Antioxidant, Hepatoprotective, and Anti-inflammatory Activity of an Aspen Bark Phenol Glycoside Complex in vitro

The aim. To study the antioxidant, hepatoprotective, and anti-inflammatory properties of an aspen bark (Populus tremula L.) phenol glycoside complex (ABPG) in vitro.

Materials and methods. ABPG was obtained by solvent extraction and characterized by high-performance liquid chromatography (HPLC). Radical scavenging activity was assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and iron chelating capacity was evaluated by the colorimetric ferrozine-based assay. Cellular effects were studied in HepG2 cells in models of lipotoxicity (oleic and palmitic acids), paracetamol-induced injury, and inflammatory stimulation with lipopolysaccharide (LPS) and interferon γ (IFNγ). Intracellular reactive oxygen species (ROS), reduced glutathione (GSH), neutral lipid content, or cell viability served as endpoints.

Results. ABPG comprised more than 70 individual constituents; its dominant components (as a share of the total HPLC peak area) were dihydroxybenzoic acid (11.5%), methoxybenzoic acid (8.5%), luteolin (7.7%), salicin (5.4%), and hydroxybenzoic acid (1.88%). The complex showed pronounced radical scavenging activity (IC₅₀ = 87.6 µg/ml in the DPPH assay) and chelated Fe²⁺ ions in the amount equivalent to 14.07 mg ethylenediaminetetraacetic acid per gram of the sample. In HepG2 cells, ABPG lowered ROS levels under lipotoxic conditions by 47.6% and prevented intracellular lipid accumulation by 40.4%. In paracetamol-induced injury, it restored cell viability to 77.9%; and under LPS/IFNγ stimulation, it decreased ROS production by 19.2% while raising GSH levels by 42.9%.

Conclusion. The aspen bark phenol glycoside complex modulates the redox state and stress responses of hepatocytes, conferring cytoprotection through redox-dependent mechanisms. These effects underlie its antioxidant, hepatoprotective, and anti-inflammatory effects in vitro.

1. Gan C., Yuan Y., Shen H., Gao J., Kong X., Che Z. et al. Liver diseases: epidemiology, causes, trends and predictions. Signal Transduction and Targeted Therapy. 2025;10(1):33. DOI: 10.1038/s41392-024-02072-z.

2. Sanfeliu-Redondo D., Gibert-Ramos A., Gracia-Sancho J. Cell senescence in liver diseases: pathological mechanism and theranostic opportunity. Nature Reviews Gastroenterology & Hepatology. 2024;21:477–492. DOI: 10.1038/s41575-024-00913-4.

3. Ezhilarasan D., Lakshmi T. A molecular insight into the role of antioxidants in nonalcoholic fatty liver diseases. Oxidative Medicine and Cellular Longevity. 2022;1(2022):9233650. DOI: 10.1155/2022/9233650.

4. Sharma P., Nandave M., Nandave D., Yadav S., Vargas-DeLa-Cruz C., Singh S. et al. Reactive oxygen species (ROS)-mediated oxidative stress in chronic liver diseases and its mitigation by medicinal plants. Am. J. Transl. Res. 2023;15(11):6321.

5. Kukavica B., Škondrić S., Knežević K., Ignjatović T., Mihajlović D., Nakarada Đ. et al. Integrated profiling of essential metals, phenolic compounds, anti-inflammatory and hydroxyl radical scavenging activities for five medicinal plants. Biometals. 2025;38:1863–1880. DOI: 10.1007/s10534-025-00735-0.

6. Gupta M., Singh N., Gulati M., Gupta R., Sudhakar K., Kapoor B. Herbal bioactives in treatment of inflammation: An overview. South African Journal of Botany. 2021;143:205–225. DOI: 10.1016/j.sajb.2021.07.027.

7. Nisca A., Tanase C. Approaches to Extracting Bioactive Compounds from Bark of Various Plants: A Brief Review. Plants. 2025;14(18):2929. DOI: 10.3390/plants14182929.

8. Писарева С.И., Юдина Н.В., Лоскутова Ю.В., Буркова В.Н., Боев С.Г. Влияние механообработки коры осины на антиоксидантные свойства водноспиртовых веществ. Химия растительного сырья. 2008;(3):45–50.

9. Орлова А., Повыдыш М. Обзор методов качественного и количественного анализа танинов в растительном сырье. Химия растительного сырья. 2019;(4):29–45. DOI: 10.14258/jcprm.2019045459.

10. Olszowy M. What is responsible for antioxidant properties of polyphenolic compounds from plants? Plant Physiology and Biochemistry. 2019;144:135–143. DOI: 10.1016/j.plaphy.2019.09.039

11. Lee S.E., Ju E.M., Kim J.H. Free radical scavenging and antioxidant enzyme fortifying activities of extracts from Smilax china root. Experimental & Molecular Medicine. 2001;33(4):263–268. DOI: 10.1038/emm.2001.43.

12. Santos J.S., Alvarenga Brizola V.R., Granato D. High-throughput assay comparison and standardization for metal chelating capacity screening: A proposal and application. Food Chemistry. 2017;214:515–522. DOI: 10.1016/j.foodchem.2016.07.091.

13. Repetto G., del Peso A., Zurita J.L. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature Protocols. 2008;3(7):1125–1131. DOI: 10.1038/nprot.2008.75

14. Yao H. R., Liu J., Plumeri D., Cao Y. B., He T., Lin L. et al. Lipotoxicity in HepG2 cells triggered by free fatty acids. Am. J. Transl. Res. 2011;3(3):284.

15. Zhang M., Lai J., Wu Q., Lai J., Su J., Zhu B. et al. Naringenin induces HepG2 cell apoptosis via ROS-mediated JAK-2/ STAT-3 signaling pathways. Molecules. 2023;28(11):4506. DOI: 10.3390/molecules28114506

16. Cubit I., Rojas S., Kulikowski V., Martinez J., Prabhakar S. Cytokine expression in HepG2 cells and the effects of Budesonide. Journal of ATE. 2024;3(2):98–106. DOI: 10.5281/zenodo.13989027.

17. Chen S., Pu J., Bai J., Yin Y., Wu K., Wang J. et al. EZH2 promotes hepatocellular carcinoma progression through modulating miR-22/galectin-9 axis. J. Exp. Clin. Cancer Res. 2018;37:3. DOI: 10.1186/s13046-017-0670-6.

18. Cox D.P., Cardozo Pelaez F. High throughput method for assessment of cellular reduced glutathione in mammalian cells. Journal of Environmental Protection Science. 2007; 1:23–28.

19. Cosme F., Aires A., Pinto T., Oliveira I., Vilela A., Gonçalves B. A comprehensive review of bioactive tannins in foods and beverages: Functional properties, health benefits, and sensory qualities. Molecules. 2025;30(4):800. DOI: 10.3390/molecules30040800.

20. Xiao J., Guo S., Wang D., An Q. Fenton-like reaction: Recenta advances and new trends. Chemistry. 2024;30(24): e202304337. DOI: 10.1002/chem.202304337.

21. Kim I.H., Choi J.W., Nam T.J. PYP1-4 peptide from Pyropia yezoensis protects against acetaminophen-induced hepatotoxicity in HepG2 cells. Exp. Ther. Med. 2020;19(2):849–860. DOI: 10.3892/etm.2019.8304.

22. Ngo V., Duennwald M.L. Nrf2 and oxidative stress: a general overview of mechanisms and implications in human disease. Antioxidants. 2022;11(12):2345. DOI: 10.3390/antiox11122345.

23. Dhande D., Dhok A., Anjankar A., Nagpure S. Silymarin as an Antioxidant Therapy in Chronic Liver Diseases: A Comprehensive Review. Cureus. 2024;16(8):e67083. DOI: 10.7759/cureus.67083.

24. Surai P.F. Silymarin as a natural antioxidant: an overview of the current evidence and perspectives. Antioxidants. 2015; 4(1):204–247. DOI: 10.3390/antiox4010204.

25. Li Y., Wu Q., Deng Y., Lv H., Qiu J., Chi G. et al. D(-)-Salicin inhibits the LPS-induced inflammation in RAW264.7 cells and mouse models. Int. Immunopharmacol. 2015;26(2): 286–294. DOI: 10.1016/j.intimp.2015.04.016