International Journal of Anatomy and Research

eISSN 2321-4287 · pISSN 2321-8967

Volume 14 · Issue 2 · Pages 9568–9582 · June 2026

Research Article

Ameliorative Effect of Ferula asafoetida on Formaldehyde-Induced Liver Damage in Male Rats: Biochemical, Histological, and Immunohistochemical Perspectives of Ki-67 Expression

Thulluri Sravani Priya1*, Karthick Selvaraj¹, Y. Divya Prafulla², Saravana Kumar. S¹

¹Department of Anatomy, Meenakshi Medical College Hospital and Research Institute, Meenakshi Academy of Higher Education and Research University, Chennai-631552, India.
²Department of Pathology, Kamineni Institute of Medical Sciences, Narketpally, Hyderabad - 508254, India.

Corresponding author: Thulluri Sravani Priya, PhD Scholar, Department of Anatomy, Meenakshi Medical College Hospital and Research Institute, Meenakshi Academy of Higher Education and Research University, Chennai-631552, India. ORCID: 0009-0001-0549-3137, E-Mail: priya.anjibabu@gmail.com

Author ORCIDs: Karthick Selvaraj: 0000-0001-5977-3305; Y. Divya Prafulla: 0000-0001-8802-8152; Saravana Kumar. S: 0000-0002-7213-2825

Received: 17 April 2026 · Revision received: 30 April 2026 · Accepted: 12 May 2026 · Published: 05 June 2026

DOI: 10.16965/ijar.2026.159

ABSTRACT

Background

Formaldehyde is a common environment and industrial toxicant that is known to cause oxidative stress-mediated hepatotoxicity. Antioxidant and cytoprotective properties of natural compounds like Ferula asafoetida have been explored to alleviate such toxic effects. This study sought to compare the hepatoprotective and anti-proliferative properties of Ferula asafoetida on formaldehyde-induced liver damage in male albino rats, using biochemical, histological, and immunohistochemical methods.

Materials and Methods

Thirty six adult male albino rats were randomly categorized into six groups (n = 6). Animals were given oral formaldehyde (10 mg/kg) and/or Ferula asafoetida extract (25 mg/kg) over a period of 42 days, after which they were allowed a 42 days recovery period in respective groups. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in serum at Days 15, 29, 43, and 85. The weight of the liver was measured at the conclusion of the experiment period. Hematoxylin and eosin staining were used to evaluate histopathological and Ki-67 immunohistochemistry was used to evaluate cellular proliferation. One-way ANOVA with Tukey post hoc test were used to conduct statistical analysis.

Results

Formaldehyde exposure led to a substantial increase in the levels of AST, ALT, and ALP suggestive of progressive hepatocellular and hepatobiliary damage. Histological examination showed that the inflammation, fibrosis and hepatocellular apoptosis were significantly increased with a rise in Ki-67 expression, which indicated an improvement in cell proliferation. A partial response was on the co-administration of Ferula asafoetida to inhibit biochemical changes, histopathological injury and decrease Ki-67. The differences in liver weights of recovery groups were not statistically significant. Although there was some improvement, after the exposure was stopped, hepatic architecture and function could not fully recover.

Conclusion

Ferula asafoetida shows moderate hepatoprotective and anti-proliferative effects against liver toxicity induced by formaldehyde, but does not completely reverse hepatic homeostasis.

Keywords: Formaldehyde; Ferula asafoetida; Hepatotoxicity; Ki-67; Immunohistochemistry; Liver weight.

INTRODUCTION

Formaldehyde is a very common chemical in industries, environment, and biomedical industries, such as its applications in disinfection, textile processing and preservation of biological specimens. Formaldehyde is a strong toxicant due to its high reactive property that can cause profound cellular and molecular damages. Long-term or chronic exposure has been linked to systemic toxicity especially in metabolically active organs like liver. Formaldehyde is particularly toxic to the liver, which is the main location of xenobiotic metabolism and detoxification. Formaldehyde exposure is reported to cause hepatocellular degeneration, inflammatory infiltration and alterations to normal hepatic architecture which eventually causes liver dysfunction [1].

Oxidative stress and inflammation are the main mediators of the pathogenesis of hepatotoxicity caused by formaldehyde. Exposure to formaldehyde stimulates the overproduction of reactive oxygen species (ROS), which cause lipid peroxidation, protein peroxidation as well as DNA damage. This oxidative imbalance disturbs cellular homeostasis and triggers inflammatory and apoptotic signaling pathways. Hepatocellular injury is, therefore, indicated by high serum biomarkers of hepatic dysfunction, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) which are sensitive markers of hepatic dysfunction [2,3]. Continued exposure also leads to hepatobiliary dysfunction and morphological changes of the liver [4,5].

Histopathological analysis offers important information on structural changes of hepatic injury. The toxicity of formaldehyde is marked with periportal inflammation, fibrosis, hepatocellular degeneration, and apoptosis causing the derailment of normal hepatic structure [1]. Besides structural changes, organ weight analysis, especially liver weight, is a significant index of toxicological effect. Immunohistochemical markers like Ki-67 (a nuclear protein expressed during active cell cycle phases) measure cellular proliferation in response to injury and regeneration [5].

Natural compounds with antioxidant and anti-inflammatory effects have been of interest in recent years as potential therapeutic agents to counteract chemical-induced toxicity. Ferula asafoetida, an oleo-gum-resin of the genus Ferula, contains bioactive constituents like ferulic acid, coumarins, and sulfur-containing sesquiterpenes, functioning as an antioxidant, anti-inflammatory, and hepatoprotective agent [1,6-9]. The aim of the current study was to test the hepatoprotective and anti-proliferative properties of Ferula asafoetida on a rat model of formaldehyde-induced toxicity combining biochemical markers (AST, ALT, ALP), liver weight, histopathology, and Ki-67 expression.

MATERIALS AND METHODS

Experimental Animals and Grouping

Thirty-six healthy adult male albino rats (200-220 g) were maintained under standard laboratory conditions (22 ± 2°C, 50-60% humidity, 12h light/dark cycle) with ad libitum access to water and standard pellet diet. Animals were randomly divided into six groups (n = 6 per group): Group 1 (G1, Negative control: distilled water for 42 days); Group 2 (G2, Formaldehyde positive control: 10 mg/kg/day orally for 42 days); Group 3 (G3, Ferula asafoetida extract: 25 mg/kg/day orally for 42 days); Group 4 (G4, Ferula asafoetida 25 mg/kg + Formaldehyde 10 mg/kg orally for 42 days); Group 5 (G5, Recovery negative control: 42 days vehicle followed by 42 days untreated recovery); Group 6 (G6, Formaldehyde recovery: 42 days formaldehyde followed by 42 days untreated recovery) [11-13].

Extract and Chemical Formulations

Ferula asafoetida oleo-gum-resin was extracted via Soxhlet extraction using 70% ethanol and concentrated under reduced pressure at 50°C. Formulations were prepared daily in 0.5% carboxymethyl cellulose sodium (Na-CMC) suspension at 2.5 mg/mL (dosing volume 10 mL/kg). Formaldehyde (37% w/v) was freshly diluted daily in distilled water to 2 mg/mL (dosing schedule 10 mg/kg/day, dose volume 5 mL/kg) [14,15].

Biochemical Analysis and Organ Weight

Blood samples collected from the retroorbital plexus at Days 15, 29, 43, and 85 were centrifuged (3000 rpm, 10 min) to harvest serum. Serum AST, ALT, and ALP activities were quantified using diagnostic enzymatic colorimetric kits on a semi-automated analyzer. At sacrifice (Day 42 for G1-G4; Day 84 for G5-G6), excised liver tissues were blotted and weighed on an analytical digital scale.

Histopathological and Ki-67 Immunohistochemical Analysis

Liver specimens fixed in 10% neutral buffered formalin (24-48 h) were paraffin-embedded, sectioned at 4-5 µm, and stained with Hematoxylin and Eosin (H&E). Semi-quantitative scoring (0 to 3: 0 = none, 1 = slight, 2 = moderate, 3 = severe) was conducted for inflammation, fibrosis, and apoptosis. For immunohistochemistry, heat-induced antigen retrieval in citrate buffer (pH 6.0) was performed on poly-L-lysine slides, endogenous peroxidases quenched with 3% H₂O₂, and sections incubated with primary anti-Ki-67 antibody (AB9260, Sigma-Aldrich/Chemicon), HRP-conjugated secondary antibody, and visualized with 3,3'-diaminobenzidine (DAB) counterstained with hematoxylin. Ki-67 positivity was scored from 0 to 4 based on positive cell percentages.

Statistical Analysis

Data were expressed as Mean ± Standard Deviation (SD). Differences among groups were evaluated using One-Way Analysis of Variance (ANOVA) followed by Tukey's post hoc test in SPSS (Version 27.0). Significance was defined as p < 0.05, and effect size (eta2) was calculated.

RESULTS

At the conclusion of the 24-week experiment period, liver weights were 11.62 ± 0.39 g in G5 (control recovery) and 10.68 ± 1.64 g in G6 (formaldehyde recovery), demonstrating no statistically significant difference (F = 1.87, p = 0.2014, eta2 = 0.16) (Figure 1A).

Serum AST, ALT, and ALP showed progressive escalation over time following formaldehyde intoxication (Figure 1B-D). AST rose significantly by Day 29 (F = 16.58, p < 0.001) and peaked at Day 43 (F = 23.33, p < 0.001) in G2 and G4 vs G1. ALT rose significantly by Day 15 (F = 10.88, p < 0.001) through Day 43 (F = 23.62, p < 0.001). ALP increased markedly on Day 29 (F = 24.04, p = 0.001) and Day 43 (F = 21.25, p < 0.001). Co-administration of Ferula asafoetida (G4) partially attenuated these elevations. During the recovery period (Day 85), G6 maintained significantly elevated AST (F = 19.62, p = 0.001), ALT (F = 19.69, p = 0.001), and ALP (F = 53.48, p < 0.001) compared to G5.

[picture]

Figure 1: Liver weight and serum liver enzyme levels across experimental groups. (A) Liver weights in the recovery groups (G5 vs G6) showed no statistically significant difference; (B-D) progressive escalation of serum AST, ALT, and ALP following formaldehyde intoxication, partially attenuated by co-administration of Ferula asafoetida.

Histopathological evaluation (Figure 2) revealed severe multifocal periportal and peribiliary inflammation, fibrous connective tissue proliferation, and apoptosis in G2 (Table 1). G4 exhibited significantly decreased inflammation (2.00 ± 0.50 vs 3.00 ± 0.00, p = 0.004) and fibrosis (1.00 ± 0.00 vs 2.50 ± 0.50, p = 0.006) compared to G2. Spontaneous recovery in G6 showed partial restoration of hepatic architecture.

[picture]

Figure 2: Histopathological evaluation of liver tissue (H&E) revealed severe multifocal periportal and peribiliary inflammation, fibrous connective tissue proliferation, and apoptosis in G2; G4 exhibited significantly decreased inflammation and fibrosis compared to G2, and spontaneous recovery in G6 showed partial restoration of hepatic architecture.

Table 1: Semi-Quantitative Histopathological Scores of Liver Tissue Across Experimental Groups.

Group | Description | Inflammation (G) Mean ± SD | Fibrosis (F) Mean ± SD | Apoptosis (A) Mean ± SD
G1 | Negative Control | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00
G2 | Formaldehyde | 3.00 ± 0.00*** | 2.50 ± 0.50*** | 2.00 ± 0.00***
G3 | Ferula asafoetida | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00
G4 | FA + Formaldehyde | 2.00 ± 0.50++ | 1.00 ± 0.00++ | 2.00 ± 0.50
G5 | Recovery Control | 1.50 ± 0.50+ | 1.50 ± 0.50+ | 1.00 ± 0.00+
G6 | Formaldehyde Recovery | 0.50 ± 0.50++ | 0.50 ± 0.50++ | 0.50 ± 0.50++

Data expressed as Mean ± SD; Statistical test: One-way ANOVA followed by Tukey's post hoc test. ***p < 0.001 vs G1 (control); +p < 0.05 vs G2 (formaldehyde group); ++p < 0.01 vs G2 (formaldehyde group).

Ki-67 immunohistochemistry demonstrated negligible expression in G1 (0.00 ± 0.00) and G3 (0.00 ± 0.00), severe immunoreactivity in G2 (2.83 ± 0.41, p < 0.001 vs G1), and significant suppression in G4 (1.17 ± 0.41, p = 0.002 vs G2) (Figures 3 and 4). Persistent elevated expression was observed in G6 (2.67 ± 0.52, p < 0.001 vs G1/G5), confirming ongoing regenerative pressure.

[picture]

Figure 3: Ki-67 immunohistochemistry demonstrated negligible expression in G1 and G3, severe immunoreactivity in G2, and significant suppression with co-administration of Ferula asafoetida in G4.

[picture]

Figure 4: Persistent elevated Ki-67 expression was observed in G6 during the recovery period, confirming ongoing regenerative pressure.

DISCUSSION

This investigation demonstrates that formaldehyde causes a progressive cascade of hepatocellular and hepatobiliary damage mediated by oxidative stress and lipid peroxidation [1,2]. Liver weight remained relatively stable, confirming that mass alone is an insensitive indicator of toxic hepatocellular injury.

The sequential elevation of AST/ALT followed by ALP reflects initial hepatocyte disruption progressing to secondary hepatobiliary damage [18]. Ferula asafoetida co-treatment partially ameliorated serum enzyme spikes, periportal inflammation, and fibrosis, attributable to its bioactive ferulic acid and coumarin constituents [1,19]. However, incomplete normalization underscores that severe xenobiotic oxidative stress may exceed the protective ceiling of single herbal regimens.

Ki-67 up-regulation in formaldehyde-treated rats indicates strong reactive proliferative pressure among hepatocytes and sinusoidal cells [5,23,24]. Down-regulation of Ki-67 in the Ferula asafoetida co-treatment group demonstrates its anti-proliferative and cytoprotective properties. Sustained biomarker and histopathological abnormalities during the recovery period (G6) indicate that chemical-induced hepatic lesions do not achieve rapid spontaneous resolution.

CONCLUSION

Ferula asafoetida shows moderate hepatoprotective and anti-proliferative effects against formaldehyde-induced liver toxicity by decreasing serum enzyme levels, attenuating inflammation and fibrosis, and modulating Ki-67 proliferative activity, though complete restoration of hepatic homeostasis is not fully achieved.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the support of the Department of Anatomy and the Institutional Animal Facility for providing the necessary infrastructure and technical assistance to conduct this study.

DECLARATIONS

Ethics approval: Approved by the Institutional Animal Ethics Committee (IAEC) (TBPL/FART/009/24 on 17/08/2024) in accordance with CPCSEA guidelines, Government of India.

Financial disclosure: This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest: None.

Author's contribution: T.S.P., K.S., Y.D.P., and S.S. conceived and planned the experiments. T.S.P., K.S., and Y.D.P. conducted the experiments. T.S.P. and K.S. performed the histomorphometric analysis and statistical evaluation. T.S.P., Y.D.P., and S.S. contributed to sample collection, tissue processing, and histopathological preparation. T.S.P. took the lead in writing the manuscript. All authors provided critical feedback and approved the final manuscript.

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