As a major metabolic organ, the liver plays pivotal roles in detoxification, protein synthesis, and the regulation of glucose levels [1]. However, although the liver has unique regenerative capacities, the excessive consumption of alcohol or drugs and ingestion of other environmental xenobiotics can potentially cause severe hepatic damage, including hepatic fibrosis, cirrhosis, and hepatocellular carcinoma [2, 3]. Oxidative stress and inflammation have been established to be the primary factors contributing to the induction and propagation of liver injury, and thus suppressing these events may provide the key to controlling and mitigating their damaging effects [4, 5, 6].

A mounting body of evidence indicates that different types of liver injury can be prevented using antioxidant-rich natural products, owing to their capacities to eliminate oxidative stress and hence suppress inflammatory responses [7, 8, 9]. In recent years, a growing number of studies have specifically demonstrated the hepatoprotective potential of plant-derived oils, phenolic extracts, and flavonoid-rich fractions in experimental models of liver toxicity [10, 11]. These phytoingredients have been shown to attenuate CCl₄-induced hepatotoxicity through multiple mechanisms, including activation of the Nrf2/Keap1 antioxidant signaling pathway, suppression of NF-$\kappa$B-mediated pro-inflammatory cytokine production, and inhibition of lipid peroxidation [12, 13]. Furthermore, comparative investigations have established that several plant-derived compounds confer hepatoprotection comparable to that of silymarin, the clinical reference standard, supporting their potential as accessible and well-tolerated alternatives [14, 15]. Ajwa (Phoenix dactylifera) seed oil (ASO) has emerged as a particularly promising therapeutic candidate. ASO contains notable amounts of phenolic compounds, flavonoids, and essential fatty acids, which contribute to combating oxidative stress, cytokine production, and an imbalance in hepatic enzymes [10, 16]. The oil has been shown to reduce lipid peroxidation, promote antioxidant enzyme activity, and restore histopathological features of the liver following petrochemical-induced liver damage [17].

Carbon tetrachloride (CCl₄) is often used experimentally to induce hepatic injury in animals for evaluation of the hepatoprotective properties of drugs of interest. The toxicity induced by CCl₄ is associated with the generation of trichloromethyl free radicals, which contribute to oxidative damage, lipid peroxidation, and death of hepatic cells [18, 19]. In this study, we assessed the capacity ASO to confer protection against CCl₄-induced acute liver damage, with the efficacy being compared with that of silymarin, a well-established hepatoprotective drug [20]. Collectively, the findings of this study will contribute to enhancing our understanding of the properties of ASO as a source of antioxidants in managing liver injury and thereby augment the growing body of evidence on the potential use of natural products as hepatoprotective agents.

The liver, as a key metabolic organ, is continuously exposed to multiple chemicals and is consequently particularly prone to oxidative stress. In this regard, CCl₄, a well-established hepatotoxic agent, is widely used in experimental models to induce liver damage and fibrosis [18, 19]. Although the flesh and seeds of Ajwa dates have been demonstrated to have antioxidant and anti-inflammatory properties [10, 16], the hepatoprotective effects of ASO against CCl₄-induced liver damage has yet to be assessed. In this study, we accordingly sought to determine the protective effects of ASO against CCl₄-induced hepatotoxicity in mice using biochemical and histological techniques.

CCl₄-treated mice were observed to have sustained substantial liver damage, characterized by elevated levels of serum parameters such as ALT, AST, LDH, cholesterol, and triglyceride, as well as reductions in albumin levels, which are features indicative of hepatocellular damage and compromised liver functions. CCl₄ also perturbed hepatic oxidative status, depleting total antioxidant capacity and inducing lipid peroxidation, thereby contributing to heightened levels of oxidative stress. Moreover, histological analyses revealed structural damage to liver tissues, characterized by cellular degeneration, elevated levels of collagen deposition, and a depletion of glycogen, changes that are consistent with liver injury and early fibrosis and are frequently seen in the study of hepatotoxicity [5, 18].

The administration of ASO was found to ameliorate CCl₄-induced liver damage at both the biochemical and histological levels, contributing to a normalization of the serum concentrations of ALT, AST, LDH, lipids, and albumin, whilst restoring hepatic antioxidant capacity. Similarly, histological evaluations of liver tissues obtained from ASO-treated mice revealed reductions in CCl₄-induced hepatocellular injury and fibrosis [22]. Furthermore, PAS staining revealed that mice receiving ASO were characterized by a well-preserved hepatic architecture with reductions in the depletion of glycogen, thereby providing evidence of the hepatoprotective efficacy of this oil.

To a large extent, the observed hepatoprotective effects of ASO can be attributed to its antioxidant properties. In this regard, previous studies have reported that the antioxidant activities of ASO are associated with its phenolic and flavonoid contents [23]. Antioxidant-rich compounds have previously been shown to protect against the hepatocellular damage induced by oxidative stress [8, 9], mitigating oxidative stress in hepatocytes, facilitating cellular recovery, and minimizing the evidence of intoxication. The hepatotoxicity attributable to CCl₄ is primarily mediated via the generation of reactive oxygen species, leading to oxidative stress [6, 24], causing lipid peroxidation, membrane damage, DNA damage, and inflammation [5, 18]. The amelioration of liver damage markers and reduction in the indicators of fibrosis observed in ASO-treated mice provides evidence that this oil facilitates hepatoprotection via mechanisms associated with the elimination of free radicals [25].

Chronic administration of hepatotoxic agents such as CCl₄ can induce hepatic damage via activation of hepatic stellate cells that promote collagen synthesis and deposition, resulting in liver fibrosis [26]. Although established hepatic fibrosis typically develops following repeated or chronic CCl₄ exposure rather than a single acute dose, the elevated collagen deposition observed at 24 hours in our model represents an early fibrogenic response, likely reflecting acute hepatic stellate cell activation and extracellular matrix remodeling rather than true fibrosis. The ability of ASO to attenuate this early collagen deposition suggests a potential anti-fibrogenic effect that warrants validation in chronic CCl₄ models with repeated dosing over several weeks [27].

In addition to the aforementioned conditions, by mediating the release of different pro-inflammatory cytokines at the site of injury, oxidative stress also plays a key role in inducing inflammation, as has previously been reported with respect to CCl₄-induced liver damage [19]. The protective effects of ASO could thus also be explained in terms of its capacity to replenish endogenous antioxidant capacity and scavenge free radicals, thereby ameliorating the inflammatory response and preventing the progression of fibrosis [16]. These findings are consistent with a growing body of recent literature demonstrating that phenolic-rich plant extracts and seed oils ameliorate CCl₄-induced hepatotoxicity through Nrf2 pathway activation, restoration of glutathione-dependent antioxidant enzymes, and suppression of NF-$\kappa$B-driven cytokine release [12, 13]. Notably, several recent studies have reported comparable hepatoprotective efficacy between natural phytoingredients and silymarin in acute CCl₄ models, lending support to our present findings [14, 15].

On the basis of our findings in this study and those reported in the existing literature, we propose a multi-faceted mechanism for the hepatoprotective effects of ASO against CCl₄-induced liver injury. Firstly, it is assumed that the phenolic compounds and flavonoids identified in ASO (gallic acid, catechin, epicatechin, and ferulic acid) act as direct free radical scavengers, neutralizing the trichloromethyl radicals generated by the metabolism of CCl₄. Secondly, ASO appears to enhance the efficacy of endogenous antioxidant defense systems, as evidenced by the restoration of total antioxidant capacity, potentially mediated via an upregulation of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. Thirdly, the high contents of unsaturated fatty acids (particularly oleic and linoleic acids) in ASO may contribute to stabilizing hepatocyte membranes and reduce the levels of lipid peroxidation, as indicated by a significant reduction in serum MDA levels. Fourthly, it is plausible that the anti-inflammatory properties of ASO are associated with a suppression of the production of pro-inflammatory cytokine and inhibition of inflammatory cell infiltration into hepatic tissues. Finally, the reductions in collagen deposition observed in ASO-treated mice indicate that this oil may prevent hepatic stellate cell activation, thereby inhibiting the early stages of fibrogenesis. Consequently, future studies employing molecular pathway analysis, including assessments of Nrf2 signaling, NF-$\kappa$B activation, and specific cytokine profiles, could provide valuable insights for confirming and further elucidating these proposed mechanisms.

Our finding that with respect to all evaluated parameters, the observed efficacy of ASO was comparable to that of the reference hepatoprotective agent silymarin, is particularly relevant. Specifically, ASO-treated animals showed no significant difference from those treated with silymarin regarding the levels of liver enzymes, lipid profiles, oxidative stress markers, and histological features (p $>$ 0.05 for all comparisons), whereas both treatments contributed to improvements in these parameters compared with CCl₄-treated mice (p 0.05). These findings thus provide a clear indication of the therapeutic potential of ASO and compelling evidence for its development as a natural hepatoprotective agent, potentially offering a safer and more accessible alternative to synthetic hepatoprotective drugs.

Although in this study we utilized multiple complementary methods to assess liver damage and protection, encompassing biochemical indicators, oxidative stress parameters, and comprehensive histological examinations, there are certain limitations that should be acknowledged.

Notably, we primarily examined the preventive effects of ASO administered prior to exposing mice to CCl₄, rather than assessing its therapeutic potential subsequent to the induction of liver damage. A key limitation of this study is the use of a single-dose acute CCl₄ model with tissue collection at 24 hours. True hepatic fibrosis, characterized by progressive collagen cross-linking and fibrous septum formation, requires repeated or chronic CCl₄ administration typically over 4–8 weeks. Therefore, the collagen deposition observed in this study should be interpreted as an early fibrogenic signal rather than established fibrosis, and the anti-fibrotic claims of ASO must be validated using a chronic liver injury model. Moreover, the molecular mechanisms underlying the hepatoprotective effects of ASO, including specific signaling pathways, gene expression profiles, and detection of the bioactive compounds associated with these effects, have yet to be thoroughly elucidated. For example, we did not examine the levels of inflammatory cytokine or markers of hepatic stellate cell activation, which would contribute to enhancing our understanding of the anti-inflammatory and anti-fibrotic mechanisms of ASO. Similarly, we have yet to address dose–response relationships or potential adverse effects associated with the prolonged administration of ASO.

In further research, it will be necessary to focus on evaluating the therapeutic efficacy of ASO in established liver injury models, conduct studies on chronic administration, perform detailed mechanistic investigations, and assess the safety profile of this oil for potential clinical translation.

In this study, we established that ASO has notable hepatoprotective effects against CCl₄-induced liver damage, as evidenced by a normalization of serum biochemical markers and restoration of hepatic architecture. The observed improvements in oxidative stress markers and histological features highlight the potential utility of ASO as a promising natural therapeutic agent for protecting the liver against oxidative stress-induced damage and subsequent fibrosis. Further studies are required to elucidate the molecular mechanisms underlying ASO-mediated hepatoprotective effects and to evaluate its long-term efficacy using chronic liver injury models.

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

THA, AFA, SWA, and FSA performed the experiments, critically revised the manuscript, and approved the final version. MHA, LAA, and AGA contributed to data analysis, revised the manuscript, and approved the final version. HMZ, AAB, and BDA, contributed to results interpretation, critically revised the manuscript, and approved the final version. AAA and HE performed experiments, wrote the original draft, and approved the final manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The research obtained ethical clearance from the Research Ethics Committee of the Faculty of Pharmacy at Taibah University in Madinah, Kingdom of Saudi Arabia (Project Reference Number COPTU-REC-122-20250128). Animal experiments adhere to the 3Rs principle: substitution, reduction, and optimization.

Not applicable.

This research received no external funding.

The authors declare no conflict of interest.