model development\u003c/h3\u003e\n\u003cp\u003eRats were treated for 84 days (12 weeks), with the NC group receiving standard chow and the HFD group maintained on a high-fat diet. The STD group received HFD plus atorvastatin (30 mg/kg, p.o.), while the TEST group received HFD plus \u003cem\u003eProsopis juliflora\u003c/em\u003e hydroalcoholic extract (500 mg/kg, p.o., in 1% CMC) administered once daily by gavage.(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eBodyweight, food intake and water intake\u003c/h3\u003e\n\u003cp\u003eChanges in body weight, food intake, and water intake were monitored weekly from Week 1 to Week 12 to assess metabolic status and NAFLD progression. Body weight of individual rats was recorded using an electronic balance, while daily food and water consumption were calculated by subtracting leftovers from the amounts provided over 24 hours. These measurements enabled evaluation of dietary effects and treatment-induced metabolic alterations. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBlood Collection and Serum Preparation\u003c/h2\u003e \u003cp\u003eOne day before euthanasia, rats were fasted for 24 h and anesthetized on day 84 with phenobarbitone (40 mg/kg, i.p.) for retro-orbital blood collection using non-heparinized tubes. Animals were then euthanized with phenobarbitone overdose (150 mg/kg, i.p.), and serum was obtained by clotting at room temperature followed by centrifugation at 2000 rpm for 15 min. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of biochemical parameters\u003c/h2\u003e \u003cp\u003eThe separated serum samples were transferred to the biochemistry laboratory for the estimation of lipid profile parameters (total cholesterol, LDL-C, HDL-C, VLDL, and triglycerides) and liver function markers (total, direct, and indirect bilirubin; SGOT; SGPT; alkaline phosphatase; albumin; and globulin) with creatine kinase-MB (CK-MB), was evaluated by using a diagnostic reagent test kit (Roche, Germany) on a Hitachi automatic analyzer 902 (Tokyo, Japan) (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOrgan Collection and Preservation\u003c/h2\u003e \u003cp\u003eAfter euthanasia, the liver was excised through an abdominal incision, rinsed with cold saline to remove blood residues, and immediately fixed in 10% neutral-buffered formalin for 48 hours. The formalin-fixed tissues were subsequently submitted to the pathology laboratory for routine histopathological processing and microscopic evaluation (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLiver pathological staining\u003c/h2\u003e \u003cp\u003eFollowing model installation and drug administration, the living was separated, washed, preserved in 10% formalin, and dried for paraffin embedding. The tissue was sliced into micrometer slices with a microtome (model TP1020, purchased from Leica), routinely stained with HE, clarified mounted, and examined under a light microscope (model ECLIPSE E 100, purchased from Nikon) for pathological abnormalities in the liver. The pathological severity of fatty lobule development, lobular inflammation, and hepatocyte ballooning was assessed using previously documented NAFLD histopathology (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003cp\u003eThe present study evaluated the longitudinal effects of a high-fat diet (HFD) and the therapeutic influence of the standard drug and test formulation on food intake, water intake, and body-weight progression over a 12-week period. Statistical comparisons were made against Week 1 values within each group, and significance patterns (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were used to interpret the physiological alterations induced during the study.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhytochemical characterization of\u003c/b\u003e \u003cb\u003eP. juliflora\u003c/b\u003e \u003cb\u003eextract\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHPTLC analysis of the hydro-alcoholic extract of \u003cem\u003eProsopis juliflora\u003c/em\u003e revealed a characteristic chromatographic fingerprint with nine distinct peaks at Rf values of 0.016, 0.42, 0.46, 0.63, 0.66, 0.74, 0.86, 0.94, and 0.98 (Table\u0026nbsp;5 in Shrivastava et al., 2024). Quantification against reference standards showed that the extract contained \u003cb\u003e0.27% quercetin\u003c/b\u003e (peak at Rf 0.47), whereas gallic acid was not detected in this extract. The presence of quercetin and other polyphenolic compounds provides a reproducible chemical profile for the test formulation used in the present study (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFood Intake\u003c/h2\u003e \u003cp\u003eA clear week-wise elevation in food intake was observed across all groups, but the magnitude and timing of significance varied. In the HFD group, food intake became highly significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) starting from Week 1 itself and remained elevated throughout the study period, indicating early and persistent hyperphagia induced by high-fat feeding. Both the standard and test groups also demonstrated significant increases (mostly p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) from mid-study onwards, suggesting an adaptive rise in energy intake during treatment. However, across several weeks (e.g., Weeks 2\u0026ndash;3), the standard and test groups showed fewer significant deviations compared to HFD, which reflects partial normalization of feeding behavior and possible appetite-modulating effects of the formulations. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows weeks evaluation for food intake.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eWeekly changes in water intake (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;6) across experimental groups.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeek\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHFD (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStd. (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTest (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 *\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 **\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 ns\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 ns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWater intake was measured weekly in normal control (NC), high-fat diet (HFD), standard formulation (STND), and test formulation (TEST) groups. Data were analyzed using \u003cb\u003etwo-way ANOVA\u003c/b\u003e (factors: treatment and time), followed by \u003cb\u003eBonferroni\u0026rsquo;s multiple comparisons test\u003c/b\u003e. Statistical significance represents comparison with Week 1 values within the same group: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, \u003cb\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/b\u003e, and ns\u0026thinsp;=\u0026thinsp;not significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eWater Intake\u003c/h2\u003e \u003cp\u003eWater intake showed significant weekly fluctuations in all groups. The HFD group exhibited consistent and early increases (mostly p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), consistent with diet-induced metabolic stress and compensatory polydipsia. The standard formulation showed marked increases in several weeks (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but unlike HFD, certain weeks showed nonsignificant changes, indicating more stable hydration patterns. The test formulation followed a similar pattern, with significant increases in multiple weeks but also nonsignificant changes at Weeks 6 and 8. These intermittent nonsignificant intervals suggest that the test formulation may help maintain water balance more effectively than HFD alone. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows weeks evaluation for water intake.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eWeekly changes in water intake (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;6) across experimental groups.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeek\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHFD (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSTND (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTEST (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 (ns)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 (ns)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 (ns)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 (ns)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42 (ns)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 **\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 (ns)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 (ns)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 (ns)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42 (ns)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 *\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWater intake was measured weekly in normal control (NC), high-fat diet (HFD), standard formulation (STND), and test formulation (TEST) groups. Data were analyzed using \u003cb\u003etwo-way ANOVA\u003c/b\u003e (factors: treatment and time), followed by \u003cb\u003eBonferroni\u0026rsquo;s multiple comparisons test\u003c/b\u003e. Statistical significance represents comparison with Week 1 values within the same group: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, \u003cb\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/b\u003e, and ns\u0026thinsp;=\u0026thinsp;not significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBody Weight\u003c/h2\u003e \u003cp\u003eBody weight was the most robustly affected parameter, with highly significant increases (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) across nearly all weeks. The HFD group showed a steep and continuous rise from Week 2 onward, confirming the successful induction of obesity. By contrast, the standard group exhibited a more moderated weight gain across weeks, remaining significantly elevated but consistently lower than the HFD group. The test formulation demonstrated a similar pattern of significant weekly gain; however, the rate of increase was slower than HFD but higher than standard treatment. These findings suggest that while both interventions produced measurable therapeutic influence, the standard drug exerted a stronger anti-obesity effect than the test formulation. The test formulation nonetheless attenuated weight progression compared to untreated HFD rats, indicating partial efficacy. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows weeks evaluation for body weight.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eWeekly changes in body weight (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;6) across experimental groups.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeek\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHFD (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStand. (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTest (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e126.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e122.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 (ns)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e116.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71 (ns)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e133.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68 (ns)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e154.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e157.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e140.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e178.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e159.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e177.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e172.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e189.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e170.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e220.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e191.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e173.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e190.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e235.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e200.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e241.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e190.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e265.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e221.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e259.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e195.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e295.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.42 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e251.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e269.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e201.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e318.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e265.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e305.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e204.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e340.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e270.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e317.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e213.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e368.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e276.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e327.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e218.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e396.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e283.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e332.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e223.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e419.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e289.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e343.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWater intake was measured weekly in normal control (NC), high-fat diet (HFD), standard formulation (STND), and test formulation (TEST) groups. Data were analyzed using \u003cb\u003etwo-way ANOVA\u003c/b\u003e (factors: treatment and time), followed by \u003cb\u003eBonferroni\u0026rsquo;s multiple comparisons test\u003c/b\u003e. Statistical significance represents comparison with Week 1 values within the same group: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, \u003cb\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/b\u003e, and ns\u0026thinsp;=\u0026thinsp;not significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical parameter investigation\u003c/h2\u003e \u003cp\u003eThe study evaluated the hepatoprotective and hypolipidemic effects of a test formulation in a high-fat diet (HFD)\u0026ndash;induced hyperlipidemia model. Biochemical parameters related to liver function and lipid metabolism were analyzed using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. Cardiac stress was evidenced by a significant rise in\u003c/p\u003e \u003cp\u003eCK-MB levels in HFD rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 vs. NC). Both treatments significantly reduced CK-MB levels, with STD restoring values to near-normal and TEST providing moderate but meaningful cardioprotection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cb\u003e(A)\u003c/b\u003e Creatine Kinase-MB (CK-MB, IU/L). Data are presented as mean; statistical significance is indicated (*P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, ns: non-significant).\u003c/p\u003e \u003cp\u003eThe HFD group showed a marked rise in indirect serum bilirubin levels were markedly elevated in HFD rats (Figure. 2A; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. NC), reflecting impaired hepatic conjugation. Both treatments significantly reduced bilirubin levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. HFD), with STD showing near-complete normalization and TEST partial but effective recovery and ALP Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) versus NC, indicating hepatic injury and cholestatic stress. Both STD and TEST treatments significantly reduced ALP levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to HFD, confirming hepatoprotective action. A mild difference between NC and TEST (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) suggested partial but effective normalization, while STD showed complete recovery with no significant difference from NC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe HFD group showed a marked rise in SGPT Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to NC, indicating hepatocellular damage from lipid-induced oxidative stress. Both STD and TEST treatments significantly lowered SGPT levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) versus HFD, confirming strong hepatoprotective effects. No significant difference between NC and STD (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) reflected near-complete enzyme normalization, while slightly elevated levels in the TEST group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) suggested partial yet effective recovery.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Serum biochemical parameters across experimental groups (NC, HFD, STD, TEST). \u003cb\u003e(A)\u003c/b\u003e Direct Bilirubin (g/dL). \u003cb\u003e(B)\u003c/b\u003e Glutamic Oxaloacetic Transaminase (SGOT, IU/L). \u003cb\u003e(C)\u003c/b\u003e Globulin (g/dL). \u003cb\u003e(D)\u003c/b\u003e Cholesterol (mg/dL). Data are presented as mean. Statistical significance is indicated (*P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ns, non-significant).\u003c/p\u003e \u003cp\u003eThe HFD group showed a significant increase in serum albumin levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. NC), indicating altered hepatic protein metabolism due to oxidative stress. Both STD and TEST treatments significantly reduced albumin levels compared to HFD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with STD achieving complete normalization and TEST showing near-normal recovery (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. NC). Direct bilirubin was also significantly increased in the HFD group. The HFD group exhibited a significant increase in total serum bilirubin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. NC), indicating hepatic dysfunction both standard (STD) and test (TEST) treatments significantly reduced bilirubin levels compared to HFD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), demonstrating hepatoprotective effects. While STD restored values to near-normal levels (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 vs. NC), the TEST formulation showed partial but effective recovery (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), highlighting its therapeutic potential in maintaining hepatic integrity and lipid homeostasis. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating biliary dysfunction, and was significantly lowered by both treatments. SGOT levels were markedly elevated in HFD rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating hepatocellular injury; STD restored values close to NC, while TEST produced significant improvement (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Serum globulin levels were also elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with STD fully normalizing and TEST partially restoring these levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Serum lipid profile across experimental groups (NC, HFD, STD, TEST). \u003cb\u003e(A)\u003c/b\u003e Triglycerides (TGs, mg/dL). \u003cb\u003e(B)\u003c/b\u003e High-Density Lipoprotein (HDL, mg/dL). \u003cb\u003e(C)\u003c/b\u003e Very Low-Density Lipoprotein (VLDL, mg/dL). Elevated TGs and VLDL in the HFD group indicate dyslipidemia. Data are presented as mean. Statistical significance is indicated (*P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eSerum triglycerides were significantly elevated. The HFD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. NC), reflecting impaired lipid metabolism and hepatic fat accumulation. Both treatments markedly reduced triglyceride levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. HFD), with STD achieving near-complete normalization and TEST showing partial restoration. Serum HDL levels were significantly increased in HFD rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. NC), possibly due to compensatory lipoprotein alterations. Both treatments normalized HDL levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. HFD), with STD showing complete recovery and TEST exhibiting slight residual elevation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. NC). HFD also caused significant elevations in VLDL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and LDL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. NC), which were effectively reduced by both treatments, with STD showing superior normalization. The HFD group exhibited a marked increase in serum cholesterol (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. NC), indicating diet-induced hyperlipidemia and hepatic lipid imbalance both the standard (STD) and test (TEST) treatments significantly reduced cholesterol levels compared to HFD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). While STD restored cholesterol to near-normal levels with no significant difference from NC (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), the TEST group showed slightly elevated values (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. NC), indicating partial but effective recovery.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePhysical examination of organ with histological validation\u003c/h2\u003e \u003cp\u003eAccording to standard experimental protocols, organs collected from each experimental group were carefully examined for gross morphological and histopathological alterations. Visual inspection revealed that livers from the disease control (HFD) group exhibited noticeable physical changes when compared with those from the normal control group, indicating diet-induced hepatic damage. In contrast, organs obtained from the treatment groups showed visibly improved morphology, with structural features approaching those of the normal control group.\u003c/p\u003e \u003cp\u003eNormal control group Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, Hepatic architecture and lobules were preserved in normal liver slices. In the major vein (d), liver cells formed orderly trabeculae separated by intact hepatic sinusoids. Hepatocytes' big, spherical nuclei (f) and prominent nucleoli (g) showed cellular activity. A intact portal triad\u0026mdash;hepatic artery branches (c), arterioles (a), and bile ducts (b) was visible. Overall, liver tissue did not show degeneration or inflammation (magnification40\u0026times;).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHFD Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC control liver slices showed significant histological changes. Trabecular and lobular architecture were significantly damaged. Inflammation surrounding the central vein indicated lobular parenchyma fatty degeneration. Many intracytoplasmic fat droplets pushed hepatocyte nuclei to the periphery (j), swelling them. Vacuolized, frothy cytoplasm and compressed nuclear chromatin characterized large hepatocytes. Kupffer cells, erythrocytes, and mononuclear inflammatory cells surrounded sinusoids. Liver parenchyma had inflammatory cells and localized hepatocellular necrosis. PV dilatation, inflammatory infiltration, ductular response, bile duct formation, and portal inflammation occurred. Data shows that a high-fat diet causes considerable liver damage and lobular disruption (magnification 40\u0026times;).\u003c/p\u003e \u003cp\u003e30 mg/kg ATO\u0026thinsp;+\u0026thinsp;HFD Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, Liver slices from HFD-fed ATO (30 mg/kg) rats showed remarkable hepatic architectural improvements. Hepatoprotection was near normal in lobular, portal, biliary, and fibrotic patterns. Visible hepatic sinusoids regulate intrahepatic blood flow. Vacuolar gaps were sometimes whitish, indicating little lipid.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Representative photomicrographs of liver histopathology (H\u0026amp;E staining, 40\u0026times; magnification). \u003cb\u003e(A)\u003c/b\u003e Liver section from the HFD\u0026thinsp;+\u0026thinsp;PC (500 mg/kg) treatment group, showing partial restoration of hepatic architecture with reduced lipid accumulation and mild inflammation. \u003cb\u003e(B)\u003c/b\u003e Normal control group exhibiting preserved hepatic lobules, intact sinusoids, and healthy hepatocytes with prominent nucleoli. \u003cb\u003e(C)\u003c/b\u003e HFD control group displaying severe steatosis, trabecular disruption, inflammatory infiltration, and hepatocellular necrosis. \u003cb\u003e(D)\u003c/b\u003e HFD\u0026thinsp;+\u0026thinsp;ATO (30 mg/kg) treatment group, showing near-normal hepatic structure with markedly reduced steatosis, intact sinusoids, and visible hepatocyte regeneration. Treatments demonstrate progressive restoration of liver morphology from diet-induced damage.\u003c/p\u003e \u003cp\u003eThere were no necrotic lesions in moderate, widespread inflammation. Steatosis decreased with rare intracytoplasmic fat droplets. Kupffer cells (l) extended along the sinusoidal lining, phagocytic and reparative. Regenerating hepatocytes (k) in the central vein led to near-normal hepatic architecture (magnification 40\u0026times;).\u003c/p\u003e \u003cp\u003ePC\u0026thinsp;+\u0026thinsp;HFD (500 mg/kg) Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, treated liver sections had some histological improvement over HFD controls. The trabecular pattern was mainly recovered with most hepatic lobules partially veiled and some obscured. Some hepatocytes had empty vacuole-like cytoplasm (n), indicating low lipid accumulation. The sinusoidal walls had numerous Kupffer cells, indicating debris removal. Mild erythrocyte and mononuclear cell infiltration surrounded sinusoids. The medicine reduced fatty globules more than the HFD control group, indicating hepatoprotection and lipid-lowering. Hepatic architecture was more typical (magnification 40\u0026times;).\u003c/p\u003e \u003cp\u003eThese histological changes clearly reflected the therapeutic effect of the treatments, highlighting a progressive restoration from the disease state toward normal hepatic structure.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAdministration of a high-fat diet (HFD) for 12 weeks successfully induced metabolic stress and obesity in rats, as evidenced by significant increases in food intake, water intake, and body weight compared with baseline and normal control (NC) animals (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Hyperphagia, polydipsia, and progressive weight gain confirmed successful model induction. Co-treatment with atorvastatin (STD, 30 mg/kg) and \u003cem\u003eProsopis juliflora\u003c/em\u003e extract (TEST, 500 mg/kg) significantly attenuated these changes, reducing weight gain and partially normalizing intake patterns, with the STD group showing a slightly stronger anti-obesity effect.\u003c/p\u003e \u003cp\u003eHFD feeding caused marked hepatic injury, demonstrated by significant elevations in SGPT, SGOT, ALP, and bilirubin fractions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. NC), along with disrupted protein metabolism and severe dyslipidemia. Both treatments exerted pronounced hepatoprotective effects, with STD achieving near-complete normalization and TEST producing significant improvement across all biochemical parameters. The TEST formulation also showed strong hypolipidemic activity, significantly reducing cholesterol, triglycerides, LDL, and VLDL, with efficacy comparable to atorvastatin. Elevated CK-MB levels in HFD rats indicated cardiac stress, which was significantly attenuated by both treatments.\u003c/p\u003e \u003cp\u003eHistopathological analysis corroborated biochemical findings, revealing severe steatosis and lobular disruption in HFD rats, while TEST and STD treatments markedly restored hepatic architecture. Collectively, these results highlight the hepatoprotective, hypolipidemic, and cardioprotective potential of \u003cem\u003eProsopis juliflora\u003c/em\u003e. Future studies should focus on molecular mechanisms, dose optimization, long-term safety, and clinical translation.\u003c/p\u003e \u003cp\u003eThe HPTLC fingerprint confirmed the presence of quercetin (0.27%) and several other phenolic constituents in the \u003cem\u003eP. juliflora\u003c/em\u003e hydro-alcoholic extract. Quercetin is known for its antioxidant, anti-inflammatory, and hepatoprotective properties (Chen et al., 2016; Xu et al., 2019), which likely contribute to the observed attenuation of HFD-induced hepatic injury, dyslipidemia, and cardiac stress. The standardized fingerprint also enhances the reproducibility of the present findings and supports the potential of \u003cem\u003eP. juliflora\u003c/em\u003e as a phytotherapeutic agent for NAFLD.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study shows that prolonged high-fat diet feeding induces metabolic dysfunction, hepatic injury, dyslipidemia, and cardiac stress, closely resembling human NAFLD/MASLD. Treatment with \u003cem\u003eProsopis juliflora\u003c/em\u003e hydro-alcoholic extract significantly improved lipid profile, liver enzymes, bilirubin fractions, CK-MB levels, and body-weight regulation. Histopathological findings confirmed marked restoration of hepatic architecture with reduced steatosis and inflammation. Overall, \u003cem\u003eProsopis juliflora\u003c/em\u003e demonstrated comparable multi-target hepatoprotective and hypolipidemic efficacy to atorvastatin, supporting its potential as a therapeutic candidate for diet-induced NAFLD.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbbreviation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFull Form\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eALP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAlkaline Phosphatase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eALT / SGPT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAlanine Aminotransferase / Serum Glutamic Pyruvic Transaminase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAST / SGOT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAspartate Aminotransferase / Serum Glutamic Oxaloacetic Transaminase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCK-MB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCreatine Kinase–Myocardial Band\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHDL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHigh-Density Lipoprotein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHFD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHigh-Fat Diet\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIU/L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInternational Units per Liter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLDL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLow-Density Lipoprotein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNAFLD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNon-Alcoholic Fatty Liver Disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNormal Control\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eStandard Error of Mean\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSTD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eStandard Drug (Atorvastatin)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTotal Cholesterol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTEST\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTest Formulation (\u003cem\u003eProsopis juliflora\u003c/em\u003e Extract)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTriglycerides\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eVLDL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVery-Low-Density Lipoprotein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003eThere is no funding declaration.\u003c/p\u003e\n\u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e\n\u003cp\u003eAll the authors declare that they have no competing financial interests or personal relationships that have influenced the work reported in the review.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eA.K.S \u0026amp; S.S: Writing – review \u0026amp; editing, Conceptualization. G.S. \u0026amp; S.K. Methodology, review \u0026amp; Conceptualization. V.A, S.P,V. \u0026amp; J.K: editing \u0026amp; Software\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eA.K.S \u0026amp; S.S\u003c/strong\u003e: Writing – review \u0026amp; editing, Conceptualization. \u003cstrong\u003eG.S. \u0026amp; S.K.\u003c/strong\u003e Methodology, review \u0026amp; Conceptualization. \u003cstrong\u003eV.A, S.P,V. \u0026amp; J.K\u003c/strong\u003e: editing \u0026amp; Software\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eHPTLC data in supplementary file.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Generative AI and AI-assisted technologies in the writing process\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no generative AI was used in the preparation of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHuang H tyng, Hewitt M, Li W, Alazawi W. 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Res J Pharm Technol 2024;17:3801\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.52711/0974-360X.2024.00590\u003c/span\u003e\u003cspan address=\"10.52711/0974-360X.2024.00590\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"comparative-clinical-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Comparative Clinical Pathology](https://link.springer.com/journal/580)","snPcode":"580","submissionUrl":"https://submission.springernature.com/new-submission/580/3","title":"Comparative Clinical Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Prosopis Juliflora, high fat diet (HFD), fatty liver, hydro-alcoholic extract, nonalcoholic fatty liver disease (NAFLD), atorvastatin (ATO).","lastPublishedDoi":"10.21203/rs.3.rs-9324986/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9324986/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHerbal remedies form a cornerstone of traditional medical systems. \u003cem\u003eProsopis juliflora\u003c/em\u003e is valued in Ayurveda and Traditional Chinese Medicine for its wound-healing, anti-inflammatory, and antioxidant properties. However, its potential role in preventing nonalcoholic fatty liver disease (NAFLD) remains largely unexplored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study aims to assess the protective effect of a hydro-alcoholic extract from \u003cem\u003eProsopis Juliflora\u003c/em\u003e aerial part against NAFLD brought on by a high-fat meal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale Wistar albino rats were administered a high-fat diet (HFD) concomitantly with the hydro-alcoholic extract of \u003cem\u003eProsopis juliflora\u003c/em\u003e aerial parts (500 mg/kg) or atorvastatin (30 mg/kg) for 84 days, beginning on Day 1. Normal control animals received standard chow with 1% CMC. At the end of the experimental period, animals were euthanized, and blood and liver tissues were collected for analysis. Serum biochemical markers were quantified, and liver specimens were processed for histopathological evaluation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHPTLC analysis revealed a characteristic fingerprint with nine peaks and a quercetin content of 0.27%. HFD feeding significantly increased body weight, liver enzymes, bilirubin, CK-MB, and dyslipidemia (p \u0026lt; 0.001). Treatment with \u003cem\u003eP. juliflora\u003c/em\u003e extract significantly attenuated these alterations (p \u0026lt; 0.001), reduced hepatic steatosis and inflammation on histopathology, and showed effects comparable to atorvastatin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HPTLC-characterized \u003cem\u003eP. juliflora\u003c/em\u003e hydro-alcoholic extract exerts potent hepatoprotective, hypolipidemic, and cardioprotective effects in HFD-fed rats, supporting its potential as a therapeutic agent for NAFLD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial registration number and date of registration – Not applicable\u003c/strong\u003e\u003c/p\u003e","manuscriptTitle":"Protective Effects of HPTLC-Characterized Prosopis juliflora Hydro-Alcoholic Extract with Quantified Quercetin on High-Fat Diet-Induced Hepatic Injury, Dyslipidemia, and Cardiac Stress in Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 19:33:06","doi":"10.21203/rs.3.rs-9324986/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-15T04:38:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"237607257532890825633908045614500112240","date":"2026-05-14T15:51:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289702839209191450106806147747954482475","date":"2026-04-26T03:28:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-23T22:02:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-17T05:25:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-17T05:24:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Comparative Clinical Pathology","date":"2026-04-05T08:32:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"comparative-clinical-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Comparative Clinical Pathology](https://link.springer.com/journal/580)","snPcode":"580","submissionUrl":"https://submission.springernature.com/new-submission/580/3","title":"Comparative Clinical Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0ae9b4b7-5f62-45c3-aa51-c9b78efd9471","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-15T04:38:09+00:00","index":49,"fulltext":""},{"type":"reviewerAgreed","content":"237607257532890825633908045614500112240","date":"2026-05-14T15:51:23+00:00","index":48,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T19:33:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 19:33:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9324986","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9324986","identity":"rs-9324986","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}