Phenolic-rich extracts of Teucrium oliverianum confer protection against thioacetamide-induced liver fibrosis in rats: Insights from metabolomics, biochemical and histopathological analysis

Abstract

Background: Hepatic fibrosis unfolds as a pathological buildup of extracellular matrix triggered by liver injury. Thioacetamide (TAA) plays a versatile role across various fields-from industrial processes and laboratory research to chemical stabilization. Teucrium plants, widely traditional plants, owing to its myriads of pharmacological activities.

Methods and findings: T. oliverianum ethanolic (TO-EtOH) and ethyl acetate (TO-EtOAc) extracts were explored for their bioactive metabolites via UHPLC-ESI-qTOF-MS/MS that yielded 48 compounds, mainly flavonoids and phenylethanoid glycosides, alongside phenolic acids, iridoid glycosides, and limonoids. Both extracts showed notable hepatoprotective effects in a thioacetamide (TAA)-induced liver injury model, supporting their therapeutic potential. The TAA group showed a significant increase in AST, ALT, ALP, MDA and TNF-α levels concurrent with a significant decrease of GSH level versus normal control group. In contrast, TO-EtOAC and TO-EtOH administered rats showed a decrease in liver enzymes, including ALT, AST, ALP, total bilirubin, and MDA, and an increase in GSH as compared to the TAA model group. Furthermore, both extracts considerably decreased the overall liver TNF-α content inferring anti-inflammatory action. The histo- and immunohistochemical assays of liver tissue of rats in TAA revealed prominent pathological alterations with bridging fibroplasia in multiple hepatic lobules. A restorative effect that improved hepatic morphology with apparent normal hepatic cells and nominal fibroplasia was evident in the administration of both extracts. Among both extracts, TO-EtOH appeared more effective than TO-EtOAC as manifested by a significant improvement in liver's biochemical parameters and structural organization.

Conclusion: This study provides robust evidence supporting the antifibrotic effects of T. oliverianum in a TAA-induced liver injury model. The anti-proliferative activity and hepatoprotective effects are likely to be mediated by its richness in phenolic acids, flavonoids and phenylethanoids.

Fig 1. UHPLC-ESI-qTOF-MS/MS base peak chromatograms of T. oliverianum above ground parts in negative ionization mode.

A) ethanol extract (TO-EtOH) (A) and ethyl acetate extract (TO-EtOAc) (B).

Fig 2. Representative structure of some metabolites identified in T. oliverianum above-ground parts using UPLC-MS.

(A) phenolic acids, (B) flavonoids/flavonoid glycosides, (C) phenylethanoid glycosides, (D) iridoid glycosides.

Fig 3. Effect of T. oliverianum extracts on liver enzymes and total bilirubin.

A) AST, B) ALT, C) ALP D) total bilirubin. Rats were administered thioacetamide (TAA) 200 mg/kg, i.p., or TO-EtOAC at doses (200 and 400 mg/kg) orally, or rats received TO-EtOH at doses (200 and 400 mg/kg) for a period of 4 weeks. The results are expressed as mean ± SD; Significance was tested at p < 0.05 using one-way ANOVA followed by Tukey’s post-hoc test for comparison (n = 8/group). a significant difference from control, b significant difference from TAA, c significant difference from two doses of TO-EtOAc, d significant difference from two high doses of different types of extract &e significant difference from two doses of TO-EtOH.

Fig 4. Effects of T. oliverianum extracts on hepatic content of GSH and MDA.

A) GSH, B) MDA. Rats were administered thioacetamide (TAA) 200 mg/kg, i.p., or TO-EtOAC at doses (200 and 400 mg/kg) orally, or rats received TO-EtOH at doses (200 and 400 mg/kg) for a period of 4 weeks. The results were expressed as mean ± SD; Significance was tested at p < 0.05 using one-way ANOVA followed by Tukey’s post-hoc test for comparison (n = 8/group). a significant difference from control, b significant difference from TAA, c significant difference from two doses of TO-EtOAc, d significant difference from two high doses of different types of extract &e significant difference from two doses of TO-EtOH.

Fig 5. Effect of T. olivarium extracts on hepatic content of TNF-α.

Rats were administered thioacetamide (TAA) 200 mg/kg, i.p., or TO-EtOAC) at doses (200 and 400 mg/kg) orally, or rats received TO-EtOH at doses (200 and 400 mg/kg) for a period of 4 weeks. The results were expressed as mean ± SD; Significance was tested at p < 0.05 using one-way ANOVA followed by Tukey’s post-hoc test for comparison (n = 8/group). a significant difference from control, b significant difference from TAA, c significant difference from two doses of TO-EtOAc, d significant difference from two high doses of different types of extract &e significant difference from two doses of TO-EtOH.

Fig 6. Photographs showing histopathological alterations in hepatic H&E-stained sections among experimental groups.

Control group showing normal hepatic cords and hepatic triad, TAA intoxicated group showing hepatocellular degeneration and necrosis with portal hepatitis and fibrosis, TO-EtOAc at doses 200 mg/kg showing portal bridging with focal hepatocellular degeneration and necrosis, TO-EtOAc at doses 400 mg/kg scattered hepatocyte suffered from necrobiotic changes with focal mononuclear inflammatory cells, TO-EtOH at doses 200 mg/kg showing nominal inflammatory cells with mild portal bridging, TO-EtOH at doses 400 mg/kg showing apparent normal hepatic cells and nominal fibroplasia, Bar chart presenting the total hepatic lesion score in different experimental groups. The values were expressed as median with interquartile range that evaluated by the Kruskal-Walli’s test followed by Dunn’s Multiple Comparison test. ^ns when P > 0.05, ^** when P < 0.01 and ^*** when P < 0.001.

Fig 7. Photomicrographs of hepatic sections stained with picrosirius red (bright field and polarized light) and hepatic immunohistochemical expression of α-SMA in different experimental groups.