INVESTIGATION OF THE HEPATOTOXIC EFFECTS OF SOME HERBAL PRODUCTS BY IN VITRO METHOD

Öz

Objective: This study investigated the hepatotoxic effects of the Ayurvedic plants Atractylis lancea, Stevia rebaudiana, and Senecio vulgaris exclusively through in vitro methods. Although many individuals use herbal products to support general health or relieve various symptoms, these products are often perceived as inherently safe due to their botanical origin. Such misconceptions may encourage their uncontrolled short- and long-term use, thereby posing potential health risks. In this context, the hepatotoxic potential of diterpene glycosides and pyrrolizidine alkaloids derived from Asteraceae species was examined in vitro using human hepatocellular carcinoma (HepG2) cell lines to provide insights into their safety profiles. Material and Method: Two complementary cytotoxicity assays—MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and LDH (lactate dehydrogenase)—were applied to HepG2 cells cultured in vitro. Both assays are well-established, widely used, and reliable for assessing hepatocellular injury. Result and Discussion: The results emphasize the necessity of systematic toxicological evaluation of herbal products widely used in traditional medicine. Clarifying the mechanisms of xenobiotic-induced hepatotoxicity through in vitro studies is essential for risk assessment and for the predicting of potential health hazards. This study provides further insight into the hepatotoxic potential of Ayurvedic herbal products and highlights the need for cautious use within public health contexts.

Anahtar Kelimeler

• Ayurvedic plants
• hepatotoxicity
• HepG2 cells
• in vitro

BAZI BİTKİSEL ÜRÜNLERİN HEPATOTOKSİK ETKİLERİNİN İN VİTRO YÖNTEMLE ARAŞTIRILMASI

Öz

Amaç: Bu çalışmada, Ayurvedik bitkiler olan Atractylis lancea, Stevia rebaudiana ve Senecio vulgaris’in hepatotoksik etkileri yalnızca in vitro yöntemlerle araştırılmıştır. Günümüzde birçok birey, genel sağlığını desteklemek veya belirli semptomları hafifletmek amacıyla bitkisel ürünleri tercih etmektedir. Ancak bu ürünler, bitkisel kökenli olmaları nedeniyle çoğunlukla tamamen güvenli oldukları yönünde yanlış bir algıyla değerlendirilmektedir. Bu yanlış inanış, kısa ve uzun vadeli kontrolsüz kullanımı teşvik ederek potansiyel sağlık risklerini artırmaktadır. Bu bağlamda, Asteraceae familyasından elde edilen diterpen glikozitler ve pirolizidin alkaloidlerin hepatotoksik potansiyeli, insan hepatoselüler karsinom (HepG2) hücre hattı kullanılarak in vitro koşullarda incelenmiş ve güvenlik profilleri hakkında bilgi sunulmuştur. Gereç ve Yöntem: İki tamamlayıcı sitotoksisite testi-MTT (3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolyum bromür) ve LDH (laktat dehidrogenaz)-in vitro koşullarda kültüre edilmiş HepG2 hücrelerine uygulanmıştır. Her iki test de hepatoselüler hasarı değerlendirmede iyi tanımlanmış ve yaygın olarak kullanılan yöntemlerdir. Sonuç ve Tartışma: Bulgular, geleneksel tıpta yaygın olarak kullanılan bitkisel ürünlerin sistematik toksikolojik açıdan değerlendirilmesinin önemini vurgulamaktadır. Kseno-biyotiklerin neden olduğu hepatotoksisitenin mekanizmalarının in vitro olarak aydınlatılması, risk değerlendirmesi ve potansiyel sağlık tehlikelerinin öngörülmesi açısından kritik öneme sahiptir. Bu çalışma, Ayurvedik bitkisel ürünlerin hepatotoksik potansiyeline dair mevcut bilgi birikimine katkı sağlamakta ve halk sağlığı bağlamında dikkatli kullanım gerekliliğini ortaya koymaktadır.

Anahtar Kelimeler

• Ayurvedik bitkiler
• hepatotoksisite
• HepG2 hücreleri
• in vitro

Kaynakça

1. 1. Rolnik, A., Olas, B. (2021). The plants of the Asteraceae family as agents in the protection of human health. International Journal of Molecular Sciences, 22(6), 3009. [Crossref]
2. 2. Jaiswal, Y.S., Williams, L.L. (2017). A glimpse of Ayurveda–The forgotten history and principles of Indian traditional medicine. Journal of Traditional and Complementary Medicine, 7(1), 50-53. [Crossref]
3. 3. Mukherjee, P.K., Bahadur, S., Chaudhary, S.K., Kar, A., Mukherjee, K. (2015). Quality related safety issue-evidence-based validation of herbal medicine farm to pharma. In: P.K. Mukherjee (Ed.), Evidence-based Validation of Herbal Medicine (pp. 1-28). Elsevier. [Crossref]
4. 4. Russmann, S., Kullak-Ublick, G.A., Grattagliano, I. (2009). Current concepts of mechanisms in drug-induced hepatotoxicity. Current Medicinal Chemistry, 16(23), 3041-3053. [Crossref]
5. 5. Jaeschke, H., Gores, G.J., Cederbaum, A.I., Hinson, J.A., Pessayre, D., Lemasters, J.J. (2002). Mechanisms of hepatotoxicity. Toxicological Sciences, 65(2), 166-176. [Crossref]
6. 6. King, P.D., Perry, M.C. (2001). Hepatotoxicity of chemotherapy. The Oncologist, 6(2), 162-176. [Crossref]
7. 7. Zorzi, D., Laurent, A., Pawlik, T.M., Lauwers, G.Y., Vauthey, J.N., Abdalla, E.K. (2007). Chemotherapy-associated hepatotoxicity and surgery for colorectal liver metastases. Journal of British Surgery, 94(3), 274-286. [Crossref]
8. 8. Xu, J., Oda, S., Yokoi, T. (2018). Cell-based assay using glutathione-depleted HepaRG and HepG2 human liver cells for predicting drug-induced liver injury. Toxicology in Vitro, 48, 286-301.[Crossref]

9. 9. Chen, X.W., Serag, E.S., Sneed, K.B., Zhou, S.F. (2011). Herbal bioactivation, molecular targets and the toxicity relevance. Chemico-Biological Interactions, 192(3), 161-176. [Crossref]
10. 10. Thompson, M., Jaiswal, Y., Wang, I., and Williams, L. (2017). Hepatotoxicity: Treatment, causes and applications of medicinal plants as therapeutic agents. Journal of Phytopharmacology, 6(3), 186-93. [Crossref]
11. 11. Han, J., Zhu, X., Gao, Z., Xiao, Y., Zhang, J., Wang, P., Fang, J., Li, Y., Zhu, Y., Li, Y., Jin, N., Lu, H., Lin, D., Liu, W. (2023). Antiviral effects of Atractyloside A on the influenza B virus (Victoria strain) infection. Frontiers in Microbiology, 13, 1067725.[Crossref]
12. 12. Allam, A.-W.I., Nassar, A.M., Besheit, S.Y. (2001). Nitrogen fertilizer requirements of Stevia rebaudiana bertoni, under egyptian conditions. Egyptian Journal of Agricultural Research, 79(3), 1005-1018. [Crossref]
13. 13. Mantovaneli, I.C.C., Ferretti, E.C., Simões, M.R., Silva, C.F.da. (2004). The effect of temperature and flow rate on the clarification of the aqueous stevia-extract in a fixed-bed column with zeolites. Brazilian Journal of Chemical Engineering, 21(3), 449-458. [Crossref]
14. 14. Debnath, M. (2007). Clonal propagation and antimicrobial activity of an endemic medicinal plant Stevia rebaudiana. Journal of Medicinal Plants Research, 2(2), 045–051.
15. 15. Pól, J., Hohnová, B., and Hyötyläinen, T. (2007). Characterisation of Stevia rebaudiana by comprehensive two-dimensional liquid chromatography time-of-flight mass spectrometry. Journal of Chromatography A, 1150(1-2), 85-92. [Crossref]
16. 16. Larrey, D. (1997). Hepatotoxicity of herbal remedies. Journal of Hepatology, 26, 47-51. [Crossref]
17. 17. Hartmann, T., Zimmer, M. (1986). Organ-specific distribution and accumulation of pyrrolizidine alkaloids during the life history of two annual Senecio species. Journal of Plant Physiology, 122(1), 67-80. [Crossref]
18. 18. Woolf, G.M. (1994). Acute Hepatitis Associated with the Chinese Herbal Product Jin Bu Huan. Annals of Internal Medicine, 121(10), 729. [Crossref]
19. 19. Nadir, A., Agrawal, S., King, P.D., Marshall, J.B. (1996). Acute hepatitis associated with the use of a Chinese herbal product, ma-huang. American Journal of Gastroenterology (Springer Nature), 91(7).
20. 20. Kessler, R.C., Davis, R.B., Foster, D.F., Van Rompay, M.I., Walters, E.E., Wilkey, S.A., Kaptchuk, T.J., Eisenberg, D.M. (2001). Long-term trends in the use of complementary and alternative medical therapies in the United States. Annals of Internal Medicine, 135(4), 262. [Crossref]
21. 21. De Smet, P.A. (2002). Herbal remedies. New England Journal of Medicine, 347(25), 2046-2056. [Crossref]
22. 22. Kaur, P., Robin, Mehta, R.G., Arora, S., Singh, B. (2018). Progression of conventional hepatic cell culture models to bioengineered HepG2 cells for evaluation of herbal bioactivities. Biotechnology Letters, 40, 881-893. [Crossref]
23. 23. Jie, M., Lin, H., He, Z., Liu, H., Li, H., Lin, J.M. (2018). An on-chip intestine-liver model for multiple drugs absorption and metabolism behavior simulation. Science China Chemistry, 61, 236-242. [Crossref]
24. 24. Khare, N., Chandra, S. (2019). Stevioside mediated chemosensitization studies and cytotoxicity assay on breast cancer cell lines MDA-MB-231 and SKBR3. Saudi Journal of Biological Sciences, 26(7), 1596-1601. [Crossref]
25. 25. Pozzobon, R.G., Rutckeviski, R., Carlotto, J., Schneider, V.S., Cordeiro, L.M.C., Mancarz, G.F.F., de Souza, L.M., Mello, R.G., Smiderle, F.R. (2023). Chemical evaluation of Liquidambar styraciflua L. fruits extracts and their potential as anticancer drugs. Molecules, 28(1), 360. [Crossref]
26. 26. Nuringtyas, T.R., Verpoorte, R., Klinkhamer, P.G., van Oers, M.M., Leiss, K.A. (2014). Toxicity of pyrrolizidine alkaloids to Spodoptera exigua using insect cell lines and injection bioassays. Journal of Chemical Ecology, 40(6), 609-616. [Crossref]
27. 27. Xiong, A., Yang, L., Ji, L., Wang, Z., Yang, X., Chen, Y., Wang, X., Wang, C., Wang, Z. (2012). UPLC-MS based metabolomics study on Senecio scandens and S. vulgaris: An approach for the differentiation of two Senecio herbs with similar morphology but different toxicity. Metabolomics, 8(4), 614-623. [Crossref]
28. 28. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65(1-2), 55-63. [Crossref]
29. 29. Prinz H. (2010). Hill coefficients, dose-response curves and allosteric mechanisms. Journal of Chemical Biology, 3(1), 37-44. [Crossref]
30. 30. Decker, T., Lohmann-Matthes, M.L. (1988). A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. Journal of Immunological Methods, 115(1), 61-69. [Crossref]
31. 31. Experimental Revolution · Micro-Curated | ELK Biotechnology’s new micro sample ELISA kit makes a grand debut!-ELK Biotechnology CO., Ltd. (n.d.). Retrieved September 14, 2025, from https://www.elkbiotech.com/. Accessed date: 20.09.2025.
32. 32. Oh, S.J., Kim, H., Liu, Y., Han, H.K., Kwon, K., Chang, K.H., Park, K., Kim, Y., Shim, K., An, S.S. A., Lee, M.Y. (2014). Incompatibility of silver nanoparticles with lactate dehydrogenase leakage assay for cellular viability test is attributed to protein binding and reactive oxygen species generation. Toxicology Letters, 225(3), 422-432. [Crossref]
33. 33. Fu, P.P., Xia, Q., Lin, G., Chou, M.W. (2004). Pyrrolizidine alkaloids-genotoxicity, metabolism enzymes, metabolic activation, and mechanisms. Drug Metabolism Reviews, 36(1), 1-55. [Crossref]
34. 34. World Health Organization (WHO). (2024). Global health statistics 2024: A comprehensive analysis of worldwide health trends. World Health Organization. https://www.who.int/publications/i/item/9789240 94703
35. 35. Akerele, O. (1993). Nature's medicinal bounty: Don’t throw it away. In World Health Forum 1993, 14(4): 390-395.
36. 36. Ekor, M. (2014). The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Frontiers in Pharmacology, 4, 177. [Crossref]
37. 37. Pozzobon, R.G., Rutckeviski, R., Carlotto, J., Schneider, V.S., Cordeiro, L.M.C., Mancarz, G.F.F., de Souza, L.M., Mello, R.G., Smiderle, F.R. (2023). Chemical evaluation of Liquidambar styraciflua L. fruits extracts and their potential as anticancer drugs. Molecules, 28(1), 360. [Crossref]
38. 38. Stewart, M.J., Steenkamp, V. (2000). The biochemistry and toxicity of atractyloside: A review. Therapeutic Drug Monitoring, 22(6), 641-649. [Crossref]
39. 39. Bender, C., Graziano, S., Zimmermann, B.F. (2015). Study of Stevia rebaudiana bertoni antioxidant activities and cellular properties. International Journal of Food Sciences and Nutrition, 66(5), 553-558. [Crossref]
40. 40. Khare, N., Chandra, S. (2019). Stevioside mediated chemosensitization studies and cytotoxicity assay on breast cancer cell lines MDA-MB-231 and SKBR3. Saudi Journal of Biological Sciences, 26(7), 1596-1601. [Crossref]
41. 41. Chen, J.M., Ding, L., Sui, X.C., Xia, Y.M., Lu,T. (2016). Production of a bioactive sweetener steviolbioside via specific hydrolyzing ester linkage of stevioside with a β-galactosidase. Food Chemistry, 196, 155-160. [Crossref]
42. 42. Yang, M., Ma, J., Ruan, J., Zhang, C., Ye, Y., Fu, P.P.C., Lin, G. (2020). Absorption difference between hepatotoxic pyrrolizidine alkaloids and their N-oxides-Mechanism and its potential toxic impact. Journal of Ethnopharmacology, 249, 112421. [Crossref]
43. 43. Field, R.A., Stegelmeier, B.L., Colegate, S.M., Brown, A.W., Green, B.T. (2015). An in vitro comparison of the cytotoxic potential of selected dehydropyrrolizidine alkaloids and some N-oxides. Toxicon, 97, 36-45. [Crossref]
44. 44. Green, C.E., Segall, H.J., Byard, J.L. (1981). Metabolism, cytotoxicity, and genotoxicity of the pyrrolizidine alkaloid senecionine in primary cultures of rat hepatocytes. Toxicology and Applied Pharmacology, 60(2), 176-185. [Crossref]
45. 45. Griffin, D.S., and Segall, H.J. (1986). Genotoxicity and cytotoxicity of selected pyrrolizidine alkaloids, a possible alkenal metabolite of the alkaloids, and related alkenals. Toxicology and Applied Pharmacology, 86(2), 227-234. [Crossref]
46. 46. Min, Z.O.U., Shuang-Shuang, Y.U., Ke, W.A.N.G., Zhang, D.Y., Xiao-Ming, W.U., Wei-Yi, H.U.A. (2013). Glycosylation of ent-kaurene derivatives and an evaluation of their cytotoxic activities. Chinese Journal of Natural Medicines, 11(3), 289-295. [Crossref]
47. 47. Republic of Türkiye Ministry of Agriculture and Forestry. (n.d.). Retrieved September 14, 2025, from https://www.tarimorman.gov.tr/. Accessed date: 14.09.2025.