The liver is the primary site for the metabolism and elimination of xenobiotics and endogenous compounds, rendering it particularly vulnerable to chemically induced injury [¹]. Drug-induced liver injury represents a major cause of acute hepatic failure, with acetaminophen overdose being one of the most extensively studied and clinically relevant models of hepatotoxicity [²]. Under therapeutic conditions, acetaminophen is predominantly metabolized through glucuronidation and sulfation pathways; however, excessive dosing leads to cytochrome P450-mediated bioactivation to the highly reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) [³]. Accumulation of NAPQI results in depletion of intracellular glutathione reserves, covalent binding to hepatic proteins, mitochondrial dysfunction, enhanced generation of reactive oxygen species, and subsequent hepatocellular necrosis [⁴]. Acetaminophen-induced liver damage is commonly associated with altered ALT, AST, ALP, bilirubin, and protein levels, which are used as biochemical indicators of liver function [⁵].

Currently available hepatoprotective therapies are limited in efficacy, necessitating the exploration of alternative agents capable of modulating oxidative, inflammatory, and metabolic pathways involved in liver damage [⁶]. While oxidative stress and inflammation are recognized contributors to acetaminophen-induced hepatotoxicity, this study focused specifically on routine serum biochemical markers-ALT, AST, ALP, bilirubin, and circulating protein fractions-as primary endpoints. These biomarkers provide reliable, accessible measures of hepatocellular and hepatobiliary function, reflecting functional impairment and recovery without requiring direct assessment of oxidative or inflammatory pathways [⁷,⁸]. In this context, medicinal plants constitute a valuable source of structurally diverse bioactive compounds with potential hepatoprotective properties [⁹].

P. spicatus (Juss.) Rohr. (Asteraceae) is a perennial herb traditionally used to manage inflammatory disorders, infectious diseases, and hepatic dysfunction [¹⁰]. Phytochemical investigations have revealed the presence of sesquiterpene lactones, triterpenoids, sterols, and related secondary metabolites, several of which exhibit antioxidant, anti-inflammatory, and cytoprotective activities [¹¹]. Experimental studies have reported moderate hepatoprotective effects of P. spicatus extracts against chemically induced liver injury, including toxin and drug-mediated models [¹²]; however, systematic evaluation of organ-specific extracts, particularly roots, in acetaminophen-induced hepatotoxicity remains limited.

Accordingly, the present study was designed to evaluate the effects of the ethanolic root extract of P. spicatus on serum biochemical markers of hepatocellular and hepatobiliary injury in a rat model of acetaminophen-induced acute hepatotoxicity. By focusing on these routine endpoints, the study aims to establish the functional hepatoprotective potential of P. spicatus before mechanistic or histopathological investigations.

Acetaminophen (APAP) is widely used as an analgesic and antipyretic, yet overdose results in severe hepatotoxicity due to metabolic activation and oxidative stress [²⁰]. Under therapeutic conditions, acetaminophen is primarily detoxified via glucuronidation and sulfation pathways; however, a minor fraction is bioactivated by cytochrome P450 enzymes to the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI), which is normally neutralized through conjugation with reduced glutathione (GSH) [²¹]. Following overdose, saturation of conjugation pathways leads to excessive NAPQI formation, hepatic GSH depletion, and covalent binding of NAPQI to cellular macromolecules, triggering lipid peroxidation, calcium dysregulation, mitochondrial dysfunction, and hepatocellular necrosis [²²,²³]. Hepatocellular damage disrupts membrane integrity, resulting in leakage of cytosolic enzymes into the circulation, making serum transaminases and alkaline phosphatase reliable biomarkers of liver injury [²⁴,²⁵]. These biomarkers reflect both hepatocellular membrane integrity and hepatobiliary functional status, and differential modulation of these parameters may indicate selective biochemical effects rather than uniform cytoprotection.

In the present study, acetaminophen administration was associated with elevated serum ALT, AST, ALP, and bilirubin levels, confirming hepatocellular and hepatobiliary dysfunction. Treatment with P. spicatus (PS) root extract attenuated these alterations, with significant reductions observed in ALP at both doses and in bilirubin in a dose-dependent manner. However, ALT and AST levels were not significantly reduced at either dose, indicating that the extract did not demonstrate statistically significant protection against hepatocellular enzyme leakage under the present experimental conditions. The higher dose produced marked reductions in ALP and bilirubin levels, suggesting a possible modulatory effect on hepatobiliary parameters rather than comprehensive hepatocellular protection [²⁶]. Because histopathological assessment and additional cholestatic markers (e.g., GGT or bile acids) were not evaluated, any inference regarding selective canalicular or biliary membrane protection should be interpreted cautiously and considered hypothesis-generating rather than definitive. Accordingly, the observed reductions in ALP and bilirubin, in the absence of significant normalization of ALT and AST, suggest selective modulation of hepatobiliary biochemical parameters rather than comprehensive hepatocellular protection under the present experimental conditions [²⁶]. Similar patterns of partial biochemical modulation, characterized by improvement in cholestatic markers with limited transaminase normalization, have been reported in certain plant-derived hepatoprotective studies, suggesting that phytochemical extracts may differentially influence hepatobiliary and hepatocellular components depending on dose, composition, and injury severity [²⁷,²⁸].

In addition to enzymatic changes, PS treatment at 1000 mg/kg significantly restored serum total protein, albumin, and globulin levels, indicating improved circulating protein levels. Notably, serum protein fractions in the 1000 mg/kg group exceeded values observed in the normal control group. Such supranormal elevations may reflect biological variability in circulating proteins after acute injury and recovery, adaptive compensatory responses of the liver, acute phase reactant changes, or transient shifts in hydration and plasma volume rather than definitive supraphysiological protein synthesis. Without histopathological correlation and comprehensive biomarker profiling, these elevated protein levels should be interpreted cautiously as exploratory observations rather than conclusive evidence of enhanced synthetic capacity [²⁹,³⁰]. Since the liver is the primary site of plasma protein synthesis [³¹], reductions in these parameters are indicative of impaired hepatocellular function in acute hepatic injury [³²]. Restoration of protein levels following PS administration suggests enhancement of hepatocyte functional integrity and biosynthetic activity [³³].

Nevertheless, because transaminase levels were not significantly normalized and structural assessment was not performed, this improvement should be interpreted as functional modulation rather than confirmed cytoprotective regeneration. Thus, the improvements in circulating protein fractions likely reflect partial restoration of hepatic synthetic function rather than confirmed structural regeneration of hepatocytes.

Overall, the findings demonstrate that P. spicatus root extract ameliorated selected biochemical alterations induced by acetaminophen, particularly ALP and bilirubin elevation, and improved hepatic synthetic parameters at the higher dose. These effects indicate selective biochemical improvement and partial functional restoration, but do not establish comprehensive hepatocellular protection. In addition to statistical significance, the magnitude of change in ALP and bilirubin relative to the acetaminophen control group indicates a biologically meaningful attenuation of hepatobiliary dysfunction. However, effect size estimation would further strengthen interpretation in future studies [³⁴,³⁵]. Although oxidative stress is central to acetaminophen-induced hepatotoxicity, oxidative stress markers, antioxidant enzyme activity, and histopathological validation were not assessed in this study; therefore, attribution of antioxidant or membrane-stabilizing mechanisms would be premature [³⁶]. Therefore, any mechanistic interpretation regarding antioxidant, anti-inflammatory, or membrane-stabilizing activity should be considered speculative and requires direct biochemical and structural validation [³⁷]. Future studies should incorporate oxidative stress profiling, molecular pathway analyses, and liver histology to clarify the mechanistic basis of the observed effects. Additionally, the relatively high doses employed (500-1000 mg/kg crude extract) necessitate careful consideration of dose optimization, extract standardization, pharmacokinetic characterization, and safety evaluation before translational extrapolation can be justified [³⁸]. Inclusion of extract-only control groups in future investigations would further help delineate intrinsic hepatic effects independent of acetaminophen-induced injury. Importantly, because an extract-only control group was not included in the present study, the extract’s independent biochemical effects in the absence of an acetaminophen challenge cannot be excluded. Another limitation of the present study is that the crude ethanolic extract was not chemically standardized using chromatographic fingerprinting or quantified marker compounds; therefore, potential variability in phytochemical composition may influence reproducibility. In addition, because the sex distribution within individual groups was not recorded, it was not possible to assess whether males and females responded differently to treatment. This remains a potential source of biological variability that could not be addressed in the present study. This represents a design limitation that should be addressed in future investigations.

In this acetaminophen-induced acute liver injury model, the ethanolic root extract of P. spicatus produced selective biochemical attenuation of hepatobiliary dysfunction, demonstrated by significant reductions in ALP and total bilirubin, as well as restoration of circulating protein fractions at 1000 mg/kg. The absence of significant normalization of ALT and AST indicates that comprehensive hepatocellular protection was not achieved under the present experimental conditions. These findings indicate partial biochemical improvement in hepatobiliary functional markers. Definitive validation will require histological examination, assessment of oxidative stress and inflammatory markers, inclusion of extract-only controls, and standardized pharmacological characterization before translational extrapolation can be made.