Abstract Plant‐based therapeutics offer a promising strategy for managing liver disorders associated with oxidative stress and inflammation. This study evaluated the dose-dependent hepatoprotective effects of an ethanol extract from Cassia rhombifolia L. fruits (CREE) in a murine model of carbon tetrachloride (CCl₄)-induced liver injury. Mice received CREE orally at 100, 200, or 300 mg/kg once daily for 8 weeks, while hepatotoxicity was induced by intraperitoneal CCl₄ injections twice weekly. Physiological indices, liver function biomarkers [aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin], oxidative stress markers [malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT)], and pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6)] were quantified, and liver histology was assessed. Dose-response relationships were analyzed using regression and correlation models. CREE produced significant, dose-dependent improvements in body weight, liver index, and liver function tests, along with reduced MDA levels and restoration of SOD and CAT activities. Circulating TNF-α, IL-1β, and IL-6 concentrations decreased progressively with increasing CREE dose. Histopathological examination revealed a graded restoration of hepatic architecture, with a near-normal appearance at 300 mg/kg. Acute oral toxicity testing showed no adverse effects at doses up to 3,000 mg/kg, indicating a favorable safety margin. Overall, these findings demonstrate that CREE confers dose-dependent hepatoprotection through coordinated antioxidant, anti-inflammatory, and cytoprotective mechanisms, supporting its potential as a safe, multi-target phytotherapeutic candidate for chemical-induced liver injury.

Keywords: Cassia rhombifolia, Hepatoprotection, Oxidative stress, Inflammatory cytokines, Carbon tetrachloride, Dose-response relationship

Citation:  Tran, T.P.N., Vu, T.H., and Bui, H.Q. 2026. Evaluation of the dose-response relationship of ethanol extract from Cassia rhombifolia L. fruits in a murine model of CCl₄-induced hepatotoxicity. Natural and Life Sciences Communications. 25(2): e2026034.

Graphical Abstract:

INTRODUCTION

Liver injury caused by chemical toxins remains a significant global health challenge, with oxidative stress and inflammation recognized as key pathogenic mechanisms (Garam et al., 2025). Carbon tetrachloride (CCl₄) is widely employed in experimental models of hepatotoxicity due to its capacity to induce centrilobular necrosis via the generation of reactive oxygen species (ROS) and pro-inflammatory cytokines (Saha et al., 2024). While conventional pharmacological therapies are available, their use is often constrained by safety concerns, cost, and limited accessibility, thereby underscoring the need for plant-derived alternatives (Sun et al., 2024).

Natural products rich in bioactive phytochemicals have garnered significant attention for their hepatoprotective properties, primarily attributed to their antioxidant and anti-inflammatory activities (Sebghatollahi et al., 2025). Cassia rhombifolia L., a species traditionally utilized in Vietnamese folk medicine, has demonstrated preliminary antioxidant potential and liver-protective effects (Khanh et al., 2023). Despite these promising indications, the in vivo dose–response relationship and the mechanistic basis of its ethanol extract (CREE) remain largely unexplored. In the development of plant-based therapeutics, establishing both efficacy and safety is critical. As such, a preliminary acute oral toxicity study was also conducted to assess the systemic tolerability of CREE in accordance with international guidelines (OECD, 2022). This step is essential for evaluating the therapeutic margin and supporting the rational use of botanical extracts in preclinical models.

This study, therefore, aimed to investigate the hepatoprotective effects of CREE in a murine model of CCl₄-induced liver injury, with a specific focus on dose-dependent outcomes. By integrating physiological, biochemical, oxidative, and immunological assessments with acute toxicity profiling and histopathological analysis, this work seeks to provide comprehensive insight into the therapeutic potential and underlying mechanisms of CREE-mediated hepatoprotection. Special emphasis was placed on linking biochemical and histological changes to the concept of hepatoprotection.

MATERIALS AND METHODS

Plant material and extract preparation

Ripe fruits of Cassia rhombifolia L. were collected from Mien Dong Farm, Cu Chi District, Ho Chi Minh City, Vietnam (Coordinates: 11°01′40″N, 106°28′59″E). The botanical identity of the plant was authenticated by a taxonomist from the Industrial University of Ho Chi Minh City, and a voucher specimen (code: CR071124VST) was deposited for future reference.

The air-dried fruits were ground into a fine powder and subjected to cold maceration in 95% ethanol for 72 hours with intermittent stirring. The extract was filtered using Whatman No.1 filter paper and concentrated under reduced pressure at 40°C using a rotary evaporator. The extraction procedure was adapted from Lezoul et al. (2020) with minor modifications in solvent strength and maceration time. The resulting crude ethanol extract (referred to as CREE) was stored at 4°C in a sealed container until further use.

Phytochemical screening and quantification

Qualitative phytochemical screening of CREE was conducted to detect the presence of major secondary metabolites, including flavonoids, polyphenols, terpenoids, alkaloids, saponins, steroids, and cardiac glycosides, using standard colorimetric and precipitation methods (Tran et al., 2023a).

Quantitative assays were performed to determine the total content of selected phytochemical groups using spectrophotometric techniques. Total polyphenol content (TPC) was determined by the Folin-Ciocalteu method and expressed as mg gallic acid equivalents per gram of extract (mg GAE/g). Total flavonoid content (TFC) was assessed using the aluminum chloride colorimetric method and expressed as mg quercetin equivalents per gram of extract (mg QE/g). Total terpenoid content (TTC) was calculated based on terpineol equivalence (mg TAE/g), and total alkaloid content (TAC) was estimated using the Harborne method and reported in mg atropine equivalents per gram (mg AE/g) (Tran et al., 2023b). All measurements were performed in triplicate.

HPLC profiling of major phytoconstituents

High-performance liquid chromatography (HPLC) was used to profile and quantify major phenolic and flavonoid constituents in CREE. The analysis was carried out on a Shimadzu HPLC system equipped with a C18 reversed-phase column (250 mm × 4.6 mm, 5 µm). A binary gradient consisting of solvent A (0.1% formic acid in water) and solvent B (acetonitrile) was applied over 35 minutes. Detection wavelengths were set at 280 nm and 360 nm for phenolic acids and flavonoids, respectively. Identification was conducted by comparing the retention times and UV spectra of the sample with those of standard compounds.

Quantitative analysis was performed using external calibration curves of authentic standards (R² > 0.998). Results were expressed as µg/g of dry extract and reported as mean ± standard deviation (SD) from triplicate determinations (Adil et al., 2024).

Experimental animals and ethical approval

Swiss albino mice with an initial body weight of 28-30 g were used in this study and were procured from the Pasteur Institute, Ho Chi Minh City, Vietnam. Animals were housed in standard polypropylene cages under controlled environmental conditions (temperature: 24 ± 2°C, humidity: 55-60%, light/dark cycle: 12 h/12 h). Mice were given free access to a commercial pellet diet and filtered water. A 7-day acclimatization period preceded experimentation.

All procedures involving animals were reviewed and approved by the Animal Ethics Committee (Approval No. 135HD/DHNN), and conducted following the International Guiding Principles for Biomedical Research Involving Animals.

Acute oral toxicity study

A modified acute oral toxicity protocol, adapted from OECD Guideline 423 (OECD, 2022), was employed to assess the maximum tolerated dose of CREE, with an extended dose range up to 3,000 mg/kg. Female Swiss albino mice (n = 6 per group) were fasted overnight and subsequently administered a single oral dose of CREE at 1,000, 2,000, or 3,000 mg/kg body weight. Animals were closely observed for the first 4 hours post-dosing and monitored daily for 14 consecutive days for clinical signs of toxicity, behavioral abnormalities, and mortality. Body weights were recorded on days 0, 7, and 14. At the end of the observation period, all animals were euthanized, and a gross pathological examination of major organs was performed to identify any treatment-related abnormalities (Nhung and Quoc, 2024a).

Experimental design and hepatotoxicity induction

Mice were randomly assigned to six groups (n = 6 per group): Group I (Normal control: distilled water + olive oil, i.p.); Group II (CCl₄ control: distilled water + CCl₄, 0.5 mL/kg, i.p.); Group III (Silymarin: 200 mg/kg/day, p.o., suspended in 0.5% CMC); Groups IV-VI (CREE at 100, 200, and 300 mg/kg/day, p.o.). All treatments lasted for 8 weeks. Except for the normal group, hepatotoxicity was induced by CCl₄ (i.p., twice weekly). Seventy-two hours after the final dose, animals were anesthetized for blood and tissue collection (Nhung and Quoc, 2024b).

Body weight gain and relative liver weight

Body weights were measured weekly using a precision balance. Body weight gain (%) was calculated as the difference between the final and initial weight. After sacrifice, livers were excised and weighed to determine the relative liver weight (RLW), calculated as (liver weight/body weight) × 100 (Kim et al., 2024).

Sample collection and biochemical analyses

Blood was collected via cardiac puncture under anesthesia, and serum was separated by centrifugation for analysis. Liver tissues were homogenized in phosphate-buffered saline for biochemical assays (Bui and Tran, 2025). Serum AST, ALT, ALP, and total bilirubin were quantified using commercial colorimetric assay kits (Sigma-Aldrich, St. Louis, MO, USA; Cat. No. MAK055, MAK052, MAK435, and MAK387, respectively).

Assessment of oxidative stress markers

Oxidative stress was evaluated by quantifying hepatic malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT). Hepatic oxidative stress biomarkers were assessed using commercial assay kits, which are based on the TBARS method for MDA, the nitroblue tetrazolium method for SOD, and H₂O₂ decomposition for CAT (Cayman Chemical, Ann Arbor, MI, USA; MDA: Cat. No. 10009055; SOD: Cat. No. 706002; CAT: Cat. No. 707002). All results were expressed per mg protein (Tran and Tran, 2024).

Evaluation of inflammatory cytokines

Plasma TNF-α, IL-1β, and IL-6 concentrations were determined using mouse ELISA kits (Abcam, Cambridge, UK; TNF-α: Cat. No. ab208348; IL-1β: Cat. No. ab197742; IL-6: Cat. No. ab222503), following the manufacturer’s instructions. Measurements were carried out in triplicate (Xia et al., 2024).

Histopathological examination

Liver tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 µm thickness. Sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope for histological alterations, including necrosis, inflammation, and fatty changes (Nhung and Quoc, 2024a).

Histopathological lesion severity was semi-quantitatively scored using a 0-4 scale adapted from standard hepatic injury scoring systems: 0 = none, 1 = minimal (<10%), 2 = mild (10-30%), 3 = moderate (30-60%), 4 = severe (>60%). Parameters evaluated included hepatocellular necrosis, ballooning degeneration, inflammatory cell infiltration, and sinusoidal/architectural disruption. Scores were obtained independently by two blinded pathologists, and mean ± SD values were used for statistical analysis (Bui and Tran, 2025).

Statistical analysis and dose-response modeling

All data are expressed as mean ± SD. Statistical comparisons were made using one-way ANOVA followed by Tukey’s post hoc test (GraphPad Prism 9.0). Dose-response relationships were assessed using linear and non-linear regression analyses, with Pearson’s r and R² values calculated. Sigmoidal dose-response curves were fitted using the Hill equation to estimate EC₅₀ values where appropriate. Differences were considered significant at P < 0.05.

Before performing ANOVA, all datasets were examined for compliance with statistical assumptions. The Shapiro-Wilk test was used to evaluate normality, and Levene’s test was applied to assess homogeneity of variances. Log-transformation was used as needed when slight deviations from normality were detected. All datasets ultimately met the normality and homogeneity criteria required for one-way ANOVA, validating the use of parametric statistical tests.

RESULTS

All variables met the assumptions of ANOVA (normal distribution and homogeneity of variance), ensuring valid statistical comparisons among groups.

Phytochemical profile of CREE

Qualitative and quantitative phytochemical analysis (Table 1) confirmed the presence of several major secondary metabolites in CREE, particularly polyphenols and flavonoids. These findings were further supported and expanded by HPLC profiling (Figure 1), which identified and quantified specific phenolic and flavonoid constituents (Table 2).

Table 1. The ethanol extract's qualitative and quantitative phytochemical composition from Cassia rhombifolia L. fruits (CREE).

Note: (+): Presence confirmed by qualitative screening.

HPLC results of major phenolic and flavonoid constituents

The most abundant compounds identified were quercetin (1,563.25 ± 24.83 µg/g) and rutin (1,121.47 ± 19.72 µg/g) among the flavonoids, followed by gallic acid (934.28 ± 17.21 µg/g), chlorogenic acid (746.12 ± 15.43 µg/g), and caffeic acid (489.77 ± 11.39 µg/g) among the phenolic acids. Ferulic acid was detected in trace amounts (143.89 ± 6.52 µg/g). (Table 2, Figure 1).

Table 2. Quantitative profile of major phytoconstituents in CREE via HPLC analysis.

Note: Content (µg/g extract, mean ± SD)

Figure 1. Representative HPLC chromatogram of the ethanol extract from Cassia rhombifolia fruit (CREE), showing the main compounds including gallic acid, chlorogenic acid, caffeic acid, ferulic acid, rutin, and quercetin at their respective retention times.

Acute oral toxicity findings

No mortality or clinical signs of toxicity were recorded in any treatment group, even at the highest dose of 3,000 mg/kg. Body weight increased steadily across all groups, and no significant deviations were observed when compared to baseline. Gross pathological examination showed no visible abnormalities in major organs. These findings suggest that CREE possesses a favorable acute safety profile, with an LD₅₀ value exceeding 3,000 mg/kg.

Body weight and relative organ weights

As presented in Table 3 and Figure 2, CREE treatment resulted in dose-dependent improvements in physiological parameters. Both final body weight and percentage weight gain increased progressively with increasing doses of CREE, showing strong positive correlations. In contrast, relative liver weight exhibited a consistent downward trend, reflecting a dose-responsive reduction in liver enlargement associated with CCl₄-induced hepatotoxicity.

Compared with the CCl₄ control group, which exhibited marked suppression of body weight gain and a pronounced increase in relative liver weight, all CREE-treated groups showed significant recovery of these parameters (P < 0.05). At 300 mg/kg, both body weight gain and liver index values approached those of the normal and silymarin-treated groups, indicating a substantial reversal of CCl₄-induced hepatomegaly.

Table 3. Correlation between CREE dose and physiological parameters in mice with CCl₄-induced hepatotoxicity.

Note: ↑↑: Strong positive correlation; ↓↓: Strong negative correlation.

Figure 2. Dose-response effects of ethanol extract from Cassia rhombifolia fruits (CREE) on physiological parameters in mice with CCl₄-induced hepatotoxicity. (A) Linear regression between CREE dose and relative liver weight in CCl₄-induced mice. (B) Bar chart representing dose-dependent changes in body weight gain (%) across CREE-treated groups. Data are presented as mean ± SD.

Serum biochemical markers

Liver function markers were significantly disrupted in the CCl₄ control group, which showed marked elevations of serum AST, ALT, ALP, and total bilirubin compared with the normal control, confirming the successful induction of hepatocellular injury. CREE administration significantly ameliorated these alterations in a dose-dependent manner (Table 4, Figure 3). All three doses of CREE reduced serum AST, ALT, ALP, and total bilirubin levels relative to the CCl₄ group (P < 0.05), with the 300 mg/kg dose yielding values that were close to those observed in the silymarin and normal control groups. Because elevations of these markers are classical indicators of hepatocellular necrosis and cholestasis, their dose-dependent reduction in CREE-treated mice reflects restoration of hepatocellular membrane integrity and biliary function, thereby highlighting the hepatoprotective effect of CREE compared with the CCl₄-only condition.

Table 4. Dose-response relationship between CREE dose and liver function biomarkers.

Note: Mean ± SD (n=6 per dose). Regression, r, R², and P were computed using individual animal data across doses (n=18). P tests whether the slope differs from zero (two-sided). Total bilirubin unit is mg/dL; if µmol/L is required, convert using mg/dL × 17.104.

Figure 3. Dose-response effects of CREE on serum AST and ALT levels in CCl₄-induced hepatotoxicity. (A) Linear regression analysis showing the inverse relationship between CREE dose and serum AST levels in mice with CCl₄-induced hepatotoxicity. (B) Bar chart representing the reduction in serum ALT levels across increasing CREE doses. Data are expressed as mean ± SD. Statistical significance between groups was determined by one-way ANOVA (P < 0.05).

Oxidative stress markers

In the CCl₄ control group, hepatic MDA levels were markedly elevated, whereas SOD and CAT activities were significantly depressed compared with the normal control, indicating severe oxidative stress (Table 5, Figure 4). CREE treatment significantly modulated these oxidative stress markers in a dose-dependent fashion. With increasing doses of CREE, hepatic MDA concentrations declined progressively, while SOD and CAT activities were markedly enhanced.

The dose-dependent reduction in lipid peroxidation, together with the concomitant increase in antioxidant enzyme activities, suggests that CREE strengthens the endogenous antioxidant defense system. This reinforcement of antioxidant capacity limits CCl₄-induced lipid peroxidation and secondary damage to hepatocytes, thereby contributing to the overall hepatoprotective effect of CREE.

Table 5. Correlation between CREE dose and oxidative stress biomarkers, including malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT), in CCl₄-induced hepatotoxicity.

Note: ↑↑: Strong positive correlation; ↓↓: Strong negative correlation.

Figure 4. Dose-dependent effects of CREE on oxidative stress biomarkers in CCl₄-induced hepatotoxicity. (A) Linear regression plot showing the inverse correlation between CREE dose and MDA levels. (B) Bar chart displaying the increase in SOD activity across treatment groups (mean ± SD).

Cytokine levels

As expected, the CCl₄ control group showed markedly elevated circulating levels of TNF-α, IL-1β, and IL-6 compared with the normal control, indicating a robust systemic inflammatory response (Table 6, Figure 5). In terms of inflammatory modulation, CREE elicited a clear dose-dependent suppression of these pro-inflammatory cytokines. All three doses of CREE significantly reduced TNF-α, IL-1β, and IL-6 levels relative to the CCl₄ group, with the 300 mg/kg dose yielding cytokine concentrations that approximated those observed in the silymarin-treated mice. Notably, regression analyses revealed strong inverse correlations between CREE dose and each cytokine marker, supporting a graded pharmacodynamic effect.

The graded suppression of pro-inflammatory cytokines together with the normalization of relative liver weight with increasing CREE doses indicates attenuation of CCl₄-induced hepatic inflammation and swelling. Such dose-dependent anti-inflammatory effects, particularly at 300 mg/kg, are consistent with a protective role of CREE in maintaining liver structure and function in the context of chemically induced hepatic injury.

Table 6. Correlation between CREE dose and inflammatory cytokines (TNF-α, IL-1β, IL-6) in CCl₄-induced hepatotoxicity.

Note: ↑↑: Strong positive correlation; ↓↓: Strong negative correlation.

Figure 5. Dose-response effects of CREE on inflammatory cytokine levels in CCl₄-induced hepatotoxicity. (A) Linear regression analysis between CREE dose and TNF-α concentration, showing a strong inverse relationship. (B) Bar chart displaying IL-1β levels (mean ± SD) across different treatment groups.

Gross and histopathological assessment of hepatic injury

(A) Macroscopic plate (a-f). (a) Normal control: smooth surface and uniform reddish-brown color; (b) CCl₄ group: marked pallor, swelling and surface irregularities; (c) Silymarin (200 mg/kg): improved color and surface features; (d-f) CREE-treated groups at 100, 200, and 300 mg/kg, respectively, showing progressive macroscopic restoration in a dose-dependent manner.

(B) Histopathological plate (a-f), H&E staining, 400×, scale bar = 50 µm. (a) Normal control: preserved lobular architecture with intact central vein (CV) and minimal inflammatory infiltration; (b) CCl₄ group: severe hepatocellular ballooning (→), confluent necrosis and dense inflammatory infiltrates; (c) Silymarin: largely preserved architecture with reduced inflammation; (d-f) CREE-treated groups at 100, 200 and 300 mg/kg, respectively, showing dose-dependent histological improvement with reduced ballooning, necrosis and inflammatory cell infiltration, and progressive restoration of lobular architecture.

Figure 6. Plate of gross and histopathological features of liver tissue in the CCl₄-induced hepatotoxicity model.

Table 7. Semi-quantitative histopathological scores (0-4 scale) in CCl₄-induced mice treated with CREE.

Note: *P < 0.05 vs CCl₄; **P < 0.01; ***P < 0.001

Macroscopic evaluation (Figure 6A) revealed that livers from the normal control group exhibited a smooth surface and uniform reddish-brown coloration, indicative of preserved structural integrity. In contrast, the CCl₄-treated group showed evident hepatomegaly, pallor, and surface irregularities, reflecting severe necrotic and inflammatory injury. CREE treatment resulted in progressive improvement in liver morphology in a dose-dependent manner. Notably, livers from the 300 mg/kg CREE group displayed near-normal gross appearance comparable to the silymarin group, while mild improvements were observed in the 100 and 200 mg/kg groups.

Semi-quantitative scoring confirmed the qualitative histological findings. As shown in Table 7, the CCl₄-only group exhibited severe necrosis, ballooning degeneration, inflammatory infiltration, and architectural collapse (scores 3.5–3.9). CREE treatment reduced all lesion scores in a clear dose-dependent manner (P < 0.05), with the 300 mg/kg dose producing near-normal values comparable to the silymarin group. These quantitative data provide objective confirmation of the histological improvement observed in CREE-treated mice.

Histological analysis (Figure 6B) supported these gross observations. Normal liver architecture, including radiating hepatic cords and central veins, was preserved in the control group. In contrast, the CCl₄ group exhibited marked hepatocellular degeneration, cytoplasmic vacuolization, sinusoidal disruption, and dense inflammatory infiltration. Treatment with CREE dose-dependently ameliorated these histopathological lesions. At 100 mg/kg, mild reductions in ballooning degeneration and inflammatory foci were noted. At 200 mg/kg, architectural restoration became more apparent with reduced necrotic areas. At 300 mg/kg, hepatic structure was substantially restored, with minimal inflammatory infiltration and reappearance of intact central veins, closely resembling the histology observed in the silymarin-treated group.

Together, these gross and microscopic observations indicate that CREE exerts a clear, dose-dependent protective effect on liver structure. The progressive reduction in hepatocellular necrosis, ballooning degeneration, and dense inflammatory infiltrates, along with partial restoration of normal lobular architecture, demonstrates preservation of the liver parenchyma in CREE-treated mice. Notably, the 300 mg/kg dose produced macroscopic and histological features that closely resembled those of the silymarin group, supporting the hepatoprotective potential of CREE in the CCl₄-induced hepatotoxicity model.

Summary

Table 8 illustrates the results of correlation analysis, revealing strong positive associations between the treatment and improvements in physiological parameters and antioxidant enzyme activities. Conversely, strong negative correlations were observed with hepatic injury markers, oxidative stress indicators, and pro-inflammatory cytokines. Collectively, these findings suggest that the treatment confers protective effects by enhancing systemic antioxidant capacity and mitigating inflammatory responses.

Table 8. Summary of correlation coefficients by parameter.

Note: ↑↑: Strong positive correlation; ↓↓: Strong negative correlation.

DISCUSSION

The present study demonstrates that the ethanol extract of Cassia rhombifolia fruits (CREE), which contains polyphenols, flavonoids, terpenoids, alkaloids, and saponins, exerts a clear, dose-dependent hepatoprotective effect in a CCl₄-induced murine model of liver injury (Bui and Tran, 2025). These phytoconstituents are widely recognized for their antioxidative, anti-inflammatory, and cytoprotective properties, all of which are critical in limiting xenobiotic-induced hepatic damage (Rodríguez-Negrete et al., 2024). The consistent dose-response relationships observed across physiological, biochemical, oxidative, and immunological parameters reinforce the pharmacological relevance of CREE as a multi-target hepatoprotective agent (Gutierrez et al., 2024).

The acute oral toxicity evaluation indicated that CREE is practically non-toxic at doses up to 3,000 mg/kg, with no mortality or overt clinical signs, in accordance with OECD classification (OECD, 2022). The absence of gross organ pathology further supports its systemic tolerability (Balkrishna et al., 2022). This favorable safety profile is likely related to its phytochemical composition, particularly flavonoids and terpenoids, which have been associated with membrane-stabilizing, antioxidant, and cytoprotective effects (Abbasi et al., 2024). These compounds are known to modulate detoxification enzymes, reduce the generation of reactive oxygen species, and prevent tissue injury during xenobiotic exposure (Vicidomini et al., 2024). Similar findings have been reported for other Cassia species, such as C. siamea and C. alata, which showed no acute toxicity at high doses in murine models (Tasiam et al., 2020; Edegbo et al., 2023), supporting the notion that Cassia-derived extracts generally possess a low inherent toxicity risk consistent with their traditional medicinal use.

The protective effect of CREE becomes particularly evident when directly comparing the CREE-treated groups with the CCl₄-only control. As expected, CCl₄ administration alone produced marked hepatotoxicity, characterized by reduced body weight gain, a pronounced increase in relative liver weight, sharp elevations of serum AST, ALT, ALP, and total bilirubin, increased hepatic MDA levels, reduced SOD and CAT activities, elevated TNF-α, IL-1β, and IL-6 concentrations, and severe histopathological lesions. In contrast, CREE treatment significantly reversed these alterations in a dose-dependent fashion. Improvements in body weight and reductions in liver index indicate attenuation of hepatic enlargement and systemic metabolic disruption commonly associated with toxin-induced liver injury (Li et al., 2024; Saha et al., 2024). At 300 mg/kg, most physiological and biochemical parameters approached the values observed in normal and silymarin-treated mice, highlighting a substantial functional recovery.

Biochemical analyses further support the hepatoprotective action of CREE. CCl₄ challenge alone caused marked increases in serum AST, ALT, ALP, and total bilirubin, reflecting hepatocellular necrosis and cholestasis. CREE administration significantly ameliorated these changes in a dose-dependent manner, with the highest dose yielding values close to those of the normal and silymarin groups. Because elevations in aminotransferases and bilirubin are classical indicators of hepatocyte membrane disruption and impaired bile excretion, their normalization by CREE indicates stabilization of hepatocellular membranes and restoration of biliary function (Fiorucci et al., 2024). The correlation data suggest that these effects are closely linked to the phytochemical profile of CREE, particularly flavonoids and terpenoids with known membrane-stabilizing and anti-cholestatic properties (Ortiz-Mendoza et al., 2023).

Oxidative stress is a central mechanism in CCl₄-induced hepatotoxicity, primarily driven by reactive metabolites that promote lipid peroxidation and cellular damage. In the present study, CCl₄ markedly elevated hepatic MDA while suppressing SOD and CAT activities, confirming severe oxidative insult. CREE treatment produced a clear, dose-dependent reduction in MDA together with a concomitant increase in SOD and CAT activities. These changes indicate that CREE reinforces the endogenous antioxidant defense system and limits lipid peroxidation. Mechanistically, such effects are consistent with activation of Nrf2-ARE signaling and subsequent upregulation of antioxidant genes, thereby enhancing hepatocyte resilience against oxidative injury through ROS scavenging and enzymatic protection (Saha et al., 2024).

Inflammatory mediators also play a pivotal role in propagating liver damage in the CCl₄ model. Here, the CCl₄-only group showed marked elevations of TNF-α, IL-1β, and IL-6, reflecting a pronounced pro-inflammatory response. CREE significantly and dose-dependently reduced these cytokines, with the 300 mg/kg dose yielding levels comparable to those in the silymarin group. These findings suggest that CREE exerts potent immunomodulatory effects, likely through inhibition of NF-κB and MAPK signaling pathways, which are key drivers of hepatic inflammation (Al-Qahtani et al., 2024), and through attenuation of the IL-6/STAT3 axis implicated in chronic inflammatory damage (Samad et al., 2025). Such anti-inflammatory actions are in line with the established pharmacological properties of plant-derived flavonoids and phenolics (Tangpradubkiat et al., 2024).

Taken together, these mechanistic data indicate that CREE protects the liver primarily through coordinated antioxidant, anti-inflammatory, and membrane-stabilizing pathways. In the canonical CCl₄ model, reactive metabolites trigger excessive ROS production, leading to lipid peroxidation (MDA ↑), disruption of hepatocyte membranes (AST/ALT/ALP ↑), cholestasis (total bilirubin ↑), and amplification of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 ↑), which ultimately drive hepatocellular necrosis and architectural collapse (Garam et al., 2025). In contrast, CREE, rich in polyphenols and flavonoids such as quercetin, rutin, gallic and chlorogenic acids, interrupts this pathological cascade by lowering MDA, restoring SOD and CAT activities, suppressing circulating cytokines and stabilizing hepatocyte membranes, thereby limiting the progression from biochemical injury to overt structural damage (Chaudhary et al., 2025; Noohi et al., 2022). This integrated antioxidant and anti-inflammatory response is consistent with the multi-target hepatoprotective profile observed in the present study.

Histopathological analyses provide anatomical confirmation of these biochemical and molecular findings. The CCl₄-only group exhibited extensive hepatocellular degeneration, ballooning, sinusoidal disruption, confluent necrosis, and dense inflammatory infiltration, together with collapse of normal lobular architecture. In contrast, CREE-treated mice displayed a dose-dependent restoration of hepatic structure: mild improvement at 100 mg/kg, more extensive architectural recovery and reduced necrotic areas at 200 mg/kg, and near-normal histology with minimal inflammatory infiltrates at 300 mg/kg, closely resembling the silymarin-treated group. These observations are consistent with elevated antioxidant enzyme activity and reduced cytokine levels (Hadi and Al-Atrakji, 2025) and are likely mediated by membrane-stabilizing, ROS-scavenging, and signaling-modulating actions of CREE phytoconstituents (Sebghatollahi et al., 2025). Similar histological recovery has been reported for related Cassia species in chemically induced liver injury models (Ding et al., 2023).

Importantly, the dose-dependent changes in these biochemical and histological parameters should not be viewed merely as numerical trends but as convergent evidence of genuine hepatoprotection. As the CREE dose increased from 100 to 300 mg/kg, serum AST, ALT, ALP, and total bilirubin progressively declined toward normal values, hepatic MDA levels were reduced, SOD and CAT activities were enhanced, pro-inflammatory cytokines were suppressed, relative liver weight normalized, and hepatic architecture was largely restored. Taken together, these consistent dose–response patterns across multiple levels of biological organization, physiological, biochemical, oxidative, immunological, and histological, demonstrate that CREE exerts a robust, dose-dependent hepatoprotective effect against CCl₄-induced liver injury, with the 300 mg/kg dose showing the most pronounced benefits.

Future studies should focus on the isolation and characterization of the specific active phytoconstituents responsible for these effects, as well as complementary in vitro and in silico investigations to further elucidate the molecular mechanisms underlying the hepatoprotective action of CREE. In addition, long-term safety evaluations, pharmacokinetic profiling, and assessment of potential synergistic interactions with existing hepatoprotective agents will be essential to support the translational development of CREE as a natural therapeutic option for liver disorders.

CONCLUSION

This study demonstrates that the ethanol extract of Cassia rhombifolia L. fruits (CREE) confers dose-dependent hepatoprotective effects in a CCl₄-induced murine model. CREE administration improved physiological and biochemical parameters, mitigated oxidative and inflammatory responses, and restored hepatic architecture in a histologically confirmed manner. Additionally, CREE exhibited no signs of acute toxicity up to 3,000 mg/kg, indicating a favorable safety profile. These findings highlight the therapeutic potential of CREE as a safe and multi-target phytotherapeutic candidate for managing toxin-induced liver injury.

ACKNOWLEDGEMENTS

The authors sincerely thank the diagnostic centers and hospitals in Ho Chi Minh City, Vietnam, for their kind collaboration and support. Special thanks are extended to the Biomedical Technology Research Group, Industrial University of Ho Chi Minh City, for their technical assistance and valuable contributions to this study.

AUTHOR CONTRIBUTIONS

Thi Phuong Nhung Tran: Conceptualization (Lead), Project Administration (Lead), Supervision (Lead), Experimental Design (Lead), Data Analysis (Lead), Interpretation (Lead), Manuscript (Lead), Writing – Original Draft (Lead), Investigation (Lead), Data Collection (Lead), Critical Revision (Lead), Correspondence (Lead); Hong Quan Bui: Validation (Lead), Data Collection (Supporting), Statistical Analysis (Lead); Thi Hoan Vu: Resources (Lead), Methodology (Lead), Visualization (Lead).

CONFLICT OF INTEREST

The authors declare that they hold no competing interests.

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